US20130195800A1 - Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof - Google Patents
Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof Download PDFInfo
- Publication number
- US20130195800A1 US20130195800A1 US13/636,473 US201113636473A US2013195800A1 US 20130195800 A1 US20130195800 A1 US 20130195800A1 US 201113636473 A US201113636473 A US 201113636473A US 2013195800 A1 US2013195800 A1 US 2013195800A1
- Authority
- US
- United States
- Prior art keywords
- disease
- protein
- sequences
- fever
- vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000013598 vector Substances 0.000 title claims abstract description 154
- 230000001225 therapeutic effect Effects 0.000 title claims abstract description 126
- 108090000623 proteins and genes Proteins 0.000 title claims description 390
- 102000004169 proteins and genes Human genes 0.000 title claims description 235
- 210000004027 cell Anatomy 0.000 claims abstract description 136
- 239000003446 ligand Substances 0.000 claims abstract description 124
- 239000002955 immunomodulating agent Substances 0.000 claims abstract description 71
- 229940121354 immunomodulator Drugs 0.000 claims abstract description 71
- 238000000034 method Methods 0.000 claims abstract description 66
- 241001465754 Metazoa Species 0.000 claims abstract description 20
- 230000003213 activating effect Effects 0.000 claims abstract description 20
- 102000040430 polynucleotide Human genes 0.000 claims description 192
- 108091033319 polynucleotide Proteins 0.000 claims description 192
- 239000002157 polynucleotide Substances 0.000 claims description 192
- 206010028980 Neoplasm Diseases 0.000 claims description 164
- 108091023040 Transcription factor Proteins 0.000 claims description 112
- 102000040945 Transcription factor Human genes 0.000 claims description 112
- 241000282414 Homo sapiens Species 0.000 claims description 111
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 107
- 201000011510 cancer Diseases 0.000 claims description 102
- 230000006870 function Effects 0.000 claims description 98
- 230000001105 regulatory effect Effects 0.000 claims description 78
- 201000010099 disease Diseases 0.000 claims description 63
- 108010065805 Interleukin-12 Proteins 0.000 claims description 59
- 102000013462 Interleukin-12 Human genes 0.000 claims description 57
- 230000001419 dependent effect Effects 0.000 claims description 48
- 208000035475 disorder Diseases 0.000 claims description 43
- 230000002584 immunomodulator Effects 0.000 claims description 40
- 241000124008 Mammalia Species 0.000 claims description 37
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 33
- -1 a-L-iduronidase Proteins 0.000 claims description 30
- 239000013612 plasmid Substances 0.000 claims description 24
- 108010074328 Interferon-gamma Proteins 0.000 claims description 23
- 108010057988 ecdysone receptor Proteins 0.000 claims description 20
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 claims description 18
- 238000001727 in vivo Methods 0.000 claims description 16
- 239000012190 activator Substances 0.000 claims description 15
- 208000015439 Lysosomal storage disease Diseases 0.000 claims description 12
- 208000010412 Glaucoma Diseases 0.000 claims description 10
- 208000015181 infectious disease Diseases 0.000 claims description 10
- 206010061218 Inflammation Diseases 0.000 claims description 9
- 230000004054 inflammatory process Effects 0.000 claims description 9
- 241000701161 unidentified adenovirus Species 0.000 claims description 9
- 241000700605 Viruses Species 0.000 claims description 8
- 201000001441 melanoma Diseases 0.000 claims description 8
- 230000004044 response Effects 0.000 claims description 8
- 102000000589 Interleukin-1 Human genes 0.000 claims description 7
- 108010002352 Interleukin-1 Proteins 0.000 claims description 7
- 239000002502 liposome Substances 0.000 claims description 7
- 206010053185 Glycogen storage disease type II Diseases 0.000 claims description 6
- 230000007812 deficiency Effects 0.000 claims description 6
- 208000019423 liver disease Diseases 0.000 claims description 6
- 208000032007 Glycogen storage disease due to acid maltase deficiency Diseases 0.000 claims description 5
- 102000006992 Interferon-alpha Human genes 0.000 claims description 5
- 108010047761 Interferon-alpha Proteins 0.000 claims description 5
- 201000004502 glycogen storage disease II Diseases 0.000 claims description 5
- 208000022018 mucopolysaccharidosis type 2 Diseases 0.000 claims description 5
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 claims description 5
- 230000003612 virological effect Effects 0.000 claims description 5
- 208000035473 Communicable disease Diseases 0.000 claims description 4
- 150000002632 lipids Chemical class 0.000 claims description 4
- 201000008482 osteoarthritis Diseases 0.000 claims description 4
- 230000009885 systemic effect Effects 0.000 claims description 4
- 241000701447 unidentified baculovirus Species 0.000 claims description 4
- 241001430294 unidentified retrovirus Species 0.000 claims description 4
- 208000024720 Fabry Disease Diseases 0.000 claims description 3
- 208000015872 Gaucher disease Diseases 0.000 claims description 3
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims description 3
- 102100033448 Lysosomal alpha-glucosidase Human genes 0.000 claims description 3
- 208000022873 Ocular disease Diseases 0.000 claims description 3
- 102000014128 RANK Ligand Human genes 0.000 claims description 3
- 108010025832 RANK Ligand Proteins 0.000 claims description 3
- 241000700584 Simplexvirus Species 0.000 claims description 3
- 230000001684 chronic effect Effects 0.000 claims description 3
- 230000003394 haemopoietic effect Effects 0.000 claims description 3
- 208000032839 leukemia Diseases 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 208000031229 Cardiomyopathies Diseases 0.000 claims description 2
- 241000701459 Caulimovirus Species 0.000 claims description 2
- 241000702421 Dependoparvovirus Species 0.000 claims description 2
- 241000233866 Fungi Species 0.000 claims description 2
- 241000702463 Geminiviridae Species 0.000 claims description 2
- 102000053187 Glucuronidase Human genes 0.000 claims description 2
- 108010060309 Glucuronidase Proteins 0.000 claims description 2
- 206010025323 Lymphomas Diseases 0.000 claims description 2
- 102000005262 Sulfatase Human genes 0.000 claims description 2
- 241000700618 Vaccinia virus Species 0.000 claims description 2
- 229920001222 biopolymer Polymers 0.000 claims description 2
- 230000017074 necrotic cell death Effects 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 210000004180 plasmocyte Anatomy 0.000 claims description 2
- 108060007951 sulfatase Proteins 0.000 claims description 2
- 201000008827 tuberculosis Diseases 0.000 claims description 2
- 230000002792 vascular Effects 0.000 claims description 2
- 206010037660 Pyrexia Diseases 0.000 claims 14
- 208000008955 Mucolipidoses Diseases 0.000 claims 8
- 208000001905 GM2 Gangliosidoses Diseases 0.000 claims 5
- 201000008905 GM2 gangliosidosis Diseases 0.000 claims 5
- 206010056886 Mucopolysaccharidosis I Diseases 0.000 claims 5
- 208000002474 Tinea Diseases 0.000 claims 4
- 230000000366 juvenile effect Effects 0.000 claims 4
- 201000002273 mucopolysaccharidosis II Diseases 0.000 claims 4
- 208000000690 mucopolysaccharidosis VI Diseases 0.000 claims 4
- 208000023504 respiratory system disease Diseases 0.000 claims 4
- 206010061393 typhus Diseases 0.000 claims 4
- 208000000230 African Trypanosomiasis Diseases 0.000 claims 3
- 102100022548 Beta-hexosaminidase subunit alpha Human genes 0.000 claims 3
- 206010005098 Blastomycosis Diseases 0.000 claims 3
- 201000003808 Cystic echinococcosis Diseases 0.000 claims 3
- 208000009366 Echinococcosis Diseases 0.000 claims 3
- 208000032982 Hemorrhagic Fever with Renal Syndrome Diseases 0.000 claims 3
- 101001045440 Homo sapiens Beta-hexosaminidase subunit alpha Proteins 0.000 claims 3
- 208000002979 Influenza in Birds Diseases 0.000 claims 3
- 206010072927 Mucolipidosis type I Diseases 0.000 claims 3
- 206010037742 Rabies Diseases 0.000 claims 3
- 208000021811 Sandhoff disease Diseases 0.000 claims 3
- 206010046851 Uveitis Diseases 0.000 claims 3
- 206010022000 influenza Diseases 0.000 claims 3
- 208000025919 mucopolysaccharidosis type 7 Diseases 0.000 claims 3
- 208000004597 Bacillary angiomatosis Diseases 0.000 claims 2
- 241000283690 Bos taurus Species 0.000 claims 2
- 206010006500 Brucellosis Diseases 0.000 claims 2
- 208000008889 California Encephalitis Diseases 0.000 claims 2
- 241000711506 Canine coronavirus Species 0.000 claims 2
- 208000003732 Cat-scratch disease Diseases 0.000 claims 2
- 206010011668 Cutaneous leishmaniasis Diseases 0.000 claims 2
- 206010012565 Developmental glaucoma Diseases 0.000 claims 2
- 206010014096 Echinococciasis Diseases 0.000 claims 2
- 241000244160 Echinococcus Species 0.000 claims 2
- 208000028506 Familial Exudative Vitreoretinopathies Diseases 0.000 claims 2
- 241000282324 Felis Species 0.000 claims 2
- 208000017462 Galactosialidosis Diseases 0.000 claims 2
- 206010061192 Haemorrhagic fever Diseases 0.000 claims 2
- 201000008327 Haverhill fever Diseases 0.000 claims 2
- 108010053317 Hexosaminidase A Proteins 0.000 claims 2
- 102000016871 Hexosaminidase A Human genes 0.000 claims 2
- 206010023126 Jaundice Diseases 0.000 claims 2
- 201000009908 La Crosse encephalitis Diseases 0.000 claims 2
- 206010024238 Leptospirosis Diseases 0.000 claims 2
- 208000016604 Lyme disease Diseases 0.000 claims 2
- 208000001344 Macular Edema Diseases 0.000 claims 2
- 201000011442 Metachromatic leukodystrophy Diseases 0.000 claims 2
- 206010072928 Mucolipidosis type II Diseases 0.000 claims 2
- 206010028095 Mucopolysaccharidosis IV Diseases 0.000 claims 2
- 206010056893 Mucopolysaccharidosis VII Diseases 0.000 claims 2
- 208000025915 Mucopolysaccharidosis type 6 Diseases 0.000 claims 2
- 208000002537 Neuronal Ceroid-Lipofuscinoses Diseases 0.000 claims 2
- 206010030348 Open-Angle Glaucoma Diseases 0.000 claims 2
- 206010035664 Pneumonia Diseases 0.000 claims 2
- 206010039491 Sarcoma Diseases 0.000 claims 2
- 206010039705 Scleritis Diseases 0.000 claims 2
- 208000000828 Sialic Acid Storage Disease Diseases 0.000 claims 2
- 201000001828 Sly syndrome Diseases 0.000 claims 2
- 206010042175 Streptobacillary fever Diseases 0.000 claims 2
- 241000282898 Sus scrofa Species 0.000 claims 2
- 201000005485 Toxoplasmosis Diseases 0.000 claims 2
- 108010039203 Tripeptidyl-Peptidase 1 Proteins 0.000 claims 2
- 102100034197 Tripeptidyl-peptidase 1 Human genes 0.000 claims 2
- 102100040247 Tumor necrosis factor Human genes 0.000 claims 2
- 208000025865 Ulcer Diseases 0.000 claims 2
- 206010047400 Vibrio infections Diseases 0.000 claims 2
- 206010047505 Visceral leishmaniasis Diseases 0.000 claims 2
- 206010000210 abortion Diseases 0.000 claims 2
- 231100000176 abortion Toxicity 0.000 claims 2
- 239000005557 antagonist Substances 0.000 claims 2
- 206010064097 avian influenza Diseases 0.000 claims 2
- 208000028104 epidemic louse-borne typhus Diseases 0.000 claims 2
- 201000006902 exudative vitreoretinopathy Diseases 0.000 claims 2
- 230000002458 infectious effect Effects 0.000 claims 2
- 208000002780 macular degeneration Diseases 0.000 claims 2
- 201000004015 melioidosis Diseases 0.000 claims 2
- 208000020460 mucolipidosis II alpha/beta Diseases 0.000 claims 2
- 201000007607 neuronal ceroid lipofuscinosis 3 Diseases 0.000 claims 2
- 208000007865 relapsing fever Diseases 0.000 claims 2
- 208000017810 streptobacillary rat-bite fever Diseases 0.000 claims 2
- 208000011580 syndromic disease Diseases 0.000 claims 2
- 231100000397 ulcer Toxicity 0.000 claims 2
- RZQQXRVPPOOCQR-UHFFFAOYSA-N 2,3-dihydro-1,3,4-oxadiazole Chemical compound C1NN=CO1 RZQQXRVPPOOCQR-UHFFFAOYSA-N 0.000 claims 1
- VSCBATMPTLKTOV-UHFFFAOYSA-N 2-tert-butylimino-n,n-diethyl-1,3-dimethyl-1,3,2$l^{5}-diazaphosphinan-2-amine Chemical compound CCN(CC)P1(=NC(C)(C)C)N(C)CCCN1C VSCBATMPTLKTOV-UHFFFAOYSA-N 0.000 claims 1
- 241000606750 Actinobacillus Species 0.000 claims 1
- 208000004142 Acute Retinal Necrosis Syndrome Diseases 0.000 claims 1
- 208000009746 Adult T-Cell Leukemia-Lymphoma Diseases 0.000 claims 1
- 208000016683 Adult T-cell leukemia/lymphoma Diseases 0.000 claims 1
- 208000029602 Alpha-N-acetylgalactosaminidase deficiency Diseases 0.000 claims 1
- 206010001935 American trypanosomiasis Diseases 0.000 claims 1
- 241001465677 Ancylostomatoidea Species 0.000 claims 1
- 102400000068 Angiostatin Human genes 0.000 claims 1
- 108010079709 Angiostatins Proteins 0.000 claims 1
- 241000243790 Angiostrongylus cantonensis Species 0.000 claims 1
- 241000359199 Angiostrongylus costaricensis Species 0.000 claims 1
- 201000002862 Angle-Closure Glaucoma Diseases 0.000 claims 1
- 208000031295 Animal disease Diseases 0.000 claims 1
- 102100022146 Arylsulfatase A Human genes 0.000 claims 1
- 102100031491 Arylsulfatase B Human genes 0.000 claims 1
- 206010068220 Aspartylglucosaminuria Diseases 0.000 claims 1
- 241000271566 Aves Species 0.000 claims 1
- 208000004429 Bacillary Dysentery Diseases 0.000 claims 1
- 208000007198 Bovine Brucellosis Diseases 0.000 claims 1
- 208000011691 Burkitt lymphomas Diseases 0.000 claims 1
- 208000033436 CLN6 disease Diseases 0.000 claims 1
- 241000701931 Canine parvovirus Species 0.000 claims 1
- 241000282465 Canis Species 0.000 claims 1
- 206010007187 Capillariasis Diseases 0.000 claims 1
- 208000002177 Cataract Diseases 0.000 claims 1
- 108010036867 Cerebroside-Sulfatase Proteins 0.000 claims 1
- 102100034480 Ceroid-lipofuscinosis neuronal protein 6 Human genes 0.000 claims 1
- 108010075016 Ceruloplasmin Proteins 0.000 claims 1
- 102100023321 Ceruloplasmin Human genes 0.000 claims 1
- 208000026368 Cestode infections Diseases 0.000 claims 1
- 241000867610 Chlorocebus pygerythrus Species 0.000 claims 1
- 241000867607 Chlorocebus sabaeus Species 0.000 claims 1
- 206010008761 Choriomeningitis lymphocytic Diseases 0.000 claims 1
- 208000005590 Choroidal Neovascularization Diseases 0.000 claims 1
- 206010060823 Choroidal neovascularisation Diseases 0.000 claims 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 claims 1
- 208000001726 Classical Swine Fever Diseases 0.000 claims 1
- 102100031162 Collagen alpha-1(XVIII) chain Human genes 0.000 claims 1
- 206010018325 Congenital glaucomas Diseases 0.000 claims 1
- 206010010741 Conjunctivitis Diseases 0.000 claims 1
- 206010010755 Conjunctivitis viral Diseases 0.000 claims 1
- 201000007336 Cryptococcosis Diseases 0.000 claims 1
- 241000221204 Cryptococcus neoformans Species 0.000 claims 1
- 208000008953 Cryptosporidiosis Diseases 0.000 claims 1
- 206010011502 Cryptosporidiosis infection Diseases 0.000 claims 1
- 206010011777 Cystinosis Diseases 0.000 claims 1
- 206010058202 Cystoid macular oedema Diseases 0.000 claims 1
- 208000011518 Danon disease Diseases 0.000 claims 1
- 201000004624 Dermatitis Diseases 0.000 claims 1
- 206010012504 Dermatophytosis Diseases 0.000 claims 1
- 206010012689 Diabetic retinopathy Diseases 0.000 claims 1
- 206010013029 Diphyllobothriasis Diseases 0.000 claims 1
- 208000000655 Distemper Diseases 0.000 claims 1
- 102100031675 DnaJ homolog subfamily C member 5 Human genes 0.000 claims 1
- 208000019878 Eales disease Diseases 0.000 claims 1
- 206010014584 Encephalitis california Diseases 0.000 claims 1
- 206010014611 Encephalitis venezuelan equine Diseases 0.000 claims 1
- 206010060742 Endocrine ophthalmopathy Diseases 0.000 claims 1
- 108010079505 Endostatins Proteins 0.000 claims 1
- 208000000966 Enoplida Infections Diseases 0.000 claims 1
- 241000224432 Entamoeba histolytica Species 0.000 claims 1
- 208000004232 Enteritis Diseases 0.000 claims 1
- 208000035751 Epidemic nephropathy Diseases 0.000 claims 1
- 206010015084 Episcleritis Diseases 0.000 claims 1
- 241000283073 Equus caballus Species 0.000 claims 1
- 208000002166 Erythema Chronicum Migrans Diseases 0.000 claims 1
- 206010062488 Erythema migrans Diseases 0.000 claims 1
- 208000001948 Farber Lipogranulomatosis Diseases 0.000 claims 1
- 208000033149 Farber disease Diseases 0.000 claims 1
- 241000711475 Feline infectious peritonitis virus Species 0.000 claims 1
- 241000714165 Feline leukemia virus Species 0.000 claims 1
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 claims 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 claims 1
- 206010017533 Fungal infection Diseases 0.000 claims 1
- 201000008892 GM1 Gangliosidosis Diseases 0.000 claims 1
- 208000005577 Gastroenteritis Diseases 0.000 claims 1
- 206010017915 Gastroenteritis shigella Diseases 0.000 claims 1
- 206010017916 Gastroenteritis staphylococcal Diseases 0.000 claims 1
- 101800001586 Ghrelin Proteins 0.000 claims 1
- 102400000442 Ghrelin-28 Human genes 0.000 claims 1
- 241000223783 Glaucoma Species 0.000 claims 1
- 208000010055 Globoid Cell Leukodystrophy Diseases 0.000 claims 1
- 102000004547 Glucosylceramidase Human genes 0.000 claims 1
- 108010017544 Glucosylceramidase Proteins 0.000 claims 1
- 208000001500 Glycogen Storage Disease Type IIb Diseases 0.000 claims 1
- 208000035148 Glycogen storage disease due to LAMP-2 deficiency Diseases 0.000 claims 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 claims 1
- 206010018691 Granuloma Diseases 0.000 claims 1
- 241000606790 Haemophilus Species 0.000 claims 1
- 208000021727 Hantavirus hemorrhagic fever with renal syndrome Diseases 0.000 claims 1
- 206010019143 Hantavirus pulmonary infection Diseases 0.000 claims 1
- 208000032969 Hemorrhagic Septicemia Diseases 0.000 claims 1
- 208000005176 Hepatitis C Diseases 0.000 claims 1
- 208000037319 Hepatitis infectious Diseases 0.000 claims 1
- 201000002563 Histoplasmosis Diseases 0.000 claims 1
- 101000710215 Homo sapiens Ceroid-lipofuscinosis neuronal protein 6 Proteins 0.000 claims 1
- 101000845893 Homo sapiens DnaJ homolog subfamily C member 5 Proteins 0.000 claims 1
- 206010020460 Human T-cell lymphotropic virus type I infection Diseases 0.000 claims 1
- 241000714260 Human T-lymphotropic virus 1 Species 0.000 claims 1
- 241000714259 Human T-lymphotropic virus 2 Species 0.000 claims 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 claims 1
- 208000015178 Hurler syndrome Diseases 0.000 claims 1
- 208000015204 Hurler-Scheie syndrome Diseases 0.000 claims 1
- 108700037017 Hyaluronidase Deficiency Proteins 0.000 claims 1
- 208000005503 Hyaluronidase deficiency Diseases 0.000 claims 1
- 206010053317 Hydrophobia Diseases 0.000 claims 1
- 102100037850 Interferon gamma Human genes 0.000 claims 1
- 208000010038 Ischemic Optic Neuropathy Diseases 0.000 claims 1
- 201000006336 Juvenile glaucoma Diseases 0.000 claims 1
- 208000016028 Korean hemorrhagic fever Diseases 0.000 claims 1
- 208000028226 Krabbe disease Diseases 0.000 claims 1
- 208000004554 Leishmaniasis Diseases 0.000 claims 1
- 206010024229 Leprosy Diseases 0.000 claims 1
- 241000589902 Leptospira Species 0.000 claims 1
- 241000589926 Leptospira interrogans serovar Hardjo Species 0.000 claims 1
- 241000692235 Lipoptena cervi Species 0.000 claims 1
- 241000213879 Lyssa Species 0.000 claims 1
- 241000701076 Macacine alphaherpesvirus 1 Species 0.000 claims 1
- 206010025415 Macular oedema Diseases 0.000 claims 1
- 208000035051 Malignant migrating focal seizures of infancy Diseases 0.000 claims 1
- 201000005505 Measles Diseases 0.000 claims 1
- 206010027234 Meningitis eosinophilic Diseases 0.000 claims 1
- 201000000090 Microsporidiosis Diseases 0.000 claims 1
- 241000893980 Microsporum canis Species 0.000 claims 1
- 241001460074 Microsporum distortum Species 0.000 claims 1
- 206010072929 Mucolipidosis type III Diseases 0.000 claims 1
- 206010072930 Mucolipidosis type IV Diseases 0.000 claims 1
- 102100026502 Mucolipin-1 Human genes 0.000 claims 1
- 208000002678 Mucopolysaccharidoses Diseases 0.000 claims 1
- 208000028781 Mucopolysaccharidosis type 1 Diseases 0.000 claims 1
- 208000000149 Multiple Sulfatase Deficiency Disease Diseases 0.000 claims 1
- 208000035032 Multiple sulfatase deficiency Diseases 0.000 claims 1
- 241000204031 Mycoplasma Species 0.000 claims 1
- 208000031888 Mycoses Diseases 0.000 claims 1
- 108010027520 N-Acetylgalactosamine-4-Sulfatase Proteins 0.000 claims 1
- 102100023282 N-acetylglucosamine-6-sulfatase Human genes 0.000 claims 1
- 108010023320 N-acetylglucosamine-6-sulfatase Proteins 0.000 claims 1
- 208000003226 Necatoriasis Diseases 0.000 claims 1
- 206010028851 Necrosis Diseases 0.000 claims 1
- 208000010359 Newcastle Disease Diseases 0.000 claims 1
- 208000014060 Niemann-Pick disease Diseases 0.000 claims 1
- 206010030113 Oedema Diseases 0.000 claims 1
- 201000005191 Oesophagostomiasis Diseases 0.000 claims 1
- 206010030924 Optic ischaemic neuropathy Diseases 0.000 claims 1
- 208000007792 Orbital Pseudotumor Diseases 0.000 claims 1
- 208000035452 Orbital pseudotumour Diseases 0.000 claims 1
- 241000283973 Oryctolagus cuniculus Species 0.000 claims 1
- 102000008108 Osteoprotegerin Human genes 0.000 claims 1
- 108010035042 Osteoprotegerin Proteins 0.000 claims 1
- 201000010183 Papilledema Diseases 0.000 claims 1
- 208000014645 Pasteurella hemorrhagic septicemia Diseases 0.000 claims 1
- 241001494479 Pecora Species 0.000 claims 1
- 208000007634 Peste-des-Petits-Ruminants Diseases 0.000 claims 1
- 206010035015 Pigmentary glaucoma Diseases 0.000 claims 1
- 206010035148 Plague Diseases 0.000 claims 1
- 206010035673 Pneumonia chlamydial Diseases 0.000 claims 1
- 206010063381 Polypoidal choroidal vasculopathy Diseases 0.000 claims 1
- 206010063664 Presumed ocular histoplasmosis syndrome Diseases 0.000 claims 1
- 208000037006 Progressive epilepsy-intellectual disability syndrome, Finnish type Diseases 0.000 claims 1
- 208000002158 Proliferative Vitreoretinopathy Diseases 0.000 claims 1
- 102100036197 Prosaposin Human genes 0.000 claims 1
- 101710152403 Prosaposin Proteins 0.000 claims 1
- 241000287531 Psittacidae Species 0.000 claims 1
- 206010037151 Psittacosis Diseases 0.000 claims 1
- 206010037688 Q fever Diseases 0.000 claims 1
- 206010064714 Radiation retinopathy Diseases 0.000 claims 1
- 206010057190 Respiratory tract infections Diseases 0.000 claims 1
- 201000001949 Retinal Vasculitis Diseases 0.000 claims 1
- 201000007527 Retinal artery occlusion Diseases 0.000 claims 1
- 206010038886 Retinal oedema Diseases 0.000 claims 1
- 208000014139 Retinal vascular disease Diseases 0.000 claims 1
- 208000007014 Retinitis pigmentosa Diseases 0.000 claims 1
- 206010038923 Retinopathy Diseases 0.000 claims 1
- 206010038933 Retinopathy of prematurity Diseases 0.000 claims 1
- 206010038934 Retinopathy proliferative Diseases 0.000 claims 1
- 201000004282 Rickettsialpox Diseases 0.000 claims 1
- 208000013608 Salla disease Diseases 0.000 claims 1
- 208000006731 Salmonella Food Poisoning Diseases 0.000 claims 1
- 208000025816 Sanfilippo syndrome type A Diseases 0.000 claims 1
- 208000025820 Sanfilippo syndrome type B Diseases 0.000 claims 1
- 208000025802 Sanfilippo syndrome type C Diseases 0.000 claims 1
- 208000025804 Sanfilippo syndrome type D Diseases 0.000 claims 1
- 201000002883 Scheie syndrome Diseases 0.000 claims 1
- 241000242678 Schistosoma Species 0.000 claims 1
- 206010040064 Septic arthritis streptobacillus Diseases 0.000 claims 1
- 208000017460 Sialidosis type 2 Diseases 0.000 claims 1
- 208000000453 Skin Neoplasms Diseases 0.000 claims 1
- 108010061312 Sphingomyelin Phosphodiesterase Proteins 0.000 claims 1
- 241001149963 Sporothrix schenckii Species 0.000 claims 1
- 208000031726 Spotted Fever Group Rickettsiosis Diseases 0.000 claims 1
- 206010041896 St. Louis Encephalitis Diseases 0.000 claims 1
- 206010042254 Strongyloidiasis Diseases 0.000 claims 1
- 206010042265 Sturge-Weber Syndrome Diseases 0.000 claims 1
- 206010043841 Tick-borne fever Diseases 0.000 claims 1
- 235000011941 Tilia x europaea Nutrition 0.000 claims 1
- 241000130764 Tinea Species 0.000 claims 1
- 206010044269 Toxocariasis Diseases 0.000 claims 1
- 206010044608 Trichiniasis Diseases 0.000 claims 1
- 241000893966 Trichophyton verrucosum Species 0.000 claims 1
- 206010067409 Trichophytosis Diseases 0.000 claims 1
- 108700001567 Type I Schindler Disease Proteins 0.000 claims 1
- 206010046306 Upper respiratory tract infection Diseases 0.000 claims 1
- 208000018756 Variant Creutzfeldt-Jakob disease Diseases 0.000 claims 1
- 208000002687 Venezuelan Equine Encephalomyelitis Diseases 0.000 claims 1
- 201000009145 Venezuelan equine encephalitis Diseases 0.000 claims 1
- 241000607598 Vibrio Species 0.000 claims 1
- 208000005914 Viral Conjunctivitis Diseases 0.000 claims 1
- 208000010094 Visna Diseases 0.000 claims 1
- 208000028207 Weil disease Diseases 0.000 claims 1
- 208000026589 Wolman disease Diseases 0.000 claims 1
- 208000003152 Yellow Fever Diseases 0.000 claims 1
- 241000607479 Yersinia pestis Species 0.000 claims 1
- 208000025087 Yersinia pseudotuberculosis infectious disease Diseases 0.000 claims 1
- KZWHEHSUEBTKJM-SLPGGIOYSA-N [(2r,3r,4s,5r)-3,4,5,6-tetrahydroxy-1-oxohexan-2-yl]sulfamic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](C=O)NS(O)(=O)=O KZWHEHSUEBTKJM-SLPGGIOYSA-N 0.000 claims 1
- NARKTLKJPPMFJF-LEJQEAHTSA-N [(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl n-[(2s)-2,6-diaminohexanoyl]sulfamate Chemical compound O[C@@H]1[C@H](O)[C@@H](COS(=O)(=O)NC(=O)[C@@H](N)CCCCN)O[C@H]1N1C2=NC=NC(N)=C2N=C1 NARKTLKJPPMFJF-LEJQEAHTSA-N 0.000 claims 1
- 230000003187 abdominal effect Effects 0.000 claims 1
- 108020002494 acetyltransferase Proteins 0.000 claims 1
- 102000005421 acetyltransferase Human genes 0.000 claims 1
- 102000010126 acid sphingomyelin phosphodiesterase activity proteins Human genes 0.000 claims 1
- 201000006966 adult T-cell leukemia Diseases 0.000 claims 1
- 201000008333 alpha-mannosidosis Diseases 0.000 claims 1
- 208000005067 anisakiasis Diseases 0.000 claims 1
- 201000007058 anterior ischemic optic neuropathy Diseases 0.000 claims 1
- 208000002507 ascaridiasis Diseases 0.000 claims 1
- FZCSTZYAHCUGEM-UHFFFAOYSA-N aspergillomarasmine B Natural products OC(=O)CNC(C(O)=O)CNC(C(O)=O)CC(O)=O FZCSTZYAHCUGEM-UHFFFAOYSA-N 0.000 claims 1
- 201000008680 babesiosis Diseases 0.000 claims 1
- 201000007032 bacterial conjunctivitis Diseases 0.000 claims 1
- 210000003323 beak Anatomy 0.000 claims 1
- 201000006486 beta-mannosidosis Diseases 0.000 claims 1
- 208000005881 bovine spongiform encephalopathy Diseases 0.000 claims 1
- 206010006451 bronchitis Diseases 0.000 claims 1
- 208000014058 canine distemper Diseases 0.000 claims 1
- 208000031406 ceroid lipofuscinosis, neuronal, 4 (Kufs type) Diseases 0.000 claims 1
- 208000024042 cholesterol ester storage disease Diseases 0.000 claims 1
- 208000013760 cholesteryl ester storage disease Diseases 0.000 claims 1
- 208000020832 chronic kidney disease Diseases 0.000 claims 1
- 208000017580 chronic wasting disease Diseases 0.000 claims 1
- 201000001891 corneal deposit Diseases 0.000 claims 1
- 208000035250 cutaneous malignant susceptibility to 1 melanoma Diseases 0.000 claims 1
- 201000010206 cystoid macular edema Diseases 0.000 claims 1
- 235000013365 dairy product Nutrition 0.000 claims 1
- 125000005077 diacylhydrazine group Chemical group 0.000 claims 1
- 208000001848 dysentery Diseases 0.000 claims 1
- 206010014599 encephalitis Diseases 0.000 claims 1
- 201000005901 endemic typhus Diseases 0.000 claims 1
- 206010014801 endophthalmitis Diseases 0.000 claims 1
- 229940007078 entamoeba histolytica Drugs 0.000 claims 1
- 201000009449 eosinophilic meningitis Diseases 0.000 claims 1
- 210000003746 feather Anatomy 0.000 claims 1
- 208000005098 feline infectious peritonitis Diseases 0.000 claims 1
- 201000008049 fucosidosis Diseases 0.000 claims 1
- 230000002538 fungal effect Effects 0.000 claims 1
- GNKDKYIHGQKHHM-RJKLHVOGSA-N ghrelin Chemical compound C([C@H](NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)CN)COC(=O)CCCCCCC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C1=CC=CC=C1 GNKDKYIHGQKHHM-RJKLHVOGSA-N 0.000 claims 1
- 201000006592 giardiasis Diseases 0.000 claims 1
- 201000008977 glycoproteinosis Diseases 0.000 claims 1
- 201000009277 hairy cell leukemia Diseases 0.000 claims 1
- 201000005648 hantavirus pulmonary syndrome Diseases 0.000 claims 1
- 230000002008 hemorrhagic effect Effects 0.000 claims 1
- 208000006454 hepatitis Diseases 0.000 claims 1
- 231100000283 hepatitis Toxicity 0.000 claims 1
- 208000005252 hepatitis A Diseases 0.000 claims 1
- 208000002672 hepatitis B Diseases 0.000 claims 1
- 208000029080 human African trypanosomiasis Diseases 0.000 claims 1
- 238000002513 implantation Methods 0.000 claims 1
- 208000016491 infection by Trypanosoma rhodesiense Diseases 0.000 claims 1
- 208000017476 juvenile neuronal ceroid lipofuscinosis Diseases 0.000 claims 1
- 208000028507 juvenile open angle glaucoma Diseases 0.000 claims 1
- 208000025014 late infantile neuronal ceroid lipofuscinosis Diseases 0.000 claims 1
- 239000004571 lime Substances 0.000 claims 1
- 201000003265 lymphadenitis Diseases 0.000 claims 1
- 208000001419 lymphocytic choriomeningitis Diseases 0.000 claims 1
- 201000010230 macular retinal edema Diseases 0.000 claims 1
- 208000004396 mastitis Diseases 0.000 claims 1
- 201000011475 meningoencephalitis Diseases 0.000 claims 1
- 208000027889 monkey disease Diseases 0.000 claims 1
- 208000005871 monkeypox Diseases 0.000 claims 1
- 201000000626 mucocutaneous leishmaniasis Diseases 0.000 claims 1
- 201000007769 mucolipidosis Diseases 0.000 claims 1
- 208000020468 mucolipidosis III alpha/beta Diseases 0.000 claims 1
- 206010028093 mucopolysaccharidosis Diseases 0.000 claims 1
- 208000012253 mucopolysaccharidosis IVA Diseases 0.000 claims 1
- 208000036710 mucopolysaccharidosis type 3A Diseases 0.000 claims 1
- 208000036709 mucopolysaccharidosis type 3B Diseases 0.000 claims 1
- 208000036707 mucopolysaccharidosis type 3C Diseases 0.000 claims 1
- 208000036725 mucopolysaccharidosis type 3D Diseases 0.000 claims 1
- 208000012226 mucopolysaccharidosis type IIIA Diseases 0.000 claims 1
- 208000012227 mucopolysaccharidosis type IIIB Diseases 0.000 claims 1
- 208000012224 mucopolysaccharidosis type IIIC Diseases 0.000 claims 1
- 208000027333 mucopolysaccharidosis type IIID Diseases 0.000 claims 1
- 208000012091 mucopolysaccharidosis type IVB Diseases 0.000 claims 1
- 201000003142 neovascular glaucoma Diseases 0.000 claims 1
- 208000021971 neovascular inflammatory vitreoretinopathy Diseases 0.000 claims 1
- 201000010756 nephropathia epidemica Diseases 0.000 claims 1
- 201000008051 neuronal ceroid lipofuscinosis Diseases 0.000 claims 1
- 201000007638 neuronal ceroid lipofuscinosis 8 Diseases 0.000 claims 1
- 201000007635 neuronal ceroid lipofuscinosis 8 northern epilepsy variant Diseases 0.000 claims 1
- XXUPLYBCNPLTIW-UHFFFAOYSA-N octadec-7-ynoic acid Chemical compound CCCCCCCCCCC#CCCCCCC(O)=O XXUPLYBCNPLTIW-UHFFFAOYSA-N 0.000 claims 1
- 201000010668 orbital plasma cell granuloma Diseases 0.000 claims 1
- 201000000901 ornithosis Diseases 0.000 claims 1
- 201000008968 osteosarcoma Diseases 0.000 claims 1
- 201000000041 pigment dispersion syndrome Diseases 0.000 claims 1
- 201000006509 pleuropneumonia Diseases 0.000 claims 1
- 230000006785 proliferative vitreoretinopathy Effects 0.000 claims 1
- 201000010108 pycnodysostosis Diseases 0.000 claims 1
- 208000010563 rat-bite fever Diseases 0.000 claims 1
- 208000020029 respiratory tract infectious disease Diseases 0.000 claims 1
- 201000011195 retinal edema Diseases 0.000 claims 1
- 208000004644 retinal vein occlusion Diseases 0.000 claims 1
- 201000000306 sarcoidosis Diseases 0.000 claims 1
- 201000005113 shigellosis Diseases 0.000 claims 1
- 208000011985 sialidosis Diseases 0.000 claims 1
- 201000000849 skin cancer Diseases 0.000 claims 1
- 201000002612 sleeping sickness Diseases 0.000 claims 1
- 201000000539 sparganosis Diseases 0.000 claims 1
- 201000004284 spotted fever Diseases 0.000 claims 1
- 201000005428 steroid-induced glaucoma Diseases 0.000 claims 1
- 208000003265 stomatitis Diseases 0.000 claims 1
- BWMISRWJRUSYEX-SZKNIZGXSA-N terbinafine hydrochloride Chemical compound Cl.C1=CC=C2C(CN(C\C=C\C#CC(C)(C)C)C)=CC=CC2=C1 BWMISRWJRUSYEX-SZKNIZGXSA-N 0.000 claims 1
- 201000006427 tick-borne relapsing fever Diseases 0.000 claims 1
- 201000004647 tinea pedis Diseases 0.000 claims 1
- 230000008359 toxicosis Effects 0.000 claims 1
- 201000006397 traumatic glaucoma Diseases 0.000 claims 1
- 208000003982 trichinellosis Diseases 0.000 claims 1
- 201000007588 trichinosis Diseases 0.000 claims 1
- 201000004978 trichostrongylosis Diseases 0.000 claims 1
- 241001529453 unidentified herpesvirus Species 0.000 claims 1
- 230000009385 viral infection Effects 0.000 claims 1
- 210000004916 vomit Anatomy 0.000 claims 1
- 230000008673 vomiting Effects 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 113
- 238000000338 in vitro Methods 0.000 abstract description 26
- 210000004443 dendritic cell Anatomy 0.000 abstract description 10
- 238000002347 injection Methods 0.000 abstract description 10
- 239000007924 injection Substances 0.000 abstract description 10
- 239000003814 drug Substances 0.000 abstract description 6
- 230000001613 neoplastic effect Effects 0.000 abstract 1
- 235000018102 proteins Nutrition 0.000 description 214
- 150000007523 nucleic acids Chemical group 0.000 description 109
- 241000699666 Mus <mouse, genus> Species 0.000 description 96
- 108091028043 Nucleic acid sequence Proteins 0.000 description 66
- 108020004414 DNA Proteins 0.000 description 60
- 241000700159 Rattus Species 0.000 description 56
- 102000039446 nucleic acids Human genes 0.000 description 56
- 108020004707 nucleic acids Proteins 0.000 description 56
- 125000003275 alpha amino acid group Chemical group 0.000 description 52
- 108090000765 processed proteins & peptides Proteins 0.000 description 51
- 229940117681 interleukin-12 Drugs 0.000 description 50
- 102000004196 processed proteins & peptides Human genes 0.000 description 47
- 229920001184 polypeptide Polymers 0.000 description 45
- 108020001756 ligand binding domains Proteins 0.000 description 38
- 108091027981 Response element Proteins 0.000 description 34
- 102000034527 Retinoid X Receptors Human genes 0.000 description 34
- 108010038912 Retinoid X Receptors Proteins 0.000 description 34
- 102000006255 nuclear receptors Human genes 0.000 description 34
- 108020004017 nuclear receptors Proteins 0.000 description 34
- 241000287828 Gallus gallus Species 0.000 description 33
- 108020005497 Nuclear hormone receptor Proteins 0.000 description 31
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 31
- 102000004127 Cytokines Human genes 0.000 description 30
- 108090000695 Cytokines Proteins 0.000 description 30
- 210000001519 tissue Anatomy 0.000 description 30
- 210000001744 T-lymphocyte Anatomy 0.000 description 28
- 239000012634 fragment Substances 0.000 description 26
- 239000002773 nucleotide Substances 0.000 description 25
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 24
- 125000003729 nucleotide group Chemical group 0.000 description 24
- 230000001939 inductive effect Effects 0.000 description 23
- 230000035897 transcription Effects 0.000 description 23
- 238000013518 transcription Methods 0.000 description 23
- 102000008070 Interferon-gamma Human genes 0.000 description 22
- 210000002865 immune cell Anatomy 0.000 description 22
- 239000000203 mixture Substances 0.000 description 22
- 102000005962 receptors Human genes 0.000 description 22
- 108020003175 receptors Proteins 0.000 description 22
- 108091026890 Coding region Proteins 0.000 description 21
- 229960003130 interferon gamma Drugs 0.000 description 20
- 108010002069 Defensins Proteins 0.000 description 19
- 108010076504 Protein Sorting Signals Proteins 0.000 description 19
- 230000000694 effects Effects 0.000 description 19
- 108020003589 5' Untranslated Regions Proteins 0.000 description 18
- 102000000541 Defensins Human genes 0.000 description 18
- 108700026244 Open Reading Frames Proteins 0.000 description 18
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 18
- 206010060862 Prostate cancer Diseases 0.000 description 17
- 108020001507 fusion proteins Proteins 0.000 description 17
- 102000037865 fusion proteins Human genes 0.000 description 17
- 238000009396 hybridization Methods 0.000 description 17
- 238000011282 treatment Methods 0.000 description 17
- 230000027455 binding Effects 0.000 description 16
- 230000004568 DNA-binding Effects 0.000 description 15
- 108091034117 Oligonucleotide Proteins 0.000 description 15
- 230000004913 activation Effects 0.000 description 13
- 238000001994 activation Methods 0.000 description 13
- 102000053602 DNA Human genes 0.000 description 12
- 102000018679 Tacrolimus Binding Proteins Human genes 0.000 description 12
- 108010027179 Tacrolimus Binding Proteins Proteins 0.000 description 12
- 239000002299 complementary DNA Substances 0.000 description 12
- 230000035772 mutation Effects 0.000 description 12
- 101000611183 Homo sapiens Tumor necrosis factor Proteins 0.000 description 11
- 102100035304 Lymphotactin Human genes 0.000 description 11
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 11
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 11
- 102000057041 human TNF Human genes 0.000 description 11
- 235000019801 trisodium phosphate Nutrition 0.000 description 11
- 102100021663 Baculoviral IAP repeat-containing protein 5 Human genes 0.000 description 10
- 108010008951 Chemokine CXCL12 Proteins 0.000 description 10
- 101001033280 Homo sapiens Cytokine receptor common subunit beta Proteins 0.000 description 10
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 10
- 108010002687 Survivin Proteins 0.000 description 10
- 230000006907 apoptotic process Effects 0.000 description 10
- 230000000295 complement effect Effects 0.000 description 10
- 102000055647 human CSF2RB Human genes 0.000 description 10
- 230000028993 immune response Effects 0.000 description 10
- 238000001890 transfection Methods 0.000 description 10
- 238000013519 translation Methods 0.000 description 10
- 206010006187 Breast cancer Diseases 0.000 description 9
- 102100025248 C-X-C motif chemokine 10 Human genes 0.000 description 9
- YZIWDEXMYWHXDR-UHFFFAOYSA-N COc1cc(cc(OC)c1CN1CC(O)C1)-c1cc(C)c(=O)n(C)c1 Chemical compound COc1cc(cc(OC)c1CN1CC(O)C1)-c1cc(C)c(=O)n(C)c1 YZIWDEXMYWHXDR-UHFFFAOYSA-N 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 108010002350 Interleukin-2 Proteins 0.000 description 9
- 102000000588 Interleukin-2 Human genes 0.000 description 9
- 102100021592 Interleukin-7 Human genes 0.000 description 9
- 108010002586 Interleukin-7 Proteins 0.000 description 9
- 235000001014 amino acid Nutrition 0.000 description 9
- 150000001413 amino acids Chemical class 0.000 description 9
- 206010012601 diabetes mellitus Diseases 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 229940100994 interleukin-7 Drugs 0.000 description 9
- 210000002540 macrophage Anatomy 0.000 description 9
- 238000002560 therapeutic procedure Methods 0.000 description 9
- 230000014616 translation Effects 0.000 description 9
- 208000026310 Breast neoplasm Diseases 0.000 description 8
- 102100036842 C-C motif chemokine 19 Human genes 0.000 description 8
- 102100036170 C-X-C motif chemokine 9 Human genes 0.000 description 8
- 102100032937 CD40 ligand Human genes 0.000 description 8
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 8
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 8
- 101000885654 Homo sapiens Beta-defensin 110 Proteins 0.000 description 8
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 description 8
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 8
- 102000003812 Interleukin-15 Human genes 0.000 description 8
- 108090000172 Interleukin-15 Proteins 0.000 description 8
- 108090000978 Interleukin-4 Proteins 0.000 description 8
- 108010002335 Interleukin-9 Proteins 0.000 description 8
- 102000000585 Interleukin-9 Human genes 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 8
- 102000004473 OX40 Ligand Human genes 0.000 description 8
- 108010042215 OX40 Ligand Proteins 0.000 description 8
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 8
- 102100038358 Prostate-specific antigen Human genes 0.000 description 8
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 8
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 8
- 230000000259 anti-tumor effect Effects 0.000 description 8
- 210000003719 b-lymphocyte Anatomy 0.000 description 8
- 238000001415 gene therapy Methods 0.000 description 8
- 230000003993 interaction Effects 0.000 description 8
- 229940118526 interleukin-9 Drugs 0.000 description 8
- 239000003550 marker Substances 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 108020004999 messenger RNA Proteins 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 230000035755 proliferation Effects 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 8
- 108010082808 4-1BB Ligand Proteins 0.000 description 7
- 102100036301 C-C chemokine receptor type 7 Human genes 0.000 description 7
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 7
- 108010029697 CD40 Ligand Proteins 0.000 description 7
- 101150013553 CD40 gene Proteins 0.000 description 7
- 108010008978 Chemokine CXCL10 Proteins 0.000 description 7
- 206010009944 Colon cancer Diseases 0.000 description 7
- 101000716065 Homo sapiens C-C chemokine receptor type 7 Proteins 0.000 description 7
- 101001019455 Homo sapiens ICOS ligand Proteins 0.000 description 7
- 101000804764 Homo sapiens Lymphotactin Proteins 0.000 description 7
- 108010000521 Human Growth Hormone Proteins 0.000 description 7
- 102000002265 Human Growth Hormone Human genes 0.000 description 7
- 239000000854 Human Growth Hormone Substances 0.000 description 7
- 102100034980 ICOS ligand Human genes 0.000 description 7
- 108090000171 Interleukin-18 Proteins 0.000 description 7
- 102000003810 Interleukin-18 Human genes 0.000 description 7
- 102100030704 Interleukin-21 Human genes 0.000 description 7
- 108010066979 Interleukin-27 Proteins 0.000 description 7
- 102100036678 Interleukin-27 subunit alpha Human genes 0.000 description 7
- 102000004388 Interleukin-4 Human genes 0.000 description 7
- 102100026236 Interleukin-8 Human genes 0.000 description 7
- 102100032101 Tumor necrosis factor ligand superfamily member 9 Human genes 0.000 description 7
- 230000037396 body weight Effects 0.000 description 7
- 208000029742 colonic neoplasm Diseases 0.000 description 7
- 210000002889 endothelial cell Anatomy 0.000 description 7
- 239000003102 growth factor Substances 0.000 description 7
- 229940040731 human interleukin-12 Drugs 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 108010074108 interleukin-21 Proteins 0.000 description 7
- 229940028885 interleukin-4 Drugs 0.000 description 7
- 210000001616 monocyte Anatomy 0.000 description 7
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000012546 transfer Methods 0.000 description 7
- 208000023275 Autoimmune disease Diseases 0.000 description 6
- 108091033380 Coding strand Proteins 0.000 description 6
- UPEZCKBFRMILAV-JNEQICEOSA-N Ecdysone Natural products O=C1[C@H]2[C@@](C)([C@@H]3C([C@@]4(O)[C@@](C)([C@H]([C@H]([C@@H](O)CCC(O)(C)C)C)CC4)CC3)=C1)C[C@H](O)[C@H](O)C2 UPEZCKBFRMILAV-JNEQICEOSA-N 0.000 description 6
- 102100037907 High mobility group protein B1 Human genes 0.000 description 6
- 101001025337 Homo sapiens High mobility group protein B1 Proteins 0.000 description 6
- 108090000467 Interferon-beta Proteins 0.000 description 6
- 102100030698 Interleukin-12 subunit alpha Human genes 0.000 description 6
- 101000597780 Mus musculus Tumor necrosis factor ligand superfamily member 18 Proteins 0.000 description 6
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 6
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 6
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 6
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 6
- UPEZCKBFRMILAV-UHFFFAOYSA-N alpha-Ecdysone Natural products C1C(O)C(O)CC2(C)C(CCC3(C(C(C(O)CCC(C)(C)O)C)CCC33O)C)C3=CC(=O)C21 UPEZCKBFRMILAV-UHFFFAOYSA-N 0.000 description 6
- 230000033228 biological regulation Effects 0.000 description 6
- 239000000306 component Substances 0.000 description 6
- 230000000139 costimulatory effect Effects 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- UPEZCKBFRMILAV-JMZLNJERSA-N ecdysone Chemical compound C1[C@@H](O)[C@@H](O)C[C@]2(C)[C@@H](CC[C@@]3([C@@H]([C@@H]([C@H](O)CCC(C)(C)O)C)CC[C@]33O)C)C3=CC(=O)[C@@H]21 UPEZCKBFRMILAV-JMZLNJERSA-N 0.000 description 6
- 239000002158 endotoxin Substances 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 108090000865 liver X receptors Proteins 0.000 description 6
- 102000004311 liver X receptors Human genes 0.000 description 6
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 6
- 201000002528 pancreatic cancer Diseases 0.000 description 6
- 208000008443 pancreatic carcinoma Diseases 0.000 description 6
- 230000008488 polyadenylation Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 210000002307 prostate Anatomy 0.000 description 6
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 6
- 230000010076 replication Effects 0.000 description 6
- 201000000980 schizophrenia Diseases 0.000 description 6
- 229960002930 sirolimus Drugs 0.000 description 6
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- 230000004614 tumor growth Effects 0.000 description 6
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 6
- 238000011144 upstream manufacturing Methods 0.000 description 6
- 108020005345 3' Untranslated Regions Proteins 0.000 description 5
- 102100025221 CD70 antigen Human genes 0.000 description 5
- 102000019034 Chemokines Human genes 0.000 description 5
- 108010012236 Chemokines Proteins 0.000 description 5
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 5
- 101000947172 Homo sapiens C-X-C motif chemokine 9 Proteins 0.000 description 5
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 description 5
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 5
- 101000655540 Homo sapiens Protransforming growth factor alpha Proteins 0.000 description 5
- 108090001007 Interleukin-8 Proteins 0.000 description 5
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 5
- 206010033128 Ovarian cancer Diseases 0.000 description 5
- 102100032350 Protransforming growth factor alpha Human genes 0.000 description 5
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000002585 base Substances 0.000 description 5
- 210000000349 chromosome Anatomy 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 230000001086 cytosolic effect Effects 0.000 description 5
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 5
- 239000003623 enhancer Substances 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 5
- 230000006801 homologous recombination Effects 0.000 description 5
- 238000002744 homologous recombination Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 229940096397 interleukin-8 Drugs 0.000 description 5
- XKTZWUACRZHVAN-VADRZIEHSA-N interleukin-8 Chemical compound C([C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@@H](NC(C)=O)CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(=O)N1[C@H](CCC1)C(=O)N1[C@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC=1C=CC(O)=CC=1)C(=O)N[C@H](CO)C(=O)N1[C@H](CCC1)C(N)=O)C1=CC=CC=C1 XKTZWUACRZHVAN-VADRZIEHSA-N 0.000 description 5
- 229920006008 lipopolysaccharide Polymers 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- 210000000822 natural killer cell Anatomy 0.000 description 5
- 210000002569 neuron Anatomy 0.000 description 5
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 5
- 230000011664 signaling Effects 0.000 description 5
- 210000002460 smooth muscle Anatomy 0.000 description 5
- 230000002103 transcriptional effect Effects 0.000 description 5
- 102100039829 Beta-defensin 110 Human genes 0.000 description 4
- 108010060434 Co-Repressor Proteins Proteins 0.000 description 4
- 102000008169 Co-Repressor Proteins Human genes 0.000 description 4
- 241000701022 Cytomegalovirus Species 0.000 description 4
- 102000052510 DNA-Binding Proteins Human genes 0.000 description 4
- 101100044298 Drosophila melanogaster fand gene Proteins 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 4
- 101150064015 FAS gene Proteins 0.000 description 4
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 4
- 101000885652 Homo sapiens Beta-defensin 112 Proteins 0.000 description 4
- 101000885656 Homo sapiens Beta-defensin 113 Proteins 0.000 description 4
- 101000885660 Homo sapiens Beta-defensin 116 Proteins 0.000 description 4
- 101000884714 Homo sapiens Beta-defensin 4A Proteins 0.000 description 4
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 description 4
- 101001010600 Homo sapiens Interleukin-12 subunit alpha Proteins 0.000 description 4
- 101000852992 Homo sapiens Interleukin-12 subunit beta Proteins 0.000 description 4
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 description 4
- 101000932478 Homo sapiens Receptor-type tyrosine-protein kinase FLT3 Proteins 0.000 description 4
- 102000003996 Interferon-beta Human genes 0.000 description 4
- 102000003814 Interleukin-10 Human genes 0.000 description 4
- 108090000174 Interleukin-10 Proteins 0.000 description 4
- 102100022883 Nuclear receptor coactivator 3 Human genes 0.000 description 4
- 206010061535 Ovarian neoplasm Diseases 0.000 description 4
- 101100335198 Pneumocystis carinii fol1 gene Proteins 0.000 description 4
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 4
- 101100386928 Rattus norvegicus Defb4 gene Proteins 0.000 description 4
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 4
- 102100020718 Receptor-type tyrosine-protein kinase FLT3 Human genes 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- 108091081024 Start codon Proteins 0.000 description 4
- 108700027336 Suppressor of Cytokine Signaling 1 Proteins 0.000 description 4
- 102100024779 Suppressor of cytokine signaling 1 Human genes 0.000 description 4
- 230000006052 T cell proliferation Effects 0.000 description 4
- 108700009124 Transcription Initiation Site Proteins 0.000 description 4
- 108700019146 Transgenes Proteins 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 210000000612 antigen-presenting cell Anatomy 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 210000004556 brain Anatomy 0.000 description 4
- 230000024245 cell differentiation Effects 0.000 description 4
- 230000000875 corresponding effect Effects 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 238000002641 enzyme replacement therapy Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 238000005734 heterodimerization reaction Methods 0.000 description 4
- 102000049262 human DEFB4A Human genes 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 210000002510 keratinocyte Anatomy 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- 208000014018 liver neoplasm Diseases 0.000 description 4
- 108010019677 lymphotactin Proteins 0.000 description 4
- 210000003712 lysosome Anatomy 0.000 description 4
- 230000001868 lysosomic effect Effects 0.000 description 4
- 230000000626 neurodegenerative effect Effects 0.000 description 4
- 230000003389 potentiating effect Effects 0.000 description 4
- 108020001580 protein domains Proteins 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- 108010085238 Actins Proteins 0.000 description 3
- 102000007469 Actins Human genes 0.000 description 3
- 208000024827 Alzheimer disease Diseases 0.000 description 3
- 102100038495 Bile acid receptor Human genes 0.000 description 3
- 101710085500 C-X-C motif chemokine 9 Proteins 0.000 description 3
- 102000001902 CC Chemokines Human genes 0.000 description 3
- 108010040471 CC Chemokines Proteins 0.000 description 3
- 108010055166 Chemokine CCL5 Proteins 0.000 description 3
- 101710096438 DNA-binding protein Proteins 0.000 description 3
- 241000255601 Drosophila melanogaster Species 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 102100039556 Galectin-4 Human genes 0.000 description 3
- 101000603876 Homo sapiens Bile acid receptor Proteins 0.000 description 3
- 101001002634 Homo sapiens Interleukin-1 alpha Proteins 0.000 description 3
- 101001033249 Homo sapiens Interleukin-1 beta Proteins 0.000 description 3
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 description 3
- 108090001061 Insulin Proteins 0.000 description 3
- 102000014150 Interferons Human genes 0.000 description 3
- 108010050904 Interferons Proteins 0.000 description 3
- 102100039064 Interleukin-3 Human genes 0.000 description 3
- 108010002386 Interleukin-3 Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 229920000057 Mannan Polymers 0.000 description 3
- 102100025082 Melanoma-associated antigen 3 Human genes 0.000 description 3
- 208000008589 Obesity Diseases 0.000 description 3
- 102000043276 Oncogene Human genes 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 102000011755 Phosphoglycerate Kinase Human genes 0.000 description 3
- 102000013674 S-100 Human genes 0.000 description 3
- 108700021018 S100 Proteins 0.000 description 3
- 208000006011 Stroke Diseases 0.000 description 3
- 108010017842 Telomerase Proteins 0.000 description 3
- 239000004098 Tetracycline Substances 0.000 description 3
- 210000004241 Th2 cell Anatomy 0.000 description 3
- 101001099217 Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) Triosephosphate isomerase Proteins 0.000 description 3
- 102000004060 Transforming Growth Factor-beta Type II Receptor Human genes 0.000 description 3
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 description 3
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 3
- 102000004243 Tubulin Human genes 0.000 description 3
- 108090000704 Tubulin Proteins 0.000 description 3
- 230000035508 accumulation Effects 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 230000000840 anti-viral effect Effects 0.000 description 3
- 230000014102 antigen processing and presentation of exogenous peptide antigen via MHC class I Effects 0.000 description 3
- 238000003782 apoptosis assay Methods 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 208000006673 asthma Diseases 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 102000012265 beta-defensin Human genes 0.000 description 3
- 108050002883 beta-defensin Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000000481 breast Anatomy 0.000 description 3
- 125000002091 cationic group Chemical group 0.000 description 3
- 230000022131 cell cycle Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 210000003714 granulocyte Anatomy 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 208000027866 inflammatory disease Diseases 0.000 description 3
- 230000028709 inflammatory response Effects 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 229940076264 interleukin-3 Drugs 0.000 description 3
- 238000001638 lipofection Methods 0.000 description 3
- 201000007270 liver cancer Diseases 0.000 description 3
- 235000020824 obesity Nutrition 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000000770 proinflammatory effect Effects 0.000 description 3
- 230000007115 recruitment Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 230000035882 stress Effects 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 229960002180 tetracycline Drugs 0.000 description 3
- 229930101283 tetracycline Natural products 0.000 description 3
- 235000019364 tetracycline Nutrition 0.000 description 3
- 150000003522 tetracyclines Chemical class 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 201000004384 Alopecia Diseases 0.000 description 2
- 241000238682 Amblyomma americanum Species 0.000 description 2
- 102000009091 Amyloidogenic Proteins Human genes 0.000 description 2
- 108010048112 Amyloidogenic Proteins Proteins 0.000 description 2
- 102100039181 Ankyrin repeat domain-containing protein 1 Human genes 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 108010074708 B7-H1 Antigen Proteins 0.000 description 2
- 102000008096 B7-H1 Antigen Human genes 0.000 description 2
- 102100037913 Beta-defensin 106 Human genes 0.000 description 2
- 102100037909 Beta-defensin 107 Human genes 0.000 description 2
- 102100039828 Beta-defensin 112 Human genes 0.000 description 2
- 102100039807 Beta-defensin 113 Human genes 0.000 description 2
- 102100039803 Beta-defensin 114 Human genes 0.000 description 2
- 102100039801 Beta-defensin 115 Human genes 0.000 description 2
- 102100039804 Beta-defensin 116 Human genes 0.000 description 2
- 208000031648 Body Weight Changes Diseases 0.000 description 2
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 2
- 102100032366 C-C motif chemokine 7 Human genes 0.000 description 2
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 2
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 102000006435 Chemokine CCL21 Human genes 0.000 description 2
- 108010083702 Chemokine CCL21 Proteins 0.000 description 2
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 2
- 208000005243 Chondrosarcoma Diseases 0.000 description 2
- 102100032218 Cytokine-inducible SH2-containing protein Human genes 0.000 description 2
- 101710132484 Cytokine-inducible SH2-containing protein Proteins 0.000 description 2
- 101150003873 DEFB10 gene Proteins 0.000 description 2
- 108010063814 DEFB106 Proteins 0.000 description 2
- 101150090151 DEFB3 gene Proteins 0.000 description 2
- 101150102996 DEFB5 gene Proteins 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 2
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 2
- 101150033466 Defb4 gene Proteins 0.000 description 2
- 101150016350 Defb9 gene Proteins 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 101800004490 Endothelin-1 Proteins 0.000 description 2
- 208000032612 Glial tumor Diseases 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 102100034221 Growth-regulated alpha protein Human genes 0.000 description 2
- 102100040896 Growth/differentiation factor 15 Human genes 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 208000031886 HIV Infections Diseases 0.000 description 2
- 208000037357 HIV infectious disease Diseases 0.000 description 2
- 101000952040 Homo sapiens Beta-defensin 1 Proteins 0.000 description 2
- 101000912247 Homo sapiens Beta-defensin 103 Proteins 0.000 description 2
- 101000951733 Homo sapiens Beta-defensin 105 Proteins 0.000 description 2
- 101000951635 Homo sapiens Beta-defensin 107 Proteins 0.000 description 2
- 101000885657 Homo sapiens Beta-defensin 114 Proteins 0.000 description 2
- 101000885661 Homo sapiens Beta-defensin 115 Proteins 0.000 description 2
- 101000762366 Homo sapiens Bone morphogenetic protein 2 Proteins 0.000 description 2
- 101000797758 Homo sapiens C-C motif chemokine 7 Proteins 0.000 description 2
- 101000608765 Homo sapiens Galectin-4 Proteins 0.000 description 2
- 101001069921 Homo sapiens Growth-regulated alpha protein Proteins 0.000 description 2
- 101000998140 Homo sapiens Interleukin-36 alpha Proteins 0.000 description 2
- 101001040964 Homo sapiens Interleukin-36 receptor antagonist protein Proteins 0.000 description 2
- 101001005719 Homo sapiens Melanoma-associated antigen 3 Proteins 0.000 description 2
- 101001024605 Homo sapiens Next to BRCA1 gene 1 protein Proteins 0.000 description 2
- 101000974356 Homo sapiens Nuclear receptor coactivator 3 Proteins 0.000 description 2
- 101000685719 Homo sapiens Protein S100-A5 Proteins 0.000 description 2
- 101000629617 Homo sapiens Protein sprouty homolog 4 Proteins 0.000 description 2
- 101000871245 Homo sapiens Putative beta-defensin 108A Proteins 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- 101000623857 Homo sapiens Serine/threonine-protein kinase mTOR Proteins 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 206010022489 Insulin Resistance Diseases 0.000 description 2
- 102100026720 Interferon beta Human genes 0.000 description 2
- 102100036701 Interleukin-12 subunit beta Human genes 0.000 description 2
- 102000013264 Interleukin-23 Human genes 0.000 description 2
- 108010065637 Interleukin-23 Proteins 0.000 description 2
- 108010002616 Interleukin-5 Proteins 0.000 description 2
- 102100039897 Interleukin-5 Human genes 0.000 description 2
- 102000004889 Interleukin-6 Human genes 0.000 description 2
- 108090001005 Interleukin-6 Proteins 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 2
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 2
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 2
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 108010009474 Macrophage Inflammatory Proteins Proteins 0.000 description 2
- 102000009571 Macrophage Inflammatory Proteins Human genes 0.000 description 2
- 102100034256 Mucin-1 Human genes 0.000 description 2
- 101000870678 Mus musculus Beta-defensin 4 Proteins 0.000 description 2
- 101000918894 Mus musculus Beta-defensin 8 Proteins 0.000 description 2
- 101100386921 Mus musculus Defb2 gene Proteins 0.000 description 2
- 101100498881 Mus musculus Defb5 gene Proteins 0.000 description 2
- 101100498884 Mus musculus Defb6 gene Proteins 0.000 description 2
- 101100498886 Mus musculus Defb7 gene Proteins 0.000 description 2
- 101001002629 Mus musculus Interleukin-1 alpha Proteins 0.000 description 2
- 101001033286 Mus musculus Interleukin-1 beta Proteins 0.000 description 2
- 101100420477 Mus musculus S100a1 gene Proteins 0.000 description 2
- 101100420483 Mus musculus S100a3 gene Proteins 0.000 description 2
- 101100420488 Mus musculus S100a4 gene Proteins 0.000 description 2
- 101100420491 Mus musculus S100a5 gene Proteins 0.000 description 2
- 101100476480 Mus musculus S100a8 gene Proteins 0.000 description 2
- 101100476484 Mus musculus S100a9 gene Proteins 0.000 description 2
- 101100532402 Mus musculus S100b gene Proteins 0.000 description 2
- 241000186359 Mycobacterium Species 0.000 description 2
- 108090001146 Nuclear Receptor Coactivator 1 Proteins 0.000 description 2
- 108090001145 Nuclear Receptor Coactivator 3 Proteins 0.000 description 2
- 102100037226 Nuclear receptor coactivator 2 Human genes 0.000 description 2
- 108010077524 Peptide Elongation Factor 1 Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 102100033214 Putative beta-defensin 108A Human genes 0.000 description 2
- 101001002626 Rattus norvegicus Interleukin-1 alpha Proteins 0.000 description 2
- 101001076393 Rattus norvegicus Interleukin-1 beta Proteins 0.000 description 2
- 101710100968 Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- 208000001647 Renal Insufficiency Diseases 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 241000714474 Rous sarcoma virus Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 102100023085 Serine/threonine-protein kinase mTOR Human genes 0.000 description 2
- 108700028909 Serum Amyloid A Proteins 0.000 description 2
- 102000054727 Serum Amyloid A Human genes 0.000 description 2
- 102100032800 Spermine oxidase Human genes 0.000 description 2
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 2
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 2
- 230000006044 T cell activation Effects 0.000 description 2
- 102100032938 Telomerase reverse transcriptase Human genes 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000006601 Thymidine Kinase Human genes 0.000 description 2
- 108020004440 Thymidine kinase Proteins 0.000 description 2
- 101710195626 Transcriptional activator protein Proteins 0.000 description 2
- 102400001320 Transforming growth factor alpha Human genes 0.000 description 2
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 2
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 2
- 102000044159 Ubiquitin Human genes 0.000 description 2
- 108090000848 Ubiquitin Proteins 0.000 description 2
- 108091023045 Untranslated Region Proteins 0.000 description 2
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 description 2
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 229960005305 adenosine Drugs 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 231100000360 alopecia Toxicity 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 235000010208 anthocyanin Nutrition 0.000 description 2
- 229930002877 anthocyanin Natural products 0.000 description 2
- 239000004410 anthocyanin Substances 0.000 description 2
- 150000004636 anthocyanins Chemical class 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 230000004579 body weight change Effects 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000011712 cell development Effects 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 230000007969 cellular immunity Effects 0.000 description 2
- 201000007455 central nervous system cancer Diseases 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 108010010918 ecdysteroid receptor Proteins 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 210000003527 eukaryotic cell Anatomy 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 239000003862 glucocorticoid Substances 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 210000002443 helper t lymphocyte Anatomy 0.000 description 2
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 2
- 230000002363 herbicidal effect Effects 0.000 description 2
- 239000004009 herbicide Substances 0.000 description 2
- 239000000833 heterodimer Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 102000046975 human DEFB1 Human genes 0.000 description 2
- 102000055779 human DEFB103A Human genes 0.000 description 2
- 102000051468 human DEFB105A Human genes 0.000 description 2
- 102000055062 human IL12A Human genes 0.000 description 2
- 102000050932 human IL12B Human genes 0.000 description 2
- 102000053863 human S100A5 Human genes 0.000 description 2
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 2
- 210000003016 hypothalamus Anatomy 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 229940079322 interferon Drugs 0.000 description 2
- 108040006870 interleukin-10 receptor activity proteins Proteins 0.000 description 2
- 229940124829 interleukin-23 Drugs 0.000 description 2
- 108090000237 interleukin-24 Proteins 0.000 description 2
- 102000003898 interleukin-24 Human genes 0.000 description 2
- 229940100602 interleukin-5 Drugs 0.000 description 2
- 229940100601 interleukin-6 Drugs 0.000 description 2
- 208000028867 ischemia Diseases 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 201000006370 kidney failure Diseases 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 210000005075 mammary gland Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000011278 mitosis Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 210000000066 myeloid cell Anatomy 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 230000001817 pituitary effect Effects 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 230000037452 priming Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000005522 programmed cell death Effects 0.000 description 2
- 208000017497 prostate disease Diseases 0.000 description 2
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 2
- 230000004043 responsiveness Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012552 review Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000002027 skeletal muscle Anatomy 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 208000017520 skin disease Diseases 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 210000001685 thyroid gland Anatomy 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- 230000005740 tumor formation Effects 0.000 description 2
- 238000010396 two-hybrid screening Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- 239000000260 (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol Substances 0.000 description 1
- HWYCFZUSOBOBIN-AQJXLSMYSA-N (2s)-2-[[(2s)-1-[(2s)-5-amino-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]-3-phenylpropanoyl]amino]-5-oxopentanoyl]pyrrolidine-2-carbonyl]amino]-n-[(2s)-1-[[(2s)-1-amino-1-oxo-3-phenylpropan-2-yl]amino]-5-(diaminome Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)C1=CC=CC=C1 HWYCFZUSOBOBIN-AQJXLSMYSA-N 0.000 description 1
- FQVLRGLGWNWPSS-BXBUPLCLSA-N (4r,7s,10s,13s,16r)-16-acetamido-13-(1h-imidazol-5-ylmethyl)-10-methyl-6,9,12,15-tetraoxo-7-propan-2-yl-1,2-dithia-5,8,11,14-tetrazacycloheptadecane-4-carboxamide Chemical compound N1C(=O)[C@@H](NC(C)=O)CSSC[C@@H](C(N)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C)NC(=O)[C@@H]1CC1=CN=CN1 FQVLRGLGWNWPSS-BXBUPLCLSA-N 0.000 description 1
- HXNBAOLVPAWYLT-NVNXTCNLSA-N (5z)-5-[[5-bromo-2-[(2-bromophenyl)methoxy]phenyl]methylidene]-2-sulfanylidene-1,3-thiazolidin-4-one Chemical compound S\1C(=S)NC(=O)C/1=C/C1=CC(Br)=CC=C1OCC1=CC=CC=C1Br HXNBAOLVPAWYLT-NVNXTCNLSA-N 0.000 description 1
- DWAKNKKXGALPNW-BYPYZUCNSA-N (S)-1-pyrroline-5-carboxylic acid Chemical compound OC(=O)[C@@H]1CCC=N1 DWAKNKKXGALPNW-BYPYZUCNSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 1
- YKBGVTZYEHREMT-KVQBGUIXSA-N 2'-deoxyguanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 YKBGVTZYEHREMT-KVQBGUIXSA-N 0.000 description 1
- CKTSBUTUHBMZGZ-SHYZEUOFSA-N 2'‐deoxycytidine Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 CKTSBUTUHBMZGZ-SHYZEUOFSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- BWRRWBIBNBVHQF-UHFFFAOYSA-N 4-(3-pyridin-2-yl-1,2,4-oxadiazol-5-yl)butanoic acid Chemical compound O1C(CCCC(=O)O)=NC(C=2N=CC=CC=2)=N1 BWRRWBIBNBVHQF-UHFFFAOYSA-N 0.000 description 1
- XAEJIFARBQJLML-UHFFFAOYSA-N 4-[2-(2-cyclopentylidenehydrazinyl)-1,3-thiazol-4-yl]benzonitrile Chemical compound C1=CC(C#N)=CC=C1C1=CSC(NN=C2CCCC2)=N1 XAEJIFARBQJLML-UHFFFAOYSA-N 0.000 description 1
- 102220490683 4-hydroxybenzoate polyprenyltransferase, mitochondrial_F63S_mutation Human genes 0.000 description 1
- 108020005029 5' Flanking Region Proteins 0.000 description 1
- 101710163881 5,6-dihydroxyindole-2-carboxylic acid oxidase Proteins 0.000 description 1
- MSLKMRUEVOYOOZ-VBYMZDBQSA-L 519-62-0 Chemical compound [Mg+2].[N-]1C2=C(C=3[C@H]([C@H](C)C(=CC=4C(=C(C=C)C(=C5)N=4)C)N=3)CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@@H](C(=O)OC)C([O-])=C2C(C)=C1C=C1C(CC)=C(C=O)C5=N1 MSLKMRUEVOYOOZ-VBYMZDBQSA-L 0.000 description 1
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 102100022089 Acyl-[acyl-carrier-protein] hydrolase Human genes 0.000 description 1
- 208000010370 Adenoviridae Infections Diseases 0.000 description 1
- 206010060931 Adenovirus infection Diseases 0.000 description 1
- 102100024394 Adipocyte enhancer-binding protein 1 Human genes 0.000 description 1
- 102400001318 Adrenomedullin Human genes 0.000 description 1
- 101800004616 Adrenomedullin Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 102100034035 Alcohol dehydrogenase 1A Human genes 0.000 description 1
- 102100036826 Aldehyde oxidase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108010079054 Amyloid beta-Protein Precursor Proteins 0.000 description 1
- 102000014303 Amyloid beta-Protein Precursor Human genes 0.000 description 1
- 206010002198 Anaphylactic reaction Diseases 0.000 description 1
- 101710122305 Ankyrin repeat domain-containing protein 1 Proteins 0.000 description 1
- 102100036824 Annexin A8 Human genes 0.000 description 1
- 108050002216 Annexin A8 Proteins 0.000 description 1
- 206010002660 Anoxia Diseases 0.000 description 1
- 241000976983 Anoxia Species 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 108020005544 Antisense RNA Proteins 0.000 description 1
- 241000256844 Apis mellifera Species 0.000 description 1
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- 101100288313 Arabidopsis thaliana KTI4 gene Proteins 0.000 description 1
- 101100377295 Arabidopsis thaliana ZHD11 gene Proteins 0.000 description 1
- 206010051113 Arterial restenosis Diseases 0.000 description 1
- 206010003594 Ataxia telangiectasia Diseases 0.000 description 1
- 102100035029 Ataxin-1 Human genes 0.000 description 1
- 108010032963 Ataxin-1 Proteins 0.000 description 1
- 206010003805 Autism Diseases 0.000 description 1
- 208000020706 Autistic disease Diseases 0.000 description 1
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 1
- 101150033765 BAG1 gene Proteins 0.000 description 1
- 108700034663 BCL2-associated athanogene 1 Proteins 0.000 description 1
- 108091007065 BIRCs Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 201000001321 Bardet-Biedl syndrome Diseases 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 102100025142 Beta-microseminoprotein Human genes 0.000 description 1
- 102100023995 Beta-nerve growth factor Human genes 0.000 description 1
- 208000020925 Bipolar disease Diseases 0.000 description 1
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 1
- 102100025423 Bone morphogenetic protein receptor type-1A Human genes 0.000 description 1
- 102100032312 Brevican core protein Human genes 0.000 description 1
- 101710140080 Brevican core protein Proteins 0.000 description 1
- 102100035875 C-C chemokine receptor type 5 Human genes 0.000 description 1
- 101710149870 C-C chemokine receptor type 5 Proteins 0.000 description 1
- 101150042405 CCN1 gene Proteins 0.000 description 1
- 102100024209 CD177 antigen Human genes 0.000 description 1
- 102100027207 CD27 antigen Human genes 0.000 description 1
- 102100021824 COP9 signalosome complex subunit 5 Human genes 0.000 description 1
- 108090000835 CX3C Chemokine Receptor 1 Proteins 0.000 description 1
- 102100039196 CX3C chemokine receptor 1 Human genes 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- 101100190541 Caenorhabditis elegans pink-1 gene Proteins 0.000 description 1
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102100039510 Cancer/testis antigen 2 Human genes 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 241000701489 Cauliflower mosaic virus Species 0.000 description 1
- 101710119334 Ceramide transfer protein Proteins 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 108010082161 Chemokine CCL19 Proteins 0.000 description 1
- 102000003805 Chemokine CCL19 Human genes 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 108020004998 Chloroplast DNA Proteins 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 208000031879 Chédiak-Higashi syndrome Diseases 0.000 description 1
- 108020004638 Circular DNA Proteins 0.000 description 1
- 208000030808 Clear cell renal carcinoma Diseases 0.000 description 1
- 101100018293 Clostridioides difficile pflE gene Proteins 0.000 description 1
- 102100024484 Codanin-1 Human genes 0.000 description 1
- 101710202267 Codanin-1 Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 206010055114 Colon cancer metastatic Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 208000002330 Congenital Heart Defects Diseases 0.000 description 1
- 206010053138 Congenital aplastic anaemia Diseases 0.000 description 1
- 108091035707 Consensus sequence Proteins 0.000 description 1
- 102100031673 Corneodesmosin Human genes 0.000 description 1
- 101710139375 Corneodesmosin Proteins 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- 101150068240 Cyb5r4 gene Proteins 0.000 description 1
- 108010068192 Cyclin A Proteins 0.000 description 1
- 108010060273 Cyclin A2 Proteins 0.000 description 1
- 102100025191 Cyclin-A2 Human genes 0.000 description 1
- 102100034741 Cyclin-dependent kinase 20 Human genes 0.000 description 1
- 102100033270 Cyclin-dependent kinase inhibitor 1 Human genes 0.000 description 1
- 108010037462 Cyclooxygenase 2 Proteins 0.000 description 1
- 241000252233 Cyprinus carpio Species 0.000 description 1
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 1
- 102100027417 Cytochrome P450 1B1 Human genes 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- YAHZABJORDUQGO-NQXXGFSBSA-N D-ribulose 1,5-bisphosphate Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)C(=O)COP(O)(O)=O YAHZABJORDUQGO-NQXXGFSBSA-N 0.000 description 1
- 102100034157 DNA mismatch repair protein Msh2 Human genes 0.000 description 1
- 102100021147 DNA mismatch repair protein Msh6 Human genes 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 108700020911 DNA-Binding Proteins Proteins 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 241000252212 Danio rerio Species 0.000 description 1
- 102000010170 Death domains Human genes 0.000 description 1
- 108050001718 Death domains Proteins 0.000 description 1
- 102000005721 Death-Associated Protein Kinases Human genes 0.000 description 1
- 108010031042 Death-Associated Protein Kinases Proteins 0.000 description 1
- 208000027219 Deficiency disease Diseases 0.000 description 1
- CKTSBUTUHBMZGZ-UHFFFAOYSA-N Deoxycytidine Natural products O=C1N=C(N)C=CN1C1OC(CO)C(O)C1 CKTSBUTUHBMZGZ-UHFFFAOYSA-N 0.000 description 1
- 102100031149 Deoxyribonuclease gamma Human genes 0.000 description 1
- 208000020401 Depressive disease Diseases 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 102100036912 Desmin Human genes 0.000 description 1
- 108010044052 Desmin Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 101100239693 Dictyostelium discoideum myoD gene Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 108090000204 Dipeptidase 1 Proteins 0.000 description 1
- 102100029707 DnaJ homolog subfamily B member 4 Human genes 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 101001048065 Drosophila melanogaster E3 ubiquitin-protein ligase HRD1 Proteins 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 101150105042 EPS8 gene Proteins 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 208000017701 Endocrine disease Diseases 0.000 description 1
- 102100037241 Endoglin Human genes 0.000 description 1
- 108010036395 Endoglin Proteins 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102100021598 Endoplasmic reticulum aminopeptidase 1 Human genes 0.000 description 1
- 102400000686 Endothelin-1 Human genes 0.000 description 1
- 102100033902 Endothelin-1 Human genes 0.000 description 1
- 102100023721 Ephrin-B2 Human genes 0.000 description 1
- 108010044090 Ephrin-B2 Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 206010015108 Epstein-Barr virus infection Diseases 0.000 description 1
- 101150099168 Erap1 gene Proteins 0.000 description 1
- 102100030910 Eyes absent homolog 4 Human genes 0.000 description 1
- 108010054218 Factor VIII Proteins 0.000 description 1
- 102000001690 Factor VIII Human genes 0.000 description 1
- 201000004939 Fanconi anemia Diseases 0.000 description 1
- 108010087894 Fatty acid desaturases Proteins 0.000 description 1
- 102100024802 Fibroblast growth factor 23 Human genes 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 102100028875 Formylglycine-generating enzyme Human genes 0.000 description 1
- 101710192607 Formylglycine-generating enzyme Proteins 0.000 description 1
- 101150094690 GAL1 gene Proteins 0.000 description 1
- 101150038242 GAL10 gene Proteins 0.000 description 1
- 102100028501 Galanin peptides Human genes 0.000 description 1
- 108010001515 Galectin 4 Proteins 0.000 description 1
- 102100024637 Galectin-10 Human genes 0.000 description 1
- 208000006442 Gastroschisis Diseases 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 0.000 description 1
- 208000007465 Giant cell arteritis Diseases 0.000 description 1
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 1
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 1
- 102100039992 Gliomedin Human genes 0.000 description 1
- 101710177565 Gliomedin Proteins 0.000 description 1
- 102000015626 Glucagon-Like Peptide-2 Receptor Human genes 0.000 description 1
- 108010024044 Glucagon-Like Peptide-2 Receptor Proteins 0.000 description 1
- 102100038958 Glutamate receptor ionotropic, NMDA 3B Human genes 0.000 description 1
- 101710195174 Glutamate receptor ionotropic, NMDA 3B Proteins 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 102000009465 Growth Factor Receptors Human genes 0.000 description 1
- 108010009202 Growth Factor Receptors Proteins 0.000 description 1
- 102000004858 Growth differentiation factor-9 Human genes 0.000 description 1
- 108090001086 Growth differentiation factor-9 Proteins 0.000 description 1
- 101710194460 Growth/differentiation factor 15 Proteins 0.000 description 1
- 102100039939 Growth/differentiation factor 8 Human genes 0.000 description 1
- 101150009006 HIS3 gene Proteins 0.000 description 1
- 102000049983 HMGA1a Human genes 0.000 description 1
- 108700039142 HMGA1a Proteins 0.000 description 1
- 108700010013 HMGB1 Proteins 0.000 description 1
- 102000055207 HMGB1 Human genes 0.000 description 1
- 101100246753 Halobacterium salinarum (strain ATCC 700922 / JCM 11081 / NRC-1) pyrF gene Proteins 0.000 description 1
- 102100027489 Helicase-like transcription factor Human genes 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 108091005904 Hemoglobin subunit beta Proteins 0.000 description 1
- 102100022816 Hemojuvelin Human genes 0.000 description 1
- 208000009889 Herpes Simplex Diseases 0.000 description 1
- 108010068250 Herpes Simplex Virus Protein Vmw65 Proteins 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 102100038720 Histone deacetylase 9 Human genes 0.000 description 1
- 102100021090 Homeobox protein Hox-A9 Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000824278 Homo sapiens Acyl-[acyl-carrier-protein] hydrolase Proteins 0.000 description 1
- 101000833122 Homo sapiens Adipocyte enhancer-binding protein 1 Proteins 0.000 description 1
- 101000780443 Homo sapiens Alcohol dehydrogenase 1A Proteins 0.000 description 1
- 101000928314 Homo sapiens Aldehyde oxidase Proteins 0.000 description 1
- 101000971171 Homo sapiens Apoptosis regulator Bcl-2 Proteins 0.000 description 1
- 101000762379 Homo sapiens Bone morphogenetic protein 4 Proteins 0.000 description 1
- 101000934638 Homo sapiens Bone morphogenetic protein receptor type-1A Proteins 0.000 description 1
- 101000980845 Homo sapiens CD177 antigen Proteins 0.000 description 1
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 description 1
- 101000868215 Homo sapiens CD40 ligand Proteins 0.000 description 1
- 101000896048 Homo sapiens COP9 signalosome complex subunit 5 Proteins 0.000 description 1
- 101000889345 Homo sapiens Cancer/testis antigen 2 Proteins 0.000 description 1
- 101000945708 Homo sapiens Cyclin-dependent kinase 20 Proteins 0.000 description 1
- 101000944380 Homo sapiens Cyclin-dependent kinase inhibitor 1 Proteins 0.000 description 1
- 101000725164 Homo sapiens Cytochrome P450 1B1 Proteins 0.000 description 1
- 101001134036 Homo sapiens DNA mismatch repair protein Msh2 Proteins 0.000 description 1
- 101000968658 Homo sapiens DNA mismatch repair protein Msh6 Proteins 0.000 description 1
- 101000866008 Homo sapiens DnaJ homolog subfamily B member 4 Proteins 0.000 description 1
- 101000938422 Homo sapiens Eyes absent homolog 4 Proteins 0.000 description 1
- 101000846394 Homo sapiens Fibroblast growth factor 19 Proteins 0.000 description 1
- 101001051973 Homo sapiens Fibroblast growth factor 23 Proteins 0.000 description 1
- 101100121078 Homo sapiens GAL gene Proteins 0.000 description 1
- 101100067756 Homo sapiens GAST gene Proteins 0.000 description 1
- 101001066129 Homo sapiens Glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 101000893549 Homo sapiens Growth/differentiation factor 15 Proteins 0.000 description 1
- 101001081105 Homo sapiens Helicase-like transcription factor Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 101000756823 Homo sapiens Hemojuvelin Proteins 0.000 description 1
- 101001032092 Homo sapiens Histone deacetylase 9 Proteins 0.000 description 1
- 101001035237 Homo sapiens Integrin alpha-D Proteins 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 101001008919 Homo sapiens Kallikrein-10 Proteins 0.000 description 1
- 101001139126 Homo sapiens Krueppel-like factor 6 Proteins 0.000 description 1
- 101000798114 Homo sapiens Lactotransferrin Proteins 0.000 description 1
- 101000608935 Homo sapiens Leukosialin Proteins 0.000 description 1
- 101000578784 Homo sapiens Melanoma antigen recognized by T-cells 1 Proteins 0.000 description 1
- 101000896484 Homo sapiens Mitotic checkpoint protein BUB3 Proteins 0.000 description 1
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 description 1
- 101000982032 Homo sapiens Myosin-binding protein C, cardiac-type Proteins 0.000 description 1
- 101000602930 Homo sapiens Nuclear receptor coactivator 2 Proteins 0.000 description 1
- 101000974343 Homo sapiens Nuclear receptor coactivator 4 Proteins 0.000 description 1
- 101000979629 Homo sapiens Nucleoside diphosphate kinase A Proteins 0.000 description 1
- 101000738901 Homo sapiens PMS1 protein homolog 1 Proteins 0.000 description 1
- 101000619708 Homo sapiens Peroxiredoxin-6 Proteins 0.000 description 1
- 101001066878 Homo sapiens Polyribonucleotide nucleotidyltransferase 1, mitochondrial Proteins 0.000 description 1
- 101001132819 Homo sapiens Protein CBFA2T3 Proteins 0.000 description 1
- 101000806511 Homo sapiens Protein DEPP1 Proteins 0.000 description 1
- 101000928406 Homo sapiens Protein diaphanous homolog 3 Proteins 0.000 description 1
- 101001048702 Homo sapiens RNA polymerase II elongation factor ELL2 Proteins 0.000 description 1
- 101000712576 Homo sapiens Ras-related C3 botulinum toxin substrate 3 Proteins 0.000 description 1
- 101000708790 Homo sapiens SPARC-related modular calcium-binding protein 2 Proteins 0.000 description 1
- 101000632270 Homo sapiens Semaphorin-3B Proteins 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 101000616761 Homo sapiens Single-minded homolog 2 Proteins 0.000 description 1
- 101000694017 Homo sapiens Sodium channel protein type 5 subunit alpha Proteins 0.000 description 1
- 101000716693 Homo sapiens Sodium/iodide cotransporter Proteins 0.000 description 1
- 101000625358 Homo sapiens Transcription initiation factor TFIID subunit 2 Proteins 0.000 description 1
- 101000844504 Homo sapiens Transient receptor potential cation channel subfamily M member 4 Proteins 0.000 description 1
- 101000653679 Homo sapiens Translationally-controlled tumor protein Proteins 0.000 description 1
- 101000845176 Homo sapiens Tsukushi Proteins 0.000 description 1
- 101000830596 Homo sapiens Tumor necrosis factor ligand superfamily member 15 Proteins 0.000 description 1
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 description 1
- 101000837574 Homo sapiens Ubiquitin-conjugating enzyme E2 W Proteins 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 208000035150 Hypercholesterolemia Diseases 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 1
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 102000000521 Immunophilins Human genes 0.000 description 1
- 108010016648 Immunophilins Proteins 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 102000055031 Inhibitor of Apoptosis Proteins Human genes 0.000 description 1
- 102000048143 Insulin-Like Growth Factor II Human genes 0.000 description 1
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 1
- 102000004374 Insulin-like growth factor binding protein 3 Human genes 0.000 description 1
- 108090000965 Insulin-like growth factor binding protein 3 Proteins 0.000 description 1
- 102100039904 Integrin alpha-D Human genes 0.000 description 1
- 102000005755 Intercellular Signaling Peptides and Proteins Human genes 0.000 description 1
- 108010070716 Intercellular Signaling Peptides and Proteins Proteins 0.000 description 1
- 102100037874 Intercellular adhesion molecule 4 Human genes 0.000 description 1
- 101710148793 Intercellular adhesion molecule 4 Proteins 0.000 description 1
- 108010011301 Interleukin-12 Subunit p35 Proteins 0.000 description 1
- 102000014154 Interleukin-12 Subunit p35 Human genes 0.000 description 1
- 108010011429 Interleukin-12 Subunit p40 Proteins 0.000 description 1
- 102000014158 Interleukin-12 Subunit p40 Human genes 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- 208000016286 Iron metabolism disease Diseases 0.000 description 1
- 102100027613 Kallikrein-10 Human genes 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 102100022260 Killin Human genes 0.000 description 1
- 101710193777 Killin Proteins 0.000 description 1
- 102100020679 Krueppel-like factor 6 Human genes 0.000 description 1
- 108010041081 Kv Channel-Interacting Proteins Proteins 0.000 description 1
- 102100033173 Kv channel-interacting protein 1 Human genes 0.000 description 1
- 102100032241 Lactotransferrin Human genes 0.000 description 1
- 206010056715 Laurence-Moon-Bardet-Biedl syndrome Diseases 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 241000238114 Leptuca pugilator Species 0.000 description 1
- 108010004098 Leucyl aminopeptidase Proteins 0.000 description 1
- 102000002704 Leucyl aminopeptidase Human genes 0.000 description 1
- 102100039564 Leukosialin Human genes 0.000 description 1
- 108010051335 Lipocalin-2 Proteins 0.000 description 1
- 102000013519 Lipocalin-2 Human genes 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 241000254022 Locusta migratoria Species 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 102100022699 Lymphoid enhancer-binding factor 1 Human genes 0.000 description 1
- 108090001093 Lymphoid enhancer-binding factor 1 Proteins 0.000 description 1
- 108010091221 Lymphotoxin beta Receptor Proteins 0.000 description 1
- 229910015837 MSH2 Inorganic materials 0.000 description 1
- 108010072582 Matrilin Proteins Proteins 0.000 description 1
- 102100033670 Matrilin-3 Human genes 0.000 description 1
- 102000000422 Matrix Metalloproteinase 3 Human genes 0.000 description 1
- 108010016160 Matrix Metalloproteinase 3 Proteins 0.000 description 1
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 1
- 101710204288 Melanoma-associated antigen 3 Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102100034028 Membrane-bound transcription factor site-1 protease Human genes 0.000 description 1
- 208000029725 Metabolic bone disease Diseases 0.000 description 1
- 102100026261 Metalloproteinase inhibitor 3 Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 108010092801 Midkine Proteins 0.000 description 1
- 102000016776 Midkine Human genes 0.000 description 1
- 108020005196 Mitochondrial DNA Proteins 0.000 description 1
- 102100040200 Mitochondrial uncoupling protein 2 Human genes 0.000 description 1
- 101710112393 Mitochondrial uncoupling protein 2 Proteins 0.000 description 1
- 102100021718 Mitotic checkpoint protein BUB3 Human genes 0.000 description 1
- 108010074633 Mixed Function Oxygenases Proteins 0.000 description 1
- 102000008109 Mixed Function Oxygenases Human genes 0.000 description 1
- 241000713333 Mouse mammary tumor virus Species 0.000 description 1
- 108010008707 Mucin-1 Proteins 0.000 description 1
- 102100023123 Mucin-16 Human genes 0.000 description 1
- 102100034263 Mucin-2 Human genes 0.000 description 1
- 108010008705 Mucin-2 Proteins 0.000 description 1
- 108010008699 Mucin-4 Proteins 0.000 description 1
- 102100022693 Mucin-4 Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100277391 Mus musculus Defb1 gene Proteins 0.000 description 1
- 101100407308 Mus musculus Pdcd1lg2 gene Proteins 0.000 description 1
- 102000013609 MutL Protein Homolog 1 Human genes 0.000 description 1
- 108010026664 MutL Protein Homolog 1 Proteins 0.000 description 1
- 241000949972 Mycobacterium bovis BCG str. Pasteur 1173P2 Species 0.000 description 1
- 101710107068 Myelin basic protein Proteins 0.000 description 1
- 108060008487 Myosin Proteins 0.000 description 1
- 102000003505 Myosin Human genes 0.000 description 1
- 102100026771 Myosin-binding protein C, cardiac-type Human genes 0.000 description 1
- 108010056852 Myostatin Proteins 0.000 description 1
- 241000721621 Myzus persicae Species 0.000 description 1
- SDVKTCLBCCAFIS-HDYLNDSGSA-N N-[(1S,2S,3R)-3-(5,6-dihydrobenzo[b][1]benzazepin-11-yl)-2-hydroxycyclohexyl]-4-(trifluoromethoxy)benzenesulfonamide Chemical compound O[C@@H]1[C@H](CCC[C@H]1N1C2=C(CCC3=C1C=CC=C3)C=CC=C2)NS(=O)(=O)C1=CC=C(OC(F)(F)F)C=C1 SDVKTCLBCCAFIS-HDYLNDSGSA-N 0.000 description 1
- 101150006989 NDEL1 gene Proteins 0.000 description 1
- 230000006051 NK cell activation Effects 0.000 description 1
- 108091061960 Naked DNA Proteins 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 102100038816 Neuronatin Human genes 0.000 description 1
- 101710194997 Neuronatin Proteins 0.000 description 1
- 102400001095 Neuropeptide FF Human genes 0.000 description 1
- 108090000913 Nitrate Reductases Proteins 0.000 description 1
- 102000006570 Non-Histone Chromosomal Proteins Human genes 0.000 description 1
- 108010008964 Non-Histone Chromosomal Proteins Proteins 0.000 description 1
- 101710202677 Non-specific lipid-transfer protein Proteins 0.000 description 1
- 102000004966 Nuclear Receptor Coactivator 1 Human genes 0.000 description 1
- 108010062309 Nuclear Receptor Interacting Protein 1 Proteins 0.000 description 1
- 102100023312 Nuclear distribution protein nudE-like 1 Human genes 0.000 description 1
- 102100037223 Nuclear receptor coactivator 1 Human genes 0.000 description 1
- 108090001144 Nuclear receptor coactivator 2 Proteins 0.000 description 1
- 102100022927 Nuclear receptor coactivator 4 Human genes 0.000 description 1
- 102100022935 Nuclear receptor corepressor 1 Human genes 0.000 description 1
- 101710153661 Nuclear receptor corepressor 1 Proteins 0.000 description 1
- 102100030569 Nuclear receptor corepressor 2 Human genes 0.000 description 1
- 101710153660 Nuclear receptor corepressor 2 Proteins 0.000 description 1
- 102100029558 Nuclear receptor-interacting protein 1 Human genes 0.000 description 1
- 101710149086 Nuclease S1 Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 102100023252 Nucleoside diphosphate kinase A Human genes 0.000 description 1
- 208000021384 Obsessive-Compulsive disease Diseases 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 102000016978 Orphan receptors Human genes 0.000 description 1
- 108070000031 Orphan receptors Proteins 0.000 description 1
- 102000004067 Osteocalcin Human genes 0.000 description 1
- 108090000573 Osteocalcin Proteins 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 108091033411 PCA3 Proteins 0.000 description 1
- 101150012394 PHO5 gene Proteins 0.000 description 1
- 102100037482 PMS1 protein homolog 1 Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 206010033892 Paraplegia Diseases 0.000 description 1
- 108700020797 Parathyroid Hormone-Related Proteins 0.000 description 1
- 102000043299 Parathyroid hormone-related Human genes 0.000 description 1
- 208000034038 Pathologic Neovascularization Diseases 0.000 description 1
- 102000010292 Peptide Elongation Factor 1 Human genes 0.000 description 1
- 102100022239 Peroxiredoxin-6 Human genes 0.000 description 1
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 1
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 1
- 102100040865 Phospholipase A2 group XV Human genes 0.000 description 1
- 101710127148 Phospholipase A2 group XV Proteins 0.000 description 1
- 102100022428 Phospholipid transfer protein Human genes 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 108010069381 Platelet Endothelial Cell Adhesion Molecule-1 Proteins 0.000 description 1
- 102000015795 Platelet Membrane Glycoproteins Human genes 0.000 description 1
- 108010010336 Platelet Membrane Glycoproteins Proteins 0.000 description 1
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 1
- 241000251574 Polyandrocarpa misakiensis Species 0.000 description 1
- 102100034410 Polyribonucleotide nucleotidyltransferase 1, mitochondrial Human genes 0.000 description 1
- 102100022810 Potassium voltage-gated channel subfamily H member 1 Human genes 0.000 description 1
- 108050000074 Potassium voltage-gated channel subfamily H member 1 Proteins 0.000 description 1
- 102100025071 Potassium voltage-gated channel subfamily H member 5 Human genes 0.000 description 1
- 108050000076 Potassium voltage-gated channel subfamily H member 5 Proteins 0.000 description 1
- 208000005107 Premature Birth Diseases 0.000 description 1
- 206010036590 Premature baby Diseases 0.000 description 1
- 102100022033 Presenilin-1 Human genes 0.000 description 1
- 108010036933 Presenilin-1 Proteins 0.000 description 1
- RJKFOVLPORLFTN-LEKSSAKUSA-N Progesterone Chemical class C1CC2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H](C(=O)C)[C@@]1(C)CC2 RJKFOVLPORLFTN-LEKSSAKUSA-N 0.000 description 1
- 108700030875 Programmed Cell Death 1 Ligand 2 Proteins 0.000 description 1
- 102100024213 Programmed cell death 1 ligand 2 Human genes 0.000 description 1
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 1
- 102100024819 Prolactin Human genes 0.000 description 1
- 108010057464 Prolactin Proteins 0.000 description 1
- 108090000612 Proline Oxidase Proteins 0.000 description 1
- 102000004177 Proline oxidase Human genes 0.000 description 1
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 1
- 102000015433 Prostaglandin Receptors Human genes 0.000 description 1
- 108010050183 Prostaglandin Receptors Proteins 0.000 description 1
- 102100029500 Prostasin Human genes 0.000 description 1
- 102100035703 Prostatic acid phosphatase Human genes 0.000 description 1
- 102100033812 Protein CBFA2T3 Human genes 0.000 description 1
- 102100037469 Protein DEPP1 Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102100036468 Protein diaphanous homolog 3 Human genes 0.000 description 1
- 102100024601 Protein tyrosine phosphatase type IVA 3 Human genes 0.000 description 1
- 101710138647 Protein tyrosine phosphatase type IVA 3 Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 102000013009 Pyruvate Kinase Human genes 0.000 description 1
- 108020005115 Pyruvate Kinase Proteins 0.000 description 1
- 102100023750 RNA polymerase II elongation factor ELL2 Human genes 0.000 description 1
- 230000004570 RNA-binding Effects 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108091030071 RNAI Proteins 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 101000611641 Rattus norvegicus Protein phosphatase 1 regulatory subunit 15A Proteins 0.000 description 1
- 101000653754 Rattus norvegicus Sphingosine 1-phosphate receptor 5 Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 102000007156 Resistin Human genes 0.000 description 1
- 108010047909 Resistin Proteins 0.000 description 1
- 241000219061 Rheum Species 0.000 description 1
- 101100394989 Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009) hisI gene Proteins 0.000 description 1
- 241000701507 Rice tungro bacilliform virus Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 108010015695 S100 calcium binding protein A10 Proteins 0.000 description 1
- 108091005487 SCARB1 Proteins 0.000 description 1
- 101700004678 SLIT3 Proteins 0.000 description 1
- 102100032724 SPARC-related modular calcium-binding protein 2 Human genes 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 101100434411 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ADH1 gene Proteins 0.000 description 1
- 101100184491 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) MNR2 gene Proteins 0.000 description 1
- 102100037118 Scavenger receptor class B member 1 Human genes 0.000 description 1
- 241000242583 Scyphozoa Species 0.000 description 1
- 102000005473 Secretory Phospholipases A2 Human genes 0.000 description 1
- 108010031873 Secretory Phospholipases A2 Proteins 0.000 description 1
- 102100027979 Semaphorin-3B Human genes 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101710192761 Serine-type anaerobic sulfatase-maturating enzyme Proteins 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- 102100025521 Serpin B7 Human genes 0.000 description 1
- 101710156145 Serpin B7 Proteins 0.000 description 1
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 102100021825 Single-minded homolog 2 Human genes 0.000 description 1
- 102100027339 Slit homolog 3 protein Human genes 0.000 description 1
- 102100029937 Smoothelin Human genes 0.000 description 1
- 101710151526 Smoothelin Proteins 0.000 description 1
- 102100027198 Sodium channel protein type 5 subunit alpha Human genes 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 101710167338 Spermine oxidase Proteins 0.000 description 1
- 102100028897 Stearoyl-CoA desaturase Human genes 0.000 description 1
- 101150112794 Stk3 gene Proteins 0.000 description 1
- 108700027337 Suppressor of Cytokine Signaling 3 Proteins 0.000 description 1
- 108010075383 Suppressor of Cytokine Signaling Proteins Proteins 0.000 description 1
- 102000008036 Suppressor of Cytokine Signaling Proteins Human genes 0.000 description 1
- 102100024283 Suppressor of cytokine signaling 3 Human genes 0.000 description 1
- 101800001271 Surface protein Proteins 0.000 description 1
- 241000282894 Sus scrofa domesticus Species 0.000 description 1
- 102000001435 Synapsin Human genes 0.000 description 1
- 108050009621 Synapsin Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 108010029625 T-Box Domain Protein 2 Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 102100038721 T-box transcription factor TBX2 Human genes 0.000 description 1
- 108010016283 TCF Transcription Factors Proteins 0.000 description 1
- 102000000479 TCF Transcription Factors Human genes 0.000 description 1
- 101710084188 TGF-beta receptor type-2 Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N THREONINE Chemical compound CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 102000005450 TIE receptors Human genes 0.000 description 1
- 108010006830 TIE receptors Proteins 0.000 description 1
- 101150080074 TP53 gene Proteins 0.000 description 1
- 101150033985 TPI gene Proteins 0.000 description 1
- 101150032817 TPI1 gene Proteins 0.000 description 1
- 102000003618 TRPM4 Human genes 0.000 description 1
- 208000001163 Tangier disease Diseases 0.000 description 1
- 108010009978 Tec protein-tyrosine kinase Proteins 0.000 description 1
- 241000254109 Tenebrio molitor Species 0.000 description 1
- 241000255588 Tephritidae Species 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical class OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 102000007501 Thymosin Human genes 0.000 description 1
- 108010046075 Thymosin Proteins 0.000 description 1
- 208000033781 Thyroid carcinoma Diseases 0.000 description 1
- 208000024799 Thyroid disease Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 108010031429 Tissue Inhibitor of Metalloproteinase-3 Proteins 0.000 description 1
- 101150085042 Tnfsf4 gene Proteins 0.000 description 1
- 102100037116 Transcription elongation factor 1 homolog Human genes 0.000 description 1
- 102100025041 Transcription initiation factor TFIID subunit 2 Human genes 0.000 description 1
- 102100022011 Transcription intermediary factor 1-alpha Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 102000006747 Transforming Growth Factor alpha Human genes 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 108060008539 Transglutaminase Proteins 0.000 description 1
- 102100029887 Translationally-controlled tumor protein Human genes 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
- 241000242582 Tripedalia Species 0.000 description 1
- 102000004987 Troponin T Human genes 0.000 description 1
- 108090001108 Troponin T Proteins 0.000 description 1
- 102100031296 Tsukushi Human genes 0.000 description 1
- 102100024587 Tumor necrosis factor ligand superfamily member 15 Human genes 0.000 description 1
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 description 1
- 102100022156 Tumor necrosis factor receptor superfamily member 3 Human genes 0.000 description 1
- 101710165474 Tumor necrosis factor receptor superfamily member 5 Proteins 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 108010037584 Type 4 Cyclic Nucleotide Phosphodiesterases Proteins 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 108091000117 Tyrosine 3-Monooxygenase Proteins 0.000 description 1
- 102000048218 Tyrosine 3-monooxygenases Human genes 0.000 description 1
- 101150050575 URA3 gene Proteins 0.000 description 1
- 102100028700 Ubiquitin-conjugating enzyme E2 W Human genes 0.000 description 1
- 102000008219 Uncoupling Protein 2 Human genes 0.000 description 1
- 108010021111 Uncoupling Protein 2 Proteins 0.000 description 1
- 102000005630 Urocortins Human genes 0.000 description 1
- 108010059705 Urocortins Proteins 0.000 description 1
- 108010075653 Utrophin Proteins 0.000 description 1
- 102000011856 Utrophin Human genes 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- 102000016549 Vascular Endothelial Growth Factor Receptor-2 Human genes 0.000 description 1
- 102100034166 Vasculin Human genes 0.000 description 1
- 108010065472 Vimentin Proteins 0.000 description 1
- 102100035071 Vimentin Human genes 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 101710087237 Whey acidic protein Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 241000269368 Xenopus laevis Species 0.000 description 1
- 101100181311 Xenopus laevis lsm14b-a gene Proteins 0.000 description 1
- 108010055615 Zein Proteins 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 208000017733 acquired polycythemia vera Diseases 0.000 description 1
- 101150102866 adc1 gene Proteins 0.000 description 1
- 208000011589 adenoviridae infectious disease Diseases 0.000 description 1
- ULCUCJFASIJEOE-NPECTJMMSA-N adrenomedullin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(=O)N[C@@H]1C(N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](CSSC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)[C@@H](C)O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=CC=C1 ULCUCJFASIJEOE-NPECTJMMSA-N 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 108010056760 agalsidase beta Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 108010050122 alpha 1-Antitrypsin Proteins 0.000 description 1
- 108010026331 alpha-Fetoproteins Proteins 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 230000036783 anaphylactic response Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000007953 anoxia Effects 0.000 description 1
- 230000003527 anti-angiogenesis Effects 0.000 description 1
- 230000000692 anti-sense effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 230000005784 autoimmunity Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 239000013602 bacteriophage vector Substances 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 108010020169 beta-microseminoprotein Proteins 0.000 description 1
- GINJFDRNADDBIN-FXQIFTODSA-N bilanafos Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCP(C)(O)=O GINJFDRNADDBIN-FXQIFTODSA-N 0.000 description 1
- 230000008275 binding mechanism Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000004641 brain development Effects 0.000 description 1
- 102100029170 cAMP-specific 3',5'-cyclic phosphodiesterase 4D Human genes 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000009400 cancer invasion Effects 0.000 description 1
- 239000012830 cancer therapeutic Substances 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 229920006317 cationic polymer Polymers 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000006369 cell cycle progression Effects 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 208000015114 central nervous system disease Diseases 0.000 description 1
- 229940049197 cerezyme Drugs 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- ATNHDLDRLWWWCB-DVXFRRMCSA-O chlorophyll a Chemical compound [Mg+2].[N-]1C(C=C2[C@H]([C@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)C(=[NH+]2)C2=C3[N-]C(=C4)C(C)=C3C(=O)[C@H]2C(=O)OC)C)=C(C)C(C=C)=C1C=C1C(C)=C(CC)C4=[NH+]1 ATNHDLDRLWWWCB-DVXFRRMCSA-O 0.000 description 1
- 229930002868 chlorophyll a Natural products 0.000 description 1
- 229930002869 chlorophyll b Natural products 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000006690 co-activation Effects 0.000 description 1
- 230000003081 coactivator Effects 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 208000028831 congenital heart disease Diseases 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 101150008232 csdA gene Proteins 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 101150077693 deaD gene Proteins 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 230000005860 defense response to virus Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 210000005045 desmin Anatomy 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 150000008037 diacylhydrazines Chemical class 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 208000037765 diseases and disorders Diseases 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 230000000464 effect on transcription Effects 0.000 description 1
- 229940012882 elaprase Drugs 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000008753 endothelial function Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 101150107963 eno gene Proteins 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 230000002461 excitatory amino acid Effects 0.000 description 1
- 239000003257 excitatory amino acid Substances 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 229940014516 fabrazyme Drugs 0.000 description 1
- 229960000301 factor viii Drugs 0.000 description 1
- 229930002886 farnesol Natural products 0.000 description 1
- 229940043259 farnesol Drugs 0.000 description 1
- 108010052621 fas Receptor Proteins 0.000 description 1
- 102000018823 fas Receptor Human genes 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- 229940044627 gamma-interferon Drugs 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000012178 germinal center formation Effects 0.000 description 1
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 1
- 206010061989 glomerulosclerosis Diseases 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000001456 gonadotroph Effects 0.000 description 1
- 210000000224 granular leucocyte Anatomy 0.000 description 1
- 230000036433 growing body Effects 0.000 description 1
- 208000037824 growth disorder Diseases 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 230000009583 hair follicle growth Effects 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 230000002489 hematologic effect Effects 0.000 description 1
- 201000000386 hemochromatosis type 2A Diseases 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 210000001320 hippocampus Anatomy 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 108010027263 homeobox protein HOXA9 Proteins 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 102000047000 human FGF19 Human genes 0.000 description 1
- 102000047486 human GAPDH Human genes 0.000 description 1
- 230000003553 hypophosphatemic effect Effects 0.000 description 1
- 108010072166 idursulfase Proteins 0.000 description 1
- 108010039650 imiglucerase Proteins 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000004968 inflammatory condition Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 230000005732 intercellular adhesion Effects 0.000 description 1
- 102000044166 interleukin-18 binding protein Human genes 0.000 description 1
- 108010070145 interleukin-18 binding protein Proteins 0.000 description 1
- 210000003963 intermediate filament Anatomy 0.000 description 1
- 208000028774 intestinal disease Diseases 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 201000008632 juvenile polyposis syndrome Diseases 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 201000005296 lung carcinoma Diseases 0.000 description 1
- 210000005265 lung cell Anatomy 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 208000037841 lung tumor Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000003810 lymphokine-activated killer cell Anatomy 0.000 description 1
- 230000002132 lysosomal effect Effects 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 108010083942 mannopine synthase Proteins 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000001806 memory b lymphocyte Anatomy 0.000 description 1
- 210000003071 memory t lymphocyte Anatomy 0.000 description 1
- 230000006996 mental state Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 108091057645 miR-15 stem-loop Proteins 0.000 description 1
- 108091027943 miR-16 stem-loop Proteins 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 239000013586 microbial product Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000002297 mitogenic effect Effects 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- 208000005264 motor neuron disease Diseases 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 108010065781 myosin light chain 2 Proteins 0.000 description 1
- 230000006654 negative regulation of apoptotic process Effects 0.000 description 1
- 230000018901 negative regulation of programmed cell death Effects 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000005044 neurofilament Anatomy 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 210000004882 non-tumor cell Anatomy 0.000 description 1
- 108010058731 nopaline synthase Proteins 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- AHLBNYSZXLDEJQ-FWEHEUNISA-N orlistat Chemical compound CCCCCCCCCCC[C@H](OC(=O)[C@H](CC(C)C)NC=O)C[C@@H]1OC(=O)[C@H]1CCCCCC AHLBNYSZXLDEJQ-FWEHEUNISA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 108700025694 p53 Genes Proteins 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 239000003614 peroxisome proliferator Substances 0.000 description 1
- 238000009520 phase I clinical trial Methods 0.000 description 1
- 108010055752 phenylalanyl-leucyl-phenylalanyl-glutaminyl-prolyl-glutaminyl-arginyl-phenylalaninamide Proteins 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 208000037244 polycythemia vera Diseases 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 208000015768 polyposis Diseases 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000030786 positive chemotaxis Effects 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000019525 primary metabolic process Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000583 progesterone congener Substances 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 229940097325 prolactin Drugs 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 210000004129 prosencephalon Anatomy 0.000 description 1
- 108010031970 prostasin Proteins 0.000 description 1
- 108010043671 prostatic acid phosphatase Proteins 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 239000003488 releasing hormone Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 150000004492 retinoid derivatives Chemical class 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 102000015340 serglycin Human genes 0.000 description 1
- 108010050065 serglycin Proteins 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000001743 silencing effect Effects 0.000 description 1
- 108010048078 site 1 membrane-bound transcription factor peptidase Proteins 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 230000001148 spastic effect Effects 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 102000005969 steroid hormone receptors Human genes 0.000 description 1
- 108020003113 steroid hormone receptors Proteins 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 230000003319 supportive effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 108010057210 telomerase RNA Proteins 0.000 description 1
- 102000055501 telomere Human genes 0.000 description 1
- 108091035539 telomere Proteins 0.000 description 1
- 210000003411 telomere Anatomy 0.000 description 1
- 206010043207 temporal arteritis Diseases 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 208000013077 thyroid gland carcinoma Diseases 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- CRDAMVZIKSXKFV-UHFFFAOYSA-N trans-Farnesol Natural products CC(C)=CCCC(C)=CCCC(C)=CCO CRDAMVZIKSXKFV-UHFFFAOYSA-N 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 108010071511 transcriptional intermediary factor 1 Proteins 0.000 description 1
- 108091008023 transcriptional regulators Proteins 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 102000003601 transglutaminase Human genes 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
- 208000025421 tumor of uterus Diseases 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 239000000225 tumor suppressor protein Substances 0.000 description 1
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 description 1
- 229940045145 uridine Drugs 0.000 description 1
- 239000000777 urocortin Substances 0.000 description 1
- ZEBBPGHOLWPSGI-KPLDDXDLSA-N urocortin ii Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@H](C(=O)N[C@@H](CS)C(N)=O)CC1=CN=CN1 ZEBBPGHOLWPSGI-KPLDDXDLSA-N 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 108090000195 villin Proteins 0.000 description 1
- 210000005048 vimentin Anatomy 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 208000016261 weight loss Diseases 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
- A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
- A61K38/1866—Vascular endothelial growth factor [VEGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/191—Tumor necrosis factors [TNF], e.g. lymphotoxin [LT], i.e. TNF-beta
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
- A61K38/208—IL-12
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
- A61K38/212—IFN-alpha
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/461—Cellular immunotherapy characterised by the cell type used
- A61K39/4615—Dendritic cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/462—Cellular immunotherapy characterized by the effect or the function of the cells
- A61K39/4622—Antigen presenting cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/463—Cellular immunotherapy characterised by recombinant expression
- A61K39/4635—Cytokines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/46—Cellular immunotherapy
- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
- A61K39/4643—Vertebrate antigens
- A61K39/4644—Cancer antigens
- A61K39/46449—Melanoma antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/06—Antiglaucoma agents or miotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5434—IL-12
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/861—Adenoviral vectors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/48—Blood cells, e.g. leukemia or lymphoma
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
- A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
- A61K2239/56—Kidney
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10041—Use of virus, viral particle or viral elements as a vector
- C12N2710/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10341—Use of virus, viral particle or viral elements as a vector
- C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
- C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Cell Biology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Biophysics (AREA)
- Hematology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Oncology (AREA)
- Virology (AREA)
- Communicable Diseases (AREA)
- Diabetes (AREA)
- Ophthalmology & Optometry (AREA)
- Toxicology (AREA)
Abstract
This invention relates to the field of therapeutics. Most specifically, the invention provides methods of generating conditionally expressing vectors for one or more immunomodulators under the control of a gene expression modulation system in the presence of activating ligand and uses for therapeutic purposes in animals. These vector may be provided to treat a variety of disorders, e.g., neoplastic disorders, through direct injection or through in vitro engineered cells, such as dendritic cells.
Description
- 1. Field of the Invention
- This invention relates to the field of gene therapy for the treatment of diseases and disorders, for example, cancer, lysosomal storage disorders, ocular diseases, liver diseases, or infectious diseases. In one embodiment, the invention provides the engineering of immune cells or therapy support cells (TSC) to express one or more therapeutic proteins (e.g., immunomodulators) and use of the cells as therapeutics. In another embodiment, the invention includes a vector, e.g., adenovirus, for conditional expression of therapeutic proteins (e.g., immunodulators) disclosed herein, e.g., IL-12, TNF-alpha and methods of using such vectors.
- 2. Background
- Interleukin-12 (IL-12) is a member of the type I cytokine family involved in contributing to a number of biological processes including, but not limited to, protective immune response and suppression of tumorigenesis (Abdi et al., 2006; Adorini, 1999; Adorini, 2001; Adorini et al., 2002; Adorini et al., 1996; Akhtar et al., 2004; Akiyama et al., 2000; Al-Mohanna et al., 2002; Aliberti et al., 1996; Allavena et al., 1994; Alli and Khar, 2004; Alzona et al., 1996; Amemiya et al., 2006; Araujo et al., 2001; Arulanandam et al., 1999; Athie et al., 2000; Athie-Morales et al., 2004; Bertagnolli et al., 1992; Bhardwaj et al., 1996; Biedermann et al., 2006; Brunda and Gately, 1994; Buchanan et al., 1995; Romani et al., 1997; Rothe et al., 1996; Satoskar et al., 2000; Schopf et al., 1999; Thomas et al., 2000; Tsung et al., 1997; Wolf et al., 1994; Yuminamochi et al., 2007). A growing body of evidence suggests that IL-12 may be a promising target to control human diseases (e.g., cancer).
- Despite the fact that IL-12 remains promising as a cancer therapeutic agent based on its potent supportive activity on Type-1 anti-tumor NK cells, CD4+ T cells and CD8+ T cells (Trinchieri, 2003), the reported toxicity of recombinant human IL-12 (rhIL-12) in patients (Atkins et al., 1997), together with limited sources of GMP-grade rhIL-12 for clinical application, have prevented successful IL-12-based therapeutic approaches. Thus it seems reasonable that gene therapy approaches may represent safer, more tenable treatment options. Indeed, phase I clinical trials implementing intra- or peri-tumoral delivery of recombinant viral- (Sangro et al., 2004; Triozzi et al., 2005) or plasmid-based IL-12 cDNA (Heinzerling et al., 2005), or IL-12 gene modified autologous fibroblasts (Kang et al., 2001) have been found safe and well-tolerated.
- However, objective clinical responses in patients with melanoma or a diverse range of carcinomas receiving these gene therapies have been rare, variable, transient and largely focused at the site of treatment (Heinzerling et al., 2005; Kang et al., 2001; Sangro et al., 2004; Triozzi et al., 2005). In cases where disease resolution was partial or complete, increased frequencies of tumor-infiltrating lymphocytes (Heinzerling et al., 2005; Sangro et al., 2004) and elevated levels of circulating tumor-specific CD8+ T cells (Heinzerling et al., 2005) have been noted, consistent with the improved cross-priming of antigen-specific T cells in these patients.
- Since the cross-priming of specific T cells is best accomplished by dendritic cells (DC) that serve as a natural but regulated source of IL-12 (Berard et al., 2000), recent reports of the superior pre-clinical efficacy of DC-based IL-12 gene therapy have been of great interest (Satoh et al., 2002; Tatsumi et al., 2003; Yamanaka et al., 2002). For example, it was shown that intratumoral (i.t.) injection of DC engineered to produce IL-12p70 (via recombinant adenovirus infection) results in the dramatically improved cross-priming of a broadly-reactive, tumor-specific CD8+ T cell repertoire in concert with tumor rejection in murine models (Tatsumi et al., 2003). Given the previous use of a recombinant adenovirus encoding mIL-12 under a CMV-based promoter (rAd.cIL12, (Tatsumi et al., 2003)), engineered DC production of IL-12 was constitutive, hence the immunologic impact of this cytokine early within the tumor lesion and later within tumor-draining lymph nodes could not be resolved with regards to therapeutic outcome. Thus, a need exists for DC engineered for conditional expression of IL-12 for the purpose of regulating both the level of transgene expression and the timing of the transgene activation. The invention provides a promising therapeutic outcome for the use of such cells.
- Many of the therapeutic proteins currently under investigation in pre-clinical or clinical trials do not exhibit harmful side effects when present in a patient prior to expression of the nucleic acid sequence in the host cell of the patient or the proper physiologic context. Some proteins, however, such as tumor necrosis factor (TNF), cause adverse effects when expressed outside the normal physiologic tissues or context (e.g., exposed to non-target tissues). Systemic or even local administration of this protein is extremely toxic to many non-tumor cell types, potentiating anaphylaxis and cachexia. In addition, prolonged exposure to TNF-alpha may yield profoundly different cellular responses than acute stimulations. For these reasons, safe and effective TNF-alpha therapies against cancer have remained elusive.
- In view of the problems associated with gene expression of genes through vector compositions containing the protein encoded by the nucleic acid sequence of interest in, there remains a need for an improved transfer vector compositions to be used for direct injection or for use in cell based therapies.
- Lysosomal storage diseases (LSDs) represent a class of inherited genetic disorders that can currently be treated only by protein therapeutics, in the form of enzyme replacement therapy.
- LSDs are a class of 49 genetically inherited disorders characterized by a deficiency of one or more lysosomal enzymes that causes accumulation of undigested macromolecules inside the lysosome. Accumulation of these waste products causes lysosomes within cells to enlarge, leading to cell damage and degeneration. Accumulated damage in organs and tissues results in progressive deterioration in physical and/or mental state, and eventually death. Diagnosis is typically made in infancy. The severity of the individual disease is variable and correlated to the amount of residual enzyme activity produced by the defective gene.
- The incidence of LSDs is about 1 in 5000 persons (130,000 cases worldwide). Severity is variable and is correlated to the amount of residual enzyme activity produced by the defective gene. Severely affected patients may live only into their teens, while less severely affected patients may survive into adulthood.
- Enzyme replacement therapy is the only method available to treat LSDs. Therapy consists of systemic infusion of active proteins that target to lysosomes and break down accumulating waste molecules. Examples of LSD protein therapeutics include Fabrazyme (Genzyme) for Fabry Disease, Elaprase (Shire) for MPSII, and Myozome (Genzyme) for Pompe Disease, and Cerezyme (Genzyme) for Gaucher Disease.
- Enzyme replacement therapy is accompanied by certain drawbacks, such as the requirement for post-translational protein modifications, the replacement enzymes exhibit short half lives in vivo, and patients develop an immune response to the replacement enzymes. Therefore, there remains a need in the art for and alternative to enzyme replacement therapy to treat lysosomal storage disease.
- The invention provides a recombinant vector encoding protein(s) having the function(s) of one or more therapeutic proteins (e.g., immunomodulators), under the control of one or more promoters. In one embodiment, the one or more promoters are conditional. In another embodiment, the one or more promoters are constitutive. In another embodiment, the vector is an adenovirus vector encoding the protein(s) driven off a promoter that can be conditionally, activated by provision of a soluble small molecule ligand such as diacylhydrazines (e.g., RG-115819, RG-115830 or RG-115932). This vector allows for the control of expression of the protein(s) from immune cells, TSC and from direct injection of the vectors comprising therapeutic proteins (e.g., immunomodulators).
- In one embodiment, the invention provides a vector for conditionally expressing protein(s) having the function(s) of one or more therapeutic proteins (e.g., immunomodulators) comprising a polynucleotide encoding a gene switch, wherein said polynucleotide encoding a gene switch comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins having the function of a therapeutic protein (e.g., immunomodulator) linked to a promoter which is activated by said ligand-dependent transcription factor. In one embodiment, the therapeutic protein (e.g., immunomodulator) is selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10R DN or a subunit thereof, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, CCL3 (MIP-1a), CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12 (SDF-1), CCL21 (6Ckine), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1, Flt3L, IFN-beta, TNF-alpha, dnFADD, TGF-alpha, PD-L1RNAi, a PD-L1 antisense oligonucleotide, TGFbRII DN, ICOS-L, S100, CD40L, p53, survivin, p53-survivin fusion, MAGE3, PSA and PSMA.
- In another embodiment, the invention provides a vector for expressing protein(s) having the function(s) of one or more therapeutic proteins (e.g., immunomodulators) and a protein having the function of IL-12, comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, (2) a polynucleotide encoding said protein(s) having the function(s) of the one or more therapeutic proteins (e.g., immunomodulators), and (3) a polynucleotide encoding a protein having the function of the IL-12; wherein at least one polynucleotide of (2) and (3) are linked to the promoter which is activated by the ligand-dependent transcription factor.
- In some embodiments, the vector of the invention conditionally expresses TNF-alpha. In certain embodiments, the vector, e.g., adenoviral vector, conditionally expressing one or more proteins having the function of a therapeutic protein (e.g., immunomodulator), e.g., TNF-alpha, further comprises a nucleic acid sequence encoding a signal peptide. The signal peptide can be codon-optimized. In other embodiments, the vector further comprises 5′ untranslated region (UTR), 3′ regulatory region, or both and improves protein expression and/or overall yield.
- The invention further provides a method of producing a population of cells, e.g., immune cells or TSC, expressing protein(s) having the function of one or more therapeutic proteins (e.g., immunomodulators), by modifying (e.g., transfecting, electroporating, etc.) the cells with a recombinant vector conditionally expressing protein(s) having the function(s) of the one or more therapeutic proteins (e.g., immunomodulators), wherein the vector comprises a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins having the function of a therapeutic protein (e.g., immunomodulator) modulator linked to a promoter which is activated by said ligand-dependent transcription factor.
- In another embodiment, the invention provides a method of producing a population of cells, e.g., immune cells or TSC, expressing proteins having the function(s) of one or more therapeutic proteins (e.g., immunomodulators) and a protein having the function of IL-12, by modifying the cells with a recombinant vector comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, (2) a polynucleotide encoding said protein(s) having the function(s) of the one or more therapeutic proteins (e.g., immunomodulators), and (3) a polynucleotide encoding a protein having the function of the IL-12; wherein at least one polynucleotide of (2) and (3) are linked to the promoter which is activated by said ligand-dependent transcription factor.
- In some embodiments, the invention provides a method of increasing expression of a therapeutic protein (e.g., immunomodulator), e.g., TNF-alpha, mRNA expression, or protein expression comprising generating the vector conditionally expressing one or more proteins having the function of a therapeutic protein (e.g., immunomodulator) and one or more regulatory sequence, wherein said one or more regulatory sequence improves expression of the therapeutic proteins (e.g., immunomodulators), e.g., TNF-alpha.
- The invention further provides a population of cells, e.g., immune cells or TSC, expressing protein(s) having the function of one or more therapeutic proteins (e.g., immunomodulators), which has been modified (e.g., transfected, electroporated, etc.) with a recombinant vector conditionally the expressing protein(s) having the function(s) of the one or more therapeutic proteins (e.g., immunomodulators), wherein the vector comprises a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins having the function of a therapeutic protein (e.g., immunomodulator) linked to the promoter which is activated by said ligand dependent transcription factor.
- In another embodiment, the invention provides a population of cells, e.g., immune cells or TSC, expressing proteins having the function(s) of one or more therapeutic proteins (e.g., immunomodulators) and a protein having the function of IL-12, which has been modified with a recombinant vector comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, (2) a polynucleotide encoding said protein(s) having the function(s) of the one or more therapeutic proteins (e.g., immunomodulators) and (3) a polynucleotide encoding a protein having the function of the IL-12; wherein at least one polynucleotide of (2) and (3) are linked to a promoter which is activated by said ligand-dependent transcription factor.
- In another embodiment, the invention provides a composition comprising two or more populations of cells of the present invention, e.g., immune cells or TSC, wherein each population of cells in the composition expresses one or more therapeutic proteins (e.g., immunomodulators) that are different from the one or more therapeutic proteins (e.g., immunomodulators) expressed in the other population(s) of cells in the composition. In one embodiment, the composition contains two populations of cells. In another embodiment, the composition contains more than two populations of cells. In another embodiment, the composition contains three populations of cells. In another embodiment, the composition contains four populations of cells.
- In another embodiment, the invention provides an in vitro engineered cell, e.g., immune cell or TSC, comprising a vector comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding a protein having the function of a therapeutic protein (e.g., immunomodulator) linked to a promoter which is activated by said ligand-dependent transcription factor. In another embodiment, the invention provides an in vitro engineered cell, e.g., immune cell or TSC, comprising a vector comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, (2) a polynucleotide encoding a protein having the function of a therapeutic protein (e.g., immunomodulator), and (3) a polynucleotide encoding a protein having the function of IL-12; wherein at least one polynucleotide of (2) and (3) are linked to a promoter which is activated by said ligand-dependent transcription factor.
- In another embodiment, the invention provides a composition comprising two or more populations of in vitro engineered cells, e.g., immune cells or TSCs, of the present invention, wherein each of the populations of in vitro engineered cells in the composition comprises a vector comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding a protein having the function of a therapeutic protein (e.g., immunomodulator) linked to a promoter which is activated by said ligand-dependent transcription factor, and wherein each population of in vitro engineered cells in the composition expresses one or more therapeutic proteins (e.g., immunomodulators) that are different from the one or more therapeutic proteins (e.g., immunomodulators) expressed in the other population(s) of in vitro engineered cell in the composition. In one embodiment, the invention provides a composition comprising two or more populations of in vitro engineered cells, e.g., immune cell or TSC, each of said populations of cells comprising a vector comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, (2) a polynucleotide encoding a protein having the function of a therapeutic protein (e.g., immunomodulator), and (3) a polynucleotide encoding a protein having the function of IL-12; wherein at least one polynucleotide of (2) and (3) are linked to a promoter which is activated by said ligand-dependent transcription factor. In one embodiment, the composition contains two populations of in vitro engineered cells. In another embodiment, the composition contains more than two populations of in vitro engineered cells. In another embodiment, the composition contains three populations of in vitro engineered cells. In another embodiment, the composition contains four populations of in vitro engineered cells.
- The invention also provides a pharmaceutical composition comprising a population of cells, e.g., immune cells or TSC, as described herein or a composition suitable for direct injection of the expression vectors absent a population of cells, i.e., directly injected.
- In one embodiment, the polynucleotide coding for the one or more proteins having the functions of the immunomodulator is under control of the promoter of the gene switch and the polynucleotide coding for a protein having the function of IL-12 is under control of a constitutive promoter. In another embodiment, both the polynucleotide coding for protein(s) having the functions of the therapeutic proteins (e.g., immunomodulators) and the polynucleotide coding for a protein having the function of IL-12 are both under control of a multicistronic promoter of the gene switch. In another embodiment, the polynucleotide coding for a protein(s) having the function of the therapeutic proteins (e.g., immunomodulators) is under control of the promoter of the gene switch and the polynucleotide coding for a protein having the function of IL-12 is under control of a conditional promoter which is different than the gene switch promoter. In a further embodiment, the gene regulation system for the polynucleotide coding for the protein(s) having the function of the therapeutic proteins (e.g., immunomodulators) and the gene regulation system for the polynucleotide having the function of IL-12 are orthogonal. In a further embodiment, the gene regulation system for each polynucleotide coding for each protein is orthogonal.
- In one embodiment, the invention also provides a treatment of cancer, such as, but not limited to, melanoma tumors, glioma tumors, renal cancer, and prostate cancers, as well as the cancers listed herein in Table 1. IL-12 gene therapy has demonstrated anti-tumor efficacy in animal model studies when applied as a recombinant cDNA vector (Faure et al., 1998; Sangro et al., 2005), but even more so, when applied in the context of gene-modified DC (Satoh et al., 2002; Svane et al., 1999; Tatsumi et al., 2003; Yamanaka et al., 2002). To date, however, human phase I trials of IL-12 gene therapy implementing plasmids or viral vectors have failed to achieve durable, objective clinical responses in the cancer setting (Heinzerling et al., 2005; Kang et al., 2001; Sangro et al., 2004; Triozzi et al., 2005) gene therapy as described herein provides a promising therapeutic modality.
- In one embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of:
-
- (a) administering intratumorally to tumor microenvironments, in the area surrounding the tumor, or systemically a population of immune cells, TSCs or vectors of the invention (or a combination thereof), which are in vitro engineered to conditionally express one or more proteins having the function of a therapeutic protein (e.g., immunomodulator); and
- (b) administering to said mammal a therapeutically effective amount of an activating ligand;
- thereby inducing expression of a protein having the function of the therapeutic protein (e.g., immunomodulator) and treating said tumor.
- In one embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of:
-
- (a) administering intratumorally to tumor microenvironments a population of immune cells or TSC, which are in vitro engineered to conditionally express one or more proteins having the function of a therapeutic protein (e.g., immunomodulator); and
- (b) administering to said mammal a therapeutically effective amount of an activating ligand;
- thereby inducing expression of a protein having the function of the therapeutic proteins (e.g., immunomodulators) and treating said tumor.
- In another embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of:
-
- (a) administering intratumorally to tumor microenvironments two or more populations of immune cells or TSCs, which are in vitro engineered to conditionally express one or more proteins having the function of a therapeutic protein (e.g., immunomodulator), wherein each population of immune cells or TSCs expresses a different set of one or more therapeutic proteins (e.g., immunomodulators); and
- (b) administering to said mammal a therapeutically effective amount of one or more activating ligands;
- thereby inducing expression of proteins having the function of the therapeutic proteins (e.g., immunomodulators) and treating said tumor.
- In another embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of:
-
- (a) administering intratumorally to tumor microenvironments a population of an immune cells or TSC, which are in vitro engineered to conditionally express one or more proteins having the function of a therapeutic protein (e.g., immunomodulator) and a protein having the function of IL-12, wherein at least one of the proteins having the function of the therapeutic protein (e.g., immunomodulator) or IL-12 is under control of a conditional promoter that is activated by a ligand; and
- (b) administering to said mammal a therapeutically effective amount of the activating ligand;
- thereby inducing expression of a protein having the function of the therapeutic protein (e.g., immunomodulator) and/or the protein having the function of IL-12 and treating said tumor.
- In another embodiment, the invention provides a method for treating a tumor in a mammal, comprising the steps of:
-
- (a) administering intratumorally to tumor microenvironments two or more populations of an immune cells or TSCs, which are in vitro engineered to conditionally express one or more proteins having the function of a therapeutic protein (e.g., immunomodulator) and a protein having the function of IL-12, wherein each population of immune cells or TSCs expresses a different set of one or more proteins having the function of a therapeutic protein (e.g., immunomodulator), wherein at least one of the proteins having the function of the therapeutic protein (e.g., immunomodulator) or IL-12 is under control of a conditional promoter that is activated by a ligand; and
- (b) administering to said mammal a therapeutically effective amount of one or more activating ligands;
- thereby inducing expression of a protein having the function of the therapeutic proteins (e.g., immunomodulator) and/or the protein having the function of IL-12 and treating said tumor.
- In another embodiment, the invention provides a method for treating a disease or disorder in a mammal, comprising the steps of:
-
- (a) administering to said mammal a population of modified cells, which are modified to conditionally express one or more proteins having the function of an therapeutic protein (e.g., immunomodulator); and
- (b) administering to said mammal a therapeutically effective amount of an activating ligand;
- thereby inducing expression of a protein having the function of the therapeutic protein (e.g., immunomodulator) and treating said disease or disorder.
- In another embodiment, the invention provides a method for treating a disease or disorder in a mammal, comprising the steps of:
-
- (a) administering to said mammal two or more populations of modified cells, which are modified to conditionally express one or more proteins having the function of a therapeutic protein (e.g., immunomodulator), wherein each population of modified cells expresses a different set of one or more therapeutic proteins (e.g., immunomodulators); and
- (b) administering to said mammal a therapeutically effective amount of one or more activating ligands;
- thereby inducing expression of proteins having the function of the therapeutic proteins (e.g., immunomodulators) and treating said disease or disorder.
- In another embodiment, the invention provides a method for treating a disease or disorder in a mammal, comprising the steps of:
-
- (a) administering to said mammal a population of a modified cells, which are modified to conditionally express one or more proteins having the function of a therapeutic protein (e.g., immunomodulator) and a protein having the function of IL-12, wherein at least one of the proteins having the function of the therapeutic protein (e.g., immunomodulator) or IL-12 is under control of a conditional promoter that is activated by a ligand; and
- (b) administering to said mammal a therapeutically effective amount of the activating ligand;
- thereby inducing expression of a protein having the function of the therapeutic protein (e.g., immunomodulator) and/or the protein having the function of IL-12 and treating said disease or disorder.
- In another embodiment, the invention provides a method for treating a disease or disorder in a mammal, comprising the steps of:
-
- (a) administering to said mammal two or more populations of modified cells, which are modified to conditionally express one or more proteins having the function of a therapeutic protein (e.g., immunomodulator) and a protein having the function of IL-12, wherein each population of modified cells expresses a different set of one or more proteins having the function of a therapeutic protein (e.g., immunomodulator), wherein at least one of the proteins having the function of the therapeutic protein (e.g., immunomodulator) or IL-12 is under control of a conditional promoter that is activated by a ligand; and
- (b) administering to said mammal a therapeutically effective amount of one or more activating ligands;
- thereby inducing expression of a protein having the function of the therapeutic proteins (e.g., immunomodulators) and/or the protein having the function of IL-12 and treating said disease or disorder.
- The invention also provides a method for determining the efficacy of engineered cell-, e.g., immune cell- or TSC-, based therapy by measuring the level of expression or activity of IFN-gamma in a patient before the start of therapy, thereby generating a control level, followed by the administration of cells engineered to express one or more proteins having the functions of a therapeutic protein (e.g., immunomodulator) and optionally a protein having the function of IL-12, administering an effective amount of an activating ligand, and then measuring the level of expression of IFN-gamma to generate a test level, and comparing the control level to the test level to determine if the therapeutic regime is effective.
- Further included is a method of treating a tumor, reducing a tumor size, or preventing a tumor formation in a mammal in need thereof comprising (a) administering a therapeutically effective amount of the vector conditionallly expressing at least one therapeutic protein (e.g., immunomodulator), e.g., IL-12, TNF-alpha, in said mammal, (b) administering to said mammal a therapeutically effective amount of one or more activating ligand, wherein said activating ligand activates expression of the protein having the function of the therapeutic protein (e.g., immunomodulator), thereby inducing expression of the protein having the function of the therapeutic protein (e.g., immunomodulator) and treating said tumor.
- In one embodiment, the invention provides a method for determining the efficacy of an in vitro engineered cell-, e.g., immune cell- or TSC-, based therapeutic regime in a patient comprising:
-
- (a) measuring the level of expression or the level of activity or both of interferon-gamma (IFN-gamma) in a first biological sample obtained from said patient in need thereof before administration of the in vitro engineered cells, thereby generating a control level;
- (b) administering to a patient in need thereof the in vitro engineered cells engineered to conditionally express one or more proteins having the functions of a therapeutic protein (e.g., immunomodulator) and optionally a protein having the function of IL-12;
- (c) administering to said patient in need thereof an effective amount of an activating ligand;
- (d) measuring the level of expression or the level of activity or both of IFN-gamma in a second biological sample obtained from said patient in need thereof following administration of in vitro engineered immune cells and activating ligand, thereby generating a test level; and
- (e) comparing the control level to the test level of IFN-gamma, wherein an increase in the test level of expression, activity or both of IFN-gamma relative to the control level indicates that the therapeutic regime is effective in said patient in need thereof.
- In one embodiment, the invention provides a method for treating a tumor, reducing a tumor size, or preventing a tumor formation in a mammal in need thereof, comprising: (a) administering intratumorally to tumor microenvironments a vector for conditionally expressing protein(s) having the function(s) of one or more therapeutic proteins (e.g., immunomodulators), the vector comprising a polynucleotide encoding a gene switch, wherein the polynucleotide comprises (1) at least one transcription factor sequence which is operably linked to a promoter, wherein the at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins having the function of a therapeutic protein (e.g., immunomodulator) operably linked to a promoter which is activated by the ligand-dependent transcription factor, wherein the one or more therapeutic proteins (e.g., immunomodulators) are selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10R DN or a subunit thereof, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, IFN-alpha 1, IFN alpha 2, IL-15-R-alpha, CCL3 (MIP-1a), CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12 (SDF-1), CCL21 (6Ckine), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1, Flt3L, IFN-beta, TNF-alpha, dnFADD, BCG, TGF-alpha, PD-L1 RNAi, a PD-L1 antisense oligonucleotide, TGFbRII DN, ICOS-L, S100, CD40L, OX40L, p53, survivin, p53-survivin fusion, MAGE3, PSA and PSMA, wherein the vector is not contained within a cell; and (b) administering to the mammal a therapeutically effective amount of one or more activating ligands; thereby inducing expression of the one or more proteins having the functions of the therapeutic protein (e.g., immunomodulator) and treating the tumor.
- The present invention also provides a method for treating a disease in a mammal in need thereof, comprising: (a) administering to said mammal a vector for conditionally expressing protein(s), said vector comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence which is operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins operably linked to a promoter which is activated by said ligand-dependent transcription factor, wherein said vector is not contained within a cell; and (b) administering to said non-human animal a therapeutically effective amount of one or more activating ligands; thereby inducing expression of the one or more proteins and treating said disease.
- The present invention also provides a method for treating a lysosomal storage disorder in a mammal in need thereof, comprising: (a) administering to said mammal a vector for conditionally expressing one or more protein(s), said vector comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence which is operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins operably linked to a promoter which is activated by said ligand-dependent transcription factor, wherein said vector is not contained within a cell prior to in vivo administration; and (b) administering to said mammal a therapeutically effective amount of one or more activating ligands; thereby inducing expression of the one or more proteins and treating said lysosomal storage disorder.
- The present invention also provides a method for treating a liver disease in a mammal in need thereof, comprising: (a) administering to said mammal a vector for conditionally expressing protein(s), said vector comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence which is operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins operably linked to a promoter which is activated by said ligand-dependent transcription factor, wherein said vector is not contained within a cell prior to in vivo administration; and (b) administering to said non-human animal a therapeutically effective amount of one or more activating ligands; thereby inducing expression of the one or more proteins and treating said liver disease.
-
FIG. 1 shows a plasmid map for a regulated promoter expression system for a bicistronic transcript encoding hIL-12. -
FIG. 2 shows a plasmid map for a regulated promoter expression system for a bicistronic transcript encoding hIL-21 and hIL-15. -
FIG. 3 shows a plasmid map for a regulated promoter expression system for a bicistronic transcript encoding mIL-12. -
FIG. 4 shows a plasmid map for a regulated promoter expression system for a bicistronic transcript encoding mIL-21 and rnIL-15. -
FIG. 5 shows a plasmid map for a regulated promoter expression system for hIL-21. -
FIG. 6 shows a plasmid map for a regulated promoter expression system for mIL-21. -
FIG. 7 shows a plasmid map for a regulated promoter expression system for a tricistronic transcript encoding hIL-12 and hIL-21. -
FIG. 8 shows a plasmid map for a regulated promoter expression system for a tricistronic transcript encoding mIL-12 and mIL-21. -
FIG. 9 shows the structure of the vector rAd.RheoIL12 in which the E1 and E3 regions have been deleted and the RheoSwitch® Therapeutic System (RTS)-IL-12 components replace the E1 region. The box labeled “IL12” represents the IL-12p40 and IL-12p35 coding sequences separated by IRES. -
FIG. 10 shows a summary chart for translation, post-transcriptional, translation, and post-translation processes. -
FIG. 11 shows modular elements with representation of production pivots. -
FIG. 12 shows a schematic diagram of adenovirus-compatible ULTRAVECTOR® backbone with switch and inducible modular synthetic gene. -
FIG. 13 shows an adenoviral vector map (Vector 43318) for a regulated promoter expressionsystem comprising TNFwt 5′UTR, TNFwtUV signal peptide, TNFwt UV open reading frame (ORF), and 3′ regulatory region of SV40e+pA. -
FIG. 14 shows an adenoviral vector map (Vector 43319) for a regulated promoter expressionsystem comprising TNFwt 5′UTR, TNFoptUV signal peptide, TNFopt UV ORF, and 3′ regulatory region of SV40e+pA. -
FIG. 15 shows an adenoviral vector map (Vector 43320) for a regulated promoter expressionsystem comprising TNFwt 5′UTR, IL-2optUV signal peptide, TNFoptUV ORF, and 3′ regulatory region of SV40e+pA. -
FIG. 16 shows an adenoviral vector map (Vector 43321) for a regulated promoter expressionsystem comprising 5U2 5′UTR, TNFwtUV signal peptide, TNFwtUV ORF, and 3′ regulatory region of SV40e+pA. -
FIG. 17 shows an adenoviral vector map (Vector 43322) for a regulated promoter expressionsystem comprising 5U2 5′UTR, TNFoptUV signal peptide, TNFoptUV ORF, and 3′ regulatory region of SV40e+pA. -
FIG. 18 shows an adenoviral vector map (Vector 43323) for a regulated promoter expressionsystem comprising 5U2 5′UTR, IL-2optUV signal peptide, TNFoptUV ORF, and 3′ regulatory region of SV40e+pA. -
FIG. 19 shows an adenoviral vector map (Vector 43324) for a regulated promoter expressionsystem comprising TNFwt 5′UTR, TNFwtUV signal peptide, TNFwtUV ORF, and 3′ regulatory region of hGH+pA. -
FIG. 20 shows an adenoviral vector map (Vector 43325) for a regulated promoter expressionsystem comprising TNFwt 5′UTR, TNFoptUV signal peptide, TNFoptUV ORF, and 3′ regulatory region of hGH+pA. -
FIG. 21 shows an adenoviral vector map (Vector 43326) for a regulated promoter expressionsystem comprising TNFwt 5′UTR, IL-2optU V signal peptide, TNFoptUV ORF, and 3′ regulatory region of hGH+pA. -
FIG. 22 shows an adenoviral vector map (Vector 43327) for a regulated promoter expressionsystem comprising 5U2 5′UTR, TNFwtUV signal peptide, TNFwtUV ORF, and 3′ regulatory region of hGH+pA. -
FIG. 23 shows an adenoviral vector map (Vector 43328) for a regulated promoter expressionsystem comprising 5U2 5′UTR, TNFwtUV signal peptide, TNFwtUV ORF, and 3′ regulatory region of hGH+pA. -
FIG. 24 shows an adenoviral vector map (Vector 43329) for a regulated promoter expressionsystem comprising 5U2 5′UTR, TNFwtUV signal peptide, TNFwtUV ORF, and 3′ regulatory region of hGH+pA. -
FIG. 25 shows an adenoviral vector map (Vector 43533) for a regulated promoter expression system comprising TNFwt5′UTR, TNFwtUV signal peptide, TNFwtUV ORF, andTNFwt 3′UTR. -
FIG. 26 shows an adenoviral vector map (Vector 43534) for a regulated promoter expressionsystem comprising TNFwt 5′UTR, TNF full-length ORF, andTNFwt 3′UTR. -
FIG. 27 shows a graph showing normalized secreted protein levels of TNF-alpha following transfection of HEK293 cells with vectors with varied PT3 components (−/+) induction via RHEOSWITCH® ligand. -
FIG. 28 shows a graph depicting normalized secreted protein levels of TNF-alpha following transfection of CHO-K1 cells with vectors with varied PT3 components (−/+) induction via RHEOSWITCH®ligand. -
FIG. 29 shows fold differences in TNF-alpha secretion following transfection of HEK293 cells. -
FIG. 30 shows protein secretion differences between5U2 5′UTR and wtUV TNF-alpha 5′UTR. -
FIG. 31 is a line graph that shows Ad-RTS-IL-12 dose response study in the mouse B16F0 melanoma model. Mice were treated onday 12 with a single injection of Ad-RTS-IL-12 at doses of 1 ee7; 1 ee8; 1 ee9; 5ee9; 1ee10; 5ee10 viral particles (vp)) and ligand was delivered using chow starting onday 11. The x-axis shows days post tumor cell inoculation and y-axis indicates tumor volume. Dose levels showed substantial anti-tumor effect. The % tumor reduction compared to control is indicated. -
FIG. 32 is a line graph that shows body weight changes over the course of the study. The changes are shown as % body weight on y-axis. -
FIGS. 33A and 33B are line graphs that show the anti-tumor activity (FIG. 33A ) and safety (FIG. 33B ) of Ad-RTS-mIL12 in the Lewis mouse lung carcinoma (LLC) model. Lewis lung tumor was grown subcutaneousely in immunocompetent C57b/6 mice. When the tumor reached desirable size, the treatment was initiated. Animals received a single dose of AdRTS-mIL12 (1e10 vp) onDay -
FIGS. 34A and 34B are line graphs that show efficacy (FIG. 34A ) and safety (FIG. 34B ) in a mouse melanoma (B16F0) model in which animals were treated with Ad-RTS-mIL12. Tumor was developed s.c. on the flank of C57b/6 mice. A single dose of Ad-RTS-mIL12 (1e10 vp) administered intratumorally (i.t.) onDay 13 post cell inoculation (arrow). The activator ligand (50, 100, 250, 500, 750 and 1000 mg/kg) was given 24 hr prior to vector injection until end of experiment. The tumor growth inhibition was indicated as percentage and compared to control animals. Ad-RTS-IL12 showed therapeutic benefit with broad activator dose window. The treatment was well tolerated. -
FIGS. 35A and 35B are line graphs that show efficacy (FIG. 35A ) and safety (FIG. 35B ) of Ad-RTS-mIL12 in a mouse colon cancer (CT26Luc) model. Murine colon tumor was grown subcutaneously on the flank region of Balb/C mice. Animals were treated intratumorally (i.t.) twice with of Ad-RTS-mIL12 at a dose level of 1e10 vp/100 ul onday -
FIGS. 36A and 36B are line graphs that show efficacy (FIG. 36A ) and safety (FIG. 36B ) Efficacy of Ad-RTS-mIL12 in a pancreatic cancer (PAN02) model. Subcutaneous PAN02 tumor bearing mice were treated intratumorally (i.t.) with a single dose of Ad-RTS-mIL12 at a dose level of 1 e10 vp/100 ul onDay 7 and 14 (arrow) post tumor cell inoculation. The activator chow was supplied to animals a day before vector administration until end of experiment. The control or vector alone group received normal rodent chow only. The result suggests the Ad-RTS-IL12 displays significant antitumor activity (97%) relative to control animals. No major body weight changes were found as a result of Ad-RTS-IL12 therapy. -
FIG. 37 is a vector map for AD-RTS-mIFN alpha. -
FIG. 38 is a vector map for AD-RTS-mTNF alpha. -
FIGS. 39A and 39B are line graphs that show efficacy (FIG. 39A ) and safety (FIG. 39B ) of Ad-RTS-mIL12 in a breast cancer (4T1) model. The 4T1 tumors were grown subcutaneously (s.c.) on the flank of BALB/C mice. The tumor bearing mice were randomized into four groups with 5 animals each; control no treatment, the activator ligand (L) alone, Ad-RTS-mIL12 alone and Ad-RTS-mIL12 with activator ligand. A single injection of Ad-RTS-m/L12 was given intratumorally (i.t.) at a dose level of 1e10 v.p./100 μl PBS at three different time points (arrow). The activator ligand was given to mice through chow 24 hr prior to vector injection until end of experiment. Tumor sizes (volume) and body weights (%) are shown as mean±SE. The animals with no treatment (control) had rapid tumor growth. Treatment with activator ligand alone or three doses of Ad-RTS-mIL12 without activator ligand had slight tumor growth inhibition of 22% and 35% respectively relative to control. Notably, the treatment with Ad-RTS-IL12 plus activator ligand led to significant tumor growth inhibition of 82% compared with control animals with no treatment. No major body weight loss was found in any treatment. -
FIG. 40 is a vector map for Interferon alpha-2a. - Cytokines
- The polynucleotide sequences of interleukin 1 (IL-1), which are cytokines important for inflammatory response against infection, are available from public databases as accession numbers M28983 (human IL-1α); M15330 (human IL-1β); AF201830 (human IL-1δ); AF201831 (human IL-1ε); AF201832 (human IL-1ζ); AF201833 (human IL-1η); NM—010554 (mouse IL-1α); NM—008361 (mouse IL-1β); NM—019451 (mouse L-1δ); □ NM—019450 (mouse IL-1f6); NM—027163 (mouse IL-1f8); NM—153511 (mouse IL-1f9); NM—204524 (chicken IL-1β); NM—017019 (rat IL-1α); and NM—031512 (rat IL-1β), sequences of which are incorporated by reference herein.
- The amino acid sequences of interleukin 1 (IL-1) are available from public databases as accession numbers AAA59134 (human IL-1α); AAA59135 (human IL-1β); AAF25210 (human IL-1δ); AAF25211 (human IL-1ε); AAF25212 (human IL-1ζ); AAF25213 (human IL-1η); NP—034684 (mouse IL-1α); NP—032387 (mouse IL-1β); NP—062324 (mouse L-1δ); □□NP—062323 (mouse IL-1f6); NP—081439 (mouse IL-1f8); NP—705731 (mouse IL-1f9); NP—989855 (chicken IL-1β); NP—058715 (rat IL-1α); and NP—113700 (rat IL-1β), sequences of which are incorporated by reference herein. Laurent et al., Psychiatr. Genet. 7: 103 (1997) identified polymorphic mutations in human interleukin-1 beta gene.
- The polynucleotide sequences of interleukin 2 (IL-2), which belongs to a family of cytokines, including IL-4, IL-7, IL-9, IL-15, and IL-21, are available from public databases as accession numbers U25676 (human); NM—008366 (mouse); NM—204153 (chicken); and NM—053836 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of interleukin 2 (IL-2) are available from public databases as accession numbers AAA70092 (human); NP—032392 (mouse); NP—989484 (chicken); and NP—446288 (rat), sequences of which are incorporated by reference herein.
- Liu et al., Appl. Biochem. Biotechnol. 133: 77 (2006) generated mutant human IL-2, and Lorberboum et al., J. Bial. Chem. 265: 16311 (1990) describes generation of chimeric IL-2.
- The polynucleotide sequences of interleukin 4 (IL-4), which is a cytokine that induces differentiation of naïve helper T cells to Th2 cells, are available from public databases as accession numbers M23442 (human); NM—021283 (mouse); NM—001007079 (chicken); and NM—201270 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of interleukin 4 (IL-4) are available from public databases as accession numbers AAA59150 (human); NP—067258 (mouse); NP—001007080 (chicken); and NP—958427 (rat), sequences of which are incorporated by reference herein.
- Kawashima et al., J. Med. Genet. 35: 502 (1998) describes polymorphisms in IL-4 gene, that are associated with atopic dermatitis.
- Interleukin 7 (IL-7) is a cytokine important for B and T cell development. The polynucleotide sequences of IL-7 are available from public databases as accession numbers J04156 (human); NM—008371 (mouse); NM—001037833 (chicken); and NM—013110 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of interleukin 7 (IL-7) are available from public databases as accession numbers AAA59156 (human); NP—032397 (mouse); NP—001032922 (chicken); and NP—037242 (rat), sequences of which are incorporated by reference herein.
- Feng et al., Genetics 175:545 (2007) have identified point mutations in IL-7 that results in functional deficiency.
- Interleukin 9 (IL-9) is a cytokine produced by T-cells and is a regulator of hematopoietic cells. The polynucleotide sequences of IL-9 are available from public databases as accession numbers NM—000590 (human); NM—008373 (mouse); NM—001037825 (chicken); and NM—001105747 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of interleukin 9 (IL-9) are available from public databases as accession numbers NP—000581 (human); NP—032399 (mouse); NP—001032914 (chicken); and NP—001099217 (rat), sequences of which are incorporated by reference herein.
- IL-12 is a cytokine that can act as a growth factor for activated T and NK cells, enhance the lytic activity of NK/lymphokine-activated Killer cells, and stimulate the production of IFN-gamma by resting peripheral blood mononuclear cells (PBMC). The polynucleotide sequences of IL-12 are available from public databases as accession numbers NM—000882 (human IL12A); NM—002187 (human IL12B); NM—008351 (mouse IL12a); NM—008352 (mouse IL12b); NM—213588 (chicken IL12A); NM—213571 (chicken IL12B); NM—053390 (rat IL12a); and NM—022611 (rat IL12b), sequences of which are incorporated by reference herein.
- The amino acid sequences of interleukin 12 (IL-12) are available from public databases as accession numbers NP—000873 (human IL12A); NP—002178 (human IL12B); NP—032377 (mouse IL12a); NP—032378 (mouse IL12b); NP—998753 (chicken IL12A); NP—998736 (chicken IL12B); NP—445842 (rat IL12a); and NP—072133 (rat IL12b), sequences of which are incorporated by reference herein.
- Interleukin 15 (IL-15) is a cytokine that regulates T and natural killer cell activation and proliferation. The polynucleotide sequences of IL-15 are available from public databases as accession numbers U14407 (human); NM—008357 (mouse); EU334509 (chicken); and AF015719 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of interleukin 15 (IL-15) are available from public databases as accession numbers AAA21551 (human); NP—032383 (mouse); ABY55312 (chicken); and AAB94536 (rat), sequences of which are incorporated by reference herein.
- Interleukin 18 (IL-18), a cytokine produced by macrophage that together with
interleukin 12 induces cell-mediated immunity following infection with microbial products. The polynucleotide sequences of IL-18 are available from public databases as accession numbers U90434 (human); NM—008360 (mouse); EU747333 (chicken); and AY258448 (rat), sequences of which are incorporated by reference herein. - The amino acid sequences of interleukin 18 (IL-18) are available from public databases as accession numbers AAB50010 (human); NP—032386 (mouse); ACE79188 (chicken); and AAP14669 (rat), sequences of which are incorporated by reference herein.
- The polynucleotide sequences of interleukin 21 (IL-21), which is a cytokine that has a potent regulatory effects on cells of the immune system, including natural killer cells and cytotoxic T cells by inducing cell proliferation, are available from public databases as accession numbers AF254069 (human); NM—021782 (mouse); NM—001024835 (chicken); and NM—001108943 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of interleukin 21 (IL-21) are available from public databases as accession numbers, AAG29348 (human); NP—068554 (mouse); NP—001020006 (chicken); and NP—001102413 (rat), sequences of which are incorporated by reference herein.
- Interleukin 27 (IL-27) is a cytokine that plays important function in regulating the activity of B and T lymphocytes. The polynucleotide sequences of IL-27 are available from public databases as accession numbers AY099296 (human); NM—145636 (mouse); and XM—344962 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of interleukin 27 (IL-27) are available from public databases as accession numbers AAM34498 (human); NP—663611 (mouse); and XP—344963 (rat), sequences of which are incorporated by reference herein.
- The polynucleotide sequences of interferon beta 1 (IFNB1), which is a member of group of interferon proteins that bind to specific cell surface receptors (IFNAR), and stimulates both macrophages and natural killer (NK) cells to elicit an anti-viral response, are available from public databases as accession numbers NM—002176 (human); NM—010510 (mouse); NM—001024836 (chicken); and NM—019127 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of interferon beta 1 (IFNB1) are available from public databases as accession numbers NP—002167 (human); NP—034640 (mouse); NP—001020007 (chicken); and NP—062000 (rat), sequences of which are incorporated by reference herein.
- Interferon gamma (IFN-gamma) is a soluble cytokine that is the only Type II interferon and has antiviral, immunoregulatory, and anti-tumor activity. The polynucleotide sequences of IFN-gamma are available from public databases as accession numbers NM—000619 (human); NM—008337 (mouse); and NM—138880 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of interferon gamma (IFN-gamma) are available from public databases as accession numbers NP—000610 (human); NP—032363 (mouse); and NP—620235 (rat) sequences of which are incorporated by reference herein.
- The polynucleotide sequences of tumor necrosis factor (TNF-alpha), which is a multifunctional proinflammatory cytokine secreted predominantly by monocytes/macrophages that has effects on lipid metabolism, coagulation, insulin resistance, and endothelial function, are available from public databases as accession numbers X02910 (human); NM—013693 (mouse); and BC107671 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of TNF-alpha are available from public databases as accession numbers CAA26669 (human); NP—038721 (mouse); and AAI07672 (rat), sequences of which are incorporated by reference herein.
- Human TNF-alpha (abbreviated herein as hTNF-alpha, or simply hTNF) is a human cytokine that exists as a 17 kD soluble form (sTNF-alpha) and a 26 kD membrane associated form (tmTNF-alpha), the biologically active form of which is composed of a trimer of noncovalently bound 17 kD molecules. The structure of hTNF-alpha is described for example, in Pennica, D., et al. (1984) Nature 312:724-729; Davis, J. M., et al. (1987) Biochemistry 26:1322-1326; and Jones, E. Y., et al. (1989) Nature 338:225-228. TNF-alpha may bind to TNF-receptor type 1 (TNFR-1) or TNF-receptor type 2 (TNFR-2) and is involved in regulating immune cells, inducing apoptosis or inflammation, or inhibiting tumorigenesis or viral replication. The cell signaling cascades produced by TNF/TNFR binding are described, e.g., in Wajant, H., et al. (2003) Cell Death Differ. 10(1): 45-65 or Chen, G., et al. (2002) Science 296: 1634-5.
- The full-length human TNF-alpha polypeptide consists of a cytoplasmic domain, a transmembrane domain, and an extracellular domain. A polypeptide sequence of 233aa was reported as a human TNF-alpha polypeptide sequence and is designated herein as SEQ ID NO: 37, which has a cytoplasmic domain of amino acids 1-35 of SEQ ID NO: 37, a transmembrane domain of amino acids 36-56 of SEQ ID NO: 37, and an extracellular domain of amino acids 57-233 of SEQ ID NO: 37. SEQ ID NO: 37 is a nucleotide sequence encoding SEQ ID NO: 35 or 36. Variants of human TNF-alpha include, but are not limited to, the polypeptides with one or more of the following mutations: L105S, R108W, L112F, A160V, S162F, V167A, E222K, F63S, PSD84-86VNR, or E183R.
- Chemokines
- Chemokine (C motif) ligand 1 (XCL1, also known as Lymphotactin) is chemotactic for CD4+ and CD8+ T cells but not for monocytes, and induces a rise in intracellular calcium in peripheral blood lymphocytes. The polynucleotide sequences of XCL1 are available from public databases as accession numbers NM—002995 (human); NM—008510 (mouse); and NM—134361 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of XCL1 are available from public databases as accession numbers NP—002986 (human); NP—032536 (mouse); and NP—599188 (rat), sequences of which are incorporated by reference herein. U.S. Pat. No. 6,022,534 discloses lymphotactin and use to either attract cytotoxic T cells and/or NK cells, and/or to induce proliferation or resident cells. Methods for isolation and usage of an anti-lymphotactin antibody, and XCL1 fusion protein are also disclosed.
- The polynucleotide sequences of CC chemokine ligand 3 (CCL3), also known as macrophage inflammatory protein-1 (MIP-1), which is a so-called monokine (a type of cytokine produced primarily by monocytes and macrophages) that is involved in the acute inflammatory state in the recruitment and activation of polymorphonuclear leukocytes, are available from public databases as accession numbers NM—002983 (human); NM—011337 (mouse); and NM—013025 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of CCL3 are available from public databases as accession numbers NP—002974 (human); NP—035467 (mouse); and NP—037157 (rat), sequences of which are incorporated by reference herein.
- The polynucleotide sequences of CCL5 (RANTES), which is a proinflammatory cytokine involved in inflammation and asthma, are available from public databases as accession numbers AF043341 (human); NM—013653 (mouse); and NM—031116 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of CCL5 are available from public databases as accession numbers AAC03541 (human); NP—038681 (mouse); and NP—112378 (rat), sequences of which are incorporated by reference herein.
- The polynucleotide sequences of CC chemokine ligand 7 (CCL7), which is a chemokine involved in macrophage recruitment during inflammation and cancer invasion, are available from public databases as accession numbers NM—006273 (human); NM—013654 (mouse); and NM—001007612 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of CCL7 are available from public databases as accession numbers NP—006264 (human); NP—038682 (mouse); and NP—001007613 (rat), sequences of which are incorporated by reference herein.
- Chemokine (CXC motif) ligand 9 (CXCL9, also known as MIG) is a T-cell chemoattractant inducible by gamma interferon. The polynucleotide sequences of CXCL9 are available from public databases as accession numbers NM—002416 (human); NM—0108599 (mouse); and NM—145672 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of CXCL9 are available from public databases as accession numbers NP—002407 (human); NP—032625 (mouse); and NP—663705 (rat), sequences of which are incorporated by reference herein.
- Chemokine (C—X—C motif) ligand 10 (CXCL10) is a small cytokine with roles in chemoattraction for cells in the immune system, adhesion of T cells to endothelial cells, anti-tumor activity and angiogenesis. The polynucleotide sequences of CXCL10 are available from public databases as accession numbers X02530 (human); NM—021274 (mouse); and BC058444 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of chemokine (C—X—C motif) ligand 10 (CXCL10) are available from public databases as accession numbers CAA26370 (human); NP—067249 (mouse); and AAH58444 (rat), sequences of which are incorporated by reference herein.
- Chemokine (C-X-C motif) ligand 12 (CXCL12), also known as stromal cell-derived factor 1 (SDF-1), is a small cytokine that belong to the intercrine family, members of which activate leukocytes and are often induced by proinflammatory stimuli such as LPS, TNF or IL1. The polynucleotide sequences of CXCL12 are available from public databases as accession numbers NM—000609 (human); NM—001012477 (mouse); NM—204510 (chicken); and NM—001033883 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of CXCL12 are available from public databases as accession numbers NP—000600 (human); NP—001012495 (mouse); NP—989841 (chicken); and NP—001029055 (rat), sequences of which are incorporated by reference herein.
- Hansson et al., Microbes and Infection 8:841 (2006) discusses that interaction between chemokine (C-C motif) receptor 7 (CCR7) and chemokine (C-C motif) ligand 19 (CCL19, also known as MIP-3β) is crucial for the generation of primary immune responses. The polynucleotide sequences of CCR7 are available from public databases as accession numbers NM—001838 (human); and NM—007719 (mouse), sequences of which are incorporated by reference herein.
- The amino acid sequences of CCR7 are available from public databases as accession numbers NP—001829 (human); and NP—031745 (mouse), sequences of which are incorporated by reference herein.
- The polynucleotide sequences of CCL19 are available from public databases as accession numbers NM—006274 (human); and NM—011888 (mouse), sequences of which are incorporated by reference herein.
- The amino acid sequences of CCL19 are available from public databases as accession numbers NP—006265 (human); and NP—036018 (mouse), sequences of which are incorporated by reference herein.
- The polynucleotide sequences of CC chemokine ligand 21 (CCL21), a well established ligand for CCR7 which is necessary for CD4+ but not CD8+ T cells to reach their steady state ‘set point’, and perturbations in the expression of CCL21 may alter susceptibility to autoimmunity, are available from public databases as accession numbers AB002409 (human); NM—011335 (mouse CCL21a); NM—011124 (mouse CCL21b); and NM—023052 (mouse CCL21c); sequences of which are incorporated by reference herein.
- The amino acid sequences of CCL21 are available from public databases as accession numbers BAA21817 (human); NP—035465 (mouse CCL21a); NP—035254 (mouse CCL21b); and NP—075539 (mouse CCL21c), sequences of which are incorporated by reference herein.
- Interleukin-8 (IL-8), is a chemokine, also called neutrophil-activating peptide-1 or SCYB8, is a tissue-derived peptide secreted by several types of cells in response to inflammatory stimuli. U.S. Pat. Nos. 6,133,426 and 6,177,980 disclose amino acid and polynucleotide sequences of humanized anti-IL-8 antibodies. The polynucleotide sequence of human IL-8 is available from public database as accession number NM—000584, sequence of which is incorporated by reference herein.
- The amino acid sequence of human IL-8 is available from public database as accession number NP—000575, sequence of which is incorporated by reference herein.
- Growth Factors
- Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a cytokine that functions as a white blood cell growth factor, stimulates stems cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. The polynucleotide sequences of GM-CSF are available from public databases as accession numbers M11734 (human); NM—009969 (mouse); EU520303 (chicken); NM—001037660 (rat Csf2ra); and NM—133555 (rat Csf2rb), sequences of which are incorporated by reference herein.
- The amino acid sequences of granulocyte/macrophage colony-stimulating factor (GM-CSF) are available from public databases as accession numbers AAA52122 (human); NP—034099 (mouse); ACB11534 (chicken); NP—001032749 (rat Csf2ra); and NP—598239 (Csf2rb), sequences of which are incorporated by reference herein.
- The polynucleotide sequences of FMS-related tyrosine kinase ligand (FLT3/FLK2 ligand, Flt3L), which may function as a growth factor receptor on hematopoietic stem cells or progenitor cells or both, are available from public databases as accession numbers U04806 (human); and NM—013520 (mouse), sequences of which are incorporated by reference herein.
- The amino acid sequences of FLT3/FLK2 ligand (Flt3L) are available from public databases as accession numbers AAA17999 (human); and NP—038548 (mouse), sequences of which are incorporated by reference herein.
- The polynucleotide sequence of transforming growth factor, alpha (TGF-alpha), which is upregulated in some human cancers can reversibly confer the transformed phenotype on cultured cells, is available from public databases as accession numbers NM—001099691 (human); NM—031199 (mouse); NM—001001614 (chicken); and NM—012671 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of TGF-alpha is available from public databases as accession numbers NP—001093161 (human); NP—112476 (mouse); NP—001001614 (chicken); and NP—036803 (rat), sequences of which are incorporated by reference herein.
- Adjuvants
- Beta-defensins are antimicrobial peptides implicated in innate immune response against many Gram-negative and Gram-positive bacteria, fungi and viruses. The polynucleotide sequences of beta-defensins are available from public databases as accession numbers X92744 (human hBD-1); AJ000152 (human hBD-2); AF217245 (human beta defensin-3); AJ314835 (human beta defensin-4); AB089180 (human hBD-5); AY122466 (human defensin beta 106, DEFB106); AF540979 (human beta defensin 107, DEFB107); AF529416 (human beta defensin, DEFB108); DQ012014 (human beta defensin 110, DEFB110); DQ012015 (human beta defensin 111, DEFB111); DQ012016 (human beta defensin 112, DEFB112); DQ012017 (human beta defensin 113, DEFB113); DQ012018 (human beta defensin 114, DEFB114); DQ012019 (human beta defensin 115, DEFB115); DQ012020 (human beta defensin 116, DEFB116); DQ012021 (human beta defensin 117, DEFB117); NM—007843 (mouse defensin beta 1); NM—010030 (mouse defensin beta 2, Defb2); NM—013756 (mouse defensin beta 3, Defb3); NM—019728 (mouse defensin beta 4, Defb4); NM—030734 (mouse defensin beta 5, Defb5); NM—054074 (mouse defensin beta 6, Defb6); NM—139220 (mouse defensin beta 7); NM—153108 (mouse defensin beta 8, Defb8); NM—139219 (mouse defensin beta 9, Defb9); and NM—139225 (mouse defensin beta 10, Defb10); sequences of which are incorporated by reference herein.
- The amino acid sequences of beta-defensins are available from public databases as accession numbers CAA63405 (human hBD-1); CAB65126 (human hBD-2); AAF73853 (human beta defensin-3); CAC85520 (human beta defensin-4); BAC10630 (human hBD-5); AAM93908 (human defensin beta 106, DEFB106); AAN33115 (human beta defensin 107, DEFB107); AAQ09525 (human beta defensin, DEFB108); AAY59750 (human beta defensin 110, DEFB110); AAY59751 (human beta defensin 111, DEFB111); AAY59752 (human beta defensin 112, DEFB112); AAY59753 (human beta defensin 113, DEFB113); AAY59754 (human beta defensin 114, DEFB114); AAY59755 (human beta defensin 115, DEFB115); AAY59756 (human beta defensin 116, DEFB116); AAY59757 (human beta defensin 117, DEFB117); NP—031869 (mouse defenin beta 1); NP—034160 (mouse defensin beta 2, Defb2); NP—038784 (mouse defensin beta 3, Defb3); NP—062702 (mouse defensin beta 4, Defb4); NP—109659 (mouse defensin beta 5, Defb5); NP—473415 (mouse defensin beta 6, Defb6); NP—631966 (mouse defensin beta 7, Defb7); NP—694748 (mouse defensin beta 8, Defb8); NP—631965 (mouse defensin beta 9, Defb9); and NP—631971 (mouse defensin beta 10, Defb10), sequences of which are incorporated by reference herein. See also U.S. Pat. No. 5,242,902 for additional human and rat defensin peptide sequences.
- High-mobility group box-1 (HMGB1) proteins are nonhistone chromosomal proteins that function as cytokines, mediating local and systemic responses to necrotic cell death and cancer, invasion by pathogens, trauma, and sepsis. The polynucleotide sequences of
HMGB 1 proteins are available from public databases as accession numbers NM—002128 (human); NM—010439 (mouse); NM—204902 (chicken); and NM—012963 (rat), sequences of which are incorporated by reference herein. - The amino acid sequences of high-mobility group box-1 (HMGB1) are available from public databases as accession numbers NP—002119 (human); NP—034569 (mouse); NP—990233 (chicken); and NP—037095 (rat), sequences of which are incorporated by reference herein.
- Phagocytic S100 proteins mediate inflammatory responses and recruit inflammatory cells to sites of tissue damage, and are members of Damage-associated molecular pattern (DAMP) molecules that are important for innate immunity. See Foell et al., J Leukocyte Biol. 81:1 (2006). The polynucleotide sequences of S100 proteins are available from public databases as accession numbers BC014392 (human S100 A1); BC002829 (human S100 A2); BC012893 (human S100 A3); BC016300 (human S100 A4); Z18954 (human S100D); BC001431 (human S100 A6); BC 034687 (human S100 A7); BC005928 (human S100 A8); BC047681 (human S100 A9); BC015973 (human S100 A10); D38583 (human clagizzarin); NM—011309 (mouse S100a1); NM—009115 (mouse S100b); NM—013650 (mouse S100a8); NM—009114 (mouse S100a9); NM—011310 (mouse S100a3); NM—011311 (mouse S100a4); and NM—011312 (mouse S100a5), sequences of which are incorporated by reference herein.
- The amino acid sequences of S100 proteins are available from public databases as accession numbers AAH14392 (human S100 A1); AAH02829 (human S100 A2); AAH12893 (human S100 A3); AAH16300 (human S100 A4); CAA79479 (human S100D); AAH01431 (human S100 A6); AAH34687 (human S100 A7); AAH05928 (human S100 A8); AAH47681 (human S100 A9); AAH15973 (human 5100 A10); BAA07597 (human clagizzarin); NP—035439 (mouse S100a1); NP—033141 (mouse S100b); NP—038678 (mouse S100a8); NP—033140 (mouse S100a9); NP—035440 (mouse S100a3); NP—035441 (mouse S100a4); and NP—035442 (mouse S100a5), sequences of which are incorporated by reference herein.
- Mannan, a plant polysaccharide, that is a polymer of the sugar mannose, is useful for generation of an immune response. U.S. Pat. No. 5,807,559, discloses immunogenic conjugates of Mannan that may be useful for generating T cell immunity against tumor-associated carbohydrate structures or against carbohydrate structures expressed on infectious agents and/or infected host cells. U.S. Pat. No. 5,773,425 discloses use of mannan to relieve symptoms and/or cure viral diseases and to enhance immune response.
- Bacille Calmette-Guerin (BCG), live attenuated Mycobacterium species, are used as vaccine against to prevent severe and fatal tuberculosis. U.S. Pat. No. 7,393,541 discloses generation of an adjuvant vaccine for producing an in vivo T-cell mediated immune response to a mycobacterium in a mammalian subject. See also Hubbard and Collins, Infect. Immun. 59(2): 570. U.S. Pat. No. 5,292,513 discloses a method for priming macrophages in vivo in patients in need of enhanced bactericidal and anti-viral activity with heat killed BCG. The complete genome sequence of BCG is available from public databases as accession number NC—008769 (M. bovis BCG str. Pasteur 1173P2, complete genome).
- Bacterial lipopolysaccharides (LPS) are endotoxins that induces a strong immune response upon infection with Gram-negative bacteria. U.S. Pat. No. 4,148,877 discloses fractionation of LPS from bacterial culture and use the fraction as a drug to induce resistance to bacterial infection. U.S. Pat. No. 5,292,513 discloses a method for priming macrophages in vivo in patients in need of enhanced bactericidal and anti-viral activity with LPS.
- Co-stimulatory Molecules (Positive)
- OX40 ligand (OX40L) belongs to tumor necrosis factor (ligand) superfamily member 4 (Tnfsf4), is expressed on dendritic cells and promotes Th2 cell differentiation. The polynucleotide sequences of OX40 ligand are available from public databases as accession numbers X79929 (human); U12763 (mouse); and AF037067 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of OX40 ligand (OX40L) are available from public databases as accession numbers CAA56284 (human); AAA21871 (mouse); and AAC67236 (rat), sequences of which are incorporated by reference herein.
- The 4-1BB ligand (4-1BBL) belongs to tumor necrosis factor (ligand) superfamily member 9 (Tnfsf9), which is a
type 2 transmembrane glycoprotein and is expressed on activated T lymphocytes. The polynucleotide sequences of 4-1BBL are available from public databases as accession numbers NM—003811 (human); NM—009404 (mouse); and AY332409 (rat), sequences of which are incorporated by reference herein. - The amino acid sequences of 4-1BB ligand (4-1BBL) are available from public databases as accession numbers NP—003802 (human); NP—033430 (mouse); and AAQ01228 (rat), sequences of which are incorporated by reference herein.
- The CD40 protein belongs to the tumor necrosis factor
receptor superfamily member 5, is essential in mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. The polynucleotide sequences of CD40 proteins are available from public databases as accession numbers X60592 (human); NM—170701 (mouse); NM—204665 (chicken); and NM—134360 (rat), sequences of which are incorporated by reference herein. - The amino acid sequences of CD40 proteins are available from public databases as accession numbers CAA43045 (human); NP—733802 (mouse); NP—989996 (chicken); and NP—599187 (rat), sequences of which are incorporated by reference herein.
- CD40L (CD40 ligand, or CD154) is primarily expressed on activated T cells and is a member of the TNF superfamily of molecules. It binds to CD40 on antigen-presenting cells. CD40L plays the role of a costimulatory molecule and induces activation in antigen-presenting cells in associate with T cell receptor stimulation my MHC molecules on the antigen-presenting cells. CD40L has three binding partners: CD40, α5β1 integrin and αIIbβ3. The CD40L sequences are available from public databases as accession numbers NM—000074 and MP—000065 (human) and NM—011616 and NP—035746 (mouse).
- The glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) can evoke effective tumor immunity via T cell stimulation. Administration of anti-GITR monoclonal antibody (mAb) can provoke potent tumor-specific immunity and eradicated established tumors without eliciting overt autoimmune disease. See Ko et al., J. Exp. Med. 7: 885 (2005). U.S. Pat. No. 6,503,184 B1 discloses an Anti-GITR antibody.
- The polynucleotide sequences of GITR ligand (GITRL) are available from public databases as accession numbers AY358868 (human); and AY359852 (mouse), sequences of which are incorporated by reference herein.
- The amino acid sequences of GITR ligand (GITRL) are available from public databases as accession numbers AAQ89227 (human); and AAQ55265 (mouse), sequences of which are incorporated by reference herein.
- Herpes virus entry mediator (HVEM) binding ligand (HSVgD), also referred to as p30, or LIGHT is a TNF family member involved in co-stimulation of T cells. LIGHT has two receptors, herpes virus entry mediator (HVEM) and lymphotoxin-βreceptor (LT-βR). Being a ligand for HVEM, HSVgD activates T cells by acting as a costimulatory factor to T cells that results in T cell proliferation and cytokine secretion. See U.S. Pat. No. 7,118,742 for polynucleotide and amino acid sequences of LIGHT. U.S. Pat. No. 5,654,174 describes a variant gD protein with deletion of carboxy terminal residues.
- CD70 is a cytokine that binds to CD27. It plays a role in T-cell activation. Induces the proliferation of costimulated T-cells and enhances the generation of cytolytic T-cells. The polynucleotide sequences of CD70 are available from public databases as accession numbers NM—001252 (human); NM—011617 (mouse); and NM—001106878 (rat), sequences of which are incorporated by reference herein.
- The amino acid sequences of CD70 are available from public databases as accession numbers NP—001243 (human); NP—035747 (mouse); and NP—001100348 (rat), sequences of which are incorporated by reference herein.
- ICOS-L is a ligand for the T-cell-specific cell surface receptor ICOS and acts as a costimulatory signal for T-cell proliferation and cytokine secretion. ICOS-L also induces B-cell proliferation and differentiation into plasma cells. ICOS-L could play an important role in mediating local tissue responses to inflammatory conditions, as well as in modulating the secondary immune response by co-stimulating memory T-cell function. The polynucleotide sequences of ICOS-L are available from public databases as accession numbers NM—015259 (human); and NM—015790 (mouse), sequences of which are incorporated by reference herein.
- The amino acid sequences of ICOS-L are available from public databases as accession numbers NP—056074 (human); and NP—056605 (mouse), sequences of which are incorporated by reference herein.
- PD-L1 (also known as CD274) protein is expressed in activated monocytes, T and B cells. PD-L1 is upregulated in monocytes upon treatment with IFN-gamma, and in dendritic cells and keratinocytes upon treatment with IFN-gamma, together with other activators. The polynucleotide sequences of PD-
L 1 proteins are available from public databases as accession numbers NM—014143 (human); and NM—021893 (mouse), sequences of which are incorporated by reference herein. - The amino acid sequences of PD-L1 proteins are available from public databases as accession numbers NP—054862 (human); and NP—068693 (mouse), sequences of which are incorporated by reference herein.
- Co-Stimulatory Molecule (Negative)
- Cytotoxic T lymphocyte-associated 4 (CTLA4) is a member of the immunoglobulin superfamily and is a costimulatory molecule expressed in activated T cells. U.S. Pat. Nos. 7,034,121 and 6,984,720 disclose methods of preparation and usage of antibodies against CTLA4. U.S. Pat. No. 6,984,720 also discloses amino acid sequences of heavy and light chain of anti-CTLA4 antibody.
- PD-1 molecules are members of the immunoglobulin gene superfamily, which binds to PD-1 ligand (PD-L1). Binding of a PD-1 receptor on a T-cell by PD-L1 transmits a costimulatory signal to the cell, which prevents the cells from progressing through the cell cycle, and increases T cell proliferation. Inhibition of an interaction between PD-L1 and receptor on the T cell with an anti-PD-L1 antibody results in the down regulation of the immune response termed as immune cell energy. U.S. Pat. No. 7,029,674 discloses methods of preparation and sequence of anti-PD-L1 antibody.
- PD-L2 is primarily known as a ligand for PD-1 (or the human homologue PDCD1). However, PD-12 has been reported to be involved in the costimulatory signal, essential for T lymphocyte proliferation and IFN-gamma production in a PDCD1-independent manner. Interaction with PDCD1 inhibit T-cell proliferation by blocking cell cycle progression, and cytokine production. Yamazaki et al., J. of Immunol. 169: 5538 (2002) and Ansari et al., J. Exp. Med. 198: 63 (2003) describe preparation of anti-PD-L2 monoclonal antibodies.
- Counter Immune Suppressants (Tolerance Inhibitors)
- Transforming growth factor-beta (TGF-β) is a multifunctional protein that regulates cell proliferation and differentiation, by interacting with one of the two transmembrane serine/threonine kinase receptors, type I and type II. See Chen et al., Science 28: 1335 (1993). TGF receptor type II (TGFR2) phosphorylate and activate type I receptors which autophosphorylate, then bind and activate SMAD transcriptional regulators. Lynch M A et al., Cancer Res. 58: 4227 (1998) describes mutations in the transforming growth factor β receptor type II gene (TGFBR2) that are associated with human ovarian carcinomas. Brand et al., J. Biol. Chem. 268:11500-11503 (1993) describes that deletion of predicted serine/theronine kinase cytoplasmic domain (nucleotides 1172-2036 of TGFfβR2 cDNA H2-3FF, available from public databases as accession number M85079 and amino acid sequence available as accession number AAA61164) impairs the all three TGF-β (1, 2 and 3) dependent gene expressions. TGF-β is produced in most human tumors and inhibits tumor antigen-specific cellular immunity. Foster et al., J. Immunother. 31:500 (2008) describes that expression of dominant negative TGFβR2 in cytotoxic T lymphocytes can lead to resistance to the inhibitory effects of TGF-β.
- TGFβ acts synergistically with TGFα in inducing transformation. It also acts as a negative autocrine growth factor. Dysregulation of TGFβ activation and signaling may result in apoptosis. Ziyadeh et al., Proc. Natl. Acad. Sci. 97: 8015 (2000) describes that administration of anti-TGFβ antibody can prevent renal insufficiency and glomerulosclerosis in the db/db mouse, a model of type II diabetes that develops overt nephropathy. Methods of generation and use of TGFβ monoclonal antibodies are described in U.S. Pat. No. 6,419,928. Barcellos-Hoff et al., Am J. Pathol. 147:5 (1995) also describes a method for generation of TGFβ antibody. Amino acid and nucleotide sequences for TGFβ fusion protein constructs are described in U.S. Pat. No. 6,756,215.
- IL-10 is a cytokine produced by activated Th2 cells, B cells, keratinocytes, monocytes, and macrophages. IL-10 inhibits the synthesis of a number of cytokines, including IFN-gamma, IL-2, IL-3, TNF and GM-CSF produced by activated macrophages and by helper T-cells. IL-10 is useful in promoting growth and differentiation of activated human B cells, inhibiting Th1 responses to prevent transplant rejection and T cell-mediated autoimmune diseases. O'Farrell et al., EMBO J. 17:1006 (1998); Kanbayashi et al., Cell Immunol. 171:153 (1996); Fukushima et al., Br. J. Ophthalmol. 90:1535 (2006); and van Lent et al., Ann. Rheum. Dis. 66:334 (2007) describe the preparation of anti-IL10 antibodies. U.S. Pat. No. 7,326,567 discloses polynucleotide sequence of IL-10 antibody. U.S. Pat. No. 5,837,232 discloses a method to treat a B-cell mediated autoimmune disorder with anti-IL-10 antibodies.
- Suppressor of cytokine signaling (SOCS) family proteins form part of a classical negative feedback system that regulates cytokine signal transduction. Alexander et al. Cell 98: 597 (1999) describes that suppressor of cytokine signaling 1 (SOCS1) is a critical inhibitor of interferon-gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Hilton et al., Proc. Natl. Acad. Sci. USA 95:114 (1999) discusses that SOCS1 is involved in negative regulation of cytokines that signal through the JAK/STAT3 pathway. Ohya et al. J. Biol. Chem. 272: 27178 (1997) describes that SOCS proteins appear to be a major regulator of signaling by interleukin 6 (IL-6) and leukemia inhibitory factor (LIF). U.S. Pat. No. 6,534,277 discloses a method for the preparation and use of anti-SOCS1 antibody, where a nucleic acid sequence encoding SOCS1 antibody is introduced into cells such that the antibody is expressed by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect. U.S. Pat. Nos. 6,323,317 and 7,049,418 also disclose anti-SOCS1 antibodies.
- TGF-α is a mitogenic polypeptide that is able to bind to the EGF receptor and to act synergistically with TGF-β to promote anchorage-independent cell proliferation in soft agar. Ellis et al., N. Engl. J. Med. 317:158 (1987) describes that TGF-α plays a role in certain paraneoplastic manifestations of melanoma. U.S. Pat. No. 4,742,003 and Xian et al., The J. of Histochem. & Cytochem. 47:949 (1999) describe methods of preparation of Anti-TGF-α antibodies.
- Both tumor necrosis factor receptor (TNFR1) and Fas contain cytoplasmic Fas-associated protein with death domain (FADD), which is essential for Fas and TNF-induced signaling for programmed cell death (apoptosis) and receptor oligomerization. A mammalian protein designated FADD having the ability to bind the cytoplasmic region or domain of the Fas receptor and inhibits FAS mediated apoptosis has been identified. The polynucleotide sequence of FADD is available from public database as accession number U24231, and the amino acid sequence as accession number AAA86517, which are incorporated by reference herein. A FADD fragment or nucleic acid encoding it which is a dominant negative inhibitor of functionally intact native FADD is described in U.S. Pat. No. 6,562,797 B1.
- p53 (also known as protein 53 or tumor protein 53), is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that is involved in preventing cancer. Amino acid and polynucleotide sequences for p53 are available as accession numbers NM—00546 and NP—000537 (human) and NM—011640 and NP—035770 (mouse).
- Survivin is a member of the inhibitor of apoptosis family. The survivin protein functions to inhibit caspase activation, thereby leading to negative regulation of apoptosis or programmed cell death. This has been shown by disruption of survivin induction pathways leading to increase in apoptosis and decrease in tumour growth. The survivin protein is expressed highly in most human tumours and fetal tissue, but is completely absent in terminally differentiated cells. This fact therefore makes survivin an ideal target for cancer therapy as cancer cells are targeted while normal cells are left alone. Survivin expression is also highly regulated by the cell cycle and is only expressed in the G2-M phase. It is known that survivin localizes to the mitotic spindle by interaction with tubulin during mitosis and may play a contributing role in regulating mitosis. Rregulation of survivin appears seems to be linked to the p53 protein. Amino acid and polynucleotide sequences for p53 are available as accession numbers NM—001012270 and NP—001012270 (human) and NM—001012273 and NP—001012273 (mouse).
- The melanoma-associated antigen—3 (MAGES) amino acid sequence is found as accession number P43357-1 (UniParc).
- Prostate-specific antigen (PSA) is a protein produced by the cells of the prostate gland. PSA is present in small quantities in the serum of men with healthy prostates, but is often elevated in the presence of prostate cancer and in other prostate disorders.
- Prostate specific membrane antigen (PSMA) is a
type 2 integral membrane glycoprotein found in prostate tissues and a few other tissues. It is a possible therapeutic target for prostate cancer. - SEQ ID NO: 1 is a polynucleotide sequence of a construct coding for mIL-12 and m-IL21.
- SEQ ID NO: 2 is a polynucleotide sequence of a construct coding for hIL-12 and hIL-21.
- SEQ ID NO: 3 is a polynucleotide sequence of a construct coding for mIL-21 and mIL-15.
- SEQ Ill NO: 4 is a polynucleotide sequence of a construct coding for mIL-12.
- SEQ ID NO: 5 is a polynucleotide sequence of a construct coding for hIL-21 and hIL-15.
- SEQ ID NO: 6 is a polynucleotide sequence of a construct coding for hIL-21.
- SEQ ID NO: 7 is a polynucleotide sequence of a construct coding for mIL-21.
- SEQ ID NO: 8 is a polynucleotide sequence of a construct coding for hIL-21.
- SEQ ID NO: 9 is a polynucleotide sequence coding for mIL-21.
- SEQ ID NO: 10 is an amino acid sequence of mIL-21.
- SEQ ID NO: 11 is a polynucleotide sequence coding for mIL-15.
- SEQ ID NO: 12 is an amino acid sequence of mIL-15.
- SEQ ID NO: 13 is a polynucleotide sequence coding for mp40 of mIL-12.
- SEQ ID NO: 14 is the amino acid sequence of mp40 of mIL-12.
- SEQ ID NO: 15 is a polynucleotide sequence coding for mp35 of mIL-12.
- SEQ ID NO: 16 is the amino acid sequence of mp35 of mIL-12.
- SEQ ID NO: 17 is a polynucleotide sequence coding for hIL-21.
- SEQ ID NO: 18 is the amino acid sequence of hIL-21.
- SEQ ID NO: 19 is a polynucleotide sequence coding for hIL-15.
- SEQ ID NO: 20 is the amino acid sequence of hIL-15.
- SEQ ID NO: 21 is a polynucleotide sequence coding for p40 of hIL-12.
- SEQ ID NO: 22 is the amino acid sequence of p40 of hIL-12.
- SEQ ID NO: 23 is a polynucleotide sequence coding for p35 of hIL-12.
- SEQ ID NO: 24 is the amino acid sequence of p35 of hIL-12.
- SEQ ID NO: 25 is a nucleic acid sequence of an ecdysone response element found in Drosophila.
- SEQ ID NO: 26 is a nucleic acid sequence of an ecdysone response element found in Drosophila melanogaster.
- SEQ ID NO: 27 is a nucleic acid sequence of an ecdysone response element found in Drosophila melanogaster.
- SEQ ID NO: 28 is a restriction site of a horning endonuclease (HE) enzyme (I-SceI)
- SEQ ID NO: 29 is a DNA sequence of adenovirus vector comprising human IL-12 coding sequence: Ad-RTS-hIL-12 (SP1-RheoIL-12).
- SEQ ID NO: 30 is a nucleic acid sequence of human TNF wild-
type 5′UTR. - SEQ ID NO: 31 is a nucleic acid sequence of
5U2 5′UTR. - SEQ ID NO: 32 is a codon-optimized nucleic acid sequence encoding IL-2 signal peptide.
- SEQ ID NO: 33 is a wild-type nucleic acid sequence encoding human TNF-alpha signal peptide.
- SEQ ID NO: 34 is a codon-optimized nucleotide sequence encoding human TNF-alpha signal peptide.
- SEQ ID NO: 35 is a wild-type nucleic acid sequence encoding human TNT-alpha.
- SEQ ID NO: 36 is a codon-optimized nucleic acid sequence encoding human TNF-alpha.
- SEQ ID NO: 37 is an amino acid sequence of human TNF-alpha.
- SEQ ID NO: 38 is a nucleic acid sequence of 3′ regulatory region comprising a nucleotide sequence encoding a SV40 polyadenylation signal.
- SEQ ID NO: 39 is a nucleic acid sequence of 3′ regulatory region comprising a nucleotide sequence encoding a human growth hormone polyadenylation signal.
- SEQ ID NO: 40 is a nucleic acid sequence comprising wild-type human TNF-
alpha 3′UTR. - SEQ ID NO: 41 is a nucleic acid sequence of human TNF-
alpha 3′ UTR AtoC mutant. - SEQ ID NO: 42 is a nucleic acid sequence of
human GAST 3′UTR. - SEQ ID NO: 43 is a nucleic acid sequence of synthetic 3′ regulatory region.
- SEQ ID NO: 44 is a nucleic acid sequence of
human GAPDH 5′UTR. - SEQ ID NO: 45 is a wild-type nucleic acid sequence of insuline SP.
- SEQ ID NO: 46 is a wild-type nucleic acid sequence encoding human FGF-19 signal peptide.
- SEQ ID NO: 47 is a nucleic acid sequence of
Vector 43318. - SEQ ID NO: 48 is a nucleic acid sequence of
Vector 43319. - SEQ ID NO: 49 is a nucleic acid sequence of
Vector 43320. - SEQ ID NO: 50 is a nucleic acid sequence of
Vector 43321. - SEQ ID NO: 51 is a nucleic acid sequence of
Vector 43322. - SEQ ID NO: 52 is a nucleic acid sequence of
Vector 43323. - SEQ ID NO: 53 is a nucleic acid sequence of
Vector 43324. - SEQ ID NO: 54 is a nucleic acid sequence of
Vector 43325. - SEQ ID NO: 55 is a nucleic acid sequence of
Vector 43326. - SEQ ID NO: 56 is a nucleic acid sequence of
Vector 43327. - SEQ ID NO: 57 is a nucleic acid sequence of
Vector 43328. - SEQ ID NO: 58 is a nucleic acid sequence of
Vector 43329. - SEQ ID NO: 59 is a nucleic acid sequence of
Vector 43533. - SEQ ID NO: 60 is a nucleic acid sequence of Vector 43534.
- SEQ ID NO: 61 is a nucleic acid sequence of Vector VVN2823 (Ad-RTS-hIL-12).
- SEQ ID NO: 62 is a nucleic acid sequence of Vector VVN2539 (Ad-RTS-mIL-12).
- Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference and understanding, and the inclusion of such definitions herein should not necessarily be construed to mean a substantial difference over what is generally understood in the art. Commonly understood definitions of molecular biology terms and/or methods and/or protocols can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; Lewin, Genes V, Oxford University Press: New York, 1994; Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001) and Ausubel et al., Current Protocols in Molecular Biology (1994). As appropriate, procedures involving the use of commercially available kits and/or reagents are generally carried out in accordance with manufacturer's guidance and/or protocols and/or parameters unless otherwise noted.
- The term “isolated” for the purposes of the invention designates a biological material (cell, nucleic acid or protein) that has been removed from its original environment (the environment in which it is naturally present). For example, a polynucleotide present in the natural state in a plant or an animal is not isolated, however the same polynucleotide separated from the adjacent nucleic acids in which it is naturally present, is considered “isolated.”
- The term “purified,” as applied to biological materials does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. It is rather a relative definition.
- “Nucleic acid,” “nucleic acid molecule,” “oligonucleotide,” “nucleotide,” and “polynucleotide” are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA.
- The term “fragment,” as applied to polynucleotide sequences, refers to a nucleotide sequence of reduced length relative to the reference nucleic acid and comprising, over the common portion, a nucleotide sequence identical to the reference nucleic acid. Such a nucleic acid fragment according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. Such fragments comprise, or alternatively consist of, oligonucleotides ranging in length from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000, or more consecutive nucleotides of a nucleic acid according to the invention.
- As used herein, an “isolated nucleic acid fragment” refers to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.
- A “gene” refers to a polynucleotide comprising nucleotides that encode a functional molecule, including functional molecules produced by transcription only (e.g., a bioactive RNA species) or by transcription and translation (e.g., a polypeptide). The term “gene” encompasses cDNA and genomic DNA nucleic acids. “Gene” also refers to a nucleic acid fragment that expresses a specific RNA, protein or polypeptide, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and/or coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A chimeric gene may comprise coding sequences derived from different sources and/or regulatory sequences derived from different sources. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism, A “foreign” gene or “heterologous” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. For example, the interleukin-12 (IL-12) gene encodes the IL-12 protein. IL-12 is a heterodimer of a 35-kD subunit (p35) and a 40-kD subunit (p40) linked through a disulfide linkage to make fully functional IL-12p70. The IL-12 gene encodes both the p35 and p40 subunits.
- “Heterologous DNA” refers to DNA not naturally located in the cell, or in a chromosomal site of the cell. The heterologous DNA may include a gene foreign to the cell.
- The term “genome” includes chromosomal as well as mitochondrial, chloroplast and viral DNA or RNA.
- A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook et al. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly
Chapter 11 and Table 11.1 therein). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. - Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm of 55°, can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5×SSC, 0.5% SDS. Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5× or 6×SSC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5× or 6×SSC.
- Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The term “complementary” is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the invention also includes isolated nucleic acid fragments that are complementary to the complete sequences as disclosed or used herein as well as those substantially similar nucleic acid sequences.
- In one embodiment of the invention, polynucleotides are detected by employing hybridization conditions comprising a hybridization step at Tm of 55° C., and utilizing conditions as set forth above. In other embodiments, the Tm is 60° C., 63° C., or 65° C.
- Post-hybridization washes also determine stringency conditions. One set of conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 minutes (min), then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. One set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS is increased to 60° C. Another set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.
- The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8).
- In one embodiment of the invention, polynucleotides are detected by employing hybridization conditions comprising a hybridization step in less than 500 mM salt and at least 37° C., and a washing step in 2×SSPE at a temperature of at least 63° C. In another embodiment, the hybridization conditions comprise less than 200 mM salt and at least 37° C. for the hybridization step. In a further embodiment, the hybridization conditions comprise 2×SSPE and 63° C. for both the hybridization and washing steps.
- In another embodiment, the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; e.g., at least about 20 nucleotides; e.g., at least 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.
- The term “probe” refers to a single-stranded nucleic acid molecule that can base pair with a complementary single stranded target nucleic acid to form a double-stranded molecule.
- As used herein, the term “oligonucleotide” refers to a short nucleic acid that is hybridizable to a genomic DNA molecule, a cDNA molecule, a plasmid DNA or an mRNA molecule. Oligonucleotides can be labeled, e.g., with 32P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated. A labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid. Oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a nucleic acid, for DNA sequencing, or to detect the presence of a nucleic acid. An oligonucleotide can also be used to form a triple helix with a DNA molecule. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
- A “primer” refers to an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double stranded nucleic acid region that can serve as an initiation point for DNA synthesis under suitable conditions. Such primers may be used in a polymerase chain reaction or for DNA sequencing.
- “Polymerase chain reaction” is abbreviated PCR and refers to an in vitro method for enzymatically amplifying specific nucleic acid sequences. PCR involves a repetitive series of temperature cycles with each cycle comprising three stages: denaturation of the template nucleic acid to separate the strands of the target molecule, annealing a single stranded PCR oligonucleotide primer to the template nucleic acid, and extension of the annealed primer(s) by DNA polymerase, PCR provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.
- “Reverse transcription-polymerase chain reaction” is abbreviated RT-PCR and refers to an in vitro method for enzymatically producing a target cDNA molecule or molecules from an RNA molecule or molecules, followed by enzymatic amplification of a specific nucleic acid sequence or sequences within the target cDNA molecule or molecules as described above. RT-PCR also provides a means to detect the presence of the target molecule and, under quantitative or semi-quantitative conditions, to determine the relative amount of that target molecule within the starting pool of nucleic acids.
- A DNA “coding sequence” or “coding region” refers to a double-stranded DNA sequence that encodes a polypeptide and can be transcribed and translated into a polypeptide in a cell, ex vivo, in vitro or in vivo when placed under the control of suitable regulatory sequences. “Suitable regulatory sequences” refers to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If the coding sequence is intended for expression in an eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.
- “Open reading frame” is abbreviated ORF and refers to a length of nucleic acid sequence, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.
- The term “head-to-head” is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-head orientation when the 5′ end of the coding strand of one polynucleotide is adjacent to the 5′ end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds away from the 5′ end of the other polynucleotide. The term “head-to-head” may be abbreviated (5′)-to-(5′) and may also be indicated by the symbols (←→) or (3′←5′5′→3′).
- The term “tail-to-tail” is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a tail-to-tail orientation when the 3′ end of the coding strand of one polynucleotide is adjacent to the 3′ end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds toward the other polynucleotide. The term “tail-to-tail” may be abbreviated (3′)-to-(3′) and may also be indicated by the symbols (→←) or (5′→3′3′←5′).
- The term “head-to-tail” is used herein to describe the orientation of two polynucleotide sequences in relation to each other. Two polynucleotides are positioned in a head-to-tail orientation when the 5′ end of the coding strand of one polynucleotide is adjacent to the 3′ end of the coding strand of the other polynucleotide, whereby the direction of transcription of each polynucleotide proceeds in the same direction as that of the other polynucleotide. The term “head-to-tail” may be abbreviated (5′)-to-(3′) and may also be indicated by the symbols (→→) or (5′→3′5′→3′).
- The term “downstream” refers to a nucleotide sequence that is located 3′ to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.
- The term “upstream” refers to a nucleotide sequence that is located 5′ to a reference nucleotide sequence. In particular, upstream nucleotide sequences generally relate to sequences that are located on the 5′ side of a coding sequence or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.
- The terms “restriction endonuclease” and “restriction enzyme” are used interchangeably and refer to an enzyme that binds and cuts within a specific nucleotide sequence within double stranded DNA.
- “Homologous recombination” refers to the insertion of a foreign DNA sequence into another DNA molecule, e.g., insertion of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
- Several methods known in the art may be used to propagate a polynucleotide according to the invention. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As described herein, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.
- A “vector” refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment may be attached so as to bring about the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. Possible vectors include, for example, plasmids or modified viruses including, for example bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives, or the Bluescript vector. Another example of vectors that are useful in the invention is the ULTRAVECTOR® Production System (Intrexon Corp., Blacksburg, Va.) as described in WO 2007/038276. For example, the insertion of the DNA fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate DNA fragments into a chosen vector that has complementary cohesive termini. Alternatively, the ends of the DNA molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) into the DNA termini. Such vectors may be engineered to contain selectable marker genes that provide for the selection of cells that have incorporated the marker into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.
- Viral vectors, and particularly retroviral vectors, have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, adenovirus, geminivirus, and caulimovirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. In addition to a nucleic acid, a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
- The term “plasmid” refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.
- A “cloning vector” refers to a “replicon,” which is a unit length of a nucleic acid, preferably DNA, that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. Cloning vectors may be capable of replication in one cell type and expression in another (“shuttle vector”). Cloning vectors may comprise one or more sequences that can be used for selection of cells comprising the vector and/or one or more multiple cloning sites for insertion of sequences of interest.
- The term “expression vector” refers to a vector, plasmid or vehicle designed to enable the expression of an inserted nucleic acid sequence. The cloned gene, i.e., the inserted nucleic acid sequence, is usually placed under the control of control elements such as a promoter, a minimal promoter, an enhancer, or the like. Initiation control regions or promoters, which are useful to drive expression of a nucleic acid in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving expression of these genes can be used in an expression vector, including but not limited to, viral promoters, bacterial promoters, animal promoters, mammalian promoters, synthetic promoters, constitutive promoters, tissue specific promoters, pathogenesis or disease related promoters, developmental specific promoters, inducible promoters, light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, alkaline phosphatase promoters (useful for expression in Saccharomyces); AOX1 promoter (useful for expression in Pichia); β-lactamase, lac, ara, tet, trp, lPL, lPR, T7, tac, and trc promoters (useful for expression in Escherichia coli); light regulated-, seed specific-, pollen specific-, ovary specific-, cauliflower mosaic virus 35S, CMV 35S minimal, cassaya vein mosaic virus (CsVMV), chlorophyll a/b binding protein, ribulose 1,5-bisphosphate carboxylase, shoot-specific, root specific, chitinase, stress inducible, rice tungro bacilliform virus, plant super-promoter, potato leucine aminopeptidase, nitrate reductase, mannopine synthase, nopaline synthase, ubiquitin, zein protein, and anthocyanin promoters (useful for expression in plant cells); animal and mammalian promoters known in the art including, but are not limited to, the SV40 early (SV40e) promoter region, the promoter contained in the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV), the promoters of the E1A or major late promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus (CMV) early promoter, the herpes simplex virus (HSV) thymidine kinase (TK) promoter, a baculovirus IE1 promoter, an elongation factor 1 alpha (EF1) promoter, a phosphoglycerate kinase (PGK) promoter, a ubiquitin (Ubc) promoter, an albumin promoter, the regulatory sequences of the mouse metallothionein-L promoter and transcriptional control regions, the ubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like), the promoters of the intermediate filaments (desmin, neurofilaments, keratin, GFAP, and the like), the promoters of therapeutic genes (of the MDR, CFTR or factor VIII type, and the like), pathogenesis or disease related-promoters, and promoters that exhibit tissue specificity and have been utilized in transgenic animals, such as the elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region active in pancreatic beta cells, immunoglobulin gene control region active in lymphoid cells, mouse mammary tumor virus control region active in testicular, breast, lymphoid and mast cells albumin gene, Apo AI and Apo AII control regions active in liver, alpha-fetoprotein gene control region active in liver, alpha 1-antitrypsin gene control region active in the liver, beta-globin gene control region active in myeloid cells, myelin basic protein gene control region active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region active in skeletal muscle, and gonadotropic releasing hormone gene control region active in the hypothalamus, pyruvate kinase promoter, villin promoter, promoter of the fatty acid binding intestinal protein, promoter of the smooth muscle cell α-actin, and the like. In addition, these expression sequences may be modified by addition of enhancer or regulatory sequences and the like.
- Vectors may be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311).
- A polynucleotide according to the invention can also be introduced in vivo by lipofection. For the past decade, there has been increasing use of liposomes for encapsulation and transfection of nucleic acids in vitro. Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al., Proc. Natl. Acad. Sci. USA. 84:7413 (1987); Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027 (1988); and Ulmer et al., Science 259:1745 (1993)). The use of cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner et al., Science 337:387 (1989)). Particularly useful lipid compounds and compositions for transfer of nucleic acids are described in WO95/18863, WO96/17823 and U.S. Pat. No. 5,459,127. The use of lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly preferred in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain. Lipids may be chemically coupled to other molecules for the purpose of targeting (Mackey et al. 1988, supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
- Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or a cationic polymer (e.g., WO95/21931).
- It is also possible to introduce a vector in vivo as a naked DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and 5,580,859). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum. Gene Ther. 3:147 (1992); and Wu et al., J. Biol. Chem. 262:4429 (1987)).
- The term “transfection” refers to the uptake of exogenous or heterologous RNA or DNA by a cell. A cell has been “transfected” by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced inside the cell. A cell has been “transformed” by exogenous or heterologous RNA or DNA when the transfected RNA or DNA effects a phenotypic change. The transforming RNA or DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.
- “Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
- In addition, the recombinant vector comprising a polynucleotide according to the invention may include one or more origins for replication in the cellular hosts in which their amplification or their expression is sought, markers or selectable markers.
- The term “selectable marker” refers to an identifying factor, usually an antibiotic or chemical resistance gene, that is able to be selected for based upon the marker gene's effect, i.e., resistance to an antibiotic, resistance to a herbicide, colorimetric markers, enzymes, fluorescent markers, and the like, wherein the effect is used to track the inheritance of a nucleic acid of interest and/or to identify a cell or organism that has inherited the nucleic acid of interest. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like.
- The term “reporter gene” refers to a nucleic acid encoding an identifying factor that is able to be identified based upon the reporter gene's effect, wherein the effect is used to track the inheritance of a nucleic acid of interest, to identify a cell or organism that has inherited the nucleic acid of interest, and/or to measure gene expression induction or transcription. Examples of reporter genes known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), and the like. Selectable marker genes may also be considered reporter genes.
- “Promoter” and “promoter sequence” are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters.” Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters.” Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as “developmentally-specific promoters” or “cell differentiation-specific promoters.” Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as “inducible promoters” or “regulatable promoters.” It is farther recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.
- In any of the vectors of the present invention, the vector optionally comprises a promoter disclosed herein. In one embodiment, the promoter is a promoter listed in Table 1 herein.
- In any of the vectors of the present invention, the vector optionally comprises a tissue-specific promoter. In one embodiment, the tissue-specific promoter is a tissue specific promoter disclosed herein. In another embodiment, the tissue-specific promoter is a tissue specific promoter listed in Table 2 herein.
- The promoter sequence is typically bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence is found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
- “Therapeutic switch promoter” (“TSP”) refers to a promoter that controls expression of a gene switch component. Gene switches and their various components are described in detail elsewhere herein. In certain embodiments a TSP is constitutive, i.e., continuously active. A consitutive TSP may be either constitutive-ubiquitous (i.e., generally functions, without the need for additional factors or regulators, in any tissue or cell) or constitutive-tissue or cell specific (i.e., generally functions, without the need for additional factors or regulators, in a specific tissue type or cell type). In certain embodiments a TSP of the invention is activated under conditions associated with a disease, disorder, or condition. In certain embodiments of the invention where two or more TSPs are involved the promoters may be a combination of constitutive and activatable promoters. As used herein, a “promoter activated under conditions associated with a disease, disorder, or condition” includes, without limitation, disease-specific promoters, promoters responsive to particular physiological, developmental, differentiation, or pathological conditions, promoters responsive to specific biological molecules, and promoters specific for a particular tissue or cell type associated with the disease, disorder, or condition, e.g. tumor tissue or malignant cells. TSPs can comprise the sequence of naturally occurring promoters, modified sequences derived from naturally occurring promoters, or synthetic sequences (e.g., insertion of a response element into a minimal promoter sequence to alter the responsiveness of the promoter).
- A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced (if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.
- “Transcriptional and translational control sequences” refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.
- The term “response element” refers to one or more cis-acting DNA elements which confer responsiveness on a promoter mediated through interaction with the DNA-binding domains of a transcription factor. This DNA element may be either palindromic (perfect or imperfect) in its sequence or composed of sequence motifs or half sites separated by a variable number of nucleotides. The half sites can be similar or identical and arranged as either direct or inverted repeats or as a single half site or multimers of adjacent half sites in tandem. The response element may comprise a minimal promoter isolated from different organisms depending upon the nature of the cell or organism into which the response element is incorporated. The DNA binding domain of the transcription factor binds, in the presence or absence of a ligand, to the DNA sequence of a response element to initiate or suppress transcription of downstream gene(s) under the regulation of this response element. Examples of DNA sequences for response elements of the natural ecdysone receptor include: RRGG/TTCANTGAC/ACYY (SEQ ID NO: 25) (see Cherbas et. al., Genes Dev. 5:120 (1991)); AGGTCAN(n)AGGTCA, where N(n) can be one or more spacer nucleotides (SEQ ID NO: 26) (see D'Avino et al., Mol. Cell. Endocrinol. 113:1 (1995)); and GGGTTGAATGAATTT (SEQ ID NO: 27) (see Antoniewski et al., Mol. Cell. Biol. 14:4465 (1994)).
- The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
- The term “expression” as used herein refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into a protein or polypeptide.
- The terms “cassette,” “expression cassette” and “gene expression cassette” refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at specific restriction sites or by homologous recombination. The segment of DNA comprises a polynucleotide that encodes a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation. “Transformation cassette” refers to a specific vector comprising a polynucleotide that encodes a polypeptide of interest and having elements in addition to the polynucleotide that facilitate transformation of a particular host cell. Cassettes, expression cassettes, gene expression cassettes and transformation cassettes of the invention may also comprise elements that allow for enhanced expression of a polynucleotide encoding a polypeptide of interest in a host cell. These elements may include, but are not limited to: a promoter, a minimal promoter, an enhancer, a response element, a terminator sequence, a polyadenylation sequence, and the like.
- For purposes of this invention, the term “gene switch” refers to the combination of a response element associated with a promoter, and a ligand-dependent transcription factor-based system which, in the presence of one or more ligands, modulates the expression of a gene into which the response element and promoter are incorporated. The term “a polynucleotide encoding a gene switch” refers to the combination of a response element associated with a promoter, and a polynucleotide encoding a ligand-dependent transcription factor-based system which, in the presence of one or more ligands, modulates the expression of a gene into which the response element and promoter are incorporated.
- The therapeutic switch promoters of the invention may be any promoter that is useful for treating, ameliorating, or preventing a specific disease, disorder, or condition. Examples include, without limitation, promoters of genes that exhibit increased expression only during a specific disease, disorder, or condition and promoters of genes that exhibit increased expression under specific cell conditions (e.g., proliferation, apoptosis, change in pH, oxidation state, oxygen level). In some embodiments where the gene switch comprises more than one transcription factor sequence, the specificity of the therapeutic methods can be increased by combining a disease- or condition-specific promoter with a tissue- or cell type-specific promoter to limit the tissues in which the therapeutic product is expressed. Thus, tissue- or cell type-specific promoters are encompassed within the definition of therapeutic switch promoter.
- As an example of disease-specific promoters, useful promoters for treating cancer include the promoters of oncogenes. Examples of classes of oncogenes include, but are not limited to, growth factors, growth factor receptors, protein kinases, programmed cell death regulators and transcription factors. Specific examples of oncogenes include, but are not limited to, sis, erb B, erb B-2, ras, abl, myc and bcl-2 and TERT. Examples of other cancer-related genes include tumor associated antigen genes and other genes that are overexpressed in neoplastic cells (e.g., MAGE-1, carcinoembryonic antigen, tyrosinase, prostate specific antigen, prostate specific membrane antigen, p53, MUC-1, MUC-2, MUC-4, HER-2/neu, T/Tn, MART-1, gp100, GM2, Tn, sTn, and Thompson Friedenreich antigen (TF)).
- Examples of promoter sequences and other regulatory elements (e.g., enhancers) that are known in the art and are useful as therapeutic switch promoters in the present invention are disclosed in the references listed in Tables 1 and 2, along with the disease/disorder (Table 1) or tissue specificity (Table 2) associated with each promoter. The promoter sequences disclosed in these references are herein incorporated by reference in their entirety.
- The polynucleotide encoding any of the proteins listed in Table 1 may also be expressed using a vector of the present invention with a promoter that is not a therapeutic promoter.
-
TABLE 1 Patent/ Published Promoter Sequence Disease/Disorder Application No. Her-2/neu (ERBB2/c-erbB-2) cancer 5,518,885 Osteocalcin calcified tumors 5,772,993 stromelysin-1 cancer 5,824,794 prostate specific antigen prostate cancer 5,919,652 human sodium-iodide symporter thyroid carcinoma 6,015,376 H19, IF-1, IGF-2 cancer 6,306,833 thymosin β15 breast, pancreatic, 6,489,463 prostate cancer T cell factor cancer 6,608,037 cartilage-derived retinoic acid- chondrosarcoma, 6,610,509 sensitive protein mammary tumor Insulin pancreatic cancer 6,716,824 PEG-3 cancer 6,737,523 telomerase reverse transcriptase cancer 6,777,203 melanoma differentiation cancer 6,841,362 associated gene-7 Prostasin cancer 6,864,093 telomerase catalytic subunit; cancer 6,936,595 cyclin-A midkine; c-erbB-2 cancer 7,030,099 prostate-specific membrane prostate cancer 7,037,647 antigen p51 cancer 7,038,028 telomerase RNA cancer 7,084,267 prostatic acid phosphatase prostate cancer 7,094,533 PCA3dd3 prostate cancer 7,138,235 DF3/MUC1 cancer 7,247,297 hex II cancer 2001/0011128 cyclooxygenase-2 cancer 2002/0107219 super PSA prostate cancer 2003/0078224 skp2 cancer 2003/0109481 PRL-3 metastatic colon cancer 2004/0126785 CA125/M17S2 ovarian cancer 2004/0126824 IAI.3B ovarian cancer 2005/0031591 CRG-L2 liver cancer 2005/0124068 TRPM4 prostate cancer 2006/0188990 RTVP glioma 2006/0216731 TARP prostate cancer, breast 2007/0032439 cancer telomere reverse transcriptase cancer 2007/0059287 A4 amyloid protein Alzheimer's disease 5,151,508 amyloid β-protein precursor Alzheimer's disease 5,643,726 precursor of the Alzheimer's Alzheimer's disease 5,853,985 Disease A4 amyloid protein neuropeptide FF CNS disorders 6,320,038 endoplasmic reticulum stress stress 7,049,132 elements urocortin II psychopathologies 7,087,385 tyrosine hydroxylase neurological disorders 7,195,910 complement factor 3; serum inflammation 5,851,822 amyloid A3 tissue inhibitor of metallo- rheumatism, cancer, 5,854,019 proteinase-3 (TIMP-3) autoimmune disease, inflammation p75 tumor necrosis factor autoimmune disease 5,959,094 receptor tumor necrosis factor-α inflammation 6,537,784 peroxisome proliferator activated inflammation 6,870,044 receptor/IIA-1 nonpancreatic secreted phospholipase A2 SOCS-3 growth disorders, 2002/0174448 autoimmune disease, inflammation SR-BI lipid disorders 5,965,790 Ob obesity 5,698,389 site-1 protease obesity, diabetes 7,045,294 TIGR glaucoma 7,138,511 VL30 anoxia 5,681,706 excitatory amino acid trans- nervous system 2004/0171108 porter-2 ischemia MDTS9 renal failure 2006/0014931 LIM, pyrroline 5-carboxylate prostate disorders 2006/0134688 reductase, SIM2 Bax apoptosis 5,744,310 Fas apoptosis 5,888,764 bbc3 apoptosis 7,202,024 PINK-1 PI-3 kinase/Akt 2006/0228776 pathway disorders -
TABLE 2 Patent/ Published Application Promoter Sequence Tissue Specificity No. troponin T skeletal muscle 5,266,488 myoD muscle 5,352,595 Actin muscle 5,374,544 smooth muscle 22α arterial smooth muscle 5,837,534 Utrophin muscle 5,972,609 Myostatin muscle 6,284,882 smooth muscle myosin smooth muscle 6,780,610 heavy chain cardiac ankyrin repeat protein cardiac muscle 7,193,075 MLP muscle 2002/0042057 Smoothelin smooth muscle 2003/0157494 MYBPC3 cardiomyocytes 2004/0175699 Tα1 α-tubulin neurons 5,661,032 intercellular adhesion neurons 5,753,502 molecule-4 (ICAM-4) γ-aminobutyric acid type hippocampus 6,066,726 A receptor β1 subunit neuronal nicotinic acetylcholine neurons 6,177,242 receptor β2-subunit presenilin-1 neurons 6,255,473 calcium-calmodulin-dependent forebrain 6,509,190 kinase IIα CRF2α receptor brain 7,071,323 nerve growth factor neurons 2003/159159 GLP-2 receptor gut, brain 2002/0045173 type I transglutaminase keratinocytes 5,643,746 K14 keratinocytes 6,596,515 stearoyl-CoA desaturase skin 2002/0151018 Megsin renal cells 6,790,617 Prolactin pituitary 5,082,779 GDF-9 ovary, testes, 7,227,013 hypothalamus, pituitary, placenta PSP94 prostate 2003/0110522 NRL; NGAL mammary gland 5,773,290 long whey acidic protein mammary gland 5,831,141 mammary associated amyloid A mammary ductal 2005/0107315 epithelial cells endothelin-1 endothelial cells 5,288,846 Serglycin hematopoietic cells 5,340,739 platelet-endothelial cell adhesion platelets, leukocytes, 5,668,012 molecule-1 (PECAM-1) endothelial cells Tie receptor tyrosine kinase endothelial cells, bone 5,877,020 marrow KDR/flk-1 endothelial cells 5,888,765 Endoglin endothelial cells 6,103,527 CCR5 myeloid and lymphoid 6,383,746 cells CD11d myeloid cells 6,881,834 platelet glycoprotein IIb hematopoietic cells 6,884,616 preproendothelin-1 endothelial cells 7,067,649 interleukin-18 binding protein mononuclear cells 2006/0239984 CD34 hematopoietic stem 5,556,954 cells Tec tyrosine kinase hematopoietic stem 6,225,459 cells, liver - Other genes that exhibit changes in expression levels during specific diseases or disorders and therefore may provide promoters that are useful in the present invention include, without limitation, the genes (along with the associated disease/disorder) listed in Table 3.
-
TABLE 3 Patent/Published Gene Disease/Disorder Application No. MLH1, MSH2, MSH6, PMS1, APC Colorectal cancer 7,148,016 LEF-1 Colon cancer 2002/0169300 F2 receptor Colon cancer 2002/0187502 TGF-β type II receptor Colon cancer 2004/0038284 EYA4 Colon cancer 2005/0003463 PCA3 Prostate cancer 7,138,235 K2 Prostate cancer 6,303,361 PROST 03 Prostate cancer metastases 2002/0009455 PCAM-1 Prostate cancer 2002/0042062 PCADM-1 Prostate cancer 2003/0100033 PCA3dd3 Prostate cancer 2003/0165850 PCAV Prostate cancer 2006/0275747 PAcP Androgen-insensitive 2006/0294615 prostate cancer SEQ ID NO: 1 of the patent Liver cancer 5,866,329 5,866,329, incorporated by reference herein SEQ ID NOS: 1, 3 of the U.S. patent Hepatocellular cancer 2002/0115094 application publication 2002/0115094, incorporated by reference herein SEQ ID NO: 1 of the patent U.S. Hepatocellular carcinoma 2005/0037372 application publication 2005/0037372, incorporated by reference herein ATB0 Hepatocellular carcinoma 2006/0280725 SEQ ID NOS: 1, 3 of the U.S. patent Liver cancer 2007/0042420 application publication 2007/0042420 CSA-1 Chondrosarcoma 2001/0016649 SEQ ID NOS: 1-15 of the U.S. patent Pancreatic cancer 2001/0016651 application publication 2001/0016651, incorporated by reference herein SEQ ID NOS: 1-15 of the U.S. patent Pancreatic cancer 2003/0212264 application publication 2003/0212264, incorporated by reference herein SYG972 Breast cancer 2002/0055107 Urb-ctf Breast cancer 2003/0143546 BCU399 Breast cancer 2003/0180728 TBX2 Breast cancer 2004/0029185 Cyr61 Breast cancer 2004/0086504 DIAPH3 Breast cancer 2005/0054826 SEQ ID NOS: 1-24 of the U.S. patent Breast cancer 2007/0134669 application publication 2007/0134669, incorporated by reference herein Human aspartyl (asparaginyl) beta- CNS cancer 2002/0102263 hydroxylase BEHAB CNS cancer 2003/0068661 IL-8 Kaposi's Sarcoma 2003/0096781 SEQ ID NOS: 1-278 of the U.S. Hematological cancers 2002/0198362 patent application publication 2002/0198362, incorporated by reference herein BLSA B-cell cancer 2003/0147887 BP1 Leukemia 2003/0171273 DAP-kinase, HOXA9 Non-small cell lung cancer 2003/0224509 ARP Clear cell renal carcinoma, 2004/0010119 inflammatory disorders Nbk Renal cancer 2005/0053931 CD43 Ovarian cancer 2006/0216231 SEQ ID NOS: 1-84 of the U.S. patent Ovarian cancer 2007/0054268 application publication 2007/0054268, incorporated by reference herein β7-hcG, β6-hCG, β6e-hCG, Uterine tumors 2006/0292567 β5-hCG, β8-hcG, β3-hCG MTA1s Hormone insensitive 2006/0204957 cancer Old-35, Old-64 Tumor proliferation 2003/0099660 LAGE-1 Cancer 6,794,131 CIF150/hTAFII150 Cancer 6,174,679 P65 oncofetal protein Cancer 5,773,215 Telomerase Cancer 2002/0025518 CYP1B1 Cancer 2002/0052013 14-3-3σ Cancer 2002/0102245 NES1 Cancer 2002/0106367 CAR-1 Cancer 2002/0119541 HMGI, MAG Cancer 2002/0120120 ELL2 Cancer 2002/0132329 Ephrin B2 Cancer 2002/0136726 WAF1 Cancer 2002/0142442 CIF130 Cancer 2002/0143154 C35 Cancer 2002/0155447 BMP2 Cancer 2002/0159986 BUB3 Cancer 2002/0160403 Polymerase kappa Cancer 2003/0017573 EAG1, EAG2 Cancer 2003/0040476 SEQ ID NOS: 18, 20, 22 of the U.S. Cancer 2003/0044813 patent application publication 2003/0044813, incorporated by reference herein HMG I Cancer 2003/0051260 HLTF Cancer 2003/0082526 Barx2 Cancer 2003/0087243 SEQ ID NOS: 18, 20, 22, 32, 34, Cancer 2003/0108920 36 of the U.S. patent application publication 2003/0108920, incorporated by reference herein Cables Cancer 2003/0109443 Pp 32r1 Cancer 2003/0129631 BMP4 Cancer 2003/0134790 TS10q23.3 Cancer 2003/0139324 Nuclear spindle-associating protein Cancer 2003/0157072 PFTAIRE Cancer 2003/0166217 SEMA3B Cancer 2003/0166557 MOGp Cancer, multiple sclerosis, 2003/0166898 inflammatory disease Fortilin Cancer 2003/0172388 SEQ ID NO: 1 of the U.S. patent Cancer 2003/0215833 application publication 2003/0215833, incorporated by reference herein IGFBP-3 Cancer 2004/0005294 Polyhomeotic 2 Cancer 2004/0006210 PNQALRE Cancer 2004/0077009 SEQ ID NOS: 1, 3 of the U.S. patent Cancer 2004/0086916 application publication 2004/0086916, incorporated by reference herein SCN5A Cancer 2004/0146877 miR15, miR16 Cancer 2004/0152112 Headpin Cancer 2004/0180371 PAOh1/SMO Cancer 2004/0229241 Hippo, Mst2 Cancer 2005/0053592 PSMA-like Cancer, neurological 2005/0064504 disorders JAB1 Cancer 2005/0069918 NF-AT Cancer 2005/0079496 P28ING5 Cancer 2005/0097626 MTG16 Cancer 2005/0107313 ErbB-2 Cancer 2005/0123538 HDAC9 Cancer 2005/0130146 GPBP Cancer 2005/0130227 MG20 Cancer 2005/0153352 KLF6 Cancer 2005/0181374 ARTS1 Cancer 2005/0266443 Dock 3 Cancer 2006/0041111 Annexin 8 Cancer 2006/0052320 MH15 Cancer 2006/0068411 DELTA-N p73 Cancer 2006/0088825 RapR6 Cancer 2006/099676 StarD10 Cancer 2006/0148032 Ciz1 Cancer 2006/0155113 HLJ1 Cancer 2006/0194235 RapR7 Cancer 2006/0240021 A34 Cancer 2006/0292154 Sef Cancer 2006/0293240 Killin Cancer 2007/0072218 SGA-1M Cancer 2007/0128593 TGFβ Type II receptor Cancer 2002/0064786 GCA-associated genes Giant cell arteritis 6,743,903 PRV-1 Polycythemia vera 6,686,153 SEQ ID NOS: 2, 4 of the U.S. Pat. No. Ischemia 5,948,637 5,948,637, incorporated by reference herein Vezf1 Vascular disorders 2002/0023277 MLP Dilatative cardiomyopathy 2002/0042057 VEGI Pathological angiogenesis 2002/0111325 PRO256 Cardiovascular disorders 2002/0123091 AOP2 Atherosclerosis 2002/0142417 Remodelin Arterial restenosis, fibrosis 2002/0161211 Phosphodiesterase 4D Stroke 2003/0054531 Prostaglandin receptor subtype EP3 Peripheral arterial 2003/0157599 occlusive disease CARP Heart disorders 2004/0014706 HOP Congenital heart disease 2004/0029158 SEQ ID NOS: 1-4 of the U.S. patent Apoplexy 2004/0087784 application publication 2004/0087784, incorporated by reference herein PLTP Atherosclerosis, vascular 2006/0252787 disease, hypercholesterolemia, Tangier's disease, familial HDL deficiency disease SEQ ID NOS: 1, 3-8, 15, 16 of the Thrombosis 2007/0160996 U.S. patent application publication 2007/0160996, incorporated by reference herein UCP-2 Stroke 2002/0172958 FLJ11011 Fanconi's Anemia 2006/0070134 Codanin-1 Anemia 2006/0154331 SEQ ID NOS: 1, 6, 8 of the U.S. Insulin-dependent diabetes 5,763,591 Pat. No. 5,763,591, incorporated by mellitus reference herein Resistin Type II diabetes 2002/0161210 Archipelin Diabetes 2003/0202976 SEQ ID NOS: 2, 7, 16, 27 of the U.S. Diabetes, hyperlipidemia 2004/0053397 patent application publication 2004/0053397, incorporated by reference herein Neuronatin Metabolic disorders 2004/0259777 Ncb5or Diabetes 2005/0031605 7B2 Endocrine disorders 2005/0086709 PTHrP, PEX Metabolic bone diseases 2005/0113303 KChIP1 Type II diabetes 2005/0196784 SLIT-3 Type II diabetes 2006/0141462 CX3CR1 Type II diabetes 2006/0160076 SMAP-2 Diabetes 2006/0210974 SEQ ID NOS: 2, 8, 12, 16, 22, 26, Type II diabetes 2006/0228706 28, 32 of the U.S. patent application publication 2006/0228706, incorporated by reference herein IC-RFX Diabetes 2006/0264611 E2IG4 Diabetes, insulin 2007/0036787 resistance, obesity SEQ ID NOS: 2, 8, 10, 14, 18, 24, Diabetes 2007/0122802 26, 30, 34, 38, 44, 50, 54, 60, 62, 68, 74, 80, 86, 92, 98, 104, 110 of the U.S. patent application publication 2007/0122802, incorporated by reference herein UCP2 Body weight disorders 2002/0127600 Ob receptor Body weight disorders 2002/0182676 Ob Bodyweight disorders 2004/0214214 Dp1 Neurodegenerative 2001/0021771 disorders NRG-1 Schizophrenia 2002/0045577 Synapsin III Schizophrenia 2002/0064811 NRG1AG1 Schizophrenia 2002/0094954 AL-2 Neuronal disorders 2002/0142444 Proline dehydrogenase Bipolar disorder, major 2002/0193581 depressive disorder, schizophrenia, obsessive compulsive disorder MNR2 Chronic neurodegenerative 2002/0197678 disease ATM Ataxia-telangiectasia 2004/0029198 Ho-1 Dementing diseases 2004/0033563 CON202 Schizophrenia 2004/0091928 Ataxin-1 Neurodegenerative 2004/0177388 disorders NR3B Motor neuron disorders 2005/0153287 NIPA-1 Hereditary spastic 2005/0164228 paraplegia DEPP, adrenomedullin, csdA Schizophrenia 2005/0227233 Inf-20 Neurodegenerative 2006/0079675 diseases EOPA Brain development and 2007/0031830 degeneration disorders SERT Autism 2007/0037194 FRP-1 Glaucoma 2002/0049177 Serum amyloid A Glaucoma 2005/0153927 BMP2 Osteoporosis 2002/0072066 BMPR1A Juvenile polyposis 2003/0072758 ACLP Gastroschisis 2003/0084464 Resistin-like molecule β Familial adenomatous 2003/0138826 polyposis, diabetes, insulin resistance, colon cancer, inflammatory bowel disorder Dlg5 Inflammatory bowel 2006/0100132 disease SEQ ID NOS: 1-82 of the U.S. patent Osteoarthritis 2002/0119452 application publication 2002/0119452, incorporated by reference herein TRANCE Immune system disorders 2003/0185820 Matrilin-3 Osteoarthritis 2003/0203380 Synoviolin Rheumatoid arthritis 2004/0152871 SEQ ID NOS: 9, 35 of the U.S. Osteoarthritis 2007/0028314 patent application publication 2007/0028314, incorporated by reference herein HIV LTR HIV infection 5,627,023 SHIVA HIV infection 2004/0197770 EBI 1, EBI 2, EBI 3 Epstein Barr virus infection 2002/0040133 NM23 family Skin/intestinal disorders 2002/0034741 SEQ ID NO: 1 of the U.S. patent Psoriasis 2002/0169127 application publication 2002/0169127, incorporated by reference herein Eps8 Skin disorders, wound 2003/0180302 healing Beta-10 Thyroid gland pathology 2002/0015981 SEQ ID NO: 2 of the U.S. patent Thyroid conditions 2003/0207403 application publication 2003/0207403, incorporated by reference herein SEQ ID NO: 3 of the U.S. patent Thyroid disorders 2007/0020275 application publication 2007/0020275, incorporated by reference herein Hair follicle growth factor Alopecia 2003/0036174 Corneodesmosin Alopecia 2003/0211065 GCR9 Asthma, lymphoma, 2003/0166150 leukemia SEQ ID NO: 1-71 of the U.S. patent Asthma 2004/0002084 application publication 2004/0002084, incorporated by reference herein Bg Chediak-Higashi syndrome 2002/0115144 SEQ ID NOS: 1-16 of the U.S. patent Endometriosis 2002/0127555 application publication 2002/0127555, incorporated by reference herein FGF23 Hypophosphatemic 2005/0156014 disorders BBSR Bardet-Biedl syndrome 2003/0152963 MIC-1 Fetal abnormalities, cancer, 2004/0053325 inflammatory disorders, miscarriage, premature birth MIA-2 Liver damage 2004/0076965 IL-17B Cartilage degenerative 2004/0171109 disorders Formylglycine generating enzyme Multiple sulfatase 2004/0229250 deficiency LPLA2 Pulmonary alveolar 2006/0008455 proteinosis CXCL1O Respiratory illnesses 2006/0040329 SEQ ID NOS: 1, 2 of the U.S. patent Nephropathy 2006/0140945 application publication 2006/0140945, incorporated by reference herein HFE2A Iron metabolism disease 2007/0166711 - Once a gene with an expression pattern that is modulated during a disease, disorder, or condition is identified, the promoter of the gene may be used in the gene switch of the invention. The sequence of many genes, including the promoter region, is known in the art and available in public databases, e.g., GenBank. Thus, once an appropriate gene is identified, the promoter sequence can be readily identified and obtained. Another aspect of the present invention is directed towards identifying suitable genes whose promoter can be isolated and placed into a gene switch. The identity of the gene, therefore, may not be critical to specific embodiments of the present invention, provided the promoter can be isolated and used in subsequent settings or environments. The current invention thus includes the use of promoters from genes that are yet to be identified. Once suitable genes are identified, it is a matter of routine skill or experimentation to determine the genetic sequences needed for promoter function. Indeed, several commercial protocols exist to aid in the determination of the promoter region of genes of interest. By way of example, Ding et al. recently elucidated the promoter sequence of the novel Sprouty4 gene (Am. J. Physiol. Lung Cell. Mol. Physiol. 287: L52 (2004), which is incorporated by reference) by progressively deleting the 5′-flanking sequence of the human Sprouty4 gene. Briefly, once the transcription initiation site was determined, PCR fragments were generated using common PCR primers to clone segments of the 5′-flanking segment in a unidirectional manner. The generated segments were cloned into a luciferase reporter vector and luciferase activity was measured to determine the promoter region of the human Sprouty4 gene.
- Another example of a protocol for acquiring and validating gene promoters includes the following steps: (1) acquire diseased and non-diseased cell/tissue samples of similar/same tissue type; (2) isolate total RNA or mRNA from the samples; (3) perform differential microarray analysis of diseased and non-diseased RNA; (4) identify candidate disease-specific transcripts; (5) identify genomic sequences associated with the disease-specific transcripts; (6) acquire or synthesize DNA sequence upstream and downstream of the predicted transcription start site of the disease-specific transcript; (7) design and produce promoter reporter vectors using different lengths of DNA from
step 6; and (8) test promoter reporter vectors in diseased and non-diseased cells/tissues, as well as in unrelated cells/tissues. - The source of the promoter that is inserted into the gene switch can be natural or synthetic, and the source of the promoter should not limit the scope of the invention described herein. In other words, the promoter may be directly cloned from cells, or the promoter may have been previously cloned from a different source, or the promoter may have been synthesized.
- The gene switch may be any gene switch that regulates gene expression by addition or removal of a specific ligand. In one embodiment, the gene switch is one in which the level of gene expression is dependent on the level of ligand that is present. Examples of ligand-dependent transcription factor complexes that may be used in the gene switches of the invention include, without limitation, members of the nuclear receptor superfamily activated by their respective ligands glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline. In one aspect of the invention, the gene switch is an EcR-based gene switch. Examples of such systems include, without limitation, the systems described in U.S. Pat. Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006/0014711, 2007/0161086, and International Published Application No. WO 01/70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038, U.S. Published Patent Application Nos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457, and 2006/0100416, and International Published Application Nos. WO 01/70816, WO 02/066612, WO 02/066613, WO 02/066014, WO 02/066615, WO 02/29075, and WO 2005/108617, each of which is incorporated by reference in its entirety. An example of a non-steroidal ecdysone agonist-regulated system is the RheoSwitch® Mammalian Inducible Expression System (New England Biolabs, Ipswich, Mass.). In another aspect of the invention, the gene switch is based on heterodimerization of FK506 binding protein (FKBP) with FKBP rapamycin associated protein (FRAP) and is regulated through rapamycin or its non-immunosuppressive analogs. Examples of such systems, include, without limitation, the ARGENT™ Transcriptional Technology (ARIAD Pharmaceuticals, Cambridge, Mass.) and the systems described in U.S. Pat. Nos. 6,015,709, 6,117,680, 6,479,653, 6,187,757, and 6,649,595.
- In one embodiment, the gene switch comprises a single transcription factor sequence encoding a ligand-dependent transcription factor complex under the control of a therapeutic switch promoter. The transcription factor sequence may encode a ligand-dependent transcription factor complex that is a naturally occurring or an artificial ligand dependent transcription factor complex. An artificial transcription factor is one in which the natural sequence of the transcription factor has been altered, e.g., by mutation of the sequence or by the combining of domains from different transcription factors. In one embodiment, the transcription factor comprises a Group H nuclear receptor ligand binding domain. In one embodiment, the Group H nuclear receptor ligand binding domain is from an ecdysone receptor, a ubiquitous receptor (UR), an orphan receptor 1 (OR-1), a steroid hormone nuclear receptor 1 (NER-1), a retinoid X receptor interacting protein-15 (RIP-15), a liver X receptor β (LXRβ), a steroid hormone receptor like protein (RLD-1), a liver X receptor (LXR), a liver X receptor α (LXRα), a farnesoid X receptor (FXR), a receptor interacting protein 14 (RIP-14), or a farnesol receptor (HRR-1). In another embodiment, the Group H nuclear receptor LBD is from an ecdysone receptor.
- A. Ecdysone-based Gene Switch
- The EcR and the other Group H nuclear receptors are members of the nuclear receptor superfamily wherein all members are generally characterized by the presence of an amino-terminal transactivation domain (AD, also referred to interchangeably as “TA” or “TD”), optionally fused to a heterodimerization partner (HP) to form a coactivation protein (CAP), a DNA binding domain (DBD), and a LBD fused to the DBD via a hinge region to form a ligand-dependent transcription factor (LTF). As used herein, the term “DNA binding domain” comprises a minimal polypeptide sequence of a DNA binding protein, up to the entire length of a DNA binding protein, so long as the DNA binding domain functions to associate with a particular response element. Members of the nuclear receptor superfamily are also characterized by the presence of four or five domains: A/B, C, D, E, and in some members F (see U.S. Pat. No. 4,981,784 and Evans, Science 240:889 (1988)). The “A/B” domain corresponds to the transactivation domain, “C” corresponds to the DNA binding domain, “D” corresponds to the hinge region, and “E” corresponds to the ligand binding domain. Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD corresponding to “F”.
- The following polypeptide sequence was reported as a polypeptide sequence of Ecdysone receptor (Ecdysteroid receptor) (20-hydroxy-ecdysone receptor) (20E receptor) (EcRH) (
Nuclear receptor subfamily 1 group H member 1) and has the accession number P34021 in Genbank. -
Ecdysone receptor (878aa) from Drosophila melanogaster (Fruit fly) (SEQ ID NO: 5) 1 mkrrwsnngg fmrlpeesss evtsssnglv lpsgvnmsps sldshdycdq dlwlcgnesg 61 sfggsnghgl sqqqqsvitl amhgcsstlp aqttiiping nangnggstn gqyvpgatnl 121 galangmlng gfngmqqqiq nghglinstt pstpttplhl qqnlggaggg giggmgilhh 181 angtpnglig vvgggggvgl gvggggvggl gmqhtprsds vnsissgrdd lspssslngy 241 sanescdakk skkgpaprvq eelclvcgdr asgyhynalt cegckgffrr svtksavycc 301 kfgracemdm ymrrkcqecr lkkclavgmr pecvvpenqc amkrrekkaq kekdkmttsp 361 ssqhggngsl asgggqdfvk keildlmtce ppqhatipll pdeilakcqa rnipsltynq 421 laviykliwy qdgyeqpsee dlrrimsqpd enesqtdvsf rhiteitilt vqlivefakg 481 lpaftkipqe dqitllkacs sevmmlrmar rydhssdsif fannrsytrd sykmagmadn 541 iedllhfcrq mfsmkvdnve yalltaivif sdrpglekaq lveaiqsyyi dtlriyilnr 601 hcgdsmslvf yakllsilte lrtlgnqnae mcfslklknr klpkfleeiw dvhaippsvq 661 shlqitqeen erleraermr asvggaitag idcdsastsa aaaaaqhqpq pqpqpqpssl 721 tqndsqhqtq pqlqpqlppq lqgqlqpqlq pqlqtqlqpq iqpqpqllpv sapvpasvta 781 pgslsavsts seymggsaai gpitpattss itaavtasst tsavpmgngv gvgvgvggnv 841 smyanaqtam almgvalhsh qeqliggvav ksehstta - The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins. The EcR, like a subset of the nuclear receptor family, also possesses less well-defined regions responsible for heterodimerization properties. Because the domains of nuclear receptors are modular in nature, the LBD, DBD, and AD may be interchanged.
- In another embodiment, the transcription factor comprises a AD, a DBD that recognizes a response element associated with the therapeutic protein or therapeutic polynucleotide whose expression is to be modulated; and a Group H nuclear receptor LBD. In certain embodiments, the Group H nuclear receptor LBD comprises a substitution mutation.
- In another embodiment, the gene switch comprises a first transcription factor sequence, e.g., a CAP, under the control of a first therapeutic switch promoter (TSP-1) and a second transcription factor sequence, e.g., a LTF, under the control of a second therapeutic switch promoter (TSP-2), wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex (LDTFC), i.e., a “dual switch”- or “two-hybrid”-based gene switch. The first and second TSPs may be the same or different. In this embodiment, the presence of two different TSPs in the gene switch that are required for therapeutic molecule expression enhances the specificity of the therapeutic method (see
FIG. 2 ).FIG. 2 also demonstrates the ability to modify the therapeutic gene switch to treat any disease, disorder, or condition simply by inserting the appropriate TSPs. - In a further embodiment, both the first and the second transcription factor sequence, e.g., a CAP or a LTF, are under the control of a single therapeutic switch promoter (e.g. TSP-1 in
FIG. 1 ). Activation of this promoter will generate both CAP and LTF with a single open reading frame. This can be achieved with the use of a transcriptional linker such as an IRES (internal ribosomal entry site). In this embodiment, both portions of the ligand-dependent transcription factor complex are synthesized upon activation of TSP-1. TSP-1 can be a constitutive promoter or only activated under conditions associated with the disease, disorder, or condition. - In a further embodiment, one transcription factor sequence, e.g. a LTF, is under the control of a therapeutic switch promoter only activated under conditions associated with the disease, disorder, or condition (e.g., TSP-2 or TSP-3 in
FIG. 4 ) and the other transcription factor sequence, e.g., CAP, is under the control of a constitutive therapeutic switch promoter (e.g., TSP-1 inFIG. 4 ). In this embodiment, one portion of the ligand-dependent transcription factor complex is constitutively present while the second portion will only be synthesized under conditions associated with the disease, disorder, or condition. - In another embodiment, one transcription factor sequence, e.g., CAP, is under the control of a first TSP (e.g., TSP-1 in
FIG. 3 ) and two or more different second transcription factor sequences, e.g., and LTF-2 are under the control of different TSPs (e.g., TSP-2 and TSP-3 inFIG. 3 ). In this embodiment, each of the LTFs may have a different DBD that recognizes a different factor-regulated promoter sequence (e.g., DBD-A binds to a response element associated with factor-regulated promoter-1 (FRP-1) and DBD-B binds to a response element associated with factor-regulated promoter-2 (FRP-2). Each of the factor-regulated promoters may be operably linked to a different therapeutic gene. In this manner, multiple treatments may be provided simultaneously. - In one embodiment, the first transcription factor sequence encodes a polypeptide comprising a AD, a DBD that recognizes a response element associated with the therapeutic product sequence whose expression is to be modulated; and a Group H nuclear receptor LBD, and the second transcription factor sequence encodes a transcription factor comprising a nuclear receptor LBD selected from a vertebrate retinoid X receptor (RXR), an invertebrate RXR, an ultraspiracle protein (USP), or a chimeric nuclear receptor comprising at least two different nuclear receptor ligand binding domain polypeptide fragments selected from a vertebrate RXR, an invertebrate RXR, and a USP (see WO 01/70816 A2 and US 2004/0096942 A1). The “partner” nuclear receptor ligand binding domain may further comprise a truncation mutation, a deletion mutation, a substitution mutation, or another modification.
- In another embodiment, the gene switch comprises a first transcription factor sequence encoding a first polypeptide comprising a nuclear receptor LBD and a DBD that recognizes a response element associated with the therapeutic product sequence whose expression is to be modulated, and a second transcription factor sequence encoding a second polypeptide comprising an AD and a nuclear receptor LBD, wherein one of the nuclear receptor LBDs is a Group H nuclear receptor LBD. In one embodiment, the first polypeptide is substantially free of an AD and the second polypeptide is substantially free of a DBD. For purposes of the invention, “substantially free” means that the protein in question does not contain a sufficient sequence of the domain in question to provide activation or binding activity.
- In another aspect of the invention, the first transcription factor sequence encodes a protein comprising a heterodimerization partner and an AD (a “CAP”) and the second transcription factor sequence encodes a protein comprising a DBD and a LBD (a “LTF”).
- When only one nuclear receptor LBD is a Group H LBD, the other nuclear receptor LBD may be from any other nuclear receptor that forms a dimer with the Group H LBD. For example, when the Group H nuclear receptor LBD is an EcR LBD, the other nuclear receptor LBD “partner” may be from an EcR, a vertebrate RXR, an invertebrate RXR, an ultraspiracle protein (USP), or a chimeric nuclear receptor comprising at least two different nuclear receptor LBD polypeptide fragments selected from a vertebrate RXR, an invertebrate RXR, or a USP (see WO 01/70816 A2, International Patent Application No. PCT/US02/05235 and US 2004/0096942 A1, incorporated herein by reference in their entirety). The “partner” nuclear receptor ligand binding domain may further comprise a truncation mutation, a deletion mutation, a substitution mutation, or another modification.
- In one embodiment, the vertebrate RXR LBD is from a human Homo sapiens, mouse Mus musculus, rat Rattus norvegicus, chicken Gallus gallus, pig Sus scrofa domestica, frog Xenopus laevis, zebrafish Danio rerio, tunicate Polyandrocarpa misakiensis, or jellyfish Tripedalia cysophora RXR.
- In one embodiment, the invertebrate RXR ligand binding domain is from a locust Locusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodid tick Amblyomma americanum RXR homolog 1 (“AmaRXR1”), an ixodid tick Amblyomma americanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celuca pugilator RXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog (“TmRXR”), a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphid Myzus persicae RXR homolog (“MpRXR”), or a non-Dipteran/non-Lepidopteran RXR homolog.
- In one embodiment, the chimeric RXR LBD comprises at least two polypeptide fragments selected from a vertebrate species RXR polypeptide fragment, an invertebrate species RXR polypeptide fragment, or a non-Dipteran/non-Lepidopteran invertebrate species RXR homolog polypeptide fragment. A chimeric RXR ligand binding domain for use in the present invention may comprise at least two different species RXR polypeptide fragments, or when the species is the same, the two or more polypeptide fragments may be from two or more different isoforms of the species RXK polypeptide fragment. Such chimeric RXR LBDs are disclosed, for example, in WO 2002/066614.
- In one embodiment, the chimeric RXR ligand binding domain comprises at least one vertebrate species RXR polypeptide fragment and one invertebrate species RXR polypeptide fragment.
- In another embodiment, the chimeric RXR ligand binding domain comprises at least one vertebrate species RXR polypeptide fragment and one non-Dipteran/non-Lepidopteran invertebrate species RXR homolog polypeptide fragment.
- The ligand, when combined with the LBD of the nuclear receptor(s), which in turn are bound to the response element of a FRP associated with a therapeutic product sequence, provides external temporal regulation of expression of the therapeutic product sequence. The binding mechanism or the order in which the various components of this invention bind to each other, that is, for example, ligand to LBD, DBD to response element, AD to promoter, etc., is not critical.
- In a specific example, binding of the ligand to the LBD of a Group H nuclear receptor and its nuclear receptor LBD partner enables expression of the therapeutic product sequence. This mechanism does not exclude the potential for ligand binding to the Group H nuclear receptor (GHNR) or its partner, and the resulting formation of active homodimer complexes (e.g. GHNR+GHNR or partner+partner). Preferably, one or more of the receptor domains is varied producing a hybrid gene switch. Typically, one or more of the three domains, DBD, LBD, and AD, may be chosen from a source different than the source of the other domains so that the hybrid genes and the resulting hybrid proteins are optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski et al., Nature 335:563 (1988)) or LexA protein from Escherichia coli (see Brent et al., Cell 43:729 (1985)), or synthetic response elements specific for targeted interactions with proteins designed, modified, and selected for such specific interactions (see, for example, Kim et al., Proc. Natl. Acad. Sci. USA, 94:3616 (1997)) to accommodate hybrid receptors. Another advantage of two-hybrid systems is that they allow choice of a promoter used to drive the gene expression according to a desired end result. Such double control may be particularly important in areas of gene therapy, especially when cytotoxic proteins are produced, because both the timing of expression as well as the cells wherein expression occurs may be controlled. When genes, operably linked to a suitable promoter, are introduced into the cells of the subject, expression of the exogenous genes is controlled by the presence of the system of this invention, Promoters may be constitutively or inducibly regulated or may be tissue-specific (that is, expressed only in a particular type of cells) or specific to certain developmental stages of the organism.
- The DNA binding domain of the first hybrid protein binds, in the presence or absence of a ligand, to the DNA sequence of a response element to initiate or suppress transcription of downstream gene(s) under the regulation of this response element.
- The functional LDTFC, e.g., an EcR complex, may also include additional protein(s) such as immunophilins. Additional members of the nuclear receptor family of proteins, known as transcriptional factors (such as DHR38 or betaFTZ-1), may also be ligand dependent or independent partners for EcR, USP, and/or RXR. Additionally, other cofactors may be required such as proteins generally known as coactivators (also termed adapters or mediators). These proteins do not bind sequence-specifically to DNA and are not involved in basal transcription. They may exert their effect on transcription activation through various mechanisms, including stimulation of DNA-binding of activators, by affecting chromatin structure, or by mediating activator-initiation complex interactions. Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuous coactivator C response element B binding protein, CBP/p300 (for review see Glass et al., Curr. Opin. Cell Biol. 9:222 (1997)). Also, protein cofactors generally known as corepressors (also known as repressors, silencers, or silencing mediators) may be required to effectively inhibit transcriptional activation in the absence of ligand. These corepressors may interact with the unliganded EcR to silence the activity at the response element. Current evidence suggests that the binding of ligand changes the conformation of the receptor, which results in release of the corepressor and recruitment of the above described coactivators, thereby abolishing their silencing activity. Examples of corepressors include N-CoR and SMRT (for review, see Horwitz et al., Mol. Endocrinol. 10:1167 (1996)). These cofactors may either be endogenous within the cell or organism, or may be added exogenously as transgenes to be expressed in either a regulated or unregulated fashion.
- B. Rapamycin Based Gene Switch
- The present invention further provides a gene switch system which utilizes FK506 binding protein as the ligand-dependent transcription factor complex and rapamycin as the ligand. In one embodiment, the construct encoding the gene switch comprises
-
- (a) a first polynucleotide encoding a first chimeric protein which binds to rapamycin or an analog thereof and which comprises at least one FK506-binding protein (FKBP) domain and at least one protein domain heterologous thereto, wherein the FKBP domain comprises a peptide sequence selected from:
- (1) a naturally occurring FKBP
- (2) a variant of a naturally occurring FKBP in which up to 10 amino acid residues have been deleted, inserted, or replaced with substitute amino acids, and
- (3) an FKBP encoded by a DNA sequence which selectively hybridizes to a DNA sequence encoding an FKBP of (1) or (2);
- (b) a second polynucleotide encoding a second chimeric protein which forms a complex with both (a) rapamycin or a rapamycin analog and (b) the first chimeric protein, and which comprises at least one FKBP:rapamycin binding (FRB) domain and at least one protein domain heterologous thereto, wherein the FRB domain comprises a peptide sequence selected from:
- (4) a naturally occurring FRB domain,
- (5) a variant of a naturally occurring FRB domain in which up to 10 amino acid residues have been deleted, inserted, or replaced with substitute amino acids, and
- (6) an FRB domain encoded by a DNA sequence which selectively hybridizes to a DNA sequence encoding an FRB of (4) or (5).
- (a) a first polynucleotide encoding a first chimeric protein which binds to rapamycin or an analog thereof and which comprises at least one FK506-binding protein (FKBP) domain and at least one protein domain heterologous thereto, wherein the FKBP domain comprises a peptide sequence selected from:
- In this gene switch system, each of the first polynucleotide and the second polynucleotide are under the control of one or more therapeutic switch promoters as described elsewhere herein. Furthermore, in certain embodiments, at least one protein domain heterologous to the FKBP and/or FRB domains in the first and second chimeric protein may be one or more “action” or “effector” domains. Effector domains may be selected from a wide variety of protein domains including DNA binding domains, transcription activation domains, cellular localization domains and signaling domains (i.e., domains which are capable upon clustering or multimerization, of triggering cell growth, proliferation, differentiation, apoptosis, gene transcription, etc.).
- In certain embodiments, one fusion protein contains at least one DNA binding domain (e.g., a GAL4 or ZFHD1 DNA-binding domain) and another fusion protein contains at least one transcription activation domain (e.g., a VP16 or p65 transcription activation domain). Ligand-mediated association of the fusion proteins represents the formation of a transcription factor complex and leads to initiation of transcription of a target gene linked to a DNA sequence recognized by (i.e., capable of binding with) the DNA-binding domain on one of the fusion proteins. Information regarding the gene expression system as well as the ligand is disclosed in U.S. Pat. Nos. 6,187,757 B1, 6,649,595 B1, 6,509,152 B1, 6,479,653 B1, and 6,117,680 B1.
- In other embodiments, the present invention provides a gene switch system which comprises polynucleotides encoding two fusion proteins which self-aggregate in the absence of a ligand, wherein (a) the first fusion protein comprises a conditional aggregation domain which binds to a selected ligand and a transcription activation domain, and (b) the second fusion protein comprising a conditional aggregation domain which binds to a selected ligand and a DNA binding domain, and (c) in the absence of ligand, the cells express a gene operably linked to regulatory DNA to which said DNA binding domain binds. Modified cells comprising the gene switch system are expanded in the presence of the ligand in an amount sufficient for repression of the gene. Ligand removal induces expression of the encoded protein that causes cell death. The nucleic acids encoding the two fusion proteins are under the control of at least one conditional promoter. The gene expression system utilizing conditional aggregation domains is disclosed in U.S. Publication No. 2002/0048792.
- C. Procaryotic Repressor/Operator Based Gene Switch System
- In one embodiment, the present invention provides gene switch system comprising (a) a first polynucleotide coding for a transactivator fusion protein comprising a prokaryotic tetracycline (“tet”) repressor and a eucaryotic transcriptional activator protein domain; and (b) a second polynucleotide coding for a therapeutic protein or therapeutic polypeptide, wherein said second polynucleotide is operably linked to a minimal promoter and at least one tet operator sequence. The first polynucleotide coding for a transactivator fusion protein may comprise therapeutic switch promoter as described elsewhere herein. The expression of the lethal protein is up-regulated in the absence of tetracycline. (see, e.g., Gossen et al. (1992) Proc. Natl. Acad. Sci. 89: 5547-5551; Gossen et al. (1993) TIBS 18: 471-475; Furth et al. (1994) Proc. Natl. Acad. Sci. 91: 9302-9306; and Shockett et al. (1995) Proc. Natl. Acad. Sci. 92: 6522-6526). The TetO expression system is disclosed in U.S. Pat. No. 5,464,758 B1.
- In another embodiment, the gene switch system comprises the lactose (“Lac”) repressor-operator systems from the bacterium Escherichia coli. The gene switch system of the present invention may also comprise (a) a first polynucleotide coding for a transactivator fusion protein comprising a prokaryotic lac I repressor and a eucaryotic transcriptional activator protein domain; and (b) a second polynucleotide coding for a therapeutic protein or therapeutic polypeptide, wherein said second polynucleotide is operably linked to a therapeutic switch promoter. In the Lac system, a lac operon is inactivated in the absence of lactose, or synthetic analogs such as isopropyl-b-D-thiogalacto side.
- Additional gene switch systems include those described in the following: U.S. Pat. No. 7,091,038; WO2004078924; EP1266015; US20010044151; US20020110861; US20020119521; US20040033600; US20040197861; US20040235097; US20060020146; US20040049437; US20040096942; US20050228016; US20050266457; US20060100416; WO2001/70816; WO2002/29075; WO2002/066612; WO2002/066613; WO2002/066614; WO2002/066615; WO2005/108617; U.S. Pat. No. 6,258,603; US20050209283; US20050228016; US20060020146; EP0965644; U.S. Pat. No. 7,304,162; U.S. Pat. No. 7,304,161; MX234742; KR10-0563143; AU765306; AU2002-248500; and AU2002-306550.
- D. Combination of the Gene Switch Systems
- The present invention provides nucleic acid compositions, modified cells, and bioreactors comprising two or more gene switch systems comprising different ligand-dependent transcription factor complexes which are activated by an effective amount of one or more ligands, wherein the two or more gene switch systems comprise a first gene switch and a second gene switch, both of which selectively induce expression of one or more therapeutic polypeptides or therapeutic polynucleotides, upon binding to one or more ligands. Within the scope of the present invention are any numbers of and/or combinations of gene switch systems.
- In one embodiment, the present invention provides a nucleic acid composition comprising:
- a. a first gene switch system which comprises:
-
- i. a first gene expression cassette comprising a polynucleotide encoding a first hybrid polypeptide which comprises:
- 1. a transactivation domain, which activates a factor-regulated promoter operably associated with a polynucleotide encoding a therapeutic polypeptide or therapeutic polynucleotide; and
- 2. a heterodimer partner domain,
- ii. a second gene expression cassette comprising a polynucleotide encoding a second hybrid polypeptide which comprises:
- 1. a DNA-binding domain, which recognizes a factor-regulated promoter operably associated with a polynucleotide encoding a therapeutic polypeptide or therapeutic polynucleotide; and
- 2. a ligand binding domain; and
- iii. a third gene expression cassette comprising a polynucleotide encoding a therapeutic polypeptide or therapeutic polynucleotide comprising:
- 1. a factor-regulated promoter, which is activated by the transactivation domain of the second hybrid polypeptide; and,
- 2. a polynucleotide encoding a therapeutic polypeptide or therapeutic polynucleotide, and
b. a second gene expression system which comprises:
- i. a first gene expression cassette comprising a polynucleotide encoding a first hybrid polypeptide which comprises:
- 1. a transactivation domain, which activates a factor-regulated promoter operably associated with a polynucleotide encoding a therapeutic polypeptide or therapeutic polynucleotide; and
- 2. a heterodimer partner domain,
- ii. a second gene expression cassette comprising a polynucleotide encoding a second hybrid polypeptide which comprises:
- 1. a DNA-binding domain, which recognizes a factor-regulated promoter operably associated with a polynucleotide encoding a therapeutic polypeptide or therapeutic polynucleotide; and
- 2. a ligand binding domain; and
- iii. a third gene expression cassette comprising a polynucleotide encoding a therapeutic polypeptide or therapeutic polynucleotide comprising:
- 1. a factor-regulated promoter, which is activated by the transactivation domain of the second hybrid polypeptide; and,
- 2. a polynucleotide encoding a therapeutic polypeptide or therapeutic polynucleotide.
- i. a first gene expression cassette comprising a polynucleotide encoding a first hybrid polypeptide which comprises:
- The multiple inducible gene expression systems provide for expression of a given therapeutic polynucleotide or therapeutic polypeptide under conditions associated with different diseases, disorders or conditions, or expression of multiple therapeutic polypeptides or therapeutic polynucleotides either under the same conditions associated with the same disease disorder or condition, or under different conditions associated with different diseases, disorders, or conditions.
- In certain embodiments, the combination of two or more gene switch systems may be (1) a dual-switch ecdysone receptor based gene expression system and (2) a single-switch ecdysone receptor based gene switch. In other embodiments, the combination may be (1) an single- or dual-switch ecdysone receptor based gene switch and (2) a rapamycin based gene switch. Alternatively, the combination of gene switch systems may be two identical rapamycin based gene switch systems disclosed above. Any possible combinations of the gene switch systems are within the scope of the invention. Examples of dual-switch ecdysone systems can be found, for example, in WO 2002/29075 and US 2002/0110861.
- As used herein, the term “ligand,” as applied to LDTFC-based gene switches e.g., EcD complex based gene switches, describes small and soluble molecules having the capability of activating a gene switch to stimulate expression of a polypeptide encoded therein. The ligand for a ligand-dependent transcription factor complex of the invention binds to the protein complex comprising one or more of the ligand binding domain, the heterodimer partner domain, the DNA binding domain, and the transactivation domain. The choice of ligand to activate the ligand-dependent transcription factor complex depends on the type of the gene switch utilized.
- Examples of ligands include, without limitation, an ecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N′-diacylhydrazines such as those disclosed in U.S. Pat. Nos. 6,013,836; 5,117,057; 5,530,028; and 5,378,726 and U.S. Published Application Nos. 2005/0209283 and 2006/0020146; oxadiazolines as described in U.S. Published Application No. 2004/0171651; dibenzoylalkyl cyanohydrazines such as those disclosed in European Application No. 461,809; N-alkyl-N,N′-diaroylhydrazines such as those disclosed in U.S. Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed in European Application No. 234,994; N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat. No. 4,985,461; arnidoketones such as those described in U.S. Published Application No. 2004/0049037; each of which is incorporated herein by reference and other similar materials including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, framesol, bile acids, 1,1-biphosphonate esters, juvenile hormone III, and the like. Examples of diacylhydrazine ligands useful in the present invention include RG-115819 (3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N′-(2-methyl-3-methoxy-benzoyl)-hydrazide), RG-115932 ((R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and RG-115830 (3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide). See, e.g., U.S. patent application Ser. No. 12/155,111, and PCT Appl. No. PCT/US2008/006757, both of which are incorporated herein by reference in their entireties.
- For example, a ligand for the edysone receptor based gene switch may be selected from any suitable ligands. Both naturally occurring ecdysone or ecdyson analogs (e.g., 20-hydroxyecdysone, muristerone A, ponasterone A, ponasterone B, ponasterone C, 26-iodoponasterone A, inokosterone or 26-mesylinokosterone) and non-steroid inducers may be used as a ligand for gene switch of the present invention. U.S. Pat. No. 6,379,945 B1, describes an insect steroid receptor isolated from Heliothis virescens (“HEcR”) which is capable of acting as a gene switch responsive to both steroid and certain non-steroidal inducers. Non-steroidal inducers have a distinct advantage over steroids, in this and many other systems which are responsive to both steroids and non-steroid inducers, for a number of reasons including, for example: lower manufacturing cost, metabolic stability, absence from insects, plants, or mammals, and environmental acceptability. U.S. Pat. No. 6,379,945 B1 describes the utility of two dibenzoylhydrazines, 1,2-dibenzoyl-1-tert-butyl-hydrazine and tebufenozide (N-(4-ethylbenzoyl)-N′-(3,5-dimethylbenzoyl)-N′-tert-butyl-hydrazine) as ligands for an ecdysone-based gene switch. Also included in the present invention as a ligand are other dibenzoylhydrazines, such as those disclosed in U.S. Pat. No. 5,117,057 B1. Use of tebufenozide as a chemical ligand for the ecdysone receptor from Drosophila melanogaster is also disclosed in U.S. Pat. No. 6,147,282. Additional, non-limiting examples of ecdysone ligands are 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, a 1,2-diacyl hydrazine, an N′-substituted-N,N′-disubstituted hydrazine, a dibenzoylalkyl cyanohydrazine, an N-substituted-N-alkyl-N,N-diaroyl hydrazine, an N-substituted-N-acyl-N-alkyl, carbonyl hydrazine or an N-aroyl-N′-alkyl-N′-aroyl hydrazine. (See U.S. Pat. No. 6,723,531).
- In one embodiment, the ligand for an ecdysone based gene switch system is a diacylhydrazine ligand or chiral diacylhydrazine ligand. The ligand used in the gene switch system may be compounds of Formula I
-
- wherein
- A is alkoxy, arylalkyloxy or aryloxy;
- B is optionally substituted aryl or optionally substituted heteroaryl; and
- R1 and R2 are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclo, optionally substituted aryl or optionally substituted heteroaryl;
- or pharmaceutically acceptable salts, hydrates, crystalline forms or amorphous forms thereof.
- In another embodiment, the ligand may be enantiomerically enriched compounds of Formula II
-
- wherein
- A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substituted aryl or optionally substituted heteroaryl;
- B is optionally substituted aryl or optionally substituted heteroaryl; and
- R1 and R2 are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclo, optionally substituted aryl or optionally substituted heteroaryl;
- with the proviso that R1 does not equal R2;
- wherein the absolute configuration at the asymmetric carbon atom bearing R1 and R2 is predominantly S;
- or pharmaceutically acceptable salts, hydrates, crystalline forms or amorphous forms thereof.
- In certain embodiments, the ligand may be enantiomerically enriched compounds of Formula III
-
- wherein
- A is alkoxy, arylalkyloxy, aryloxy, arylalkyl, optionally substituted aryl or optionally substituted heteroaryl;
- B is optionally substituted aryl or optionally substituted heteroaryl; and
- R1 and R2 are independently optionally substituted alkyl, arylalkyl, hydroxyalkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclo, optionally substituted aryl or optionally substituted heteroaryl;
- with the proviso that R1 does not equal R2;
- wherein the absolute configuration at the asymmetric carbon atom bearing R1 and R2 is predominantly R;
- or pharmaceutically acceptable salts, hydrates, crystalline forms or amorphous forms thereof.
- In one embodiment, a ligand may be (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide having an enantiomeric excess of at least 95% or a pharmaceutically acceptable salt, hydrate, crystalline form or amorphous form thereof.
- The diacylhydrazine ligands of Formula I and chiral diacylhydrazine ligands of Formula II or III, when used with an ecdysone-based gene switch system, provide the means for external temporal regulation of expression of a therapeutic polypeptide or therapeutic polynucleotide of the present invention. See U.S. application Ser. No. 12/155,111, filed May 29, 2008, which is fully incorporated by reference herein.
- The ligands used in the present invention may form salts. The term “salt(s)” as used herein denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases. In addition, when a compound of Formula I, II or III contains both a basic moiety and an acidic moiety, zwitterions (“inner salts”) may be formed and are included within the term “salt(s)” as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are used, although other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation. Salts of the compounds of Formula I, II or III may be formed, for example, by reacting a compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
- The ligands which contain a basic moiety may form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, 2-hydroxyethanesulfonates, lactates, maleates (formed with maleic acid), methanesulfonates (formed with methanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates such as tosylates, undecanoates, and the like.
- The ligands which contain an acidic moiety may form salts with a variety of organic and inorganic bases. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and salts with amino acids such as arginine, lysine and the like.
- Non-limiting examples of the ligands for the inducible gene expression system utilizing the FK506 binding domain are FK506, Cyclosporin A, or Rapamycin. FK506, rapamycin, and their analogs are disclosed in U.S. Pat. Nos. 6,649,595 B2 and 6,187,757. See also U.S. Pat. Nos. 7,276,498 and 7,273,874.
- The ligands described herein may be administered alone or as part of a pharmaceutical composition comprising a pharmaceutically acceptable carrier. In one embodiment, the pharmacetical compoistion are in the form of solutions, suspensions, tablets, capsules, ointments, elixirs, or injectable compositions.
- In one embodiment, the vector and methods of the present invention can be used to express a polynucleotide that encodes a protein including, but not limited to, a cytokine, an immunomodulator, a clotting factor, an antibody or a fragment of an antibody, a tumor necrosis factor receptor (TNFR), such as Ertanercept, an erythropoietin, alpha-1 antitrypsin, an interferon (IFN), interferon alpha, interferon beta, interferon gamma, interferon-beta-1a, interferon-beta-1b, Factor VII, Factor VIII, Factor IX, antithrombin III, a hepatitis B virus protein, a hormone, for example, a growth hormone (GH), human growth hormone (hGH), parathyroid hormone (PH), thyroid stimulating hormone (TSH), GCSF or fragment thereof, GM-CSF or a fragment thereof.
- In one embodiment, the polynucleotide encoding an antibody encodes a monoclonal antibody.
- In another embodiment, the vector and methods of the present invention can be used to express nucleic acid as a vaccine. The present invention also provides a vaccine composition comprising a vector or expression system of the present invention. In another embodiment, the vaccine comnposition comprises an adjuvant.
- The term “ecdysone receptor-based,” with respect to a gene switch, refers to a gene switch comprising at least a functional part of a naturally occurring or synthetic ecdysone receptor ligand binding domain and which regulates gene expression in response to a ligand that binds to the ecdysone receptor ligand binding domain. Examples of ecdysone-responsive systems are described in U.S. Pat. Nos. 7,091,038 and 6,258,603. In one embodiment, the system is the RheoSwitch® Therapeutic System (RTS), which contains two fusion proteins, the DEF domains of a mutagenized ecdysone receptor (EcR) fused with a Gal4 DNA binding domain and the EF domains of a chimeric RXR fused with a VP16 transcription activation domain, expressed under a constitutive promoter as illustrated in
FIG. 1 . - The terms “modulate” and “modulates” mean to induce, reduce or inhibit nucleic acid or gene expression, resulting in the respective induction, reduction or inhibition of protein or polypeptide production.
- The polynucleotides or vectors according to the invention may further comprise at least one promoter suitable for driving expression of a gene in a host cell.
- Enhancers that may be used in embodiments of the invention include but are not limited to: an SV40 enhancer, a cytomegalovirus (CMV) enhancer, an elongation factor 1 (EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.
- Termination control regions, i.e., terminator or polyadenylation sequences, may also be derived from various genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included. In one embodiment of the invention, the termination control region may be comprised or be derived from a synthetic sequence, synthetic polyadenylation signal, an SV40 late polyadenylation signal, an SV40 polyadenylation signal, a bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, or the like.
- The terms “3′ non-coding sequences” or “3′ untranslated region (UTR)” refer to DNA sequences located downstream (3′) of a coding sequence and may comprise polyadenylation [poly(A)] recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor.
- “Regulatory region” refers to a nucleic acid sequence that regulates the expression of a second nucleic acid sequence. A regulatory region may include sequences which are naturally responsible for expressing a particular nucleic acid (a homologous region) or may include sequences of a different origin that are responsible for expressing different proteins or even synthetic proteins (a heterologous region). In particular, the sequences can be sequences of prokaryotic, eukaryotic, or viral genes or derived sequences that stimulate or repress transcription of a gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, promoters, enhancers, transcriptional termination sequences, and signal sequences which direct the polypeptide into the secretory pathways of the target cell.
- A regulatory region from a “heterologous source” refers to a regulatory region that is not naturally associated with the expressed nucleic acid. Included among the heterologous regulatory regions are regulatory regions from a different species, regulatory regions from a different gene, hybrid regulatory sequences, and regulatory sequences which do not occur in nature, but which are designed by one having ordinary skill in the art.
- “RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to a double-stranded DNA that is complementary to and derived from mRNA. “Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. “Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that is not translated yet has an effect on cellular processes.
- “Polypeptide,” “peptide” and “protein” are used interchangeably and refer to a polymeric compound comprised of covalently linked amino acid residues.
- An “isolated polypeptide,” “isolated peptide” or “isolated protein” refer to a polypeptide or protein that is substantially free of those compounds that are normally associated therewith in its natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds, or the presence of impurities which do not interfere with biological activity, and which may be, for example, due to incomplete purification, addition of stabilizers, or compounding into a pharmaceutically acceptable preparation.
- A “substitution mutant polypeptide” or a “substitution mutant” will be understood to mean a mutant polypeptide comprising a substitution of at least one wild-type or naturally occurring amino acid with a different amino acid relative to the wild-type or naturally occurring polypeptide. A substitution mutant polypeptide may comprise only one wild-type or naturally occurring amino acid substitution and may be referred to as a “point mutant” or a “single point mutant” polypeptide. Alternatively, a substitution mutant polypeptide may comprise a substitution of two or more wild-type or naturally occurring amino acids with two or more amino acids relative to the wild-type or naturally occurring polypeptide. According to the invention, a Group H nuclear receptor ligand binding domain polypeptide comprising a substitution mutation comprises a substitution of at least one wild-type or naturally occurring amino acid with a different amino acid relative to the wild-type or naturally occurring Group H nuclear receptor ligand binding domain polypeptide.
- When the substitution mutant polypeptide comprises a substitution of two or more wild-type or naturally occurring amino acids, this substitution may comprise either an equivalent number of wild-type or naturally occurring amino acids deleted for the substitution, i.e., 2 wild-type or naturally occurring amino acids replaced with 2 non-wild-type or non-naturally occurring amino acids, or a non-equivalent number of wild-type amino acids deleted for the substitution, i.e., 2 wild-type amino acids replaced with 1 non-wild-type amino acid (a substitution+deletion mutation), or 2 wild-type amino acids replaced with 3 non-wild-type amino acids (a substitution+insertion mutation).
- Substitution mutants may be described using an abbreviated nomenclature system to indicate the amino acid residue and number replaced within the reference polypeptide sequence and the new substituted amino acid residue. For example, a substitution mutant in which the twentieth (20th) amino acid residue of a polypeptide is substituted may be abbreviated as “x20z”, wherein “x” is the amino acid to be replaced, “20” is the amino acid residue position or number within the polypeptide, and “z” is the new substituted amino acid. Therefore, a substitution mutant abbreviated interchangeably as “E20A” or “Glu20Ala” indicates that the mutant comprises an alanine residue (commonly abbreviated in the art as “A” or “Ala”) in place of the glutamic acid (commonly abbreviated in the art as “E” or “Glu”) at
position 20 of the polypeptide. - A substitution mutation may be made by any technique for mutagenesis known in the art, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem. 253:6551 (1978); Zoller et al., DNA 3:479 (1984); Oliphant et al., Gene 44:177 (1986); Hutchinson et al., Proc. Natl. Acad. Sci. USA 83:710 (1986)), use of TAB® linkers (Pharmacia), restriction endonuclease digestion/fragment deletion and substitution, PCR-mediated/oligonucleotide-directed mutagenesis, and the like. PCR-based techniques are preferred for site-directed mutagenesis (see Higuchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press,
Chapter 6, pp. 61-70). - The term “fragment,” as applied to a polypeptide, refers to a polypeptide whose amino acid sequence is shorter than that of the reference polypeptide and which comprises, over the entire portion with these reference polypeptides, an identical amino acid sequence. Such fragments may, where appropriate, be included in a larger polypeptide of which they are a part. Such fragments of a polypeptide according to the invention may have a length of at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300 or more amino acids.
- A “variant” of a polypeptide or protein refers to any analogae, fragment, derivative, or mutant which is derived from a polypeptide or protein and which retains at least one biological property of the polypeptide or protein. Different variants of the polypeptide or protein may exist in nature. These variants may be allelic variations characterized by differences in the nucleotide sequences of the structural gene coding for the protein, or may involve differential splicing or post-translational modification. The skilled artisan can produce variants having single or multiple amino acid substitutions, deletions, additions, or replacements. These variants may include, inter alia: (a) variants in which one or more amino acid residues are substituted with conservative or non-conservative amino acids, (b) variants in which one or more amino acids are added to the polypeptide or protein, (c) variants in which one or more of the amino acids includes a substituent group, and (d) variants in which the polypeptide or protein is fused with another polypeptide such as serum albumin. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to persons having ordinary skill in the art. In one embodiment, a variant polypeptide comprises at least about 14 amino acids.
- The term “homology” refers to the percent of identity between two polynucleotide or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known to the art. For example, homology can be determined by a direct comparison of the sequence information between two polypeptide molecules by aligning the sequence information and using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s) and size determination of the digested fragments.
- As used herein, the term “homologous” in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a “common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al., Cell 50:667 (1987)). Such proteins (and their encoding genes) have sequence homology, as reflected by their high degree of sequence similarity. However, in common usage and in the application, the term “homologous,” when modified with an adverb such as “highly,” may refer to sequence similarity and not a common evolutionary origin.
- Accordingly, the term “sequence similarity” in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., Cell 50:667 (1987)). In one embodiment, two DNA sequences are “substantially homologous” or “substantially similar” when at least about 50% (e.g., at least about 75%, 90%, or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by compar'ng the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art (see e.g., Sambrook et al., 1989, supra).
- As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases do not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the invention such as deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript. It is therefore understood that the invention encompasses more than the specific exemplary sequences. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.
- Moreover, the skilled artisan recognizes that substantially similar sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS), with the sequences exemplified herein. Substantially similar nucleic acid fragments of the invention are those nucleic acid fragments whose DNA sequences are at least about 70%, 80%, 90% or 95% identical to the DNA sequence of the nucleic acid fragments reported herein.
- Two amino acid sequences are “substantially homologous” or “substantially similar” when greater than about 40% of the amino acids are identical, or greater than 60% are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package,
Version 7, Madison, Wis.) pileup program. - The term “corresponding to” is used herein to refer to similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured. A nucleic acid or amino acid sequence alignment may include spaces. Thus, the term “corresponding to” refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
- A “substantial portion” of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol. Biol. 215:403 (1993)); available at ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.
- The term “percent identity,” as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “identity” and “similarity” can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using sequence analysis software such as the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences may be performed using the Clustal method of alignment (Higgins et al., CABIOS. 5:151 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method may be selected:
KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. - The term “sequence analysis software” refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. “Sequence analysis software” may be commercially available or independently developed. Typical sequence analysis software includes, but is not limited to, the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol. 215:403 (1990)), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison, Wis. 53715 USA). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the “default values” of the program referenced, unless otherwise specified. As used herein “default values” will mean any set of values or parameters which originally load with the software when first initialized.
- “Chemically synthesized,” as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.
- As used herein, two or more individually operable gene regulation systems are said to be “orthogonal” when: a) modulation of each of the given systems by its respective ligand, at a chosen concentration, results in a measurable change in the magnitude of expression of the gene of that system, and b) the change is statistically significantly different than the change in expression of all other systems simultaneously operable in the cell, tissue, or organism, regardless of the simultaneity or sequentiality of the actual modulation. Preferably, modulation of each individually operable gene regulation system effects a change in gene expression at least 2-fold greater than all other operable systems in the cell, tissue, or organism, e.g., at least 5-fold, 10-fold, 100-fold, or 500-fold greater. Ideally, modulation of each of the given systems by its respective ligand at a chosen concentration results in a measurable change in the magnitude of expression of the gene of that system and no measurable change in expression of all other systems operable in the cell, tissue, or organism. In such cases the multiple inducible gene regulation system is said to be “fully orthogonal.” Useful orthogonal ligands and orthogonal receptor-based gene expression systems are described in US 2002/0110861 A1.
- The term “exogenous gene” means a gene foreign to the subject, that is, a gene which is introduced into the subject through a transformation process, an unmutated version of an endogenous mutated gene or a mutated version of an endogenous unmutated gene. The method of transformation is not critical to this invention and may be any method suitable for the subject known to those in the art. Exogenous genes can be either natural or synthetic genes which are introduced into the subject in the form of DNA or RNA which may function through a DNA intermediate such as by reverse transcriptase. Such genes can be introduced into target cells, directly introduced into the subject, or indirectly introduced by the transfer of transformed cells into the subject.
- The term “therapeutic product” refers to a therapeutic polypeptide or therapeutic polynucleotide which imparts a beneficial function to the host cell in which such product is expressed. Therapeutic polypeptides may include, without limitation, peptides as small as three amino acids in length, single- or multiple-chain proteins, and fusion proteins. Therapeutic polynucleotides may include, without limitation, antisense oligonucleotides, small interfering RNAs, ribozymes, and RNA external guide sequences. The therapeutic product may comprise a naturally occurring sequence, a synthetic sequence or a combination of natural and synthetic sequences.
- The term “ligand-dependent transcription factor complex” or “LDTFC” refers to a transcription factor comprising one or more protein subunits, which complex can regulate gene expression driven by a “factor-regulated promoter” as defined herein. A model LDTFC is an “ecdysone receptor complex” generally refers to a heterodimeric protein complex having at least two members of the nuclear receptor family, ecdysone receptor (“EcR”) and ultraspiracle (“USP”) proteins (see Yao et al., Nature 366:476 (1993)); Yao et al., Cell 71:63 (1992)). A functional LDTFC such as an EcR complex may also include additional protein(s) such as immunophilins. Additional members of the nuclear receptor family of proteins, known as transcriptional factors (such as DHR38, betaFTZ-1 or other insect homologs), may also be ligand dependent or independent partners for EcR and/or USP. A LDTFC such as an EcR complex can also be a heterodimer of EcR protein and the vertebrate homolog of ultraspiracle protein, retinoic acid-X-receptor (“RXR”) protein or a chimera of USP and RXR. The terms “LDTFC” and “EcR complex” also encompass homodimer complexes of the EcR protein or USP, as well as single polypeptides or trimers, tetramer, and other multimers serving the same function.
- A LDTFC such as an EcR complex can be activated by an active ecdysteroid or non-steroidal ligand bound to one of the proteins of the complex, inclusive of EcR, but not excluding other proteins of the complex. A LDTFC such as an EcR complex includes proteins which are members of the nuclear receptor superfamily wherein all members are characterized by the presence of one or more polypeptide subunits comprising an amino-terminal transactivation domain (“AD,” “TD,” or “TA,” used interchangeably herein), a DNA binding domain (“DBD”), and a ligand binding domain (“LBD”). The AD may be present as a fusion with a “heterodimerization partner” or “HP.” A fusion protein comprising an AD and HP of the invention is referred to herein as a “coactivation protein” or “CAP.” The DBD and LBD may be expressed as a fusion protein, referred to herein as a “ligand-inducible transcription factor (“LTF”). The fusion partners may be separated by a linker, e.g., a hinge region. Some members of the LTF family may also have another transactivation domain on the carboxy-terminal side of the LBD. The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for ecdysone response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins.
- The DNA sequences making up the exogenous gene, the response element, and the LDTFC, e.g., EcR complex, may be incorporated into archaebacteria, procaryotic cells such as Escherichia coli, Bacillus subtilis, or other enterobacteria, or eucaryotic cells such as plant or animal cells. However, because many of the proteins expressed by the gene are processed incorrectly in bacteria, eucaryotic cells are preferred. The cells may be in the form of single cells or multicellular organisms. The nucleotide sequences for the exogenous gene, the response element, and the receptor complex can also be incorporated as RNA molecules, preferably in the form of functional viral RNAs such as tobacco mosaic virus. Of the eucaryotic cells, vertebrate cells are preferred because they naturally lack the molecules which confer responses to the ligands of this invention for the EcR. As a result, they are “substantially insensitive” to the ligands of this invention. Thus, the ligands useful in this invention will have negligible physiological or other effects on transformed cells, or the whole organism. Therefore, cells can grow and express the desired product, substantially unaffected by the presence of the ligand itself.
- The term “ecdysone receptor complex” generally refers to a heterodimeric protein complex having at least two members of the nuclear receptor family, ecdysone receptor (“EcR”) and ultraspiracle (“USP”) proteins (see Yao et al., Nature 366:476 (1993)); Yao et al., Cell 71:63 (1992)). The functional EcR complex may also include additional protein(s) such as immunophilins. Additional members of the nuclear receptor family of proteins, known as transcriptional factors (such as DHR38, betaFTZ-1 or other insect homologs), may also be ligand dependent or independent partners for EcR and/or USP. The EcR complex can also be a heterodimer of EcR protein and the vertebrate homolog of ultraspiracle protein, retinoic acid-X-receptor (“RXR”) protein or a chimera of USP and RXR. The term EcR complex also encompasses homodimer complexes of the EcR protein or USP.
- An EcR complex can be activated by an active ecdysteroid or non-steroidal ligand bound to one of the proteins of the complex, inclusive of EcR, but not excluding other proteins of the complex. As used herein, the term “ligand,” as applied to EcR-based gene switches, describes small and soluble molecules having the capability of activating a gene switch to stimulate expression of a polypeptide encoded therein. Examples of ligands include, without limitation, an ecdysteroid, such as ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, and the like, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N′-diacylhydrazines such as those disclosed in U.S. Pat. Nos. 6,013,836; 5,117,057; 5,530,028; and 5,378,726 and U.S. Published Application Nos. 2005/0209283 and 2006/0020146; oxadiazolines as described in U.S. Published Application No. 2004/0171651; dibenzoylalkyl cyanohydrazines such as those disclosed in European Application No. 461,809; N-alkyl-N,N′-diaroylhydrazines such as those disclosed in U.S. Pat. No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those disclosed in European Application No. 234,994; N-aroyl-N-alkyl-N′-aroylhydrazines such as those described in U.S. Pat. No. 4,985,461; amidoketones such as those described in U.S. Published Application No. 2004/0049037; and other similar materials including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylharpagide, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, framesol, bile acids, 1,1-biphosphonate esters, juvenile hormone III, and the like. Examples of diacylhydrazine ligands useful in the invention include RG-115819 (3,5-Dimethyl-benzoic acid N-(1-ethyl-2,2-dimethyl-propyl)-N′-(2-methyl-3-methoxy-benzoyl)-hydrazide), RG-115932 ((R)-3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and RG-115830 (3,5-Dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide). See U.S. Appln. 12/155,111, filed May 29, 2008, and PCT/US2008/006757 filed May 29, 2008, for additional diacylhydrazines that are useful in the practice of the invention.
- The EcR complex includes proteins which are members of the nuclear receptor superfamily wherein all members are characterized by the presence of an amino-terminal transactivation domain (“TA”), a DNA binding domain (“DBD”), and a ligand binding domain (“LBD”) separated by a hinge region. Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD. The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for ecdysone response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins.
- The DNA sequences making up the exogenous gene, the response element, and the EcR complex may be incorporated into archaebacteria, procaryotic cells such as Escherichia coli, Bacillus subtilis, or other enterobacteria, or eucaryotic cells such as plant or animal cells. However, because many of the proteins expressed by the gene are processed incorrectly in bacteria, eucaryotic cells are preferred. The cells may be in the form of single cells or multicellular organisms. The nucleotide sequences for the exogenous gene, the response element, and the receptor complex can also be incorporated as RNA molecules, preferably in the form of functional viral RNAs such as tobacco mosaic virus. Of the eucaryotic cells, vertebrate cells are preferred because they naturally lack the molecules which confer responses to the ligands of this invention for the EcR. As a result, they are “substantially insensitive” to the ligands of this invention. Thus, the ligands useful in this invention will have negligible physiological or other effects on transformed cells, or the whole organism. Therefore, cells can grow and express the desired product, substantially unaffected by the presence of the ligand itself.
- EcR ligands, when used with the EcR complex which in turn is bound to the response element linked to an exogenous gene (e.g., IL-12), provide the means for external temporal regulation of expression of the exogenous gene. The order in which the various components bind to each other, that is, ligand to receptor complex and receptor complex to response element, is not critical. Typically, modulation of expression of the exogenous gene is in response to the binding of the EcR complex to a specific control, or regulatory, DNA element. The EcR protein, like other members of the nuclear receptor family, possesses at least three domains, a transactivation domain, a DNA binding domain, and a ligand binding domain. This receptor, like a subset of the nuclear receptor family, also possesses less well-defined regions responsible for heterodimerization properties. Binding of the ligand to the ligand binding domain of EcR protein, after heterodimerization with USP or RXR protein, enables the DNA binding domains of the heterodimeric proteins to bind to the response element in an activated form, thus resulting in expression or suppression of the exogenous gene. This mechanism does not exclude the potential for ligand binding to either EcR or USP, and the resulting formation of active homodimer complexes (e.g., EcR+EcR or USP+USP). In one embodiment, one or more of the receptor domains can be varied producing a chimeric gene switch. Typically, one or more of the three domains may be chosen from a source different than the source of the other domains so that the chimeric receptor is optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski et al., Nature 335:563 (1988) or LexA protein from E. coli (see Brent et al., Cell 43:729 (1985)) to accommodate chimeric EcR complexes. Another advantage of chimeric systems is that they allow choice of a promoter used to drive the exogenous gene according to a desired end result. Such double control can be particularly important in areas of gene therapy, especially when cytotoxic proteins are produced, because both the timing of expression as well as the cells wherein expression occurs can be controlled. When exogenous genes, operatively linked to a suitable promoter, are introduced into the cells of the subject, expression of the exogenous genes is controlled by the presence of the ligand of this invention. Promoters may be constitutively or inducibly regulated or may be tissue-specific (that is, expressed only in a particular type of cell) or specific to certain developmental stages of the organism.
- In certain embodiments, the therapeutic switch promoter described in the methods is consititutive. In certain embodiments, the therapeutic switch promoter is activated under conditions associated with a disease, disorder, or condition, e.g., the promoter is activated in response to a disease, in response to a particular physiological, developmental, differentiation, or pathological condition, and/or in response to one or more specific biological molecules; and/or the promoter is activated in particular tissue or cell types. In certain embodiments, the disease, disorder, or condition is responsive to the therapeutic polypeptide or polynucleotide. For example in certain non-limiting embodiments the therapeutic polynucleotide or polypeptide is useful to treat, prevent, ameliorate, reduce symptoms, prevent progression, or cure the disease, disorder or condition, but need not accomplish any one or all of these things. In certain embodiments, the first and second polynucleotides are introduced so as to permit expression of the ligand-dependent transcription factor complex under consitions associated with a disease, disorder or condition. In one embodiment, the therapeutic methods are carried out such that the therapeutic polypeptide or therapeutic polynucleotide is expressed and disseminated through the subject at a level sufficient to treat, ameliorate, or prevent said disease, disorder, or condition. As used herein, “disseminated” means that the polypeptide is expressed and released from the modified cell sufficiently to have an effect or activity in the subject. Dissemination may be systemic, local or anything in between. For example, the therapeutic polypeptide or therapeutic polynucleotide might be systemically disseminated through the bloodstream or lymph system. Alternatively, the therapeutic polypeptide or therapeutic polynucleotide might be disseminated locally in a tissue or organ to be treated.
- Numerous genomic and cDNA nucleic acid sequences coding for a variety of polypeptides, such as transcription factors and reporter proteins, are well known in the art. Those skilled in the art have access to nucleic acid sequence information for virtually all known genes and can either obtain the nucleic acid molecule directly from a public depository, the institution that published the sequence, or employ routine methods to prepare the molecule. See for example the description of the sequence accession numbers, infra.
- The gene switch may be any gene switch system that regulates gene expression by addition or removal of a specific ligand. In one embodiment, the gene switch is one in which the level of gene expression is dependent on the level of ligand that is present. Examples of ligand-dependent transcription factors that may be used in the gene switches of the invention include, without limitation, members of the nuclear receptor superfamily activated by their respective ligands (e.g., glucocorticoid, estrogen, progestin, retinoid, ecdysone, and analogs and mimetics thereof) and rTTA activated by tetracycline. In one aspect of the invention, the gene switch is an EcR-based gene switch. Examples of such systems include, without limitation, the systems described in U.S. Pat. Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos. 2006/0014711, 2007/0161086, and International Published Application No. WO 01/70816. Examples of chimeric ecdysone receptor systems are described in U.S. Pat. No. 7,091,038, U.S. Published Patent Application Nos. 2002/0110861, 2004/0033600, 2004/0096942, 2005/0266457, and 2006/0100416, and International Published Application Nos. WO 01/70816, WO 02/066612, WO 02/066613, WO 02/066614, WO 02/066615, WO 02/29075, and WO 2005/108617. An example of a non-steroidal ecdysone agonist-regulated system is the RheoSwitch® Mammalian Inducible Expression System (New England Biolabs, Ipswich, Mass.).
- In one embodiment, a polynucleotide encoding the gene switch comprises a single transcription factor sequence encoding a ligand-dependent transcription factor under the control of a promoter. The transcription factor sequence may encode a ligand-dependent transcription factor that is a naturally occurring or an artificial transcription factor. An artificial transcription factor is one in which the natural sequence of the transcription factor has been altered, e.g., by mutation of the sequence or by the combining of domains from different transcription factors. In one embodiment, the transcription factor comprises a Group H nuclear receptor ligand binding domain (LBD). In one embodiment, the Group H nuclear receptor LBD is from an EcR, a ubiquitous receptor, an
orphan receptor 1, a NER-1, a steroid hormonenuclear receptor 1, a retinoid X receptor interacting protein-15, a liver X receptor β, a steroid hormone receptor like protein, a liver X receptor, a liver X receptor α, a farnesoid X receptor, areceptor interacting protein 14, or a farnesol receptor. In another embodiment, the Group H nuclear receptor LBD is from an ecdysone receptor. - The EcR and the other Group H nuclear receptors are members of the nuclear receptor superfamily wherein all members are generally characterized by the presence of an amino-terminal transactivation domain (TD), a DNA binding domain (DBD), and a LBD separated from the DBD by a hinge region. As used herein, the term “DNA binding domain” comprises a minimal polypeptide sequence of a DNA binding protein, up to the entire length of a DNA binding protein, so long as the DNA binding domain functions to associate with a particular response element. Members of the nuclear receptor superfamily are also characterized by the presence of four or five domains: A/B, C, D, E, and in some members F (see U.S. Pat. No. 4,981,784 and Evans, Science 240:889 (1988)). The “A/B” domain corresponds to the transactivation domain, “C” corresponds to the DNA binding domain, “D” corresponds to the hinge region, and “E” corresponds to the ligand binding domain. Some members of the family may also have another transactivation domain on the carboxy-terminal side of the LBD corresponding to “F”.
- The DBD is characterized by the presence of two cysteine zinc fingers between which are two amino acid motifs, the P-box and the D-box, which confer specificity for response elements. These domains may be either native, modified, or chimeras of different domains of heterologous receptor proteins. The EcR, like a subset of the nuclear receptor family, also possesses less well-defined regions responsible for heterodimerization properties. Because the domains of nuclear receptors are modular in nature, the LBD, DBD, and TD may be interchanged.
- In another embodiment, the transcription factor comprises a TD, a DBD that recognizes a response element associated with the exogenous gene whose expression is to be modulated; and a Group H nuclear receptor LBD. In certain embodiments, the Group H nuclear receptor LBD comprises a substitution mutation.
- In another embodiment, a polynucleotide encoding the gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor, i.e., a “dual switch”- or “two-hybrid”-based gene switch. The first and second promoters may be the same or different.
- In certain embodiments, the polynucleotide encoding a gene switch comprises a first transcription factor sequence and a second transcription factor sequence under the control of a promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor, i.e., a “single gene switch”. The first transcription factor sequence and a second transcription factor sequence may be connected by an internal ribosomal entry site (IRES). The IRES may be an EMCV IRES.
- In one embodiment, the first transcription factor sequence encodes a polypeptide comprising a TD, a DBD that recognizes a response element associated with the exogenous gene whose expression is to be modulated; and a Group H nuclear receptor LBD, and the second transcription factor sequence encodes a transcription factor comprising a nuclear receptor LBD selected from a vertebrate RXR LBD, an invertebrate RXR LBD, an ultraspiracle protein LBD, and a chimeric LBD comprising two polypeptide fragments, wherein the first polypeptide fragment is from a vertebrate RXR LBD, an invertebrate RXR LBD, or an ultraspiracle protein LBD, and the second polypeptide fragment is from a different vertebrate RXR LBD, invertebrate RXR LBD, or ultraspiracle protein LBD.
- In another embodiment, the gene switch comprises a first transcription factor sequence encoding a first polypeptide comprising a nuclear receptor LBD and a DBD that recognizes a response element associated with the exogenous gene whose expression is to be modulated, and a second transcription factor sequence encoding a second polypeptide comprising a TD and a nuclear receptor LBD, wherein one of the nuclear receptor LBDs is a Group H nuclear receptor LBD. In one embodiment, the first polypeptide is substantially free of a TD and the second polypeptide is substantially free of a DBD. For purposes of the invention, “substantially free” means that the protein in question does not contain a sufficient sequence of the domain in question to provide activation or binding activity.
- In another aspect of the invention, the first transcription factor sequence encodes a protein comprising a heterodimer partner and a TD and the second transcription factor sequence encodes a protein comprising a DBD and a LBD.
- When only one nuclear receptor LBD is a Group H LBD, the other nuclear receptor LBD may be from any other nuclear receptor that forms a dimer with the Group H LBD. For example, when the Group H nuclear receptor LBD is an EcR LBD, the other nuclear receptor LBD “partner” may be from an EcR, a vertebrate RXR, an invertebrate RXR, an ultraspiracle protein (USP), or a chimeric nuclear receptor comprising at least two different nuclear receptor LBD polypeptide fragments selected from a vertebrate RXR, an invertebrate RXR, and a USP (see WO 01/70816 A2, international Patent Application No. PCT/US02/05235 and US 2004/0096942 A1). The “partner” nuclear receptor ligand binding domain may further comprise a truncation mutation, a deletion mutation, a substitution mutation, or another modification.
- In one embodiment, the vertebrate RXR LBD is from a human Homo sapiens, mouse Mus musculus, rat Rattus norvegicus, chicken Gallus gallus, pig Sus scrofa domestica, frog Xenopus laevis, zebrafish Danio rerio, tunicate Polyandrocarpa misakiensis, or jellyfish Tripedalia cysophora RXR.
- In one embodiment, the invertebrate RXR ligand binding domain is from a locust Locusta migratoria ultraspiracle polypeptide (“LmUSP”), an ixodid tick Amblyomma americanum RXR homolog 1 (“AmaRXR1”), an ixodid tick Amblyomma americanum RXR homolog 2 (“AmaRXR2”), a fiddler crab Celuca pugilator RXR homolog (“CpRXR”), a beetle Tenebrio molitor RXR homolog (“TmRXR”), a honeybee Apis mellifera RXR homolog (“AmRXR”), an aphid Myzus persicae RXR homolog (“MpRXR”), or a non-Dipteran/non-Lepidopteran RXR homolog.
- In one embodiment, the chimeric RXR LBD comprises at least two polypeptide fragments selected from a vertebrate species RXR polypeptide fragment, an invertebrate species RXR polypeptide fragment, and a non-Dipteran/non-Lepidopteran invertebrate species RXR homolog polypeptide fragment. A chimeric RXR ligand binding domain for use in the invention may comprise at least two different species RXR polypeptide fragments, or when the species is the same, the two or more polypeptide fragments may be from two or more different isoforms of the species RXR polypeptide fragment.
- In one embodiment, the chimeric RXR ligand binding domain comprises at least one vertebrate species RXR polypeptide fragment and one invertebrate species RXR polypeptide fragment.
- In another embodiment, the chimeric RXR ligand binding domain comprises at least one vertebrate species RXR polypeptide fragment and one non-Dipteran/non-Lepidopteran invertebrate species RXR homolog polypeptide fragment.
- The ligand, when combined with the LBD of the nuclear receptor(s), which in turn are bound to the response element linked to the exogenous gene, provides external temporal regulation of expression of the exogenous gene. The binding mechanism or the order in which the various components of this invention bind to each other, that is, for example, ligand to LBD, DBD to response element, TD to promoter, etc., is not critical.
- In a specific example, binding of the ligand to the LBD of a Group H nuclear receptor and its nuclear receptor LBD partner enables expression of the exogenous gene. This mechanism does not exclude the potential for ligand binding to the Group H nuclear receptor (GHNR) or its partner, and the resulting formation of active homodimer complexes (e.g., GHNR+GHNR or partner+partner). Preferably, one or more of the receptor domains is varied producing a hybrid gene switch. Typically, one or more of the three domains, DBD, LBD, and TD, may be chosen from a source different than the source of the other domains so that the hybrid genes and the resulting hybrid proteins are optimized in the chosen host cell or organism for transactivating activity, complementary binding of the ligand, and recognition of a specific response element. In addition, the response element itself can be modified or substituted with response elements for other DNA binding protein domains such as the GAL-4 protein from yeast (see Sadowski et al., Nature 335:563 (1988)) or LexA protein from Escherichia coli (see Brent et al., Cell 43:729 (1985)), or synthetic response elements specific for targeted interactions with proteins designed, modified, and selected for such specific interactions (see, for example, Kim et al., Proc. Natl. Acad. Sci. USA, 94:3616 (1997)) to accommodate hybrid receptors.
- The functional EcR complex may also include additional protein(s) such as immunophilins. Additional members of the nuclear receptor family of proteins, known as transcriptional factors (such as DHR38 or betaFTZ-1), may also be ligand dependent or independent partners for EcR, USP, and/or RXR. Additionally, other cofactors may be required such as proteins generally known as coactivators (also termed adapters or mediators). These proteins do not bind sequence-specifically to DNA and are not involved in basal transcription. They may exert their effect on transcription activation through various mechanisms, including stimulation of DNA-binding of activators, by affecting chromatin structure, or by mediating activator-initiation complex interactions. Examples of such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70, SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/AIB1/RAC3/pCIP as well as the promiscuous coactivator C response element B binding protein, CBP/p300 (for review see Glass et al., Curr. Opin. Cell Biol. 9:222 (1997)). Also, protein cofactors generally known as corepressors (also known as repressors, silencers, or silencing mediators) may be required to effectively inhibit transcriptional activation in the absence of ligand. These corepressors may interact with the unliganded EcR to silence the activity at the response element. Current evidence suggests that the binding of ligand changes the conformation of the receptor, which results in release of the corepressor and recruitment of the above described coactivators, thereby abolishing their silencing activity. Examples of corepressors include N-CoR and SMRT (for review, see Horwitz et al., Mol. Endocrinol. 10:1167 (1996)). These cofactors may either be endogenous within the cell or organism, or may be added exogenously as transgenes to be expressed in either a regulated or unregulated fashion.
- The exogenous gene is operably linked to a promoter comprising at least one response element that is recognized by the DBD of the ligand-dependent transcription factor encoded by the gene switch. In one embodiment, the promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more copies of the response element. Promoters comprising the desired response elements may be naturally occurring promoters or artificial promoters created using techniques that are well known in the art, e.g., one or more response elements operably linked to a minimal promoter.
- A gene encoding an immunomodulator, e.g., IL-12, TNF-alpha, signal peptides, or any transcription factors herein can also be codon-optimized. In one embodiment, a coding region of an immunomodulator, e.g., IL-12, TNF-alpha, a signal peptide, or a transcription factor is codon-optimized for expression in human. As appreciated by one of ordinary skill in the art, various nucleic acid coding regions will encode the same polypeptide due to the redundancy of the genetic code. Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The “genetic code” which shows which codons encode which amino acids is reproduced herein as Table 4. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the polypeptides encoded by the DNA.
-
TABLE 4 The Standard Genetic Code T C A G T TTT Phe (F) TCT Ser (S) TAT Tyr (Y) TGT Cys (C) TTC Phe (F) TCC Ser (S) TAC Tyr (Y) TGC TTA Leu (L) TCA Ser (S) TAA Ter TGA Ter TTG Leu (L) TCG Ser (S) TAG Ter TGG Trp (W) C CTT Leu (L) CCT Pro (P) CAT His (H) CGT Arg (R) CTC Leu (L) CCC Pro (P) CAC His (H) CGC Arg (R) CTA Leu (L) CCA Pro (P) CAA Gln (Q) CGA Arg (R) CTG Leu (L) CCG Pro (P) CAG Gln (Q) CGG Arg (R) A ATT Ile (I) ACT Thr (T) AAT Asn (N) AGT Ser (S) ATC Ile (I) ACC Thr (T) AAC Asn (N) AGC Ser (S) ATA Ile (I) ACA Thr (T) AAA Lys (K) AGA Arg (R) ATG Met (M) ACG Thr (T) AAG Lys (K) AGG Arg (R) G GTT Val (V) GCT Ala (A) GAT Asp (D) GGT Gly (G) GTC Val (V) GCC Ala (A) GAC Asp (D) GGC Gly (G) GTA Val (V) GCA Ala (A) GAA Glu (E) GGA Gly (G) GTG Val (V) GCG Ala (A) GAG Glu (E) GGG Gly (G) - It is to be appreciated that any polynucleotide that encodes a polypeptide in accordance with the invention falls within the scope of this invention, regardless of the codons used.
- Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing polypeptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.
- The polynucleotides are prepared by incorporating codons preferred for use in the genes of a given species into the DNA sequence.
- Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at http://www.kazusa.or.jp/codon/ (visited May 30, 2006), and these tables can be adapted in a number of ways. See Nakamura, Y., et al., “Codon usage tabulated from the international DNA sequence databases: status for the
year 2000” Nucl. Acids Res. 28:292 (2000). Codon usage tables for humans calculated from GenBank Release 151.0, are reproduced below as Table 5 (from http://www.kazusa.or.jp/codon/supra). These tables use mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. The tables have been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons. -
TABLE 5 Codon Usage Table for Human Genes (Homo sapiens) Amino Acid Codon Frequency of Usage Phe UUU 0.4525 UUC 0.5475 Leu UUA 0.0728 UUG 0.1266 CUU 0.1287 CUC 0.1956 CUA 0.0700 CUG 0.4062 Ile AUU 0.3554 AUC 0.4850 AUA 0.1596 Met AUG 1.0000 Val GUU 0.1773 GUC 0.2380 GUA 0.1137 GUG 0.4710 Ser UCU 0.1840 UCC 0.2191 UCA 0.1472 UCG 0.0565 AGU 0.1499 AGC 0.2433 Pro CCU 0.2834 CCC 0.3281 CCA 0.2736 CCG 0.1149 Thr ACU 0.2419 ACC 0.3624 ACA 0.2787 ACG 0.1171 Ala GCU 0.2637 GCC 0.4037 GCA 0.2255 GCG 0.1071 Tyr UAU 0.4347 UAC 0.5653 His CAU 0.4113 CAC 0.5887 Gln CAA 0.2541 CAG 0.7459 Asn AAU 0.4614 AAC 0.5386 Lys AAA 0.4212 AAG 0.5788 Asp GAU 0.4613 GAC 0.5387 Glu GAA 0.4161 GAG 0.5839 Cys UGU 0.4468 UGC 0.5532 Trp UGG 1.0000 Arg CGU 0.0830 CGC 0.1927 CGA 0.1120 CGG 0.2092 AGA 0.2021 AGG 0.2011 Gly GGU 0.1632 GGC 0.3438 GGA 0.2459 GGG 0.2471 - By utilizing these or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species.
- A number of options are available for synthesizing codon-optimized coding regions designed by any of the methods described above, using standard and routine molecular biological manipulations well known to those of ordinary skill in the art.
- In one embodiment, the coding region encoding the immunomodulator, e.g., TNF-alpha, in the vector of the invention is codon-optimized. In another embodiment, the coding region is codon-optimized for expression in human. In a particular embodiment, TNF-alpha in the invention is encoded by a codon-optimized nucleic acid sequence.
- To introduce the polynucleotides into the cells in vivo or ex vivo, a vector can be used. The vector may be, for example, a plasmid vector or a single-or double-stranded RNA or DNA viral vector. Such vectors may be introduced into cells of a subject in need thereof, e.g., mammal, by well-known techniques for introducing DNA and RNA into cells. Viral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells. As used herein, the term “host cell” or “host” is used to mean a cell of the invention that is harboring one or more polynucleotides of the invention.
- Thus, at a minimum, the vectors must include the polynucleotides of the invention. Other components of the vector may include, but are not limited to, selectable markers, chromatin modification domains, additional promoters driving expression of other polypeptides that may also be present on the vector (e.g., a lethal polypeptide), genomic integration sites, recombination sites, and molecular insertion pivots. The vectors may comprise any number of these additional elements, either within or not within the polynucleotides, such that the vector can be tailored to the specific goals of the therapeutic methods desired.
- In one embodiment of the invention, the vectors that are introduced into the cells further comprise a “selectable marker gene” which, when expressed, indicates that the gene switch construct of the invention has been integrated into the genome of the host cell. In this manner, the selector gene can be a positive marker for the genome integration. While not critical to the methods of the invention, the presence of a selectable marker gene allows the practitioner to select for a population of live cells where the vector construct has been integrated into the genome of the cells. Thus, certain embodiments of the invention comprise selecting cells where the vector has successfully been integrated. As used herein, the term “select” or variations thereof, when used in conjunction with cells, is intended to mean standard, well-known methods for choosing cells with a specific genetic make-up or phenotype. Typical methods include, but are not limited to, culturing cells in the presence of antibiotics, such as G418, neomycin and ampicillin. Other examples of selectable marker genes include, but are not limited to, genes that confer resistance to dihydrofolate reductase, hygromycin, or mycophenolic acid. Other methods of selection include, but are not limited to, a selectable marker gene that allows for the use of thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase or adenine phosphoribosyltransferase as selection agents. Cells comprising a vector construct comprising an antibiotic resistance gene or genes would then be capable of tolerating the antibiotic in culture. Likewise, cells not comprising a vector construct comprising an antibiotic resistance gene or genes would not be capable of tolerating the antibiotic in culture.
- As used herein, a “chromatin modification domain” (CMD) refers to nucleotide sequences that interact with a variety of proteins associated with maintaining and/or altering chromatin structure, such as, but not limited to, DNA insulators. See Ciavatta et al., Proc. Nat'l Acad. Sci. U.S.A., 103:9958 (2006). Examples of CMDs include, but are not limited to, the chicken β-globulin insulator and the chicken hypersensitive site 4 (cHS4). The use of different CMD sequences between one or more gene programs (i.e., a promoter, coding sequence, and 3′ regulatory region), for example, can facilitate the use of the differential CMD DNA sequences as “mini homology arms” in combination with various microorganism or in vitro recombineering technologies to “swap” gene programs between existing multigenic and monogenic shuttle vectors. Other examples of chromatin modification domains are known in the art or can be readily identified.
- Polynucleotide and nucleic acid coding regions in the vector of the invention can be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of an immunomodulator, e.g., TNF-alpha. According to the signal hypothesis, proteins secreted by mammalian cells have a signal peptide or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Polypeptides secreted by vertebrate cells generally have a signal peptide fused to the N-terminus of the polypeptide, which is cleaved from the complete or “full length” polypeptide to produce a secreted or “mature” form of the polypeptide.
- In one embodiment, a vector of the invention comprises a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence which is operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins having the function of an immunomodulator operably linked to a promoter which is activated by said ligand-dependent transcription factor, wherein said polynucleotide encoding one or more proteins having the function of an immunomodulator further comprises a nucleic acid sequence encoding a signal peptide. In another embodiment, the signal peptide increase secretion of the immunomodulator, TNF-alpha, encoded by the vector compared to a vector comprising the immunomodulator's native signal peptide gene, e.g., TNF-alpha wild-type signal peptide gene. In particular, the signal peptide used in the invention can be codon-optimized. In a specific embodiment, the signal peptide is encoded by IL-2 wild-type signal peptide gene. In a further specific embodiment, the signal peptide is encoded by codon-optimized IL-2 signal peptide gene.
- The vector of the invention can comprise various regulatory regions, for example, 5′ untranslated region (5′UTR), 3′ UTR, or both. The present invention is also directed. to using various regulatory regions to induce improved secretion, protein translation, post-translation, mRNA transcription, or post-transcription process. As used herein, the “5′ untranslated region” or “5′UTR” of a gene is to be understood as that part of a gene which is transcribed into a primary RNA transcript (pre-mRNA) and which part is located upstream of the coding sequence. The primary transcript is the initial RNA product, containing introns and exons, produced by transcription of DNA. Many primary transcripts must undergo RNA processing to form the physiologically active RNA species. The processing into a mature mRNA may comprise trimming of the ends, removal of introns, capping and/or cutting out of individual rRNA molecules from their precursor RNAs. The 5′UTR of an mRNA is thus that part of the mRNA which is not translated into protein and which is located upstream of the coding sequence. In a genomic sequence, the 5′UTR is typically defined as the region between the transcription initiation site and the start codon. The 5′ untranslated regions (5′UTRs) of vertebrate mRNAs may be a few tens of bases to several hundred bases in length (Crowe et al., 2006 BMC Genomics 7:16). The 5′UTR used herein may occur naturally or be modified to contain one or more nucleic acid sequences not contiguous in nature (chimeric sequences), and/or may encompass substitutions, insertions, and deletions and combinations thereof. In one embodiment, the 5′UTR sequence is derived from the wild-type TNF-alpha sequence or 5U2 sequence. In another embodiment, the 5′UTR sequence is 5′UTR of 5U2. In some embodiments, the 5′UTR induces improved protein expression, e.g, mRNA transcription, pre-transcription, or post-transcription.
- The 3′ untranslated region (UTR) used in the invention refer to DNA sequences located downstream (3′) of a coding sequence and may comprise polyadenylation [poly(A)] recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. Any suitable polyadenylation sequence can be used, including a synthetic optimized sequence, as well as the polyadenylation sequence of BGH (Bovine Growth Hormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), and the papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus). In a particular embodiment, a 3′ regulatory region is the SV40e (human Sarcoma Virus-40) polyadenylation sequence. In another particular embodiment, a 3′ regulatory region is the polyadenylation sequence of human growth hormone.
- In certain embodiments, the signal peptide and/or the regulatory region alone or in combination can improve the protein secretion, transcription, or translation at least two fold, three fold, four fold, five fold, six fold, seven fold, eight fold, nine fold, 10 fold, 50 fold, 100 fold, 200 fold, 300 fold, 400 fold, or 500 fold compared to a control, which does not contain the signal peptide and/or the regulatory region. The secretion level of a protein, e.g., TNF-alpha, can be normalized to the protein expression encoded by a vector having a wild-type gene. In another specific embodiment of the present invention, the signal peptide and/or the regulatory region alone or in combination increase productivity of the immunomodulator, e.g., TNF-alpha, about 5% to about 10%, about 11% to about 20%, about 21% to about 30%, about 31% to about 40%, about 41% to about 50%, about 51% to about 60%, about 61% to about 70%, about 71% to about 80%, about 81% to about 90%, about 91% to about 100%, about 101% to about 149%, about 150% to about 199%, about 200% to about 299%, about 300% to about 499%, or about 500% to about 1000%. In a specific embodiment, the present invention comprises a vector conditionally expressing an immunomodulator, e.g. TNF-alpha, wherein said vector comprises 5′ UTR of 5U2, a codon-optimized nucleic acid sequence encoding IL-2 signal peptide, a codon-optimized coding region encoding an immunomodulator. TNF alpha, and a polyadenylation signal of SV40e or human growth hormone.
- In a further embodiment, the vector of the invention comprises a. polynucleotide sequence selected from the group consisting of SEQ ID NO: 47 (Vector 43318), SEQ ID NO: 48 (Vector 43319), SEQ ID NO: 49 (Vector 43320), SEQ ID NO: 50 (Vector 43321), SEQ ID NO: 51 (Vector 43322), SEQ ID NO: 52 (Vector 43323), SEQ ID NO: 53 (Vector 43324), SEQ ID NO: 54 (Vector 43325), SEQ ID NO: 55 (Vector 43326), SEQ ID NO: 56 (Vector 43327), SEQ ID NO: 57 (Vector 43328), and SEQ. ID NO: 58 (Vector 43329). In a still specific embodiment, the vector comprises a polynucleotide sequence of SEQ ID NO: 52 (vector 43323) or SEQ ID NO: 58 (vector 43329).
- Particular vectors for use with the invention are expression vectors that code for proteins or polynucleotides. Generally, such vectors comprise cis-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
- A great variety of expression vectors can be used to express proteins or polynucleotides. Such vectors include chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as adeno-associated viruses, lentiviruses, baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. All may be used for expression in accordance with this aspect of the invention. Generally, any vector suitable to maintain, propagate or express polynucleotides or proteins in a host may be used for expression in this regard.
- Suitable viral vectors used in the invention include, but not limited to, adenovirus-based vectors, retroviral vectors, herpes simplex virus (HSV)-based vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV)-based vectors, and AAV-adenoviral chimeric vectors. These viral vectors can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994).
- In one embodiment, a viral vector of the invention is an adenoviral vector.
- Adenovirus (Ad) is a 36 kb double-stranded DNA virus that efficiently transfers DNA in vivo to a variety of different target cell types. The adenoviral vector can be produced in high titers and can efficiently transfer DNA to replicating and non-replicating cells. The adenoviral vector genome can be generated using any species, strain, subtype, mixture of species, strains, or subtypes, or chimeric adenovirus as the source of vector DNA. Adenoviral stocks that can be employed as a source of adenovirus can be amplified from the
adenoviral serotypes 1 through 51, which are currently available from the American Type Culture Collection (ATCC, Manassas, Va.), or from any other serotype of adenovirus available from any other source. For instance, an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41). or any other adenoviral serotype. Given that the human adenovirus serotype 5 (Ad5) genome has been completely sequenced, the adenoviral vector of the invention is described herein with respect to the Ad5 serotype. The adenoviral vector can be any adenoviral vector capable of growth in a cell, which is in some significant part (although not necessarily substantially) derived from or based upon the genome of an adenovirus. The adenoviral vector can be based on the genome of any suitable wild-type adenovirus. In certain embodiments, the adenoviral vector is derived from the genome of a wild-type adenovirus of group C, especially ofserotype - In other embodiments, the adenoviral vector is replication-deficient. The term “replication-deficient” used herein means that the adenoviral vector comprises a genome that lacks at least one replication-essential gene function. A deficiency in a gene, gene function, or gene or genomic region, as used herein, is defined as a deletion of sufficient genetic material of the viral genome to impair or obliterate the function of the gene whose nucleic acid sequence was deleted in whole or in part. Replication-essential gene functions are those gene functions that are required for replication (i.e., propagation) of a replication-deficient adenoviral vector. Replication-essential gene functions are encoded by, for example, the adenoviral early regions (e.g., the E1, E2, and E4 regions), late regions (e.g., the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., VA-RNA I and/or VA-RNA II). In still other embodiments, the replication-deficient adenoviral vector comprises an adenoviral genome deficient in at least one replication-essential gene function of one or more regions of an adenoviral genome (e.g., two or more regions of an adenoviral genome so as to result in a multiply replication-deficient adenoviral vector). The one or more regions of the adenoviral genome are selected from the group consisting of the E1, E2, and E4 regions. The replication-deficient adenoviral vector can comprise a deficiency in at least one replication-essential gene function of the E1 region (denoted an E1-deficient adenoviral vector), particularly a deficiency in a replication-essential gene function of each of the adenoviral E1A region and the adenoviral E1B region. In addition to such a deficiency in the E1 region, the recombinant adenovirus also can have a mutation in the major late promoter (MLP), as discussed in International Patent Application WO 00/00628. In a particular embodiment, the vector is deficient in at least one replication-essential gene function of the E1 region and at least part of the nonessential E3 region (e.g., an Xba I deletion of the E3 region) (denoted an E1/E3-deficient adenoviral vector).
- In certain embodiments, the adenoviral vector is “multiply deficient,” meaning that the adenoviral vector is deficient in one or more gene functions required for viral replication in each of two or more regions of the adenoviral genome. For example, the aforementioned E1-deficient or E1/E3-deficient adenoviral vector can be further deficient in at least one replication-essential gene function of the E4 region (denoted an E1/E4-deficient adenoviral vector). An adenoviral vector deleted of the entire E4 region can elicit a lower host immune response.
- Alternatively, the adenoviral vector lacks replication-essential gene functions in all or part of the E1 region and all or part of the E2 region (denoted an E1/E2-deficient adenoviral vector). Adenoviral vectors lacking replication-essential gene functions in all or part of the E1 region, all or part of the E2 region, and all or part of the E3 region also are contemplated herein. If the adenoviral vector of the invention is deficient in a replication-essential gene function of the E2A region, the vector does not comprise a complete deletion of the E2A region, which is less than about 230 base pairs in length. Generally, the E2A region of the adenovirus codes for a DBP (DNA binding protein), a polypeptide required for DNA replication. DBP is composed of 473 to 529 amino acids depending on the viral serotype. It is believed that DBP is an asymmetric protein that exists as a prolate ellipsoid consisting of a globular Ct with an extended Nt domain. Studies indicate that the Ct domain is responsible for DBP's ability to bind to nucleic acids, bind to zinc, and function in DNA synthesis at the level of DNA chain elongation. However, the Nt domain is believed to function in late gene expression at both transcriptional and post-transcriptional levels, is responsible for efficient nuclear localization of the protein, and also may be involved in enhancement of its own expression. Deletions in the Nt domain between
amino acids 2 to 38 have indicated that this region is important for DBP function (Brough et al., Virology, 196, 269-281 (1993)). While deletions in the E2A region coding for the Ct region of the DBP have no effect on viral replication, deletions in the E2A region which code foramino acids 2 to 38 of the Nt domain of the DBP impair viral replication. In one embodiment, the multiply replication-deficient adenoviral vector contains this portion of the E2A region of the adenoviral genome. In particular, for example, the desired portion of the E2A region to be retained is that portion of the E2A region of the adenoviral genome which is defined by the 5′ end of the E2A region, specifically positions Ad5(23816) to Ad5(24032) of the E2A region of the adenoviral genome of serotype Ad5. - The adenoviral vector can be deficient in replication-essential gene functions of only the early regions of the adenoviral genome, only the late regions of the adenoviral genome, and both the early and late regions of the adenoviral genome. The adenoviral vector also can have essentially the entire adenoviral genome removed, in which case at least either the viral inverted terminal repeats (ITRs) and one or more promoters or the viral ITRs and a packaging signal are left intact (i.e., an adenoviral amplicon). The larger the region of the adenoviral genome that is removed, the larger the piece of exogenous nucleic acid sequence that can be inserted into the genome. For example, given that the adenoviral genome is 36 kb, by leaving the viral ITRs and one or more promoters intact, the exogenous insert capacity of the adenovirus is approximately 35 kb. Alternatively, a multiply deficient adenoviral vector that contains only an ITR and a packaging signal effectively allows insertion of an exogenous nucleic acid sequence of approximately 37-38 kb. Of course, the inclusion of a spacer element in any or all of the deficient adenoviral regions will decrease the capacity of the adenoviral vector for large inserts. Suitable replication-deficient adenoviral vectors, including multiply deficient adenoviral vectors, are disclosed in U.S. Pat. Nos. 5,851,806 and 5,994,106 and International Patent Applications WO 95/34671 and WO 97/21826. In one embodiment, the vector for use in the present inventive method is that described in International Patent Application PCT/US01/20536.
- It should be appreciated that the deletion of different regions of the adenoviral vector can alter the immune response of the mammal. In particular, the deletion of different regions can reduce the inflammatory response generated by the adenoviral vector. Furthermore, the adenoviral vector's coat protein can be modified so as to decrease the adenoviral vector's ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in International Patent Application WO 98/40509.
- The adenoviral vector, when multiply replication-deficient, especially in replication-essential gene functions of the E1 and E4 regions, can include a spacer element to provide viral growth in a complementing cell line similar to that achieved by singly replication deficient adenoviral vectors, particularly an adenoviral vector comprising a deficiency in the E1 region. The spacer element can contain any sequence or sequences which are of the desired length. The spacer element sequence can be coding or non-coding and native or non-native with respect to the adenoviral genome, but does not restore the replication-essential function to the deficient region. In the absence of a spacer, production of fiber protein and/or viral growth of the multiply replication-deficient adenoviral vector is reduced by comparison to that of a singly replication-deficient adenoviral vector. However, inclusion of the spacer in at least one of the deficient adenoviral regions, preferably the E4 region, can counteract this decrease in fiber protein production and viral growth. The use of a spacer in an adenoviral vector is described in U.S. Pat. No. 5,851,806.
- Construction of adenoviral vectors is well understood in the art. Adenoviral vectors can be constructed and/or purified using the methods set forth, for example, in U.S. Pat. No. 5,965,358 and International Patent Applications WO 98/56937, WO 99/15686, and WO 99/54441. The production of adenoviral gene transfer vectors is well known in the art, and involves using standard molecular biological techniques such as those described in, for example, Sambrook et al., supra, Watson et al., supra, Ausubel et al., supra, and in several of the other references mentioned herein.
- Replication-deficient adenoviral vectors are typically produced in complementing cell lines that provide gene functions not present in the replication-deficient adenoviral vectors, but required for viral propagation, at appropriate levels in order to generate high titers of viral vector stock. In one embodiment, a cell line complements for at least one and/or all replication-essential gene functions not present in a replication-deficient adenovirus. The complementing cell line can complement for a deficiency in at least one replication-essential gene function encoded by the early regions, late regions, viral packaging regions, virus-associated RNA regions, or combinations thereof, including all adenoviral functions (e.g., to enable propagation of adenoviral amplicons, which comprise minimal adenoviral sequences, such as only inverted terminal repeats (ITRs) and the packaging signal or only ITRs and an adenoviral promoter). In another embodiment, the complementing cell line complements for a deficiency in at least one replication-essential gene function (e.g., two or more replication-essential gene functions) of the E1 region of the adenoviral genome, particularly a deficiency in a replication-essential gene function of each of the E 1A and E1B regions. In addition, the complementing cell line can complement for a deficiency in at least one replication-essential gene function of the E2 (particularly as concerns the adenoviral DNA polymerase and terminal protein) and/or E4 regions of the adenoviral genome. Desirably, a cell that complements for a deficiency in the E4 region comprises the E4-ORF6 gene sequence and produces the E4-ORF6 protein. Such a cell desirably comprises at least ORF6 and no other ORF of the E4 region of the adenoviral genome. The cell line preferably is further characterized in that it contains the complementing genes in a non-overlapping fashion with the adenoviral vector, which minimizes, and practically eliminates, the possibility of the vector genome recombining with the cellular DNA. Accordingly, the presence of replication competent adenoviruses (RCA) is minimized if not avoided in the vector stock, which, therefore, is suitable for certain therapeutic purposes, especially gene therapy purposes. The lack of RCA in the vector stock avoids the replication of the adenoviral vector in non-complementing cells. The construction of complementing cell lines involves standard molecular biology and cell culture techniques, such as those described by Sambrook et al., supra, and Ausubel et al., supra. Complementing cell lines for producing the gene transfer vector (e.g., adenoviral vector) include, but are not limited to, 293 cells (described in, e.g., Graham et al., J. Gen. Virol., 36, 59-72 (1977)), PER.C6 cells (described in, e.g., International Patent Application WO 97/00326, and U.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (described in, e.g., International Patent Application WO 95/34671 and Brough et al., J. Virol., 71, 9206-9213 (1997)). The insertion of a nucleic acid sequence into the adenoviral genome (e.g., the E1 region of the adenoviral genome) can be facilitated by known methods, for example, by the introduction of a unique restriction site at a given position of the adenoviral genome.
- Retrovirus is an RNA virus capable of infecting a wide variety of host cells. Upon infection, the retroviral genome integrates into the genome of its host cell and is replicated along with host cell DNA, thereby constantly producing viral RNA and any nucleic acid sequence incorporated into the retroviral genome. As such, long-term expression of a therapeutic factor(s) is achievable when using retrovirus. Retroviruses contemplated for use in gene therapy are relatively non-pathogenic, although pathogenic retroviruses exist. When employing pathogenic retroviruses, e.g., human immunodeficiency virus (HIV) or human T-cell lymphotrophic viruses (HTLV), care must be taken in altering the viral genome to eliminate toxicity to the host. A retroviral vector additionally can be manipulated to render the virus replication-deficient. As such, retroviral vectors are considered particularly useful for stable gene transfer in vivo. Lentiviral vectors, such as HIV-based vectors, are exemplary of retroviral vectors used for gene delivery. Unlike other retroviruses, HIV-based vectors are known to incorporate their passenger genes into non-dividing cells and, therefore, can be of use in treating persistent forms of disease.
- An HSV-based viral vector is suitable for use as a gene transfer vector to introduce a nucleic acid into numerous cell types. The mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a linear double-stranded DNA molecule that is 152 kb. Most replication-deficient HSV vectors contain a deletion to remove one or more intermediate-early genes to prevent replication. Advantages of the HSV vector are its ability to enter a latent stage that can result in long-term DNA expression and its large viral DNA genome that can accommodate exogenous DNA inserts of up to 25 kb. Of course, the ability of HSV to promote long-term production of exogenous protein is potentially disadvantageous in terms of short-term treatment regimens. However, one of ordinary skill in the art has the requisite understanding to determine the appropriate vector for a particular situation. HSV-based vectors are described in, for example, U.S. Pat. Nos. 5,837,532, 5,846,782, 5,849,572, and 5,804,413, and International Patent Applications WO 91/02788, WO 96/04394, WO 98/15637, and WO 99/06583.
- AAV vectors are viral vectors of particular interest for use in gene therapy protocols. AAV is a DNA virus, which is not known to cause human disease. The AAV genome is comprised of two genes, rep and cap, flanked by inverted terminal repeats (ITRs), which contain recognition signals for DNA replication and packaging of the virus. AAV requires co-infection with a helper virus (i.e., an adenovirus or a herpes simplex virus), or expression of helper genes, for efficient replication. AAV can be propagated in a wide array of host cells including human, simian, and rodent cells, depending on the helper virus employed. An AAV vector used for administration of a nucleic acid sequence typically has approximately 96% of the parental genome deleted, such that only the ITRs remain. This eliminates immunologic or toxic side effects due to expression of viral genes. If desired, the AAV rep protein can be co-administered with the AAV vector to enable integration of the AAV vector into the host cell genome. Host cells comprising an integrated AAV genome show no change in cell growth or morphology (see, e.g., U.S. Pat. No. 4,797,368). As such, prolonged expression of therapeutic factors from AAV vectors can be useful in treating persistent and chronic diseases.
- The polynucleotide sequence in the expression vector is operatively linked to appropriate expression control sequence(s) including, for instance, a promoter to direct mRNA transcription. Representatives of additional promoters include, but are not limited to, constitutive promoters and tissue specific or inducible promoters. Examples of constitutive eukaryotic promoters include, but are not limited to, the promoter of the mouse metallothionein I gene (Hamer et al., J. Mol. Appl. Gen. 1:273 (1982)); the TK promoter of Herpes virus (McKnight, Cell 31:355 (1982)); the SV40 early promoter (Benoist et al., Nature 290:304 (1981)); and the vaccinia virus promoter. Additional examples of the promoters that could be used to drive expression of a protein or polynucleotide include, but are not limited to, tissue-specific promoters and other endogenous promoters for specific proteins, such as the albumin promoter (hepatocytes), a proinsulin promoter (pancreatic beta cells) and the like. In general, expression constructs will contain sites for transcription, initiation and termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs may include a translation initiating AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
- In addition, the constructs may contain control regions that regulate, as well as engender expression. Generally, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.
- Examples of eukaryotic vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, and pCMV-EGFP available from Clontech. Many other vectors are well-known and commercially available.
- Particularly usefal vectors, which comprise molecular insertion pivots for rapid insertion and removal of elements of gene programs, are described in United States Published Patent Application No. 2004/0185556, U.S. patent application Ser. No. 11/233,246 and International Published Application Nos. WO 2005/040336 and WO 2005/116231. An example of such vectors is the ULTRAVECTOR® Production System (Intrexon Corp., Blacksburg, Va.), as described in WO 2007/038276. As used herein, a “gene program” is a combination of genetic elements comprising a promoter (P), an expression sequence (E) and a 3′ regulatory sequence (3), such that “PE3” is a gene program. The elements within the gene program can be easily swapped between molecular pivots that flank each of the elements of the gene program. A molecular pivot, as used herein, is defined as a polynucleotide comprising at least two non-variable rare or uncommon restriction sites arranged in a linear fashion. In one embodiment, the molecular pivot comprises at least three non-variable rare or uncommon restriction sites arranged in a linear fashion. Typically any one molecular pivot would not include a rare or uncommon restriction site of any other molecular pivot within the same gene program. Cognate sequences of greater than 6 nucleotides upon which a given restriction enzyme acts are referred to as “rare” restriction sites. There are, however, restriction sites of 6 bp that occur more infrequently than would be statistically predicted, and these sites and the endonucleases that cleave them are referred to as “uncommon” restriction sites. Examples of either rare or uncommon restriction enzymes include, but are not limited to, AsiS I, Pac I, Sbf I, Fse I, Asc I, Mlu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I, Sfo I, Sgr AI, AflIII, Pvu I, Ngo MIV, Ase I, Flp I, Pme I, Sda I, Sgf I, Srf I, Nru I, Acl I, Cla I, Csp45 I, Age I, Bst1107 I, BstB I, Hpa I, Aat II, EcoR V, Nhe I, Spe I, Avi II, Avr II, Mfe I, Afe I, Fsp I, Kpn I, Sca I, BspE I, Nde I, Bfr I, Xno I, Pml I, ApaL I, Kas I, Xma I, BsrB I, Nsi I, Sac II, Sac I, Blp I, PspoM I, Pci I, Stu I, Sph I, BamH I, Bsu36 I, Xba I, BbvC I, Bgl II, Nco I, Hind III, EcoR I, BsrG I and Sse8781 I.
- The vector may also comprise restriction sites for a second class of restriction enzymes called homing endonuclease (HE) enzymes. HE enzymes have large, asymmetric restriction sites (12-40 base pairs), and their restriction sites are infrequent in nature. For example, the HE known as I-SceI has an 18 bp restriction site (5′TAGGGATAACAGGGTAAT3′ (SEQ ID NO: 28)), predicted to occur only once in every 7×1010 base pairs of random sequence. This rate of occurrence is equivalent to only one site in a genome that is 20 times the size of a mammalian genome. The rare nature of HE sites greatly increases the likelihood that a genetic engineer can cut a gene program without disrupting the integrity of the gene program if HE sites are included in appropriate locations in a cloning vector plasmid.
- Selection of appropriate vectors and promoters for expression in a host cell is a well-known procedure, and the requisite techniques for vector construction and introduction into the host, as well as its expression in the host are routine skills in the art.
- The introduction of the polynucleotides into the cells can be a transient transfection, stable transfection, or can be a locus-specific insertion of the vector. Transient and stable transfection of the vectors into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986); Keown et al., 1990, Methods Enzymol. 185: 527-37; Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y. These stable transfection methods result in random insertion of the vector into the genome of the cell. Further, the copy number and orientation of the vectors are also, generally speaking, random.
- In one embodiment of the invention, the vector is inserted into a bio-neutral site in the genome. A bio-neutral site is a site in the genome where insertion of the polynucleotides interferes very little, if any, with the normal function of the cell. Bio-neutral sites may be analyzed using available bioinformatics. Many bio-neutral sites are known in the art, e.g., the ROSA-equivalent locus. Other bio-neutral sites may be identified using routine techniques well known in the art. Characterization of the genomic insertion site(s) is performed using methods known in the art. To control the location, copy number and/or orientation of the polynucleotides when introducing the vector into the cells, methods of locus-specific insertion may be used. Methods of locus-specific insertion are well-known in the art and include, but are not limited to, homologous recombination and recombinase-mediated genome insertion. Of course, if locus-specific insertion methods are to be used in the methods of the invention, the vectors may comprise elements that aid in this locus-specific insertion, such as, but not limited to, homologous recombination. For example, the vectors may comprise one, two, three, four or more genomic integration sites (GISs). As used herein, a “genomic integration site” is defined as a portion of the vector sequence which nucleotide sequence is identical or nearly identical to portions of the genome within the cells that allows for insertion of the vector in the genome. In particular, the vector may comprise two genomic insertion sites that flank at least the polynucleotides. Of course, the GISs may flank additional elements, or even all elements present on the vector.
- In another embodiment, locus-specific insertion may be carried out by recombinase-site specific gene insertion. Briefly, bacterial recombinase enzymes, such as, but not limited to, PhiC31 integrase can act on “pseudo” recombination sites within the human genome. These pseudo recombination sites can be targets for locus-specific insertion using the recombinases. Recombinase-site specific gene insertion is described in Thyagarajan et al., Mol. Cell. Biol. 21:3926 (2001). Other examples of recombinases and their respective sites that may be used for recombinase-site specific gene insertion include, but are not limited to, serine recombinases such as R4 and TP901-1 and recombinases described in WO 2006/083253.
- In a further embodiment, the vector may comprise a chemo-resistance gene, e.g., the multidrug resistance gene mdr1, dihydrofolate reductase, or O6-alkylguanine-DNA alkyltransferase. The chemo-resistance gene may be under the control of a constitutive (e.g., CMV) or inducible (e.g., RheoSwitch®) promoter. In this embodiment, if it is desired to treat a disease in a subject while maintaining the modified cells within the subject, a clinician may apply a chemotherapeutic agent to destroy diseased cells while the modified cells would be protected from the agent due to expression of a suitable chemo-resistance gene and may continue to be used for treatment, amelioration, or prevention of a disease or disorder. By placing the chemo-resistance gene under an inducible promoter, the unnecessary expression of the chemo-resistance gene can be avoided, yet it will still be available in case continued treatment is needed. If the modified cells themselves become diseased, they could still be destroyed by inducing expression of a lethal polypeptide as described below.
- The methods of the invention are carried out by introducing the polynucleotides encoding the gene switch and the exogenous gene into cells of a subject. Any method known for introducing a polynucleotide into a cell known in the art, such as those described above, can be used.
- When the polynucleotides are to be introduced into cells ex vivo, the cells may be obtained from a subject by any technique known in the art, including, but not limited to, biopsies, scrapings, and surgical tissue removal. The isolated cells may be cultured for a sufficient amount of time to allow the polynucleotides to be introduced into the cells, e.g., 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, hours or more. Methods for culturing primary cells for short periods of time are well known in the art. For example, cells may be cultured in plates (e.g., in microwell plates) either attached or in suspension.
- For ex vivo therapeutic methods, cells are isolated from a subject and cultured under conditions suitable for introducing the polynucleotides into the cells. Once the polynucleotides have been introduced into the cells, the cells are incubated for a sufficient period of time to allow the ligand-dependent transcription factor to be expressed, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours or more. At some point after the introduction of the polynucleotides into the cells (either before or after significant levels of the ligand-dependent transcription factor is expressed), the cells are introduced back into the subject. Reintroduction may be carried out by any method known in the art, e.g., intravenous infusion or direct injection into a tissue or cavity. In one embodiment, the presence of the polynucleotides in the cells is determined prior to introducing the cells back into the subject. In another embodiment, cells containing the polynucleotides are selected (e.g., based on the presence of a selectable marker in the polynucleotides) and only those cells containing the polynucleotides are reintroduced into the subject. After the cells are reintroduced to the subject, ligand is administered to the subject to induce expression of the therapeutic polypeptide or therapeutic polynucleotide. In an alternative embodiment, the ligand may be added to the cells even before the cells are reintroduced to the subject such that the therapeutic polypeptide or therapeutic polynucleotide is expressed prior to reintroduction of the cells. The ligand may be administered by any suitable method, either systemically (e.g., orally, intravenously) or locally intraperitoneally, intrathecally, intraventricularly, direct injection into the tissue or organ where the cells are reintroduced). The optimal timing of ligand administration can be determined for each type of cell and disease or disorder using only routine techniques.
- The in vivo therapeutic methods of the invention involve direct in vivo introduction of the polynucleotides, e.g., adenoviral vector, into the cells of the subject. The polynucleotides may be introduced into the subject systemically or locally (e.g., at the site of the disease or disorder). Once the polynucleotides have been introduced to the subject, the ligand may be administered to induce expression of the therapeutic polypeptide or therapeutic polynucleotide. The ligand may be administered by any suitable method, either systemically (e.g., orally, intravenously) or locally (e.g., intraperitoneally, intrathecally, intraventricularly, direct injection into the tissue or organ where the disease or disorder is occurring). The optimal timing of ligand administration can be determined for each type of cell and disease or disorder using only routine techniques.
- For in vivo use, the ligands described herein may be taken up in pharmaceutically acceptable carriers, such as, for example, solutions, suspensions, tablets, capsules, ointments, elixirs, and injectable compositions. Pharmaceutical compositions may contain from 0.01% to 99% by weight of the ligand. Compositions may be either in single or multiple dose forms. The amount of ligand in any particular pharmaceutical composition will depend upon the effective dose, that is, the dose required to elicit the desired gene expression or suppression.
- Suitable routes of administering the pharmaceutical preparations include oral, rectal, topical (including dermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, intratumoral, intradermal, intrathecal and epidural), intravitreal, and by naso-gastric tube. It will be understood by those skilled in the art that the route of administration will depend upon the condition being treated and may vary with factors such as the condition of the recipient.
- As used herein, the term “rAD.RheoIL12” refers to an adenoviral polynucleotide vector harboring the IL-12 gene under the control of a gene switch of the RheoSwitch® Therapeutic System (RTS), which is capable of producing IL-12 protein in the presence of activating ligand. As used herein, the term “rAd.cIL12” refers to an adenoviral polynucleotide control vector containing the IL-12 gene under the control of a constitutive promoter.
- As used herein, the term “IL-12p70” refers to IL-12 protein, which naturally has two subunits commonly referred to as p40 and p35. The term IL-12p70 encompasses fusion proteins comprising the two subunits of IL-12 (p40 and p35), wherein the fusion protein may include linker amino acids between subunits.
- As used herein, the term “a protein having the function of an immunomodulator” refers to a protein that has at least 20% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80% or 90%) of any bioactivity of an immunomodulator selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10R or a subunit thereof DN, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, CCL3 (MIP-1a), CCLS (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12 (SDF-1), CCL21 (6Ckine), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1, F1t3L, IFN-beta, TNF-alpha, dnFADD, BCG, TGF-alpha, PD-L1, TGFbRII DN, ICOS-L and S100. Likewise, the term “a protein having the function of IL-12” refers to a protein that has at least 20% (e.g., at least 30%, 40%, 50%, 60%, 70%, 80% or 90%) of any bioactivity of human IL-12. The bioactivities of such immunomodulators are well known. See the following Table.
-
TABLE 6 Immunomodulators and their functions Immunomodulator Function Cytokines Interleukin-1 (IL-1) IL-1 is a cytokine produced by activated macrophages. IL-1 stimulates thymocyte proliferation by inducing IL-2 release, B- cell maturation and proliferation, and fibroblast growth factor activity. IL-1 proteins are involved in the inflammatory response. Interleukin-2 (IL-2) IL-2 is a family of cytokines, that is produced by T-cells in response to antigenic or mitogenic stimulation, this protein is required for T-cell proliferation and other activities crucial to regulation of the immune response. IL-2 can stimulate B-cells, monocytes, lymphokine-activated killer cells, natural killer cells, and glioma cells. Interleukin-3 (IL-3) IL-3 stimulates the proliferation of hematopoietic pluripotent progenitor cells. It is secreted by activated T cells to support growth and differentiation of T cells from the bone marrow in an immune response. The combined intratumoral Ad-mIL-3 gene therapy in combination with radiation therapy was shown to significantly suppress tumor growth (Oh 2004). Interleukin-4 (IL-4) IL-4 is a cytokine that participates in at least several B-cell activation processes as well as of other cell types. It is a costimulator of DNA-synthesis. It induces the expression of class II MHC molecules on resting B-cells. It enhances both secretion and cell surface expression of IgE and IgG1. It also regulates the expression of the low affinity Fc receptor for IgE (CD23) on both lymphocytes and monocytes. Interleukin-5 (IL-5) IL-5 stimulates B cell growth and increase immunoglobulin secretion and induce tumor suppression (Nakashima 1993, Wu 1992). Interleukin-7 (IL-7) IL-7 is a cytokine that is a hematopoietic growth factor capable of stimulating the proliferation of lymphoid progenitors. It is important for proliferation during certain stages of B-cell maturation. Interleukin-9 (IL-9) IL-9 supports IL-2 independent and IL-4 independent growth of helper T-cells. Interleukin-15 (IL-15) IL-15 is a cytokine that stimulates the proliferation of T- lymphocytes. Stimulation by IL-15 requires interaction of IL-15 with components of IL-2R, including IL-2R beta and probably IL-2R gamma but not IL-2R alpha. Interleukin-18 (IL-18) IL-18 augments natural killer cell activity in spleen cells and stimulates interferon gamma production in T-helper type I cells. Interleukin-21 (IL-21) IL-21 is a cytokine with immunoregulatory activity. IL-21 may promote the transition between innate and adaptive immunity. Interleukin-23 (IL-23) IL-23 acts directly on DC to promote immunogenic presentation of tumor peptide and can I resulted in robust intratumoral CD8(+) and CD4(+) T-cell infiltration and induced a specific TH1-type response to the tumor in regional lymph nodes and spleen. (Hu 2006). Interleukin-27 (IL-27) IL-27 is a cytokine with pro- and anti-inflammatory properties, that can regulate T helper cell development, suppress T-cell proliferation, stimulate cytotoxic T cell activity, induce isotype switching in B-cells, and that has diverse effects on innate immune cells. Intereukin-24 (IL-23) IL-24 has been shown to suppress tumor growth (Susan 2004, Fisher 2003). INF-alpha (IFNα) IFN-alpha has anti-tumor function (Taqliaferri 2005). Interferon beta 1 (IFNB1) IFNB1 is a member of group of interferon proteins that bind to specific cell surface receptors (IFNAR), and stimulates both macrophages and natural killer (NK) cells to elicit an antiviral, antibacterial and anticancer activities. Interferon gamma (IFN- IFN-gamma is produced by lymphocytes activated by specific gamma) antigens or mitogens. IFN-gamma, in addition to having antiviral activity, has important immunoregulatory functions. It is a potent activator of macrophages, it has antiproliferative effects on transformed cells and it can potentiate the antiviral and antitumor effects of the type I interferons. Tumor necrosis factor (TNF- TNF-α is mainly secreted by macrophages and can induce cell alpha) death of certain tumor cell lines. It is a potent pyrogen, causing fever by direct action or by stimulation of interleukin-1 secretion. Chemokines Chemokine (C motif) ligand Chemokine (C motif) ligand 1 (XCL1, also known as 1 (XCL1) Lymphotactin) is chemotactic for CD4+ and CD8+ T cells but not for monocytes, and induces a rise in intracellular calcium in peripheral blood lymphocytes. The combination of XCL1 with IL-2 and IL-12 can enhance immunotherapy and augment the antitumor response (Emtage 1999, Wang 2002). CC chemokine ligand 3CC chemokine ligand 3 (CCL3), also known as macrophage (CCL3) inflammatory protein-1 (MIP-1), which is a so-called monokine (a type of cytokine produced primarily by monocytes and macrophages) that is involved in the acute inflammatory state in the recruitment and activation of polymorphonuclear leukocytes. CCL5 (RANTES) CCL5 (RANTES), is a chemoattractant for blood monocytes, memory T-helper cells and eosinophils. Causes the release of histamine from basophils and activates eosinophils. Binds to CCR1, CCR3, CCR4 and CCR5. One of the major HIV- suppressive factors produced by CD8+ T-cells. CC chemokine ligand 7CCL7 is a chemotactic factor that attracts monocytes and (CCL7) eosinophils, but not neutrophils. CCL7 also augments monocyte anti-tumor activity. Also induces the release of gelatinase B. Chemokine (CXC motif) CXCL9 is a cytokine that affects the growth, movement, or ligand 9 (CXCL9) activation state of cells that participate in immune and inflammatory response. Chemotactic for activated T-cells. Chemokine (C—X—C motif) Chemokine (C—X—C motif) ligand 10 (CXCL10) is a small ligand 10 (CXCL10) cytokine with roles in chemoattraction for cells in the immune system, adhesion of T cells to endothelial cells, anti-tumor activity and angiogenesis. Chemokine (C—X—C motif) Chemokine (C—X—C motif) ligand 12 (CXCL12), also known as ligand 12 (CXCL12) stormal cell-derived factor 1 (SDF-1), is a small cytokine that belong to the intercrine family, members of which activate leukocytes and are often induced by proinflammatory stimuli such as LPS, TNF or IL1. Chemokine (C-C motif) CCR7 is the receptor for the MIP-3-beta chemokine. Probable receptor 7 (CCR7) mediator of EBV effects on B-lymphocytes or of normal lymphocyte functions. Chemokine (C-C motif) CCL19 plays a role not only in inflammatory and immunological ligand 19 (CCL19, also responses but also in normal lymphocyte recirculation and known as MIP-3β) homing. CCL19 has an important role in trafficking of T-cells in thymus, and T-cell and B-cell migration to secondary lymphoid organs. It specifically binds to chemokine receptor CCR7. CC chemokine ligand 21CCL21 inhibits hemopoiesis and stimulates chemotaxis. CCL21 (CCL21) is chemotactic in vitro for thymocytes and activated T-cells, but not for B-cells, macrophages, or neutrophils. Interleukin-8 (IL-8) IL-8 is a chemotactic factor that attracts neutrophils, basophils, and T-cells, but not monocytes. It is also involved in neutrophil activation. It is released from several cell types in response to an inflammatory stimulus. Growth Factors Granulocyte/macrophage GM-CSF is a cytokine that stimulates the growth and colony-stimulating factor differentiation of hematopoietic precursor cells from various (GM-CSF) lineages, including granulocytes, macrophages, eosinophils and erythrocytes. FMS-related tyrosine kinase FMS-related tyrosine kinase ligand (FLT3/FLK2 ligand, Flt3L), ligand (FLT3/FLK2 ligand, which may function as a growth factor receptor on hematopoietic Flt3L) stem cells or progenitor cells or both. TGFA TGF alpha is a mitogenic polypeptide that is able to bind to the EGF receptor and to act synergistically with TGF beta to promote anchorage-independent cell proliferation in soft agar. Adjuvants Beta-defensin Beta-defensins are antimicrobial peptides implicated in innate immune response against many Gram-negative and Gram- positive bacteria, fungi and viruses. High-mobility group box-1 High-mobility group box-1 (HMGB1) proteins are nonhistone (HMGB1) chromosomal proteins that function as cytokines, mediating local and systemic responses to necrotic cell death and cancer, invasion by pathogens, trauma, and sepsis. S100 Phagocytic S100 proteins mediate inflammatory responses and recruit inflammatory cells to sites of tissue damage, and are members of Damage-associated molecular pattern (DAMP) molecules that are important for innate immunity. Mannan Mannan, a plant polysaccharide, that is a polymer of the sugar mannose, is useful for generation of a immune response. Bacille Calmette-Guerin Bacille Calmette-Guerin (BCG), live attenuated Mycobacterium (BCG) species, are used as vaccine against to prevent severe and fatal tuberculosis. Bacterial lipopolysaccharides Bacterial lipopolysaccharides (LPS) are endotoxins that induces (LPS) a strong immune response upon infection with Gram-negative bacteria. Co-stimulatory Molecule (Positive) OX40 ligand OX40 ligand (OX40L) belongs to tumor necrosis factor (ligand) superfamily member 4 (Tnfsf4), is expressed on dendritic cells and promotes Th2 cell differentiation. 4-1BB ligand (4-1BBL) 4-1BB ligand (4-1BBL) belongs to tumor necrosis factor (ligand) superfamily member 9 (Tnfsf9), which is a type 2transmembrane glycoprotein and is expressed on activated T lymphocytes. 4-1BBL induces the proliferation of activated peripheral blood T-cells, and has a role in activation-induced cell death (AICD). CD40 The CD40 protein belongs to the tumor necrosis factor receptor superfamily member 5, is essential in mediating a broad variety of immune and inflammatory responses including T cell- dependent immunoglobulin class switching, memory B cell development, and germinal center formation. Glucocorticoid-induced GITR can evoke effective tumor immunity via T cell stimulation. tumor necrosis factor receptor Administration of anti-GITR monoclonal antibody (mAb) can family-related protein (GITR) provoke potent tumor-specific immunity and eradicated established tumors without eliciting overt autoimmune disease. GITR Ligand (GITRL) GITRL is the ligand for GITR. CD70 CD70 is a cytokine that binds to CD27. It plays a role in T-cell activation. Induces the proliferation of costimulated T-cells and enhances the generation of cytolytic T-cells. LIGHT (HSVgD) Herpes virus entry mediator (HVEM) binding ligand (HSVgD), also referred to as p30, or LIGHT is a TNF family member involved in co-stimulation of T cells. PD-L1 (also known as PD-L1 (also known as CD274) protein is expressed in activated CD274) monocytes, T and B cells. PD-L1 is upregulated in monocytes upon treatment with IFN-gamma, and in dendritic cells and keratinocytes upon treatment with IFN-gamma, together with other activators. ICOS-L ICOS-L is a ligand for the T-cell-specific cell surface receptor ICOS and acts as a costimulatory signal for T-cell proliferation and cytokine secretion; induces also B-cell proliferation and differentiation into plasma cells. Co-stimulatory Molecule (Negative) Anti-CTLA4 Cytotoxic T lymphocyte-associated 4 (CTLA4) is a member of the immunoglobulin superfamily and is a costimulatory molecule expressed in activated T cells. Anti-PD-L1 Binding of a PD-1 receptor on a T-cell by PD-L1 transmits a negative costimulatory signal to the cell, which prevents the cells to progress through the cell cycle, and increases T cell proliferation. Inhibition of an interaction between PD-L1 and receptor on the T cell with an anti-PD-L1 antibody results in the downregulation of the immune response termed as immune cell anergy. Anti-PD-L2 PD-L2 is involved in the costimulatory signal, essential for T lymphocyte proliferation and IFN-gamma production in a PDCD1-independent manner, but the ligand is known to primarily act through PD-1 resulting in anergic responses. Counter Immune Suppressant (Tolerance Inhibitors) TGFR2DN On ligand binding, TGFR2 forms a receptor complex consisting of two type II and two type I transmembrane serine/threonine kinases. Type II receptors phosphorylate and activate type I receptors which autophosphorylate, then bind and activate SMAD transcriptional regulators. Receptor for TGF-beta. Deletion of predicted serine/theronine kinase cytoplasmic domain (nucleotides 1172-2036 of TGFβR2 cDNA H2-3FF, available from public databases as accession number M85079 and amino acid sequence available as accession number AAA61164) impairs the all three TGF-β (1, 2 and 3) dependent gene expressions. Anti-TGFβ TGFβ is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. TGFβ acts synergistically with TGFα in inducing transformation. It also acts as a negative autocrine growth factor. Dysregulation of TGFβ activation and signaling may result in apoptosis. administration of anti-TGFβ antibody can prevent renal insufficiency and glomerulosclerosis in the db/db mouse, a model of type II diabetes that develops overt nephropathy. Anti-IL10 IL-10 is a cytokine produced by activated Th2 cells, B cells, keratinocytes, monocytes, and macrophages. IL-10 is useful in promoting growth and differentiation of activated human B cells, inhibiting Th1 responses to prevent transplant rejection and T cell-mediated autoimmune diseases. Anti-Suppressor of cytokine Suppressor of cytokine signaling1 (SOCS1) is a critical inhibitor signaling1 (SOCS1) of interferon-gamma signaling and prevents the potentially fatal neonatal actions of this cytokine. Anti-TGF-α TGF-α is a mitogenic polypeptide that is able to bind to the EGF receptor and to act synergistically with TGF-β to promote anchorage-independent cell proliferation in soft agar. Fas contain cytoplasmic Fas- FADD is essential for Fas and TNF-induced signaling for associated protein with death programmed cell death (apoptosis) and receptor oligomerization. domain (FADD) - The bioactivities of IL-12 are also well known and include, without limitation, differentiation of naive T cells into Th1 cells, stimulation of the growth and function of T cells, production of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-α) from T and natural killer (NK) cells, reduction of IL-4 mediated suppression of IFN-gamma, enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes, stimulation of the expression of IL-12R-β1 and IL-12R-β2, facilitation of the presentation of tumor antigens through the upregulation of MHC I and II molecules, and anti-angiogenic activity. The term “a protein having the function of IL-12” encompasses mutants of a wild type IL-12 sequence, wherein the wild type sequence has been altering by one or more of addition, deletion, or substitution of amino acids, as well as non-IL-12 proteins that mimic one or more of the bioactivities of IL-12.
- As used herein, the terms “activating” or “activate” refer to any measurable increase in cellular activity of a gene switch, resulting in expression of a gene of interest (e.g., selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10R or a subunit thereof DN, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, CCL3 (MIP-1a), CCL5 (RANTES), CCL7 (MCPS), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12 (SDF-1), CCL21 (6Ckine), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1, F1t3L, IFN-beta, TNF-alpha, dnFADD, TGF-alpha, PD-L1 RNAi, a PD-L1 antisense oligonucleotide, TGFbRII DN, ICOS-L and S100.
- As used herein, the terms “treating” or “treatment” of a disease refer to executing a protocol, which may include administering one or more drugs or in vitro engineered cells to a mammal (human or non-human), in an effort to alleviate signs or symptoms of the disease. Thus, “treating” or “treatment” should not necessarily be construed to require complete alleviation of signs or symptoms, does not require a cure, and specifically includes protocols that have only marginal effect on the subject.
- As used herein, “immune cells” include dendritic cells, macrophages, neurophils, mast cells, eosinophils, basophils, natural killer cells and lymphocytes (e.g., B and T cells).
- As used herein, the terms “dendritic cells” and “DC” are interchangeably used.
- As used herein, the term “therapy support cells” (TSC) are cells that can be modified (e.g., transfected, electroporated, etc.) with the vector of the invention to deliver the one or more proteins having the function of an immunomodulator and, optionally, a protein having the function of IL-12, to tumor microenvironments. Such TSC include, but are not limited to, stem cells, fibroblasts, endothelial cells and keratinocytes.
- As used herein, the terms “in vitro engineered immune cells” or “in vitro engineered population of immune cells” or “a population of engineered immune cells” or “immune cells expressing an immunomodulator” or “immune cells expressing IL-12” refer to immune cells, e.g., dendritic cells, conditionally expressing an immunomodulator and/or IL-12 as the case may be under the control of a gene switch, which can be activated by an activating ligand.
- As used herein, the terms “in vitro engineered TSC” or “in vitro engineered population of TSC” or “a population of engineered TSC” or “TSC expressing an immunomodulator” or “TSC expressing IL-12” refer to therapy support cells, e.g., stem cells, fibroblasts, endothelial cells and keratinocytes, conditionally expressing an immunomodulator and/or IL-12 as the case may be under the control of a gene switch, which can be activated by activating ligand.
- As used herein, the term “modified cell” refers to cells which have been altered by a process including, but not limited to, transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation and lipofection (lysosome fusion).
- As used herein, the terms “MOI” or “Multiplicity of Infection” refer to the average number of adenovirus particles that infect a single cell in a specific experiment (e.g., recombinant adenovirus or control adenovirus)
- As used herein, the term “tumor” refers to all benign or malignant cell growth and proliferation either in vivo or in vitro, whether precancerous or cancerous cells and/or tissues.
- In another embodiment, the vector and methods of the present invention can be used to treat disease.
- In another embodiment, the vector and methods of the present invention can be used to treat a cancer. Non-limiting examples of cancers that can be treated according to the invention include breast cancer, prostate cancer, lymphoma=skin cancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic granulocytic leukemia, acute granulocytic leukemia, hairy cell leukemia, neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, soft-tissue sarcoma, mesothelioma, osteogenic sarcoma, primary macroglobulinemia, and retinoblastoma, and the like. In another embodiment, the vector and methods of the present invention can be used to treat a metabolic-related disorder including, but not limited to, a disorder selected from the group consisting of dyslipidemia, atherosclerosis, insulin resistance, diabetes (e.g. diabetes type I, diabetes type II, MODY, and gestational diabetes), obesity, impaired glucose tolerance, atheromatous disease, hypertension, heart disease (which includes, but is not limited to, coronary heart disease, stroke, cardiac insufficiency, coronary insufficiency, and high blood pressure), hyperlipidemia, glucose intolerance, insulin resistance, hyperglycemia, hyperinsulinemia, metabolic syndrome X (or syndrome X, or insulin resistance syndrome, or Reaven's syndrome, or the metabolic cardiovascular risk syndrome), hypertension, chronic fatigue, accelerated aging, degenerative disease, endocrine deficiencies of aging, Gm1, gangliosidosis, Morquio-B disease, Krabbe's disease, Fabry's disease, Gaucher's disease, Tay-Sachs disease, Sandhoff disease, fucosidosis, disorders of carbohydrate metabolism (e.g. glycogen storage disease), disorders of amino acid metabolism (e.g. phenylketonuria, maple syrup urine disease, glutaric acidernia type 1), disorders of organic acid metabolism (e.g. alcaptonuria), disorders of fatty acid oxidation and mitochondrial metabolism (e.g. medium chain acyl dehydrogenase deficiency), disorders of porphyrin metabolism (e.g. acute intermittent porphyria), disorders of purine or pyrimidine metabolism (e.g. Lesch-Nyhan syndrome), disorders of steroid metabolism (e.g. congenital adrenal hyperplasia), disorders of mitochondrial function (e.g. Kearns-Sayre syndrome), and disorders of peroxisomal function (e.g. Zellweger syndrome).
- In another embodiment, the vector and methods of the present invention can be used to treat an autoimmune disorder including, but not limited to, a disorder selected from the group consisting of Achlorhydra Autoimmune Active Chronic Hepatitis, Acute Disseminated Encephalomyelitis, Acute hemorrhagic leukoencephalitis, Addison's Disease, gammaglobulinemia, Agammaglobulinemia, Alopecia greata, Amyotrophic Lateral Sclerosis, Ankylosing Spondylitis, Anti-GBM/TBM Nephritis, Antiphospholipid syndrome, Antisynthetase syndrome, Arthritis, Atopic allergy, Atopic Dermatitis, Aplastic Anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome Types I, II, & III, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune uveitis, Balo disease/Balo concentric sclerosis, Bechets Syndrome, Berger's disease, Bickerstaff s encephalitis, Blau syndrome, Bullous Pemphigoid, Castleman's disease, Chronic Fatigue Immune Dysfunction Syndrome, chronic inflammatory demyelinating polyneuropathy, Chronic recurrent multifocal ostomyelitis, Churg-Strauss syndrome, Cicatricial Pemphigoid, Coeliac Disease, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Cranial arteritis, CREST syndrome, Crohns Disease, Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemic sclerosis, Dressler's syndrome, Discoid lupus erythematosus, eczema, Enthesitis-related arthritis, Eosinophilic fasciitis, Epidermolysis bullosa acquisita, Erythema nodosum, Essential mixed cryogiobulinemia, Evan's syndrome, Fibrodysplasia ossificans progressiva, Fibromyositis, Fibrosing aveolitis, Gastritis, Gastrointestinal pemphigoid, Giant cell arteritis, Goodpasture's syndrome, Graves disease, Guillain-Barré syndrome (GBS), Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anaemia, Henoch-Schonlein purpura, Herpes gestationis, Hughes syndrome (or Antiphospholipid syndrome), Hypogammaglobulinemia, Idiopathic Inflammatory Demyelinating Diseases, Idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, IgA nephropathy (or Berger's disease), Inclusion body myositis, ory demyelinating polyneuopathy, Juvenile idiopathic arthritis, Juvenile rheumatoid arthritis, Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lou Gehrig's Disease, Lupoid hepatitis, Lupus erythematosus, Majeed syndrome, Ménière's disease, Microscopic polyangiitis, Miller-Fisher syndrome, Mixed Connective Tissue Disease, Mucha-Habermann disease, Muckle-Wells syndrome, Multiple Myeloma, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (also Devic's Disease), Occular cicatricial pemphigoid, Ord thyroiditis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paraneoplastic cerebellar degeneration, Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis, Pemphigus, Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis, POEMS syndrome, Polyarteritis nodosa, Polymyalgia rheumatica, Polymyositis, Primary biliary cirrhosis, psoriasis, psoriatic arthritis, Pyoderma gangrenosum, pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatoid fever, Schmidt syndrome, Schnitzler syndrome, Scleritis, Sjögren's syndrome, Spondyloarthropathy, sticky blood syndrome, Still's Disease, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sweet syndrome, Sydenham Chorea, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis, Tolosa-Hunt syndrome, Transverse Myelitis, Ulcerative Colitis, Undifferentiated connective tissue disease, Undifferentiated spondyloarthropathy, vasculitis, Wegener's granulomatosis, Wilson's syndrome, and Wiskott-Aldrich syndrome.
- In another embodiment, vector and the methods of the present invention can be used to treat an ocular disorder that includes, but is not limited to, a disorder selected from the group consisting of glaucoma including Open Angle Glaucoma (e.g., Primary Open Angle Glaucoma, Pigmentary Glaucoma, and Exfoliative Glaucoma, Low Tension Glaucoma), Angle Closure Glaucoma (also known clinically as closed angle glaucoma, narrow angle glaucoma, pupillary block glaucoma, and ciliary block glaucoma) (e.g., Acute Angle Closure Glaucoma and Chronic Angle Closure Glaucoma), Aniridic Glaucoma, Congenital Glaucoma, Juvenile Glaucoma, Lens-Induced Glaucoma, Neovascular Glaucoma (e.g., using vectors composed of Vascular Endothelial Growth Factor (VEGF) decoy, Pigment Derived Growth Factor (PDGF), Endostatin, Angiostatin, or Angiopoetin-1), Post-Traumatic Glaucoma, Steroid-Induced Glaucoma, Sturge-Weber Syndrome Glaucoma, and Uveitis-Induced Glaucoma, diabetic retinopathy (e.g., using vectors composed of VEGF decoy, PDGF, Endostatin, Angiostatin, or Angiopoetin-1), macular degeneration (e.g. vectors composed of VEGF decoy, PDGF, Endostatin, Angiostatin, Angiopoetin-1, ATP Binding Casette Subfamily A Member 4), macular degeneration (e.g., using vectors composed of VEGF decoy, PDGF, Endostatin, Angiostatin, Angiopoetin-1, ATP Binding Casette Subfamily A Member 4), choroidal neovascularization, (e.g., using vectors composed of VEGF decoy, PDGF, Endostatin, Angiostatin, or Angiopoetin-1), vascular leak, and/or retinal edema, bacterial conjunctivitis, fungal conjunctivitis, viral conjunctivitis, uveitis, keratic precipitates, macular edema (e.g., using vectors composed of VEGF decoy, PDGF, Endostatin, Angiostatin, or Angiopoetin-1), inflammation response after intra-ocular lens implantation, uveitis syndromes (for example, chronic iridocyclitis or chronic endophthalmitis), retinal vasculitis (for example, as seen in rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythymatosus, progressive systemic sclerosis, polyarteritis nodosa, Wegener's granulomatosis, termporal arteritis, Adamantiades Bechcet disease, Sjorgen's, relapsing polychondritis and HLA-B27 associated spondylitis), sarcoidosis, Eales disease, acute retinal necrosis, Vogt Koyanaki Harada syndrome, occular toxoplasmosis, radiation retinopathy, proliferative vitreoretinopathy, endophthalmitis, ocular glaucomas (for example, inflammatory glaucomas), optic neuritis, ischemic optic neuropathy (e.g. vectors composed of Allotopic NADH dehydrogenase Unit 4), thyroid associated orbitopathy, orbital pseudotumor, pigment dispersion syndrome (pigmentary glaucoma), scleritis, episcleritis choroidopathies (for example, “White-dot” syndromes including, but not limited to, acute multifocal posterior placoid), retinopathies (for example, cystoid macular edema, central serous choroidopathy and presumed ocular histoplasmosis syndrome (e.g., vectors composed of Glial Cell Derived Neurotropic Factor, Peripherin-2)), retinal vascular disease (tor example, diabetic retinopathy, Coat's disease and retinal arterial macroaneurysm), retinal artery occlusions, retinal vein occlusions, retinopathy of prematurity, retinitis pigmentosa (e.g. vectors composed of Retinal Pigment Specific 65 kDa protein), familial exudative vitreoretinopathy (FEVR), idiopathic polypoidal choroidal vasculopathy, epiretinal macular membranes and cataracts.
- In another embodiment, the vector and methods of the present invention can be used to treat a blood disorder that includes, but is not limited to, a blood disorder selected from the group consisting of anemia, bleeding and clotting disorders (e.g., disseminated intravascular coagulation (DIC), hemophilia, Henoch-Schonlien Purpura, hereditary hemorrhagic telangiectasia, thrombocytopenia (ITP, TTP), thrombophilia, Von Willebrand's disease), leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia), lymphomas (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma), myeloproliferative disorders (e.g., myelofibrosis, Polycythemia Vera, thrombocythemia), plasma cell disorders (e.g., macroglobulinemia, monoclonal gammopathies of undetermined significance, multiple lyeloma), spleen disorders, white blood cell disorders (e.g., basophilic disorder, eosinophilic disorder, lymphocytopenia, monocyte disorders, neutropenia, neutrophillic leukocytosis), thrombosis, deep vein thrombosis (DVT), hemochromatosis, menorrhagia, sickle cell disease, and thalassemia.
- In another embodiment, the vector and methods of the present invention can be used to treat a neurological disorder that includes, but is not limited to, a neurological disorders selected from the group consisting of Gaucher disease, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Huntington's disease, Fredrich's ataxia, Mild Cognitive Impairment, Cerebral Amyloid Angiopathy, Parkinsonism Disease, Lewy Body Disease, Frontotemporal Dementia (FTD) Multiple System Atrophy (MSA), Progressive Supranuclear Palsy, and movement disorders (including ataxia, cerebral palsy, choreoathetosis, dystonia, Tourette's syndrome, kernicterus) and tremor disorders, and leukodystrophies (including adrenoleukodystrophy, metachromatic leukodystrophy, Canavan disease, Alexander disease, Pelizaeus-Merzbacher disease), neuronal ceroid lipofucsinoses, ataxia telangectasia, Rett Syndrome, alpha.-synucleinopathy (e.g., Lewy Body Disease, Multiple System Atrophy, Hallervorden-Spatz disease, or Frontotemporal Dementia), Niemann-Pick Type C disease (NPCD),
spinocerebellar ataxia Type 1,Type 2, andType 3, and dentatorubral pallidoluysian atrophy (DRLPA). - In another embodiment, the vector and methods of the present invention can be used to treat a lung disorder that includes, but is not limited to, a lung disorder selected from the group consisting of asthma, atelectasis, bronchitis, COPD (chronic obstructive pulmonary disease), emphysema, Lung cancer, mesothelioma, pneumonia, asbestosis, Aspergilloma, Aspergillosis, Aspergillosis—acute invasive, bronchiectasis, bronchiolitis obliterans organizing pneumonia (BOOP), eosinophilic pneumonia, necrotizing pneumonia, ral effusion, pneumoconiosis, pneumothorax, pulmonary actinomycosis, monary alveolar proteinosis, pulmonary anthrax, pulmonary arteriovenous malformation, pulmonary fibrosis, pulmonary embolus, pulmonary histiocytosis X (eosinophilic granuloma), pulmonary hypertension, pulmonary edema, pulmonary hemorrhage, pulmonary nocardiosis, pulmonary tuberculosis, pulmonary veno-occlusive disease, rheumatoid lung disease, sarcoidosis, radiation fibrosis, hypersensitivity pneumonitis, acute respiratory distress syndrome (ARDS), infant respiratory distress syndrome, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonia, lymphangioleiomyomatosis, pulmonary Langerhans' cell histiocytosis, pulmonary alveolar proteinosis, sinusitis, tonsillitis, otitis media, pharyngitis, laryngitis, Pulmonary hamartoma, pulmonary sequestration, congenital cystic adenomatoid malformation (CCAM), and cystic fibrosis.
- In another embodiment, the vector and methods of the present invention can be used to treat a rheumatologic disorder that includes, but is not limited to, a rheumatic disorder selected from the group consisting of systemic lupus erythematosus, dermatomyositis, scleroderma, systemic necrotizing arteritis, cutaneous necrotizing venulitis, rheumatoid arthritis, Sjogren's Syndrome, Raynaud's phenomenon, Reiter's syndrome, arthritis, psoriatic arthritis, seronegative spondyloarthropathies, Sjogren's syndrome, systemic sclerosis, dermatomyositis/polymyositis, mixed connective tissue disease, and ankylosing spondylitis.
- In another embodiment, the vector and methods of the present invention can be used to treat an infectious disease in a human that includes, but is not limited to, an infectious disease selected from the group consisting of fungal diseases such as dermatophytosis (e.g., trichophytosis, ringworm or tinea infections), athletes foot, paronychia, pityriasis versicolor, erythrasma, intertrigo, fungal diaper rash, candida vulvitis, candida balanitis, otitis externa, candidiasis (cutaneous and mucocutaneous), chronic mucocandidiasis (e.g. thrush and vaginal candidiasis), cryptococcosis, geotrichosis, trichosporosis, aspergillosis, penicilliosis, fusariosis, zygomycosis, sporotrichosis, chromomycosis, coccidioidomycosis, histoplasmosis, blastomycosis, paracoccidioidomycosis, pseudallescheriosis, mycetoma, mycotic keratitis, otomycosis, pneumocystosis, and fungemia, Acinetobacter infections, Actinomycosis, African sleeping sickness, AIDS (Acquired immune deficiency syndrome), Amebiasis, Anaplasmosis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, atrovirus infection, Babesiosis, Bacillus cereus infection, Bacterial pneumonia, Bacterial vaginosis (BV), Bacteroides infection, Balantidiasis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Borrelia infection, Botulism (and Infant botulism), Brazilian hemorrhagic fever, Brucellosis, Burkholderia infection, Burali ulcer, Calcivirus infection (Norovirus and Sapovirus), Candidiasis, Cat-scratch disease, Cellulitis, Chagas Disease (American trypanosomiasis), Chancroid, Chickenpox, Chlamydia, Cholera, Chromoblastomycosis, Clonorchiasis, Clostridium difficile, Coccidioidomycosis, Colorado tick fever (CTF), Common cold (Acute viral rhinopharyngitis; Acute coryza), Creutzfeldt-Jakob disease (CJD), Cryptococcosis, Cryptosporidiosis, ous larva migrans (CLM), Dengue fever, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Diphyllobothriasis, Dracunculiasis, Ebola hemorrhagic fever, Echinococcosis, Ehrlichiosis, Enterobiasis (Pinworm infection), Enterococcus infection, Enterovirus infection, Epidemic typhus, Erythema infectiosum, Exanthem subitum, Fasciolopsiasis, Fasciolosis, Fatal familial insomnia (FFI), Filariasis, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Geotrichosis, Gerstmann-Sträussler-Scheinker syndrome (GSS), Giardiasis Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale (Donovanosis), Group A streptococcal infection, Group B streptococcal infection, Haemophilus influenzae, Hand, foot and mouth disease (HFMD), Hantavirus Pulmonary Syndrome (HPS) Helicobacter pylori infection, ic-uremic syndrome (HUS), Hemorrhagic fever with renal syndrome (HFRS), Hepatitis A, B, C, D, E, Herpes simplex, Histoplasmosis, Hookworm infection, n bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis (HGA), Human granulocytic anaplasmosis (HGA), Human monocytic ehrlichiosis, Human papillomavirus (HPV) infection, Human parainfluenza virus infection, Hymenolepiasis, Epstein-Barr Virus Infectious Mononucleosis (Mono), Influenza (flu), Isosporiasis, Kawasaki disease, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Legionellosis (Legionnaires' disease), Legionellosis (Pontiac fever), Leishmaniasis, Leprosy, Leptospirosis, Listeriosis, Lyne disease (Lyme borreliosis), Lymphatic filariasis (Elephantiasis), Lymphocytic choriomeningitis, Malaria, Marburg hemorrhagic fever (MHF), Measles, Melioidosis (Whitmore's disease), Meningitis, Meningococcal disease, Metagonimiasis, Microsporidiosis, Molluscum contagiosum (MC), Mumps, Murine typhus (Endemic typhus), Mycoplasma pneumonia, Mycetoma, Myiasis, Neonatal conjunctivitis (Ophthalmia neonatorum), (New) Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Nocardiosis, Onchocerciasis (River blindness), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Pasteurellosis, Pediculosis capitis (Head lice), Pediculosis corporis (Body lice), Pediculosis pubis (Pubic lice, Crab lice), Pelvic inflammatory disease (PID), Pertussis (Whooping cough), Plague, Pneumococcal infection, Pneumocystis pneumonia (PCP), Pneumonia, Poliomyelitis, Poliomyelitis, Prevotella infection, mary amoebic meningoencephalitis (PAM), Progressive multifocal leukoencephalopathy, Psittacosis, Q fever, Rabies, Rat-bite fever, Respiratory syncytial virus infection, Rhinosporidiosis, inovirus infection, Rickettsial infection, Rickettsialpox, Rift Valley fever (RVF), Rocky mountain spotted fever (RMSF), Rotavirus infection, Rubella, Salmonellosis, SARS (Severe Acute Respiratory Syndrome), Scabies, Schistosomiasis, Sepsis, Shigellosis (Bacillary dysentery), Shingles (Herpes zoster), Smallpox (Variola), Sporotrichosis, Staphylococcal food poisoning, Staphylococcal infection, Strongyloidiasis, Syphilis, Taeniasis, tanus (Lockjaw), Tinea barbae (Barber's itch), Tinea capitis (Ringworm of the Scalp), Tinea corporis (Ringworm of the Body), Tinea cruris (Jock itch), Tinea manuum (Ringworm of the Hand), Tinea nigra, Tinea unguiurn (Onychomycosis), Tinea versicolor (Pityriasis versicolor), Toxocariasis (Visceral Larva Migrans (VLM)), Toxoplasmosis, Trichinellosis, Trichomoniasis, Trichuriasis (Whipworm infection), Tuberculosis, Tularemia, Ureaplasma urealyticum infection, Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, viral pneumonia, West Nile Fever, White piedra (Tinea blanca), Yersinia pseudoniberculosis infection, Yersiniosis, Yellow fever, and Zygomycosis.
- In another embodiment, the vector and methods of the present invention can be used to treat one or more diseases in a mammal. In one aspect, the mammal is a human. In another aspect, the mammal is a non-human animal. One can readily contemplate a variety of diseases that can be treated using the teachings of the present invention. These diseases include, but are not limited to chronic renal disease, osteoarthritis, oncology, viral upper respiratory infection, feline plasma cell stomatitis, feline eosinophillic granulomas, feline leukemia virus infection, canine distemper infection, systemic fungal infections, cardiomyopathy, mucopolysaccharidosis VII, and infectious disease.
- In one aspect, disease that is treated is an infectious diseases in an animal, and such infectious disease include, but are not limited to, Bovine respiratory disease, Porcine respiratory disease, Avian influenza, Avian infectious bronchitis, Bovine spongiform encephalopathy, Canine leishmaniasis, Chronic wasting disease, human immune deficiciency virus (HIV), hepatitis, hepatitis A, hepatitis B, hepatitis C, Classical swine fever, Echinococcus, Enzootic pneumonia, FIP, Foot-and-mouth disease, Jaagsiekte, Maedi-Visna, Mastitis in animals, Microsporum canis, Orf (animal disease), Peste des petits ruminants, Pox diseases, Psittacine beak and feather disease, Rabies, Mediterranean fever (Brucellosis) or Bang's disease or undulant fever, Malta fever, contagious abortion, epizootic abortion, Salmonella food poisoning, enteric paratyphosis, Bacillary dysentery, Pseudotuberculosis, plague, pestilential fever, Tuberculosis, Vibrios, Circling disease, Weil's disease (Leptospirosis) or canicola fever, Hemorrhagic jaundice (Leptospira icterohaemorrhagiae), dairy worker fever (L. hardjo), Relapsing fever, tick-borne relapsing fever, spirochetal fever, vagabond fever, famine fever, Lyme arthritis, Bannworth's syndrome (lime disease), tick-borne meningopolyneuritis, erythema chronicum migrans, Vibriosis, Colibacteriosis, colitoxemia, white scours, gut edema of swine, enteric paratyphosis, Staphylococcal alimentary toxicosis, staphylococcal gastroenteritis, Canine Corona Virus (CCV) or canine parvovirus enteritis, feline infectious peritonitis virus, transmissible gastroenteritis (TGE) virus, Hagerman Redmouth Disease (ERMD), Infectious Hematopoietic necrosis (IHN), porcine Actinobacillus (Haemophilus) pleuropneumonia, Hansen's disease, Streptotrichosis, Mycotic Dermatitis of Sheep, Pseudoglanders, Whitmore's disease, Francis' disease, deer-fly fever, rabbit fever, O'Hara disease, Streptobacillary fever, Haverhill fever, epidemic arthritic erythema, sodoku, Shipping or transport fever, hemorrhagic septicemia, Ornithosis, Parrot Fever, Chlamydiosis, North American blastomycosis, Chicago disease, Gilchrist's disease, Cat Scratch Fever, Benign Lymphoreticulosis, Benign nonbacterial Lymphadenitis, Bacillary Angiomatosis, Bacillary Peliosis Hepatis, Query fever, Balkan influenza, Balkan grippe, abattoir fever, Tick-borne fever, pneumorickettsiosis, American Tick Typhus, Tick-borne Typhus Fever, Vesicular Rickettsiosis, Kew Gardens Spotted Fever, Flea-borne Typhus Fever, Endemic Typhus Fever, Urban Typhus, Ringworm, Dermatophytosis, Tinea, Trichophytosis, Microsporosis, Jock Itch, Athlete's Foot, Sporothrix schenckii, dimorphic fungus, Cryptococcosis and histoplasmosis, Benign Epidermal Monkeypox, BEMP, Herpesvirus simiae, Simian B Disease, Venezuelan equine encephalitis, Type C lethargic encephalitis, Yellow fever, Black Vomit, hantavirus pulmonary syndrome, Korean Hemorrhagic Fever, Nephropathia Epidemica, Epidemic Hemorrhagic Fever, Hemorrhagic Nephrosonephritis, lymphocytic choriomeningitis, California encephalitis/La crosse encephalitis, African Hemorrhagic Fever, Green or Vervet Monkey Disease, Hydrophobia, Lyssa, Infectious hepatitis, Epidemic hepatitis, Epidemic jaundice, Rubeola, Morbilli, Swine and Equine Influenza, Fowl Plague, Newcastle disease, Piroplasmosis, toxoplasmosis, African Sleeping Sickness, Gambian Trypanosomiasis, Rhodesian Trypanosomiasis, Chagas's Disease, Chagas-Mazza Disease, South American Trypanosomiasis, Entamoeba histolytica, Balantidial dysentery, cryptosporidiosis, giardiasis, Cutaneous leishmaniasis: Chiclero ulcer, espundia, pianbols, uta, and buba (in the Americas); oriental sore, Aleppo boil (in the Old World); Bagdad boil, Delhi boil, Bauru ulcer, Visceral leishmaniasis: kala-azar, Microsporidiosis, Anisakiasis, Trichinosis, Angiostrongylosis, eosinophilic meningitis or meningoencephalitis (A. cantonensis), abdominal angiostrongylosis (A. costaricensis), Uncinariasis, Necatoriasis, Hookworm Disease, Capillariasis, Brugiasis, Toxocariasis, Oesophagostomiasis, Strongyloidiasis, Trichostrongylosis, Ascaridiasis, Diphyllobothriasis, Sparganosis, Hydatidosis, Hydatid Disease, Echinococcus granulosis, Cystic hydatid disease, Tapeworm Infection, Schistosoma and the like.
- Treatment of malignant diseases caused by infectious pathogens are contemplated as well. Examples of such diseases include, but are not limited to, osteosarcoma, leukemia, lymphoma, Burkitt lymphoma caused by EBV, Rous sarcoma caused by Rous retrovirus, Kaposi' sarcoma caused by
herpes virus type 8, adult T-cell leukemia caused by HTLV-I retrovirus, or hairy cell leukemia caused by HTLV-II, and many other tumors and leukemias caused by infectious agents and viruses. - In one embodiment, the one or more proteins used to treat one or more of the above diseases includes, but is not limited to, erythropoetin, ghrelin, osteoprotegerin, RANKL, RANKL decoy, TNF-αantagonist, an IL-1 antagonist, G-CSF, GM-CSF, IFN-α, IFN-γ, angiostatin, endostatin, TNF-α, PP1DCY-LSRLOC, β-glucuronidase, and IL-12. In another embodiment, the one or more proteins of the invention includes, but is not limited to, IL-1, IL-2, IL-12, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10R DN or a subunit thereof, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, IFN-
alpha 1,IFN alpha 2, IL-15-R-alpha, CCL3 (MIP-1a), CCL5 (RANTES), CCL7 (MCPS), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12 (SDF-1), CCL21 (6Ckine), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1, Flt3L, IFN-beta, TNF-alpha, dnFADD, BCG, TGF-alpha, PD-L1 RNAi, a PD-L1 antisense oligonucleotide, TGFbRII DN, ICOS-L, and S100. - In one embodiment, the vector administered to the mammal afflicted with one or more of the disclosed diseases is an adenoviral vector. In one embodiment, the vector comprises a polynucleotide encoding a gene switch. In one aspect, the gene switch is an EcR-based gene switch. In another embodiment, the polynucleotide encoding a gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. In one aspect, the ligand is a diacylhydrazine. In another aspect, the ligand is selected from RG-115819, RG-115932, and RG-115830. In yet another aspect, the ligand is an amidoketone or an oxadiazoline.
- In another embodiment, the present invention can be used to treat one or more lysosomal storage diseases in a mammal. In one aspect, the mammal is a human. In another aspect, the mammal is a non-human animal. Examples of lysosmal storage diseases that can be treated according to the invention include, but are not limited to, Pompe disease/Glycogen storage disease type II, Gaucher Disease (Type I, Type II, Type III), Fabry disease, Mucopolysaccharidosis II (Hunter syndrome), Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), Mucopolysaccharidosis I, Metachromatic Leukodystrophy, Neuronal Ceroid Lipofuscinoses or CLN6 disease (Atypical Late Infantile, Late Onset variant, Early Juvenile, Finnish Variant Late Infantile CLN5, Jansky-Bielschowsky disease/Late infantile CLN2/TPP1 Disease, Kufs/Adult-onset NCL/CLN4 disease, Northern Epilepsy/variant late infantile CLN8, Santavuori-Haltia/Infantile CLN1/PPT disease, Beta-mannosidosis), Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease, Sanfilippo syndrome Type A, Sanfilippo syndrome Type B, Sanfilippo syndrome Type C, Sanfilippo syndrome Type D, MPSI Hurler Syndrome, Niemann-Pick Disease (Type A, Type B, Type C, Type D), Activator Deficiency/GM2 Gangliosidosis, Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease, Farber disease, Fucosidosis, Galactosialidosis (Goldberg Syndrome), GM1 gangliosidosis (Infantile, Late infantile/Juvenile, Adult/Chronic), I-Cell disease/Mucolipidosis II, Infantile Free Sialic Acid Storage Disease/ISSD, Juvenile Hexosaminidase A Deficiency, Krabbe disease (Infantile Onset, Late Onset), Mucopolysaccharidoses disorders (Pseudo-Hurler polydystrophy/Mucolipidosis IIIA, Scheie Syndrome, MPS I Hurler-Scheie Syndrome, Morquio Type A/MPS IVA, Morquio Type B/MPS IVB, MPS IX Hyaluronidase Deficiency, Sly Syndrome (MPS VII), Mucolipidosis I/Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV), Multiple sulfatase deficiency, Pycnodysostosis, Sandhoff disease/Adult Onset/GM2 gangliosidosis, Sandhoff disease/GM2 gangliosidosis—Infantile, Sandhoff disease/GM2 gangliosidosis—Juvenile, Schindler disease, Salla disease, Infantile Sialic Acid Storage Disease, Tay-Sachs/GM2 gangliosidosis, Wolman disease, Asparylglucosaminuria, and prosaposin.
- It will be appreciated that Sanfilippo syndrome Type A is synonymous with Sanfilippo syndrome Type A/MPS IIIA, Sanfilippo syndrome Type B is synonymous with Sanfilippo syndrome Type B/MPS IIIB, Sanfilippo syndrome Type C is synonymous with Sanfilippo syndrome Type C/MPS IIIC, Sanfilippo syndrome Type D is synonymous with Sanfilippo syndrome Type D/MPS IIID.
- In one embodiment, the one or more proteins expressed by the vector of the invention used to treat one or more of the above lysosomal storage diseases includes, but is not limited to, a-galactosidase A, Arylsulfatase A, α-glucosidase, b-glucosidase, glucocerebrosidase, CLN6 protein, Juvenile associated with CLN3, N-sulfoglucosamine sulfohyrolase (SGSH), a-N-acetylglucosaminidase, acetyl-CoA-glucosaminide acetyltransferase, N-acetylglucosamine-6-sulfatase, α-L-iduronidase, arylsulfatase B, acid sphingomyelinase, and iuduronate sulfatase.
- In one embodiment, the vector administered to the mammal afflicted with one or more of the disclosed lysosomal storage diseases is an adenoviral vector. In one embodiment, the vector comprises a polynucleotide encoding a gene switch. In one aspect, the gene switch is an EcR-based gene switch. In another embodiment, the polynucleotide encoding a gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. In one aspect, the ligand is a diacylhydrazine. In another aspect, the ligand is selected from RG-115819, RG-115932, and RG-115830. In yet another aspect, the ligand is an amidoketone or an oxadiazoline.
- In another embodiment, the present invention can be used to treat one or more liver disease in a mammal. In one aspect, the mammal is a human. In another aspect, the mammal is a non-human animal. In one aspect, the liver disease is Hepatitis B. In another aspect, the liver disease is Hepatitis C. In one embodiment, the protein expressed by a vector of the invention is IFN-α. In another embodiment, the protein expressed by a vector of the invention is one or more of the liver diseases comprises ceruloplasmin.
- A non-limiting example of a human liver chimeric mouse model for hepatitis B and C virus infection and treatment is disclosed in Bissig, K. D. et al., J. Clin. Investigation 120: 924 (2010). Another non-limiting example of a human hepatocyte model is the humanized mouse system marketed by Yecuris™ (Portion, Oreg.).
- A non-lmiting example of an encephalitis model useful for evaluating antiviral/antiinfective treatment is disclosed in O'Brien, L. et al., J. General Virology 90: 874-882 (2009).
- Non-limiting examples influenza models useful for evaluating antiviral/antiinfective treatment is disclosed in Beilharz, M. W. et al., Biochemical Biophysical Research Communications 355: 740-744 (2007); and Koerner, I. et al., J. Virology 81: 2025-2030 (2007).
- In one embodiment, the vector administered to the mammal afflicted with one or more of the disclosed liver diseases is an adenoviral vector. In another embodiment, the vector is not an adenoviral vector. In another embodiment, the vector is a plasmid. In one embodiment, the vector comprises a polynucleotide encoding a gene switch. In one aspect, the gene switch is an EcR-based gene switch. In another embodiment, the polynucleotide encoding a gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. one aspect, the ligand is a diacylhydrazine. In another aspect, the ligand is selected from RG-115819, RG-115932, and RG-115830. In yet another aspect, the ligand is an amidoketone or an oxadiazoline.
- The invention provides engineering of cells, e.g., immune cells and TSC, to conditionally express a protein having the function of an immunomodulator and, optionally, IL-12 and therapeutic uses and/or applications for the treatment of cancer or tumors or both. In vitro engineered immune cells and TSC that conditionally express a protein having the function of an immunomodulator and optionally IL-12 are a safe improvement over constitutive production of the protein(s). Additionally, the ability to control the timing and level of immunomodulator and optionally IL-12 expression provides improved control of the efficacy of the treatment. Therefore, in vitro engineered immune cells and TSC may be formulated into pharmaceutical compositions as therapeutics for the treatment of a cancer or a tumor in a human or a non-human organism. Alternatively, in vitro engineered populations of immune cells, TSC or subsets thereof may be used as vehicles to conditionally deliver an immunomodulator and optionally IL-12 protein production to a specific area (normal tissue, cancer, or tumor) in the body of a human or non-human organism. The immune cells may be autologous or non-autologous dendritic cells. The dendritic cells may be isolated from bone marrow or from peripheral blood circulation. In human patients, dendritic cell populations may be isolated via a leukophoresis procedure, where a white blood cell fraction is isolated and removed and other blood components are re-infused to the patient.
- In another embodiment, the dendritic cells may be prepared by transfecting human hematopoietic stem cells with a vector of the invention expressing a protein having the function of an immunomodulator and optionally a protein having the function of IL-12, and differentiating the transfected stem cell to give a dendritic cell. See U.S. Pat. No. 6,734,014.
- In one embodiment, a nucleic acid adenoviral vector is provided containing a gene switch, wherein the coding sequences for VP16-RXR and Gal4-EcR are separated by the EMCV internal ribosome entry site (IRES) sequence are inserted into the adenoviral shuttle vector under the control of the human ubiquitin C promoter. For example, the coding sequences for the p40 and p35 subunits of IL12 separated by an IRES sequence, and placed under the control of a synthetic inducible promoter, are inserted upstream of the ubiquitin C promoter. In another example, the coding sequence of TNF-alpha, which is placed under the control of a synthetic inducible promoter, is inserted upstream of the ubiquitin C promoter.
- In another embodiment, the invention provides a shuttle vector carrying transcription units (VP16-RXR and Gal4-EcR) for the two fusion proteins and inducible IL-12 or TNF-alpha subunits recombined with the adenoviral backbone (AdEasy 1) in E. coli BJ5183 cells. After verifying the recombinant clone, the plasmid carrying the rAd.RheoIL12 genome is grown in and purified from XL10-Gold cells, digested off the plasmid backbone and packaged by transfection into HEK 293 cells or CHO cells.
- Purification of the vector to enhance the concentration can be accomplished by any suitable method, such as by density gradient purification (e.g., cesium chloride (CsCl)) or by chromatography techniques (e.g., column or batch chromatography). For example, the vector of the invention can be subjected to two or three CsCl density gradient purification steps. The vector, e.g., a replication-deficient adenoviral vector, is desirably purified from cells infected with the replication-deficient adenoviral vector using a method that comprises lysing cells infected with adenovirus, applying the lysate to a chromatography resin, eluting the adenovirus from the chromatography resin, and collecting a fraction containing adenovirus.
- In a particular embodiment, the resulting primary viral stock is amplified by re-infection of HEK 293 cells or CHO cells and is purified by CsCl density-gradient centrifugation.
- In one embodiment the immunomodulator, e.g., TNF-alpha, and/or IL-12 gene is a wld-type gene sequence. In another embodiment, the immunomodulator, e.g., TNF-alpha, and/or IL-12 gene is a modified gene sequence, e.g., a chimeric sequence or a sequence that has been modified to use preferred codons.
- In one embodiment, the immunomodulator, e.g., TNF-alpha, and/or IL-12 gene is the human wild type sequence. In another embodiment, the sequence is at least 85% identical to wild type human sequence, e.g., at least 90%, 95%, or 99% identical to wild type human sequence. In a further embodiment, the gene sequence encodes the human polypeptide. In another embodiment, the gene encodes a polypeptide that is at least 85% identical to wild type human polypeptide e.g., at least 90%, 95%, or 99% identical to wild type human polypeptide.
- In one embodiment, the IL-12 gene is the wild type mouse IL-12 sequence. In another embodiment, the sequence is at least 85% identical to wild type mouse IL-12, e.g., at least 90%, 95%, or 99% identical to wild type mouse IL-12. In a further embodiment, the IL-12 gene sequence encodes the mouse IL-12 polypeptide. In another embodiment, the gene encodes a polypeptide that is at least 85% identical to wild type mouse IL-12, e.g., at least 90%, 95%, or 99% identical to wild type mouse IL-12.
- DC may be isolated from bone marrow from humans, mice, or other mammals. The dendritic cells may be isolated from the blood of humans, mice or other mammals. In human patients, dendritic cell populations may be isolated via a leukophoresis procedure as is known in the art, where a white blood cell fraction is isolated and removed and other blood components are re-infused to the patient. In one embodiment, DC are derived from murine bone marrow as previously described (Tatsumi et al., 2003). Briefly, wild-type or EGFP Tg mouse bone marrow (BM) is cultured in conditioned medium (CM) supplemented with 1000 units/ml recombinant murine granulocyte/macrophage colony-stimulating factor and recombinant mIL-4 (Peprotech, Rocky Hill, N.J.) at 37° C. in a humidified, 5% CO2 incubator for 7 days. CD11c+ DC are then isolated, e.g., using specific MACS™ beads, per the manufacturer's instructions (Miltenyi Biotec, Auburn, Calif.). CD11c+ DC produced in this manner are >95% pure based on morphology and co-expression of the CD11b, CD40, CD80, and class I and class II MHC antigens.
- One embodiment of the invention provides engineered immune cells and TSC conditionally expressing a protein having the function of an immunomodulator and optionally IL-12 suitable for therapeutic applications for the treatment of cancer, or tumors or both as gene therapy in human or non-human organism. In an embodiment, the invention provides engineered immune cells and TSC containing the gene switch.
- In another embodiment, the invention provides engineered immune cells and TSC containing at least a portion of an ecdysone receptor. In another embodiment, the invention provides engineered immune cells and TSC containing an ecdysone receptor-based gene switch. In another embodiment, the invention provides engineered immune cells and TSC containing RheoSwitch. In another embodiment, the invention provides a kit comprising engineered immune cells and TSC containing a gene switch and a ligand that modulates the gene switch. In another embodiment, the kits further comprise a diacylhydrazine ligand. In another embodiment, the kit further comprises RG-115830 or RG-115932.
- In one embodiment, the invention provides an engineered population of immune cells and TSC. In one embodiment,
day 7 cultured DC are treated with recombinant adenovirus encoding an immunomodulator and/or IL-12 driven off a constitutive or inducible promoter, or are infected with mock, control adenovirus vector (rAdψ5), over a range of multiplicity of infection (MOIs). After 48 h, infected DC are harvested and analyzed for phenotype and for production of an immunomodulator and/or IL-12 using a specific ELISA kit (BD-PharMingen, San Diego, Calif.), with a lower level of detection of 62.5 pg/ml. - In another embodiment, the invention provides in vitro engineered population of immune cells and TSC comprising a vector, e.g., a DNA vector, having a gene switch capable of conditionally expressing a protein having the function of an immunomodulator and/or IL-12, and further comprising activating ligand. In a farther embodiment, the invention provides a method of treating cancer, e.g., melanoma or glioma, by administering engineered DC to a patient and then administering an activating ligand, such as RG-115819, RG-115830 or RG-115932, to said patient. In certain embodiments, the invention is directed to a method of treating cancer, e.g., melanoma or prostate cancer, comprising administering an adenovirus comprising a polynucleotide conditionally expressing an immunomodulator, e.g., TNF-alpha, and administering an activating ligand. The patient may be a human or an animal with cancer. The treatment methods and products, engineered cells, kits, and ligands have application in human therapy and in veterinary animal therapy. Therefore, the products and methods are contemplated to be used for human and veterinary animal purposes.
- Thus, in one embodiment, the polynucleotide expressing the immunomodulator, e.g., TNF-alpha, and activating ligand are co-administered to a patient having a cancer. The activating ligand is generally administered over a number of days, e.g., before and after administeration of the polynucleotide. If systemic toxicity due to the immunomodulator, e.g., TNF-alpha, develops, then administration of the activating ligand can be reduced or eliminated in an effort to attenuated the side effects.
- In another embodiment, the polynucleotide expressing the immunomodulator, e.g., TNF-alpha, and activating ligand are co-administered to a patient suffering from one one or more lysosomal storage diseases, or one or more liver diseases. The activating ligand is generally administered over a number of days, e.g., before and after administeration of the polynucleotide. If systemic toxicitydevelops, then administration of the activating ligand can be reduced or eliminated in an effort to attenuated the side effects.
- In certain embodiments, the invention provides a method of reducing a tumor size comprising administering an adenoviral vector, which comprises a polynucleotide conditionally expressing an immunomodulator, e.g., TNF-alpha, and administering an activating ligand. Also provided is a method of preventing a tumor formation comprising administering an adenoviral vector, which comprises a polynucleotide conditionally expressing an immunomodulator, e.g., TNF-alpha, and administering an activating ligand. In some embodiments, the invention provides a method of reducing or ameliorating one or more symptom of a neoplastic disorder comprising administering an adenoviral vector, which comprises a polynucleotide conditionally expressing an immunomodulator, e.g., TNF-alpha, and administering an activating ligand. In particular, the composition comprising the vector, e.g., adenoviral vector, conditinally expressing an immunomodulator can reduce, prevent, or ameliorate systemic toxicity in the treated subject compared to a vector that constitutively expresses the immunomodulator.
- In certain embodiments, the invention provides a method of treating one or more disease or one or more lysosomal storage disease, or one or more liver disease in mammals comprising administering an adenoviral vector, which comprises a polynucleotide conditionally expressing one or more proteins and administering an activating ligand. In some embodiments, the invention provides a method of reducing or ameliorating one or more symptom of one or more disease or one or more lysosomal storage disease, or one or more liver disease in mammals comprising administering an adenoviral vector, which comprises a polynucleotide conditionally expressing an immunomodulator, e.g., TNF-alpha, and administering an activating ligand.
- Protein-based tags reduce or eliminate the need for highly specific post-translational modifications for effective targeting. Useful protein-based tags include, but are not limited to, IGF2R targeting (IGF2 (GILT)/IGF2 engineering), transferrin receptor targeting (transferrin, TfR-targeting peptides), and Tat protein (in which cell surface heparin sulfate proteoglycans (HSPGs) mediate internalization of Tat).
- Other proteins that target to the lysosome than can be used as a tag include, but are not limited to, Vitamin D binding protein, folate binding protein, lactotransferrin, sex hormone binding globulin, transthyretin, pro saposin, retinol binding protein, Apo lipoprotein B, Apo lipoprotein E, prolactin, receptor associated protein (in one embodiment, without the HNEL sequence), native transferrin, and mutant transferring (e.g., the K225E/R651A mutant or the K225E/K553A mutant).
- In one embodiment, the expression construct also encodes one or more of a reporter sequence, a localization tag sequence, and a detection tag sequence. It will further be appreciated that the composition of the invention or the methods of using the composition can be combined with any chemotherapeutic agent or agents (e.g., to provide a combined therapeutic regimen) that eliminates, reduces, inhibits or controls the growth of neoplastic cells or tumors in vivo. As used herein the terms “chemotherapeutic agent” or “chemotherapeutics” shall be held to mean any therapeutic compound that is administered to treat or prevent the growth of tumors in vivo. In particular, chemotherapeutic agents compatible with the invention comprise both “traditional” chemotherapeutic agents such as small molecules and more recently developed biologics such as antibodies, cytokines, antisense molecules, etc. that are used to reduce or retard the growth of malignant cells.
- In one aspect, the invention provides a pharmaceutical composition suitable for administration to a human or a non-human comprising a population of in vitro engineered immune cells or TSC or a vector, e.g., an adenoviral vector, expressing a protein having the function of an immunomodulator, e.g., TNF-alpha, and/or IL-12, wherein the formulation is suitable for administration by intratumoral administration. In another embodiment, a composition, e.g., pharmaceutical compositon, comprises a vector conditionally expressing an immunomodulator, e.g., TNF-alpha. In some embodiments, the composition comprises about 1×105 or more particle units (pu) of the gene transfer vector. A “particle unit” is a single vector particle. In certain embodiments, the composition comprises about 1×106 particle units of the gene transfer vector (e.g., about 1×107 or more particle units, about 1×108 or more particle units, or about 1×109 or more particle units). In other embodiments, the composition comprises about 1×1010 or more pu, 1×1011 or more pu, 1×1012 or more pu, 1×1013 or more pu, 1×1014 or more pu, or 1×1015 or more pu of the gene transfer vector, especially of a viral vector, such as a replication-deficient adenoviral vector. The number of particle units of the gene transfer vector in the composition can be determined using any suitable method known, such as by comparing the absorbance of the composition with the absorbance of a standard solution of gene transfer vector (i.e., a solution of known gene transfer vector concentration) as described further herein.
- The invention further provides a pharmaceutical composition comprising an activating ligand, such as RG-115819, RG-115830 or RG-115932, wherein the composition is suitable for administration by intraperitoneal, oral, or subcutaneous administration.
- A composition of the invention, e.g., a composition comprising an engineered DC, a vector (e.g., an adenoviral vector), or an activating ligand, can further comprise a pharmaceutically acceptable carrier. The carrier can be any suitable carrier for the an engineered dendritic cells, gene transfer vector, or activating ligand. Suitable carriers for the composition are described in U.S. Pat. No. 6,225,289. The carrier typically will be liquid, but also can be solid, or a combination of liquid and solid components. The carrier desirably is a pharmaceutically acceptable (e.g., a physiologically or pharmacologically acceptable) carrier (e.g., excipient or diluent). Pharmaceutically acceptable carriers are well known and are readily available. The choice of carrier will be determined, at least in part, by the particular components in the composition and the particular method used to administer the composition. The composition can further comprise any other suitable components, especially for enhancing the stability of the composition and/or its end-use. Accordingly, there is a wide variety of suitable formulations of the composition of the invention.
- Formulations suitable for oral administration include (a) liquid solutions, such as an effective amount of the active ingredient dissolved in diluents, such as water, saline, or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as solids or granules, (c) suspensions in an appropriate liquid, and (d) suitable emulsions. Tablet forms can include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base (such as gelatin and glycerin, or sucrose and acacia), and emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art.
- For example, the composition comprising the vector, the population of the immune cells or TSCs, or the in vitro engineered cells can comprise a buffering agent, e.g., TRIS. In one embodiment, the composition can comprise TRIS and/or glycerin. In another embodiment, the composition also comprises acidifiers, anionic or nonionic surfactants, compatibility agents, and/or diluents.
- Formulations suitable for administration via inhalation include aerosol formulations. The aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also can be formulated as non-pressurized preparations, for delivery from a nebulizer or an atomizer.
- Formulations suitable for parenteral administration include aqueous and nonaqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
- Formulations suitable for anal administration can be prepared as suppositories by mixing the active ingredient with a variety of bases such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate.
- In addition, the composition can comprise additional therapeutic or biologically-active agents. For example, therapeutic factors useful in the treatment of a particular indication can be present. Factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the gene transfer vector and physiological distress. Immune system suppressors can be administered with the composition method to reduce any immune response to the gene transfer vector itself or associated with a disorder. Altern-atively, immune enhancers can be included in the composition to upregulate the body's natural defenses against disease. Moreover, cytokines can be administered with the composition to attract immune effector cells to the tumor site.
- In the particular embodiment described herein, the invention provides a method for treating a tumor, comprising the steps in order of:
-
- a. administering intratumorally in a mammal a population of an in vitro engineered immune cells or TSC; and
- b. administering to said mammal a therapeutically effective amount of an activating ligand.
- In one embodiment, the activating ligand is administered at substantially the same time as the composition comprising the in vitro engineered immune cells or TSC or the vector, e.g., adenoviral vector, e.g., within one hour before or after administration of the cells or the vector compositions. In another embodiment, the activating ligand is administered at or less than about 24 hours after administration of the in vitro engineered immune cells or TSC or the vector. In still another embodiment, the activating ligand is administered at or less than about 48 hours after the in vitro engineered immune cells or TSC or the vector. In another embodiment, the ligand is RG-115932. In another embodiment, the ligand is administered at a dose of about 1 to 50 mg/kg/day. In another embodiment, the ligand is administered at a dose of about 30 mg/kg/day. In another embodiment, the ligand is administered daily for a period of 7 to 28 days. In another embodiment, the ligand is administered daily for a period of 14 days. In another embodiment, about 1×106 to 1×108 cells are administered. In another embodiment, about 1×107 cells are administered.
- In one embodiment, dendritic cells are engineered to conditionally express IL-2 and IL-12. IL-2 exerts potent immunoregulatory effects on effector and regulatory T, NK and NK-T cells. It is expected that expressing IL-2 and IL-12 in cells will result in reciprocal upregulation of each others receptor and induce different by complementary biological effects by virtue of separate signaling pathways. It is also expected that the combination of IL-2 and IL-12 will lengthen the duration of immune stimulation and reduce the effective dose of cells that may be more tolerated by the animal. See Dietrich 2002, Wigginton 2002, 2001, 1996 and Koyama, 1997, McDermott and Atkins 2008; Berntsen et al 2008; Tarhini et al 2008; Heemskerk et al 2008; Horton et al 2008. The polynucleotide sequences of IL-2 are available under accession numbers U25676 (human); NM—008366 (mouse); NM—204153 (chicken); and NM—053836 (rat). The polynucleotide sequences of IL-12 are available under accession numbers NM—000882 (human IL12A); NM—002187 (human IL12B); NM—008351 (mouse ILl2a); NM—008352 (mouse IL12b); NM—213588 (chicken IL12A); NM—213571 (chicken IL12B); NM—053390 (rat IL12a); and NM—022611 (rat IL12b). SEQ ID NOS: 13, 15, 21 and 23 code for human and mouse IL-12 and subunits thereof.
- In another embodiment, dendritic cells are engineered to conditionally express IL-18 and IL-12. IL-18 induces IFN-gamma production and promotes T helper cell development and NK activation. In addition, IL-18 can augment GM-CSF production and decrease IL-10 production. It is expected that expressing IL-18 and IL-12 will overcome the limitations observed when either cytokine is administered alone. It is expected that expression of IL-12 and IL-18 in dendritic cells will stimulate more vigorous tumor antigen-specific Th1 responses than when dendritic cells are transduced with either cytokine alone.
- The intratumoral injection of DCs engineered to secrete both IL-12 and IL-18 mediated the highest levels of INF-γ production and complete tumor rejection (Tatsumi 2003). See, Vujanovic, 2006. See also Coughlin, 1998, Subleski, 206, Tatsumi, 2003, and Sabel, 2007; Shiratori et al 2007; Lian et al 2007; Iinuma et al 2006. See above for IL-12 polynucleotide sequences. The polynucleotide sequences of IL-18 are available under accession numbers U90434 (human); NM—008360 (mouse); EU747333 (chicken); and AY258448 (rat).
- In another embodiment, dendritic cells are engineered to conditionally express IL-15 and IL-12. IL-15 shares some biologic activities with IL-2 that also makes it potentially useful for therapies against cancer. IL-15 stimulates the proliferation of NK cells and activated T cells, and supports the expansion of effector T cells. It has been reported that IL-15 presentation synergized with IL-12 for enhanced IFN-gamma production by NK cells. Koka, 2004; Basak 2008; Lasek et al 2004. Intratumoral delivery of IL-15 and IL-12 induced significant tumor regression in a melanoma model (Lasek 1999). See above for the IL-12 polynucleotide sequences. SEQ ID NOS: 11 and 19 code for the human and mouse IL-15.
FIGS. 2 and 4 are plasmid maps for expression systems which may be used for the human and mouse IL-12 and IL-15. - In another embodiment, dendritic cells are engineered to conditionally express IL-21 and IL-12. IL-21 and its receptor shares sequence homology with IL-2 and IL-15. IL-21 promotes the expansion and maturation of NK cells. The biologic effects of IL-21 potentially synergize with IL-12 as treatment of NK cells with IL-21 results in a significant upregulation of IL-12 receptor. In addition, IL-21 can enhance IL-12 signal transduction and cooperated for increased IFN-gamma production. See above for IL-12 polynucleotide sequences. The polynucleotide sequences of IL-21 are available under accession numbers AF254069 (human); NM—021782 (mouse); NM—001024835 (chicken); and NM—001108943 (rat). SEQ ID NOS: 6, 7, 8, 9, and 17 code for human and mouse IL-21. SEQ ID NOS: 1 and 2 are polynucleotide constructs that code for mouse and human IL-12 and IL-21.
FIGS. 7 and 8 are plasmid maps for expression systems which may be used to express human and mouse IL-12 and IL-21, respectively. - In another embodiment, dendritic cells are engineered to conditionally express TNF-alpha and IL-12. TNF-alpha is a potent activator of immune cells and mediates antitumor properties. In addition, TNF-alpha can synergize with IL-12 for enhanced expression of IFN-gamma and IL-12 receptor on T cells. In an animal study, application of both IL-12 and TNF-alpha resulted in tumor infiltration by DN8++ T cells, significant IFN-gamma production, and subsequent tumor regression. See Sabel, 2003, 2004, 2007, Taniguchi, 1998, Lasek, 2000; and Xia et al 2008. See above for IL-12 polynucleotide sequences. The polynucleotide sequences coding for TNF-alpha are available from under accession numbers X02910 (human); NM—013693 (mouse); and BC107671 (rat).
- In another embodiment, dendritic cells are engineered to conditionally express IL-7 and IL-12. IL-7 is a member of the IL-2 family and is important for T cell and B cell lympophoiesis. IL-7 regulates the homeostasis of survival and proliferation of naïve and memory CD8+ T cells. IL-7 has been proved to enhance CTL generation against tumors. In addition, IL-12 acts directed on CD8+ T cells to enhance IL-7 mediated proliferation. Further, it has been reported that IL-7 and IL-12 synergistically enhance CD8+ T cell cytotoxicity. Mehrotra, 1995; Sharma et al 2003; Tirapu et al 2002. Thus, it is expected that IL-7 and IL-12 coexpression will provide more optimal antitumor responses. See above for polynucleotide sequences coding for IL-12. The polynucleotide sequences coding for IL-7 are available under accession numbers J04156 (human); NM—008371 (mouse); NM—001037833 (chicken); and NM—013110 (rat).
- In another embodiment, dendritic cells are engineered to conditionally express GM-CSF and IL-12. GM-CSF regulates hematopoietic progenitor cell differentiation and proliferation, and plays a particularly important role in the maturation of professional antigen presenting cells (APC) such as dendritic cells. GM-CSF also enhances the capacity of dendritic cells to process and present antigens. GM-CSF functions differently than IL-12 and both elicit significant antitumor responses in animal studies. The combination of IL-12 (T cell activation) and GM-CSF (dendritic cell activation) is expected to result in more potent antitumor immunity. In animal studies, GM-CSF in combination with IL-12 treatment significantly suppressed tumor growth in multiple cancer models. Wang, 2001; Chang, 2007; Jean, 2004; Nair, 2006; Hill 2002; Small et al 2007. In human trials, GM-CSF+IL-12 were used successfully for treating myeloma patients, where the combined actions of both cytokines led to a reduction in circulating B cells. Rasmussen, 2003; Hansson, 2007; Abdalla, 2007. It is expected that coexpression of GM-CSF and IL-12 in a single cell will avoid unwanted systemic effects such as reductions in circulating B cells. See above for polynucleotide sequences coding for IL-12. The polynucleotide sequences of GM-CSF are available under accession numbers M11734 (human); NM—009969 (mouse); EU520303 (chicken); NM—001037660 (rat Csf2ra); and NM—133555 (rat Csf2rb).
- In another embodiment, dendritic cells are engineered to conditionally express a chemokine (e.g., CCL3 (MIP-1a), CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12 (SDF-1), or CCL21 (6Ckine)) and IL-12. Chemokines are chemoattractant cytokines that regulate the trafficking and activation of leukocytes and other cell types under a variety of inflammatory and noninflammatory conditions. Inflammatory cytokines control the recruitment of leukocytes in inflammation and tissue injury. Homeostatic chemokines fulfill housekeeping functions such as navigating leukocytes (e.g., dendritic cells) to and within secondary lymphoid organs as well as in bone marrow and the thymus during hematopoiesis. In animal studies, intratumoral co-injection of two separate adenoviruses expressing IL-12 and
CXCL 10 led to 100% regression of tumor nodules derived from the CT26 murine colorectal adenocarcinoma cell line. Narvaiza et al., 2000. Emtage et al., 1999, describe two double recombinant adenovirus vectors expressing either IL2 and XCL1 (lymphotactin) or IL-12 and XCL1. Intratumoral injection of the vectors breast adenocarcinoma tumors in mice elicited potent antitumor responses and gave rise to protective immunity. In other animal studies, co transduction of adenoviral vectors expressing IL-12 and CCL27 resulted in tumor regression and long term specific immunity. Gao et al., 2007. Thus, it is expected that the coexpression of a chemokine and IL-12 according to the invention will result in synergistic antitumor activity. - In another embodiment, dendritic cells are engineered to conditionally express an antiangiogenic cytokine (e.g., IP-10 and Mig) and IL-12. IP-10 and Mig are chemoattractants for T cells and NK cells and their ability to inhibit angiogenesis is dependent on NK cells. Animal studies have shown that combination therapy with two adenoviruses, one expressing IP10 and another expressing IL-12, resulted in marked antitumoral synergy. Narvaiza et al., 2000. In other studies, adenovirus vectors expressing IP10 or MIG and/or IL-12 were administered intratumorally in a murine model of mammary adenocarcinoma and fibrosarcoma. It was found that administration of IP-10 or MIG in combination with IL-12 resulted in considerable tumor regression and increased survival time of tumor-bearing animals as compared to IP10, MIG, IL-12 alone or control treated animals, with the IP-10, IL12 combination being most effective. Palmer, 2001. See also Mazzolini, 2003; and Huang 2004. Thus, it is expected that the coexpression of an antiangiogenic cytokine and IL-12 will result in synergistic antitumor activity.
- To demonstrate an effective IL-12-mediated gene therapy, a conditional cDNA expression system is used that allows one to turn on an immunomodulator and/or IL-12 production by immune cells or TSC at various time points post-intratumoral injection. Based on the results in the aggressive B16 melanoma model in C57BL/6 mice, the following conclusions are made: 1) elevated levels of IL-12 are secreted from DC.RheoIL12 in the presence of the activating ligand RG-115830 but not in the absence of the ligand; 2) intratumoral DC.RheoIL12-based therapy is as effective as intratumoral DC.cIL12-based therapy as long as RG-115830 is administered to treated animals within 24 h of DC injection (and at later time points of ligand provision, RG-115830 therapy fails); 3) IL-12 expression in DC appears to prolong the survival of these cells in the tumor microenvironment and is associated with higher numbers of intratumorally-injected DC that migrate to tumor-draining lymph nodes; and 4) the strongest immune correlate to therapy outcome is the level of tumor-specific CD8+ T cells cross-primed by the therapy and not the number of injected DC sustained in the tumor microenvironment. Overall, these data suggest that DC.IL12-based therapies likely succeed based on their positive influence on the afferent (cross-priming) of Type-1 CD8+ T cell effectors and not on later efferent events, such as injected DC-mediated recruitment of anti-tumor T cells into the tumor microenvironment, etc.
- Prior to intratumoral injection, the cells (immune cells or TSC) may be treated with a factor to stimulate the activity of the cells. For example, the cells may be treated with a co-stimulatory molecule such as positive co-stimulatory molecule including OX40L, 4-1 BBL, CD40, CD40L, GITRL, CD70, LIGHT or ICOS-L or a negative co-stimulatory molecule such as anti-CTLA4, anti-PD-L1 or anti-PD-L2 antibodies. For example, the cells (e.g., immune cells or TSC) may be incubated with a cell expressing one or more co-stimulatory molecule, e.g., J588 lymphoma cells expressing CD40 ligand molecule. In another embodiment, the cells (immune cells or TSC) may be treated with a counter immune suppressant molecule (tolerance inhibitor) such as anti-TGF-beta antibodies (for inhibiting TGF signaling within the microenvironment), anti-IL10 antibodies, TGFbRII DN (to inhibit TGF signaling within gene modified cells), IL-10R DN, dnFADD (to inhibit cell death pathways within the cells), anti-SOCS1 antibodies, siRNA or decoy (to inhibit suppressive cytokine signaling within the cells), or anti-TGFa antibodies.
- The recombinant adenoviruses carrying the polynucleotide sequences shown in
FIGS. 1-8 are produced. For example, hIL-21 is produced by cotransfection of the hIL-21 expression vector, linearized by restriction digestion at a site upstream of the left ITR, and the appropriate (example E3 deleted) adenoviral backbone in a permissive cell line such as HEK293 cells. The adenoviral vector carrying the murine immunomodulatory genes is used for transduction of murine dendritic cells or TSC for use in murine therapeutic models. For human therapeutic application, a polynucleotide encoding the human homologue of the immunomodulatory gene is inserted in the appropriate vector. The adenoviral vector for human therapeutic application is produced under GMP conditions. Example of a treatment outline (clinical trial) for stage III/IV melanoma patients is as follows: The treatment in this case involves an intratumoral injection of the adenoviral transduced dendritic cells and 14 daily oral administration of the activator drug (ligand). Subjects are screened 30 days to one week prior to the clinical trial. Each subject is asked to sign an informed consent before any procedures are initiated. The investigator will inform all subjects of the nature, aims, duration, potential hazards, and procedures to be performed during the trial and the possibility that their medical records may be reviewed by FDA. Subjects (a total of 16 to 20) are randomly grouped into 4 cohorts. All cohorts will receive an intratumoral injection of up to 5×107 transduced dendritic cells approximately 3 hours after the first dose of oral administration of the ligand. The 4 cohorts differ in the daily oral dose of ligand received:example cohort 1=0.01 mg/kg;cohort 2=0.3 mg/kg;cohort 3=1 mg/kg;cohort 4=3 mg/kg. During the course of the treatment, blood is drawn at specified time intervals for evaluation of single dose and steady state pharmacokinetics of the Activator Drug and its major metabolites. Also, blood is drawn at specified time points for the evaluation of humoral and cellular immune responses against the viral vector, RTS components and the tumor. Urine is collected and blood drawn at specific time points for serum chemistry, urinalysis, and hematology (safety profile). Tumor and/or draining lymph node biopsies are taken at specified time points to assess the transgene expression and the immune response to the tumor as a result of the therapy. Criteria for early termination are established for patients in case of adverse events, and the adverse events are recorded. The patients are followed up at 1, 2, 3 and 4 months for adverse events and therapeutic outcome. - In another embodiment, a subject in need of treatment of a tumor is (a) administered dendritic cells engineered to express an immunomodulator, for example, an immunomodulator disclosed herein, either consitutively or conditionally, and/or (b) a vector expressing an immunomodulator, for example, an immunomodulator disclosed herein, either constitutively or conditionally, is injected intratamorally to the subject. In one embodiment, the dentritic cells are engineered to express an Ad-immunomodulator vector, and particularly the Ad-RTS-immunomodulator vector. In another embodiment, the vector that is injected intratumorally to the subject is an Ad-immunomodulator vector, and particularly the Ad-RTS-immunomodulator vector.
- In another embodiment, a subject in need of treatment of a tumor is (a) administered dendritic cells engineered to express IL-12, either consitutively or conditionally, and (b) a vector expressing IL-12, either constitutively or conditionally, is injected intratamorally to the subject. In one embodiment, the dentritic cells are engineered to express an Ad-IL-12 vector, and particularly the Ad-RTS-IL-12 vector. In another embodiment, the vector that is injected intratumorally to the subject is an Ad-IL-12 vector, and particularly the Ad-RTS-IL-12 vector.
- In another embodiment, a subject in need of treatment of a tumor is (a) administered dendritic cells engineered to express IL-12, either consitutively or conditionally, and (b) the subject is administered one or more anticancer chemotherapeutic agents. In one embodiment, the engineered dentritic cells are engineered to express an Ad-IL-12 vector, and particularly the Ad-RTS-IL-12 vector. The one or more anticancer chemotherapeutic agents can be administered prior to the engineered dendritic cells are administered, after the engineered dendritic cells are administered, or concurrently with the administration of the engineered dendritic cells. In another embodiment, the anticancer chemotherapeutic is paclitaxel, a paclitaxel derivative or analog, temozolomide, a temozolomide derivative or analog, sunitinib, a sunitinib derivative or analog, gemcitabine, or a gemcitabine derivative or analog.
- In another embodiment, a subject in need of treatment of a tumor is (a) administered dendritic cells engineered to express IL-12, either consitutively or conditionally, (b) a vector expressing IL-12, either constitutively or conditionally, is injected intratumorally to the subject, and (c) the subject is administered one or more anticancer chemotherapeutic agents. In one embodiment, the dentritic cells are engineered to express an Ad-IL-12 vector, and particularly the Ad-RTS-IL-12 vector. In another embodiment, the vector that is injected intratumorally to the subject is an Ad-IL-12 vector, and particularly the Ad-RTS-IL-12 vector. The one or more anticancer chemotherapeutic agents can be administered prior to the engineered dendritic cells and the vector expressing IL-12 are administered, after the engineered dendritic cells and vector expressing IL-12 are administered, or concurrently with the administration of the engineered dendritic cells and the vector expressing IL-12. In one embodiment, the anticancer chemotherapeutic is paclitaxel, a paclitaxel derivative or analog, temozolomide, a temozolomide derivative or analog, sunitinib, a sunitinib derivative or analog, gemcitabine, or a gemcitabine derivative or analog.
- In another embodiment, a subject in need of treatment of a tumor is (a) administered dendritic cells engineered to express an immunomodulator, for example, an immunomodulator disclosed herein, either consitutively or conditionally, and (b) the subject is administered one or more anticancer chemotherapeutic agents. In one embodiment, the engineered dentritic cells are engineered to express an Ad-immunomodulator vector, and particularly the Ad-RTS-immunomodulator vector. The one or more anticancer chemotherapeutic agents can be administered prior to the engineered dendritic cells are administered, after the engineered dendritic cells are administered, or concurrently with the administration of the engineered dendritic cells. In one embodiment, the anticancer chemotherapeutic is paclitaxel, a paclitaxel derivative or analog, temozolomide, a temozolomide derivative or analog, sunitinib, a sunitinib derivative or analog, gemcitabine, or a gemcitabine derivative or analog.
- In another embodiment, a subject in need of treatment of a tumor is (a) administered dendritic cells engineered to express an immunomodulator, for example, an immunomodulator disclosed herein, either consitutively or conditionally, (b) a vector expressing an immunomodulator, for example, an immunomodulator disclosed herein, either constitutively or conditionally, is injected intratumorally to the subject, and (c) the subject is administered one or more anticancer chemotherapeutic agents. In one embodiment, the dentritic cells are engineered to express an Ad-immunomodulator vector, and particularly the Ad-RTS-immunomodulator vector. In another embodiment, the vector that is injected intratumorally to the subject is an Ad-immunomodulator vector, and particularly the Ad-RTS-immunomodulator vector. The one or more anticancer chemotherapeutic agents can be administered prior to the engineered dendritic cells and the vector expressing the immunomodulator are administered, after the engineered dendritic cells and vector expressing the immunomodulator are administered, or concurrently with the administration of the engineered dendritic cells and the vector expressing the immunomodulator. In one embodiment, the anticancer chemotherapeutic is paclitaxel, a paclitaxel derivative or analog, temozolomide, a temozolomide derivative or analog, sunitinib, a sunitinib derivative or analog, gemcitabine, or a gemcitabine derivative or analog.
- In any of the methods of the present invention, the disease or disorder may be a disease or disorder disclosed in the present application. In one embodiment, the disease or disorder is a disease or disorder listed in Table 1 herein. In another embodiment, the disease or disorder is a disease or disorder listed in Table 3 herein.
- In any of the methods of the present invention, the cancer or tumor may be a disease or disorder disclosed in the present application. In one embodiment, the cancer or tumor is a cancer or tumor listed in Table 1 herein. In another embodiment, the cancer or tumor is a cancer or tumor listed in Table 3 herein.
- It is possible to measure the effect of an immunomodulator and/or IL-12 expression on a population of cells by measuring the level of expression or activity of the Th1/Tc1 type cytokine, IFN-gamma in a biological sample from a patient.
- For the purposes of the invention, the invention provides a method for determining the efficacy of an in vitro engineered immune- or TSC-based therapeutic regimen in a cancer patient, comprising:
-
- a. measuring the level of expression or the level of activity or both of interferon-gamma (IFN-gamma) in a first biological sample obtained from a human patient before administration of in vitro engineered cells, e.g., immune cells or TSC, thereby generating a control level;
- b. administering intratumorally to said patient the in vitro engineered cells;
- c. administering to said patient an effective amount of activating ligand;
- d. measuring the level of expression or the level of activity or both of IFN-gamma in a second biological sample obtained from said patient at a time following administration of said activating ligand, thereby generating data for a test level; and
- e. comparing the control level to the test level of IFN-gamma, wherein data showing an increase in the level of expression, activity, or both of IFN-gamma in the test level relative to the control level indicates that the therapeutic treatment regimen is effective in said patient. The invention may also optionally comprise the additional steps of
- f. taking biopsy and counting tumor infiltrating lymphocytes (TIL) and/or
- g. observing tumor regression in response to the treatment.
- The term “subject” means an intact insect, plant or animal. It is also anticipated that the ligands will work equally well when the subject is a fungus or yeast. Animals for use with the invention include, but are not limited to, vertebrates, e.g., mammals such as humans, rodents, monkeys, and other animals, with humans or mice being more preferred. Other animals include veterinary animals such as dogs, cats, horses, cattle, sheep, goats, pigs and the like.
- The invention further provides a method of increasing expression of the immunomodulator, e.g., TNF-alpha, by introducing into the vector, e.g., a replication-deficient adenoviral vector, one or more regulatory sequence and optionally a nucleic acid encoding a signal peptide, wherein the vector conditionally express the immunomodulator. As used herein, the term “protein expression” includes without limitation transcription, post-transcription, translation, and/or post-translation. Also included in the invention is a method of increasing mRNA or protein expression of an immunomodulator, e.g., TNF-alpha, comprising generating a vector conditionally expressing TNF-alpha, wherein said vector further comprises one or more regulatory sequences connected to the polynucleotide sequence encoding said TNF-alpha, and adding an activating ligand, thereby inducing expression of the immunomodulator, wherein said one or more regulatory sequences and/or signal peptides improves expression of said TNF-alpha. Various regulatory regions for the invention including, but not limited to, 5′ untranslated region (5′UTR), 3′ UTR, or both have been described. In one embodiment, the 5′ UTR is 5U2. 5U2 is a fusion
canine SERCA2 intron 2 with a mutated putative consensus poly-A site, withexon 2 splice donor flanking on the 5′ end andexon 3 splice acceptor flanking on the 3′ end followed by a portion of the portion ofbovine casein 5′UTR. In another embodiment, the 3′ regulatory region is a polyadenylation signal of SV40 or hGH. - In certain embodiments, the method of the invention is also directed to improving TNF-alpha secretion by generating a vector conditionally expressing TNF-alpha, wherein said vector further comprises a signal peptide, thereby increasing secretion of TNF-alpha compared to a vector comprising the TNF-alpha native signal peptide gene, e.g., TNF-alpha wild-type signal peptide. In particular, the signal peptide used in the invention is codon-optimized. In a specific embodiment, the signal peptide is encoded by IL-2 wild-type signal peptide gene. In a further specific embodiment, the signal peptide is encoded by codon-optimized IL-2 signal peptide gene.
- Without wishing to be bound by theory, it is expected that the invention will support the use of intratumorally administered in vitro engineered immune- and TSC based gene therapy in the clinical setting, focusing on the objective clinical response as a primary study endpoint, and cross-primed anti-tumor CD8+ T cells (producing IFN-gamma) as a secondary study endpoint. The ability to turn the immunomodulator and/or IL-12 expression on and off in vivo adds an element of safety and therapeutic control to the treatment in that both the timing and level of protein expression may be controlled by the administration of ligand, and further that the timing of immunomodulator and/or IL-12 expression is expected to be critical to the therapeutic effectiveness of the method.
- The invention further supports the therapeutic applications of in vitro engineered cells with conditionally expressed genes of interest as innovative approaches for the effective and efficient treatment of human diseases.
- The present invention also provides methods for treating a tumor, reducing a tumor size, or preventing a tumor formation in a mammal in need thereof, in which a vector for conditionally expressing protein(s) having the function(s) of one or more immunomodulators that is not contained within a cell, is administered intratumorally to tumor microenvironments. In this embodiment, the vector is administered to the tumor without being packaged in a cell, such as as immune cell or a TSC. The present invention also provides methods for treating a disease in a mammal in need thereof, in which a vector for conditionally expressing protein(s) having the function(s) of one or more immunomodulators that is not contained within a cell, is administered to said mammal. In this embodiment, the vector is administered to the tumor without being packaged in a cell, such as immune cell or a TSC.
- In one embodiment, immune cells, TSC, dendritic cells, or bone marrow dendritic cells are not administered intratumorally with the vector.
- In another embodiment, a vector of the invention that is not contained within a cells is administered intratumorally simulataneously with, before, or after immune cells, TSC, dendritic cells, or bone marrow dendritic cells are are administered intratumorally.
- In one embodiment, the vector of the invention that is not contained within a cell is administered intratumorally to the same lesion as the immune cells or TSC are administered. In another embodiment, the vector of the invention that is not contained within a cell is administered intratumorally to a different lesion than the immune cells or TSC are administered.
- In one embodiment, the vector is administered to the same lesion(s) in each cycle of administration. In another embodiment, the vector that is administered is not administered to the same lesion(s) in each cycle of administration.
- In one embodiment, the tumor is a tumor of any of the cancers listed herein, e.g., in Tables 1 and 3. In another embodiment, the tumor is a melanoma tumor, a colorectal tumor, a pancreatic tumor, a breast tumor, a lung tumor or a renal tumor. In another embodiment, the tumor is a malignant melanoma. In a another embodiment, the tumor is a Stage III C or a Stage IV malignant melanoma.
- In one embodiment, the intratumoral dosage is at least about 1.0×109 viral particles per cycle of vector administration. In another embodiment, the intratumoral dosage is at least about 1.0×1010 viral particles per cycle of vector administration. In another embodiment, the intratumoral dosage is about 1.0×109 to about 1.0×1013 viral particles per cycle of vector administration. In another embodiment, the intratumoral dosage is about 1.0×1010 to about 1.0×1013 viral particles per cycle of vector administration. In another embodiment, the intratumoral dosage is about 1.0×1010, about 1.0×1011, about 1.0×1012 or about 1.0×1013 viral particles per cycle of vector administration. In one embodiment, the vector is AD-RTS-IL-12.
- In another embodiment, the present invention further provides methods for treating a liver disease in a mammal in need thereof, in which a vector for conditionally expressing protein(s) that is not contained within a cell, is administered to said mammal.
- In another embodiment, the present invention further provides methods for treating a lysosomoal storage disease in a mammal in need thereof, in which a vector for conditionally expressing protein(s) that is not contained within a cell, is administered to said mammal.
- In another embodiment, the present invention further provides methods for treating a disease in a non-human mammal in need thereof, in which a vector for conditionally expressing protein(s) that is not contained within a cell, is administered to said mammal.
- The activating ligand dosage is about 5-100 mg/day, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mg/day. In one embodiment, the activating ligand is administered at least once a day. In another embodiment, the activating ligand is administered once a day for about 14 days.
- In one embodiment, at least two dosages of the vector (e.g., about 1×1011 and 1×1012) are used in combination with at least three different dosage levels of the activating ligand (e.g., about 5 mg/day to about 100 mg/day).
- One of ordinary skill in the art will be able to optimize dosages in order to provide range of effective plasma levels of the vector, for various degrees of activating ligand activation.
- In one embodiment, the dosage of activating ligand administered to the subject is changed over the period of administration of the activating ligand within the cycle of intratumoral vector administration. In another embodiment, the dosage of activating ligand administered to the subject is decreased over the period of administration of the activating ligand within the cycle of intratumoral vector administration. In another embodiment, the dosage of activating ligand administered to the subject is increased (escalated) over the period of administration of the activating ligand within the cycle of intratumoral vector administration.
- In one embodiment, the subject is treated with 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles of intratumoral vector administration. In another embodiment, the subject is treated with 3-7 cycles of intratumoral vector administration. In another embodiment, the subject is treated with 4-6 cycles of intratumoral vector administration. In another embodiment, the subject is treated with 5 or 6 cycles of intratumoral vector administration. In another embodiment, the subject is treated with 6 cycles of intratumoral vector administration.
- In one embodiment, each cycle of intratumoral vector administration is performed 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks apart. In another embodiment, each cycle of intratumoral vector administration is performed 4 weeks apart.
- In one embodiment, the dosage of the vector is changed in each subsequent cycle of intratumoral vector administration. In another embodiment, the dosage of the vector is decreased in each subsequent cycle of intratumoral vector administration. In another embodiment, the dosage of the vector is increased in each subsequent cycle of intratumoral vector administration.
- In one embodiment, the dosages of vector and activating ligand, the number and length of the cycles of intratumoral vector administration, the frequency of vector administration and the frequency of activating ligand administration is set forth in Table 8 in Example 11 herein.
- In one embodiment, the invention also provides a pharmaceutical composition comprising pharmaceutically acceptable carrier and a vector of the invention that is not contained within a cell. Suitable carriers include, but are not limited to, saline, distilled water, sodium chloride solutions, the mixtures of sodium chloride and inorganic salts or their similar mixtures, the solutions of materials such as mannitol, lactose, dextran, and glucose, amino acid solutions such as glycine and arginine, the mixtures of organic acid solutions or salt solutions and glucose solutions, aqueous and nonaqueous, isotonic sterile injection solutions, which can contain antioxidants, chelating agents, buffers, bacteriostats, and solutes that render the formulation isotonic, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit dose or multidose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, for injections, immediately prior to use.
- In the event of conflict between any teaching or suggestion of any reference cited herein and the specification, the latter shall prevail, for purposes of the invention.
- All patents, patent applications and publications cited herein are fully incorporated by reference in their entireties.
- It is to be understood that the foregoing described embodiments and exemplifications are not intended to be limiting in any respect to the scope of the invention, and that the claims presented herein are intended to encompass all embodiments and exemplifications whether or not explicitly presented herein.
- U.S. application Ser. No. 12/247,738, entitled “Engineered Dendritic Cells And Uses For Treatment Of Cancer,” filed Oct. 8, 2008, is hereby incorporated by reference in its entirety. U.S. application Ser. No. 12/241,018, entitled “Therapeutic Gene-Switch Constructs And Bioreactors For The Expression Of Biotherapeutic Molecules, And Uses Thereof,” filed Sep. 29, 2008, is also hereby incorporated by reference in its entirety.
- A study is undertaken to determine the dose of dendritic cells and the most effective cytokine that is able to induce tumor-specific immune responses and antitumor activty in a Renca renal cell cancer tumor model
- Two tumor cell lines are used in this study: Renca and Renca-HA. The latter cell line is made by transfection of Renca cells with influenza virus hemagglutinin (HA). The advantage of Renca-HA model is the ability to trace antigen-specific T cells, since both CD8 and CD4 specific HA-derived epitopes are known and have been used.
- Specific Aim—determine the induction of HA-specific immune responses after intratumoral administration of dendritic cells.
- The Renca-HA tumor is established subcutaneously in BALB/c mice. When the tumor becoms palpable, dendritic cells are injected intratumorally. Dendritic cell administration is be repeated twice at 7-day intervals, for a total of 3 administrations.
- The following groups of mice are used (each group includes 3 mice):
- 1. Untreated mice;
- 2. Mice treated with 5×105 dendritic cells transduced with control plasmid;
- 3. Mice treated with 106 dendritic cells transuced with control plasmid;
- 4. Mice treated with 5×106 dendritic cells transduced with control plasmid;
- 5. The same as groups 2-4 using dendritic cells transduced with IL-12;
- 6. The same as groups 2-4 using dendritic cells transduced with IL-15; and
- 7. The same as groups 2-4 using dendritic cells transduced with IL-21.
- To test the effect of combination of different cytokines, mice are treated simultaneously with:
- 8. 5×105 dendritic cells transduced with IL-12, and 5×105 dendritic cells transduced with IL-15,
- 9. 5×105 dendritic cells transduced with IL-12 and 5×105 dendritic cells transduced with IL-21, and
- 10. 5×105 dendritic cells transduced with IL-15 and 5×105 dendritic cells transduced with IL-21.
- Four days after the last administration, lymph nodes of tumor-bearing mice are collected, and cells are stimulated with either MHC class I matched peptide (to detect CD8+ T cell responses) or MHC class II matched peptide (to detect CD4+ T cell responses).
- The following assays are used:
- 1. ELISPOT IFN-γ and IL-2;
- 2. T-cell proliferation;
- 3. Detection of TNFα, IL-10, IL-4, and GM-CSF release by lymph node cells.
- In addition, NK activity of lymph node cells is evaluated using YAC cells as targets.
- In parallel, cells are stimulated with anti-CD3/CD28 antibodies to evaluate non-specific response of T cells.
- The most effective dose of dendritic cells capable of inducing antigen-specific immune responses are determined.
-
Specific Aim 2—evaluate antitumor activity of dendritic cells transduced with cytokine genes. - Only those cytokine transduced dendritic cells that demonstrated statistically significant induction of immune responses are used in further experiments.
- Treatment of Renca-HA tumor-bearing mice is performed as described in
specific aim 1. One dose of DCs transduced with cytokines that shows specific activity in previous experiments is used. As a control, dendritic cells transduced with control adenovirus are used. To achieve statistical significance, each group includes 10 mice. - Tumor growth is evaluated. Renca-HA tumor contains an immunogeneic epitope that is useful for immunological monitoring and intitial testing of antitumor effect. However, to verify potential antitumor activity of the treatment non-transfected tumor cells needs to be used. Therefore, the experiments described above are repeated using the Renca tumor model.
- The safety, tolerance, transgene function, and immunological effects of intratumoral injection(s) of adenoviral transduced autologous dendritic cells engineered to express hIL-12 and one or more other immunodulators under control of the RTS, in subjects with stage III and IV melanoma will be evaluated through procedures such as those described below.
- A study involving study subjects with stage III and IV melanoma will be conducted in 4 cohorts (groups) of subjects each subject receiving a single intratumoral injection (into a melanoma tumor) of adenoviral transduced autologous (reinserted into the same subject that they came from) dendritic cells (DCs) engineered to express human interleukin-12 (hIL-12), and one or more other immunodulators, at a dose of 5×107 in combination with daily oral doses of activator drug (activating ligand). The study will use injections of dendritic cells transduced ex vivo (after the cells are removed from the subjects) with adenoviral vector for inducible expression of human IL-12 and one or more other immunodulators. The production off IL-12 and the one or more or other immunomodulators is “turned on” (induced) from the injected DCs through the activation of the RTS by the oral administration of the activator drug (RG-115932). Safety and tolerance will be assessed through physical examinations (including ECOG performance status), vital signs measurements, serum chemistry, urinalysis, hematology, adverse events “side-effects”, and antibodies and cellular immune response to the adenovirus, components of RTS, and the Activator Drug. To evaluate progress, single dose and steady-state pharmacokinetics/ADME of oral Activator Drug and its major metabolites, analysis of hIL-12 levels, other immunomodulator levels, and cellular immune response (T cells) in biopsies of the target tumors, draining lymph nodes, and peripheral circulation, as well as a serum cytokine profile will be measured.
- For instance, 16 subjects with stage III and IV melanoma are divided into four cohorts with
cohorts cohorts - The subjects will receive a single daily oral dose of activator drug (cohort 1: 0.01 mg/kg, cohort 2: 0.1 mg/kg, cohort 3: 1.0 mg/kg or cohort 4: 3 mg/kg) the first dose starting approximately 3 hours prior to the DC injection on
day 1 and continuing for 13 more consecutive days. Additional injection(s) of adenovirally transduced autologous dendritic cells in combination with 14 single (once) daily oral doses of activator drag may be administered to eligible subjects who meet the criteria for retreatment. Safety, tolerance, and dendritic cell function are assessed for all subjects in each group ofcohort 1 for up to one month after injection of the in vitro engineered dendritic cells before enrolling subjects to receive the next highest dose of the activator drug. The safety assessment will continue in all subjects for 3 months after the initial injection of the engineered dendritic cells with the possibility of extending the follow-up period to a total of six months to monitor subject safety if toxicity is observed or the subject receives additional injection(s) of the dendritic cells. - Such a study demonstrates the safety and tolerance of a single or multiple intratumoral injection(s) of adenoviral transduced autologous dendritic cells in combination with an oral activator drug in subjects with melanoma. The study provides steady-state pharmacokinetics/ADME of the oral activator drug. The study demonstrates functionality of the RTS in subjects by measuring hIL-12 expression and the expression of the one or more other immunomodulators of adenovirus transduced autologous dendritic cells in target tumor and/or draining lymph nodes in response to the activation of the RTS by the oral administration of the activator drug. Furthermore, the study demonstrates the immunological effects of the adenoviral transduced autologous dendritic cells in terms of the cellular immune response in the target tumor, draining lymph nodes, and peripheral circulation following oral administration of the activator drag.
- Melanoma is selected as an exemplary cancer, particularly with respect to melanoma. Melanoma in particular among solid tumors has been shown to respond to immunotherapy approaches, and melanoma tumors are readily accessible for intratumoral injection and biopsy. The subjects included in the study have unresectable stage III or IV melanoma, which has at least 0.5 cm in diameter, any tumor thickness, any number of lymph node involvement, in-transit metastases, or distant metastases.
- The recombinant DNA is transferred to dendritic cells (DC) by ex vivo adenoviral vector transduction. The recombinant DNA is used to express human IL-12(p70) and one or more other immunodulators from intratumorally injected immature dendritic cells which confers survival and stimulates maturation of DC in the tumor environment resulting in their subsequent migration to the draining lymph nodes. This leads to a bias toward the differentiation of T helper cells to Th1 type and also activation of tumor-specific cytotoxic T cells by cross priming with the tumor antigens.
- The recombinant DNA used as the recombinant adenoviral vector allows the expression of human IL-12 and one or more other immunodulators under the control of the RheoSwitch® Therapeutic System (RTS). The RTS comprises a bicistronic message expressed from the human Ubiquitin C promoter and codes for two fusion proteins: Gal4-EcR and VP16-RXR. Gal4-EcR is a fusion between the DNA binding domain (amino acids 1-147) of yeast Gal4 and the DEF domains of the ecdysone receptor from the insect Choristoneura fumiferana. In another embodiment, the RTS consists of a bicistronic message expressed from the human Ubiquitin C promoter and codes for two fusion proteins: Gal4-EcR and VP16-RXR. Gal4-EcR is a fusion between the DNA binding domain (amino acids 1-147) of yeast Gal4 and the DEF domains of the ecdysone receptor from the insect Choristoneura fumiferana. VP16-RXR is a fusion between the transcription activation domain of HSV-VP16 and the EF domains of a chimeric RXR derived from human and locust sequences. These Gal4-EcR and VP16-RXR sequences are separated by an internal ribosome entry site (IRES) from EMCV. These two fusion proteins dimerize when Gal4-EcR binds to a small molecule drug (RG-115932) and activate transcription of hIL-12 and one or more other immunodulators from a Gal4-responsive promoter that contains six Gal4-binding sites and a synthetic minimal promoter. The RTS transcription unit described above is placed downstream of the hIL-12 and one or more other immunodulators transcription units. This whole RTS-hIL12-immunomodualtor cassette is incorporated into the
adenovirus 5 genome at the site where the E1 region has been deleted. The adenoviral backbone also lacks the E3 gene. A map for the adenoviral vector Ad-RTS-hIL-12 is shown in FIG. 8 of US 2009/0123441 A1. - The recombinant adenoviral vector used in this study contains the following exemplary regulatory elements in addition to the viral vector sequences: Human Ubiquitin C promoter, Internal ribosome entry site derived from EMCV, an inducible promoter containing 6 copies of Gal4-binding site, 3 copies of SP-1 binding sites, and a synthetic minimal promoter sequence, SV40 polyadenylation sites, and a transcription termination sequence derived from human alpha-globin gene. It should be understood that other regulatory elements could be utilized as alternatives.
- An exemplary recombinant adenoviral vector Ad-RTS-hIL-12-immunomodulator(s) is produced in the following manner. The coding sequences for the receptor fusion proteins, VP16-RXR and Gal4-EcR separated by the EMCV-IRES (internal ribosome entry site), are inserted into the adenoviral shuttle vector under the control of the human ubiquitin C promoter (constitutive promoter). Subsequently, the coding sequences for the p40 and p35 subunits of hIL-12 separated by IRES, and one or more other immunomodulators, is placed under the control of a synthetic inducible promoter containing 6 copies of Gal4-binding site are inserted upstream of the ubiquitin C promoter and the receptor sequences. The shuttle vector contains the
adenovirus serotype 5 sequences from the left end to map unit 16 (multi), from which the E1 sequences are deleted and replaced by the RTS, IL-12 and one or more other immunomodulator sequences (RTS-hIL-12). The shuttle vector carrying the RTS-hIL-12-immunodulator(s) is tested by transient transfection in HT-1080 cells for Activator Drug-dependent IL-12 and other immunomodulator(s) expression. The shuttle vector is then recombined with the adenoviral backbone by cotransfection into HEK 293 cells to obtain recombinant adenovirus Ad-RTS-hIL-12-immunomodulator(s). The adenoviral backbone contains sequence deletions ofmu 0 to 9.2 at the left end of the genome and the E3 gene. The shuttle vector and the adenoviral backbone contain the overlapping sequence from mu 9.2 tomu 16 that allows the recombination between them and production of the recombinant adenoviral vector. Since the recombinant adenoviral vector is deficient in the E1 and E3 regions, the virus is replication-deficient in normal mammalian cells. However, the virus can replicate in HEK 293 cells that harbor the adenovirus-5 E1 region and hence provide the E1 function in trans. - An exemplary recombinant adenoviral vector is produced in the following manner: The linearized shuttle vector carrying the DNA elements for inducible expression of human IL12 and one or more other immunomodulators, and the adenoviral backbone are co-transfected into HEK293 cells. Recombination between the overlapping sequences on the shuttle vector and the viral backbone results in the production of recombinant adenovirus and is packaged into viral particles in the HEK293 cells. The HEK293 cells are grown in DMEM containing fetal bovine serum.
- The virus used for the proposed study was purified by CsCl density gradient centrifugation. The recombinant adenovirus undergoes two rounds of plaque purification and the resulting seed stock is used to produce a master viral bank (MVB) by amplification in HEK293 cells from a fully characterized master cell bank. The MVB undergoes extensive cGMP/GLP release tests including replication competent adenovirus (RCA), sterility, mycoplasma, adventitious viruses, retrovirus, human viruses HIV1/2, HTLV1/2, HAV, HBV, HCV, EBV, B19, CMV, HHV-6, 7 and 8, bovine and porcine virus, complete vector sequencing and functional testing by AD-induced expression of IL-12 and one or more other immunomodulators in human cell lines.
- The virus from MVB may be used for production of the purified virus in a cGMP facility and may again undergo release tests including identity, RCA, sterility, mycoplasma, adventitious viruses, viral particle-to-infectious units ratio, contamination of host cell DNA, endotoxin and proteins and functional testing by AD-induced expression of IL-12 and one or more other immunomodulators in human cell lines.
- A suitable method for producing recombinant adenovirus is also set forth in Anderson, R. D., Gene Therapy 7: 1034-1038 (2000).
- A suitable method for recombinant adenovirus into host cells is set forth in Komita, H. et al., Cancer Gene Therapy 16: 883-891 (2009)
- Transduction of Autologous Dendritic Cells by Adenovirus Containing hIL-12 Transgene and One or More Other Immunodulators and RheoSwitch® Therapeutic System (RTS)
- Dendritic cells derived from the human subjects are transduced ex vivo and injected into the tumor. The DC will be characterized before viral transduction for viability, purity (typically >80% cells showing DC phenotype), sterility, mycoplasma and endotoxin. After viral transduction, the cells are washed repeatedly to remove any unabsorbed virus. Supernatant from the last wash will be tested for the content of residual virus by PCR. Since the DCs are transduced ex vivo by adenoviral vector (non-integrating virus) and the life span of DCs after intratumoral injection and the subsequent migration to draining lymph nodes is short, it is not expected that the viral DNA will be incorporated into any non-target cells. The protocol used for adenoviral transduction of DCs is expected to yield 80-90% transduction and is considered very efficient.
- Harvesting of PBMC by Leukapheresis:
- Subjects undergo a standard 90 to 120 minutes leukapheresis at the Apheresis Unit of the UPCI Outpatient. The leukapheresis procedure involves the removal of blood from a vein in one arm; the passage of blood through a centrifuge (cell separator), where its components are separated and one or more components are removed; and the return of the remaining components to the subject's vein in the same or other arm. No more than 15% of the subject's total blood volume is withdrawn at any one time as blood is processed through the cell separator device. In the cell separator, blood is separated into plasma, platelets, white cells and red blood cells. White blood cells (WBC) are removed and all the other components are returned into the subject's circulation. Every attempt is made to use two peripheral IV lines for this procedure. If that is not possible, a central line may be necessary. The subject has to be cleared by physician to undergo leukapheresis, and is routinely screened for vital signs (including blood pressure) prior to the procedure.
- Processing:
- After collection, the leukapack is delivered by hand to the CPL, and is immediately processed by centrifugal elutriation in ELUTRAT™. This is a closed system validated for clinical use. The monocyte fraction is recovered, and after the recovery and viability of cells are established, they are transferred to an Aastrom cartridge for 6-day culture in the presence of IL-4 and GM-CSF. All processing and washing procedures are performed under sterile conditions.
- Initial Plating:
- Monocytes recovered from a single leukapack are counted in the presence of a trypan blue dye to determine the number of viable cells. Monocytes are evaluated for purity by flow cytometry. Monocytes are resuspended at 5 to 10×106 cells/mL in serum-free and antibiotic-free CellGenix medium, containing 1,000 IU/mL of IL-4 and 1,000 IU/mL of GM-CSF per SOP-CPL-0166, and placed in an Aastrom cartridge. A minimum loading volume of 50 ml and a minimum cell number are required for cassette inoculation.
- Culture:
- The Aastrom cartridge is placed in the incubator in the Replicell System, a fully closed, cGMP-compatible automated culture device for immature DC generation.
- Immature DC Harvest:
- On
day 6, the Aastrom cartridge is removed from the incubator and immature DCs are harvested. The cells are recovered by centrifugation at 1,500 rpm, washed in CellGenix medium, counted in the presence of a trypan blue dye and checked for morphologic and phenotypic characteristics. - Viability:
- This is determined by performing hemocytometer cell counts in the presence of trypan blue. Generally, >95% of harvested cells are viable, i.e., exclude a trypan blue dye. If viability is less than 70% the immature DCs will be discarded.
- Phenotyping:
- The cells generated in culture are counted by microscopic observation on a hemocytometer, and a preliminary differential count (DC vs. lymphocytes) is obtained using a trypan blue dye. Confirmation of the differential count is made by flow cytometry, gating on DC vs. lymphocytes and using high forward and side scatter properties of immature DC as the criterion for their identification. Immature DCs routinely contain >80% of cells with dendritic cell morphology and have DC phenotype.
- IL-12p70 Potency Assay:
- It has been established that mature DCs (mDCs) have the ability to produce IL-12p70 spontaneously or upon activation with CD40L with or without addition of innate immunity signals (e.g., LPS). A standardized IL-12p70 production assay was recently established and is applicable to small samples or large lots of DC vaccines generated under a variety of conditions. The current potency assay consists of two distinct steps, the first involving co-incubation of responder DCs with J588 lymphoma cells stably transfected with the human CD40 ligand gene as stimulators. The second step involves testing of supernatants from these co-cultures for levels of IL-12p70 secreted by DCs stimulated with J558/CD40L+/−LPS in the Luminex system. This potency assay has an inter-assay CV of 18.5% (n=30) and a broad dynamic range, which facilitates evaluation of various DC products characterized by vastly different levels of IL-12p70 production. The normal range for the assay established using DC products generated from monocytes of 13 normal donors was 8-999 pg/mL, with a mean of 270 pg/mL
- Production and Release Criteria for Dendritic Cells
- Each lot of the in vitro generated dendritic cells is tested for the presence of microbial contaminants (aerobic and anaerobic bacteria, fungi and mycoplasma), as well as endotoxin and are phenotypically and functionally characterized. All dendritic cells to be injected into subjects will be fresh and will not undergo croypreservation.
- Quality Assurance Testing of DC:
- DC generated as described above are evaluated for sterility, viability, purity, Potency and stability. Criteria for release of the cellular product are established and rigorously followed.
- Viability:
- The cells generated in culture are counted by microscopic observation on a hemacytometer, and a differential count (DC vs. lymphocytes) is obtained using a trypan blue dye. This count provides the percentage of viable cells in the tested culture. More than 70% cell viability by trypan blue exclusion and minimum 70% cells expressing HLA-DR and CD86 as the monocyte-derived DC markers are required for passing the release criteria. Additional markers may be included for exploratory analysis such as CD83 and CCR7 for assessing the DC maturation status, and CD3 and CD19 to assess the lymphocytes contamination.
- Purity:
- Two-color flow cytometry analysis of cells stained with FITC- and PE-conjugated mAbs is used to determine that the DC population identified morphologicallly expresses the surface antigens defined for DC and lack the monocyte and T and B cell lineage antigens. For vaccine preparation, the DC generated must express HLA-DR and CD86 and must not express CD3, CD19, or CD14. To be considered as mDC, the cells must express CD83+ and CCR7+.
- Potency:
- To define a measure of potency for the DC, we determine their ability to produce IL-12p70 as described above.
- Sterility:
- DC are tested by bacterial (Aerobic and anaerobic) and fungal cultures using the BD Bactec system (Becton Dickinson Co., Sparks, Md.) at the University of Pittsburgh Medical Center Microbiology Laboratory. Final results of the microbial cultures are available in 14 days. Prior to release of the DC for vaccine use, a gram stain is performed and must be negative for the presence of microorganisms.
- The IMCPL tests for mycoplasma by the use of the Gen-Probe Mycoplasma Tissue Culture Rapid Detection System (Gen-Probe, Inc. San Diego, Calif.), which is based on nucleic acid hybridization technology. Endotoxin testing is performed using the Limulus Amoebocyte Lysate Pyrogen Plus assay (Bio Whittaker, Inc., Walkerville, Md.). Endotoxin testing is performed on the cell culture at the time of harvest and prior to release of the final product. The acceptable endotoxin level is <5EU/kg body weight. Untransduced and transduced dendritic cells will be cryopreserved for future analysis.
- It is expected that all the transduced cells will express the transgene. More than 80% of the DCs are expected to be transduced. The product will be biologically active since the native coding sequence is maintained in the transgene. The viral-transduced DCs injected into the tumor are of immature DC phenotype and do not express IL-12 and one or more other immunomodulators until they undergo maturation, and hence at this stage, the expression of IL-12 and one or more other immunomodulators is mostly from the transgene. Since the expression of the IL-12 and one or more other immunomodulators transgene is induced by the small molecule activator drug RG-115932 in a dose dependent way, one can control the level of transgene expression in the transduced DCs to the desired levels. A small portion of the transduced DCs prepared for administration to the human subjects may be tested in vitro for the activator drug-dependent induction of expression of IL12 and one or more other immunomodulators. Expression of IL-12 and one or more other immunomodulators may be assayed by ELISA with a sensitivity of 4 ng/ml.
- It is expected that in vitro induction of IL-12 and one or more other immunomodulators from cells transduced by the vector used in the proposed study yields about 500 ng IL-12 and one or more other immunomodulators per 106 cells in 24 hours, determined by ELISA. In preelinical studies using mouse model of melanoma, intratumoral injection of 106 or more transduced DCs show efficacy. However, it is expected that the required intratumoral injection may show efficacy at levels below this amount and therefore injections of 5×107 transduced DCs may be utilized as a starting point to determine if less or greater amounts are required.
- For instance, in vitro, human and mouse cell lines and primary dendritic cells transduced with recombinant adenoviral vector carrying the genes for IL12 and one or more other immunomodulators show induction of IL12 expression in response to the activator drug in a dose dependent way.
- The activator drug used herein is formulated in any one of the following formulations:
- (1) 100% Labrasol;
- (2) Listerine flavored Labrasol (Latitude Pharmaceuticals Inc., USA) comprising (a) menthol, (b) thymol, (c) eucalyptol, (d) aspartame, (e) sodium saccharine, (f) citric acid, (g) peppermint flavor, (h) cream flavor, (i) labrasol;
- (3) Miglyol 812 and phospholipon 90G (Latitude Pharmaceuticals Inc., USA); or
- (4) Miglyol 812, phospholipon 90G and Vitamin E tocopheryl polyethylene glycol succinate (Latitude Pharmaceuticals Inc., USA).
- While a variety of concentrations and specific protocols may be imagined, one example for treating patients would include patients receiving intratumoral injection(s) of transduced autologous dendritic cells (AdDCs) at a concentration of 5×107 suspended in sterile saline engineered to express hIL-12 (human interleukin 12) and one or more other immunodulators under control of the RTS, in combination with the oral activator drug (RG-115932).
- Initial Treatment
-
Day 1 Inpatient Visit: Onday 1, a baseline physical examination (including vital signs, weight, and ECOG status) is performed. Urine is collected and blood drawn for baseline serum chemistry, urinanalysis, and hematology (safety profile). Approximately 3 hours before the intratumoral injection of the in vitro engineered dendritic cells, each subject is dosed with an activator drug (cohort 1—0.01 mg/kg, 0.3 mg/kg, 1.0 mg/kg, and 3 mg/kg) immediately after a meal. Blood is drawn at specified time intervals (predose, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 16, and 24 hours after the AD dose) onday 1 for evaluation of single dose pharmacokinetics of the activator drug and its major metabolites. Each subject receives a single intratumoral injection of adenoviral transduced autologous dendritic cells at a concentration of 5×107 cells, engineered to express hIL-12 and one or more other immunomodulators under the control of the RTS. The subjects are carefully monitored for local injection site reactions and/or hypersensitivity reactions.Day 2 through 14 Inpatient Visit: Ondays 2 through 14, each subject is dosed with the activator drug immediately after a meal. Vital signs and adverse events are collected daily ondays 2 through 14. Onday 4±24 hours, biopsies of the tumor and/or draining lymph nodes are removed from approximately 50% of the subjects for measurement of hIL-12 and cellular immune response. Onday 8, weight is measured. Onday 8±24 hours, biopsies of the tumor and/or draining lymph nodes are removed from subjects who did not have a biopsy performed onday 4 for measurement of hIL-12 and one or more other immunomodulators and cellular immune response. Blood is drawn onday 4±24 hours andday 8±24 hours for assay of potential antibodies and cellular immune response against the adenovirus and/or the RTS components. A serum cytokine profile is also obtained to determine if the expression of other cytokines is affected by treatment with the hIL-12 and one or more other immunomodulators transgene. Onday 8, urine is collected and blood is drawn for baseline serum chemistry, urine analysis, and hematology (safety profile). OnDay 8, blood is drawn at specified time intervals (predose, 0.5, 1, 2, 4, 6, 8, 12, 16, and 24 hours after the AD dose) for evaluation of steady-state pharmacokinetics/ADME of the activator drug and its major metabolites. -
Day 14 Inpatient Visit: Onday 14, each subject is dosed with the Activator Drug immediately after a meal. Each subject receives a physical examination (including vital signs, height, weight and ECOG status). Urine is collected and blood is drawn for serum chemistry, urinalysis, and hematology (safety profile). Blood is drawn onday 14±24 hours for assay of potential antibodies and cellular immune response against the adenovirus and/or the RTS components. A serum cytokine profile is also obtained to determine if the expression of other cytokines is affected. - Blood is collected from the subjects at specified inpatient and outpatient visits to measure potential antibodies and cellular immune response to the adenovirus and components of the RTS. Blood is obtained for a baseline serum cytokine profile. The AdVeGFP infectivity blocking type assay is used to detect an antibody response to the adenoviral vector (Gambotto, Robins et al. 2004). Antibody response to the RTS components will be assessed by western blot and/or ELISA using serum from the patient and the RTS proteins produced from an expression vector. In addition, multiplex cytokine testing will be done in the serum by Luminex for IL-12, IFN-gamma, IP-10, and other Th1/Th2 cytokines such as IL-2, TNF-alpha, IL-4, IL-5, and IL-10. These antibody and cytokine assays will need about 10 ml of blood.
- Potential Antibody and Cellular Immune Response to Adenovirus and/or Components of the RTS: Blood will be collected from the subjects at specified inpatient and outpatient visits to evaluate the potential antibody and cellular immune response to the adenovirus and components of the RTS and tumor antigens. The AdVeGFP infectivity blocking type assay will be used to detect an antibody response to the adenoviral vector (Nwanegbo, et al. 2004). Antibody response to the RTS components will be assessed by western blot and/or ELISA using serum from the subjects and the RTS proteins produced from an expression vector. In addition, multiplex cytokine testing will be done in the serum by Luminex for IL-12, UN-gamma, IP-10, and other Th1/Th2 cytokines such as IL-2, TNFa, IL-4, IL-5 and IL-10. These antibody and cytokine assays will need about 10 ml of blood.
- The cellular immune response assays use about 50-60 ml blood and CD4 and CD8 T cell subsets will be separated from it. The separated T cells will be mixed with autologous DCs transduced with empty AdV vector, AdV-RTS, or AdV-RTS-hIL12-immunomodulator(s) vectors in an ELISPOT assay for IFN-gamma production by the T cells activated by the AdV- and RTS-derived antigens, if any. Similar assays will be performed using the tumor cells as such and/or DCs expressing shared melanoma antigens to assess the early immune response to the tumor. Additional assays may also be performed as necessary.
- PREGNANCY TESTING: Females of childbearing potential is administered a urine pregnancy test at the screening visit and before the first inpatient visit of the retreatment phase. The testing is performed at least 72, 48, 24, or 12 hours prior to the administration of Activator Drug during both the initial treatment and all retreatment periods. If the urine pregnancy test is positive, then confirmation will be obtained with a serum pregnancy test. If pregnancy is confirmed, the subject will not be allowed to enter the trial or continue into the retreatment phase. The pregnancy testing may be reperformed as many times as necessary.
- CONCOMITANT MEDICATION INQUIRY: At screening, and before the first inpatient visit of the retreatment phase, each subject will be asked to provide a list of concurrent medications to determine any possible relationship to adverse events that occur during the trial and follow-up phase.
- RETREATMENT CRITERIA: If a subject has tolerated prior AdDC inoculation without adverse reactions that are limiting, and has shown no progression of disease or sy nptomatic decline at the time of potential retreatment, they will be considered for retreatment. If, in the opinion of the principal investigator, and treating physician there is a potential clinical benefit for additional intratumoral injection(s) of AdDCs in combination with Activator Drug (maximum tolerated dose from cohort 1) for 14 consecutive days, retreatment will be offered to the subject, provided the following criteria are met:
- 1. There have been no limiting toxicities,
2. The subject's disease is stable or showing clinical or subjective signs of improvement, and
3. There is no evidence of antibody or cellular immune response to adenovirus components of RheoSwitch® Therapeutic System. - ASSESSMENT OF TRANSGENE FUNCTION AND IMMUNOLOGICAL EFFECTS: Punch or excisional biopsies of the tumor and associated draining lymph nodes will be collected during screening (day −12 to day −7),
day 4,day 8 andday 14 of the trial and atmonth 1 of the follow-up for in vivo assessment of transgene expression of hIL-12 and one or more other immunomodulators, and cellular immune response. Fine needle aspiration biopsies of the tumor and associated draining lymph nodes will be collected on day −12 to −7 andday 14 of the retreatment period for in vivo assessment of transgene expression of hIL-12 and one or more other immunomodulators, and cellular immune response. Biopsies will be evaluated by standard light microscopy and immunohistochemistry to assess cellular infiltration of T cells into the tumor and draining lymph nodes. Biopsy sections will be read by a pathologist unaware of study subject background. To distinguish between endogenous and induced IL-12 expression by DCs in the tumor and draining lymph nodes, RT-PCR on RNA will be used with appropriately designed primers. Blood will be drawn for a serum cytokine profile at screening,day 4,day 8 andday 14 of the trial, atmonth 1 of the follow-up and on day −12 to −7,day 8 andday 14 of the retreatment period. A serum cytokine profile will be obtained to determine if the expression of other cytokines is affected by treatment with the hIL-12 transgene. Multiplex cytokine testing will be done in the serum by Luminex for IL-12, IFN-gamma, IP-10, and other Th1/Th2 cytokines such as IL-2, TNFa, IL-4, IL-5 and IL-10. These antibody and cytokine assays will need about 10 ml of blood. - SINGLE DOSE AND STEADY-STATE PHARMACOKINETICS OF ACTIVATOR DRUG: Blood will be drawn at specified time intervals (predose, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 16, and 24 hours after the morning dose) on
day 1 of the trial for evaluation of single dose pharmacokinetics and onday 8 of the trial for measurement of steady state pharmacokinetics/ADME of the Activator Drug and its major metabolites. Plasma will be evaluated by HPLC to obtain the following steady-state pharmacokinetic endpoints of the Activator Drug and major metabolites: Cmax (maximum observed plasma concentration), Tmax (time to maximum observed plasma concentration), Ctrough (minimum observed plasma concentration computed as the average of the concentrations at 0 and 24 hours), C24 h (plasma concentration at 24 hours), AUC24 h (area under plasma concentration-time curve fromtime 0 to 24 hours), Ke (apparent elimination rate), and T112 (apparent half-life). - It is to be understood that the foregoing described embodiments and exemplifications are not intended to be limiting in any respect to the scope of the invention, and that the claims presented herein are intended to encompass all embodiments and exemplifications whether or not explicitly presented herein.
- A matrix of rationally-selected, modular gene components can be rapidly assembled into DNA expression constructs through application of a combinatorial transgene technology such as ULTRAVECTOR®. To demonstrate that assembled gene components, which individually affect transcription, post-transcription, translation, and post-translational process, can together impact gene expression level, RheoSwitch technology was used, optionally, in combination with artificial 5′UTRs, various 3′Reg+poly(A) signals (SV40 and hGH), signal peptides (TNF-alpha and IL-2), and codon optization (+/−) schemes to modulate transcription increases the capacity of a cell to produce and secrete TNF-alpha.
FIG. 11 illustrates the modular elements used, and graphically represents each modular element flanked by unique restriction sites to provide a method for the precise assembly of modular combinations. - Modular assembly was carried out in the context of a DNA backbone designed to accept synthetic genes. One example of an ULTRAVECTOR® backbone engineered for adenoviral packaging is depicted in
FIG. 12 Combinatorial modular design combines well with adenoviral delivery of a therapeutic as it allows for the use of compact regulatory sequences which can be shorter than those found in nature. -
-
TABLE 7 A matrix yielding 11 test vectors and DNA assembled Vector 5′UTR Signal Peptide ORF (CDS) 3′Reg FIG. 43318 (17) TNFwt (1) TNFwtUV (4) TNFwtUV SV40e + pA FIG. 13 (6) (8) 43319 (18) TNFwt (1) TNFOptUV (1,2)(5) TNFoptUV SV40e + pA FIG. 14 (7) (8) 43320 (19) TNFwt (1) IL-2optUV (3) TNFoptUV SV40e + pA FIG. 15 (7) (8) 43321 (20) 5U2 (2) TNFwtUV (4) TNFwtUV SV40e + pA FIG. 16 (6) (8) 43322 (21) 5U2 (2) TNFOptUV (1,2)(5) TNFoptUV SV40e + pA FIG. 17 (7) (8) 43323 (22) 5U2 (2) IL-2optUV (3) TNFoptUV SV40e + pA FIG. 18 (7) (8) 43324 (23) TNFwt (1) TNFwtUV (4) TNFwtUV hGH + pA FIG. 19 (6) (9) 43325 (24) TNFwt (1) TNFOptUV (1,2)(5) TNFoptUV hGH + pA FIG. 20 (7) (9) 43326 (25) TNFwt (1) IL-2optUV (3) TNFoptUV hGH + pA FIG. 21 (7) (9) 43327 (26) 5U2 (2) TNFwtUV (4) TNFwtUV hGH + pA FIG. 22 (6) (9) 43329 (28) 5U2 (2) IL-2optUV (3) TNFoptUV hGH + pA FIG. 24 (7) (9) - Vectors were transiently transfected into the HEK293T cell line to assess which modular combinations result in increased TNF-alpha output. To induce the expression of TNF-alpha, ligand or vehicle control was administered to cells. Supernatant was collected and TNF-alpha levels were measured via ELISA. To set a baseline approximating wild type,
vector 43318 contains the UV conformed wild type TNF-alpha 5′UTR, signal peptide, coding sequence, andSV40pA 3′Reg. Individually, module changes of Opt(1,2) codon optimization of the TNF-alpha signal peptide or mature protein coding sequence result in incremental increases in protein secretion (vectors 43319, 43320). Additional modular substitution of the5U2 5′UTR for the TNF-wt 5′UTR further elevates secretion levels (vectors 43322, 43323). Highest secretion of TNF-alpha is achieved when thewild type 5′UTR, signal peptide, and coding sequence modules are substituted with 5U2, IL2, and TNFOptUV respective mods (vector 43329). - To demonstrate that the increased secretion of TNF-alpha is not cell type dependent, the 11 experimental vectors were transfected into CHO-K1 cells and 2 control vectors were added (see
FIG. 27 ). Vector 43534 (FIG. 26 ) and vector 43533 (FIG. 25 )) contain wild type TNF-alpha modules to serve as controls. Vector 43534 is composed of the wild type TNF-alpha sequence without the ULTRAVECTOR® assembly pivots present. Notably, the presence of ULTRAVECTOR® assembly pivots does not adversely effect the production and secretion of THF-alpha. In this data set we also demonstrate that substitution of the TNF-alphawild type 3′Reg with either the SV40e or hGH containng polyA modules results in an increase in TNF-alpha secretion. Maximal TNF-alpha output was achieved with the 5U2, IL2 signal peptide, TNFOptUV, and hGHpA combination. The data from CHO-K1 cells exhibits the same trend of incremental increase with each module substitution of 5U2 in the 5′UTR, IL-2 signal peptide, and TNFOptUV coding sequence. Interestingly, the magnitude of increase is slightly different in the two cell types. This illustrates that while the modules perform similarly in each cell type, physiologic differences in specifc cell or tissue type may influence the magnitude of modular substitution effect. Increasing the combinatorial matrix to include more modules in each category may allow for identification of superior combination depending cell type or tissue tested. Examples of additional modules that could be included in a larger matrix are included as SEQ ID NOs: 41-46. - To demonstrate the effectiveness of inducible optimized TNF-alpha constructs to treat cancer, for instance, prostrate cancer or head and neck cancer, a head and neck cancer mouse model of the disease can be employed. A single gene knockout of Smad4 has been demonstrated to yield a spontaneous model of malignant human head and neck squamous cell carcinoma (HNSCC). (PMID: 19841536)
- in the absence of such a mouse strain, human derived HNSCC tumor cells can be implanted into nude mice. Post tumor establishment the optimized TNF-alpha constructs can be introduced into the tumor with adenovirus. Varying doses of ligand can be administered to the mouse to regulate the level of optimized TNF-alpha produced. Tumor burden will be measured and tumor necrosis assessed to identify potential therapeutic candidates from the optimized TNF-alpha constructs.
- An engineered TNF-a□lpha transgene is administered to a patient by intratumoral injection of a non-replicative adenovirus DNA vector. This gene program encodes the mammalian-codon optimized mature cytokine, fused with a codon-optimized signal peptide for IL-2. In turn, the transgene cds (IL-2 SP+TNF-alpha) is flanked by a wild-type TNF-
alpha 5′UTR and theSV40 3′ Reg+poly(A) signal, and its expression is controlled by RheoSwitch technology via administration of a cautiously “dialed-in” dose of activator ligand (i.e., the DNA embodied in vector VVN-43320, seeFIG. 15 ). Preliminary data show that this combination of DNA elements yields the highest possible induction of secreted TNF-alpha while still affording “tight” and “non-leaky” control of its expression. That is, the uninduced, basal level of expression remains low in this transgene configuration, and would be less likely to exert uncontrolled, off-target effects on the patient. - In an alternative embodiment of the invention, the engineered TNF-a□lpha transgene is administered by adenovirus to a patient in a modular DNA configuration similar to vector VVN-43329 (see
FIG. 24 ), which exhibits both high basal expression and highest transgene expression. This DNA employs the artificially engineered 5′U2 and the hGH poly(A) signal, as well as full mammalian codon optimization and the IL-2 signal peptide. Control of systemic toxicity in the patient is achieved by use of a low adenovirus MOI in the intratumoral injection. To a lesser extent, expression level and temporal control is also modulated through the RheoSwitch activator ligand. Additional control of this gene product's distribution could be achieved through incorporation of tissue-specific miRNA response elements that could prohibit off-target expression in the vital organs, or through artificially engineering the adenoviral capsid for enhanced tropism. - In an additional embodiment of the invention, the engineered TNF-alpha □transgene is similar to the DNA in VVN-43328 (
FIG. 23 ), which is hypothesized to confer high stability to the artificial mRNA through the 5′U2 element. However, this construct does not make use of an IL-2 signal peptide for enhanced secretion, and it retains wild-type sequence from DNA that encodes the N′-terminus of TNF-alpha. For unknown reasons, high level secretion of artificial TNF-alpha□ could well prove to be detrimental to patient outcomes regardless of context, whereas the natural mechanism of “cytokine shedding” via metalloproteinases might limit patient toxicity by confining the factor to its tumor milieu. If natural shedding of exogenous TNF-alpha □still demonstrates off-target effects, its ectodomain stalk could be truncated by mutatior to prohibit solublization by native proteases, and the factor would potentiate activity through cell-cell contacts as a de facto Type II transmembrane protein. Alternatively, a transgene similar to the construct described by vector VVN-43328 could encode a constitutively expressed TNF-a□lpha with a mutated stalk ectodomain containing a cleavage site for an exogenous protease. This exogenous protease, would, in turn be under a RheoSwitch technology-controlled promoter element, only to be expressed in the presence of the activator ligand. Thus, cleavage and in vivo solubility (but not surface expression) of the TNF-a□lpha will be controlled through modular transgene elements. - The anti-tumor effect of Ad-RTS-IL-12 has been evaluated in a series of murine tumor models of melanoma, colorectal, pancreatic, breast, lung and renal cancers. An exemplary dose response experiment is shown below. Immuno-competent female C57bl/6 mice (6-8 week old) were inoculated subcutaneously with B16F0 murine melanoma cancer cells. Eleven days after tumor cell inoculation, when macroscopic tumor nodules were evident (tumor volumes averaged approx. 40 mm3), the mice were separated into groups of 5 animals each. There were 9 groups including: control saline treated; activator ligand treated; Ad-RTS-mIL12 (1ee10 vp) alone and 6 groups were treated with different doses of vector Ad-RTS-mIL-12 (1e7, 1e8, 1e9, 5e9, 1e10, 5e10 viral particles) plus activator ligand. Mice in the +ligand groups were provided 100 mg/kg activator ligand in 2018
Teklad Global 18% Protein Rodent Diet (Harlan Laboratories) chow (1000 mg ligand/kg chow) one day before the vector administration. The mice in control groups received 2018Teklad Global 18% Protein Rodent Diet chow. A single administration of Ad-RTS-mIL12 in 100u1 PBS was injected into tumor onday 12. Tumor volume and body weights were measured every 2-3 days using calipers and a weight scale, and the animals were followed until control tumors reached 2000 mm3. Data were uploaded into Study Log animal study software. - As shown in
FIG. 31 , substantial anti-tumor effect was observed at Ad-RTS-IL-12 doses above 1ee8 vp (range 73-99%). The lowest dose of Ad-RTS-IL-12 tested, 1ee7 vp, did not demonstrate anti-tumor effect. In the absence of activator ligand, high dose Ad-RTS-IL-12 at 1ee10 vp showed no effect, illustrating the requirement for combination of both Ad-RTS-IL-12 and activator ligand. Treatment of ligand by itself showed no effect. Therefore, this study illustrates the potent anti-tumor effects mediated by Ad RTS-IL-12 in combination with activator ligand. - Body weight analyses are presented in
FIG. 32 . Animals treated at the highest dose level (5ee10 vp) of Ad-RTS-IL-12 showed transient weight loss onday 19, but recovered byday 26. Animals in the treatment other groups did not show any clear dose response relationship and only minor changes in weight gain were observed. - Female, 6- to 8-week-old C57b/6 immunocompetent mice were inoculated subcutaneously (s.c.) with murine Lewis lung carcinoma cells (LLC). Five days post cell inoculation, the mice were randomized and assigned to treatment and control groups (n=5) for a total of four groups—no treatment (control), activator (RG-115932) alone, Ad-RTS-mIL12 alone and Ad-RTS-mILl2 plus activator. The cohorts receiving activator (L) were fed (2018
Teklad Global 18% Protein Rodent Diet (Harlan Laboratories) chow blended with activator (1000 mg/kg chow) ad libitum. Cohorts receiving treatment with Ad-RTS-mIL12 alone or no treatment continued to receive a regular diet. Treatment was initiated when the tumor reached 28±6 mm3. The Ad-RTS-mIL12 (1e10 vp/100 ul in PBS) was given to mice through intratumoral (i.t.) injection onDay - The post treatment tumor volume is shown in
FIG. 33A . The Lewis lung tumor bearing mice in control and activator (L) alone groups displayed approximately similar tumor growth kinetics. Three doses of Ad-RTS-mIL12 alone led to intermediate tumor growth. Importantly, Ad-RTS-mIL12 with activator (L) produced marked tumor growth inhibition (78%) relative to control group. This data suggests Ad-RTS-mIL12 in the presence of activator inhibits Lewis lung tumor growth. Body weight was monitored as an indicator of toxicity. No major body weight loss was found during the course of the experiment. - Female, 6- to 8-week-old C57b/6 immunocompetent mice were inoculated subcutaneously (s.c.) with murine melanoma cancer cells (B16F0). Ten days post cell inoculation, the mice were randomly assigned to treatment and control groups (n=5) for a total of nine groups: no treatment (control), activator (L) (RG-115932) alone, and Ad-RTS-mIL12 alone, and Ad-RTS-mIL12 with different activator dose (50, 100, 250, 500 and 1000 mg/kg) of ligand. The cohorts receiving activator (L) were fed rodent 2018
Teklad Global 18% Protein Rodent Diet chow blended with activator (1000 mg/kg chow) ad libitum. Cohorts receiving treatment with Ad-RTS-mIL12 alone or no treatment continued to receive a regular 2018Teklad Global 18% Protein Rodent Diet chow diet. Treatment was initiated when the tumor reached 56±18 mm3. A single dose of Ad-RTS-mIL12 (1e10 vp/100u1 in PBS) was given to mice through intratumoral (i.t.) injection onDay 13 post tumor cell inoculation. The activator (L) chow was given to mice 24 hr prior to vector injection. Tumor size and body weight of each mouse were monitored three times a week using calipers and a weight scale until the end of experiment. The experiment was terminated when the tumor size exceeded >2000 mm3. Data were uploaded into Study Log animal study software. - The post treatment tumor volume and body weight changes are shown in
FIGS. 34A and 34B . The melanoma tumor bearing mice in control and activator (L) alone groups showed similar aggressive tumor growth. The tumor growth kinetics indicated that the activator chow did not have anti-tumor activity. A slight tumor growth inhibition (12%) was observed onday 26 when animals received a single dose Ad-RTS-mIL12 (1e10 vp) without ligand. Treatment with Ad-RTS-mIL 12 plus activator (L) resulted in tumor growth inhibition (73-98%) compared to control mice. A single dose Ad-RTS-mIL12 with 50 mg/kg activator (L) produced significant tumor reduction relative to control tumors. Notably, significant anti-tumor activity (90-98%) was evident as dose activator chow increased from 100-1000 mg/kg, compared to 50 mg/kg activator chow. This data clearly show that Ad-RTS-mIL12 is active in the melanoma model and exhibits a broad therapeutic activator ligand dose window. Body weight was monitored as an indicator of toxicity. OnDay - Female, 6- to 8-week-old Balb/C immunocompetent mice were inoculated subcutaneously (s.c.) with luciferase expressing stable murine colon cancer cells (CT26Luc). Ten days post cell inoculation, the mice were randomly assigned to treatment and control groups (n=5) for a total of three groups—no treatment (control), activator (L) (RG-115932) alone, and Ad-RTS-mIL12 plus activator. Tne cohorts receiving activator (L) were fed 2018
Teklad Global 18% Protein Rodent Diet chow (Harlan Laboratories) blended with activator (1000 mg/kg chow) ad libitum. Cohorts receiving no treatment continued to receive 2018Teklad Global 18% Protein Rodent Diet chow. Treatment was initiated when the tumor reached 40±17 mm3. The Ad-RTS-mIL12 (1e10 vp/100 ul in PBS) was given to mice through intratumoral (i.t.) injection onDay - The post treatment tumor volume and body weight changes are shown in
FIGS. 35A and 35B . The colon carcinoma bearing mice in control and activator (L) alone groups showed similar aggressive tumor growth. The tumor growth kinetics indicated that the activator chow did not inhibit tumor growth. Two doses of Ad-RTS-mIL12 plus activator (L) alone resulted in complete regression and tumor growth inhibition (100%) compared to control mice. Notably, five out of five animals were completely tumor free as a result of Ad-RTS-mIL12 treatment. Mice rendered tumor-free following Ad-RTS-mIL12 treatment were re-challenged with parental CT26Luc cells. Five nave Balb/c animals were also inoculated subcutaneously with CT26Luc as control group. The control animals developed tumor nodules as expected. Importantly, tumors did not develop in all the rechallenged animals at four weeks post rechallenge. This study indicates that the Ad-RTS-mILl2 therapy developed strong anti-tumor immunity against the aggressive colon cancer model. Body weight was monitored as an indicator of toxicity. No major body weight loss was found during the course of the experiment. - Female, 6- to 8-week-old C57b/6 immuno competent mice were inoculated subcutaneously (s.c.) with syngenic PAN02 pancreatic cancer cells (ATCC). Six days post cell inoculation, the mice were randomized into groups of five animals each in four groups—no treatment, activator (RG-115932) alone, Ad-RTS-mIL12 alone and Ad-RTS-
mIL 12 plus activator. The cohorts receiving activator ligand were fed 2018Teklad Global 18% Protein Rodent Diet chow (Harlan Laboratories) blended with activator (1000 mg/kg chow) ad libitum. Cohorts receiving treatment with Ad-RTS-mIL12 alone or no treatment continued to receive 2018Teklad Global 18% Protein Rodent Diet chow. Mice received treatment with a single intratumoral (i.t.) injection of Ad-RTS-mIL12 at dose level of 1e10 vp/100 ul in PBS, onDay 7 andDay 14 post tumor cell implantation. The tumor size averaged STGT mm3 at the time of vector treatment initiation. - Tumor size and body weight of each mouse were monitored three times a week until the end of experiment. The experiment was terminated when the mice tumor size exceeded 600 mm3. Since pancreatic tumors grow very slowly, we defined as the termination of the experiment. The tumor growth in mice receiving no treatment was normal.
- In this tumor model, minor tumor growth delay was noticed in mice receiving treatment with either activator alone or Ad-RTS-mIL12 alone. In contrast, tumor growth in all Ad-RTS-
mIL 12 treated mice was dramatically inhibited (97%) in comparison with that in the control mice that received no treatment. Body weight was measured throughout the experiment using calipers and a weigh scale as a measure of toxicity. The body weight of animals injected with Ad-RTS-mILl2 showed no significant body weight decrease following administration except a transient body weight decrease (<5%) on Day 12-13. In addition, no pathological behavior (lethargy, ruffle fur, limping, dehydration, hunched posture etc) was observed in any animals. Tumor regression was maintained untilday 37, when control animals were sacrificed. Data were uploaded into Study Log animal study software. - The results are shown in
FIGS. 36A and 36B . - The aim of this study was to evaluate the intratumoral treatment with Ad-RTS-mIL12 for its efficacy and toxicity in murine breast cancer model.
- Six- to eight-week-old female BalbC mice were purchased from Charles River Laboratories or Harlan (USA). Animal care and experimental procedure were performed according to the Intrexon's Institutional Animal Care and Use Committee guideline.
- Murine breast carcinoma (4T1) cell lines were purchased from ATCC (Manassas, Va.). The 4T1 cells were grown in Roswell Park Memorial Institute medium (RPMI) 1640 (ATCC, Manassas, Va. The medium was supplemented with heat-inactivated fetal calf serum (FCS) 10% v/v, 2-mM L-glutamine (Atlanta Biologicals, Inc, Lawrenceville, Ga.), 100 IU/ml penicillin G, and 100 μg/ml streptomycin. The cells were grown at 37° C. in 5% CO2. All cell lines were routinely tested and found to be free of mycoplasma.
- Female, 6- to 8-week-old BALB/c immune-competent mice were inoculated subcutaneously (s.c.) with syngenic breast cancer (4T1) cells, 1e5 cells/50 ul. Eight days post cell inoculation, the mice were randomized into groups of five animals each in four groups—no treatment, activator alone, Ad-RTS-mIL12 alone and Ad-RTS-mIL12 plus activator. The cohorts receiving activator ligand were fed rodent chow blended with activator (1000 mg/kg) ad libitum. Cohorts receiving treatment with Ad-RTS-mIL12 alone or no treatment continued to receive standard diet (Harlan Laboratories, USA). Activator ligand is administered through a custom diet created from Harlan Teklad (a custom diet division of Harlan) formulated at 1000 mg of activator ligand to 1 Kg of the same chow which is administered to the control animals. Mice received treatment with a single intratumoral (i.t.) injection of Ad-RTS-mIL12 at dose level of 1e10 vp/100 ul in PBS, on
Day - Eight days post inoculation with breast cancer 4T1 cells, mice were randomized and assigned to treatment and control groups (n=5/group) for a total of four groups—no treatment (control), activator alone (L), Ad-RTS-mIL12 alone and Ad-RTS-mIL12 plus activator, as shown in the Table below.
-
Treatment Design for Breast (4T1) Tumor Model Treatment Tumor Activator Regular Intratumoral initiated Size, Ligand (L) Rodent Admin- after cell Body Group N Chow Chow istration inoculation Weight 1 5 No Yes Mon, Wed and Fri 2 5 Yes No Mon, Wed and Fri 3 5 No Yes Ad-RTS- Day 9,Mon, mIL12 12, 14 Wed and Fri 4 5 Yes No Ad-RTS- Day 9,Mon, mIL12 12, 14 Wed and Fri - Treatment was initiated when the tumor reached mean volume of 36 mm3. The post treatment tumor volume is shown in
FIG. 39A . The 4T1 tumor bearing mice in control, Ad-RTS-IL-12 treated and activator ligand (L) alone groups displayed tumor growth inhibition ˜20% and 35% respectively on Day 26 (FIG. 39A ). Importantly, three doses of Ad-RTS-mIL12 with activator ligand led to marked tumor growth inhibition (82%), relative to control group (p<0.005). This data suggests Ad-RTS-mIL12 in the presence of activator exhibits potent anti-tumor activity in breast cancer (4T 1) model. Body weight was monitored as an indicator of toxicity. No major body weight loss or deaths were found during the course of the experiment (FIG. 39B ). - The results demonstrate that direct intratumoral injection of Ad-RTS-mIL12 plus activator ligand is highly effective for inducing tumor regression and is safe in breast cancer model. The anti-tumor activity was significant (p<0.005) in this model.
- Following is a clinical protocol that can be used to practice the invention in the form of the administration of Ad-RTS-IL-12 vector for the treatment of unresectable stage III C or IV malignant melanoma.
- The objectives of this phase 1b clinical trial are to assess safety and objective response, tumor response rate, and immunological and other biological activities of six treatment cycles of intratumoral injections of Ad-RTS-IL-12 in combination with 14 daily oral doses of activating ligand. Ad-RTS-IL-12 dose will initially (first cycle) be administered at 1×1011 viral particles (vp) together with a 5 mg/day dose of activating ligand. The doses of both virus particles and activating ligand will then be escalated for each repeat treatment cycle for each patient, according to a fixed schedule (Table 8), provided that the preceding treatment cycle was tolerated by the patient.
- The objectives of this phase 1b stady are as follows:
- 1. Evaluate safety and tolerance of repeated treatment cycles of intratumoral injections of AD-RTS-IL-12 in an intra-patient escalating dose in combination with escalating doses of activating ligand in patients with unresectable Stage III C or IV malignant melanoma.
- 2. Obtain indications of efficacy by using diagnostic CT scans (Response Evaluation Criteria In Solid Tumors (RECIST 1.1) criteria), PET scans and photographs (as applicable).
- 3. Evaluate functionality of the RheoSwitch Therapeutic System™ (RTS™) in patients by evaluating the immunological effects of AD-RTS-IL-12 in combination with activating ligand, in terms of cellular immune response (particularly gene expression of IL-12 and other cytokines, frequency of cytotoxic T lymphocytes and Tregs) and other biological activities (e.g. apoptosis and immune cell infiltration) in the injected target tumor(s), tumor involved draining lymph nodes (if accessible) and in the peripheral circulation, and to correlate changes in the immunological and other biological parameters with prior activating ligand dose and with tumor response.
- 4. Evaluate the extent of the uptake of AD-RTS-IL-12 into tumor cells and into dendritic cells and macrophages in the tumor, to determine which cells uptake the virus whether the extent of uptake is dependent on the AD-RTS-IL-12 dose. Determine the inflammatory response and immune responses (cellular, such as cytotoxic lymphocytes and Tregs, and induction of cytokines) in the tumor, tumor-involved draining lymph nodes (if accessible) and in the peripheral circulation. The changes in the immunological and other biological parameters will be correlated with AD-RTS-IL-12 and activating ligand dose and with tumor response.
- 5. Evaluate the pharmacokinetic profile during steady state in each cycle on Days 8-9 in a subset of patients.
- 6. Evaluate QT/QTc intervals in ECGs obtained by Holter monitoring, in the patients who will undergo PK evaluation.
- Indication:
- Unresectable Stage III C (in transit), Stage IV (M1a, M1b or M1c (LDH≦2×ULN) malignant melanoma with at least 4 accessible lesions.
- Study Design:
- Phase 1b, open label, single arm, multicenter evaluation of safety, tolerance, tumor response (RECIST 1.1), and immunological and other biological effects, of six treatment cycles, each lasting for 28 days, each with an intra-tumoral injection of Ad-RTS-IL-12 in combination with 14 once daily, oral doses of activating ligand. The dose of both AD-RTS-IL-12 and activating ligand will be escalated according to
FIG. 1 and Table 1 for all patients who tolerated the preceding treatment cycle. - Study Population:
- Males and females of all races, ≧18 years of age, with unresectable Stage III C or IV malignant melanoma with ECOG performance status of 0-1, who have a minimum of 4 accessible lesions (longest diameter≦3 cm; shortest diameter≧1 cm) or palpable tumor-involved lymph nodes (longest diameter≦5 cm; shortest≧1.5 cm) for intra-tumoral injections or biopsies.
- Sample Size:
- A minimum of 12 and a maximum of 28 patients, with Stage III C or IV melanoma will be entered into this study. All patients in this protocol will be entered into a single arm, with intra-patient dose escalation of AD-RTS-IL-12 and activating ligand with each repeated treatment cycle according to
FIG. 1 and Table 1, providing that the preceding treatment cycle was well tolerated. - Test Product:
- During each cycle, the patients will be treated with a combination of the oral activating ligand and an intra-tumoral injection of a gene therapy (Ad-RTS-IL-12) engineered to express inducible hIL-12 in a dose-dependent response to activating ligand. AD-RTS-IL-12 will be prepared at a central manufacturing site and then frozen and sent to the appropriate clinical site. All patients will receive intra-tumoral injections (one per cycle for up to six cycles, 4 weeks apart) of AD-RTS-IL-12 (approximately 1.0×1011 and 1.0×1012 total viral particles per injection). The patients will also receive a single daily oral dose of activating ligand for 14 consecutive days during each cycle. The AD-RTS-IL-12 and/or the activating ligand dose will be escalated intra-patient at the start of
cycles 2 to 6 (see Table 8), in all patients who tolerated the preceding treatment cycle. AD-RTS-IL-12 will be injected into a different lesion at each cycle, and if the number of lesions is limited, the injections will be done in sequential rotation. One of the minimum of four accessible lesions will not be injected as that lesion will be used to evaluate the systemic effect of AD-RTS-IL-12. Patient dosing will be staggered at least 24 hours apart. Each intra-tumoral injection will occur once during a cycle, approximately 3 hours (±30 minutes) after the first dose of activating ligand. - Dosage:
- Activating Ligand: lowest dose/day: 5 mg; intermediate dose/day: 20 mg; highest dose/day: 100 mg. Activating Ligand will be administered during the first 14 days of each cycle.
- Ad-RTS-IL-12: dose: approximately 1.0×1011 or 1.0×1012 viral particles/tumor per injection suspended in a total volume of 0.5 ml sterile solution, with the injection volume distributed throughout the lesion, especially in the area of the tumor margin.
-
TABLE 8 Dosing Schedule Activating Number of AD-RTS-IL-12 dose Ligand Activating (intratumorally) No. mg/daily Ligand Cycle Viral Particles (VP) Inj/cycle dose (oral) doses/ cycle 1 1.0 × 1011 1 (Day 1) 5 14 (Days 1-14) 2 1.0 × 1011 1 (Day 1) 20 14 (Days 1-14) 3 1.0 × 1011 1 (Day 1) 100 14 (Days 1-14) 4 1.0 × 1012 1 (Day 1) 5 14 (Days 1-14) 5 1.0 × 1012 1 (Day 1) 20 14 (Days 1-14) 6 1.0 × 1012 1 (Day 1) 100 14 (Days 1-14) - Treatment with the next higher dose level will not begin until the safety and tolerability of the preceding treatments have been confirmed. If MTD is defined, no further escalation will occur.
- Route of Administration:
- Activating Ligand: solution in a soft gelatin capsule taken orally within 30 minutes of a meal;
- AD-RTS-IL-12:
- To be injected on the first day of each cycle into one accessible tumor lesions or tumor-involved (palpable) draining lymph nodes when necessary.
- Method of Patient Assignment:
- All patients will receive treatment according to Table 8 and be entered into one arm. Safety and tolerance will be rigorously assessed for all patients, during and after each treatment cycle. Dose escalation can only take place if the preceding cycle treatment was tolerated.
- Trial Duration:
- This study will last for each patient for approximately 28 weeks after screening.
- After a period of up to 23 days for screening evaluation (Days −30 to −7), the patients will be approved for participation into the study. On Days −6 to −2, baseline biopsies will be performed and on
Day 0, the baseline evaluation of cardiac function using Holter monitoring will be done in the patients who are evaluated for PK. OnDay 1 of each cycle, the approved patient will start receiving the experimental treatments (one intra-tumoral injection of AD-RTS-IL-12 and one oral dose of activating ligand). Activating ligand treatment in each cycle will continue for a total of 14 days, followed by 14 days of washout and observation for safety. The study treatment consists of 6 cycles, each lasting a total of 28 days including 14 days of follow-up. A post-treatment follow-up evaluation will be performed at 6 weeks after the last injection (4 weeks after the last dose of activating ligand). Viral DNA in blood will be determined. If viral DNA is present at 6 weeks after the last injection, further viral DNA assessments will be continued. However, if two consecutive negative results by Q-PCR for each source is demonstrated, no more tests will be necessary. - Primary Endpoints:
- Safety and tolerance will be assessed by physical examinations (including ECOG performance status), QT/QTc interval in ECGs (in PK patients), vital signs, serum chemistry, urinalysis, hematology, and by reports of patients of any adverse events. Objective response and response rate, as assessed by CT scans.
- Secondary Endpoints:
- Steady-state pharmacokinetics of activating ligand, in a subset of eight patients (four per AD-RTS-IL-12 dose level).
- b. Extent of inflammatory and immune response (cellular, such as cytotoxic lymphocytes and Tregs, and induction of cytokines) in the tumor, in tumor-involved draining lymph nodes (if accessible) and in the peripheral circulation, as a result of the treatment.
- c. Correlate changes in the immunological and other biological parameters with AD-RTS-IL-12 and activating ligand dose and with tumor response.
- d. Efficacy also assessed by PET scans and photographs.
- e. Long-term follow-up will occur for up to 5 years. Patients will be contacted annually by the investigator.
- Inclusion Criteria:
- a. Males or females of all races≧18 years of age;
- b. Unresectable Stage III C (in transit) or Stage IV melanoma (M1a, M1b, M1c with LDH≦2×ULN), arising from primary cutaneous, mucosal, or subungal melanoma of any tumor thickness or from an unknown primary site;
- c. A minimum of 4 accessible nonvisceral lesions (longest diameter≦3 cm; shortest≧1 cm) or palpable tumor-involved lymph nodes (longest diameter≦5 cm; shortest≧1.5 cm) for intra-tumoral injections or biopsies. At least one lesion will not be injected.
- d. ECOG performance status of 0 or 1;
- e. Patients without visible brain metastases as assessed by contrast-enhanced MRI scan at the time of screening or within 30 days prior to study entry;
- f. Adequate baseline hematological and organ function, assessed by laboratory values within 30 days prior to treatment with study treatments and prior to repeat treatment cycles and activating ligand dose escalation as follows: hemoglobin≧10 g/L, granulocytes >2500/mm3, lymphocytes >1000/mm3, platelets >100,000/mm3, serum creatinine <1.5×ULN, AST, ALT, alkaline phosphatase <2.5×ULN, LDH≦2×ULN, serum bilirubin <1.5×ULN, absolute neutrophils >500/mm3;
- g. An expected survival of at least approximately 6 months in the opinion of the investigator (as assessed mainly on performance status);
- h. Females must be post-menopausal or surgically sterile or practice effective contraception; Men who are not surgically sterile and whose partners are not post-menopausal or surgically sterile must practice effective contraception;
- i. Normal coagulation parameters as measured by PT/PTT;
- j. Signed, IRB-approved voluntary informed consent.
- Exclusion Criteria:
- a. Active, acute viral, bacterial, or fungal infections requiring specific therapy;
- b. HIV-infection due to concerns about ability to mount an effective immune response;
- c. Active autoimmune disease requiring steroids (>10 mg prednisolone or comparable) or other immunosuppressive therapy;
- d. Patients with detectable brain metastases at the time of screening (or within 30 days prior to study entry), as assessed by contrast-enhanced MRI scans;
- e. Patients with lesions >3 cm (LD) or palpable, tumor-involved lymph nodes >5 cm (LD);
- f. Patients with a hemoglobin of <10 g/L;
- g. Presence of Stage 1V visceral metastases or other distant metastases if LDH >2×ULN;
- h. Patients who have previously been treated with AD-RTS-IL-12 or activating ligand;
- i. Patients who have previously been treated with intratumoral gene therapy.
- J. Recipients of organ allografts;
- k. Other concurrent clinically active malignant disease, with the exception of other cancers of the skin;
- l. Less than 30 days (before the first dose of study medication) have elapsed since the completion of prior chemotherapy, hormonal therapy, radiotherapy, immunotherapy, or any first line therapy;
- m. Clinically significant cerebrovascular disease;
- n. History of or concurrent severe cardiac insufficiency (New York Heart Association Class III or IV) or coronary artery disease;
- o. Acute medical conditions such as ischemic heart or lung disease that may be considered an unacceptable anesthetic or operative risk;
- p. History of or current bleeding or clotting disorders;
- q. Concurrent immunosuppressive therapy such as corticosteroids (>10 mg prednisolone or comparable) and cyclosporin A;
- r. Concurrent investigational treatments, or treatment with any investigational treatment within the past 30 days (prior to the first dose of study medication);
- s. Concurrent medications that are metabolized by the CYP450 3A4 pathway;
- t. Females who are lactating or pregnant;
- u. Patients who have a history of hypersensitivity that may relate to any component of the product, e.g. to benzoic acid that might be related to activating ligand, which contains two benzene rings;
- v. Any medical or psychiatric condition which, in the opinion of the investigator, would unacceptably reduce the safety or delivery of the proposed treatment, or would preclude obtaining voluntary informed consent.
- Statistical Methods:
- Objective response (CR+PR) will be based on changes in size of injected and un-injected tumor lesion(s) as well as palpable tumor-involved lymph nodes by CT scans [utilizing Response Evaluation Criteria In Solid Tumors (RECIST 1.1)]. PET scans and/or photographs will be used to evaluate changes in metabolic activity or size (cutaneous lesions), respectively.
- The primary analysis of OS and ORR will include confidence interval, and will be performed when sample size reaches 12, 16, 20, 24 patients, and at 6 weeks after treatment of the last patient.
- Demographic, immunologic and biologic activity measures, as well as safety parameters including adverse event rates and laboratory values, will be analyzed descriptively at end of follow-up. The results will be summarized in tables, graphs and patient-by-patient listings.
- Descriptive statistics, including mean, median, standard deviation and histogram, will be used to summarize continuous measures. Frequency counts will be used for categorical variables, including objective tumor response. Immunological and biological activities will be correlated with the antitumor effect. These statistics will be provided by stratum (size of tumor lesions: ≦1 cm longest diameter [LD], >1 cm LD; size of involved DLN: ≧3 cm ID, >3 cm LD; location of lesions: visceral, non-visceral; Injection status of lesions: injected, non-injected.) The statistics will be performed by end of each treatment cycle and overall. For overall analysis, observations will be combined across strata but not across cycles.
- Compliance:
- The trials are performed in compliance with current Good Clinical Practice (cGCP).
-
- Abdalla, 2007.
- Abdi K, Singh N, Matzinger P (2006). T-cell control of IL-12p75 production. Scand J Immunol 64: 83-92.
- Adorini L (1999). Interleukin-12, a key cytokine in Th1-mediated autoimmune diseases. Cell Mol Life Sci 55: 1610-25.
- Adorini L (2001).
Interleukin 12 and autoimmune diabetes. Nat Genet. 27: 131-2. - Adorini L, Gregori S, Harrison LC (2002). Understanding autoimmune diabetes: insights from mouse models. Trends Mol Med 8: 31-8.
- Adorini L, Gregori S, Magram J, Trembleau S (1996). The role of IL-12 in the pathogenesis of Th1 cell-mediated autoimmune diseases. Ann N Y Acad Sci 795: 208-15.
- Akhtar N, Padilla M L, Dickerson E B, Steinberg H, Breen M, Auerbach R et al (2004). Interleukin-12 inhibits tumor growth in a novel angiogenesis canine hemangiosarcoma xenograft model. Neoplasia 6: 106-16.
- Akiyama Y, Watanabe M, Maruyama K, Ruscetti F W, Wiltrout R H, Yamaguchi K (2000). Enhancement of antitumor immunity against B16 melanoma tumor using genetically modified dendritic cells to produce cytokines. Gene Ther 7: 2113-21.
- Al-Mohanna F, Saleh S, Parhar R S, Collison K (2002). IL-12-dependent nuclear factor-kappaB activation leads to de novo synthesis and release of IL-8 and TNF-alpha in human neutrophils. J Leukoc Biol 72: 995-1002.
- Aliberti J C, Cardoso M A, Martins G A, Gazzinelli R T, Vieira L Q, Silva J S (1996). Interleukin-12 mediates resistance to Trypanosoma cruzi in mice and is produced by murine macrophages in response to live trypomastigotes. Infect Immun 64: 1961-7.
- Allavena P, Paganin C, Zhou D, Bianchi G, Sozzani S, Mantovani A (1994). Interleukin-12 is chemotactic for natural killer cells and stimulates their interaction with vascular endothelium. Blood 84: 2261-8.
- Alli R S, Khar A (2004). Interleukin-12 secreted by mature dendritic cells mediates activation of NK cell function. FEBS Lett 559: 71-6.
- Alzona M, Jack H M, Simms P E, Ellis T M (1996). Interleukin-12 activates interferon-gamma production by targeted activation of CD30+ T cells. Ann N Y Acad Sci 795: 127-36.
- Amemiya K, Meyers J L, Trevino S R, Chanh T C, Norris S L, Waag D M (2006). Interleukin-12 induces a Th1-like response to Burkholderia mallei and limited protection in BALB/c mice. Vaccine 24: 1413-20.
- Araujo M I, Bliss S K, Suzuki Y, Alcaraz A, Denkers E Y, Pearce E J (2001). Interleukin-12 promotes pathologic liver changes and death in mice coinfected with Schistosoma mansoni and Toxoplasma gondii. Infect Immun 69: 1454-62.
- Arulanandam B P, Van Cleave V H, Metzger D W (1999). IL-12 is a potent neonatal vaccine adjuvant. Eur J Immunol 29: 256-64.
- Athie M V, Flotow H, Hilyard K L, Cantrell D A (2000). IL-12 selectively regulates STAT4 via phosphatidylinositol 3-kinase and Ras-independent signal transduction pathways. Eur J Immunol 30: 1425-34.
- Athie-Morales V, Smits H H, Cantrell D A, Hilkens C M (2004). Sustained IL-12 signaling is required for Th1 development. J Immunol 172: 61-9.
- Atkins M B, Robertson M J, Gordon M, Lotze M T, DeCoste M, DuBois J S et al (1997). Phase I evaluation of intravenous recombinant
human interleukin 12 in patients with advanced malignancies. Clin Cancer Res 3: 409-17. - Berard F, Blanco P, Davoust J, Neidhart-Berard E M, Nouri-Shirazi M, Taquet N et al (2000). Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells. J Exp Med 192: 1535-44.
- Bertagnolli M M, Lin B Y, Young D, Hellmann S H (1992). IL-12 augments antigen-dependent proliferation of activated T lymphocytes. J Immunol 149: 3778-83.
- Bhardwaj N, Seder R A, Reddy A, Feldman M V (1996). IL-12 in conjunction with dendritic cells enhances antiviral CD8+ CTL responses in vitro. J Clin Invest 98: 715-22.
- Biedermann T, Lametschwandtner G, Tangemann K, Kund J, Hinteregger S, Carballido-Perrig N et al (2006). IL-12 instructs skin homing of human Th2 cells. J Immunol 177: 3763-70.
- Brunda M J, Gately M K (1994). Antitumor activity of interleukin-12. Clin Immunol Immunopathol 71: 253-5.
- Buchanan J M, Vogel L A, Van Cleave V H, Metzger D W (1995).
Interleukin 12 alters the isotype-restricted antibody response of mice to hen eggwhite lysozyme. Int Immunol 7: 1519-28. - Chang, 2007.
- Coughlin, 1998.
- Dietrich 2002.
- Emtage et al., “Adenoviral Vectors Expressing Lymphotactin and
Interleukin 2 or Lymphotactin andInterleukin 12 Synergize to Facilitate Tumor Regression in Murine Breast Cancer Models,” Hum. Gene Ther. 10:697 (1999). - Faure F, Even J, Kourilsky P (1998). Tumor-specific immune response: current in vitro analyses may not reflect the in vivo immune status. Crit. Rev Immunol 18: 77-86.
- Gao et al., “Cotransduction of CCL27 gene can improve the efficacy and safety of IL-12 gene therapy for cancer,” Gene Ther. 14:491-502 (2007)
- Hansson, 2007.
- Heinzerling L, Burg G, Dummer R, Maier T, Oberholzer P A, Schultz J et al (2005). Intratumoral injection of DNA encoding
human interleukin 12 into patients with metastatic melanoma: clinical efficacy. Hum Gene Ther 16: 35-48. - Hill 2002.
- Itoh T, Storkus W J, Gorelik E, Lotze M T (1994). Partial purification of murine tumor-associated peptide epitopes common to histologically distinct tumors, melanoma and sarcoma, that are presented by H-2 Kb molecules and recognized by CD8+ tumor-infiltating lymphocytes. J Immunol 153: 1202-15.
- Jean, 2004.
- Kang W K, Park C, Yoon H L, Kim W S, Yoon S S, Lee M H et al (2001).
Interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase I study. Hum Gene Ther 12: 671-84. - Koka, 2004.
- Koyama, 1997
- Lasek, 2000.
- Mehrotra, 1995.
- Narvaiza et al., “Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-gamma-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy,” J. Immunol. 164:3112 (2000).
- Nair, 2006.
- Narvaiza et al., Intratumoral Coinjection of Two Adenoviruses, One Encoding the Chemokine IFN-γ-Inducible Protein-10 and Another Encoding IL-12, Results in Marked Antitumoral Synergy,” J. Immunol. 164:3112-3122 (2000).
- Palmer et al., “Combined CXC chemokine and interleukin-12 gene transfer enhances antitumor activity,” Gene Ther. 8:282-290 (2001).
- Rasmussen, 2003.
- Romani L, Puccetti P, Bistoni F (1997). Interleukin-12 in infectious diseases. Clin Microbiol Rev 10: 611-36.
- Rothe H, Burkart V, Faust A, Kolb H (1996). Interleukin-12 gene expression mediates the accelerating effect of cyclophosphamide in autoimmune disease. Ann N Y Acad Sci 795: 397-9.
- Sabel, 2003, 2004, 2007.
- Sangro B, Mazzolini G, Ruiz J, Herraiz M, Quiroga J, Herrero I et al (2004). Phase I trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors. J Clin Oncol 22: 1389-97.
- Sangro B, Melero I, Qian C, Prieto J (2005). Gene therapy of cancer based on
interleukin 12. Curr Gene Ther 5: 573-81. - Satoh Y, Esche C, Gambotto A, Shurin G V, Yurkovetsky Z R, Robbins P D et al (2002). Local administration of IL-12-transfected dendritic cells induces antitumor immune responses to colon adenocarcinoma in the liver in mice. J Exp Ther Oncol 2: 337-49.
- Satoskar A R, Rodig S, Telford S R, 3rd, Satoskar A A, Ghosh S K, von Lichtenberg F et al (2000). IL-12 gene-deficient C57BL/6 mice are susceptible to Leishmania donovani but have diminished hepatic immunopathology. Eur J Immunol 30: 834-9.
- Schopf L R, Bliss J L, Lavigne L M, Chung C L, Wolf S F, Sypek J P (1999). Interleukin-12 is capable of generating an antigen-specific Th1-type response in the presence of an ongoing infection-driven Th2-type response. Infect Immun 67: 2166-71.
- Subleski, 2006.
- Svane I M, Boesen M, Engel A M (1999). The role of cytotoxic T-lymphocytes in the prevention and immune surveillance of tumors—lessons from normal and immunodeficient mice. Med Oncol 16: 223-38.
- Taniguchi, 1998.
- Tatsumi T, Huang J, Gooding W E, Gambotto A, Robbins P D, Vujanovic N L et al (2003). Intratumoral delivery of dendritic cells engineered to secrete both interleukin (IL)-12 and IL-18 effectively treats local and distant disease in association with broadly reactive Tc1-type immunity. Cancer Res 63: 6378-86.
- Thomas G R Chen Z, Enamorado I, Bancroft C, Van Waes C (2000). IL-12- and IL-2-induced tumor regression in a new murine model of oral squamous-cell carcinoma is promoted by expression of the CD80 co-stimulatory molecule and interferon-gamma. Int J Cancer 86: 368-74.
- Trinchieri G (2003). Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 3: 133-46.
- Triozzi P L, Allen K O, Carlisle R R, Craig M, LoBuglio A F, Conry R M (2005). Phase I study of the intratumoral administration of recombinant canarypox viruses expressing B7.1 and
interleukin 12 in patients with metastatic melanoma. Clin Cancer Res 11: 4168-75. - Tsung K, Meko J B, Peplinski G R, Tsung Y L, Norton J A (1997). IL-12 induces T helper 1-directed antitumor response. J Immunol 158: 3359-65.
- Vujanovic, 2006.
- Wang, 2001.
- Wigginton 2002, 2001, 1996
- Wolf S F, Sieburth D, Sypek J (1994). Interleukin 12: a key modulator of immune function. Stem Cells 12: 154-68.
- Yamanaka R, Zullo S A, Ramsey J, Yajima N, Tsuchiya N, Tanaka R et al (2002). Marked enhancement of antitumor immune responses in mouse brain tumor models by genetically modified dendritic cells producing Semliki Forest virus-mediated interleukin-12. J Neurosurg 97: 611-8.
- Yuminamochi E, Koike T, Takeda K, Horiuchi I, Okumura K (2007). Interleukin-12- and interferon-gamma-mediated natural killer cell activation by Agaricus blazei Murill. Immunology.
- McDermott, D. F. and Atkins, M. B. (2008) Immunotherapy of metastatic renal cell carcinoma.
- Cancer J. 14, 320-324.
- Berntsen, A., Trepiakas, R., Wenandy, L., Geertsen, P. F., thor Straten, P., Andersen, M. H., Pedersen, A. E., Claesson, M. H., Lorentzen, T., Johansen, J. S, and Svane, I. M. (2008) Therapeutic dendritic cell vaccination of patients with metastatic renal cell carcinoma: a
clinical phase 1/2 trial. J. Immunother. 31, 771-780. - Tarhini, A. A., Kirkwood, J. M., Gooding, W. E., Moschos, S, and Agarwala, S. S. (2008) A
phase 2 trial of sequential temozolomide chemotherapy followed by high-dose interleukin 2 immunotherapy for metastatic melanoma. Cancer. 113, 1632-1640. - Heemskerk, B., Liu, K., Dudley, M. E., Johnson, L. A., Kaiser, A., Downey, S., Zheng, Z., Shelton, T. E., Matsuda, K., Robbins, P. F., Morgan, R. A., Rosenberg, S. A. (2008) Adoptive cell therapy for patients with melanoma, using tumor-infiltrating lymphocytes genetically engineered to secrete interleukin-2. Hum Gene Ther. 19, 496-510.
- Horton, H. M., Lalor, P. A. and Rolland, A. P. (2008) IL-2 plasmid electroporation: from preclinical studies to phase I clinical trial. Methods Mol. Biol. 423, 361-372.
- Shiratori, I., Suzuki, Y., Oshiumi, H., Begum, N. A., Ebihara, T., Matsumoto, M., Hazeki, K., Kodama, K., Kashiwazaki, Y. and Seya, T. (2007) Recombinant interleukin-12 and interleukin-18 antitumor therapy in a guinea-pig hepatoma cell implant model.
- Cancer Sci. 98, 1936-1942.
- Lian H, Jin N, Li X, Mi Z, Zhang J, Sun L, Li X, Zheng H, Li P. (2007) Induction of an effective anti-tumor immune response and tumor regression by combined administration of IL-18 and Apoptin. Cancer Immunol Immunother. 56, 181-192.
- Iinuma, H., Okinaga, K., Fukushima, R., Inaba, T., Iwasaki, K., Okinaga, A., Takahashi, I. and Kaneko, M. (2006) Superior protective and therapeutic effects of IL-12 and IL-18 gene-transduced dendritic neuroblastoma fusion cells on liver metastasis of murine neuroblastoma. J. Immunol. 176, 3461-3469.
- Basak, G. W., Zapala, L., Wysocki, P. J., Mackiewicz, A., Jakóbisiak, M. and Lasek, W. (2008)
Interleukin 15 augments antitumor activity of cytokine gene-modified melanoma cell vaccines in a murine model. Oncol Rep. 19, 1173-1179. - Lasek, W., Basak, G., Switaj, T., Jakubowska, A. B., Wysocki, P. J., Mackiewicz, A., Drela, N., Jalili, A., Kaminski, R., Kozar, K. and Jakobisiak, M. (2004) Complete tumour regressions induced by vaccination with IL-12 gene-transduced tumour cells in combination with IL-15 in a melanoma model in mice. Cancer Immunol Immunother. 53, 363-372.
- Xia, Y., Dai, J., Lu, P., Huang, Y., Zhu, Y. and Zhang, X. (2008) Distinct effect of CD40 and TNF-signaling on the chemokine/chemokine receptor expression and function of the human monocyte-derived dendritic cells. Cell Mol. Immunol. 5, 121-131.
- Sharma, S., Bata, R. K., Yang, S. C., Hillinger, S., Zhu, L., Atianzar, K., Stricter, R. M., Riedl, K., Huang, M. and Dubinett, S. M. (2003) Interleukin-7 gene-modified dendritic cells reduce pulmonary tumor burden in spontaneous murine bronchoalveolar cell carcinoma. Hum Gene Ther. 14, 1511-1524.
- Tirapu, I., Rodriguez-Calvillo, M., Qian, C., Duarte, M., Smerdou, C., Palencia, B., Mazzolini, G., Prieto, J. and Melero, I. (2002) Cytokine gene transfer into dendritic cells for cancer treatment. Curr. Gene Ther. 2, 79-89.
- Small, E. J., Sacks, N., Nemunaitis, J., Urba, W. J., Dula, E., Centeno, A. S., Nelson, W. G., Ando, D., Howard, C., Borellini, F., Nguyen, M., Hege, K. and Simons, J. W. (2007) Granulocyte macrophage colony-stimulating factor—secreting allogeneic cellular immunotherapy for hormone-refractory prostate cancer. Clin Cancer Res. 13, 3883-3891.
- Huang, H. and Xiang, J. (2004) Synergistic effect of lymphotactin and interferon gamma-inducible protein-10 transgene expression in T-cell localization and adoptive T-cell therapy of tumors. Int. J. Cancer. 109, 817-825.
Claims (50)
1. A vector for conditionally expressing protein(s) having the function(s) of one or more therapeutic protein comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence which is operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins having the function of an therapeutic protein operably linked to a promoter which is activated by said ligand-dependent transcription factor, wherein said vector is not contained within a cell upon in vivo administration.
2. (canceled)
3. (canceled)
4. The vector of claim 1 , wherein said vector is selected from the group consisting of plasmid, adenovirus, retrovirus, adeno-associated virus, pox virus, baculovirus, vaccinia virus, herpes simplex virus, Epstein-Barr virus, adenovirus, geminivirus, caulimovirus, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.
5. The vector of claim 4 , wherein said vector is an adenoviral vector.
6. The vector of claim 1 , further comprising a polynucleotide encoding a protein having the function of IL-12.
7. The vector of claim 6 , wherein said polynucleotide encoding said one or more proteins having the functions of the immunomodulator and said polynucleotide encoding said protein(s) having the function of IL-12 are under control of a regulated promoter of said gene switch.
8. The vector of claim 1 , wherein said gene switch is an ecdysone receptor (EcR)-based gene switch.
9. The vector of claim 1 , wherein said polynucleotide encoding a gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor.
10. The vector of claim 1 , wherein said polynucleotide encoding a gene switch comprises a first transcription factor sequence and a second transcription factor sequence under the control of a promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor.
11. (canceled)
12. (canceled)
13. The vector of claim 6 , wherein said polynucleotide encoding the protein having the function of IL-12 encodes human IL-12.
14.-38. (canceled)
39. A pharmaceutical composition comprising the vector of claim 1 .
40.-49. (canceled)
50. A method of treating a disease or disorder, comprising administering (1) the vector of claim 1 , and (2) a therapeutically effective amount of one or more activating ligands to a mammal in need thereof.
51. The method of claim 50 , wherein said disease or disorder is a tumor in said mammal.
52. The method of claim 50 or claim 51 , wherein said treating is reducing a size of a tumor or preventing a tumor in said mammal.
53.-59. (canceled)
60. The method of claim 51 , wherein said tumor is a benign tumor.
61. The method of claim 50 , wherein said tumor is a malignant tumor.
62. The method of claim 50 , wherein said tumor is a melanoma.
63. The method of claim 50 , wherein said tumor is a malignant melanoma skin cancer.
64. The method of claim 50 , wherein said ligand is a diacylhydrazine.
65. The method of claim 50 , wherein said ligand is selected from RG-115819, RG-115932, and RG-115830.
66. The method of claim 50 , wherein said ligand is an amidoketone or oxadiazoline.
67.-70. (canceled)
71. A kit comprising the vector of any one of claims 1 , 4 -10 and 13, and (b) a ligand that activates the gene switch.
72. The kit of claim 71 , wherein the ligand is RG-115819, RG-115830 or RG-115932.
73. (canceled)
74. (canceled)
75. The method of claim 50 , wherein the disease is selected from the group consisting of chronic renal disease, osteoarthritis, oncology, viral upper respiratory infection, feline plasma cell stomatitis, feline eosinophillic granulomas, feline leukemia virus infection, canine distemper infection, systemic fungal infections, cardiomyopathy, mucopolysaccharidosis VII, and infectious disease.
76. The method of claim 50 , wherein the infectious disease is selected from the group consisting of Bovine respiratory disease, Porcine respiratory disease, Avian influenza, Avian infectious bronchitis, Bovine spongiform encephalopathy, Canine leishmaniasis, Chronic wasting disease, Classical swine fever, Echinococcus, Enzootic pneumonia, FIP, Foot-and-mouth disease, Jaagsiekte, Maedi-Visna, Mastitis in animals, Microsporum canis, Orf (animal disease), Peste des petits ruminants, Pox diseases, Psittacine beak and feather disease, Rabies, Mediterranean fever (Brucellosis) or Bang's disease or undulant fever, Malta fever, contagious abortion, epizootic abortion, Salmonella food poisoning, enteric paratyphosis, Bacillary dysentery, Pseudotuberculosis, plague, pestilential fever, Tuberculosis, Vibrios, Circling disease, Weil's disease (Leptospirosis) or canicola fever, Hemorrhagic jaundice (Leptospira icterohaemorrhagiae), dairy worker fever (L. hardjo), Relapsing fever, tick-borne relapsing fever, spirochetal fever, vagabond fever, famine fever, Lyme arthritis, Bannworth's syndrome (lime disease), tick-borne meningopolyneuritis, erythema chronicum migrans, Vibriosis, Colibacteriosis, colitoxemia, white scours, gut edema of swine, enteric paratyphosis, Staphylococcal alimentary toxicosis, staphylococcal gastroenteritis, Canine Corona Virus (CCV) or canine parvovirus enteritis, feline infectious peritonitis virus, transmissible gastroenteritis (TGE) virus, Hagerman Redmouth Disease (ERMD), Infectious Hematopoietic necrosis (IHN), porcine Actinobacillus (Haemophilus) pleuropneumonia, Hansen's disease, Streptotrichosis, Mycotic Dermatitis of Sheep, Pseudoglanders, Whitmore's disease, Francis' disease, deer-fly fever, rabbit fever, O'Hara disease, Streptobacillary fever, Haverhill fever, epidemic arthritic erythema, sodoku, Shipping or transport fever, hemorrhagic septicemia, Ornithosis, Parrot Fever, Chlamydiosis, North American blastomycosis, Chicago disease, Gilchrist's disease, Cat Scratch Fever, Benign Lymphoreticulosis, Benign nonbacterial Lymphadenitis, Bacillary Angiomatosis, Bacillary Peliosis Hepatis, Query fever, Balkan influenza, Balkan grippe, abattoir fever, Tick-borne fever, pneumorickettsiosis, American Tick Typhus, Tick-borne Typhus Fever, Vesicular Rickettsiosis, Kew Gardens Spotted Fever, Flea-borne Typhus Fever, Endemic Typhus Fever, Urban Typhus, Ringworm, Dermatophytosis, Tinea, Trichophytosis, Microsporosis, Jock Itch, Athlete's Foot, Sporothrix schenckii, dimorphic fungus, Cryptococcosis and histoplasmosis, Benign Epidermal Monkeypox, BEMP, Herpesvirus simiae, Simian B Disease, Type C lethargic encephalitis, Yellow fever, Black Vomit, hantavirus pulmonary syndrome, Korean Hemorrhagic Fever, Nephropathia Epidemica, Epidemic Hemorrhagic Fever, Hemorrhagic Nephrosonephritis, lymphocytic choriomeningitis, Venezuelan equine encephalitis, California encephalitis/La crosse encephalitis, African Hemorrhagic Fever, Green or Vervet Monkey Disease, Hydrophobia, Lyssa, Infectious hepatitis, Epidemic hepatitis, Epidemic jaundice, Rubeola, Morbilli, Swine and Equine Influenza, Fowl Plague, Newcastle disease, Piroplasmosis, toxoplasmosis, African Sleeping Sickness, Gambian Trypanosomiasis, Rhodesian Trypanosomiasis, Chagas's Disease, Chagas-Mazza Disease, South American Trypanosomiasis, Entamoeba histolytica, Balantidial dysentery, cryptosporidiosis, giardiasis, Cutaneous leishmaniasis: Chiclero ulcer, espundia, pianbols, uta, and buba (in the Americas); oriental sore, Aleppo boil (in the Old World); Bagdad boil, Delhi boil, Baum ulcer, Visceral leishmaniasis: kala-azar, Microsporidiosis, Anisakiasis, Trichinosis, Angiostrongylosis, eosinophilic meningitis or meningoencephalitis (A. cantonensis), abdominal angiostrongylosis (A. costaricensis), Uncinariasis, Necatoriasis, Hookworm Disease, Capillariasis, Brugiasis, Toxocariasis, Oesophagostomiasis, Strongyloidiasis, Trichostrongylosis, Ascaridiasis, Diphyllobothriasis, Sparganosis, Hydatidosis, Hydatid Disease, Echinococcus granulosis, Cystic hydatid disease, Tapeworm Infection, Schistosoma, Burkitt lymphoma caused by EBV, Rous sarcoma caused by Rous retrovirus, Kaposi' sarcoma caused by herpes virus type 8, adult T-cell leukemia caused by HTLV-I retrovirus, and hairy cell leukemia caused by HTLV-II.
77. The method of claim 75 , wherein the infectious disease is selected from the group consisting of Bovine respiratory disease, Porcine respiratory disease, and Avian influenza.
78. The method of claim 75 , wherein the oncology is selected from the group consisting of osteosarcoma, leukemia, and lymphoma.
79. The method of claim 50 , wherein the one or more proteins is selected from the group consisting of erythropoetin, ghrelin, osteoprotegerin, RANKL, RANKL decoy, TNF-a antagonist, an IL-1 antagonist, G-CSF, GM-CSF, IFN-α, IFN-γ, angiostatin, endostatin, TNF-α, PP1DCY-LSRLOC, β-glucuronidase, and IL-12.
80. (canceled)
81. The method of claim 50 , wherein said disease or disorder is a lysosomal storage disorder.
82. The method of claim 81 , wherein the lysosomal storage disorder is selected from the group consisting of Pompe disease/Glycogen storage disease type II, Gaucher Disease (Type I, Type II, Type III), Fabry disease, Mucopolysaccharidosis II (Hunter syndrome), Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), Mucopolysaccharidosis I, Metachromatic Leukodystrophy, Neuronal Ceroid Lipofuscinoses or CLN6 disease (Atypical Late Infantile, Late Onset variant, Early Juvenile, Finnish Variant Late Infantile CLN5, Jansky-Bielschowsky disease/Late infantile CLN2/TPP1 Disease, Kufs/Adult-onset NCL/CLN4 disease, Northern Epilepsy/variant late infantile CLN8, Santavuori-Haltia/Infantile CLN1/PPT disease, Beta-mannosidosis), Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease, Sanfilippo syndrome Type A, Sanfilippo syndrome Type B, Sanfilippo syndrome Type C, Sanfilippo syndrome Type D, MPSI Hurler Syndrome, Niemann-Pick Disease (Type A, Type B, Type C, Type D), Activator Deficiency/GM2 Gangliosidosis, Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease, Farber disease, Fucosidosis, Galactosialidosis (Goldberg Syndrome), GM1 gangliosidosis (Infantile, Late infantile/Juvenile, Adult/Chronic), I-Cell disease/Mucolipidosis II, Infantile Free Sialic Acid Storage Disease/ISSD, Juvenile Hexosaminidase A Deficiency, Krabbe disease (Infantile Onset, Late Onset), Mucopolysaccharidoses disorders (Pseudo-Hurler polydystrophy/Mucolipidosis IIIA, Scheie Syndrome, MPS I Hurler-Scheie Syndrome, Morquio Type A/MPS IVA, Morquio Type B/MPS IVB, MPS IX Hyaluronidase Deficiency, Sly Syndrome (MPS VII), Mucolipidosis I/Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV), Multiple sulfatase deficiency, Pycnodysostosis, Sandhoff disease/Adult Onset/GM2 gangliosidosis, Sandhoff disease/GM2 gangliosidosis—Infantile, Sandhoff disease/GM2 gangliosidosis—Juvenile, Schindler disease, Salla disease, Infantile Sialic Acid Storage Disease, Tay-Sachs/GM2 gangliosidosis, Wolman disease, Asparylglucosaminuria, and prosaposin.
83. The method of claim 82 , wherein the lysosomal storage disorder is selected from the group consisting of Pompe disease/Glycogen storage disease type II, Gaucher Disease (Type I, Type II, Type III), Fabry disease, Mucopolysaccharidosis II (Hunter syndrome), Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), Mucopolysaccharidosis I, and Metachromatic Leukodystrophy.
84. The method of claim 81 , wherein the one or more proteins is selected from the group consisting of a-galactosidase A, Arylsulfatase A, a-glucosidase, b-glucosidase, glucocerebrosidase, CLN6 protein, Juvenile associated with CLN3, N-sulfoglucosamine sulfohyrolase (SGSH), a-N-acetylglucosaminidase, acetyl-CoA-glucosaminide acetyltransferase, N-acetylglucosamine-6-sulfatase, a-L-iduronidase, arylsulfatase B, acid sphingomyelinase, and iuduronate sulfatase.
85. The method claim 50 , wherein said disease or disorder is a liver disease.
86. The method of claim 85 , wherein the liver disease is Hepatitis B.
87. The method of claim 85 , wherein the liver disease is Hepatitis C.
88. The method of claim 85 , wherein the protein is by IFN-α.
89. The method of claim 85 , wherein the protein is ceruloplasmin.
90. The method claim 50 wherein said disease or disorder is an ocular disease.
91. The method of claim 90 , wherein said ocular disease is selected from the group consisting of: glaucoma, Open Angle Glaucoma, Angle Closure Glaucoma, Aniridic Glaucoma, Congenital Glaucoma, Juvenile Glaucoma, Lens-Induced Glaucoma, Neovascular Glaucoma, Post-Traumatic Glaucoma, Steroid-Induced Glaucoma, Sturge-Weber Syndrome Glaucoma, and Uveitis-Induced Glaucoma, diabetic retinopathy, macular degeneration, macular degeneration, choroidal neovascularization, vascular leak, and/or retinal edema, bacterial conjunctivitis, fungal conjunctivitis, viral conjunctivitis, uveitis, keratic precipitates, macular edema, inflammation response after intra-ocular lens implantation, uveitis syndromes, retinal vasculitis, sarcoidosis, Eales disease, acute retinal necrosis, Vogt Koyanaki Harada syndrome, occular toxoplasmosis, radiation retinopathy, proliferative vitreoretinopathy, endophthalmitis, ocular glaucomas, ischemic optic neuropathy, thyroid associated orbitopathy, orbital pseudotumor, pigment dispersion syndrome (pigmentary glaucoma), scleritis, episcleritis choroidopathies, retinopathies (for example, cystoid macular edema, central serous choroidopathy and presumed ocular histoplasmosis syndrome, retinal vascular disease, retinal artery occlusions, retinal vein occlusions, retinopathy of prematurity, retinitis pigmentosa, familial exudative vitreoretinopathy (FEVR), idiopathic polypoidal choroidal vasculopathy, epiretinal macular membranes and cataracts.
92.-102. (canceled)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/636,473 US20130195800A1 (en) | 2010-03-23 | 2011-03-23 | Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US31679210P | 2010-03-23 | 2010-03-23 | |
US36673110P | 2010-07-22 | 2010-07-22 | |
US201161431364P | 2011-01-10 | 2011-01-10 | |
US13/636,473 US20130195800A1 (en) | 2010-03-23 | 2011-03-23 | Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof |
PCT/US2011/029682 WO2011119773A1 (en) | 2010-03-23 | 2011-03-23 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2011/029682 A-371-Of-International WO2011119773A1 (en) | 2010-03-23 | 2011-03-23 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/982,815 Continuation US20160208285A1 (en) | 2010-03-23 | 2015-12-29 | Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
US20130195800A1 true US20130195800A1 (en) | 2013-08-01 |
Family
ID=44673605
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/636,473 Abandoned US20130195800A1 (en) | 2010-03-23 | 2011-03-23 | Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof |
US14/982,815 Abandoned US20160208285A1 (en) | 2010-03-23 | 2015-12-29 | Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof |
US15/648,623 Active US10584351B2 (en) | 2010-03-23 | 2017-07-13 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof |
US16/738,015 Abandoned US20200239906A1 (en) | 2010-03-23 | 2020-01-09 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof |
Family Applications After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/982,815 Abandoned US20160208285A1 (en) | 2010-03-23 | 2015-12-29 | Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof |
US15/648,623 Active US10584351B2 (en) | 2010-03-23 | 2017-07-13 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof |
US16/738,015 Abandoned US20200239906A1 (en) | 2010-03-23 | 2020-01-09 | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof |
Country Status (13)
Country | Link |
---|---|
US (4) | US20130195800A1 (en) |
EP (2) | EP3284821B1 (en) |
JP (4) | JP2013527753A (en) |
KR (3) | KR20180108886A (en) |
CN (2) | CN107519499A (en) |
AU (4) | AU2011232435B2 (en) |
BR (1) | BR112012024166A2 (en) |
CA (1) | CA2794196A1 (en) |
HK (1) | HK1250518A1 (en) |
MX (2) | MX362513B (en) |
RU (1) | RU2612788C2 (en) |
TW (2) | TW201823463A (en) |
WO (1) | WO2011119773A1 (en) |
Cited By (34)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105349578A (en) * | 2015-11-30 | 2016-02-24 | 肇庆大华农生物药品有限公司 | Chicken GM-CSF protein, and preparation method and application thereof |
WO2016191755A1 (en) * | 2015-05-28 | 2016-12-01 | Adrian Bot | Diagnostic methods for t cell therapy |
WO2017062953A1 (en) | 2015-10-10 | 2017-04-13 | Intrexon Corporation | Improved therapeutic control of proteolytically sensitive, destabilized forms of interleukin-12 |
WO2017062708A1 (en) * | 2015-10-09 | 2017-04-13 | Virginia Commonwealth University | T cell delivery of mda-7/il-24 to improve therapeutic eradication of cancer and generate protective antitumor immunity |
WO2017087723A1 (en) * | 2015-11-19 | 2017-05-26 | The Regents Of The University Of California | Conditionally repressible immune cell receptors and methods of use thereof |
WO2017180536A1 (en) * | 2016-04-11 | 2017-10-19 | Harrison Noah James | Technique to kill pathogens in vivo |
WO2017214333A1 (en) | 2016-06-08 | 2017-12-14 | Intrexon Corporation | Cd33 specific chimeric antigen receptors |
US9855298B2 (en) | 2015-05-28 | 2018-01-02 | Kite Pharma, Inc. | Methods of conditioning patients for T cell therapy |
WO2018022574A2 (en) | 2016-07-25 | 2018-02-01 | Intrexon Corporation | Control of phenotype in plants |
WO2018026872A1 (en) * | 2016-08-01 | 2018-02-08 | Virogin Biotech Canada Ltd | Oncolytic herpes simplex virus vectors expressing immune system-stimulatory molecules |
US20180161458A1 (en) * | 2015-06-12 | 2018-06-14 | Vaxart, Inc. | Formulations for small intestinal delivery of rsv and norovirus antigens |
WO2018132494A1 (en) | 2017-01-10 | 2018-07-19 | Intrexon Corporation | Modulating expression of polypeptides via new gene switch expression systems |
US10046047B2 (en) * | 2015-02-06 | 2018-08-14 | Heat Biologics, Inc. | Vector co-expressing vaccine and costimulatory molecules |
US20190262474A1 (en) * | 2012-08-01 | 2019-08-29 | Nationwide Children's Hospital | Intrathecal delivery of recombinant adeno-associated virus 9 |
WO2020023670A1 (en) * | 2018-07-24 | 2020-01-30 | University Of Virginia Patent Foundation | Compositions and methods for combating multidrug-resistant bacteria |
US10632152B2 (en) | 2013-02-15 | 2020-04-28 | The Regents Of The University Of California | Chimeric antigen receptor and methods of use thereof |
US20200239535A1 (en) * | 2017-04-24 | 2020-07-30 | Imperial Innovations Limited | Cancer Treatment |
WO2020206056A1 (en) | 2019-04-04 | 2020-10-08 | Greenvenus Llc | Peptides and methods of plant protection |
WO2020223322A1 (en) * | 2019-04-29 | 2020-11-05 | The University Of North Carolina At Chapel Hill | Intrathecal and intravenous combination gene therapy for the treatment of infantile batten disease |
US10888594B2 (en) | 2016-05-30 | 2021-01-12 | National University Corporation Tottori University | Genetically engineered vaccinia viruses |
US11007265B2 (en) * | 2014-10-01 | 2021-05-18 | The Trustees Of The University Of Pennsylvania | Vaccines having an antigen and interleukin-21 as an adjuvant |
US11103596B2 (en) | 2015-05-11 | 2021-08-31 | Ucl Business Plc | Fabry disease gene therapy |
US11118168B2 (en) | 2017-06-07 | 2021-09-14 | Precigen, Inc. | Expression of novel cell tags |
US11136562B2 (en) | 2016-01-08 | 2021-10-05 | The Regents Of The University Of California | Conditionally active heterodimeric polypeptides and methods of use thereof |
US11154622B2 (en) | 2015-11-11 | 2021-10-26 | Precigen, Inc. | Compositions and methods for expression of multiple biologically active polypeptides from a single vector for treatment of cardiac conditions and other pathologies |
US20210380966A1 (en) * | 2014-04-30 | 2021-12-09 | Qiagen Gmbh | Method for isolating poly(a) nucleic acids |
US11219696B2 (en) | 2008-12-19 | 2022-01-11 | Nationwide Children's Hospital | Delivery of polynucleotides using recombinant AAV9 |
WO2022006332A3 (en) * | 2020-06-30 | 2022-02-03 | Chan Zuckerberg Biohub, Inc. | Gene therapy for immuno-oncology applications |
US11344589B2 (en) | 2016-05-30 | 2022-05-31 | National University Corporation Tottori University | Genetically engineered vaccinia viruses |
US11548930B2 (en) | 2017-04-04 | 2023-01-10 | Heat Biologics, Inc. | Intratumoral vaccination |
US11590210B2 (en) | 2011-06-08 | 2023-02-28 | Nationwide Children's Hospital, Inc. | Methods for delivery of polynucleotides by adeno-associated virus for lysosomal storage disorders |
US11608362B2 (en) | 2018-03-06 | 2023-03-21 | Precigen, Inc. | Hepatitis B vaccines and uses of the same |
US11638730B2 (en) | 2018-09-26 | 2023-05-02 | Astellas Pharma Inc. | Cancer therapy by combination use of oncolytic vaccinia virus and immune checkpoint inhibitor, and pharmaceutical composition and combination medicine for use in the cancer therapy |
US11666649B2 (en) | 2016-10-11 | 2023-06-06 | University Of Miami | Vectors and vaccine cells for immunity against Zika virus |
Families Citing this family (57)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9492482B2 (en) | 2008-10-08 | 2016-11-15 | Intrexon Corporation | Engineered cells expressing multiple immunomodulators and uses thereof |
EP3284821B1 (en) | 2010-03-23 | 2022-01-05 | Precigen, Inc. | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof |
EP3450568A3 (en) | 2011-03-04 | 2019-04-24 | Intrexon Corporation | Vectors conditionally expressing protein |
US11951157B2 (en) | 2011-10-11 | 2024-04-09 | Universitat Zurich | Methods of treating malignant tumour with IL-12 and anti-PD-1 antibody |
ES2888249T3 (en) | 2011-10-11 | 2022-01-03 | Univ Zuerich | Combination drug comprising IL-12 and an agent for blocking T-cell inhibitory molecules for tumor therapy |
CN102660567A (en) * | 2012-05-31 | 2012-09-12 | 程明荣 | Method for constructing pGM (granulocyte-macrophage) CSF (colony-stimulating factor)-IRES (internal ribosome entry site)-Rae1-IL (interleukin)21 vector |
WO2014145252A2 (en) | 2013-03-15 | 2014-09-18 | Milone Michael C | Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy |
WO2015090229A1 (en) | 2013-12-20 | 2015-06-25 | Novartis Ag | Regulatable chimeric antigen receptor |
CA2927862C (en) * | 2013-12-30 | 2024-01-23 | Curevac Ag | Artificial nucleic acid molecules |
MX2016011690A (en) * | 2014-03-21 | 2016-11-07 | Univ Leland Stanford Junior | Genome editing without nucleases. |
WO2016040643A1 (en) * | 2014-09-10 | 2016-03-17 | The Uab Research Foundation | Amyotrophic lateral sclerosis (als) biomarkers and uses thereof |
BR112017005390A2 (en) | 2014-09-17 | 2017-12-12 | Novartis Ag | target cytotoxic cells with chimeric receptors for adoptive immunotherapy |
EP3250682A4 (en) * | 2015-01-26 | 2018-08-01 | Fate Therapeutics, Inc. | Cells with increased immuno-regulatory properties and methods for their use and manufacture |
AU2016222887B2 (en) | 2015-02-24 | 2022-07-14 | The Regents Of The University Of California | Binding-triggered transcriptional switches and methods of use thereof |
CN104906104A (en) * | 2015-05-15 | 2015-09-16 | 铜仁学院 | Applications of a TGF-beta1 receptor blocker in preparation of medicines treating Hydatid disease |
CN106349388B (en) * | 2015-07-17 | 2021-04-02 | 上海佳文英莉生物技术有限公司 | Antibody for promoting programmed cell necrosis and application thereof |
MY190565A (en) * | 2016-03-17 | 2022-04-27 | Univ Yamaguchi | Immunocompetent cell and expression vector expressing regulatory factors of immune function |
CN109477085B (en) * | 2016-04-22 | 2023-08-01 | 萨鲁德医疗公司 | Methods and compositions for enhancing anti-inflammatory effects of interleukin 10 |
HRP20211145T1 (en) * | 2016-05-30 | 2021-12-24 | Astellas Pharma Inc. | New genetically-modified vaccinia virus |
WO2018022608A2 (en) | 2016-07-26 | 2018-02-01 | Biomarin Pharmaceutical Inc. | Novel adeno-associated virus capsid proteins |
CN106591368A (en) * | 2016-10-12 | 2017-04-26 | 郑州大学 | B subgroup adenovirus 11 vector carrying IL-15R/IL-15 fusion genes and construction and application of the same |
NZ752941A (en) | 2016-11-09 | 2023-04-28 | Intrexon Corp | Frataxin expression constructs |
CN108531497B (en) * | 2016-12-26 | 2021-06-15 | 中国农业科学院兰州兽医研究所 | Preparation method of recombinant protein P35 |
CA3052090A1 (en) * | 2017-01-30 | 2018-08-02 | Epicentrx, Inc. | Multiple transgene recombinant adenovirus |
AU2018213407A1 (en) * | 2017-01-30 | 2019-08-08 | Unum Therapeutics Inc. | Improved antibody-coupled T cell receptor constructs and therapeutic uses thereof |
TW201842921A (en) | 2017-02-28 | 2018-12-16 | 法商賽諾菲公司 | Therapeutic rna |
JP2020515564A (en) * | 2017-03-30 | 2020-05-28 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | Methods and pharmaceutical compositions for reducing episomal virus persistence and expression |
JP2020517615A (en) * | 2017-04-21 | 2020-06-18 | プレシゲン,インコーポレイテッド | Delivery of autologous cells containing matrix metalloproteinases for the treatment of scleroderma |
US20210324354A1 (en) * | 2017-05-12 | 2021-10-21 | The Children's Hospital Of Philadelphia | Sulfamidase (sgsh) variants, vectors, compositions and methods and uses for treating mucopolysaccharidosis type iiia (mps iiia) |
KR102008407B1 (en) * | 2017-09-05 | 2019-08-08 | 한국생명공학연구원 | Codon optimized il-21 and uses thereof |
WO2019090355A1 (en) * | 2017-11-06 | 2019-05-09 | Children's National Medical Center | Cells expressing antibodies and methods of treatment using the same |
WO2019097454A1 (en) | 2017-11-15 | 2019-05-23 | Friedrich Miescher Institute For Biomedical Research | Primate retinal pigment epithelium cell-specific promoter |
CN108060135A (en) * | 2017-12-26 | 2018-05-22 | 东莞赛尔生物科技有限公司 | A kind of T cell, the preparation method and application of high efficient expression P53 cancer suppressor proteins |
CN110016464A (en) * | 2018-01-08 | 2019-07-16 | 张晋宇 | For screening the system and method for antitumorigenic substance |
JP7102670B2 (en) * | 2018-01-25 | 2022-07-20 | アイ-エムエービー バイオファーマ ユーエス リミテッド | Fusion of anti-PD-L1 antibody and IL-7 |
CA3093086A1 (en) | 2018-03-05 | 2019-09-12 | The Schepens Eye Research Institute, Inc. | A therapy for glaucoma and optic neuropathy by targeting colony stimulating factors |
CA3092937A1 (en) * | 2018-03-06 | 2019-09-12 | PGEN Therapeutics, Inc. | Human papillomavirus vaccines and uses of the same |
CN108588278A (en) * | 2018-04-20 | 2018-09-28 | 咸阳职业技术学院 | A kind of parrot beak ptilosis virus PCR diagnostic kit and its detection method |
SG11202010645VA (en) * | 2018-04-27 | 2020-11-27 | Medical College Wisconsin Inc | Use of lentivector-transduced t-rapa cells for amelioration of lysosomal storage disorders |
EP3790627A2 (en) | 2018-05-09 | 2021-03-17 | BioMarin Pharmaceutical Inc. | Methods of treating phenylketonuria |
TW202005978A (en) | 2018-05-14 | 2020-02-01 | 美商拜奧馬林製藥公司 | Novel liver targeting adeno-associated viral vectors |
WO2019232253A1 (en) * | 2018-05-30 | 2019-12-05 | The Regents Of The University Of California | Gene editing of monogenic disorders in human hematopoietic stem cells -- correction of x-linked agammaglobulinemia (xla) |
JP7438988B2 (en) | 2018-06-13 | 2024-02-27 | ノバルティス アーゲー | BCMA chimeric antigen receptor and its use |
AU2019291069A1 (en) * | 2018-06-19 | 2021-01-28 | Fondazione Centro San Raffaele | Production of engineered dendritic cells and uses thereof |
WO2020006098A2 (en) * | 2018-06-26 | 2020-01-02 | Children's National Medical Center | Tumor-associated markers for detection of minimal residual disease using digital droplet pcr |
CA3116138A1 (en) * | 2018-10-17 | 2020-04-23 | Senti Biosciences, Inc. | Combinatorial cancer immunotherapy |
TW202039537A (en) * | 2018-11-27 | 2020-11-01 | 美商昂科賽克醫療公司 | Plasmid constructs for treating cancer and methods of use |
KR102524247B1 (en) * | 2018-12-21 | 2023-04-21 | 가톨릭대학교 산학협력단 | p40-EBI3 COMPLEX AND USES THEREOF |
CN109701019B (en) * | 2019-01-04 | 2021-07-16 | 中国人民解放军第二军医大学 | Novel long-chain non-coding RNA (lnc-Dpf 3), sequence, immune effect and application thereof |
TW202043256A (en) | 2019-01-10 | 2020-12-01 | 美商健生生物科技公司 | Prostate neoantigens and their uses |
GB201900741D0 (en) * | 2019-01-18 | 2019-03-06 | Synpromics Ltd | Liver-specifc inducible prometers and methods of use thereof |
KR20220024508A (en) * | 2019-06-13 | 2022-03-03 | 노봄 바이오테크놀로지스, 인크. | Biologically Contained Bacteria and Their Uses |
WO2021119539A1 (en) * | 2019-12-12 | 2021-06-17 | Senti Biosciences, Inc. | Method and compositions for regulated armoring of cells |
WO2021163874A1 (en) * | 2020-02-18 | 2021-08-26 | 苏州工业园区唯可达生物科技有限公司 | Recombinant viral vector, immunogenic composition comprising same, and uses |
JP2021182873A (en) * | 2020-05-21 | 2021-12-02 | 学校法人帝京大学 | Thermostable glucocerebrosidase |
CN113173983B (en) * | 2021-04-20 | 2023-04-18 | 西南大学 | Method for large-scale production of fluorescent protein by using silkworm silk gland |
WO2022266396A1 (en) * | 2021-06-16 | 2022-12-22 | Senti Biosciences, Inc. | Armed chimeric receptors and methods of use thereof |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20030082722A1 (en) * | 2001-08-08 | 2003-05-01 | Bingliang Fang | Method for amplifying expression from a cell specific promoter |
US7138511B1 (en) * | 1997-01-28 | 2006-11-21 | The Regents Of The University Of California | Nucleic acids, kits and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders |
US20090136465A1 (en) * | 2007-09-28 | 2009-05-28 | Intrexon Corporation | Therapeutic Gene-Switch Constructs and Bioreactors for the Expression of Biotherapeutic Molecules, and Uses Thereof |
US20100184838A1 (en) * | 2007-04-13 | 2010-07-22 | Tufts University | Compositions and methods for retinal transduction and photoreceptor specific transgene expression |
US20100292307A1 (en) * | 2006-03-28 | 2010-11-18 | Isis Innovation Limited | Construct |
US20110268766A1 (en) * | 2008-10-08 | 2011-11-03 | Intrexon Corporation | Engineered Cells Expressing Multiple Immunomodulators And Uses Thereof |
Family Cites Families (119)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4148877A (en) | 1975-12-29 | 1979-04-10 | Choay S. A. | Fraction capable of inducing in vivo a resistance to bacterial infections, process for obtaining said fraction from bacteria and drugs containing said fraction |
US5308838A (en) | 1982-05-07 | 1994-05-03 | Carrington Laboratories, Inc. | Uses of aloe products |
EP0154434B1 (en) | 1984-02-17 | 1993-01-27 | Genentech, Inc. | Human transforming growth factor and precursor or fragment thereof, cells, dna, vectors and methods for their production, compositions and products containing them, and related antibodies and diagnostic methods |
US4797368A (en) | 1985-03-15 | 1989-01-10 | The United States Of America As Represented By The Department Of Health And Human Services | Adeno-associated virus as eukaryotic expression vector |
US4985461A (en) | 1985-10-21 | 1991-01-15 | Rohm And Haas Company | Insecticidal N'-substituted-N,N'-diacylhydrazines |
US5117057A (en) | 1985-10-21 | 1992-05-26 | Rohm And Haas Company | Insecticidal N' substituted-N-N'-disubstituted-hydrazines |
FR2593630B1 (en) | 1986-01-27 | 1988-03-18 | Maurice Francois | DRAIN RESISTANCE ACTIVE MATRIX DISPLAY SCREEN AND METHODS OF MAKING SAME |
US5225443A (en) | 1986-05-01 | 1993-07-06 | Rohm And Haas Company | Insecticidal N'-substituted-N'-substituted N,N'-diacylhydrazines |
US4981784A (en) | 1987-12-02 | 1991-01-01 | The Salk Institute For Biological Studies | Retinoic acid receptor method |
US5571714A (en) | 1988-12-22 | 1996-11-05 | Celtrix Pharmaceuticals, Inc. | Monoclonal antibodies which bind both transforming growth factors β1 and β2 and methods of use |
US5354844A (en) | 1989-03-16 | 1994-10-11 | Boehringer Ingelheim International Gmbh | Protein-polycation conjugates |
US5693622A (en) | 1989-03-21 | 1997-12-02 | Vical Incorporated | Expression of exogenous polynucleotide sequences cardiac muscle of a mammal |
US5703055A (en) | 1989-03-21 | 1997-12-30 | Wisconsin Alumni Research Foundation | Generation of antibodies through lipid mediated DNA delivery |
GB8918616D0 (en) | 1989-08-15 | 1989-09-27 | Univ Glasgow | Herpes simplex virus type 1 mutant |
US5242902A (en) | 1989-09-06 | 1993-09-07 | The Regents Of The University Of California | Defensin peptide compositions and methods for their use |
US5264618A (en) | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
CA2043775A1 (en) | 1990-06-14 | 1991-12-15 | Dat P. Le | Dibenzoylakylcyanohydrazines |
US5849572A (en) | 1990-10-10 | 1998-12-15 | Regents Of The University Of Michigan | HSV-1 vector containing a lat promoter |
IL100643A (en) | 1991-01-25 | 1996-10-31 | Nippon Kayaku Kk | Hydrazine derivatives and pesticidal compositions comprising such derivatives as effective component |
US5833976A (en) | 1991-08-06 | 1998-11-10 | Schering Corporation | Use of interleukin-10 (IL-10) to treat endotoxin- or superantigen-induced toxicity |
US5530028A (en) | 1992-11-23 | 1996-06-25 | Rohm And Haas Company | Insecticidal N'-substituted-N,N'-diacylhydrazines |
US6013836A (en) | 1992-02-28 | 2000-01-11 | Rohm And Haas Company | Insecticidal N'-substituted-N,N'-disubstitutedhydrazines |
IL105503A (en) | 1992-04-28 | 1999-05-09 | Astra Ab | Peptide-carbohydrate conjugates capable of generating t cell immunity |
US5292513A (en) | 1992-05-18 | 1994-03-08 | Anthony G. Gristina | Method for nonspecific cellular immune stimulation |
US5804413A (en) | 1992-07-31 | 1998-09-08 | University Of Pittsburgh Of The Commonwealth System Of Higher Education | Herpes simplex virus strains for gene transfer |
US5464758A (en) | 1993-06-14 | 1995-11-07 | Gossen; Manfred | Tight control of gene expression in eucaryotic cells by tetracycline-responsive promoters |
FR2714830B1 (en) | 1994-01-10 | 1996-03-22 | Rhone Poulenc Rorer Sa | Composition containing nucleic acids, preparation and uses. |
FR2715847B1 (en) | 1994-02-08 | 1996-04-12 | Rhone Poulenc Rorer Sa | Composition containing nucleic acids, preparation and uses. |
US6150137A (en) | 1994-05-27 | 2000-11-21 | Ariad Pharmaceuticals, Inc. | Immunosuppressant target proteins |
US5851806A (en) | 1994-06-10 | 1998-12-22 | Genvec, Inc. | Complementary adenoviral systems and cell lines |
DE69535178T2 (en) | 1994-06-10 | 2006-12-14 | Genvec, Inc. | ADENOVER VECTOR SYSTEMS AND CELL LINES |
GB9415319D0 (en) | 1994-07-29 | 1994-09-21 | Medical Res Council | HSV viral vector |
US5846782A (en) | 1995-11-28 | 1998-12-08 | Genvec, Inc. | Targeting adenovirus with use of constrained peptide motifs |
US5965541A (en) | 1995-11-28 | 1999-10-12 | Genvec, Inc. | Vectors and methods for gene transfer to cells |
US5559099A (en) | 1994-09-08 | 1996-09-24 | Genvec, Inc. | Penton base protein and methods of using same |
US5962311A (en) | 1994-09-08 | 1999-10-05 | Genvec, Inc. | Short-shafted adenoviral fiber and its use |
FR2727679B1 (en) | 1994-12-05 | 1997-01-03 | Rhone Poulenc Rorer Sa | NEW TRANSFECTION AGENTS AND THEIR PHARMACEUTICAL APPLICATIONS |
FR2730637B1 (en) | 1995-02-17 | 1997-03-28 | Rhone Poulenc Rorer Sa | PHARMACEUTICAL COMPOSITION CONTAINING NUCLEIC ACIDS, AND USES THEREOF |
US6127525A (en) | 1995-02-21 | 2000-10-03 | Cornell Research Foundation, Inc. | Chimeric adenoviral coat protein and methods of using same |
CA2213340C (en) | 1995-03-03 | 2011-07-26 | Novartis Ag | Control of gene expression in plants by receptor mediated transactivation in the presence of a chemical ligand |
US6747138B1 (en) | 1995-04-03 | 2004-06-08 | Regents Of The University Of Michigan | Methods and compositions for regulating Fas-associated apoptosis |
AU711391B2 (en) | 1995-05-26 | 1999-10-14 | Syngenta Limited | A gene switch comprising an ecdysone receptor |
US6187757B1 (en) | 1995-06-07 | 2001-02-13 | Ariad Pharmaceuticals, Inc. | Regulation of biological events using novel compounds |
DE69638058D1 (en) | 1995-06-15 | 2009-11-26 | Crucell Holland Bv | Packaging systems for human recombinant adenoviruses for gene therapy |
US5654174A (en) | 1995-07-07 | 1997-08-05 | Competitive Technologies, Inc. | Herpes simplex virus glycoprotein D variants |
US5837511A (en) | 1995-10-02 | 1998-11-17 | Cornell Research Foundation, Inc. | Non-group C adenoviral vectors |
US6734014B1 (en) | 1996-02-08 | 2004-05-11 | The United States Of America As Represented By The Department Of Health And Human Services | Methods and compositions for transforming dendritic cells and activating T cells |
US6723531B2 (en) | 1996-04-05 | 2004-04-20 | The Salk Institute For Biological Studies | Method for modulating expression of exogenous genes in mammalian systems, and products related thereto |
US6022534A (en) | 1996-10-24 | 2000-02-08 | Schering Corporation | Lymphotactin uses |
JP2001504690A (en) * | 1996-10-29 | 2001-04-10 | バレンティス・インコーポレーテッド | Modified steroid hormones for gene therapy and their use |
US6323317B1 (en) | 1996-11-01 | 2001-11-27 | The Walter And Eliza Hall Institute Of Medical Research | Therapeutic and diagnostics proteins comprising a SOCS box |
US6177980B1 (en) | 1997-02-20 | 2001-01-23 | Kenneth C. Johnson | High-throughput, maskless lithography system |
US6133426A (en) | 1997-02-21 | 2000-10-17 | Genentech, Inc. | Humanized anti-IL-8 monoclonal antibodies |
US6020191A (en) | 1997-04-14 | 2000-02-01 | Genzyme Corporation | Adenoviral vectors capable of facilitating increased persistence of transgene expression |
ATE256196T1 (en) | 1997-06-09 | 2003-12-15 | Genvec Inc | CHIMERIC VECTORS CONTAINING THE PACKAGING REGION OF A PHAGE GENOME AND PART OF THE GENOME OF A EUKARYONTIC VIRUS |
US7118742B2 (en) | 1997-07-07 | 2006-10-10 | La Jolla Institute For Allergy And Immunology | Ligand for herpes simplex virus entry mediator and methods of use |
AU8605598A (en) | 1997-07-31 | 1999-02-22 | University Of Pittsburgh | Targeted hsv vectors |
CA2300376A1 (en) | 1997-08-26 | 1999-03-04 | Ariad Gene Therapeutics, Inc. | Fusion proteins comprising a dimerization, trimerization or tetramerization domain and an additional heterologous transcription activation, transcription repression, dna binding or ligand binding domain |
US6015709A (en) | 1997-08-26 | 2000-01-18 | Ariad Pharmaceuticals, Inc. | Transcriptional activators, and compositions and uses related thereto |
US20020048792A1 (en) | 1997-08-26 | 2002-04-25 | Ariad Gene Therapeutics, Inc. | Methods and materials for regulated production of proteins |
US6479653B1 (en) | 1997-08-26 | 2002-11-12 | Ariad Gene Therapeutics, Inc. | Compositions and method for regulation of transcription |
WO1999015686A1 (en) | 1997-09-23 | 1999-04-01 | Genvec, Inc. | Plasmids for construction of eukaryotic viral vectors |
US6503184B1 (en) | 1997-10-21 | 2003-01-07 | Human Genome Sciences, Inc. | Human tumor necrosis factor receptor-like proteins TR11, TR11SV1 and TR11SV2 |
US5981225A (en) | 1998-04-16 | 1999-11-09 | Baylor College Of Medicine | Gene transfer vector, recombinant adenovirus particles containing the same, method for producing the same and method of use of the same |
JP2002512361A (en) | 1998-04-22 | 2002-04-23 | ジェンベク、インコーポレイティッド | Efficient purification of adenovirus |
US6333318B1 (en) | 1998-05-14 | 2001-12-25 | The Salk Institute For Biological Studies | Formulations useful for modulating expression of exogenous genes in mammalian systems, and products related thereto |
US6258603B1 (en) | 1998-06-17 | 2001-07-10 | Rohm And Haas Company | Ligands for modulating the expression of exogenous genes via an ecdysone receptor complex |
US6113913A (en) | 1998-06-26 | 2000-09-05 | Genvec, Inc. | Recombinant adenovirus |
US5965358A (en) | 1998-08-26 | 1999-10-12 | Genvec, Inc. | Method for assessing the relative purity of viral gene transfer vector stocks |
US6225289B1 (en) | 1998-12-10 | 2001-05-01 | Genvec, Inc. | Methods and compositions for preserving adenoviral vectors |
US6696272B1 (en) * | 1999-06-02 | 2004-02-24 | Hsc Research & Development Limited Partnership | Products and methods for gaucher disease therapy |
JP2003501445A (en) * | 1999-06-08 | 2003-01-14 | ユーエイビー リサーチ ファンデーション | Herpes simplex virus expressing foreign gene and method of treating cancer with the same |
CA2375490A1 (en) * | 1999-06-18 | 2000-12-28 | Ariad Gene Therapeutics, Inc. | Chimeric oca-b transcription factors |
CA2589418A1 (en) | 1999-08-24 | 2001-03-01 | Medarex, Inc. | Human ctla-4 antibodies and their uses |
US7034121B2 (en) | 2000-01-27 | 2006-04-25 | Genetics Institue, Llc | Antibodies against CTLA4 |
WO2001070816A2 (en) | 2000-03-22 | 2001-09-27 | Rohm And Haas Company | Ecdysone receptor-based inducible gene expression system |
US20040033600A1 (en) | 2001-03-21 | 2004-02-19 | Palli Subba Reddy | Ecdysone receptor-based inducible gene expression system |
WO2001079555A2 (en) | 2000-04-14 | 2001-10-25 | Millennium Pharmaceuticals, Inc. | Roles of jak/stat family members in tolerance induction |
US8105825B2 (en) | 2000-10-03 | 2012-01-31 | Intrexon Corporation | Multiple inducible gene regulation system |
US6756215B1 (en) | 2000-10-20 | 2004-06-29 | The United States Of America As Represented By The Secretary Of The Department Of Health & Human Services | Functionalized TGF-β fusion proteins |
EP3470520A2 (en) | 2001-02-20 | 2019-04-17 | Intrexon Corporation | Novel substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system |
ES2424812T3 (en) | 2001-02-20 | 2013-10-08 | Intrexon Corporation | Chimeric X retinoid receptors and their use in an inducible gene expression system based on novel ecdysone receptors |
ES2385598T3 (en) | 2001-02-20 | 2012-07-27 | Intrexon Corporation | Novel inducible gene expression system based on the ecdysone receptor / X retinoid invertebrate receptor |
WO2002066615A2 (en) | 2001-02-20 | 2002-08-29 | Rheogene, Inc. | Novel substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system |
AR036993A1 (en) | 2001-04-02 | 2004-10-20 | Wyeth Corp | USE OF AGENTS THAT MODULATE THE INTERACTION BETWEEN PD-1 AND ITS LINKS IN THE SUBMODULATION OF IMMUNOLOGICAL ANSWERS |
US20030180719A1 (en) * | 2001-04-13 | 2003-09-25 | Thomas Herget | Human cellular protein gastrointestinal glutathione peroxidase as target for medical intervention against hepatitis C virus infections |
US20040235169A1 (en) * | 2001-04-20 | 2004-11-25 | Evans Ronald M. | Inducible expression of transfected genes |
CA2459827C (en) | 2001-09-26 | 2013-09-03 | Subba Reddy Palli | Leafhopper ecdysone receptor nucleic acids, polypeptides, and uses thereof |
EP1490686A2 (en) | 2001-09-26 | 2004-12-29 | RheoGene Holdings Inc. | Whitefly ecdysone receptor nucleic acids, polypeptides, and uses thereof |
NZ532060A (en) | 2001-10-02 | 2005-11-25 | Inst Clayton De La Rech | Methods and compositions relating to restricted expression lentiviral vectors and their applications |
EP1467771B1 (en) | 2001-11-14 | 2008-04-16 | Novavax, Inc. | Mycobacterial vaccine |
ATE446106T1 (en) * | 2001-11-29 | 2009-11-15 | Dandrit Biotech As | PHARMACEUTICAL COMPOSITION FOR TRIGGERING AN IMMUNE RESPONSE IN A HUMAN OR AN ANIMAL |
US20050228016A1 (en) | 2002-06-13 | 2005-10-13 | Enrique Michelotti | Tetrahydroquinolines for modulating the expression of exogenous genes via an ecdysone receptor complex |
US7375093B2 (en) | 2002-07-05 | 2008-05-20 | Intrexon Corporation | Ketone ligands for modulating the expression of exogenous genes via an ecdysone receptor complex |
US20040049437A1 (en) | 2002-09-11 | 2004-03-11 | Govone Solutions, Lp | Method, system and computer program product for automating transaction tax calculation |
US7785871B2 (en) | 2002-10-09 | 2010-08-31 | Intrexon Corporation | DNA cloning vector plasmids and methods for their use |
US20080050808A1 (en) | 2002-10-09 | 2008-02-28 | Reed Thomas D | DNA modular cloning vector plasmids and methods for their use |
EP1608384A2 (en) | 2003-01-28 | 2005-12-28 | Shanghai Sunway Biotech Co Ltd | Therapy for primary and metastatic cancers |
US7304161B2 (en) | 2003-02-10 | 2007-12-04 | Intrexon Corporation | Diaclhydrazine ligands for modulating the expression of exogenous genes in mammalian systems via an ecdysone receptor complex |
US7304162B2 (en) | 2003-02-21 | 2007-12-04 | Intrexon Corporation | Oxadiazoline ligands for modulating the expression of exogenous genes via an ecdysone receptor complex |
US7456315B2 (en) | 2003-02-28 | 2008-11-25 | Intrexon Corporation | Bioavailable diacylhydrazine ligands for modulating the expression of exogenous genes via an ecdysone receptor complex |
CA2545755A1 (en) | 2003-11-12 | 2005-05-26 | Schering Corporation | Plasmid system for multigene expression |
US20050267027A1 (en) * | 2004-04-05 | 2005-12-01 | Lounsbury Karen M | Use of erythropoietin for treatment of cancer |
US7935510B2 (en) | 2004-04-30 | 2011-05-03 | Intrexon Corporation | Mutant receptors and their use in a nuclear receptor-based inducible gene expression system |
EP2484772B1 (en) | 2004-05-18 | 2016-08-17 | Intrexon Corporation | Methods for dynamic vector assembly of DNA cloning vector plasmids |
ES2292271B1 (en) | 2004-05-20 | 2009-02-16 | Proyecto De Biomedicina Cima, S.L. | AN ADENOVIRUS-ALFAVIRUS HYBRID VECTOR FOR THE EFFECTIVE ADMINISTRATION AND EXPRESSION OF THERAPEUTIC GENES IN TUMOR CELLS. |
CA2586107A1 (en) | 2004-11-03 | 2006-05-18 | Introgen Therapeutics Inc. | A novel method for the production and purification of adenoviral vectors |
HN2005035605A (en) | 2004-12-20 | 2010-08-05 | Wyeth Corp | RAPAMYCIN ANALOGS AND USES OF THE SAME IN THE TREATMENT OF NEUROLOGICAL, PROLIFERATIVE AND INFLAMMATORY DISORDERS |
RU2007119585A (en) | 2004-12-20 | 2009-01-27 | Вайет (Us) | Derivatives of rapamycin and their use in the treatment of neurological diseases |
US9034650B2 (en) | 2005-02-02 | 2015-05-19 | Intrexon Corporation | Site-specific serine recombinases and methods of their use |
US20080076729A1 (en) * | 2005-05-19 | 2008-03-27 | Schering Aktiengesellachaft | Interferon-beta gene therapy using an improved, regulated expression system |
EP1885858A2 (en) * | 2005-05-19 | 2008-02-13 | Bayer Schering Pharma Aktiengesellschaft | Treatment of disease using an improved regulated expression system |
US20080260690A1 (en) * | 2005-11-18 | 2008-10-23 | Ares Trading S.A. | Interferon in Influenza |
CN101235387A (en) * | 2007-02-02 | 2008-08-06 | 钱程 | Adenoids viral vectors for constructing endogenesis promoter to regulate and control exogenous gene |
US20080241100A1 (en) * | 2007-03-27 | 2008-10-02 | Stefan Strobl | Pharmaceutical composition comprising a cytokine |
WO2009009115A2 (en) * | 2007-07-11 | 2009-01-15 | Nevsky Pharmaceutical Development | Liposome compositions for treatment of hepatitis c |
ES2531522T3 (en) * | 2007-10-08 | 2015-03-16 | Intrexon Corporation | Engineered modified dendritic cells and uses for cancer treatment |
CN101565718A (en) * | 2009-06-11 | 2009-10-28 | 浙江理工大学 | Construction method of three-target mosaic type oncolytic adenovirus Ad5/F11 carrier and use thereof |
EP3284821B1 (en) | 2010-03-23 | 2022-01-05 | Precigen, Inc. | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof |
-
2011
- 2011-03-23 EP EP17184774.2A patent/EP3284821B1/en active Active
- 2011-03-23 EP EP11760175.7A patent/EP2550359B1/en active Active
- 2011-03-23 BR BR112012024166A patent/BR112012024166A2/en not_active Application Discontinuation
- 2011-03-23 US US13/636,473 patent/US20130195800A1/en not_active Abandoned
- 2011-03-23 CA CA2794196A patent/CA2794196A1/en active Pending
- 2011-03-23 KR KR1020187027412A patent/KR20180108886A/en active Search and Examination
- 2011-03-23 CN CN201710724929.2A patent/CN107519499A/en active Pending
- 2011-03-23 WO PCT/US2011/029682 patent/WO2011119773A1/en active Application Filing
- 2011-03-23 RU RU2012140395A patent/RU2612788C2/en active
- 2011-03-23 AU AU2011232435A patent/AU2011232435B2/en not_active Ceased
- 2011-03-23 TW TW106131444A patent/TW201823463A/en unknown
- 2011-03-23 KR KR1020177031625A patent/KR102049161B1/en active IP Right Grant
- 2011-03-23 CN CN2011800256991A patent/CN103038343A/en active Pending
- 2011-03-23 KR KR1020127027532A patent/KR20130010121A/en active Application Filing
- 2011-03-23 MX MX2016004897A patent/MX362513B/en unknown
- 2011-03-23 JP JP2013501440A patent/JP2013527753A/en not_active Withdrawn
- 2011-03-23 TW TW106128457A patent/TWI688395B/en active
-
2012
- 2012-09-21 MX MX2018008829A patent/MX2018008829A/en unknown
-
2015
- 2015-12-29 US US14/982,815 patent/US20160208285A1/en not_active Abandoned
-
2016
- 2016-04-18 AU AU2016202426A patent/AU2016202426B2/en active Active
- 2016-05-25 JP JP2016104570A patent/JP6419107B2/en active Active
-
2017
- 2017-07-13 US US15/648,623 patent/US10584351B2/en active Active
- 2017-07-25 JP JP2017143711A patent/JP6732704B2/en active Active
-
2018
- 2018-07-31 HK HK18109887.7A patent/HK1250518A1/en unknown
- 2018-09-26 AU AU2018236775A patent/AU2018236775A1/en not_active Abandoned
-
2020
- 2020-01-09 US US16/738,015 patent/US20200239906A1/en not_active Abandoned
- 2020-03-04 JP JP2020036359A patent/JP2020110156A/en not_active Withdrawn
-
2021
- 2021-06-15 AU AU2021203961A patent/AU2021203961A1/en not_active Abandoned
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7138511B1 (en) * | 1997-01-28 | 2006-11-21 | The Regents Of The University Of California | Nucleic acids, kits and methods for the diagnosis, prognosis and treatment of glaucoma and related disorders |
US20030082722A1 (en) * | 2001-08-08 | 2003-05-01 | Bingliang Fang | Method for amplifying expression from a cell specific promoter |
US20100292307A1 (en) * | 2006-03-28 | 2010-11-18 | Isis Innovation Limited | Construct |
US20100184838A1 (en) * | 2007-04-13 | 2010-07-22 | Tufts University | Compositions and methods for retinal transduction and photoreceptor specific transgene expression |
US20090136465A1 (en) * | 2007-09-28 | 2009-05-28 | Intrexon Corporation | Therapeutic Gene-Switch Constructs and Bioreactors for the Expression of Biotherapeutic Molecules, and Uses Thereof |
US20110268766A1 (en) * | 2008-10-08 | 2011-11-03 | Intrexon Corporation | Engineered Cells Expressing Multiple Immunomodulators And Uses Thereof |
Non-Patent Citations (5)
Title |
---|
Campochiaro et al, Adenoviral Vector-Delivered Pigment Epithelium-Derived Factor for Neovascular Age-Related Macular Degeneration: Results of a Phase I Clinical Trial, HUMAN GENE THERAPY 17:167-176 (February 2006) * |
Conley et al, Non-Viral Ocular Gene Therapy: Assessment and Future Directions, Curr Opin Mol Ther. 2008 October ; 10(5): 456-463. * |
Green and Seymour, Review: Adenoviral vectors: Systemic delivery and tumor targeting, Cancer Gene Therapy (2002) 9, 1036 - 1042 * |
Orkin et al, Report and Recommendations of the Panel to Assess the NIH Investment in Resaerch on Gene Therapy, 1995, pages 1-50 * |
Raty et al, Gene Therapy: The First Approved Gene-Based Medicines, Molecular Mechanisms and Clinical Indication, Current Molecular Pharmacology, 2008, 1, 13-23 * |
Cited By (51)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11219696B2 (en) | 2008-12-19 | 2022-01-11 | Nationwide Children's Hospital | Delivery of polynucleotides using recombinant AAV9 |
US11590210B2 (en) | 2011-06-08 | 2023-02-28 | Nationwide Children's Hospital, Inc. | Methods for delivery of polynucleotides by adeno-associated virus for lysosomal storage disorders |
US20190262474A1 (en) * | 2012-08-01 | 2019-08-29 | Nationwide Children's Hospital | Intrathecal delivery of recombinant adeno-associated virus 9 |
US11738094B2 (en) * | 2012-08-01 | 2023-08-29 | Nationwide Children's Hospital | Intrathecal delivery of recombinant adeno-associated virus 9 |
US11730829B2 (en) | 2012-08-01 | 2023-08-22 | Nationwide Children's Hospital | Intrathecal delivery of recombinant adeno-associated virus 9 |
US11413357B2 (en) | 2012-08-01 | 2022-08-16 | Nationwide Children's Hospital | Intrathecal delivery of recombinant adeno-associated virus 9 |
US11311634B2 (en) | 2012-08-01 | 2022-04-26 | Nationwide Children's Hospital | Intrathecal delivery of recombinant Adeno-associated virus 9 |
US11040116B2 (en) | 2012-08-01 | 2021-06-22 | Nationwide Children's Hospital | Intrathecal delivery of recombinant adeno-associated virus 9 |
US11478510B2 (en) | 2013-02-15 | 2022-10-25 | The Regents Of The University Of California | Chimeric antigen receptor and methods of use thereof |
US10888581B2 (en) | 2013-02-15 | 2021-01-12 | The Regents Of The University Of California | Chimeric antigen receptor and methods of use thereof |
US10632152B2 (en) | 2013-02-15 | 2020-04-28 | The Regents Of The University Of California | Chimeric antigen receptor and methods of use thereof |
US20210380966A1 (en) * | 2014-04-30 | 2021-12-09 | Qiagen Gmbh | Method for isolating poly(a) nucleic acids |
US11007265B2 (en) * | 2014-10-01 | 2021-05-18 | The Trustees Of The University Of Pennsylvania | Vaccines having an antigen and interleukin-21 as an adjuvant |
US10046047B2 (en) * | 2015-02-06 | 2018-08-14 | Heat Biologics, Inc. | Vector co-expressing vaccine and costimulatory molecules |
US10758611B2 (en) | 2015-02-06 | 2020-09-01 | Heat Biologics, Inc. | Vector co-expressing vaccine and costimulatory molecules |
US10780161B2 (en) | 2015-02-06 | 2020-09-22 | Heat Biologics, Inc. | Vector co-expressing vaccine and costimulatory molecules |
US11103596B2 (en) | 2015-05-11 | 2021-08-31 | Ucl Business Plc | Fabry disease gene therapy |
US10322146B2 (en) | 2015-05-28 | 2019-06-18 | Kite Pharma, Inc. | Methods of conditioning patients for T cell therapy |
US11779601B2 (en) | 2015-05-28 | 2023-10-10 | Kite Pharma, Inc. | Diagnostic methods for T cell therapy |
WO2016191755A1 (en) * | 2015-05-28 | 2016-12-01 | Adrian Bot | Diagnostic methods for t cell therapy |
US9855298B2 (en) | 2015-05-28 | 2018-01-02 | Kite Pharma, Inc. | Methods of conditioning patients for T cell therapy |
US11491187B2 (en) | 2015-05-28 | 2022-11-08 | Kite Pharma, Inc. | Methods of conditioning patients for T cell therapy |
US20180161458A1 (en) * | 2015-06-12 | 2018-06-14 | Vaxart, Inc. | Formulations for small intestinal delivery of rsv and norovirus antigens |
US11433146B2 (en) * | 2015-06-12 | 2022-09-06 | Vaxart, Inc. | Formulations for small intestinal delivery of RSV and norovirus antigens |
WO2017062708A1 (en) * | 2015-10-09 | 2017-04-13 | Virginia Commonwealth University | T cell delivery of mda-7/il-24 to improve therapeutic eradication of cancer and generate protective antitumor immunity |
WO2017062953A1 (en) | 2015-10-10 | 2017-04-13 | Intrexon Corporation | Improved therapeutic control of proteolytically sensitive, destabilized forms of interleukin-12 |
US11154622B2 (en) | 2015-11-11 | 2021-10-26 | Precigen, Inc. | Compositions and methods for expression of multiple biologically active polypeptides from a single vector for treatment of cardiac conditions and other pathologies |
WO2017087723A1 (en) * | 2015-11-19 | 2017-05-26 | The Regents Of The University Of California | Conditionally repressible immune cell receptors and methods of use thereof |
CN105349578A (en) * | 2015-11-30 | 2016-02-24 | 肇庆大华农生物药品有限公司 | Chicken GM-CSF protein, and preparation method and application thereof |
US11136562B2 (en) | 2016-01-08 | 2021-10-05 | The Regents Of The University Of California | Conditionally active heterodimeric polypeptides and methods of use thereof |
US11185555B2 (en) | 2016-04-11 | 2021-11-30 | Noah James Harrison | Method to kill pathogenic microbes in a patient |
GB2565670A (en) * | 2016-04-11 | 2019-02-20 | James Harrison Noah | Technique to kill pathogens in vivo |
WO2017180536A1 (en) * | 2016-04-11 | 2017-10-19 | Harrison Noah James | Technique to kill pathogens in vivo |
US10888594B2 (en) | 2016-05-30 | 2021-01-12 | National University Corporation Tottori University | Genetically engineered vaccinia viruses |
US11344589B2 (en) | 2016-05-30 | 2022-05-31 | National University Corporation Tottori University | Genetically engineered vaccinia viruses |
WO2017214333A1 (en) | 2016-06-08 | 2017-12-14 | Intrexon Corporation | Cd33 specific chimeric antigen receptors |
WO2018022574A2 (en) | 2016-07-25 | 2018-02-01 | Intrexon Corporation | Control of phenotype in plants |
US11555197B2 (en) | 2016-07-25 | 2023-01-17 | Greenvenus, Llc | Control of phenotype in plants |
WO2018026872A1 (en) * | 2016-08-01 | 2018-02-08 | Virogin Biotech Canada Ltd | Oncolytic herpes simplex virus vectors expressing immune system-stimulatory molecules |
US11666649B2 (en) | 2016-10-11 | 2023-06-06 | University Of Miami | Vectors and vaccine cells for immunity against Zika virus |
WO2018132494A1 (en) | 2017-01-10 | 2018-07-19 | Intrexon Corporation | Modulating expression of polypeptides via new gene switch expression systems |
US11548930B2 (en) | 2017-04-04 | 2023-01-10 | Heat Biologics, Inc. | Intratumoral vaccination |
US20200239535A1 (en) * | 2017-04-24 | 2020-07-30 | Imperial Innovations Limited | Cancer Treatment |
US11820792B2 (en) * | 2017-04-24 | 2023-11-21 | Imperial College Innovations Limited | Cancer treatment |
US11118168B2 (en) | 2017-06-07 | 2021-09-14 | Precigen, Inc. | Expression of novel cell tags |
US11608362B2 (en) | 2018-03-06 | 2023-03-21 | Precigen, Inc. | Hepatitis B vaccines and uses of the same |
WO2020023670A1 (en) * | 2018-07-24 | 2020-01-30 | University Of Virginia Patent Foundation | Compositions and methods for combating multidrug-resistant bacteria |
US11638730B2 (en) | 2018-09-26 | 2023-05-02 | Astellas Pharma Inc. | Cancer therapy by combination use of oncolytic vaccinia virus and immune checkpoint inhibitor, and pharmaceutical composition and combination medicine for use in the cancer therapy |
WO2020206056A1 (en) | 2019-04-04 | 2020-10-08 | Greenvenus Llc | Peptides and methods of plant protection |
WO2020223322A1 (en) * | 2019-04-29 | 2020-11-05 | The University Of North Carolina At Chapel Hill | Intrathecal and intravenous combination gene therapy for the treatment of infantile batten disease |
WO2022006332A3 (en) * | 2020-06-30 | 2022-02-03 | Chan Zuckerberg Biohub, Inc. | Gene therapy for immuno-oncology applications |
Also Published As
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20200239906A1 (en) | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof | |
US10046049B2 (en) | Engineered cells expressing multiple immunomodulators and uses thereof | |
RU2711606C2 (en) | Modified dendritic cells and use thereof in treating malignant tumours | |
MX2012010920A (en) | Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: INTREXON CORPORATION, VIRGINIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:REED, CHARLES;ROETH, JEREMIAH;CUTHBERTSON, BRANDON;AND OTHERS;SIGNING DATES FROM 20130110 TO 20130324;REEL/FRAME:030279/0070 |
|
AS | Assignment |
Owner name: INTREXON CORPORATION, VIRGINIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:REED, CHARLES;ROETH, JEREMIAH;CUTHBERTSON, BRANDON;AND OTHERS;SIGNING DATES FROM 20131009 TO 20131025;REEL/FRAME:031538/0833 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |