US20020162126A1 - Methods and compositions for RNA interference - Google Patents
Methods and compositions for RNA interference Download PDFInfo
- Publication number
- US20020162126A1 US20020162126A1 US09/866,557 US86655701A US2002162126A1 US 20020162126 A1 US20020162126 A1 US 20020162126A1 US 86655701 A US86655701 A US 86655701A US 2002162126 A1 US2002162126 A1 US 2002162126A1
- Authority
- US
- United States
- Prior art keywords
- cell
- gene
- dsrna
- rna
- cells
- 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
- 238000000034 method Methods 0.000 title claims abstract description 75
- 230000009368 gene silencing by RNA Effects 0.000 title claims description 85
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 title description 82
- 239000000203 mixture Substances 0.000 title description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 266
- 102000040650 (ribonucleotides)n+m Human genes 0.000 claims abstract description 265
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 217
- 230000014509 gene expression Effects 0.000 claims abstract description 99
- 239000002773 nucleotide Substances 0.000 claims abstract description 68
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 57
- 210000004027 cell Anatomy 0.000 claims description 252
- 230000000694 effects Effects 0.000 claims description 99
- 101000907904 Homo sapiens Endoribonuclease Dicer Proteins 0.000 claims description 97
- 102100023387 Endoribonuclease Dicer Human genes 0.000 claims description 87
- 102000004169 proteins and genes Human genes 0.000 claims description 36
- 230000004044 response Effects 0.000 claims description 31
- 150000007523 nucleic acids Chemical group 0.000 claims description 29
- 102000039446 nucleic acids Human genes 0.000 claims description 24
- 108020004707 nucleic acids Proteins 0.000 claims description 24
- 238000003556 assay Methods 0.000 claims description 23
- 108091026890 Coding region Proteins 0.000 claims description 20
- 108020004414 DNA Proteins 0.000 claims description 20
- 241001465754 Metazoa Species 0.000 claims description 20
- 102000053602 DNA Human genes 0.000 claims description 19
- 102100034170 Interferon-induced, double-stranded RNA-activated protein kinase Human genes 0.000 claims description 16
- 101710089751 Interferon-induced, double-stranded RNA-activated protein kinase Proteins 0.000 claims description 16
- 239000003795 chemical substances by application Substances 0.000 claims description 16
- 108700019146 Transgenes Proteins 0.000 claims description 15
- 230000002401 inhibitory effect Effects 0.000 claims description 15
- 239000003112 inhibitor Substances 0.000 claims description 10
- 241000282414 Homo sapiens Species 0.000 claims description 9
- 241000124008 Mammalia Species 0.000 claims description 9
- 230000000295 complement effect Effects 0.000 claims description 9
- 210000004748 cultured cell Anatomy 0.000 claims description 9
- 210000004962 mammalian cell Anatomy 0.000 claims description 8
- 230000001225 therapeutic effect Effects 0.000 claims description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 6
- 230000001717 pathogenic effect Effects 0.000 claims description 6
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 6
- 230000006907 apoptotic process Effects 0.000 claims description 5
- 210000005260 human cell Anatomy 0.000 claims description 5
- 230000009261 transgenic effect Effects 0.000 claims description 5
- 108091000080 Phosphotransferase Proteins 0.000 claims description 4
- 241000288906 Primates Species 0.000 claims description 4
- 244000052769 pathogen Species 0.000 claims description 4
- 102000020233 phosphotransferase Human genes 0.000 claims description 4
- 231100000419 toxicity Toxicity 0.000 claims description 4
- 230000001988 toxicity Effects 0.000 claims description 4
- 230000006378 damage Effects 0.000 claims description 3
- 238000009826 distribution Methods 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 2
- 230000002238 attenuated effect Effects 0.000 claims description 2
- 238000009509 drug development Methods 0.000 claims description 2
- 238000007876 drug discovery Methods 0.000 claims description 2
- 210000004602 germ cell Anatomy 0.000 claims description 2
- 108091030071 RNAI Proteins 0.000 claims 2
- 210000001082 somatic cell Anatomy 0.000 claims 1
- 229920002477 rna polymer Polymers 0.000 description 73
- 239000000284 extract Substances 0.000 description 51
- 108020004999 messenger RNA Proteins 0.000 description 48
- 101710163270 Nuclease Proteins 0.000 description 38
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 37
- 108090000257 Cyclin E Proteins 0.000 description 36
- 102000003909 Cyclin E Human genes 0.000 description 36
- 230000005764 inhibitory process Effects 0.000 description 32
- 102000004190 Enzymes Human genes 0.000 description 30
- 108090000790 Enzymes Proteins 0.000 description 30
- 229940088598 enzyme Drugs 0.000 description 30
- 235000018102 proteins Nutrition 0.000 description 29
- 239000000758 substrate Substances 0.000 description 29
- 239000013598 vector Substances 0.000 description 28
- 102000000574 RNA-Induced Silencing Complex Human genes 0.000 description 26
- 108010016790 RNA-Induced Silencing Complex Proteins 0.000 description 26
- 238000001727 in vivo Methods 0.000 description 25
- 238000001890 transfection Methods 0.000 description 24
- 239000013612 plasmid Substances 0.000 description 23
- 230000000875 corresponding effect Effects 0.000 description 22
- 238000000338 in vitro Methods 0.000 description 22
- 230000030279 gene silencing Effects 0.000 description 21
- 241000196324 Embryophyta Species 0.000 description 20
- 101150066555 lacZ gene Proteins 0.000 description 20
- 108090000765 processed proteins & peptides Proteins 0.000 description 19
- 238000013518 transcription Methods 0.000 description 19
- 230000035897 transcription Effects 0.000 description 19
- 230000006870 function Effects 0.000 description 18
- 108091028043 Nucleic acid sequence Proteins 0.000 description 17
- 230000001105 regulatory effect Effects 0.000 description 17
- 108060001084 Luciferase Proteins 0.000 description 16
- 102000004196 processed proteins & peptides Human genes 0.000 description 16
- 230000004913 activation Effects 0.000 description 15
- 229920001184 polypeptide Polymers 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 108020005004 Guide RNA Proteins 0.000 description 14
- 230000002068 genetic effect Effects 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 13
- 238000003776 cleavage reaction Methods 0.000 description 13
- 230000001419 dependent effect Effects 0.000 description 13
- 239000013604 expression vector Substances 0.000 description 12
- 239000012133 immunoprecipitate Substances 0.000 description 12
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 12
- 102000008682 Argonaute Proteins Human genes 0.000 description 11
- 108010088141 Argonaute Proteins Proteins 0.000 description 11
- 108060004795 Methyltransferase Proteins 0.000 description 11
- 230000012010 growth Effects 0.000 description 11
- 210000001161 mammalian embryo Anatomy 0.000 description 11
- 230000007246 mechanism Effects 0.000 description 11
- 230000008569 process Effects 0.000 description 11
- 230000007017 scission Effects 0.000 description 11
- 108090000331 Firefly luciferases Proteins 0.000 description 10
- 239000005089 Luciferase Substances 0.000 description 10
- 241000242743 Renilla reniformis Species 0.000 description 10
- 239000003184 complementary RNA Substances 0.000 description 10
- 238000012226 gene silencing method Methods 0.000 description 10
- 239000006166 lysate Substances 0.000 description 10
- -1 monoQ Chemical compound 0.000 description 10
- 238000013519 translation Methods 0.000 description 10
- 230000014616 translation Effects 0.000 description 10
- 230000000692 anti-sense effect Effects 0.000 description 9
- 239000002299 complementary DNA Substances 0.000 description 9
- 238000006731 degradation reaction Methods 0.000 description 9
- 210000002257 embryonic structure Anatomy 0.000 description 9
- 210000003527 eukaryotic cell Anatomy 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 230000002103 transcriptional effect Effects 0.000 description 9
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 8
- 238000000636 Northern blotting Methods 0.000 description 8
- 102000003661 Ribonuclease III Human genes 0.000 description 8
- 108010057163 Ribonuclease III Proteins 0.000 description 8
- 241000700605 Viruses Species 0.000 description 8
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 230000035772 mutation Effects 0.000 description 8
- 230000036961 partial effect Effects 0.000 description 8
- 239000013589 supplement Substances 0.000 description 8
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 7
- 108020005544 Antisense RNA Proteins 0.000 description 7
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 7
- 241000233866 Fungi Species 0.000 description 7
- 108700008625 Reporter Genes Proteins 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 238000005194 fractionation Methods 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 230000001629 suppression Effects 0.000 description 7
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 6
- 241000206602 Eukaryota Species 0.000 description 6
- 101000780643 Homo sapiens Protein argonaute-2 Proteins 0.000 description 6
- 241000254158 Lampyridae Species 0.000 description 6
- 102100034207 Protein argonaute-2 Human genes 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 230000015556 catabolic process Effects 0.000 description 6
- 230000009977 dual effect Effects 0.000 description 6
- 239000003623 enhancer Substances 0.000 description 6
- 230000000977 initiatory effect Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 230000006798 recombination Effects 0.000 description 6
- 238000005215 recombination Methods 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 5
- 241000244206 Nematoda Species 0.000 description 5
- 241000254064 Photinus pyralis Species 0.000 description 5
- 108010052090 Renilla Luciferases Proteins 0.000 description 5
- 230000004075 alteration Effects 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 102000005936 beta-Galactosidase Human genes 0.000 description 5
- 108010005774 beta-Galactosidase Proteins 0.000 description 5
- 239000001110 calcium chloride Substances 0.000 description 5
- 229910001628 calcium chloride Inorganic materials 0.000 description 5
- 239000013592 cell lysate Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 210000001236 prokaryotic cell Anatomy 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- 241000219194 Arabidopsis Species 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 108700020472 CDC20 Proteins 0.000 description 4
- 102000028756 CDC20 Human genes 0.000 description 4
- 102000004039 Caspase-9 Human genes 0.000 description 4
- 108090000566 Caspase-9 Proteins 0.000 description 4
- 241000701022 Cytomegalovirus Species 0.000 description 4
- 108091070648 Dicer family Proteins 0.000 description 4
- 241000255925 Diptera Species 0.000 description 4
- 108700020954 Drosophila CycE Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 108091060211 Expressed sequence tag Proteins 0.000 description 4
- 108010059724 Micrococcal Nuclease Proteins 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 239000001506 calcium phosphate Substances 0.000 description 4
- 229910000389 calcium phosphate Inorganic materials 0.000 description 4
- 235000011010 calcium phosphates Nutrition 0.000 description 4
- 230000022131 cell cycle Effects 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000005090 green fluorescent protein Substances 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 238000001114 immunoprecipitation Methods 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 231100000518 lethal Toxicity 0.000 description 4
- 230000001665 lethal effect Effects 0.000 description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000010076 replication Effects 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 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 4
- 238000001262 western blot Methods 0.000 description 4
- 108010031677 Anaphase-Promoting Complex-Cyclosome Proteins 0.000 description 3
- 102000005446 Anaphase-Promoting Complex-Cyclosome Human genes 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 241000244203 Caenorhabditis elegans Species 0.000 description 3
- 108010068192 Cyclin A Proteins 0.000 description 3
- 102100025191 Cyclin-A2 Human genes 0.000 description 3
- 108010066133 D-octopine dehydrogenase Proteins 0.000 description 3
- 102000040623 Dicer family Human genes 0.000 description 3
- 108700041096 Drosophila Dcr-1 Proteins 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108700024394 Exon Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 206010071602 Genetic polymorphism Diseases 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 3
- 239000007995 HEPES buffer Substances 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 3
- 102000003945 NF-kappa B Human genes 0.000 description 3
- 108010057466 NF-kappa B Proteins 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 108091081021 Sense strand Proteins 0.000 description 3
- 241000700584 Simplexvirus Species 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 230000031018 biological processes and functions Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 231100000225 lethality Toxicity 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 108010058731 nopaline synthase Proteins 0.000 description 3
- 210000000287 oocyte Anatomy 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000001124 posttranscriptional effect Effects 0.000 description 3
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 239000011535 reaction buffer Substances 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 125000002652 ribonucleotide group Chemical group 0.000 description 3
- 210000003705 ribosome Anatomy 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 108010000700 Acetolactate synthase Proteins 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 101100301219 Arabidopsis thaliana RDR6 gene Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091032955 Bacterial small RNA Proteins 0.000 description 2
- 241000701822 Bovine papillomavirus Species 0.000 description 2
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 description 2
- 240000003259 Brassica oleracea var. botrytis Species 0.000 description 2
- 102000011727 Caspases Human genes 0.000 description 2
- 108010076667 Caspases Proteins 0.000 description 2
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 2
- 108010077544 Chromatin Proteins 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 108020004394 Complementary RNA Proteins 0.000 description 2
- 235000009854 Cucurbita moschata Nutrition 0.000 description 2
- 240000001980 Cucurbita pepo Species 0.000 description 2
- 108050006400 Cyclin Proteins 0.000 description 2
- 102000016736 Cyclin Human genes 0.000 description 2
- 101150091263 E3L gene Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108091029865 Exogenous DNA Proteins 0.000 description 2
- 108060002716 Exonuclease Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 2
- 102000005548 Hexokinase Human genes 0.000 description 2
- 108700040460 Hexokinases Proteins 0.000 description 2
- 241000243251 Hydra Species 0.000 description 2
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 2
- 241000713333 Mouse mammary tumor virus Species 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 101000983522 Mus musculus Caspase-9 Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 241000208422 Rhododendron Species 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- 241000714474 Rous sarcoma virus Species 0.000 description 2
- 108091027967 Small hairpin RNA Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108020004566 Transfer RNA Proteins 0.000 description 2
- 241000397921 Turbellaria Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 244000078534 Vaccinium myrtillus Species 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 238000003314 affinity selection Methods 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 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 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 238000003491 array Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000002306 biochemical method Methods 0.000 description 2
- 108091092328 cellular RNA Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000003483 chromatin Anatomy 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000000975 co-precipitation Methods 0.000 description 2
- 238000012761 co-transfection Methods 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000000287 crude extract Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 102000013165 exonuclease Human genes 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 102000051308 human DICER1 Human genes 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 239000012742 immunoprecipitation (IP) buffer Substances 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 230000001965 increasing effect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000012194 insect media Substances 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000004777 loss-of-function mutation Effects 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 230000010534 mechanism of action Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 150000003833 nucleoside derivatives Chemical class 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 238000000053 physical method Methods 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 238000011533 pre-incubation Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000000392 somatic effect Effects 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000001052 transient effect Effects 0.000 description 2
- 230000010474 transient expression Effects 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 239000012130 whole-cell lysate Substances 0.000 description 2
- 101710194665 1-aminocyclopropane-1-carboxylate synthase Proteins 0.000 description 1
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 1
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- OSJPPGNTCRNQQC-UWTATZPHSA-N 3-phospho-D-glyceric acid Chemical compound OC(=O)[C@H](O)COP(O)(O)=O OSJPPGNTCRNQQC-UWTATZPHSA-N 0.000 description 1
- 108020005029 5' Flanking Region Proteins 0.000 description 1
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 1
- 108010011619 6-Phytase Proteins 0.000 description 1
- WFPZSXYXPSUOPY-ROYWQJLOSA-N ADP alpha-D-glucoside Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H]1O)O)N1C=2N=CN=C(C=2N=C1)N)OP(O)(=O)OP(O)(=O)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O WFPZSXYXPSUOPY-ROYWQJLOSA-N 0.000 description 1
- WFPZSXYXPSUOPY-UHFFFAOYSA-N ADP-mannose Natural products C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COP(O)(=O)OP(O)(=O)OC1OC(CO)C(O)C(O)C1O WFPZSXYXPSUOPY-UHFFFAOYSA-N 0.000 description 1
- 108091006112 ATPases Proteins 0.000 description 1
- 240000005020 Acaciella glauca Species 0.000 description 1
- 108010051457 Acid Phosphatase Proteins 0.000 description 1
- 102000013563 Acid Phosphatase Human genes 0.000 description 1
- 241001133760 Acoelorraphe Species 0.000 description 1
- 235000009434 Actinidia chinensis Nutrition 0.000 description 1
- 244000298697 Actinidia deliciosa Species 0.000 description 1
- 235000009436 Actinidia deliciosa Nutrition 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 1
- 102100036664 Adenosine deaminase Human genes 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 244000291564 Allium cepa Species 0.000 description 1
- 235000002732 Allium cepa var. cepa Nutrition 0.000 description 1
- 244000144725 Amygdalus communis Species 0.000 description 1
- 235000011437 Amygdalus communis Nutrition 0.000 description 1
- 244000144730 Amygdalus persica Species 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 244000099147 Ananas comosus Species 0.000 description 1
- 235000007119 Ananas comosus Nutrition 0.000 description 1
- 241001147657 Ancylostoma Species 0.000 description 1
- 240000007087 Apium graveolens Species 0.000 description 1
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 description 1
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- 235000010591 Appio Nutrition 0.000 description 1
- 108700028919 Arabidopsis DCL1 Proteins 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 241000204727 Ascaridia Species 0.000 description 1
- 241000244186 Ascaris Species 0.000 description 1
- 244000003416 Asparagus officinalis Species 0.000 description 1
- 235000005340 Asparagus officinalis Nutrition 0.000 description 1
- 235000000832 Ayote Nutrition 0.000 description 1
- 102100021631 B-cell lymphoma 6 protein Human genes 0.000 description 1
- 108091012583 BCL2 Proteins 0.000 description 1
- 102000052609 BRCA2 Human genes 0.000 description 1
- 108700020462 BRCA2 Proteins 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 241000335053 Beta vulgaris Species 0.000 description 1
- 235000018185 Betula X alpestris Nutrition 0.000 description 1
- 235000018212 Betula X uliginosa Nutrition 0.000 description 1
- 241000219495 Betulaceae Species 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000167854 Bourreria succulenta Species 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000011301 Brassica oleracea var capitata Nutrition 0.000 description 1
- 235000017647 Brassica oleracea var italica Nutrition 0.000 description 1
- 235000001169 Brassica oleracea var oleracea Nutrition 0.000 description 1
- 101150008921 Brca2 gene Proteins 0.000 description 1
- 235000004936 Bromus mango Nutrition 0.000 description 1
- 241000931178 Bunostomum Species 0.000 description 1
- 241000244202 Caenorhabditis Species 0.000 description 1
- 101100048437 Caenorhabditis elegans unc-22 gene Proteins 0.000 description 1
- 240000001548 Camellia japonica Species 0.000 description 1
- 241000253350 Capillaria Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 108090000489 Carboxy-Lyases Proteins 0.000 description 1
- 102000004031 Carboxy-Lyases Human genes 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 235000009467 Carica papaya Nutrition 0.000 description 1
- 240000006432 Carica papaya Species 0.000 description 1
- 235000003255 Carthamus tinctorius Nutrition 0.000 description 1
- 244000020518 Carthamus tinctorius Species 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 102000016938 Catalase Human genes 0.000 description 1
- 108010084185 Cellulases Proteins 0.000 description 1
- 102000005575 Cellulases Human genes 0.000 description 1
- 241000893172 Chabertia Species 0.000 description 1
- 108030000630 Chalcone synthases Proteins 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 235000007516 Chrysanthemum Nutrition 0.000 description 1
- 244000189548 Chrysanthemum x morifolium Species 0.000 description 1
- 244000241235 Citrullus lanatus Species 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
- 235000008733 Citrus aurantifolia Nutrition 0.000 description 1
- 235000005979 Citrus limon Nutrition 0.000 description 1
- 244000131522 Citrus pyriformis Species 0.000 description 1
- 241000675108 Citrus tangerina Species 0.000 description 1
- 240000000560 Citrus x paradisi Species 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 235000013162 Cocos nucifera Nutrition 0.000 description 1
- 244000060011 Cocos nucifera Species 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 235000006481 Colocasia esculenta Nutrition 0.000 description 1
- 244000205754 Colocasia esculenta Species 0.000 description 1
- 241000218631 Coniferophyta Species 0.000 description 1
- 241001126268 Cooperia Species 0.000 description 1
- 235000007466 Corylus avellana Nutrition 0.000 description 1
- 240000003211 Corylus maxima Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 108020004703 Cruciform DNA Proteins 0.000 description 1
- 241000219112 Cucumis Species 0.000 description 1
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 1
- 240000008067 Cucumis sativus Species 0.000 description 1
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 description 1
- 235000009852 Cucurbita pepo Nutrition 0.000 description 1
- 235000009804 Cucurbita pepo subsp pepo Nutrition 0.000 description 1
- 241000219130 Cucurbita pepo subsp. pepo Species 0.000 description 1
- 235000003954 Cucurbita pepo var melopepo Nutrition 0.000 description 1
- 108010076010 Cystathionine beta-lyase Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 101100112682 Danio rerio ccne1 gene Proteins 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108700029231 Developmental Genes Proteins 0.000 description 1
- 235000009355 Dianthus caryophyllus Nutrition 0.000 description 1
- 240000006497 Dianthus caryophyllus Species 0.000 description 1
- 241001147667 Dictyocaulus Species 0.000 description 1
- 208000035240 Disease Resistance Diseases 0.000 description 1
- 241000399934 Ditylenchus Species 0.000 description 1
- 102000000331 Double-stranded RNA-binding domains Human genes 0.000 description 1
- 108050008793 Double-stranded RNA-binding domains Proteins 0.000 description 1
- 108700017247 Drosophila DCR-2 Proteins 0.000 description 1
- 101100084900 Drosophila melanogaster Rpn11 gene Proteins 0.000 description 1
- 101100072149 Drosophila melanogaster eIF2alpha gene Proteins 0.000 description 1
- 241001136566 Drosophila suzukii Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102100035813 E3 ubiquitin-protein ligase CBL Human genes 0.000 description 1
- 108050002772 E3 ubiquitin-protein ligase Mdm2 Proteins 0.000 description 1
- 102000012199 E3 ubiquitin-protein ligase Mdm2 Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 108010067770 Endopeptidase K 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
- 241000701959 Escherichia virus Lambda Species 0.000 description 1
- 108700039887 Essential Genes Proteins 0.000 description 1
- 102100034169 Eukaryotic translation initiation factor 2-alpha kinase 1 Human genes 0.000 description 1
- 101710196289 Eukaryotic translation initiation factor 2-alpha kinase 1 Proteins 0.000 description 1
- 102100038195 Exonuclease mut-7 homolog Human genes 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 235000016623 Fragaria vesca Nutrition 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 235000011363 Fragaria x ananassa Nutrition 0.000 description 1
- 102100021265 Frizzled-2 Human genes 0.000 description 1
- 101710140946 Frizzled-2 Proteins 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 102000013446 GTP Phosphohydrolases Human genes 0.000 description 1
- 102100029974 GTPase HRas Human genes 0.000 description 1
- 102100039788 GTPase NRas Human genes 0.000 description 1
- 108091006109 GTPases Proteins 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 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
- 229930182566 Gentamicin Natural products 0.000 description 1
- 241001442498 Globodera Species 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 108010021582 Glucokinase Proteins 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 240000008669 Hedera helix Species 0.000 description 1
- 244000020551 Helianthus annuus Species 0.000 description 1
- 235000003222 Helianthus annuus Nutrition 0.000 description 1
- 241001148481 Helicotylenchus Species 0.000 description 1
- 241000258937 Hemiptera Species 0.000 description 1
- 241000920462 Heterakis Species 0.000 description 1
- 241001480224 Heterodera Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 1
- 101000971234 Homo sapiens B-cell lymphoma 6 protein Proteins 0.000 description 1
- 101000851181 Homo sapiens Epidermal growth factor receptor Proteins 0.000 description 1
- 101000958030 Homo sapiens Exonuclease mut-7 homolog Proteins 0.000 description 1
- 101000584633 Homo sapiens GTPase HRas Proteins 0.000 description 1
- 101000744505 Homo sapiens GTPase NRas Proteins 0.000 description 1
- 101001012669 Homo sapiens Melanoma inhibitory activity protein 2 Proteins 0.000 description 1
- 101000876829 Homo sapiens Protein C-ets-1 Proteins 0.000 description 1
- 101000573199 Homo sapiens Protein PML Proteins 0.000 description 1
- 101000861454 Homo sapiens Protein c-Fos Proteins 0.000 description 1
- 101000579425 Homo sapiens Proto-oncogene tyrosine-protein kinase receptor Ret Proteins 0.000 description 1
- 101000857677 Homo sapiens Runt-related transcription factor 1 Proteins 0.000 description 1
- 101000800488 Homo sapiens T-cell leukemia homeobox protein 1 Proteins 0.000 description 1
- 101000837626 Homo sapiens Thyroid hormone receptor alpha Proteins 0.000 description 1
- 101000813738 Homo sapiens Transcription factor ETV6 Proteins 0.000 description 1
- 101000912503 Homo sapiens Tyrosine-protein kinase Fgr Proteins 0.000 description 1
- 101001022129 Homo sapiens Tyrosine-protein kinase Fyn Proteins 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 102000012330 Integrases Human genes 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 235000010254 Jasminum officinale Nutrition 0.000 description 1
- 240000005385 Jasminum sambac Species 0.000 description 1
- 240000007049 Juglans regia Species 0.000 description 1
- 235000009496 Juglans regia Nutrition 0.000 description 1
- 241000721662 Juniperus Species 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 235000003228 Lactuca sativa Nutrition 0.000 description 1
- 240000008415 Lactuca sativa Species 0.000 description 1
- 241000255777 Lepidoptera Species 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- OJMMVQQUTAEWLP-UHFFFAOYSA-N Lincomycin Natural products CN1CC(CCC)CC1C(=O)NC(C(C)O)C1C(O)C(O)C(O)C(SC)O1 OJMMVQQUTAEWLP-UHFFFAOYSA-N 0.000 description 1
- 235000004431 Linum usitatissimum Nutrition 0.000 description 1
- 240000006240 Linum usitatissimum Species 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 102000003820 Lipoxygenases Human genes 0.000 description 1
- 108090000128 Lipoxygenases Proteins 0.000 description 1
- 241001220360 Longidorus Species 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 108700012912 MYCN Proteins 0.000 description 1
- 101150022024 MYCN gene Proteins 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 241000220225 Malus Species 0.000 description 1
- 235000011430 Malus pumila Nutrition 0.000 description 1
- 235000015103 Malus silvestris Nutrition 0.000 description 1
- 235000014826 Mangifera indica Nutrition 0.000 description 1
- 240000007228 Mangifera indica Species 0.000 description 1
- 240000004658 Medicago sativa Species 0.000 description 1
- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 description 1
- 102100029778 Melanoma inhibitory activity protein 2 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
- 241000907681 Morpho Species 0.000 description 1
- 102100025725 Mothers against decapentaplegic homolog 4 Human genes 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101100025919 Mus musculus Ncoa6 gene Proteins 0.000 description 1
- 240000005561 Musa balbisiana Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- 108700026495 N-Myc Proto-Oncogene Proteins 0.000 description 1
- 102100030124 N-myc proto-oncogene protein Human genes 0.000 description 1
- 241000201433 Nacobbus Species 0.000 description 1
- 241001481166 Nautilus Species 0.000 description 1
- 241001137882 Nematodirus Species 0.000 description 1
- 102000007530 Neurofibromin 1 Human genes 0.000 description 1
- 108010085793 Neurofibromin 1 Proteins 0.000 description 1
- 241000221960 Neurospora Species 0.000 description 1
- 102000004108 Neurotransmitter Receptors Human genes 0.000 description 1
- 108090000590 Neurotransmitter Receptors Proteins 0.000 description 1
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 241000510960 Oesophagostomum Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241001465803 Orgyia pseudotsugata Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000243795 Ostertagia Species 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 241000904715 Oxyuris Species 0.000 description 1
- 101150012394 PHO5 gene Proteins 0.000 description 1
- 239000007990 PIPES buffer Substances 0.000 description 1
- 241000244187 Parascaris Species 0.000 description 1
- 241001148650 Paratylenchus Species 0.000 description 1
- 235000000370 Passiflora edulis Nutrition 0.000 description 1
- 244000288157 Passiflora edulis Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N Phosphinothricin Natural products CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 108010073135 Phosphorylases Proteins 0.000 description 1
- 102000009097 Phosphorylases Human genes 0.000 description 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 240000003889 Piper guineense Species 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 235000003447 Pistacia vera Nutrition 0.000 description 1
- 240000006711 Pistacia vera Species 0.000 description 1
- 102000041193 Piwi family Human genes 0.000 description 1
- 108091061182 Piwi family Proteins 0.000 description 1
- 108091036407 Polyadenylation Proteins 0.000 description 1
- 108010059820 Polygalacturonase Proteins 0.000 description 1
- 241000219000 Populus Species 0.000 description 1
- 241000183024 Populus tremula Species 0.000 description 1
- 241000193943 Pratylenchus Species 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 108090000459 Prostaglandin-endoperoxide synthases Proteins 0.000 description 1
- 102000004005 Prostaglandin-endoperoxide synthases Human genes 0.000 description 1
- 102100035251 Protein C-ets-1 Human genes 0.000 description 1
- 102100026375 Protein PML Human genes 0.000 description 1
- 102100027584 Protein c-Fos Human genes 0.000 description 1
- 102100028286 Proto-oncogene tyrosine-protein kinase receptor Ret Human genes 0.000 description 1
- 235000009827 Prunus armeniaca Nutrition 0.000 description 1
- 244000018633 Prunus armeniaca Species 0.000 description 1
- 235000006029 Prunus persica var nucipersica Nutrition 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 244000017714 Prunus persica var. nucipersica Species 0.000 description 1
- 241000508269 Psidium Species 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 240000001987 Pyrus communis Species 0.000 description 1
- 241000219492 Quercus Species 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 241000201377 Radopholus Species 0.000 description 1
- 244000088415 Raphanus sativus Species 0.000 description 1
- 235000006140 Raphanus sativus var sativus Nutrition 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 102000004389 Ribonucleoproteins Human genes 0.000 description 1
- 108010081734 Ribonucleoproteins Proteins 0.000 description 1
- 108010003581 Ribulose-bisphosphate carboxylase Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 235000017848 Rubus fruticosus Nutrition 0.000 description 1
- 240000007651 Rubus glaucus Species 0.000 description 1
- 235000011034 Rubus glaucus Nutrition 0.000 description 1
- 235000009122 Rubus idaeus Nutrition 0.000 description 1
- 102100025373 Runt-related transcription factor 1 Human genes 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- 101150019443 SMAD4 gene Proteins 0.000 description 1
- 102000001332 SRC Human genes 0.000 description 1
- 108060006706 SRC Proteins 0.000 description 1
- 241000209056 Secale Species 0.000 description 1
- 235000007238 Secale cereale Nutrition 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 240000002751 Sideroxylon obovatum Species 0.000 description 1
- 108010052160 Site-specific recombinase Proteins 0.000 description 1
- 108700031298 Smad4 Proteins 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 240000003829 Sorghum propinquum Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 235000009337 Spinacia oleracea Nutrition 0.000 description 1
- 244000300264 Spinacia oleracea Species 0.000 description 1
- 235000009184 Spondias indica Nutrition 0.000 description 1
- 108010039811 Starch synthase Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000122932 Strongylus Species 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 102100033111 T-cell leukemia homeobox protein 1 Human genes 0.000 description 1
- 101710137500 T7 RNA polymerase Proteins 0.000 description 1
- 244000299461 Theobroma cacao Species 0.000 description 1
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 1
- 235000005767 Theobroma cacao ssp. sphaerocarpum Nutrition 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 102100028702 Thyroid hormone receptor alpha Human genes 0.000 description 1
- 235000011941 Tilia x europaea Nutrition 0.000 description 1
- 240000006909 Tilia x europaea Species 0.000 description 1
- 101710183280 Topoisomerase Proteins 0.000 description 1
- 241000607216 Toxascaris Species 0.000 description 1
- 241000244031 Toxocara Species 0.000 description 1
- 102100039580 Transcription factor ETV6 Human genes 0.000 description 1
- 241000243797 Trichostrongylus Species 0.000 description 1
- 241001489151 Trichuris Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000015098 Tumor Suppressor Protein p53 Human genes 0.000 description 1
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 description 1
- 108010040002 Tumor Suppressor Proteins Proteins 0.000 description 1
- 102000001742 Tumor Suppressor Proteins Human genes 0.000 description 1
- 241001540447 Tylenchus Species 0.000 description 1
- 102100026150 Tyrosine-protein kinase Fgr Human genes 0.000 description 1
- 102100035221 Tyrosine-protein kinase Fyn Human genes 0.000 description 1
- 241001106462 Ulmus Species 0.000 description 1
- 241000571986 Uncinaria Species 0.000 description 1
- 101900001372 Vaccinia virus RNA-binding protein E3 Proteins 0.000 description 1
- 235000003095 Vaccinium corymbosum Nutrition 0.000 description 1
- 240000001717 Vaccinium macrocarpon Species 0.000 description 1
- 235000012545 Vaccinium macrocarpon Nutrition 0.000 description 1
- 235000017537 Vaccinium myrtillus Nutrition 0.000 description 1
- 235000002118 Vaccinium oxycoccus Nutrition 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 101001001642 Xenopus laevis Serine/threonine-protein kinase pim-3 Proteins 0.000 description 1
- 108010084455 Zeocin Proteins 0.000 description 1
- 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 1
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 1
- LPQOADBMXVRBNX-UHFFFAOYSA-N ac1ldcw0 Chemical compound Cl.C1CN(C)CCN1C1=C(F)C=C2C(=O)C(C(O)=O)=CN3CCSC1=C32 LPQOADBMXVRBNX-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 108010036419 acyl-(acyl-carrier-protein)desaturase Proteins 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000020224 almond Nutrition 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229940025131 amylases Drugs 0.000 description 1
- 230000031016 anaphase Effects 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 239000003957 anion exchange resin Substances 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000013473 artificial intelligence Methods 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 235000021029 blackberry Nutrition 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 235000021014 blueberries Nutrition 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 235000001046 cacaotero Nutrition 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000025084 cell cycle arrest Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 235000019693 cherries Nutrition 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 239000013611 chromosomal DNA Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 235000018597 common camellia Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 235000004634 cranberry Nutrition 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000021186 dishes Nutrition 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 108010037623 eIF-2 Kinase Proteins 0.000 description 1
- 102000010982 eIF-2 Kinase Human genes 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000003372 endocrine gland Anatomy 0.000 description 1
- 230000002616 endonucleolytic effect Effects 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 241001233957 eudicotyledons Species 0.000 description 1
- 210000003499 exocrine gland Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 244000037666 field crops Species 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000010448 genetic screening Methods 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 210000003630 histaminocyte Anatomy 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 108010090785 inulinase Proteins 0.000 description 1
- 235000011073 invertase Nutrition 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 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
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 229940043355 kinase inhibitor Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000004571 lime Substances 0.000 description 1
- OJMMVQQUTAEWLP-KIDUDLJLSA-N lincomycin Chemical compound CN1C[C@H](CCC)C[C@H]1C(=O)N[C@H]([C@@H](C)O)[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](SC)O1 OJMMVQQUTAEWLP-KIDUDLJLSA-N 0.000 description 1
- 229960005287 lincomycin Drugs 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000003593 megakaryocyte Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- BQJCRHHNABKAKU-KBQPJGBKSA-N morphine Chemical compound O([C@H]1[C@H](C=C[C@H]23)O)C4=C5[C@@]12CCN(C)[C@@H]3CC5=CC=C4O BQJCRHHNABKAKU-KBQPJGBKSA-N 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 230000009753 muscle formation Effects 0.000 description 1
- 210000000107 myocyte Anatomy 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000037434 nonsense mutation Effects 0.000 description 1
- 230000005937 nuclear translocation Effects 0.000 description 1
- 235000014571 nuts Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 231100000255 pathogenic effect Toxicity 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 108020004410 pectinesterase Proteins 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 239000003757 phosphotransferase inhibitor Substances 0.000 description 1
- 235000020233 pistachio Nutrition 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000007542 postnatal development Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 235000015136 pumpkin Nutrition 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 235000003499 redwood Nutrition 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000003584 silencer Effects 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 235000020354 squash Nutrition 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- OFVLGDICTFRJMM-WESIUVDSSA-N tetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O OFVLGDICTFRJMM-WESIUVDSSA-N 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000005030 transcription termination Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 230000034512 ubiquitination Effects 0.000 description 1
- 238000010798 ubiquitination Methods 0.000 description 1
- 101150003485 unc-22 gene Proteins 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 230000017613 viral reproduction Effects 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 101150061422 yip5 gene Proteins 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
-
- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/102—Mutagenizing nucleic acids
-
- 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
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/50—Physical structure
- C12N2310/53—Physical structure partially self-complementary or closed
-
- 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
- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
- C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function
Definitions
- RNA interference post-transcriptional gene silencing
- quelling these different names describe similar effects that result from the overexpression or misexpression of transgenes, or from the deliberate introduction of double-stranded RNA into cells (reviewed in Fire A (1999) Trends Genet 15:358-363; Sharp PA (1999) Genes Dev 13:139-141; Hunter C (1999) Curr Biol 9:R440-R442; Baulcombe DC (1999) Curr Biol 9:R599-R601; Vaucheret et al. (1998) Plant J 16:651-659).
- RNA interference commonly referred to as RNAi, offers a way of specifically and potently inactivating a cloned gene, and is proving a powerful tool for investigating gene function. But the phenomenon is interesting in its own right; the mechanism has been rather mysterious, but recent research—the latest reported by Smardon et al. (2000) Curr Biol 10:169-178—is beginning to shed light on the nature and evolution of the biological processes that underlie RNAi.
- RNAi was discovered when researchers attempting to use the antisense RNA approach to inactivate a C. elegans gene found that injection of sense-strand RNA was actually as effective as the antisense RNA at inhibiting gene function. Guo et al. (1995) Cell 81:611-620. Further investigation revealed that the active agent was modest amounts of double-stranded RNA that contaminate in vitro RNA preparations. researchers quickly determined the ‘rules’ and effects of RNAi. Exon sequences are required, whereas introns and promoter sequences, while ineffective, do not appear to compromise RNAi (though there may be gene-specific exceptions to this rule). RNAi acts systemically—injection into one tissue inhibits gene function in cells throughout the animal. The results of a variety of experiments, in C. elegans and other organisms, indicate that RNAi acts to destabilize cellular RNA after RNA processing.
- Double-stranded RNAs can provoke gene silencing in numerous in vivo contexts including Drosophila, Caenorhabditis elegans , planaria, hydra, trypanosomes, fungi and plants.
- dsRNAs Double-stranded RNAs
- One aspect of the present invention provides a method for attenuating expression of a target gene in cultured cells, comprising introducing double stranded RNA (dsRNA) into the cells in an amount sufficient to attenuate expression of the target gene, wherein the dsRNA comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene.
- dsRNA double stranded RNA
- Another aspect of the present invention provides a method for attenuating expression of a target gene in a mammalian cell, comprising
- dsRNA double stranded RNA
- the cell is suspended in culture; while in other embodiments the cell is in a whole animal, such as a non-human mammal.
- the cell is engineered with (i) a recombinant gene encoding a Dicer activity, (ii) a recombinant gene encoding an Argonaut activity, or (iii) both.
- the recombinant gene may encode, for a example, a protein which includes an amino acid sequence at least 50 percent identical to SEQ ID No. 2 or 4; or be defined by a coding sequence hybridizes under wash conditions of 2 ⁇ SSC at 22° C. to SEQ ID No. 1 or 3.
- the recombinant gene may encode, for a example, a protein which includes an amino acid sequence at least 50 percent identical to the Argonaut sequence shown in FIG. 24.
- an endogenous Dicer gene or Argonaut gene can be activated, e.g, by gene activation technology, expression of activated transcription factors or other signal transduction protein, which induces expression of the gene, or by treatment with an endogenous factor which upregualtes the level of expression of the protein or inhibits the degradation of the protein.
- the target gene is an endogenous gene of the cell.
- the target gene is an heterologous gene relative to the genome of the cell, such as a pathogen gene, e.g., a viral gene.
- the cell is treated with an agent that inhibits protein kinase RNA-activated (PKR) apoptosis, such as by treatment with agents which inhibit expression of PKR, cause its destruction, and/or inhibit the kinase activity of PKF.
- PPKR protein kinase RNA-activated
- the cell is a primate cell, such as a human cell.
- the length of the dsRNA is at least 20, 21 or 22 nucleotides in length, e.g., corresponding in size to RNA products produced by Dicer-dependent cleavage.
- the dsRNA construct is at least 25, 50, 100, 200, 300 or 400 bases. In certain embodiments, the dsRNA construct is 400-800 bases in length.
- expression of the target gene is attenuated by at least 5 fold, and more preferably at least 10, 20 or even 50 fold, e.g., relative to the untreated cell or a cell treated with a dsRNA construct which does not correspond to the target gene.
- Yet another aspect of the present invention provides a method for attenuating expression of a target gene in cultured cells, comprising introducing an expression vector having a “coding sequence” which, when transcribed, produces double stranded RNA (dsRNA) the cell in an amount sufficient to attenuate expression of the target gene, wherein the dsRNA comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene.
- the vector includes a single coding sequence for the dsRNA which is operably linked to (two) transcriptional regulatory sequences which cause transcription of in both directions (to form complementary transcripts of the coding sequence.
- the vector includes two coding sequences which, respectively, give rise to the two complementary sequences which form the dsRNA when annealed.
- the vectors are episomal, e.g., and transfection is transient.
- the vectors are chromosomally integrated, e.g., to produce a stably transfected cell line. Preferred vectors for forming such stable cell lines are the described in U.S. Pat. No. 6,025,192 and PCT publication WO/9812339, which are incorporated by reference herein.
- Still another aspect of the present invention provides an assay for identifying nucleic acid sequences responsible for conferring a particular phenotype in a cell, comprising
- Yet another aspect of the present invention provides a method of conducting a drug discovery business comprising:
- step (iii) conducting therapeutic profiling of agents identified in step (b), or further analogs thereof, for efficacy and toxicity in animals;
- step (iv) formulating a pharmaceutical preparation including one or more agents identified in step (iii) as having an acceptable therapeutic profile.
- the method may include an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.
- Another aspect of the present invention provides a method of conducting a target discovery business comprising:
- Another aspect of the invention provides a method for inhibiting RNAi by inhibiting the expression or activity of an RNAi enzyme.
- the subject method may include inhibiting the acitivity of Dicer and/or the 22-mer RNA.
- Still another aspect relates to the a method for altering the specificity of an RNAi by modifying the sequence of the RNA component of the RNAi enzyme.
- Another aspect of the invention relates to purified or semi-purified preparations of the RNAi enzyme or components thereof.
- the preparations are used for identifying compounds, especially small organic molecules, which inhibit or potentiate the RNAi activity.
- Small molecule inhibitors for example, can be used to inhibit dsRNA responses in cells which are purposefully being transfected with a virus which produces double stranded RNA.
- the dsRNA construct may comprise one or more strands of polymerized ribonucleotide. It may include modifications to either the phosphate-sugar backbone or the nucleoside.
- the double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell.
- the dsRNA construct may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses of double-stranded material may yield more effective inhibition. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
- dsRNA constructs containing a nucleotide sequences identical to a portion of the target gene is preferred for inhibition.
- RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
- sequence identity may optimized by alignment algorithms known in the art and calculating the percent difference between the nucleotide sequences.
- the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript.
- Yet another aspect of the invention pertains to transgenic non-human mammals which include a transgene encoding a dsRNA construct, preferably which is stably integrated into the genome of cells in which it occurs.
- the animals can be derived by oocyte microinjection, for example, in which case all of the nucleated cells of the animal will include the transgene, or can be derived using embryonic stem (ES) cells which have been transfected with the transgene, in which case the animal is a chimera and only a portion of its nucleated cells will include the transgene.
- ES embryonic stem
- the sequence-independent dsRNA response e.g., the PKR response, is also inhibited in those cells including the transgene.
- dsRNA itself can be introduced into an ES cell in order to effect gene silencing, and that phenotype will be carried for at least several rounds of division, e.g., into the progeny of that cell.
- FIG. 1 RNAi in S2 cells.
- Drosophila S2 cells were transfected with a plasmid that directs lacZ expression from the copia promoter in combination with dsRNAs corresponding to either human CD8 or lacZ, or with no dsRNA, as indicated.
- S2 cells were co-transfected with a plasmid that directs expression of a GFP-US9 fusion protein (12) and dsRNAs of either lacZ or cyclin E, as indicated.
- Upper panels show FACS profiles of the bulk population.
- Lower panels show FACS profiles from GFP-positive cells.
- Total RNA was extracted from cells transfected with lacZ, cyclin E, fizzy or cyclin A dsRNAs, as indicated. Northern blots were hybridized with sequences not present in the transfected dsRNAs.
- FIG. 2 RNAi in vitro.
- E600 Drosophila cyclin E
- Z800 first 800 nucleotides of lacZ
- cycE cyclin E
- Time points were 0, 10, 20, 30, 40 and 60 min for cyclin E and 0, 10, 20, 30 and 60 min for lacZ.
- Transcripts were incubated in an extract of S2 cells that had been transfected with cyclin E dsRNA (cross-hatched box, below).
- Transcripts corresponded to the first 800 nucleotides of lacZ or the first 600, 300, 220 or 100 nucleotides of cyclin E, as indicated.
- Eout is a transcript derived from the portion of the cyclin E cDNA not contained within the transfected dsRNA.
- E-ds is identical to the dsRNA that had been transfected into S2 cells. Time points were 0 and 30 min.
- Synthetic transcripts complementary to the complete cyclin E cDNA (Eas) or the final 600 nucleotides (Eas600) or 300 nucleotides (Eas300) were incubated in extract for 0 or 30 min.
- FIG. 3 Substrate requirements of the RISC. Extracts were prepared from cells transfected with cyclin E dsRNA. Aliquots were incubated for 30 min at 30° C. before the addition of either the cyclin E (E600) or lacZ (Z800) substrate. Individual 20- ⁇ l aliquots, as indicated, were pre-incubated with 1 mM CaCl 2 and 5 mM EGTA, 1 mM CaCl 2 , 5 mM EGTA and 60 U of micrococcal nuclease, 1 mM CaCl 2 and 60 U of micrococcal nuclease or 10 U of DNase I (Promega) and 5 mM EGTA. After the 30-min pre-incubation, EGTA was added to those samples that lacked it. Yeast tRNA (1 ⁇ g) was added to all samples. Time points were at 0 and 30 min.
- FIG. 4 The RISC contains a potential guide RNA.
- a Northern blots of RNA from either a crude lysate or the S100 fraction (containing the soluble nuclease activity, see Methods) were hybridized to a riboprobe derived from the sense strand of the cyclin E mRNA.
- b Soluble cyclin-E-specific nuclease activity was fractionated as described in Methods. Fractions from the anion-exchange resin were incubated with the lacZ, control substrate (upper panel) or the cyclin E substrate (centre panel). Lower panel, RNA from each fraction was analysed by northern blotting with a uniformly labelled transcript derived from sense strand of the cyclin E cDNA. DNA oligonucleotides were used as size markers.
- FIG. 5 Generation of 22 mers and degradation of mRNA are carried out by distinct enzymatic complexes.
- A Extracts prepared either from 0-12 hour Drosophila embryos or Drosophila S2 cells (see Methods) were incubated 0, 15, 30, or 60 minutes (left to right) with a uniformly-labeled double-stranded RNA corresponding to the first 500 nucleotides of the Drosophila cyclin E coding region.
- M indicates a marker prepared by in vitro transcription of a synthetic template. The template was designed to yield a 22 nucleotide transcript. The doublet most probably results from improper initiation at the +1 position.
- B Extracts prepared either from 0-12 hour Drosophila embryos or Drosophila S2 cells (see Methods) were incubated 0, 15, 30, or 60 minutes (left to right) with a uniformly-labeled double-stranded RNA corresponding to the first 500 nucleotides of the Drosophila cyclin E coding region.
- FIG. 6 Production of 22 mers by recombinant CG4792/Dicer.
- Drosophila S2 cells were transfected with plasmids that direct the expression of T7-epitope tagged versions of Drosha, CG4792/Dicer-1 and Homeless. Tagged proteins were purified from cell lysates by immunoprecipitation and were incubated with cyclin E dsRNA. For comparison, reactions were also performed in Drosophila embryo and S2 cell extracts. As a negative control, immunoprecipitates were prepared from cells transfected with a ⁇ -galactosidase expression vector. Pairs of lanes show reactions performed for 0 or 60 minutes.
- the synthetic marker (M) is as described in the legend to FIG. 1.
- B Diagrammatic representations of the domain structures of CG4792/Dicer-1, Drosha and Homeless are shown.
- Drosophila S2 cells were transfected with uniformly, 32P-labelled dsRNA corresponding to the first 500 nt. of GFP.
- RISC complex was affinity purified using a histidine-tagged version of D.m. Ago-2, a recently identified component of the RISC complex (Hammond et al., in prep).
- RISC was isolated either under conditions in which it remains ribosome associated (ls, low salt) or under conditions that extract it from the ribosome in a soluble form (hs, high salt) 6 .
- the spectrum of labelled RNAs in the total lysate is shown.
- Guide RNAs produced by incubation of dsRNA with a Dicer immunoprecipitate are compared to guide RNAs present in a affinity-purified RISC complex. These precisely comigrate on a gel that has single-nucleotide resolution.
- the lane labelled control is an affinity selection for RISC from cell that had been transfected with labeled dsRNA but not with the epitope-tagged D.m. Ago-2.
- FIG. 7 Dicer participates in RNAi.
- FIG. 8 Dicer is an evolutionarily conserved ribonuclease.
- A A model for production of 22 mers by Dicer. Based upon the proposed mechanism of action of Ribonuclease III, we propose that Dicer acts on its substrate as a dimer. The positioning of the two ribonuclease domains (RIIIa and RIIIb) within the enzyme would thus determine the size of the cleavage product.
- RIIIa and RIIIb ribonuclease domains within the enzyme would thus determine the size of the cleavage product.
- An equally plausible alternative model could be derived in which the RIIIa and RIIIb domains of each Dicer enzyme would cleave in concert at a single position. In this model, the size of the cleavage product would be determined by interaction between two neighboring Dicer enzymes.
- B A model for production of 22 mers by Dicer. Based upon the proposed mechanism of action of Ribonuclease III, we propose that Dicer acts on its substrate as
- ZAP domains are more similar within each of the Dicer and ARGONAUTE families than they are between the two groups.
- C An alignment of the ZAP domains in selected Dicer and Argonaute family members is shown. The alignment was produced using ClustalW.
- FIG. 9 Purification strategy for RISC. (second step in RNAi model).
- FIG. 10 Fractionation of RISC activity over sizing column. Activity fractionates as 500 KD complex. Also, antibody to dm argonaute 2 cofractionates with activity.
- FIGS. 11 - 13 Fractionation of RISC over monoS, monoQ, Hydroxyapatite columns. Dm argonaute 2 protein also cofactionates.
- FIG. 14 Alignment of dm argonaute 2 with other family members.
- FIG. 15 Confirmation of dm argonaute 2. S2 cells were transfected with labeled dsRNA and His tagged argonaute. Argonaute was isolated on nickel agarose and RNA component was identified on 15% acrylamide gel.
- FIG. 16 S2 cell and embryo extracts were assayed for 22 mer generating activity.
- FIG. 17 RISC can be separated from 22 mer generating activity (dicer). Spinning extracts (S100) can clear RISC activity from supernatant (left panel) however, S100 spins still contain dicer activity (right panel).
- FIG. 18 Dicer is specific for dsRNA and prefers longer substrates.
- FIG. 19 Dicer was fractionated over several columns.
- FIG. 20 Identification of dicer as enzyme which can process dsRNA into 22 mers.
- Various RNaseIII family members were expressed with n terminal tags, immunoprecipitated, and assayed for 22 mer generating activity (left panel).
- antibodies to dicer could also precipitate 22 mer generating activity.
- FIG. 21 Dicer requires ATP.
- FIG. 22 Dicer produces RNAs that are the same size as RNAs present in RISC.
- FIG. 23 Human dicer homolog when expressed and immunoprecipitated has 22 mer generating activity.
- FIG. 24 Sequence of dm argonaute 2. Peptides identified by microsequencing are shown in underline.
- FIG. 25 Molecular charaterization of dm argonaute 2. The presence of an intron in coding sequence was determined by northern blotting using intron probe. This results in a different 5′ reading frame that that published genome seqeunce. Number of polyglutaine repeats was determined by genomic PCR.
- FIG. 26 Dicer activity can be created in human cells by expression of human dicer gene. Host cell was 293. Crude extracts had dicer activity, while activity was absent from untransfected cells. Activity is not dissimilar to that seen in drosophila embryo extracts.
- FIG. 27 An ⁇ 500 nt. fragment of the gene that is to be silenced (X) is inserted into the modified vector as a stable direct repeat using standard cloning procedures. Treatment with commercially available cre recombinase reverses sequences within the loxP sites (L) to create an inverted repeat. This can be stably maintained and amplified in an sbc mutant bacterial strain (DL759). Transcription in vivo from the promoter of choice (P) yields a hairpin RNA that causes silencing. A zeocin resistance marker is included to insure maintenance of the direct and inverted repeat structures; however this is non-essential in vivo and could be removed by pre-mRNA splicing if desired. Smith, N. A. et al. Total silencing by intron-spliced hairpin RNAs. Nature 407, 319-20 (2000).
- FIG. 28 Hela, Chinese hamster ovary, and P19 (pluripotent, mouse embryonic carcinoma) cell lines transfected with plasmids expressing Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases and with dsRNA 500 mers (400 ng), either homologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP). Dual luciferase assays were carried out using an Analytical Scientific Instruments model 3010 Luminometer. In this assay Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity.
- FIG. 29 P19 (a pluripontent, mouse embryonic cell line) cells transfected with plasmids expressing Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases and with dsRNA 500 mers (500ng), either homologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP). Dual luciferase assays were carried out using an Analytical Scientific Instruments model 3010 Luminometer. In this assay Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity.
- FIG. 30 S10 fractions from P19 cell lysates were used for in vitro translations of mRNA coding for Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases. Translation reactions were programmed with various amounts of dsRNA 500 mers, either homologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP). Reactions were carried out at 30 degrees for 1 hour, after which dual luciferase assays were carried out using an Analytical Scientific Instruments model 3010 Luminometer.
- dsLUC firefly luciferase mRNA
- dsGFP non-homologous
- Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity.
- FIG. 31 S10 fractions from P19 cell lysates were used for in vitro translations of mRNA coding for Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases. Translation reactions were programmed with dsRNA or asRNA 500 mers, either complementary to firefly luciferase mRNA (asLUC and dsLUC) or non-complementary (dsGFP). Reactions were carried out at 30 degrees for 1 hour, after a 30 min preincubation with dsRNA or asRNA. Dual luciferase assays were carried out using an Analytical Scientific Instruments model 3010 Luminometer.
- Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity.
- dsRNA double-stranded RNA
- asRNA anti-sense RNA
- FIG. 32 P19 cells were grown in 6-well tissue culture plates to approximately 60% confluence. Various amounts of dsRNA, either homologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP), were added to each well and incubated for 12 hrs under normal tissue culture conditions. Cells were then transfected with plasmids expressing Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases and with dsRNA 500 mers (500 ng). Dual luciferase assays were carried out 12 hrs post-transfection using an Analytical Scientific Instruments model 3010 Luminometer.
- dsLUC firefly luciferase mRNA
- dsGFP non-homologous luciferases
- Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity.
- FIG. 33 Is a graph illustrating the relative rate of expression luciferase in cells which are treated with various antisense and dsRNA constructs.
- the present invention provides methods for attenuating gene expression in a cell using gene-targeted double stranded RNA (dsRNA).
- dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the “target” gene).
- RNAi can in fact be accomplished in cultured cells, rather than whole organisms as described in the art.
- Another salient feature of the present invention concerns the ability to carry out RNAi in higher eukaryotes, particularly in non-oocytic cells of mammals, e.g., cells from adult mammals as an example.
- the present invention(s) are based on the discovery that the RNAi phenomenum is mediated by a set of enzyme activities, including an essential RNA component, that are evolutionarily conserved in eukaryotes ranging from plants to mammals.
- RISC nuclease a multi-component nuclease co-fractionates with a discrete, 22-nucleotide RNA species which may confer specificity to the nuclease through homology to the substrate mRNAs.
- the short RNA molecules are generated by a processing reaction from the longer input dsRNA.
- these 22 mer guide RNAs may serve as guide sequences that instruct the RISC nuclease to destroy specific mRNAs corresponding to the dsRNA sequences.
- double stranded forms of the 22-mer guide RNA can be sufficient in length to induce sequence-dependent dsRNA inhibition of gene expression.
- dsRNA contructs are administered to cells having a recombinant luciferase reporter gene.
- the control cell e.g., no exogeneously added RNA
- the level of expression of the luciferase reporter is normalized to be the value of “1”.
- both long (500-mer) and short (22-mer) dsRNA constructs complementary to the luciferase gene could inhibit expression of that gene product relative to the control cell.
- Dicer an enzyme that can produce the putative guide RNAs.
- Dicer is a member of the RNAse III family of nucleases that specifically cleave dsRNA and is evolutionarily conserved in worms, flies, plants, fungi and, as described herein, mammals.
- the enzyme has a distinctive structure which includes a helicase domain and dual RNAse III motifs.
- Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family, which have been genetically linked to RNAi in lower eukaryotes.
- Dicer activation of, or overexpression of Dicer may be sufficient in many cases to permit RNA interference in otherwise non-receptive cells, such as cultured eukaryotic cells, or mammalian (non-oocytic) cells in culture or in whole organisms.
- the cells can be treated with an agent(s) that inhibits the general double-stranded RNA response(s) by the host cells, such as may give rise to sequence-independent apoptosis.
- the cells can be treated with agents that inhibit the dsRNA-dependent protein kinase known as PKR (protein kinase RNA-activated).
- PKR protein kinase RNA-activated
- Double stranded RNAs in mammalian cells typically activate protein kinase PKR and leads to apoptosis.
- the mechanism of action of PKR includes phosphorylation and inactivation eIF2a (Fire (1999) Trends Genet 15:358).
- PKR response can be overcome in favor of the sequence-specific RNAi response.
- agents which inhibit expression of PKR, cause its destruction, and/or inhibit the kinase activity of PKF are specifically contemplated for use in the present method.
- overexpression of or agents which ectopic activate IF2 ⁇ a. can be used.
- agents which can be used to suppress the PKR response include inhibitors of IKK phosphorylation of I ⁇ B, inhibitors of I ⁇ B ubiquitination, inhibitors of I ⁇ B degradation, inhibitors of NF- ⁇ B nuclear translocation, and inhibitors of NF- ⁇ B interaction with ⁇ B response elements.
- E3L vaccinia virus
- the E3L gene product contains two distinct domains. A conserved carboxy-terminal domain has been shown to bind double-stranded RNA (dsRNA) and inhibit the antiviral dsRNA response by cells. Expression of at least that portion of the E3L gene in the host cell, or the use of polypeptide or peptidomimetics thereof, can be used to suppress the general dsRNA response.
- Caspase inhibitors sensitized cells to killing by double-stranded RNA. Accordingly, ectopic expression or activated of caspases in the host cell can be used to suppress the general dsRNA response.
- the subject method is carried out in cells which have little or no general response to double stranded RNA, e.g., have no PKR-dependent dsRNA response, at least under the culture conditions.
- CHO and P19 cells can be used without having to inhibit PKR or other general dsRNA responses.
- the present invention provides a process and compositions for inhibiting expression of a target gene in a cell, expecially a mammalian cell.
- the process comprises introduction of RNA (the “dsRNA construct”) with partial or fully double-stranded character into the cell or into the extracellular environment.
- Inhibition is specific in that a nucleotide sequence from a portion of the target gene is chosen to produce the dsRNA construct.
- the method utilizes a cell in which Dicer and/or Argonaute activities are recombinantly expressed or otherwise ectopically activated. This process can be (1) effective in attenuating gene expression, (2) specific to the targeted gene, and (3) general in allowing inhibition of many different types of target gene.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked.
- a genomic integrated vector or “integrated vector”, which can become integrated into the chromsomal DNA of the host cell.
- an episomal vector i.e., a nucleic acid capable of extra-chromosomal replication.
- vectors capable of directing the expression of genes to that they are operatively linked are referred to herein as “expression vectors”.
- expression vectors In the present specification, “plasmid” and “vector” are used interchangeably unless otherwise clear from the context.
- nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- the term should also be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
- the term “gene” or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide of the present invention, including both exon and (optionally) intron sequences.
- a “recombinant gene” refers to nucleic acid encoding such regulatory polypeptides, that may optionally include intron sequences that are derived from chromosomal DNA.
- the term “intron” refers to a DNA sequence present in a given gene that is not translated into protein and is generally found between exons.
- the term “transfection” means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
- a “protein coding sequence” or a sequence that “encodes” a particular polypeptide or peptide is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences.
- 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′ (carboxy) terminus.
- a coding sequence can include, but is not limited to, cDNA from procaryotic or eukaryotic mRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, and even synthetic DNA sequences.
- a transcription termination sequence will usually be located 3′ to the coding sequence.
- encodes unless evident from its context, will be meant to include DNA sequences that encode a polypeptide, as the term is typically used, as well as DNA sequences that are transcribed into inhibitory antisense molecules.
- loss-of-function refers to genes inhibited by the subject RNAi method, refers a diminishment in the level of expression of a gene when compared to the level in the absense of dsRNA constructs.
- expression refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein.
- expression of a protein coding sequence results from transcription and translation of the coding sequence.
- Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- cultured cells refers to cells suspended in culture, e.g., dispersed in culture or in the form tissue. It does not, however, include oocytes or whole embryos (including blastocysts and the like) which may be provided in culture. In certain embodiments, the cultured cells are adults cells, e.g., non-embryonic.
- recombinant virus is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
- transduction and “transfection” are art recognized and mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
- Transformation refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a dsRNA contruct.
- Transient transfection refers to cases where exogenous DNA does not integrate into the genome of a transfected cell, e.g., where episomal DNA is transcribed into mRNA and translated into protein.
- a cell has been “stably transfected” with a nucleic acid construct when the nucleic acid construct is capable of being inherited by daughter cells.
- a “reporter gene construct” is a nucleic acid that includes a “reporter gene” operatively linked to at least one transcriptional regulatory sequence. Transcription of the reporter gene is controlled by these sequences to which they are linked. The activity of at least one or more of these control sequences can be directly or indirectly regulated by the target receptor protein. Exemplary transcriptional control sequences are promoter sequences.
- a reporter gene is meant to include a promoter-reporter gene construct that is heterologously expressed in a cell.
- transformed cells refers to cells that have spontaneously converted to a state of unrestrained growth, i.e., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control.
- transformed phenotype of malignant mammalian cells and “transformed phenotype ” are intended to encompass, but not be limited to, any of the following phenotypic traits associated with cellular transformation of mammalian cells: immortalization, morphological or growth transformation, and tumorigenicity, as detected by prolonged growth in cell culture, growth in semi-solid media, or tumorigenic growth in immuno-incompetent or syngeneic animals.
- proliferating and “proliferation” refer to cells undergoing mitosis.
- immortalized cells refers to cells that have been altered via chemical, genetic, and/or recombinant means such that the cells have the ability to grow through an indefinite number of divisions in culture.
- the “growth state” of a cell refers to the rate of proliferation of the cell and the state of differentiation of the cell.
- One aspect of the invention provides a method for potentiating RNAi by induction or ectopic activation of an RNAi enzyme in a cell (in vivo or in vitro) or cell-free mixtures.
- the RNAi activity is activated or added to a mammalian cell, e.g., a human cell, which cell may be provided in vitro or as part of a whole organism.
- the subject method is carried out using eukaryotic cells generally (except for oocytes) in culture.
- the Dicer enzyme may be activated by virtue of being recombinantly expressed or it may be activated by use of an agent which (i) induces expression of the endogenous gene, (ii) stabilizes the protein from degradation, and/or (iii) allosterically modies the enzyme to increase its activity (by altering its Kcat, Km or both).
- the present method provides for ectopic activation of Dicer.
- Dicer refers to a protein which (a) mediates an RNAi response and (b) has an amino acid sequence at least 50 percent identical, and more preferablty at least 75, 85, 90 or 95 percent identical to SEQ ID No. 2 or 4, and/or which can be encoded by a nucleic acid which hybridizes under wash conditions of 2 ⁇ SSC at 22° C., and more preferably 0.2 ⁇ SSC at 65° C., to a nucleotide represented by SEQ ID No. 1 or 3.
- the method may comprise introducing a dsRNA contruct into a cell in which Dicer has been recombinantly expressed or otherwise ectopically activated.
- At least one of the activated RNAi enzymes is Argonaut, or a homolog thereof.
- the present method provides for ectopic activation of Argonaut.
- the term “Argonaut” refers to a protein which (a) mediates an RNAi response and (b) has an amino acid sequence at least 50 percent identical, and more preferablty at least 75, 85, 90 or 95 percent identical to the amino acid sequence shown in FIG. 24. Accordingly, the method may comprise introducing a dsRNA contruct into a cell in which Argonaut has been recombinantly expressed or otherwise ectopically activated.
- This invention also provides expression vectors containing a nucleic acid encoding a Dicer or Argonaut polypeptides, operably linked to at least one transcriptional regulatory sequence.
- Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence.
- Regulatory sequences are art-recognized and are selected to direct expression of the subject Dicer or Argonaut proteins.
- transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
- any of a wide variety of expression control sequences, sequences that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding Dicer or Argonaut polypeptides of this invention.
- useful expression control sequences include, for example, a viral LTR, such as the LTR of the Moloney murine leukemia virus, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage ⁇ , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the bac
- the vector's copy number should also be considered.
- the recombinant Dicer or Argonaut genes can be produced by ligating nucleic acid encoding a Dicer or Argonaut polypeptide into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both.
- Expression vectors for production of recombinant forms of the subject Dicer or Argonaut polypeptides include plasmids and other vectors.
- suitable vectors for the expression of a Dicer or Argonaut polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
- YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein).
- These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the yeast 2 micron plasmid.
- a Dicer or Argonaut polypeptide is produced recombinantly utilizing an expression vector generated by sub-cloning the coding sequence of a Dicer or Argonaut gene.
- the preferred mammalian expression vectors contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
- the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
- vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
- derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
- BBV-1 bovine papillomavirus
- pHEBo Epstein-Barr virus
- the various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.
- suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.
- the subject invention provides a “gene activation” construct which, by homologous recombination with a genomic DNA, alters the transcriptional regulatory sequences of an endogenous Dicer or Argonaut gene.
- the gene activation construct can replace the endogenous promoter of a Dicer or Argonaut gene with a heterologous promoter, e.g., one which causes constitutive expression of the Dicer or Argonaut gene or which causes inducible expression of the gene under conditions different from the normal expression pattern of Dicer or Argonaut.
- a variety of different formats for the gene activation constructs are available. See, for example, the Transkaryotic Therapies, Inc PCT publications WO93/09222, WO95/31560, WO96/29411, WO95/31560 and WO94/12650.
- the nucleotide sequence used as the gene activation construct can be comprised of (1) DNA from some portion of the endogenous Dicer or Argonaut gene (exon sequence, intron sequence, promoter sequences, etc.) which direct recombination and (2) heterologous transcriptional regulatory sequence(s) which is to be operably linked to the coding sequence for the genomic Dicer or Argonaut gene upon recombination of the gene activation construct.
- the construct may further include a reporter gene to detect the presence of the knockout construct in the cell.
- the gene activation construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to provide the heterologous regulatory sequences in operative association with the native Dicer or Argonaut gene.
- Such insertion occurs by homologous recombination, i.e., recombination regions of the activation construct that are homologous to the endogenous Dicer or Argonaut gene sequence hybridize to the genomic DNA and recombine with the genomic sequences so that the construct is incorporated into the corresponding position of the genomic DNA.
- recombination region or “targeting sequence” refer to a segment (i.e., a portion) of a gene activation construct having a sequence that is substantially identical to or substantially complementary to a genomic gene sequence, e.g., including 5′ flanking sequences of the genomic gene, and can facilitate homologous recombination between the genomic sequence and the targeting transgene construct.
- replacement region refers to a portion of a activation construct which becomes integrated into an endogenous chromosomal location following homologous recombination between a recombination region and a genomic sequence.
- the heterologous regulatory sequences can include one or more of a variety elements, including: promoters (such as constitutive or inducible promoters), enhancers, negative regulatory elements, locus control regions, transcription factor binding sites, or combinations thereof.
- Promoters/enhancers which may be used to control expression of the targeted gene in vivo include, but are not limited to, the cytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989, J. Exp. Med., 169:13), the human ⁇ -actin promoter (Gunning et al. (1987) PNAS 84:4831-4835), the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al. (1984) Mol. Cell Biol.
- CMV cytomegalovirus
- MMTV LTR mouse mammary tumor virus long terminal repeat
- MoLV LTR Moloney murine leukemia virus
- SV40 early or late region promoter Moloney murine leukemia virus
- the replacement region merely deletes a negative transcriptional control element of the native gene, e.g., to activate expression, or ablates a positive control element, e.g., to inhibit expression of the targeted gene.
- the cell with the target gene may be derived from or contained in any organism (e.g., plant, animal, protozoan, virus, bacterium, or fungus).
- the dsRNA construct may be synthesized either in vivo or in vitro. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro.
- a regulatory region may be used to transcribe the RNA strand (or strands).
- the cell with the target gene may be derived from or contained in any organism.
- the organism may a plant, animal, protozoan, bacterium, virus, or fungus.
- the plant may be a monocot, dicot or gymnosperm; the animal may be a vertebrate or invertebrate.
- Preferred microbes are those used in agriculture or by industry, and those that are pathogenic for plants or animals. Fungi include organisms in both the mold and yeast morphologies.
- Plants include arabidopsis; field crops (e.g., alfalfa, barley, bean, com, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops (e.g., asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish, spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g., almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, faJoa, filbert, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut, and watermelon); and ornament
- vertebrate animals examples include fish, mammal, cattle, goat, pig, sheep, rodent, hamster, mouse, rat, primate, and human.
- Invertebrate animals include nematodes, other worms, drosophila, and other insects.
- Representative generae of nematodes include those that infect animals (e.g., Ancylostoma, Ascaridia, Ascaris, Bunostomum, Caenorhabditis, Capillaria, Chabertia, Cooperia, Dictyocaulus, Haernonchus, Heterakis, Nematodirus, Oesophagostomum, Ostertagia, Oxyuris, Parascaris, Strongylus, Toxascaris, Trichuris, Trichostrongylus, Tflichonema, Toxocara, Uncinaria) and those that infect plants (e.g., B ursaphalenchus, Criconerriella, Diiylenchus, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Melodoigyne
- the cell having the target gene may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like.
- the cell may be a stem cell or a differentiated cell.
- Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands.
- the target gene may be a gene derived from the cell, an endogenous gene, a transgene, or a gene of a pathogen which is present in the cell after infection thereof.
- the procedure may provide partial or complete loss of function for the target gene.
- Lower doses of injected material and longer times after administration of dsRNA may result in inhibition in a smaller fraction of cells.
- Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein.
- “Inhibition of gene expression” refers to the absence (or observable decrease) in the level of protein and/or mRNA product from a target gene. “Specificity” refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioImmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
- reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.
- AHAS acetohydroxyacid synthase
- AP alkaline phosphatase
- LacZ beta galactosidase
- GUS beta glucoronidase
- CAT
- quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present invention.
- Lower doses of injected material and longer times after administration of dsRNA may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%,90%, or 95% of targeted cells).
- Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein.
- the efficiency of inhibition may be determined by assessing the amount of gene product in the cell: mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.
- the present invention may is not limited to any type of target gene or nucleotide sequence.
- target genes e.g., adhesion molecules, cyclin kinase inhibitors, Writ family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors
- developmental genes e.g., adhesion molecules, cyclin kinase inhibitors, Writ family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors
- oncogenes e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETS 1 , ETV6, FGR, FOS, FYN
- the dsRNA construct may comprise one or more strands of polymerized ribonucleotide. It may include modifications to either the phosphate-sugar backbone or the nucleoside. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general panic response in some organisms which is generated by dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase.
- the dsRNA construct may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
- the dsRNA construct may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing an organism in a solution containing RNA.
- Methods for oral introduction include direct mixing of RNA with food of the organism, as well as engineered approaches in which a species that is used as food is engineered to express an RNA, then fed to the organism to be affected.
- Physical methods of introducing nucleic, acids include injection directly into the cell or extracellular injection into the organism of an RNA solution.
- the double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands.
- RNA duplex formation may be initiated either inside or outside the cell.
- the RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition; lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
- dsRNA constructs containing a nucleotide sequences identical to a portion of the target gene are preferred for inhibition.
- RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
- sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred.
- the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing).
- the length of the dsRNA is at least 20, 21 or 22 nucleotides in length, e.g., corresponding in size to RNA products produced by Dicer-dependent cleavage.
- the dsRNA construct is at least 25, 50, 100, 200, 300 or 400 bases.
- the dsRNA construct is 400-800 bases in length.
- RNA and the target gene are not required to practice the present invention.
- the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
- the dsRNA construct may be synthesized either in vivo or in vitro. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro.
- a regulatory region e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation
- a regulatory region may be used to transcribe the dsRNA strand (or strands).
- Inhibition may be targeted by specific transcription in an organ, tissue, or cell type; stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and/or engineering transcription at a developmental stage or age.
- the RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus.
- the dsRNA construct may be chemically or enzymatically synthesized by manual or automated reactions.
- the dsRNA construct may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6).
- RNA may be purified prior to introduction into the cell.
- RNA can be punified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography or a combination thereof.
- the dsRNA construct may be used with no or a minimum of purification to avoid losses due to sample processing.
- the dsRNA construct may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
- Physical methods of introducing nucleic acids include injection of a solution containing the dsRNA construct, bombardment by particles covered by the dsRNA construct, soaking the cell or organism in a solution of the RNA, or electroporation of cell membranes in the presence of the dsRNA construct.
- a viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of dsRNA construct encoded by the expression construct.
- Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemicalmediated transport, such as calcium phosphate, and the like.
- the dsRNA construct may be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or other-wise increase inhibition of the target gene.
- One utility of the present invention is as a method of identifying gene function in an organism, especially higher eukaryotes comprising the use of double-stranded RNA to inhibit the activity of a target gene of previously unknown function.
- functional genomics would envision determining the function of uncharacterized genes by employing the invention to reduce the amount and/or alter the timing of target gene activity.
- the invention could be used in determining potential targets for pharmaceutics, understanding normal and pathological events associated with development, determining signaling pathways responsible for postnatal development/aging, and the like.
- the increasing speed of acquiring nucleotide sequence information from genomic and expressed gene sources, including total sequences for mammalian genomes, can be coupled with the invention to determine gene function in a cell or in a whole organism.
- the preference of different organisms to use particular codons, searching sequence databases for related gene products, correlating the linkage map of genetic traits with the physical map from which the nucleotide sequences are derived, and artificial intelligence methods may be used to define putative open reading frames from the nucleotide sequences acquired in such sequencing projects.
- a simple assay would be to inhibit gene expression according to the partial sequence available from an expressed sequence tag (EST). Functional alterations in growth, development, metabolism, disease resistance, or other biological processes would be indicative of the normal role of the EST's gene product.
- EST expressed sequence tag
- duplex RNA can be produced by an amplification reaction using primers flanking the inserts of any gene library derived from the target cell or organism. Inserts may be derived from genomic DNA or mRNA (e.g., cDNA and cRNA). Individual clones from the library can be replicated and then isolated in separate reactions, but preferably the library is maintained in individual reaction vessels (e.g., a 96 well microtiter plate) to minimize the number of steps required to practice the invention and to allow automation of the process.
- mRNA e.g., cDNA and cRNA
- the subject invention provides an arrayed library of RNAi constructs.
- the array may in the form of solutions, such as multi-well plates, or may be “printed” on solid substrates upon which cells can be grown.
- solutions containing duplex RNAs that are capable of inhibiting the different expressed genes can be placed into individual wells positioned on a microtiter plate as an ordered array, and intact cells/organisms in each well can be assayed for any changes or modifications in behavior or development due to inhibition of target gene activity.
- the subject method uses an arrayed library of RNAi constructs to screen for combinations of RNAi that is lethal to host cells.
- Synthetic lethality is a bedrock principle of experimental genetics. A synthetic lethality describes the properties of two mutations which, individually, are tolerated by the organism but which, in combination, are lethal.
- the subject arrays can be used to identify loss-of-function mutations that are lethal in combination with alterations in other genes, such as activated oncogenes or loss-of-function mutations to tumor suppressors. To achieve this, one can create “phenotype arrays” using cultured cells. Expression of each of a set of genes, such as the host cell's genome, can be individually systematically disrupted using RNA interference. Combination with alterations in oncogene and tumor suppressor pathways can be used to identify synthetic lethal interactions that may identify novel therapeutic targets.
- the RNAi constructs can be fed directly to, injected into, the cell/organism containing the target gene.
- the duplex RNA can be produced by in vivo or in vitro transcription from an expression construct used to produce the library.
- the construct can be replicated as individual clones of the library and transcribed to produce the RNA; each clone can then be fed to, or injected into, the cell/organism containing the target gene.
- the function of the target gene can be assayed from the effects it has on the cell/organism when gene activity is inhibited. This screening could be amenable to small subjects that can be processed in large number, for example, tissue culture cells derived from mammals, especially primates, and most preferably humans.
- a characteristic of an organism is determined to be genetically linked to a polymorphism through RFLP or QTL analysis
- the present invention can be used to gain insight regarding whether that genetic polymorphism might be directly responsible for the characteristic. For example, a fragment defining the genetic polymorphism or sequences in the vicinity of such a genetic polymorphism can be amplified to produce an RNA, the duplex RNA can be introduced to the organism or cell, and whether an alteration in the charactenstic is correlated with inhibition can be determined.
- inhibition of the target gene causes lethality
- inhibition of the target gene may not result in any observable alteration
- the fragment contains nucleotide sequences that are not capable of inhibiting the target gene, or the target gene's activity is redundant.
- the present invention may be useful in allowing the inhibition of essential genes. Such genes may be required for cell or organism viability at only particular stages of development or cellular compartments.
- the functional equivalent of conditional mutations may be produced by inhibiting activity of the target gene when or where it is not required for viability.
- the invention allows addition of RNA at specific times of development and locations in the organism without introducing permanent mutations into the target genome.
- the present invention can target inhibition through the appropriate exons to specifically inhibit or to distinguish among the functions of family members.
- a hormone that contained an alternatively spliced transmembrane domain may be expressed in both membrane bound and secreted forms.
- the functional consequences of having only secreted hormone can be determined according to the invention by targeting the exon containing the transmembrane domain and thereby inhibiting expression of membrane-bound hormone.
- the present invention may be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples or subjects.
- Preferred components are the dsRNA and a vehicle that promotes introduction of the dsRNA.
- Such a kit may also include instructions to allow a user of the kit to practice the invention.
- an organism may be engineered to produce dsRNA which produces commercially or medically beneficial results, for example, resistance to a pathogen or its pathogenic effects, improved growth, or novel developmental patterns.
- RNA interference may provide anti-viral defence, modulate transposition or regulate gene expression 1, 6, 8-10 .
- RNA interference may provide anti-viral defence, modulate transposition or regulate gene expression 1, 6, 8-10 .
- ‘loss-of-function’ phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs.
- Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA.
- This enzyme contains an essential RNA component.
- the sequence-specific nuclease co-fractionates with a discrete, ⁇ 25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.
- dsRNAs double-stranded RNAs
- Drosophila 11, 12 the mechanisms underlying this phenomenon have remained mostly unknown. We therefore wanted to establish a biochemically tractable model in which such mechanisms could be investigated.
- RNA interference RNA interference
- Double-stranded RNAs of 540 and 400 nucleotides were quite effective, whereas dsRNAs of 200 and 300 nucleotides were less potent.
- Double-stranded cyclin E RNAs of 50 or 100 nucleotides were inert in our assay, and transfection with a single-stranded, antisense cyclin E RNA had virtually no effect.
- RNAi RNA-induced RNAi
- cyclin E dsRNA Bulk population
- cyclin A anaphase-promoting complex
- S, G2 and M a cyclin that acts in S, G2 and M
- RNAi may be a generally applicable method for probing gene function in cultured Drosophila cells.
- RNAi The decrease in mRNA levels observed upon transfection of specific dsRNAs into Drosophila cells could be explained by effects at transcriptional or post-transcriptional levels. Data from other systems have indicated that some elements of the dsRNA response may affect mRNA directly (reviewed in refs 1 and 6 ). We therefore sought to develop a cell-free assay that reflected, at least in part, RNAi.
- S2 cells were transfected with dsRNAs corresponding to either cyclin E or lacZ.
- Cellular extracts were incubated with synthetic mRNAs of lacZ or cyclin E. Extracts prepared from cells transfected with the 540-nucleotide cyclin E dsRNA efficiently degraded the cyclin E transcript; however, the lacZ transcript was stable in these lysates (FIG. 2 a ).
- lysates from cells transfected with the lacZ dsRNA degraded the lacZ transcript but left the cyclin E mRNA intact.
- RISC RNA-induced silencing complex
- transcripts that had no homology with the transfected dsRNA ( FIG. 2 b, Eout; FIG. 2 c, as300) were not degraded.
- Double-stranded RNAs homologous to an upstream cistron have little or no effect on a linked downstream cistron, despite the fact that unprocessed, polycistronic mRNAs can be readily detected 17, 18 .
- the nuclease was inactive against a dsRNA identical to that used to provoke the RNAi response in vivo (FIG. 2 b ).
- a 5′ cap nor a poly(A) tail was required, as such transcripts were degraded as efficiently as uncapped and non-polyadenylated RNAs.
- RNAi nuclease is a ribonucleoprotein, requiring both RNA and protein components.
- Biochemical fractionation is consistent with these components being associated in extract rather than being assembled on the target mRNA after its addition.
- RNAi nuclease fractionated with ribosomes in a high-speed centrifugation step.
- Activity could be extracted by treatment with high salt, and ribosomes could be removed by an additional centrifugation step. Chromatography of soluble nuclease over an anion-exchange column resulted in a discrete peak of activity (FIG. 4 b, cyclin E). This retained specificity as it was inactive against a heterologous mRNA (FIG. 4 b, lacZ). Active fractions also contained an RNA species of 25 nucleotides that is homologous to the cyclin E target (FIG. 4 b, northern).
- the band observed on northern blots may represent a family of discrete RNAs because it could be detected with probes specific for both the sense and antisense cyclin E sequences and with probes derived from distinct segments of the dsRNA (data not shown). At present, we cannot determine whether the 25-nucleotide RNA is present in the nuclease complex in a double-stranded or single-stranded form.
- RNA interference allows an adaptive defence against both exogenous and endogenous dsRNAs, providing something akin to a dsRNA immune response.
- Our data, and that of others 19 is consistent with a model in which dsRNAs present in a cell are converted, either through processing or replication, into small specificity determinants of discrete size in a manner analogous to antigen processing.
- Our results suggest that the post-transcriptional component of dsRNA-dependent gene silencing is accomplished by a sequence-specific nuclease that incorporates these small RNAs as guides that target specific messages based upon sequence recognition.
- dsRNAs provoke not only post-transcriptional gene silencing but also chromatin remodelling and transcriptional repression 20, 21 . It is now critical to determine whether conservation of gene-silencing mechanisms also exists at the transcriptional level and whether chromatin remodelling can be directed in a sequence-specific fashion by these same dsRNA-derived guide sequences.
- S2 (ref. 22) cells were cultured at 27° C. in 90% Schneider's insect media (Sigma), 10% heat inactivated fetal bovine serum (FBS). Cells were transfected with dsRNA and plasmid DNA by calcium phosphate co-precipitation 23 . Identical results were observed when cells were transfected using lipid reagents (for example, Superfect, Qiagen). For FACS analysis, cells were additionally transfected with a vector that directs expression of a green fluorescent protein (GFP)-US9 fusion protein 13 . These cells were fixed in 90% ice-cold ethanol and stained with propidium iodide at 25 ⁇ g ml ⁇ 1 .
- GFP green fluorescent protein
- Log-phase S2 cells were plated on 15-cm tissue culture dishes and transfected with 30 ⁇ g dsRNA and 30 ⁇ g carrier plasmid DNA. Seventy-two hours after transfection, cells were harvested in PBS containing 5 mM EGTA washed twice in PBS and once in hypotonic buffer (10 mM HEPES pH 7.3, 6 mM ⁇ -mercaptoethanol). Cells were suspended in 0.7 packed-cell volumes of hypotonic buffer containing Complete protease inhibitors (Boehringer) and 0.5 units ml ⁇ 1 of RNasin (Promega).
- Extracts were centrifuged at 200,000 g for 3 h and the resulting pellet (containing ribosomes) was extracted in hypotonic buffer containing also 1 mM MgCl 2 and 300 mM KOAc. The extracted material was spun at 100,000 g for 1 h and the resulting supernatant was fractionated on Source 15Q column (Pharmacia) using a KCl gradient in buffer A (20 mM HEPES pH 7.0, 1 mM dithiothreitol, 1 mM MgCl 2 ). Fractions were assayed for nuclease activity as described above.
- RNA-dependent RNA polymerases e.g. EGO-1, QDE1, SGS2/SDE1 13-16 . While potential roles have been proposed, none of these genes has been assigned a definitive function in the silencing process. Biochemical studies have suggested that PTGS is accomplished by a multicomponent nuclease that targets mRNAs for degradation 6,8,17 .
- RNAs have been produced during RNAi in vitro using an extract prepared from Drosophila embryos 8 . Putative guide RNAs can also be produced in extracts from Drosophila S2 cells (FIG. 5 a ). With the goal of understanding the mechanism of post-transcriptional gene silencing, we have undertaken both biochemical fractionation and candidate gene approaches to identify the enzymes that execute each step of RNAi.
- RISC nuclease
- RNAse III family members are among the few nucleases that show specificity for double-stranded RNA 18 .
- Analysis of the Drosophila and C. elegans genomes reveals several types of RNAse III enzymes.
- First is the canonical RNAse III which contains a single RNAse III signature motif and a double-stranded RNA binding domain (dsRBD; e.g. RNC_CAEEL).
- Second is a class represented by Drosha 19 , a Drosophila enzyme that contains two RNAse III motifs and a dsRBD (CeDrosha in C. elegans ).
- a third class contains two RNAse III signatures and an amino terminal helicase domain (e.g. Drosophila CG4792, CG6493, C. elegans K12H4.8), and these had previously been proposed by Bass as candidate RNAi nucleases 20 .
- Representatives of all three classes were tested for the ability to produce discrete, ⁇ 22 nt. RNAs from dsRNA substrates.
- CG4792 may carry out the initiation step of RNA interference by producing ⁇ 22 nt. guide sequences from dsRNAs. Because of its ability to digest dsRNA into uniformly sized, small RNAs, we have named this enzyme Dicer (Dcr). Dicer mRNA is expressed in embryos, in S2 cells, and in adult flies, consistent with the presence of functional RNAi machinery in all of these contexts (see Supplement 3).
- Dicer might be the nuclease responsible for the production of guide RNAs from dsRNAs prompted us to raise an antiserum directed against the carboxy-terminus of the Dicer protein (Dicer-1, CG4792). This antiserum could immunoprecipitate a nuclease activity from either Drosophila embryo extracts or from S2 cell lysates that produced ⁇ 22 nt. RNAs from dsRNA substrates (FIG. 6C). The putative guide RNAs that are produced by the Dicer-1 enzyme precisely comigrate with 22 mers that are produced in extract and with 22 mers that are associated with the RISC enzyme (FIGS. 6 D,F).
- RNA interference in C. elegans and in Drosophila has several requirements.
- the initiating RNA must be double-stranded, and it must be several hundred nucleotides in length.
- Dicer was inactive against single stranded RNAs regardless of length (see Supplement 4). The enzyme could digest both 200 and 500 nucleotide dsRNAs but was significantly less active with shorter substrates (see Supplement 4). Double-stranded RNAs as short as 35 nucleotides could be cut by the enzyme, albeit very inefficiently (data not shown).
- E. coli RNAse III could digest to completion dsRNAs of 35 or 22 nucleotides (not shown). This suggests that the substrate preferences of the Dicer enzyme may contribute to but not wholly determine the size dependence of RNAi.
- RNAi RNA-dependent RNA polymerases
- RNA interference can be divided into at least two distinct steps. According to this model, initiation of PTGS would occur upon processing of a double-stranded RNA by Dicer into ⁇ 22 nucleotide guide sequences, although we cannot formally exclude the possibility that another, Dicer-associated nuclease may participate in this process. These guide RNAs would be incorporated into a distinct nuclease complex (RISC) that targets single-stranded mRNAs for degradation.
- RISC nuclease complex
- the structure of the Dicer enzyme provokes speculation on the mechanism by which the enzyme might produce discretely sized fragments irrespective of the sequence of the dsRNA (see Supplement 1, FIG. 8 a ). It has been established that bacterial RNAse III acts on its substrate as a dimer 18,22,23 . Similarly, a dimer of Dicer enzymes may be required for cleavage of dsRNAs into ⁇ 22 nt. pieces. According to one model, the cleavage interval would be determined by the physical arrangement of the two RNAse III domains within Dicer enzyme (FIG. 8 a ). A plausible alternative model would dictate that cleavage was directed at a single position by the two RIII domains in a single Dicer protein.
- the 22 nucleotide interval could be dictated by interaction of neighboring Dicer enzymes or by translocation along the mRNA substrate.
- the presence of an integral helicase domain suggests that the products of Dicer cleavage might be single-stranded 22 mers that are incorporated into the RISC enzyme as such.
- a notable feature of the Dicer family is its evolutionary conservation. Homologs are found in C. elegans (K12H4.8), Arabidopsis (e.g., CARPEL FACTORY 24 , T25K16.4, AC012328 — 1), mammals (Helicase-MOI 25 ) and S. pombe (YC9A_SCHPO) (FIG. 8 b, see Supplements 6,7 for sequence comparisons).
- the human Dicer family member is capable of generating ⁇ 22 nt. RNAs from dsRNA substrates (Supplement 5) suggesting that these structurally similar proteins may all share similar biochemical functions. It has been demonstrated that exogenous dsRNAs can affect gene function in early mouse embryos 29 , and our results suggest that this regulation may be accomplished by an evolutionarily conserved RNAi machinery.
- each Dicer family member also contains a ZAP domain (FIG. 8 c ) 27 .
- This sequence was defined based solely upon its conservation in the Zwille/ARGONAUTE/Piwi family that has been implicated in RNAi by mutations in C. elegans (Rde-1) 9 and Neurospora (Qde-2) 10 . Although the function of this domain is unknown, it is intriguing that this region of homology is restricted to two gene families that participate in dsRNA-dependent silencing. Both the ARGONAUTE and Dicer families have also been implicated in common biological processes, namely the determination of stem-cell fates.
- a hypomorphic allele of carpel factory a member of the Dicer family in Arabidopsis, is characterized by increased proliferation in floral meristems 24 .
- This phenotype and a number of other characteristic features are also shared by Arabidopsis ARGONAUTE (ago1-1) mutants 26 (C. Kidner and R. Martiennsen, pers. comm.).
- Dicer as a catalyst of the initiation step of RNAi, we have begun to unravel the biochemical basis of this unusual mechanism of gene regulation. It will be of critical importance to determine whether the conserved family members from other organisms, particularly mammals, also play a role in dsRNA-mediated gene regulation.
- Plasmid constructs A full-length cDNA encoding Drosha was obtained by PCR from an EST sequenced by the Berkeley Drosophila genome project. The Homeless clone was a gift from Gillespie and Berg (Univ. Washington). The T7 epitope-tag was added to the amino terminus of each by PCR, and the tagged cDNAs were cloned into pRIP, a retroviral vector designed specifically for expression in insect cells (E. Bernstein, unpublished). In this vector, expression is driven by the Orgyia pseudotsugata IE2 promoter (Invitrogen).
- a genomic clone was amplified from a bacmid (BACR23F10; obtained from the BACPAC Resource Center in the Dept. of Human Genetics at the Roswell Park Cancer Institute). Again, during amplification, a T7 epitope tag was added at the amino terminus of the coding sequence.
- the human Dicer gene was isolated from a cDNA library prepared from HaCaT cells (GJH, unpublished). A T7-tagged version of the complete coding sequence was cloned into pCDNA3 (Invitrogen) for expression in human cells (LinX-A).
- Drosophila embryos were reared in fly cages by standard methodologies and were collected every 12 hours. The embryos were dechorionated in 50% chlorox bleach and washed thoroughly with distilled water. Lysis buffer (10 mM Hepes, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM EGTA, 10 mM ⁇ -glycerophosphate, 1 mM DTT, 0.2 mM PMSF) was added to the embryos, and extracts were prepared by homogenization in a tissue grinder. Lysates were spun for two hours at 200,000 ⁇ g and were frozen at ⁇ 80° C. LinX-A cells, a highly-transfectable derivative of human 293 cells, (Lin Xie and GJH, unpublished) were maintained in DMEM/10%FCS.
- Lysis buffer (10 mM Hepes, 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM EGTA, 10 mM
- IP buffer 125 mM KOAc, 1 mM MgOAc, 1 mM CaCl 2 , 5 mM EGTA, 20 mM Hepes pH 7.0, 1 mM DTT, 1% NP-40 plus Complete protease inhibitors (Roche)). Lysates were spun for 10 minutes at 14,000 ⁇ g and supernatants were added to T7 antibody-agarose beads (Novagen). Antibody binding proceeded for 4 hours at 4° C. Beads were centrifuged and washed in lysis buffer three times, and once in reaction buffer. The Dicer antiserum was raised in rabbits using a KLH-conjugated peptide corresponding to the C-terminal 8 amino acids of Drosophila Dicer-1 (CG4792).
- RNA preparation Templates to be transcribed into dsRNA were generated by PCR with forward and reverse primers, each containing a T7 promoter sequence. RNAs were produced using Riboprobe (Promega) kits and were uniformly labeling during the transcription reaction with 32 P-UTP. Single-stranded RNAs were purified from 1% agarose gels. dsRNA cleavage. Five microliters of embryo or S2 extracts were incubated for one hour at 30° C. with dsRNA in a reaction containing 20 mM Hepes pH 7.0, 2 mM MgOAc, 2 mM DTT, 1 mM ATP and 5% Superasin (Ambion).
- Immunoprecipitates were treated similarly except that a minimal volume of reaction buffer (including ATP and Superasin) and dsRNA were added to beads that had been washed in reaction buffer (see above).
- reaction buffer including ATP and Superasin
- dsRNA were added to beads that had been washed in reaction buffer (see above).
- ATP depletion Drosophila embryo extracts were incubated for 20 minutes at 30° C. with 2 mM glucose and 0.375 U of hexokinase (Roche) prior to the addition of dsRNA.
- T7-tagged proteins were immunoprecipitated from whole cell lysates in IP buffer using anti-T7-antibody-agarose conjugates. Proteins were released from the beads by boiling in Laemmli buffer and were separated by electrophoresis on 8% SDS PAGE. Following transfer to nitrocellulose, proteins were visualized using an HRP-conjugated anti-T7 antibody (Novagen) and chemiluminescent detection (Supersignal, Pierce).
- RNAi of Dicer Drosophila S2 cells were transfected either with a dsRNA corresponding to mouse caspase 9 or with a mixture of two dsRNAs corresponding to Drosophila Dicer-1 and Dicer-2 (CG4792 and CG6493). Two days after the initial transfection, cells were again transfected with a mixture containing a GFP expression plasmid and either luciferase dsRNA or GFP dsRNA as previously described 6 . Cells were assayed for Dicer activity or fluorescence three days after the second transfection. Quantification of fluorescent cells was done on a Coulter EPICS cell sorter after fixation. Control transfections indicated that Dicer activity was not affected by the introduction of caspase 9 dsRNA.
- RNAi double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25-33 (2000).
- RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101, 543-53 (2000).
- Bohmert, K. et al AGO1 defines a novel locus of Arabidopsis controlling leaf development. Embo J 17, 170-80 (1998).
- RNA interference In numerous model organisms, double stranded RNAs have been shown to cause effective and specific suppression of gene function (ref. 1). This response, termed RNA interference or post-transcriptional gene silencing, has evolved into a highly effective reverse genetic tool in C. elegans, Drosophila, plants and numerous other systems. In these cases, double-stranded RNAs can be introduced by injection, transfection or feeding; however, in all cases, the response is both transient and systemic. Recently, stable interference with gene expression has been achieved by expression of RNAs that form snap-back or hairpin structures (refs 2-7).
- hairpin structures often induces plasmid rearrangement, in part due to the E. coli sbc proteins that recognize and cleave cruciform DNA structures (ref 8).
Abstract
Description
- This application is a continuation-in-part of PCT application PCT/US01/08435, filed Mar. 16, 2001, and claims the benefit of U.S. Provisional applications U.S. Ser. No. 60/189,739 filed Mar. 16, 2000 and U.S. Ser. No. 60/243,097 filed Oct. 24, 2000. The specifications of such applications are incorporated by reference herein.
- [0002] Work described herein was supported by National Institutes of Health Grant R01-GM62534. The United States Government may have certain rights in the invention.
- “RNA interference”, “post-transcriptional gene silencing”, “quelling”—these different names describe similar effects that result from the overexpression or misexpression of transgenes, or from the deliberate introduction of double-stranded RNA into cells (reviewed in Fire A (1999)Trends Genet 15:358-363; Sharp PA (1999) Genes Dev 13:139-141; Hunter C (1999) Curr Biol 9:R440-R442; Baulcombe DC (1999) Curr Biol 9:R599-R601; Vaucheret et al. (1998) Plant J 16:651-659). The injection of double-stranded RNA into the nematode Caenorhabditis elegans, for example, acts systemically to cause the post-transcriptional depletion of the homologous endogenous RNA (Fire et al. (1998) Nature 391: 806-811; and Montgomery et al. (1998) PNAS 95:15502-15507). RNA interference, commonly referred to as RNAi, offers a way of specifically and potently inactivating a cloned gene, and is proving a powerful tool for investigating gene function. But the phenomenon is interesting in its own right; the mechanism has been rather mysterious, but recent research—the latest reported by Smardon et al. (2000) Curr Biol 10:169-178—is beginning to shed light on the nature and evolution of the biological processes that underlie RNAi.
- RNAi was discovered when researchers attempting to use the antisense RNA approach to inactivate aC. elegans gene found that injection of sense-strand RNA was actually as effective as the antisense RNA at inhibiting gene function. Guo et al. (1995) Cell 81:611-620. Further investigation revealed that the active agent was modest amounts of double-stranded RNA that contaminate in vitro RNA preparations. Researchers quickly determined the ‘rules’ and effects of RNAi. Exon sequences are required, whereas introns and promoter sequences, while ineffective, do not appear to compromise RNAi (though there may be gene-specific exceptions to this rule). RNAi acts systemically—injection into one tissue inhibits gene function in cells throughout the animal. The results of a variety of experiments, in C. elegans and other organisms, indicate that RNAi acts to destabilize cellular RNA after RNA processing.
- The potency of RNAi inspired Timmons and Fire (1998Nature 395: 854) to do a simple experiment that produced an astonishing result. They fed to nematodes bacteria that had been engineered to express double-stranded RNA corresponding to the C. elegans unc-22 gene. Amazingly, these nematodes developed a phenotype similar to that of unc-22 mutants that was dependent on their food source. The ability to conditionally expose large numbers of nematodes to gene-specific double-stranded RNA formed the basis for a very powerful screen to select for RNAi-defective C. elegans mutants and then to identify the corresponding genes.
- Double-stranded RNAs (dsRNAs) can provoke gene silencing in numerous in vivo contexts including Drosophila,Caenorhabditis elegans, planaria, hydra, trypanosomes, fungi and plants. However, the ability to recapitulate this phenomenon in higher eukaryotes, particularly mammalian cells, has not be accomplished in the art. Nor has the prior art demonstrated that this phenomena can be observe in cultured eukaryotes cells.
- One aspect of the present invention provides a method for attenuating expression of a target gene in cultured cells, comprising introducing double stranded RNA (dsRNA) into the cells in an amount sufficient to attenuate expression of the target gene, wherein the dsRNA comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene.
- Another aspect of the present invention provides a method for attenuating expression of a target gene in a mammalian cell, comprising
- (i) activating one or both of a Dicer activity or an Argonaut activity in the cell, and
- (ii) introducing into the cell a double stranded RNA (dsRNA) in an amount sufficient to attenuate expression of the target gene, wherein the dsRNA comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene.
- In certain embodiments, the cell is suspended in culture; while in other embodiments the cell is in a whole animal, such as a non-human mammal.
- In certain preferred embodiments, the cell is engineered with (i) a recombinant gene encoding a Dicer activity, (ii) a recombinant gene encoding an Argonaut activity, or (iii) both. For instance, the recombinant gene may encode, for a example, a protein which includes an amino acid sequence at least 50 percent identical to SEQ ID No. 2 or 4; or be defined by a coding sequence hybridizes under wash conditions of 2×SSC at 22° C. to SEQ ID No. 1 or 3. In certain embodiments, the recombinant gene may encode, for a example, a protein which includes an amino acid sequence at least 50 percent identical to the Argonaut sequence shown in FIG. 24.
- In certain embodiments, rather than use a heterologous expression construct(s), an endogenous Dicer gene or Argonaut gene can be activated, e.g, by gene activation technology, expression of activated transcription factors or other signal transduction protein, which induces expression of the gene, or by treatment with an endogenous factor which upregualtes the level of expression of the protein or inhibits the degradation of the protein.
- In certain preferred embodiments, the target gene is an endogenous gene of the cell. In other embodiments, the target gene is an heterologous gene relative to the genome of the cell, such as a pathogen gene, e.g., a viral gene.
- In certain embodiments, the cell is treated with an agent that inhibits protein kinase RNA-activated (PKR) apoptosis, such as by treatment with agents which inhibit expression of PKR, cause its destruction, and/or inhibit the kinase activity of PKF.
- In certain preferred embodiments, the cell is a primate cell, such as a human cell.
- In certain preferred embodiments, the length of the dsRNA is at least 20, 21 or 22 nucleotides in length, e.g., corresponding in size to RNA products produced by Dicer-dependent cleavage. In certain embodiments, the dsRNA construct is at least 25, 50, 100, 200, 300 or 400 bases. In certain embodiments, the dsRNA construct is 400-800 bases in length.
- In certain preferred embodiments, expression of the target gene is attenuated by at least 5 fold, and more preferably at least 10, 20 or even 50 fold, e.g., relative to the untreated cell or a cell treated with a dsRNA construct which does not correspond to the target gene.
- Yet another aspect of the present invention provides a method for attenuating expression of a target gene in cultured cells, comprising introducing an expression vector having a “coding sequence” which, when transcribed, produces double stranded RNA (dsRNA) the cell in an amount sufficient to attenuate expression of the target gene, wherein the dsRNA comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene. An certain embodiments, the vector includes a single coding sequence for the dsRNA which is operably linked to (two) transcriptional regulatory sequences which cause transcription of in both directions (to form complementary transcripts of the coding sequence. In other embodiments, the vector includes two coding sequences which, respectively, give rise to the two complementary sequences which form the dsRNA when annealed. In certain embodiments, the vectors are episomal, e.g., and transfection is transient. In other embodiments, the vectors are chromosomally integrated, e.g., to produce a stably transfected cell line. Preferred vectors for forming such stable cell lines are the described in U.S. Pat. No. 6,025,192 and PCT publication WO/9812339, which are incorporated by reference herein.
- Still another aspect of the present invention provides an assay for identifying nucleic acid sequences responsible for conferring a particular phenotype in a cell, comprising
- (i) constructing a variegated library of nucleic acid sequences from a cell in an orientation relative to a promoter to produce double stranded DNA;
- (ii) introducing the variegated dsRNA library into a culture of target cells, which cells have an activated Dicer activity or Argonaut activity;
- (iii) identifying members of the library which confer a particular phenotype on the cell, and identifying the sequence from a cell which correspond, such as being identical or homologous, to the library member.
- Yet another aspect of the present invention provides a method of conducting a drug discovery business comprising:
- (i) identifying, by the subject assay, a target gene which provides a phenotypically desirable response when inhibited by RNAi;
- (ii) identifying agents by their ability to inhibit expression of the target gene or the activity of an expression product of the target gene;
- (iii) conducting therapeutic profiling of agents identified in step (b), or further analogs thereof, for efficacy and toxicity in animals; and
- (iv) formulating a pharmaceutical preparation including one or more agents identified in step (iii) as having an acceptable therapeutic profile.
- The method may include an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.
- Another aspect of the present invention provides a method of conducting a target discovery business comprising:
- (i) identifying, by the subject assay, a target gene which provides a phenotypically desirable response when inhibited by RNAi;
- (ii) (optionally) conducting therapeutic profiling of the target gene for efficacy and toxicity in animals; and
- (iii). licensing, to a third party, the rights for further drug development of inhibitors of the target gene.
- Another aspect of the invention provides a method for inhibiting RNAi by inhibiting the expression or activity of an RNAi enzyme. Thus, the subject method may include inhibiting the acitivity of Dicer and/or the 22-mer RNA.
- Still another aspect relates to the a method for altering the specificity of an RNAi by modifying the sequence of the RNA component of the RNAi enzyme.
- Another aspect of the invention relates to purified or semi-purified preparations of the RNAi enzyme or components thereof. In certain embodiments, the preparations are used for identifying compounds, especially small organic molecules, which inhibit or potentiate the RNAi activity. Small molecule inhibitors, for example, can be used to inhibit dsRNA responses in cells which are purposefully being transfected with a virus which produces double stranded RNA.
- The dsRNA construct may comprise one or more strands of polymerized ribonucleotide. It may include modifications to either the phosphate-sugar backbone or the nucleoside. The double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell. The dsRNA construct may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses of double-stranded material may yield more effective inhibition. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition. dsRNA constructs containing a nucleotide sequences identical to a portion of the target gene is preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Thus, sequence identity may optimized by alignment algorithms known in the art and calculating the percent difference between the nucleotide sequences. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript.
- Yet another aspect of the invention pertains to transgenic non-human mammals which include a transgene encoding a dsRNA construct, preferably which is stably integrated into the genome of cells in which it occurs. The animals can be derived by oocyte microinjection, for example, in which case all of the nucleated cells of the animal will include the transgene, or can be derived using embryonic stem (ES) cells which have been transfected with the transgene, in which case the animal is a chimera and only a portion of its nucleated cells will include the transgene. In certain instances, the sequence-independent dsRNA response, e.g., the PKR response, is also inhibited in those cells including the transgene.
- In still other embodiments, dsRNA itself can be introduced into an ES cell in order to effect gene silencing, and that phenotype will be carried for at least several rounds of division, e.g., into the progeny of that cell.
- FIG. 1: RNAi in S2 cells. a, Drosophila S2 cells were transfected with a plasmid that directs lacZ expression from the copia promoter in combination with dsRNAs corresponding to either human CD8 or lacZ, or with no dsRNA, as indicated. b, S2 cells were co-transfected with a plasmid that directs expression of a GFP-US9 fusion protein (12) and dsRNAs of either lacZ or cyclin E, as indicated. Upper panels show FACS profiles of the bulk population. Lower panels show FACS profiles from GFP-positive cells. c, Total RNA was extracted from cells transfected with lacZ, cyclin E, fizzy or cyclin A dsRNAs, as indicated. Northern blots were hybridized with sequences not present in the transfected dsRNAs.
- FIG. 2: RNAi in vitro. a, Transcripts corresponding to either the first 600 nucleotides of Drosophila cyclin E (E600) or the first 800 nucleotides of lacZ (Z800) were incubated in lysates derived from cells that had been transfected with either lacZ or cyclin E (cycE) dsRNAs, as indicated. Time points were 0, 10, 20, 30, 40 and 60 min for cyclin E and 0, 10, 20, 30 and 60 min for lacZ. b, Transcripts were incubated in an extract of S2 cells that had been transfected with cyclin E dsRNA (cross-hatched box, below). Transcripts corresponded to the first 800 nucleotides of lacZ or the first 600, 300, 220 or 100 nucleotides of cyclin E, as indicated. Eout is a transcript derived from the portion of the cyclin E cDNA not contained within the transfected dsRNA. E-ds is identical to the dsRNA that had been transfected into S2 cells. Time points were 0 and 30 min. c, Synthetic transcripts complementary to the complete cyclin E cDNA (Eas) or the final 600 nucleotides (Eas600) or 300 nucleotides (Eas300) were incubated in extract for 0 or 30 min.
- FIG. 3: Substrate requirements of the RISC. Extracts were prepared from cells transfected with cyclin E dsRNA. Aliquots were incubated for 30 min at 30° C. before the addition of either the cyclin E (E600) or lacZ (Z800) substrate. Individual20-μl aliquots, as indicated, were pre-incubated with 1 mM CaCl2 and 5 mM EGTA, 1 mM CaCl2, 5 mM EGTA and 60 U of micrococcal nuclease, 1 mM CaCl2 and 60 U of micrococcal nuclease or 10 U of DNase I (Promega) and 5 mM EGTA. After the 30-min pre-incubation, EGTA was added to those samples that lacked it. Yeast tRNA (1 μg) was added to all samples. Time points were at 0 and 30 min.
- FIG. 4: The RISC contains a potential guide RNA. a, Northern blots of RNA from either a crude lysate or the S100 fraction (containing the soluble nuclease activity, see Methods) were hybridized to a riboprobe derived from the sense strand of the cyclin E mRNA. b, Soluble cyclin-E-specific nuclease activity was fractionated as described in Methods. Fractions from the anion-exchange resin were incubated with the lacZ, control substrate (upper panel) or the cyclin E substrate (centre panel). Lower panel, RNA from each fraction was analysed by northern blotting with a uniformly labelled transcript derived from sense strand of the cyclin E cDNA. DNA oligonucleotides were used as size markers.
- FIG. 5: Generation of 22 mers and degradation of mRNA are carried out by distinct enzymatic complexes. A. Extracts prepared either from 0-12 hour Drosophila embryos or Drosophila S2 cells (see Methods) were incubated 0, 15, 30, or 60 minutes (left to right) with a uniformly-labeled double-stranded RNA corresponding to the first 500 nucleotides of the Drosophila cyclin E coding region. M indicates a marker prepared by in vitro transcription of a synthetic template. The template was designed to yield a 22 nucleotide transcript. The doublet most probably results from improper initiation at the +1 position. B. Whole-cell extracts were prepared from S2 cells that had been transfected with a dsRNA corresponding to the first 500 nt. of the luciferase coding region. S10 extracts were spun at 30,000×g for 20 minutes which represents our standard RISC extract6. S100 extracts were prepared by further centrifugation of S10 extracts for 60 minutes at 100,000×g. Assays for mRNA degradation were carried out as described previously6 for 0, 30 or 60 minutes (left to right in each set) with either a single-stranded luciferase mRNA or a single-stranded cyclin E mRNA, as indicated. C. S10 or S100 extracts were incubated with cyclin E dsRNAs for 0, 60 or 120 minutes (L to R).
- FIG. 6: Production of 22 mers by recombinant CG4792/Dicer. A. Drosophila S2 cells were transfected with plasmids that direct the expression of T7-epitope tagged versions of Drosha, CG4792/Dicer-1 and Homeless. Tagged proteins were purified from cell lysates by immunoprecipitation and were incubated with cyclin E dsRNA. For comparison, reactions were also performed in Drosophila embryo and S2 cell extracts. As a negative control, immunoprecipitates were prepared from cells transfected with a β-galactosidase expression vector. Pairs of lanes show reactions performed for 0 or 60 minutes. The synthetic marker (M) is as described in the legend to FIG. 1. B. Diagrammatic representations of the domain structures of CG4792/Dicer-1, Drosha and Homeless are shown. C. Immunoprecipitates were prepared from detergent lysates of S2 cells using an antiserum raised against the C-
terminal 8 amino acids of Drosophila Dicer-1 (CG4792). As controls, similar preparations were made with a pre-immune serum and with an immune serum that had been pre-incubated with an excess of antigenic peptide. Cleavage reactions in which each of these precipitates was incubated with an ˜500 nt. fragment of Drosophila cyclin E are shown. For comparsion, an incubation of the substrate in Drosophila embryo extract was electrophoresed in parallel. D. Dicer immunoprecipitates were incubated with dsRNA substrates in the presence or absence of ATP. For comparison, the same substrate was incubated with S2 extracts that either contained added ATP or that were depleted of ATP using glucose and hexokinase (see methods). E. Drosophila S2 cells were transfected with uniformly, 32P-labelled dsRNA corresponding to the first 500 nt. of GFP. RISC complex was affinity purified using a histidine-tagged version of D.m. Ago-2, a recently identified component of the RISC complex (Hammond et al., in prep). RISC was isolated either under conditions in which it remains ribosome associated (ls, low salt) or under conditions that extract it from the ribosome in a soluble form (hs, high salt)6. For comparison, the spectrum of labelled RNAs in the total lysate is shown. F. Guide RNAs produced by incubation of dsRNA with a Dicer immunoprecipitate are compared to guide RNAs present in a affinity-purified RISC complex. These precisely comigrate on a gel that has single-nucleotide resolution. The lane labelled control is an affinity selection for RISC from cell that had been transfected with labeled dsRNA but not with the epitope-tagged D.m. Ago-2. - FIG. 7: Dicer participates in RNAi. A. Drosophila S2 cells were transfected with dsRNAs corresponding to the two Drosophila Dicers (CG4792 and CG6493) or with a control dsRNA corresponding to murine caspase 9. Cytoplasmic extracts of these cells were tested for Dicer activity. Transfection with Dicer dsRNA reduced activity in lysates by 7.4-fold. B. The Dicer-1 antiserum (CG4792) was used to prepare immunoprecipitates from S2 cells that had been treated as described above. Dicer dsRNA reduced the activity of Dicer-1 in this assay by 6.2-fold. C. Cells that had been transfected two days previously with either mouse caspase 9 dsRNA or with Dicer dsRNA were cotransfected with a GFP expression plasmid and either control, luciferase dsRNA or GFP dsRNA. Three independent experiments were quantified by FACS. A comparison of the relative percentage of GFP-positive cells is shown for control (GFP plasmid plus luciferase dsRNA) or silenced (GFP plamsid plus GFP dsRNA) populations in cells that had previously been transfected with either control (caspase 9) or Dicer dsRNAs.
- FIG. 8: Dicer is an evolutionarily conserved ribonuclease. A. A model for production of 22 mers by Dicer. Based upon the proposed mechanism of action of Ribonuclease III, we propose that Dicer acts on its substrate as a dimer. The positioning of the two ribonuclease domains (RIIIa and RIIIb) within the enzyme would thus determine the size of the cleavage product. An equally plausible alternative model could be derived in which the RIIIa and RIIIb domains of each Dicer enzyme would cleave in concert at a single position. In this model, the size of the cleavage product would be determined by interaction between two neighboring Dicer enzymes. B. Comparison of the domain structures of potential Dicer homologs in various organisms (Drosophila—CG4792, CG6493,C. elegans—K12H4.8, Arabidopsis—CARPEL FACTORY24, T25K16.4,
AC012328 —1, human Helicase-MOI25 and S. pombe—YC9A_SCHPO). The ZAP domains were identified both by analysis of individual sequences with Pfam27 and by Psi-blast28 searches. The ZAP domain in the putative S. pombe Dicer is not detected by PFAM but is identified by Psi-Blast and is thus shown in a different color. For comparison, a domain structure of the RDE1/QDE2/ARGONAUTE family is shown. It should be noted that the ZAP domains are more similar within each of the Dicer and ARGONAUTE families than they are between the two groups. C. An alignment of the ZAP domains in selected Dicer and Argonaute family members is shown. The alignment was produced using ClustalW. - FIG. 9: Purification strategy for RISC. (second step in RNAi model).
- FIG. 10: Fractionation of RISC activity over sizing column. Activity fractionates as 500 KD complex. Also, antibody to
dm argonaute 2 cofractionates with activity. - FIGS.11-13: Fractionation of RISC over monoS, monoQ, Hydroxyapatite columns.
Dm argonaute 2 protein also cofactionates. - FIG. 14: Alignment of
dm argonaute 2 with other family members. - FIG. 15: Confirmation of
dm argonaute 2. S2 cells were transfected with labeled dsRNA and His tagged argonaute. Argonaute was isolated on nickel agarose and RNA component was identified on 15% acrylamide gel. - FIG. 16: S2 cell and embryo extracts were assayed for 22 mer generating activity.
- FIG. 17: RISC can be separated from 22 mer generating activity (dicer). Spinning extracts (S100) can clear RISC activity from supernatant (left panel) however, S100 spins still contain dicer activity (right panel).
- FIG. 18: Dicer is specific for dsRNA and prefers longer substrates.
- FIG. 19: Dicer was fractionated over several columns.
- FIG. 20: Identification of dicer as enzyme which can process dsRNA into 22 mers. Various RNaseIII family members were expressed with n terminal tags, immunoprecipitated, and assayed for 22 mer generating activity (left panel). In right panel, antibodies to dicer could also precipitate 22 mer generating activity.
- FIG. 21: Dicer requires ATP.
- FIG. 22: Dicer produces RNAs that are the same size as RNAs present in RISC.
- FIG. 23: Human dicer homolog when expressed and immunoprecipitated has 22 mer generating activity.
- FIG. 24: Sequence of
dm argonaute 2. Peptides identified by microsequencing are shown in underline. - FIG. 25: Molecular charaterization of
dm argonaute 2. The presence of an intron in coding sequence was determined by northern blotting using intron probe. This results in a different 5′ reading frame that that published genome seqeunce. Number of polyglutaine repeats was determined by genomic PCR. - FIG. 26: Dicer activity can be created in human cells by expression of human dicer gene. Host cell was 293. Crude extracts had dicer activity, while activity was absent from untransfected cells. Activity is not dissimilar to that seen in drosophila embryo extracts.
- FIG. 27: An ˜500 nt. fragment of the gene that is to be silenced (X) is inserted into the modified vector as a stable direct repeat using standard cloning procedures. Treatment with commercially available cre recombinase reverses sequences within the loxP sites (L) to create an inverted repeat. This can be stably maintained and amplified in an sbc mutant bacterial strain (DL759). Transcription in vivo from the promoter of choice (P) yields a hairpin RNA that causes silencing. A zeocin resistance marker is included to insure maintenance of the direct and inverted repeat structures; however this is non-essential in vivo and could be removed by pre-mRNA splicing if desired. Smith, N. A. et al. Total silencing by intron-spliced hairpin RNAs.Nature 407, 319-20 (2000).
- FIG. 28: Hela, Chinese hamster ovary, and P19 (pluripotent, mouse embryonic carcinoma) cell lines transfected with plasmids expressing Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases and with
dsRNA 500 mers (400 ng), either homologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP). Dual luciferase assays were carried out using an Analytical Scientific Instruments model 3010 Luminometer. In this assay Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity. These data demonstrate that 500 mer dsRNA can specifically suppress cognate gene expression in vivo. - FIG. 29: P19 (a pluripontent, mouse embryonic cell line) cells transfected with plasmids expressing Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases and with
dsRNA 500 mers (500ng), either homologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP). Dual luciferase assays were carried out using an Analytical Scientific Instruments model 3010 Luminometer. In this assay Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity. These data further demonstrate that 500 mer dsRNA can specifically suppress cognate gene expression in vivo and that the effect is stable over time . - FIG. 30: S10 fractions from P19 cell lysates were used for in vitro translations of mRNA coding for Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases. Translation reactions were programmed with various amounts of
dsRNA 500 mers, either homologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP). Reactions were carried out at 30 degrees for 1 hour, after which dual luciferase assays were carried out using an Analytical Scientific Instruments model 3010 Luminometer. In this assay Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity. These data demonstrate that 500 mer dsRNA can specifically suppress cognate gene expression in vitro in a manner consistent with post-transcriptional gene silencing. Anti-sense firefly RNA did not differ significantly from dsGFP control (approximately 10%) (data not shown). - FIG. 31: S10 fractions from P19 cell lysates were used for in vitro translations of mRNA coding for Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases. Translation reactions were programmed with dsRNA or
asRNA 500 mers, either complementary to firefly luciferase mRNA (asLUC and dsLUC) or non-complementary (dsGFP). Reactions were carried out at 30 degrees for 1 hour, after a 30 min preincubation with dsRNA or asRNA. Dual luciferase assays were carried out using an Analytical Scientific Instruments model 3010 Luminometer. In this assay Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity. These data demonstrate that 500 mer double-stranded RNA (dsRNA) but not anti-sense RNA (asRNA) suppresses cognate gene expression in vitro in a manner consistent with post-transcriptional gene silencing. - FIG. 32: P19 cells were grown in 6-well tissue culture plates to approximately 60% confluence. Various amounts of dsRNA, either homologous to firefly luciferase mRNA (dsLUC) or non-homologous (dsGFP), were added to each well and incubated for 12 hrs under normal tissue culture conditions. Cells were then transfected with plasmids expressing Photinus pyralis (firefly) Renilla reniformis (sea pansy) luciferases and with
dsRNA 500 mers (500 ng). Dual luciferase assays were carried out 12 hrs post-transfection using an Analytical Scientific Instruments model 3010 Luminometer. In this assay Renilla luciferase serves as an internal control for dsRNA-specific suppression of firefly luciferase activity. These data show that 500 mer dsRNA can specifically suppress cognate gene expression in vivo without transfection under normal tissue culture conditions. - FIG. 33: Is a graph illustrating the relative rate of expression luciferase in cells which are treated with various antisense and dsRNA constructs.
- The present invention provides methods for attenuating gene expression in a cell using gene-targeted double stranded RNA (dsRNA). The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the “target” gene).
- A significant aspect to certain embodiments of the present invention relates to the demonstration in the present application that RNAi can in fact be accomplished in cultured cells, rather than whole organisms as described in the art.
- Another salient feature of the present invention concerns the ability to carry out RNAi in higher eukaryotes, particularly in non-oocytic cells of mammals, e.g., cells from adult mammals as an example.
- As described in further detail below, the present invention(s) are based on the discovery that the RNAi phenomenum is mediated by a set of enzyme activities, including an essential RNA component, that are evolutionarily conserved in eukaryotes ranging from plants to mammals.
- One enzyme contains an essential RNA component. After partial purification, a multi-component nuclease (herein “RISC nuclease”) co-fractionates with a discrete, 22-nucleotide RNA species which may confer specificity to the nuclease through homology to the substrate mRNAs. The short RNA molecules are generated by a processing reaction from the longer input dsRNA. Without wishing to be bound by any particular theory, these 22 mer guide RNAs may serve as guide sequences that instruct the RISC nuclease to destroy specific mRNAs corresponding to the dsRNA sequences.
- As illustrated in FIG. 33, double stranded forms of the 22-mer guide RNA can be sufficient in length to induce sequence-dependent dsRNA inhibition of gene expression. In the illustrated example, dsRNA contructs are administered to cells having a recombinant luciferase reporter gene. The control cell, e.g., no exogeneously added RNA, the level of expression of the luciferase reporter is normalized to be the value of “1”. As illustrated, both long (500-mer) and short (22-mer) dsRNA constructs complementary to the luciferase gene could inhibit expression of that gene product relative to the control cell. On the other hand, similarly sized dsRNA complementary to the coding sequence for another protein, green fluorescence protein (GFP), did not significantly effect the expression of luciferase—indicating that the inhibitory phenomena was in each case sequence-dependent. Likewise, single stranded 22-mers of luciferase did not inhibit expression of that gene—indicating that the inhibitory phenomena is double stranded-dependent.
- The appended examples also identify an enzyme, Dicer, that can produce the putative guide RNAs. Dicer is a member of the RNAse III family of nucleases that specifically cleave dsRNA and is evolutionarily conserved in worms, flies, plants, fungi and, as described herein, mammals. The enzyme has a distinctive structure which includes a helicase domain and dual RNAse III motifs. Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family, which have been genetically linked to RNAi in lower eukaryotes. Indeed, activation of, or overexpression of Dicer may be sufficient in many cases to permit RNA interference in otherwise non-receptive cells, such as cultured eukaryotic cells, or mammalian (non-oocytic) cells in culture or in whole organisms.
- In certain embodiments, the cells can be treated with an agent(s) that inhibits the general double-stranded RNA response(s) by the host cells, such as may give rise to sequence-independent apoptosis. For instance, the cells can be treated with agents that inhibit the dsRNA-dependent protein kinase known as PKR (protein kinase RNA-activated). Double stranded RNAs in mammalian cells typically activate protein kinase PKR and leads to apoptosis. The mechanism of action of PKR includes phosphorylation and inactivation eIF2a (Fire (1999)Trends Genet 15:358). It has also been reported that induction of NF-κB by PKR is involved in apoptosis commitment and this process is mediated through activation of the IKK complex. This sequence-independent response may reflect a form of primitive immune response, since the presence of dsRNA is a common feature of many viral lifecycles.
- As described herein, Applicants have demonstrated that the PKR response can be overcome in favor of the sequence-specific RNAi response. However, in certain instances, it can be desirable to treat the cells with agents which inhibit expression of PKR, cause its destruction, and/or inhibit the kinase activity of PKF are specifically contemplated for use in the present method. Likewise, overexpression of or agents which ectopic activate IF2αa. can be used. Other agents which can be used to suppress the PKR response include inhibitors of IKK phosphorylation of IκB, inhibitors of IκB ubiquitination, inhibitors of IκB degradation, inhibitors of NF-κB nuclear translocation, and inhibitors of NF-κB interaction with κB response elements.
- Other inhibitors of sequence-independent dsRNA response in cells include the gene product of the vaccinia virus E3L. The E3L gene product contains two distinct domains. A conserved carboxy-terminal domain has been shown to bind double-stranded RNA (dsRNA) and inhibit the antiviral dsRNA response by cells. Expression of at least that portion of the E3L gene in the host cell, or the use of polypeptide or peptidomimetics thereof, can be used to suppress the general dsRNA response. Caspase inhibitors sensitized cells to killing by double-stranded RNA. Accordingly, ectopic expression or activated of caspases in the host cell can be used to suppress the general dsRNA response.
- In other embodiments, the subject method is carried out in cells which have little or no general response to double stranded RNA, e.g., have no PKR-dependent dsRNA response, at least under the culture conditions. As illustrated in FIGS.28-32, CHO and P19 cells can be used without having to inhibit PKR or other general dsRNA responses.
- Thus, the present invention provides a process and compositions for inhibiting expression of a target gene in a cell, expecially a mammalian cell. In certain embodiments, the process comprises introduction of RNA (the “dsRNA construct”) with partial or fully double-stranded character into the cell or into the extracellular environment. Inhibition is specific in that a nucleotide sequence from a portion of the target gene is chosen to produce the dsRNA construct. In preferred embodiments, the method utilizes a cell in which Dicer and/or Argonaute activities are recombinantly expressed or otherwise ectopically activated. This process can be (1) effective in attenuating gene expression, (2) specific to the targeted gene, and (3) general in allowing inhibition of many different types of target gene.
- For convenience, certain terms employed in the specification, examples, and appended claims are collected here.
- As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to that it has been linked. One type of vector is a genomic integrated vector, or “integrated vector”, which can become integrated into the chromsomal DNA of the host cell. Another type of vector is an episomal vector, i.e., a nucleic acid capable of extra-chromosomal replication. Vectors capable of directing the expression of genes to that they are operatively linked are referred to herein as “expression vectors”. In the present specification, “plasmid” and “vector” are used interchangeably unless otherwise clear from the context.
- As used herein, the term “nucleic acid” refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.
- As used herein, the term “gene” or “recombinant gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide of the present invention, including both exon and (optionally) intron sequences. A “recombinant gene” refers to nucleic acid encoding such regulatory polypeptides, that may optionally include intron sequences that are derived from chromosomal DNA. The term “intron” refers to a DNA sequence present in a given gene that is not translated into protein and is generally found between exons. As used herein, the term “transfection” means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
- A “protein coding sequence” or a sequence that “encodes” a particular polypeptide or peptide, is a nucleic acid sequence that is transcribed (in the case of DNA) and is translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. 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′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from procaryotic or eukaryotic mRNA, genomic DNA sequences from procaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence will usually be located 3′ to the coding sequence.
- Likewise, “encodes”, unless evident from its context, will be meant to include DNA sequences that encode a polypeptide, as the term is typically used, as well as DNA sequences that are transcribed into inhibitory antisense molecules.
- The term “loss-of-function”, as it refers to genes inhibited by the subject RNAi method, refers a diminishment in the level of expression of a gene when compared to the level in the absense of dsRNA constructs.
- The term “expression” with respect to a gene sequence refers to transcription of the gene and, as appropriate, translation of the resulting mRNA transcript to a protein. Thus, as will be clear from the context, expression of a protein coding sequence results from transcription and translation of the coding sequence.
- “Cells,” “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
- The term “cultured cells” refers to cells suspended in culture, e.g., dispersed in culture or in the form tissue. It does not, however, include oocytes or whole embryos (including blastocysts and the like) which may be provided in culture. In certain embodiments, the cultured cells are adults cells, e.g., non-embryonic.
- By “recombinant virus” is meant a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the particle.
- As used herein, the terms “transduction” and “transfection” are art recognized and mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer. “Transformation”, as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a dsRNA contruct.
- “Transient transfection” refers to cases where exogenous DNA does not integrate into the genome of a transfected cell, e.g., where episomal DNA is transcribed into mRNA and translated into protein.
- A cell has been “stably transfected” with a nucleic acid construct when the nucleic acid construct is capable of being inherited by daughter cells.
- As used herein, a “reporter gene construct” is a nucleic acid that includes a “reporter gene” operatively linked to at least one transcriptional regulatory sequence. Transcription of the reporter gene is controlled by these sequences to which they are linked. The activity of at least one or more of these control sequences can be directly or indirectly regulated by the target receptor protein. Exemplary transcriptional control sequences are promoter sequences. A reporter gene is meant to include a promoter-reporter gene construct that is heterologously expressed in a cell.
- As used herein, “transformed cells” refers to cells that have spontaneously converted to a state of unrestrained growth, i.e., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control. For purposes of this invention, the terms “transformed phenotype of malignant mammalian cells” and “transformed phenotype ” are intended to encompass, but not be limited to, any of the following phenotypic traits associated with cellular transformation of mammalian cells: immortalization, morphological or growth transformation, and tumorigenicity, as detected by prolonged growth in cell culture, growth in semi-solid media, or tumorigenic growth in immuno-incompetent or syngeneic animals.
- As used herein, “proliferating” and “proliferation” refer to cells undergoing mitosis.
- As used herein, “immortalized cells” refers to cells that have been altered via chemical, genetic, and/or recombinant means such that the cells have the ability to grow through an indefinite number of divisions in culture.
- The “growth state” of a cell refers to the rate of proliferation of the cell and the state of differentiation of the cell.
- One aspect of the invention provides a method for potentiating RNAi by induction or ectopic activation of an RNAi enzyme in a cell (in vivo or in vitro) or cell-free mixtures. In preferred embodiments, the RNAi activity is activated or added to a mammalian cell, e.g., a human cell, which cell may be provided in vitro or as part of a whole organism. In other embodiments, the subject method is carried out using eukaryotic cells generally (except for oocytes) in culture. For instance, the Dicer enzyme may be activated by virtue of being recombinantly expressed or it may be activated by use of an agent which (i) induces expression of the endogenous gene, (ii) stabilizes the protein from degradation, and/or (iii) allosterically modies the enzyme to increase its activity (by altering its Kcat, Km or both).
- A. Dicer and Argonaut Activities
- In certain embodiment, at least one of the activated RNAi enzymes is Dicer, or a homolog thereof. In certain preferred embodiments, the present method provides for ectopic activation of Dicer. As used herein, the term “Dicer” refers to a protein which (a) mediates an RNAi response and (b) has an amino acid sequence at least 50 percent identical, and more preferablty at least 75, 85, 90 or 95 percent identical to SEQ ID No. 2 or 4, and/or which can be encoded by a nucleic acid which hybridizes under wash conditions of 2×SSC at 22° C., and more preferably 0.2×SSC at 65° C., to a nucleotide represented by SEQ ID No. 1 or 3. Accordingly, the method may comprise introducing a dsRNA contruct into a cell in which Dicer has been recombinantly expressed or otherwise ectopically activated.
- In certain embodiment, at least one of the activated RNAi enzymes is Argonaut, or a homolog thereof. In certain preferred embodiments, the present method provides for ectopic activation of Argonaut. As used herein, the term “Argonaut” refers to a protein which (a) mediates an RNAi response and (b) has an amino acid sequence at least 50 percent identical, and more preferablty at least 75, 85, 90 or 95 percent identical to the amino acid sequence shown in FIG. 24. Accordingly, the method may comprise introducing a dsRNA contruct into a cell in which Argonaut has been recombinantly expressed or otherwise ectopically activated.
- This invention also provides expression vectors containing a nucleic acid encoding a Dicer or Argonaut polypeptides, operably linked to at least one transcriptional regulatory sequence. Operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are art-recognized and are selected to direct expression of the subject Dicer or Argonaut proteins. Accordingly, the term transcriptional regulatory sequence includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). For instance, any of a wide variety of expression control sequences, sequences that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding Dicer or Argonaut polypeptides of this invention. Such useful expression control sequences, include, for example, a viral LTR, such as the LTR of the Moloney murine leukemia virus, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage λ, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein desired to be expressed.
- Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
- The recombinant Dicer or Argonaut genes can be produced by ligating nucleic acid encoding a Dicer or Argonaut polypeptide into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both. Expression vectors for production of recombinant forms of the subject Dicer or Argonaut polypeptides include plasmids and other vectors. For instance, suitable vectors for the expression of a Dicer or Argonaut polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such asE. coli.
- A number of vectors exist for the expression of recombinant proteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs intoS. cerevisiae (see, for example, Broach et al. (1983) in Experimental Manipulation of Gene Expression, ed. M. Inouye Academic Press, p. 83, incorporated by reference herein). These vectors can replicate in E. coli due the presence of the pBR322 ori, and in S. cerevisiae due to the replication determinant of the
yeast 2 micron plasmid. In addition, drug resistance markers such as ampicillin can be used. In an illustrative embodiment, a Dicer or Argonaut polypeptide is produced recombinantly utilizing an expression vector generated by sub-cloning the coding sequence of a Dicer or Argonaut gene. - The preferred mammalian expression vectors contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Some of these vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells. The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17.
- In yet another embodiment, the subject invention provides a “gene activation” construct which, by homologous recombination with a genomic DNA, alters the transcriptional regulatory sequences of an endogenous Dicer or Argonaut gene. For instance, the gene activation construct can replace the endogenous promoter of a Dicer or Argonaut gene with a heterologous promoter, e.g., one which causes constitutive expression of the Dicer or Argonaut gene or which causes inducible expression of the gene under conditions different from the normal expression pattern of Dicer or Argonaut. A variety of different formats for the gene activation constructs are available. See, for example, the Transkaryotic Therapies, Inc PCT publications WO93/09222, WO95/31560, WO96/29411, WO95/31560 and WO94/12650.
- In preferred embodiments, the nucleotide sequence used as the gene activation construct can be comprised of (1) DNA from some portion of the endogenous Dicer or Argonaut gene (exon sequence, intron sequence, promoter sequences, etc.) which direct recombination and (2) heterologous transcriptional regulatory sequence(s) which is to be operably linked to the coding sequence for the genomic Dicer or Argonaut gene upon recombination of the gene activation construct. For use in generating cultures of Dicer or Argonaut producing cells, the construct may further include a reporter gene to detect the presence of the knockout construct in the cell.
- The gene activation construct is inserted into a cell, and integrates with the genomic DNA of the cell in such a position so as to provide the heterologous regulatory sequences in operative association with the native Dicer or Argonaut gene. Such insertion occurs by homologous recombination, i.e., recombination regions of the activation construct that are homologous to the endogenous Dicer or Argonaut gene sequence hybridize to the genomic DNA and recombine with the genomic sequences so that the construct is incorporated into the corresponding position of the genomic DNA.
- The terms “recombination region” or “targeting sequence” refer to a segment (i.e., a portion) of a gene activation construct having a sequence that is substantially identical to or substantially complementary to a genomic gene sequence, e.g., including 5′ flanking sequences of the genomic gene, and can facilitate homologous recombination between the genomic sequence and the targeting transgene construct.
- As used herein, the term “replacement region” refers to a portion of a activation construct which becomes integrated into an endogenous chromosomal location following homologous recombination between a recombination region and a genomic sequence.
- The heterologous regulatory sequences, e.g., which are provided in the replacement region, can include one or more of a variety elements, including: promoters (such as constitutive or inducible promoters), enhancers, negative regulatory elements, locus control regions, transcription factor binding sites, or combinations thereof.
- Promoters/enhancers which may be used to control expression of the targeted gene in vivo include, but are not limited to, the cytomegalovirus (CMV) promoter/enhancer (Karasuyama et al., 1989,J. Exp. Med., 169:13), the human β-actin promoter (Gunning et al. (1987) PNAS 84:4831-4835), the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTV LTR) (Klessig et al. (1984) Mol. Cell Biol. 4:1354-1362), the long terminal repeat sequences of Moloney murine leukemia virus (MuLV LTR) (Weiss et al. (1985) RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), the SV40 early or late region promoter (Bernoist et al. (1981) Nature 290:304-310; Templeton et al. (1984) Mol. Cell Biol., 4:817; and Sprague et al. (1983) J. Virol., 45:773), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (RSV) (Yamamoto et al., 1980, Cell, 22:787-797), the herpes simplex virus (HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS 82:3567-71), and the herpes simplex virus LAT promoter (Wolfe et al. (1992) Nature Genetics, 1:379-384).
- In still other embodiments, the replacement region merely deletes a negative transcriptional control element of the native gene, e.g., to activate expression, or ablates a positive control element, e.g., to inhibit expression of the targeted gene.
- B. Cell/Organism
- The cell with the target gene may be derived from or contained in any organism (e.g., plant, animal, protozoan, virus, bacterium, or fungus). The dsRNA construct may be synthesized either in vivo or in vitro. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. For generating double stranded transcripts from a transgene in vivo, a regulatory region may be used to transcribe the RNA strand (or strands).
- Furthermore, genetic manipulation becomes possible in organisms that are not classical genetic models. Breeding and screening programs may be accelerated by the ability to rapidly assay the consequences of a specific, targeted gene disruption. Gene disruptions may be used to discover the function of the target gene, to produce disease models in which the target gene are involved in causing or preventing a pathological condition, and to produce organisms with improved economic properties.
- The cell with the target gene may be derived from or contained in any organism. The organism may a plant, animal, protozoan, bacterium, virus, or fungus. The plant may be a monocot, dicot or gymnosperm; the animal may be a vertebrate or invertebrate. Preferred microbes are those used in agriculture or by industry, and those that are pathogenic for plants or animals. Fungi include organisms in both the mold and yeast morphologies.
- Plants include arabidopsis; field crops (e.g., alfalfa, barley, bean, com, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops (e.g., asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish, spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g., almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, faJoa, filbert, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut, and watermelon); and ornamentals (e.g., alder, ash, aspen, azalea, birch, boxwood, camellia, carnation, chrysanthemum, elm, fir, ivy, jasmine, juniper, oak, palm, poplar, pine, redwood, rhododendron, rose, and rubber).
- Examples of vertebrate animals include fish, mammal, cattle, goat, pig, sheep, rodent, hamster, mouse, rat, primate, and human.
- Invertebrate animals include nematodes, other worms, drosophila, and other insects. Representative generae of nematodes include those that infect animals (e.g., Ancylostoma, Ascaridia, Ascaris, Bunostomum, Caenorhabditis, Capillaria, Chabertia, Cooperia, Dictyocaulus, Haernonchus, Heterakis, Nematodirus, Oesophagostomum, Ostertagia, Oxyuris, Parascaris, Strongylus, Toxascaris, Trichuris, Trichostrongylus, Tflichonema, Toxocara, Uncinaria) and those that infect plants (e.g.,B ursaphalenchus, Criconerriella, Diiylenchus, Ditylenchus, Globodera, Helicotylenchus, Heterodera, Longidorus, Melodoigyne, Nacobbus, Paratylenchus, Pratylenchus, Radopholus, Rotelynchus, Tylenchus, and Xiphinerna). Representative orders of insects include Coleoptera, Diptera, Lepidoptera, and Homoptera.
- The cell having the target gene may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands.
- C. Targeted Genes
- The target gene may be a gene derived from the cell, an endogenous gene, a transgene, or a gene of a pathogen which is present in the cell after infection thereof. Depending on the particular target gene and the dose of double stranded RNA material delivered, the procedure may provide partial or complete loss of function for the target gene. Lower doses of injected material and longer times after administration of dsRNA may result in inhibition in a smaller fraction of cells. Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein.
- “Inhibition of gene expression” refers to the absence (or observable decrease) in the level of protein and/or mRNA product from a target gene. “Specificity” refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioImmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). For RNA-mediated inhibition in a cell line or whole organism, gene expression is conveniently assayed by use of a reporter or drug resistance gene whose protein product is easily assayed. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.
- Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present invention. Lower doses of injected material and longer times after administration of dsRNA may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%,90%, or 95% of targeted cells). Quantitation of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein. As an example, the efficiency of inhibition may be determined by assessing the amount of gene product in the cell: mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.
- As disclosed herein, the present invention may is not limited to any type of target gene or nucleotide sequence. But the following classes of possible target genes are listed for illustrative purposes: developmental genes (e.g., adhesion molecules, cyclin kinase inhibitors, Writ family members, Pax family members, Winged helix family members, Hox family members, cytokines/lymphokines and their receptors, growth/differentiation factors and their receptors, neurotransmitters and their receptors); oncogenes (e.g., ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM 1, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressor genes (e.g., APC, BRCA 1, BRCA2, MADH4, MCC, NF 1, NF2, RB 1, TP53, and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases, cellulases, chalcone synthases, chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases, glucanases, glucose oxidases, granule-bound starch synthases, GTPases, helicases, hemicellulases, integrases, inulinases, invertases, isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes, nopaline synthases, octopine synthases, pectinesterases, peroxidases, phosphatases, phospholipases, phosphorylases, phytases, plant growth regulator synthases, polygalacturonases, proteinases and peptidases, pullanases, recombinases, reverse transcriptases, RUBISCOs, topoisomerases, and xylanases).
- D. dsRNA constructs
- The dsRNA construct may comprise one or more strands of polymerized ribonucleotide. It may include modifications to either the phosphate-sugar backbone or the nucleoside. For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. Modifications in RNA structure may be tailored to allow specific genetic inhibition while avoiding a general panic response in some organisms which is generated by dsRNA. Likewise, bases may be modified to block the activity of adenosine deaminase. The dsRNA construct may be produced enzymatically or by partial/total organic synthesis, any modified ribonucleotide can be introduced by in vitro enzymatic or organic synthesis.
- The dsRNA construct may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing an organism in a solution containing RNA. Methods for oral introduction include direct mixing of RNA with food of the organism, as well as engineered approaches in which a species that is used as food is engineered to express an RNA, then fed to the organism to be affected. Physical methods of introducing nucleic, acids include injection directly into the cell or extracellular injection into the organism of an RNA solution.
- The double-stranded structure may be formed by a single self-complementary RNA strand or two complementary RNA strands. RNA duplex formation may be initiated either inside or outside the cell. The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of double-stranded material may yield more effective inhibition; lower doses may also be useful for specific applications. Inhibition is sequence-specific in that nucleotide sequences corresponding to the duplex region of the RNA are targeted for genetic inhibition.
- dsRNA constructs containing a nucleotide sequences identical to a portion of the target gene are preferred for inhibition. RNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Thus, sequence identity may optimized by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein) and calculating the percent difference between the nucleotide sequences by, for example, the Smith-Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g., University of Wisconsin Genetic Computing Group). Greater than 90% sequence identity, or even 100% sequence identity, between the inhibitory RNA and the portion of the target gene is preferred. Alternatively, the duplex region of the RNA may be defined functionally as a nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing). In certain preferred embodiments, the length of the dsRNA is at least 20, 21 or 22 nucleotides in length, e.g., corresponding in size to RNA products produced by Dicer-dependent cleavage. In certain embodiments, the dsRNA construct is at least 25, 50, 100, 200, 300 or 400 bases. In certain embodiments, the dsRNA construct is 400-800 bases in length.
- 100% sequence identity between the RNA and the target gene is not required to practice the present invention. Thus the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
- The dsRNA construct may be synthesized either in vivo or in vitro. Endogenous RNA polymerase of the cell may mediate transcription in vivo, or cloned RNA polymerase can be used for transcription in vivo or in vitro. For transcription from a transgene in vivo or an expression construct, a regulatory region (e.g., promoter, enhancer, silencer, splice donor and acceptor, polyadenylation) may be used to transcribe the dsRNA strand (or strands). Inhibition may be targeted by specific transcription in an organ, tissue, or cell type; stimulation of an environmental condition (e.g., infection, stress, temperature, chemical inducers); and/or engineering transcription at a developmental stage or age. The RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus. The dsRNA construct may be chemically or enzymatically synthesized by manual or automated reactions. The dsRNA construct may be synthesized by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6). The use and production of an expression construct are known in the art 32,33,34 (see also WO 97/32016; U.S. Pat. Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693; and the references cited therein). If synthesized chemically or by in vitro enzymatic synthesis, the RNA may be purified prior to introduction into the cell. For example, RNA can be punified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography or a combination thereof. Alternatively, the dsRNA construct may be used with no or a minimum of purification to avoid losses due to sample processing. The dsRNA construct may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to promote annealing, and/or stabilization of the duplex strands.
- Physical methods of introducing nucleic acids include injection of a solution containing the dsRNA construct, bombardment by particles covered by the dsRNA construct, soaking the cell or organism in a solution of the RNA, or electroporation of cell membranes in the presence of the dsRNA construct. A viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of dsRNA construct encoded by the expression construct. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemicalmediated transport, such as calcium phosphate, and the like. Thus the dsRNA construct may be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or other-wise increase inhibition of the target gene.
- E. Illustrative Uses
- One utility of the present invention is as a method of identifying gene function in an organism, especially higher eukaryotes comprising the use of double-stranded RNA to inhibit the activity of a target gene of previously unknown function. Instead of the time consuming and laborious isolation of mutants by traditional genetic screening, functional genomics would envision determining the function of uncharacterized genes by employing the invention to reduce the amount and/or alter the timing of target gene activity. The invention could be used in determining potential targets for pharmaceutics, understanding normal and pathological events associated with development, determining signaling pathways responsible for postnatal development/aging, and the like. The increasing speed of acquiring nucleotide sequence information from genomic and expressed gene sources, including total sequences for mammalian genomes, can be coupled with the invention to determine gene function in a cell or in a whole organism. The preference of different organisms to use particular codons, searching sequence databases for related gene products, correlating the linkage map of genetic traits with the physical map from which the nucleotide sequences are derived, and artificial intelligence methods may be used to define putative open reading frames from the nucleotide sequences acquired in such sequencing projects.
- A simple assay would be to inhibit gene expression according to the partial sequence available from an expressed sequence tag (EST). Functional alterations in growth, development, metabolism, disease resistance, or other biological processes would be indicative of the normal role of the EST's gene product.
- The ease with which the dsRNA construct can be introduced into an intact cell/organism containing the target gene allows the present invention to be used in high throughput screening (HTS). For example, duplex RNA can be produced by an amplification reaction using primers flanking the inserts of any gene library derived from the target cell or organism. Inserts may be derived from genomic DNA or mRNA (e.g., cDNA and cRNA). Individual clones from the library can be replicated and then isolated in separate reactions, but preferably the library is maintained in individual reaction vessels (e.g., a 96 well microtiter plate) to minimize the number of steps required to practice the invention and to allow automation of the process.
- In an exemplary embodiment, the subject invention provides an arrayed library of RNAi constructs. The array may in the form of solutions, such as multi-well plates, or may be “printed” on solid substrates upon which cells can be grown. To illustrate, solutions containing duplex RNAs that are capable of inhibiting the different expressed genes can be placed into individual wells positioned on a microtiter plate as an ordered array, and intact cells/organisms in each well can be assayed for any changes or modifications in behavior or development due to inhibition of target gene activity.
- In one embodiment, the subject method uses an arrayed library of RNAi constructs to screen for combinations of RNAi that is lethal to host cells. Synthetic lethality is a bedrock principle of experimental genetics. A synthetic lethality describes the properties of two mutations which, individually, are tolerated by the organism but which, in combination, are lethal. The subject arrays can be used to identify loss-of-function mutations that are lethal in combination with alterations in other genes, such as activated oncogenes or loss-of-function mutations to tumor suppressors. To achieve this, one can create “phenotype arrays” using cultured cells. Expression of each of a set of genes, such as the host cell's genome, can be individually systematically disrupted using RNA interference. Combination with alterations in oncogene and tumor suppressor pathways can be used to identify synthetic lethal interactions that may identify novel therapeutic targets.
- In certain embodiments, the RNAi constructs can be fed directly to, injected into, the cell/organism containing the target gene. Alternatively, the duplex RNA can be produced by in vivo or in vitro transcription from an expression construct used to produce the library. The construct can be replicated as individual clones of the library and transcribed to produce the RNA; each clone can then be fed to, or injected into, the cell/organism containing the target gene. The function of the target gene can be assayed from the effects it has on the cell/organism when gene activity is inhibited. This screening could be amenable to small subjects that can be processed in large number, for example, tissue culture cells derived from mammals, especially primates, and most preferably humans.
- If a characteristic of an organism is determined to be genetically linked to a polymorphism through RFLP or QTL analysis, the present invention can be used to gain insight regarding whether that genetic polymorphism might be directly responsible for the characteristic. For example, a fragment defining the genetic polymorphism or sequences in the vicinity of such a genetic polymorphism can be amplified to produce an RNA, the duplex RNA can be introduced to the organism or cell, and whether an alteration in the charactenstic is correlated with inhibition can be determined. Of course, there may be trivial explanations for negative results with this type of assay, for example: inhibition of the target gene causes lethality, inhibition of the target gene may not result in any observable alteration, the fragment contains nucleotide sequences that are not capable of inhibiting the target gene, or the target gene's activity is redundant.
- The present invention may be useful in allowing the inhibition of essential genes. Such genes may be required for cell or organism viability at only particular stages of development or cellular compartments. The functional equivalent of conditional mutations may be produced by inhibiting activity of the target gene when or where it is not required for viability. The invention allows addition of RNA at specific times of development and locations in the organism without introducing permanent mutations into the target genome.
- If alternative splicing produced a family of transcripts that were distinguished by usage of characteristic exons, the present invention can target inhibition through the appropriate exons to specifically inhibit or to distinguish among the functions of family members. For example, a hormone that contained an alternatively spliced transmembrane domain may be expressed in both membrane bound and secreted forms. Instead of isolating a nonsense mutation that terminates translation before the transmembrane domain, the functional consequences of having only secreted hormone can be determined according to the invention by targeting the exon containing the transmembrane domain and thereby inhibiting expression of membrane-bound hormone.
- The present invention may be used alone or as a component of a kit having at least one of the reagents necessary to carry out the in vitro or in vivo introduction of RNA to test samples or subjects. Preferred components are the dsRNA and a vehicle that promotes introduction of the dsRNA. Such a kit may also include instructions to allow a user of the kit to practice the invention.
- Alternatively, an organism may be engineered to produce dsRNA which produces commercially or medically beneficial results, for example, resistance to a pathogen or its pathogenic effects, improved growth, or novel developmental patterns.
- The invention, now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.
- In a diverse group of organisms that includesCaenorhabditis elegans, Drosophila, planaria, hydra, trypanosomes, fungi and plants, the introduction of double-stranded RNAs inhibits gene expression in a sequence-specific manner1-7. These responses, called RNA interference or post-transcriptional gene silencing, may provide anti-viral defence, modulate transposition or regulate gene expression1, 6, 8-10. We have taken a biochemical approach towards elucidating the mechanisms underlying this genetic phenomenon. Here we show that ‘loss-of-function’ phenotypes can be created in cultured Drosophila cells by transfection with specific double-stranded RNAs. This coincides with a marked reduction in the level of cognate cellular messenger RNAs. Extracts of transfected cells contain a nuclease activity that specifically degrades exogenous transcripts homologous to transfected double-stranded RNA. This enzyme contains an essential RNA component. After partial purification, the sequence-specific nuclease co-fractionates with a discrete, ˜25-nucleotide RNA species which may confer specificity to the enzyme through homology to the substrate mRNAs.
- Although double-stranded RNAs (dsRNAs) can provoke gene silencing in numerous biological contexts including Drosophila11, 12, the mechanisms underlying this phenomenon have remained mostly unknown. We therefore wanted to establish a biochemically tractable model in which such mechanisms could be investigated.
- Transient transfection of cultured, Drosophila S2 cells with a lacZ expression vector resulted in β-galactosidase activity that was easily detectable by an in situ assay (FIG. 1a). This activity was greatly reduced by co-transfection with a dsRNA corresponding to the first 300 nucleotides of the lacZ sequence, whereas co-transfection with a control dsRNA (CD8) (FIG. 1a) or with single-stranded RNAs of either sense or antisense orientation (data not shown) had little or no effect. This indicated that dsRNAs could interfere, in a sequence-specific fashion, with gene expression in cultured cells.
- To determine whether RNA interference (RNAi) could be used to target endogenous genes, we transfected S2 cells with a dsRNA corresponding to the first 540 nucleotides of Drosophila cyclin E, a gene that is essential for progression into S phase of the cell cycle. During log-phase growth, untreated S2 cells reside primarily in G2/M (FIG. 1b). Transfection with lacZ dsRNA had no effect on cell-cycle distribution, but transfection with the cyclin E dsRNA caused a G1-phase cell-cycle arrest (FIG. 1b). The ability of cyclin E dsRNA to provoke this response was length-dependent. Double-stranded RNAs of 540 and 400 nucleotides were quite effective, whereas dsRNAs of 200 and 300 nucleotides were less potent. Double-stranded cyclin E RNAs of 50 or 100 nucleotides were inert in our assay, and transfection with a single-stranded, antisense cyclin E RNA had virtually no effect.
- One hallmark of RNAi is a reduction in the level of mRNAs that are homologous to the dsRNA. Cells transfected with the cyclin E dsRNA (bulk population) showed diminished endogenous cyclin E mRNA as compared with control cells (FIG. 1c). Similarly, transfection of cells with dsRNAs homologous to fizzy, a component of the anaphase-promoting complex (APC) or cyclin A, a cyclin that acts in S, G2 and M, also caused reduction of their cognate mRNAs (FIG. 1c). The modest reduction in fizzy mRNA levels in cells transfected with cyclin A dsRNA probably resulted from arrest at a point in the division cycle at which fizzy transcription is low14, 15. These results indicate that RNAi may be a generally applicable method for probing gene function in cultured Drosophila cells.
- The decrease in mRNA levels observed upon transfection of specific dsRNAs into Drosophila cells could be explained by effects at transcriptional or post-transcriptional levels. Data from other systems have indicated that some elements of the dsRNA response may affect mRNA directly (reviewed in
refs 1 and 6). We therefore sought to develop a cell-free assay that reflected, at least in part, RNAi. - S2 cells were transfected with dsRNAs corresponding to either cyclin E or lacZ. Cellular extracts were incubated with synthetic mRNAs of lacZ or cyclin E. Extracts prepared from cells transfected with the 540-nucleotide cyclin E dsRNA efficiently degraded the cyclin E transcript; however, the lacZ transcript was stable in these lysates (FIG. 2a). Conversely, lysates from cells transfected with the lacZ dsRNA degraded the lacZ transcript but left the cyclin E mRNA intact. These results indicate that RNAi ablates target mRNAs through the generation of a sequence-specific nuclease activity. We have termed this enzyme RISC (RNA-induced silencing complex). Although we occasionally observed possible intermediates in the degradation process (see FIG. 2), the absence of stable cleavage end-products indicates an exonuclease (perhaps coupled to an endonuclease). However, it is possible that the RNAi nuclease makes an initial endonucleolytic cut and that non-specific exonucleases in the extract complete the degradation process16. In addition, our ability to create an extract that targets lacZ in vitro indicates that the presence of an endogenous gene is not required for the RNAi response.
- To examine the substrate requirements for the dsRNA-induced, sequence-specific nuclease activity, we incubated a variety of cyclin-E-derived transcripts with an extract derived from cells that had been transfected with the 540-nucleotide cyclin E dsRNA (FIG. 2b, c). Just as a length requirement was observed for the transfected dsRNA, the RNAi nuclease activity showed a dependence on the size of the RNA substrate. Both a 600-nucleotide transcript that extends slightly beyond the targeted region (FIG. 2b) and an ˜1-kilobase (kb) transcript that contains the entire coding sequence (data not shown) were completely destroyed by the extract. Surprisingly, shorter substrates were not degraded as efficiently. Reduced activity was observed against either a 300- or a 220-nucleotide transcript, and a 100-nucleotide transcript was resistant to nuclease in our assay. This was not due solely to position effects because ˜100-nucleotide transcripts derived from other portions of the transfected dsRNA behaved similarly (data not shown). As expected, the nuclease activity (or activities) present in the extract could also recognize the antisense strand of the cyclin E mRNA. Again, substrates that contained a substantial portion of the targeted region were degraded efficiently whereas those that contained a shorter stretch of homologous sequence (˜130 nucleotides) were recognized inefficiently (FIG. 2c, as600). For both the sense and antisense strands, transcripts that had no homology with the transfected dsRNA ( FIG. 2b, Eout; FIG. 2c, as300) were not degraded. Although we cannot exclude the possibility that nuclease specificity could have migrated beyond the targeted region, the resistance of transcripts that do not contain homology to the dsRNA is consistent with data from C. elegans. Double-stranded RNAs homologous to an upstream cistron have little or no effect on a linked downstream cistron, despite the fact that unprocessed, polycistronic mRNAs can be readily detected17, 18. Furthermore, the nuclease was inactive against a dsRNA identical to that used to provoke the RNAi response in vivo (FIG. 2b). In the in vitro system, neither a 5′ cap nor a poly(A) tail was required, as such transcripts were degraded as efficiently as uncapped and non-polyadenylated RNAs.
- Gene silencing provoked by dsRNA is sequence specific. A plausible mechanism for determining specificity would be incorporation of nucleic-acid guide sequences into the complexes that accomplish silencing19. In accord with this idea, pre-treatment of extracts with a Ca2+-dependent nuclease (micrococcal nuclease) abolished the ability of these extracts to degrade cognate mRNAs (FIG. 3). Activity could not be rescued by addition of non-specific RNAs such as yeast transfer RNA. Although micrococcal nuclease can degrade both DNA and RNA, treatment of the extract with DNAse I had no effect (FIG. 3). Sequence-specific nuclease activity, however, did require protein (data not shown). Together, our results support the possibility that the RNAi nuclease is a ribonucleoprotein, requiring both RNA and protein components. Biochemical fractionation (see below) is consistent with these components being associated in extract rather than being assembled on the target mRNA after its addition.
- In plants, the phenomenon of co-suppression has been associated with the existence of small (˜25-nucleotide) RNAs that correspond to the gene that is being silenced19. To address the possibility that a similar RNA might exist in Drosophila and guide the sequence-specific nuclease in the choice of substrate, we partially purified our activity through several fractionation steps. Crude extracts contained both sequence-specific nuclease activity and abundant, heterogeneous RNAs homologous to the transfected dsRNA (FIGS. 2 and 4a). The RNAi nuclease fractionated with ribosomes in a high-speed centrifugation step. Activity could be extracted by treatment with high salt, and ribosomes could be removed by an additional centrifugation step. Chromatography of soluble nuclease over an anion-exchange column resulted in a discrete peak of activity (FIG. 4b, cyclin E). This retained specificity as it was inactive against a heterologous mRNA (FIG. 4b, lacZ). Active fractions also contained an RNA species of 25 nucleotides that is homologous to the cyclin E target (FIG. 4b, northern). The band observed on northern blots may represent a family of discrete RNAs because it could be detected with probes specific for both the sense and antisense cyclin E sequences and with probes derived from distinct segments of the dsRNA (data not shown). At present, we cannot determine whether the 25-nucleotide RNA is present in the nuclease complex in a double-stranded or single-stranded form.
- RNA interference allows an adaptive defence against both exogenous and endogenous dsRNAs, providing something akin to a dsRNA immune response. Our data, and that of others19, is consistent with a model in which dsRNAs present in a cell are converted, either through processing or replication, into small specificity determinants of discrete size in a manner analogous to antigen processing. Our results suggest that the post-transcriptional component of dsRNA-dependent gene silencing is accomplished by a sequence-specific nuclease that incorporates these small RNAs as guides that target specific messages based upon sequence recognition. The identical size of putative specificity determinants in plants19 and animals predicts a conservation of both the mechanisms and the components of dsRNA-induced, post-transcriptional gene silencing in diverse organisms. In plants, dsRNAs provoke not only post-transcriptional gene silencing but also chromatin remodelling and transcriptional repression20, 21. It is now critical to determine whether conservation of gene-silencing mechanisms also exists at the transcriptional level and whether chromatin remodelling can be directed in a sequence-specific fashion by these same dsRNA-derived guide sequences.
- Methods
- Cell Culture and RNA Methods
- S2 (ref. 22) cells were cultured at 27° C. in 90% Schneider's insect media (Sigma), 10% heat inactivated fetal bovine serum (FBS). Cells were transfected with dsRNA and plasmid DNA by calcium phosphate co-precipitation23. Identical results were observed when cells were transfected using lipid reagents (for example, Superfect, Qiagen). For FACS analysis, cells were additionally transfected with a vector that directs expression of a green fluorescent protein (GFP)-US9 fusion protein13. These cells were fixed in 90% ice-cold ethanol and stained with propidium iodide at 25 μg ml−1. FACS was performed on an Elite flow cytometer (Coulter). For northern blotting, equal loading was ensured by over-probing blots with a control complementary DNA (RP49). For the production of dsRNA, transcription templates were generated by polymerase chain reaction such that they contained T7 promoter sequences on each end of the template. RNA was prepared using the RiboMax kit (Promega). Confirmation that RNAs were double stranded came from their complete sensitivity to RNAse III (a gift from A. Nicholson). Target mRNA transcripts were synthesized using the Riboprobe kit (Promega) and were gel purified before use.
- Extract Preparation
- Log-phase S2 cells were plated on 15-cm tissue culture dishes and transfected with 30 μg dsRNA and 30 μg carrier plasmid DNA. Seventy-two hours after transfection, cells were harvested in PBS containing 5 mM EGTA washed twice in PBS and once in hypotonic buffer (10 mM HEPES pH 7.3, 6 mM β-mercaptoethanol). Cells were suspended in 0.7 packed-cell volumes of hypotonic buffer containing Complete protease inhibitors (Boehringer) and 0.5 units ml−1 of RNasin (Promega). Cells were disrupted in a dounce homogenizer with a type B pestle, and lysates were centrifuged at 30,000 g for 20 min. Supernatants were used in an in vitro assay containing 20 mM HEPES pH 7.3, 110 mM KOAc, 1 mM Mg(OAc)2, 3 mM EGTA, 2 mM CaCl2, 1 mM DTT. Typically, 5 μl extract was used in a 10 μl assay that contained also 10,000 c.p.m. synthetic mRNA substrate.
- Extract Fractionation
- Extracts were centrifuged at 200,000 g for 3 h and the resulting pellet (containing ribosomes) was extracted in hypotonic buffer containing also 1 mM MgCl2 and 300 mM KOAc. The extracted material was spun at 100,000 g for 1 h and the resulting supernatant was fractionated on Source 15Q column (Pharmacia) using a KCl gradient in buffer A (20 mM HEPES pH 7.0, 1 mM dithiothreitol, 1 mM MgCl2). Fractions were assayed for nuclease activity as described above. For northern blotting, fractions were proteinase K/SDS treated, phenol extracted, and resolved on 15% acrylamide 8M urea gels. RNA was electroblotted onto Hybond N+ and probed with strand-specific riboprobes derived from cyclin E mRNA. Hybridization was carried out in 500 mM NaPO4 pH 7.0, 15% formamide, 7% SDS, 1% BSA. Blots were washed in 1 SSC at 37-45° C.
- 1. Sharp, P. A. RNAi and double-strand RNA.Genes Dev. 13, 139-141 (1999).
- 2. Sanchez-Alvarado, A. & Newmark, P. A. Double-stranded RNA specifically disrupts gene expression during planarian regeneration.Proc. Natl Acad. Sci. USA 96, 5049-5054 (1999).
- 3. Lohmann, J. U., Endl, I. & Bosch, T. C. Silencing of developmental genes in Hydra.Dev. Biol. 214, 211-214 (1999).
- 4. Cogoni, C. & Macino, G. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase.Nature 399, 166-169 (1999).
- 5. Waterhouse, P. M., Graham, M. W. & Wang, M. B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA.Proc. Natl Acad. Sci. USA 95, 13959-13964 (1998).
- 6. Montgomery, M. K. & Fire, A. Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression.Trends Genet. 14, 225-228 (1998).
- 7. Ngo, H., Tschudi, C., Gull, K. & Ullu, E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei.Proc. Natl Acad. Sci. USA 95, 14687-14692 (1998).
- 8. Tabara, H. et al. The rde-1 gene, RNA interference, and transposon silencing inC. elegans. Cell 99, 123-132 (1999).
- 9. Ketting, R. F., Haverkamp, T. H. A., van Luenen, H. G. A. M. & Plasterk, R. H. A. mut-7 ofC. elegans, required for transposon silencing and RNA interference, is a homolog of Werner Syndrome helicase and RnaseD. Cell 99, 133-141 (1999).
- 10. Ratcliff, F., Harrison, B. D. & Baulcombe, D. C. A similarity between viral defense and gene silencing in plants.Science 276, 1558-1560 (1997).
- 11. Kennerdell, J. R. & Carthew, R. W. Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.Cell 95, 1017-1026 (1998).
- 12. Misquitta, L. & Paterson, B. M. Targeted disruption of gene function in Drosophila by RNA interference: a role for nautilus in embryonic somatic muscle formation.Proc. Natl Acad. Sci. USA 96, 1451-1456 (1999).
- 13. Kalejta, R. F., Brideau, A. D., Banfield, B. W. & Beavis, A. J. An integral membrane green fluorescent protein marker, Us9-GFP, is quantitatively retained in cells during propidium iodine-based cell cycle analysis by flow cytometry.Exp. Cell. Res. 248, 322-328 (1999).
- 14. Wolf, D. A. & Jackson, P. K. Cell cycle: oiling the gears of anaphase.Curr. Biol. 8, R637-R639 (1998).
- 15. Kramer, E. R., Gieffers, C., Holz, G., Hengstschlager, M. & Peters, J. M. Activation of the human anaphase-promoting complex by proteins of the CDC20/fizzy family.Curr. Biol. 8, 1207-1210 (1998).
- 16. Shuttleworth, J. & Colman, A. Antisense oligonucleotide-directed cleavage of mRNA in Xenopus oocytes and eggs.EMBO J. 7, 427-434 (1988).
- 17. Tabara, H., Grishok, A. & Mello, C. C. RNAi inC. elegans: soaking in the genome sequence. Science 282, 430-432 (1998).
- 18. Bosher, J. M., Dufourcq, P., Sookhareea, S. & Labouesse, M. RNA interference can target pre-mRNA. Consequences for gene expression in aCaenorhabditis elegans operon. Genetics 153, 1245-1256 (1999).
- 19. Hamilton, J. A. & Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants.Science 286, 950-952 (1999).
- 20. Jones, L. A., Thomas, C. L. & Maule, A. J. De novo methylation and co-suppression induced by a cytoplasmically replicating plant RNA virus.EMBO J. 17, 6385-6393 (1998).
- 21. Jones, L. A. et al. RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing.Plant Cell 11, 2291-2301 (1999).
- 22. Schneider, I. Cell lines derived from late embryonic stages ofDrosophila melanogaster. J. Embryol. Exp. Morpho. 27, 353-365 (1972).
- 23. Di Nocera, P. P. & Dawid, I. B. Transient expression of genes introduced into cultured cells of Drosophila.Proc. Natl
Acad. Sci. USA 80, 7095-7098 (1983). - Genetic approaches in worms, fungi and plants have identified a group of proteins that are essential for double-stranded RNA-induced gene silencing. Among these are ARGONAUTE family members (e.g. RDE1, QDE2)9,10,30, recQ-family helicases (MUT-7, QDE3)11,12, and RNA-dependent RNA polymerases (e.g. EGO-1, QDE1, SGS2/SDE1)13-16. While potential roles have been proposed, none of these genes has been assigned a definitive function in the silencing process. Biochemical studies have suggested that PTGS is accomplished by a multicomponent nuclease that targets mRNAs for degradation6,8,17. We have shown that the specificity of this complex may derive from the incorporation of a small guide sequence that is homologous to the mRNA substrate6. Originally identified in plants that were actively silencing transgenes7, these ˜22 nt. RNAs have been produced during RNAi in vitro using an extract prepared from Drosophila embryos8. Putative guide RNAs can also be produced in extracts from Drosophila S2 cells (FIG. 5a). With the goal of understanding the mechanism of post-transcriptional gene silencing, we have undertaken both biochemical fractionation and candidate gene approaches to identify the enzymes that execute each step of RNAi.
- Our previous studies resulted in the partial purification of a nuclease, RISC, that is an effector of RNA interference. See Example 1. This enzyme was isolated from Drosophila S2 cells in which RNAi had been initiated in vivo by transfection with dsRNA. We first sought to determine whether the RISC enzyme and the enzyme that initiates RNAi via processing of dsRNA into 22 mers are distinct activities. RISC activity could be largely cleared from extracts by high-speed centrifugation (100,000×g for 60 min.) while the activity that produces 22 mers remained in the supernatant (FIG. 5b,c). This simple fractionation indicated that RISC and the 22 mer-generating activity are separable and thus distinct enzymes. However, it seems likely that they might interact at some point during the silencing process.
- RNAse III family members are among the few nucleases that show specificity for double-stranded RNA18. Analysis of the Drosophila and C. elegans genomes reveals several types of RNAse III enzymes. First is the canonical RNAse III which contains a single RNAse III signature motif and a double-stranded RNA binding domain (dsRBD; e.g. RNC_CAEEL). Second is a class represented by Drosha19, a Drosophila enzyme that contains two RNAse III motifs and a dsRBD (CeDrosha in C. elegans). A third class contains two RNAse III signatures and an amino terminal helicase domain (e.g. Drosophila CG4792, CG6493, C. elegans K12H4.8), and these had previously been proposed by Bass as candidate RNAi nucleases20. Representatives of all three classes were tested for the ability to produce discrete, ˜22 nt. RNAs from dsRNA substrates.
- Partial digestion of a 500 nt. cyclin E dsRNA with purified, bacterial RNAse III produced a smear of products while nearly complete digestion produced a heterogeneous group of ˜11-17 nucleotide RNAs (not shown). In order to test the dual-RNAse III enzymes, we prepared T7 epitope-tagged versions of Drosha and CG4792. These were expressed in transfected S2 cells and isolated by immunoprecipitation using antibody-agarose conjugates. Treatment of the dsRNA with the CG4792 immunoprecipitate yielded ˜22 nt. fragments similar to those produced in either S2 or embryo extracts (FIG. 6a). Neither activity in extract nor activity in immunoprecipitates depended on the sequence of the RNA substrate since dsRNAs derived from several genes were processed equivalently (see Supplement 1). Negative results were obtained with Drosha and with immunoprecipitates of a DExH box helicase (Homeless21; see FIGS. 6a,b). Western blotting confirmed that each of the tagged proteins was expressed and immunoprecipitated similarly (see Supplement 2). Thus, we conclude that CG4792 may carry out the initiation step of RNA interference by producing ˜22 nt. guide sequences from dsRNAs. Because of its ability to digest dsRNA into uniformly sized, small RNAs, we have named this enzyme Dicer (Dcr). Dicer mRNA is expressed in embryos, in S2 cells, and in adult flies, consistent with the presence of functional RNAi machinery in all of these contexts (see Supplement 3).
- The possibility that Dicer might be the nuclease responsible for the production of guide RNAs from dsRNAs prompted us to raise an antiserum directed against the carboxy-terminus of the Dicer protein (Dicer-1, CG4792). This antiserum could immunoprecipitate a nuclease activity from either Drosophila embryo extracts or from S2 cell lysates that produced ˜22 nt. RNAs from dsRNA substrates (FIG. 6C). The putative guide RNAs that are produced by the Dicer-1 enzyme precisely comigrate with 22 mers that are produced in extract and with 22 mers that are associated with the RISC enzyme (FIGS. 6 D,F). It had previously been shown that the enzyme that produced guide RNAs in Drosophila embryo extracts was ATP-dependent8. Depletion of this cofactor resulted in an ˜6-fold lower rate of dsRNA cleavage and in the production of RNAs with a slightly lower mobility. Of interest was the fact that both Dicer-1 immunoprecipitates and extracts from S2 cells require ATP for the production of 22 mers (FIG. 6D). We do not observe the accumulation of lower mobility products in these cases, although we do routinely observe these in ATP-depleted embryo extracts. The requirement of this nuclease for ATP is a quite unusual property. We hypothesize that this requirement could indicate that the enzyme may act processively on the dsRNA, with the helicase domain harnessing the energy of ATP hydrolysis both for unwinding guide RNAs and for translocation along the substrate.
- Efficient induction of RNA interference inC. elegans and in Drosophila has several requirements. For example, the initiating RNA must be double-stranded, and it must be several hundred nucleotides in length. To determine whether these requirements are dictated by Dicer, we characterized the ability of extracts and of immunoprecipitated enzyme to digest various RNA substrates. Dicer was inactive against single stranded RNAs regardless of length (see Supplement 4). The enzyme could digest both 200 and 500 nucleotide dsRNAs but was significantly less active with shorter substrates (see Supplement 4). Double-stranded RNAs as short as 35 nucleotides could be cut by the enzyme, albeit very inefficiently (data not shown). In contrast, E. coli RNAse III could digest to completion dsRNAs of 35 or 22 nucleotides (not shown). This suggests that the substrate preferences of the Dicer enzyme may contribute to but not wholly determine the size dependence of RNAi.
- To determine whether the Dicer enzyme indeed played a role in RNAi in vivo, we sought to deplete Dicer activity from S2 cells and test the effect on dsRNA-induced gene silencing. Transfection of S2 cells with a mixture of dsRNAs homologous to the two Drosophila Dicer genes (CG4792 and CG6493) resulted in an ˜6-7 fold reduction of Dicer activity either in whole cell lysates or in Dicer-1 immunoprecipitates (FIGS. 7A,B). Transfection with a control dsRNA (murine caspase 9) had no effect. Qualitatively similar results were seen if Dicer was examined by Northern blotting (not shown). Depletion of Dicer in this manner substantially compromised the ability of cells to silence subsequently an exogenous, GFP transgene by RNAi (FIG. 7C). These results indicate that Dicer is involved in RNAi in vivo. The lack of complete inhibition of silencing could result from an incomplete suppression of Dicer (which is itself required for RNAi) or could indicate that in vivo, guide RNAs can be produced by more than one mechanism (e.g. through the action of RNA-dependent RNA polymerases).
- Our results indicate that the process of RNA interference can be divided into at least two distinct steps. According to this model, initiation of PTGS would occur upon processing of a double-stranded RNA by Dicer into ˜22 nucleotide guide sequences, although we cannot formally exclude the possibility that another, Dicer-associated nuclease may participate in this process. These guide RNAs would be incorporated into a distinct nuclease complex (RISC) that targets single-stranded mRNAs for degradation. An implication of this model is that guide sequences are themselves derived directly from the dsRNA that triggers the response. In accord with this model, we have demonstrated that32P-labeled, exogenous dsRNAs that have been introduced into S2 cells by transfection are incorporated into the RISC enzyme as 22 mers (FIG. 7E). However, we cannot exclude the possibility that RNA-dependent RNA polymerases might amplify 22 mers once they have been generated or provide an alternative method for producing guide RNAs.
- The structure of the Dicer enzyme provokes speculation on the mechanism by which the enzyme might produce discretely sized fragments irrespective of the sequence of the dsRNA (see
Supplement 1, FIG. 8a). It has been established that bacterial RNAse III acts on its substrate as a dimer18,22,23. Similarly, a dimer of Dicer enzymes may be required for cleavage of dsRNAs into ˜22 nt. pieces. According to one model, the cleavage interval would be determined by the physical arrangement of the two RNAse III domains within Dicer enzyme (FIG. 8a). A plausible alternative model would dictate that cleavage was directed at a single position by the two RIII domains in a single Dicer protein. The 22 nucleotide interval could be dictated by interaction of neighboring Dicer enzymes or by translocation along the mRNA substrate. The presence of an integral helicase domain suggests that the products of Dicer cleavage might be single-stranded 22 mers that are incorporated into the RISC enzyme as such. - A notable feature of the Dicer family is its evolutionary conservation. Homologs are found inC. elegans (K12H4.8), Arabidopsis (e.g., CARPEL FACTORY24, T25K16.4, AC012328—1), mammals (Helicase-MOI25) and S. pombe (YC9A_SCHPO) (FIG. 8b, see
Supplements 6,7 for sequence comparisons). In fact, the human Dicer family member is capable of generating ˜22 nt. RNAs from dsRNA substrates (Supplement 5) suggesting that these structurally similar proteins may all share similar biochemical functions. It has been demonstrated that exogenous dsRNAs can affect gene function in early mouse embryos29, and our results suggest that this regulation may be accomplished by an evolutionarily conserved RNAi machinery. - In addition to RNAseIII and helicase motifs, searches of the PFAM database indicate that each Dicer family member also contains a ZAP domain (FIG. 8c)27. This sequence was defined based solely upon its conservation in the Zwille/ARGONAUTE/Piwi family that has been implicated in RNAi by mutations in C. elegans (Rde-1)9 and Neurospora (Qde-2)10. Although the function of this domain is unknown, it is intriguing that this region of homology is restricted to two gene families that participate in dsRNA-dependent silencing. Both the ARGONAUTE and Dicer families have also been implicated in common biological processes, namely the determination of stem-cell fates. A hypomorphic allele of carpel factory, a member of the Dicer family in Arabidopsis, is characterized by increased proliferation in floral meristems24. This phenotype and a number of other characteristic features are also shared by Arabidopsis ARGONAUTE (ago1-1) mutants26 (C. Kidner and R. Martiennsen, pers. comm.). These genetic analyses begin to provide evidence that RNAi may be more than a defensive response to unusual RNAs but may also play important roles in the regulation of endogenous genes.
- With the identification of Dicer as a catalyst of the initiation step of RNAi, we have begun to unravel the biochemical basis of this unusual mechanism of gene regulation. It will be of critical importance to determine whether the conserved family members from other organisms, particularly mammals, also play a role in dsRNA-mediated gene regulation.
- Methods
- Plasmid constructs. A full-length cDNA encoding Drosha was obtained by PCR from an EST sequenced by the Berkeley Drosophila genome project. The Homeless clone was a gift from Gillespie and Berg (Univ. Washington). The T7 epitope-tag was added to the amino terminus of each by PCR, and the tagged cDNAs were cloned into pRIP, a retroviral vector designed specifically for expression in insect cells (E. Bernstein, unpublished). In this vector, expression is driven by theOrgyia pseudotsugata IE2 promoter (Invitrogen). Since no cDNA was available for CG4792/Dicer, a genomic clone was amplified from a bacmid (BACR23F10; obtained from the BACPAC Resource Center in the Dept. of Human Genetics at the Roswell Park Cancer Institute). Again, during amplification, a T7 epitope tag was added at the amino terminus of the coding sequence. The human Dicer gene was isolated from a cDNA library prepared from HaCaT cells (GJH, unpublished). A T7-tagged version of the complete coding sequence was cloned into pCDNA3 (Invitrogen) for expression in human cells (LinX-A).
- Cell culture and extract preparation. S2 and embryo culture. S2 cells were cultured at 27° C. in 5% CO2 in Schneider's insect media supplemented with 10% heat inactivated fetal bovine serum (Gemini) and 1% antibiotic-antimycotic solution (Gibco BRL). Cells were harvested for extract preparation at 10×106 cells/ml. The cells were washed 1× in PBS and were resuspended in a hypotonic buffer (10 mM Hepes pH 7.0, 2 mM MgCl2, 6 mM βME) and dounced. Cell lysates were spun 20,000×g for 20 minutes. Extracts were stored at −80° C. Drosophila embryos were reared in fly cages by standard methodologies and were collected every 12 hours. The embryos were dechorionated in 50% chlorox bleach and washed thoroughly with distilled water. Lysis buffer (10 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EGTA, 10 mM β-glycerophosphate, 1 mM DTT, 0.2 mM PMSF) was added to the embryos, and extracts were prepared by homogenization in a tissue grinder. Lysates were spun for two hours at 200,000×g and were frozen at −80° C. LinX-A cells, a highly-transfectable derivative of human 293 cells, (Lin Xie and GJH, unpublished) were maintained in DMEM/10%FCS.
- Transfections and immunoprecipitations. S2 cells were transfected using a calcium phosphate procedure essentially as previously described6. Transfection rates were ˜90% as monitored in controls using an in situ β-galactosidase assay. LinX-A cells were also transfected by calcium phosphate co-precipitation. For immunoprecipitations, cells (˜5×106 per IP) were transfected with various clones and lysed three days later in IP buffer (125 mM KOAc, 1 mM MgOAc, 1 mM CaCl2, 5 mM EGTA, 20 mM Hepes pH 7.0, 1 mM DTT, 1% NP-40 plus Complete protease inhibitors (Roche)). Lysates were spun for 10 minutes at 14,000×g and supernatants were added to T7 antibody-agarose beads (Novagen). Antibody binding proceeded for 4 hours at 4° C. Beads were centrifuged and washed in lysis buffer three times, and once in reaction buffer. The Dicer antiserum was raised in rabbits using a KLH-conjugated peptide corresponding to the C-
terminal 8 amino acids of Drosophila Dicer-1 (CG4792). - Cleavage reactions. RNA preparation. Templates to be transcribed into dsRNA were generated by PCR with forward and reverse primers, each containing a T7 promoter sequence. RNAs were produced using Riboprobe (Promega) kits and were uniformly labeling during the transcription reaction with32P-UTP. Single-stranded RNAs were purified from 1% agarose gels. dsRNA cleavage. Five microliters of embryo or S2 extracts were incubated for one hour at 30° C. with dsRNA in a reaction containing 20 mM Hepes pH 7.0, 2 mM MgOAc, 2 mM DTT, 1 mM ATP and 5% Superasin (Ambion). Immunoprecipitates were treated similarly except that a minimal volume of reaction buffer (including ATP and Superasin) and dsRNA were added to beads that had been washed in reaction buffer (see above). For ATP depletion, Drosophila embryo extracts were incubated for 20 minutes at 30° C. with 2 mM glucose and 0.375 U of hexokinase (Roche) prior to the addition of dsRNA.
- Northern and Western analysis. Total RNA was prepared from Drosophila embryos (0-12 hour), from adult flies, and from S2 cells using Trizol (Lifetech). Messenger RNA was isolated by affinity selection using magnetic oligo-dT beads (Dynal). RNAs were electrophoresed on denaturing formaldehyde/agarose gels, blotted and probed with randomly primed DNAs corresponding to Dicer. For Western analysis, T7-tagged proteins were immunoprecipitated from whole cell lysates in IP buffer using anti-T7-antibody-agarose conjugates. Proteins were released from the beads by boiling in Laemmli buffer and were separated by electrophoresis on 8% SDS PAGE. Following transfer to nitrocellulose, proteins were visualized using an HRP-conjugated anti-T7 antibody (Novagen) and chemiluminescent detection (Supersignal, Pierce).
- RNAi of Dicer. Drosophila S2 cells were transfected either with a dsRNA corresponding to mouse caspase 9 or with a mixture of two dsRNAs corresponding to Drosophila Dicer-1 and Dicer-2 (CG4792 and CG6493). Two days after the initial transfection, cells were again transfected with a mixture containing a GFP expression plasmid and either luciferase dsRNA or GFP dsRNA as previously described6. Cells were assayed for Dicer activity or fluorescence three days after the second transfection. Quantification of fluorescent cells was done on a Coulter EPICS cell sorter after fixation. Control transfections indicated that Dicer activity was not affected by the introduction of caspase 9 dsRNA.
- 1. Baulcombe, D. C. RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants.Plant Mol Biol 32, 79-88 (1996).
- 2. Wassenegger, M. & Pelissier, T. A model for RNA-mediated gene silencing in higher plants.Plant Mol Biol 37, 349-62 (1998).
- 3. Montgomery, M. K. & Fire, A. Double-stranded RNA as a mediator in sequence-specific genetic silencing and co-suppression [see comments].Trends Genet 14, 255-8 (1998).
- 4. Sharp, P. A. RNAi and double-strand RNA.Genes Dev 13, 139-41 (1999).
- 5. Sijen, T. & Kooter, J. M. Post-transcriptional gene-silencing: RNAs on the attack or on the defense? [In Process Citation].
Bioessays 22, 520-31 (2000). - 6. Hammond, S. M., Bernstein, E., Beach, D. & Hannon, G. J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature 404, 293-6 (2000).
- 7. Hamilton, A. J. & Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants [see comments].Science 286, 950-2 (1999).
- 8. Zamore, P. D., Tuschl, T., Sharp, P. A. & Bartel, D. P. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Cell 101, 25-33 (2000).
- 9. Tabara, H. et al. The rde-1 gene, RNA interference, and transposon silencing inC. elegans. Cell 99, 123-32 (1999).
- 10. Catalanotto, C., Azzalin, G., Macino, G. & Cogoni, C. Gene silencing in worms and fungi.Nature 404, 245 (2000).
- 11. Ketting, R. F., Haverkamp, T. H., van Luenen, H. G. & Plasterk, R. H. Mut-7 ofC. elegans, required for transposon silencing and RNA interference, is a homolog of Werner syndrome helicase and RNaseD. Cell 99, 133-41 (1999).
- 12. Cogoni, C. & Macino, G. Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase.Science 286, 2342-4 (1999).
- 13. Cogoni, C. & Macino, G. Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase.Nature 399, 166-9 (1999).
- 14. Smardon, A. et al. EGO-1 is related to RNA-directed RNA polymerase and functions in germ-line development and RNA interference inC. elegans [published erratum appears in Curr Biol May 18, 2000;10(10):R393-4].
Curr Biol 10, 169-78 (2000). - 15. Mourrain, P. et al. Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance.Cell 101, 533-42 (2000).
- 16. Dalmay, T., Hamilton, A., Rudd, S., Angell, S. & Baulcombe, D. C. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus.Cell 101, 543-53 (2000).
- 17. Tuschl, T., Zamore, P. D., Lehmann, R., Bartel, D. P. & Sharp, P. A. Targeted mRNA degradation by double-stranded RNA in vitro.Genes Dev 13, 3191-7 (1999).
- 18. Nicholson, A. W. Function, mechanism and regulation of bacterial ribonucleases.FEMS Microbiol Rev 23, 371-90 (1999).
- 19. Filippov, V., Solovyev, V., Filippova, M. & Gill, S. S. A novel type of RNase III family proteins in eukaryotes.Gene 245, 213-21 (2000).
- 20. Bass, B. L. Double-stranded RNA as a template for gene silencing.Cell 101, 235-8 (2000).
- 21. Gillespie, D. E. & Berg, C. A. Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases.Genes Dev 9, 2495-508 (1995).
- 22. Robertson, H. D., Webster, R. E. & Zinder, N. D. Purification and properties of ribonuclease III fromEscherichia coli. J Biol Chem 243, 82-91 (1968).
- 23. Dunn, J. J. RNase III cleavage of single-stranded RNA. Effect of ionic strength on the fideltiy of cleavage.J Biol Chem 251, 3807-14 (1976).
- 24. Jacobsen, S. E., Running, M. P. & Meyerowitz, E. M. Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems.Development 126, 5231-43 (1999).
- 25. Matsuda, S. et al. Molecular cloning and characterization of a novel human gene (HERNA) which encodes a putative RNA-helicase.Biochim Biophys Acta 1490, 163-9 (2000).
- 26. Bohmert, K. et al AGO1 defines a novel locus of Arabidopsis controlling leaf development.Embo J 17, 170-80 (1998).
- 27. Sonnhammer, E. L., Eddy, S. R. & Durbin, R. Pfam: a comprehensive database of protein domain families based on seed alignments.Proteins 28, 405 -20 (1997).
- 28. Altschul, S. F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucleic Acids Res 25, 3389-402 (1997). - 29. Wianny, F. and Zernicka-Goetz, M. Specific interference with gene function by double-stranded RNA in early mouse development.Nature Cell Biol. 2, 70-75 (2000).
- 30. Fagard, M., Boutet, S., Morel, J.-B., Bellini, C. and Vaucheret, H. Ago-1, Qde-2 and Rde-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals.Proc. Natl. Acad. Sci. USA 97, 11650-11654 (2000).
- In numerous model organisms, double stranded RNAs have been shown to cause effective and specific suppression of gene function (ref. 1). This response, termed RNA interference or post-transcriptional gene silencing, has evolved into a highly effective reverse genetic tool inC. elegans, Drosophila, plants and numerous other systems. In these cases, double-stranded RNAs can be introduced by injection, transfection or feeding; however, in all cases, the response is both transient and systemic. Recently, stable interference with gene expression has been achieved by expression of RNAs that form snap-back or hairpin structures (refs 2-7). This has the potential not only to allow stable silencing of gene expression but also inducible silencing as has been observed in trypanosomes and adult Drosophila (
refs - The presence of hairpin structures often induces plasmid rearrangement, in part due to theE. coli sbc proteins that recognize and cleave cruciform DNA structures (ref 8). We have developed a method for the construction of hairpins that does not require cloning of inverted repeats, per se. Instead, the fragment of the gene that is to be silenced is cloned as a direct repeat, and the inversion is accomplished by treatment with a site-specific recombinase, either in vitro (or potentially in vivo) (see FIG. 27). Following recombination, the inverted repeat structure is stable in a bacterial strain that lacks an intact SBC system (DL759). We have successfully used this strategy to construct numerous hairpin expression constructs that have been successfully used to provoke gene silencing in Drosophila cells.
- 1. Bosher, J. M. & Labouesse, M. RNA interference: genetic wand and genetic watchdog.
Nat Cell Biol 2, E31-6 (2000). - 2. Fortier, E. & Belote, J. M. Temperature-dependent gene silencing by an expressed inverted repeat in Drosophila [published erratum appears in Genesis; May 27, 2000; (1):47]. Genesis 26, 240-4 (2000).
- 3. Kennerdell, J. R. & Carthew, R. W. Heritable gene silencing in Drosophila using double-stranded RNA.Nat Biotechnol 18, 896-8 (2000).
- 4. Lam, G. & Thummel, C. S. Inducible expression of double-stranded RNA directs specific genetic interference in Drosophila [In Process Citation].
Curr Biol 10, 957-63 (2000). - 5. Shi, H. et al. Genetic interference inTrypanosoma brucei by heritable and inducible double-stranded RNA.
Rna 6, 1069-76 (2000). - 6. Smith, N. A. et al Total silencing by intron-spliced hairpin RNAs.Nature 407, 319-20 (2000).
- 7. Tavernarakis, N., Wang, S. L., Dorovkov, M., Ryazanov, A. & Driscoll, M. Heritable and inducible genetic interference by double-stranded RNA encoded by transgenes.
Nat Genet 24, 180-3 (2000). - 8. Connelly, J. C. & Leach, D. R. The sbcC and sbcD genes ofEscherichia coli encode a nuclease involved in palindrome inviability and genetic recombination.
Genes Cells 1, 285-91 (1996). - Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
- All of the above-cited references and publications are hereby incorporated by reference.
Claims (25)
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/866,557 US20020162126A1 (en) | 2000-03-16 | 2001-05-24 | Methods and compositions for RNA interference |
US10/350,798 US20040086884A1 (en) | 2000-03-16 | 2003-01-24 | Methods and compositions for RNA interference |
US10/997,086 US8202846B2 (en) | 2000-03-16 | 2004-11-23 | Methods and compositions for RNA interference |
US11/894,676 US8153776B2 (en) | 2000-03-16 | 2007-08-20 | Methods and compositions for RNA interference |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18973900P | 2000-03-16 | 2000-03-16 | |
US24309700P | 2000-10-24 | 2000-10-24 | |
PCT/US2001/008435 WO2001068836A2 (en) | 2000-03-16 | 2001-03-16 | Methods and compositions for rna interference |
US09/866,557 US20020162126A1 (en) | 2000-03-16 | 2001-05-24 | Methods and compositions for RNA interference |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2001/008435 Continuation-In-Part WO2001068836A2 (en) | 2000-03-16 | 2001-03-16 | Methods and compositions for rna interference |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/055,797 Continuation-In-Part US20030084471A1 (en) | 2000-03-16 | 2002-01-22 | Methods and compositions for RNA interference |
US10/350,798 Continuation-In-Part US20040086884A1 (en) | 2000-03-16 | 2003-01-24 | Methods and compositions for RNA interference |
Publications (1)
Publication Number | Publication Date |
---|---|
US20020162126A1 true US20020162126A1 (en) | 2002-10-31 |
Family
ID=26885458
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/858,862 Expired - Fee Related US7732417B2 (en) | 2000-03-16 | 2001-05-16 | Methods and compositions for RNA interference using recombinant Dicer and Argonaut |
US09/866,557 Abandoned US20020162126A1 (en) | 2000-03-16 | 2001-05-24 | Methods and compositions for RNA interference |
US10/350,798 Abandoned US20040086884A1 (en) | 2000-03-16 | 2003-01-24 | Methods and compositions for RNA interference |
US12/152,837 Expired - Lifetime US8383599B2 (en) | 2000-03-16 | 2008-05-16 | Methods and compositions for RNA interference |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/858,862 Expired - Fee Related US7732417B2 (en) | 2000-03-16 | 2001-05-16 | Methods and compositions for RNA interference using recombinant Dicer and Argonaut |
Family Applications After (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/350,798 Abandoned US20040086884A1 (en) | 2000-03-16 | 2003-01-24 | Methods and compositions for RNA interference |
US12/152,837 Expired - Lifetime US8383599B2 (en) | 2000-03-16 | 2008-05-16 | Methods and compositions for RNA interference |
Country Status (7)
Country | Link |
---|---|
US (4) | US7732417B2 (en) |
EP (1) | EP1272630A2 (en) |
JP (1) | JP2003526367A (en) |
AU (1) | AU2001245793A1 (en) |
CA (1) | CA2403397A1 (en) |
IL (1) | IL151781A0 (en) |
WO (1) | WO2001068836A2 (en) |
Cited By (128)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020007051A1 (en) * | 1999-12-10 | 2002-01-17 | David Cheo | Use of multiple recombination sites with unique specificity in recombinational cloning |
US20020086356A1 (en) * | 2000-03-30 | 2002-07-04 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US20020114784A1 (en) * | 1999-01-28 | 2002-08-22 | Medical College Of Georgia Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA |
US20030068799A1 (en) * | 1995-06-07 | 2003-04-10 | Invitrogen Corporation | Recombinational cloning using engineered recombination sites |
US20030124555A1 (en) * | 2001-05-21 | 2003-07-03 | Invitrogen Corporation | Compositions and methods for use in isolation of nucleic acid molecules |
US20030166282A1 (en) * | 2002-02-01 | 2003-09-04 | David Brown | High potency siRNAS for reducing the expression of target genes |
US20030169748A1 (en) * | 2002-03-11 | 2003-09-11 | Weyman Raphael J. | Stackable network units with resiliency facility |
US20030186233A1 (en) * | 2000-05-21 | 2003-10-02 | Invitrogen Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites |
US20030190654A1 (en) * | 2002-01-22 | 2003-10-09 | Ribopharma | Double-stranded RNA (dsRNA) and method of use for inhibiting expression of a fusion gene |
US20030224432A1 (en) * | 2002-05-03 | 2003-12-04 | Jason Myers | Methods and compositions for use in preparing siRNAs |
US20040002077A1 (en) * | 2001-11-28 | 2004-01-01 | Center For Advanced Science And Technology Incubation, Ltd. | siRNA expression system and method for producing functional gene knock-down cell using the system |
US20040001811A1 (en) * | 2001-01-09 | 2004-01-01 | Ribopharma Ag | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US20040014113A1 (en) * | 2002-05-31 | 2004-01-22 | The Regents Of The University Of California | Method for efficient RNA interference in mammalian cells |
US20040018176A1 (en) * | 2002-07-24 | 2004-01-29 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiogenesis |
US20040033602A1 (en) * | 2002-06-12 | 2004-02-19 | Ambion, Inc. | Methods and compositions relating to polypeptides with RNase III domains that mediate RNA interference |
WO2004015062A2 (en) | 2002-08-12 | 2004-02-19 | New England Biolabs, Inc. | Methods and compositions relating to gene silencing |
US20040053875A1 (en) * | 1999-01-30 | 2004-03-18 | Ribopharma Ag | Method and medicament for inhibiting the expression of a given gene |
US20040063207A1 (en) * | 1995-06-07 | 2004-04-01 | Invitrogen Corporation | Recombinational cloning using nucleic acids having recombination sites |
US20040086911A1 (en) * | 2002-06-24 | 2004-05-06 | Baylor College Of Medicine | Inhibition of gene expression in vertebrates using double-stranded RNA (RNAi) |
US20040126791A1 (en) * | 2001-10-26 | 2004-07-01 | Ribopharma Ag | Compositions and methods for treating trail-resistant cancer cells |
US20040175703A1 (en) * | 1999-11-24 | 2004-09-09 | Ribopharma Ag | Compositions and methods for inhibiting expression of a target gene |
US20040203145A1 (en) * | 2002-08-07 | 2004-10-14 | University Of Massachusetts | Compositions for RNA interference and methods of use thereof |
US20040221337A1 (en) * | 1999-10-27 | 2004-11-04 | Baulcombe David C. | Gene silencing |
US20040242518A1 (en) * | 2002-09-28 | 2004-12-02 | Massachusetts Institute Of Technology | Influenza therapeutic |
US20040248174A1 (en) * | 2003-04-18 | 2004-12-09 | Thetrustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiopoietin 1and 2 and their receptor Tie2 |
US20040248094A1 (en) * | 2002-06-12 | 2004-12-09 | Ford Lance P. | Methods and compositions relating to labeled RNA molecules that reduce gene expression |
US20040259247A1 (en) * | 2000-12-01 | 2004-12-23 | Thomas Tuschl | Rna interference mediating small rna molecules |
US20050014264A1 (en) * | 2002-12-11 | 2005-01-20 | University Of Massachusetts | Method of introducing siRNA into adipocytes |
WO2005016504A2 (en) | 2003-06-23 | 2005-02-24 | Pioneer Hi-Bred International, Inc. | Disruption of acc synthase genes to delay senescence in plants |
US20050059044A1 (en) * | 2003-06-03 | 2005-03-17 | Graham Michael Wayne | Double-stranded nucleic acid |
US20050059028A1 (en) * | 2002-11-18 | 2005-03-17 | Genospectra, Inc. | RNAi-based sensors, caged interfering RNAs, and methods of use thereof |
US20050069918A1 (en) * | 2003-05-29 | 2005-03-31 | Francois Claret | JAB1 as a prognostic marker and a therapeutic target for human cancer |
US20050074757A1 (en) * | 2001-10-12 | 2005-04-07 | Ribopharma Ag | Compositions and methods for inhibiting expression of a mutant gene |
US20050112763A1 (en) * | 2003-08-21 | 2005-05-26 | Cold Spring Harbor Laboratory | RNAI-based modification of heterochromatin |
US20050176667A1 (en) * | 2001-01-09 | 2005-08-11 | Alnylam Europe Ag | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US20050260756A1 (en) * | 2003-01-28 | 2005-11-24 | Troy Carol M | Complex for facilitating delivery of dsRNA into a cell and uses thereof |
WO2005019410A3 (en) * | 2003-04-25 | 2005-12-08 | Intradigm Corp | Rnai agents for anti-sars coronavirus therapy |
US20050282203A1 (en) * | 2002-11-18 | 2005-12-22 | Genospectra, Inc. | Caged RNAs and methods of use thereof |
US20060003915A1 (en) * | 2002-04-18 | 2006-01-05 | Karina Drumm | Means and methods for the specific modulation of target genes in the cns and the eye and methods for their identification |
US20060084621A1 (en) * | 2001-01-09 | 2006-04-20 | Hans-Peter Vornlocher | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US20060115455A1 (en) * | 2004-10-22 | 2006-06-01 | Reed Kenneth C | Therapeutic RNAi agents for treating psoriasis |
US20060142228A1 (en) * | 2004-12-23 | 2006-06-29 | Ambion, Inc. | Methods and compositions concerning siRNA's as mediators of RNA interference |
US20060166919A1 (en) * | 2004-12-23 | 2006-07-27 | Alcon, Inc. | RNAi inhibition of CTGF for treatment of ocular disorders |
US20060172965A1 (en) * | 2005-02-01 | 2006-08-03 | Alcon, Inc. | RNAi-mediated inhibition of ocular targets |
US20060172961A1 (en) * | 2004-12-23 | 2006-08-03 | Alcon, Inc. | RNAi inhibition of serum amyloid a for treatment of glaucoma |
US20060211637A1 (en) * | 2002-08-06 | 2006-09-21 | Intradigm Corporation | Methods of down regulating target gene expression in vivo by introduction of interfering rna |
US20060223773A1 (en) * | 2005-03-11 | 2006-10-05 | Alcon, Inc. | RNAi-mediated inhibition of Frizzled Related Protein-1 for treatment of glaucoma |
US20060252035A1 (en) * | 2001-08-06 | 2006-11-09 | Lori Friedman | Sphks as modifiers of the p53 pathway and methods of use |
US20070015722A1 (en) * | 2003-06-20 | 2007-01-18 | Kraynack Brian A | Double stranded compositions comprising a 3'-endo modified strand for use in gene modulation |
US20070025969A1 (en) * | 2005-02-03 | 2007-02-01 | Roelvink Petrus W | RNAi expression constructs |
US20070036740A1 (en) * | 2004-10-06 | 2007-02-15 | Reed Kenneth C | Modulation of hair growth |
US20070081982A1 (en) * | 2005-04-28 | 2007-04-12 | Elisabeth Evertsz | Multiple RNAi expression cassettes for simultaneous delivery of RNAi agents related to heterozygotic expression patterns |
US20070105113A1 (en) * | 2003-08-14 | 2007-05-10 | Takara Bio Inc. | Methods of degrading dsrna and synthesizing rna |
US7223576B2 (en) | 1995-06-07 | 2007-05-29 | Invitrogen Corporation | Recombinational cloning using engineered recombination sites |
US20070218524A1 (en) * | 2003-09-30 | 2007-09-20 | Jun Tomono | Polypeptide Having Rnase III Activity |
US20070275919A1 (en) * | 2003-11-04 | 2007-11-29 | Sergei Gryaznov | Rna Amidates and Thioamidates for Rnai |
US20080152654A1 (en) * | 2006-06-12 | 2008-06-26 | Exegenics, Inc., D/B/A Opko Health, Inc. | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS |
US20080200420A1 (en) * | 2001-07-12 | 2008-08-21 | Zamore Phillip D | In vivo production of small interfering RNAs that mediate gene silencing |
US7422741B2 (en) | 2004-03-05 | 2008-09-09 | Vegenics Limited | VEGFR-3 fusion proteins |
US20080234941A1 (en) * | 2003-10-27 | 2008-09-25 | Jackson Aimee L | Method of Designing Sirnas for Gene Silencing |
US20090023671A1 (en) * | 2005-01-06 | 2009-01-22 | Brashears Sarah J | Rnai Agents for Maintenance of Stem Cells |
US20090048192A1 (en) * | 2004-06-03 | 2009-02-19 | Isis Pharmaceuticals, Inc. | Double Strand Compositions Comprising Differentially Modified Strands for Use in Gene Modulation |
US20090061487A1 (en) * | 2006-09-08 | 2009-03-05 | Samuel Jotham Reich | Sirna and methods of manufacture |
US7521431B2 (en) | 2002-11-01 | 2009-04-21 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of HIF-1 alpha |
US20090104260A1 (en) * | 2003-01-16 | 2009-04-23 | The Trustees Of The University Of Pennsylvania | Compositions and methods for sirna inhibition of icam-1 |
US20090137514A1 (en) * | 2002-04-26 | 2009-05-28 | Nucleonics, Inc. | Methods and compositions for silencing genes without inducing toxicity |
US20090175871A1 (en) * | 2005-11-25 | 2009-07-09 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for demonstrating presence or absence of markers associated with the presence and/or the chemosensitivity of tumors |
US20090203055A1 (en) * | 2005-04-18 | 2009-08-13 | Massachusetts Institute Of Technology | Compositions and methods for RNA interference with sialidase expression and uses thereof |
US7579451B2 (en) | 2004-07-21 | 2009-08-25 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a modified or non-natural nucleobase |
US7615618B2 (en) | 2004-06-30 | 2009-11-10 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a non-phosphate backbone linkage |
US7626014B2 (en) | 2004-04-27 | 2009-12-01 | Alnylam Pharmaceuticals | Single-stranded and double-stranded oligonucleotides comprising a 2-arylpropyl moiety |
US7632932B2 (en) | 2004-08-04 | 2009-12-15 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a ligand tethered to a modified or non-natural nucleobase |
US20100009856A1 (en) * | 2002-06-21 | 2010-01-14 | Sinogenomax Sompany LTD. | Randomized dna libraries and double-stranded rna libraries, use and method of production thereof |
US7670823B1 (en) | 1999-03-02 | 2010-03-02 | Life Technologies Corp. | Compositions for use in recombinational cloning of nucleic acids |
US7674778B2 (en) | 2004-04-30 | 2010-03-09 | Alnylam Pharmaceuticals | Oligonucleotides comprising a conjugate group linked through a C5-modified pyrimidine |
US7695902B2 (en) | 1996-06-06 | 2010-04-13 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides and ribonucleases for cleaving RNA |
US7745418B2 (en) | 2001-10-12 | 2010-06-29 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting viral replication |
US7812149B2 (en) | 1996-06-06 | 2010-10-12 | Isis Pharmaceuticals, Inc. | 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations |
US20110003378A1 (en) * | 2004-03-05 | 2011-01-06 | Benitec, Inc. | MULTIPLE PROMOTER EXPRESSION CASSETTES FOR SIMULTANEOUS DELIVERY OF RNAi AGENTS |
WO2011004003A1 (en) | 2009-07-10 | 2011-01-13 | Schmuelling Thomas | Disruption of ckx3 and at least one other ckx gene in a plant or plant cell leads to improved traits |
US7884086B2 (en) | 2004-09-08 | 2011-02-08 | Isis Pharmaceuticals, Inc. | Conjugates for use in hepatocyte free uptake assays |
EP2292739A1 (en) | 2006-03-24 | 2011-03-09 | Institut National De La Recherche Agronomique | Method for preparing differentiated avian cells and genes involved in the maintenance of pluripotency |
US20110070627A1 (en) * | 2008-04-29 | 2011-03-24 | Eberhard-Karls-Universitaet Tuebingen Universitaetsklinikum | Composition for the cultivation of sophisticated bacteria |
US8030066B2 (en) | 2000-12-11 | 2011-10-04 | Life Technologies Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites |
US8058448B2 (en) | 2004-04-05 | 2011-11-15 | Alnylam Pharmaceuticals, Inc. | Processes and reagents for sulfurization of oligonucleotides |
US8084422B2 (en) | 2007-04-04 | 2011-12-27 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method of treating insulin resistance with a selective inhibitor of CB2 receptor activity |
EP2423304A1 (en) | 2010-08-30 | 2012-02-29 | IMBA-Institut für Molekulare Biotechnologie GmbH | Use of a RNA ligase |
EP2433644A1 (en) | 2010-09-22 | 2012-03-28 | IMBA-Institut für Molekulare Biotechnologie GmbH | Breast cancer therapeutics |
WO2012084715A1 (en) | 2010-12-20 | 2012-06-28 | Schmuelling Thomas | Disruption of ahp6 gene leads to plants with improved seed yield |
US8304189B2 (en) | 2003-12-01 | 2012-11-06 | Life Technologies Corporation | Nucleic acid molecules containing recombination sites and methods of using the same |
EP2548578A1 (en) | 2006-05-17 | 2013-01-23 | The Ludwig Institute for Cancer Research | Targeting VEGF-B regulation of fatty acid transporters to modulate human diseases |
US8394947B2 (en) | 2004-06-03 | 2013-03-12 | Isis Pharmaceuticals, Inc. | Positionally modified siRNA constructs |
US20130160156A1 (en) * | 2010-06-08 | 2013-06-20 | Devgen Private Limited | Method for down-regulating gene expression in fungi |
US8470792B2 (en) | 2008-12-04 | 2013-06-25 | Opko Pharmaceuticals, Llc. | Compositions and methods for selective inhibition of VEGF |
US20130180001A1 (en) * | 2009-11-30 | 2013-07-11 | Jean-Philippe Vielle-Calzada | Plants that reproduce via unreduced gametes |
US20130197207A1 (en) * | 2010-06-01 | 2013-08-01 | Universidty of Kentucky Research Foundation | Method of inhibiting alu rna and therapeutic uses thereof |
US8569474B2 (en) | 2004-03-09 | 2013-10-29 | Isis Pharmaceuticals, Inc. | Double stranded constructs comprising one or more short strands hybridized to a longer strand |
WO2013160291A2 (en) | 2012-04-23 | 2013-10-31 | Imba - Institut Für Molekulare Biotechnologie Gmbh | Archease as rna ligase complex member |
US8604183B2 (en) | 2002-11-05 | 2013-12-10 | Isis Pharmaceuticals, Inc. | Compositions comprising alternating 2′-modified nucleosides for use in gene modulation |
EP2700949A1 (en) | 2012-08-24 | 2014-02-26 | IMG Institut für medizinische Genomforschung Planungsgesellschaft M.B.H. | Use of biliverdin reductase proteins as cancer marker |
US8664189B2 (en) | 2008-09-22 | 2014-03-04 | Rxi Pharmaceuticals Corporation | RNA interference in skin indications |
US8815818B2 (en) | 2008-07-18 | 2014-08-26 | Rxi Pharmaceuticals Corporation | Phagocytic cell delivery of RNAI |
US8815821B2 (en) | 2002-02-01 | 2014-08-26 | Life Technologies Corporation | Double-stranded oligonucleotides |
EP2806025A1 (en) | 2002-09-05 | 2014-11-26 | California Institute of Technology | Use of zinc finger nucleases to stimulate gene targeting |
US9074211B2 (en) | 2008-11-19 | 2015-07-07 | Rxi Pharmaceuticals Corporation | Inhibition of MAP4K4 through RNAI |
US9074213B2 (en) | 2001-01-09 | 2015-07-07 | Alnylam Pharmacuticals, Inc. | Compositions and methods for inhibiting expression of a target gene |
US9080171B2 (en) | 2010-03-24 | 2015-07-14 | RXi Parmaceuticals Corporation | Reduced size self-delivering RNAi compounds |
US9095504B2 (en) | 2010-03-24 | 2015-08-04 | Rxi Pharmaceuticals Corporation | RNA interference in ocular indications |
US9096636B2 (en) | 1996-06-06 | 2015-08-04 | Isis Pharmaceuticals, Inc. | Chimeric oligomeric compounds and their use in gene modulation |
US9340786B2 (en) | 2010-03-24 | 2016-05-17 | Rxi Pharmaceuticals Corporation | RNA interference in dermal and fibrotic indications |
WO2016099918A1 (en) | 2014-12-17 | 2016-06-23 | Pioneer Hi Bred International Inc | Modulation of yep6 gene expression to increase yield and other related traits in plants |
US9493774B2 (en) | 2009-01-05 | 2016-11-15 | Rxi Pharmaceuticals Corporation | Inhibition of PCSK9 through RNAi |
EP3124610A1 (en) | 2010-10-28 | 2017-02-01 | Benitec Biopharma Limited | Hbv treatment |
US20170067050A1 (en) * | 2002-07-10 | 2017-03-09 | Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. | Rna-interference by single-stranded rna molecules |
US9745574B2 (en) | 2009-02-04 | 2017-08-29 | Rxi Pharmaceuticals Corporation | RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality |
US9777275B2 (en) | 2002-02-01 | 2017-10-03 | Life Technologies Corporation | Oligonucleotide compositions with enhanced efficiency |
US10131904B2 (en) | 2008-02-11 | 2018-11-20 | Rxi Pharmaceuticals Corporation | Modified RNAi polynucleotides and uses thereof |
WO2018229251A1 (en) | 2017-06-16 | 2018-12-20 | Imba - Institut Für Molekulare Biotechnologie Gmbh | Blood vessel organoid, methods of producing and using said organoids |
US10240153B2 (en) | 2014-07-16 | 2019-03-26 | Arrowhead Pharmaceuticals, Inc. | Organic compositions to treat APOC3-related diseases |
EP3693384A1 (en) | 2014-03-11 | 2020-08-12 | Cellectis | Method for generating t-cells compatible for allogenic transplantation |
US10808247B2 (en) | 2015-07-06 | 2020-10-20 | Phio Pharmaceuticals Corp. | Methods for treating neurological disorders using a synergistic small molecule and nucleic acids therapeutic approach |
US10900039B2 (en) | 2014-09-05 | 2021-01-26 | Phio Pharmaceuticals Corp. | Methods for treating aging and skin disorders using nucleic acids targeting Tyr or MMP1 |
US10934550B2 (en) | 2013-12-02 | 2021-03-02 | Phio Pharmaceuticals Corp. | Immunotherapy of cancer |
US11001845B2 (en) | 2015-07-06 | 2021-05-11 | Phio Pharmaceuticals Corp. | Nucleic acid molecules targeting superoxide dismutase 1 (SOD1) |
US11021707B2 (en) | 2015-10-19 | 2021-06-01 | Phio Pharmaceuticals Corp. | Reduced size self-delivering nucleic acid compounds targeting long non-coding RNA |
US11214801B2 (en) | 2017-09-11 | 2022-01-04 | Arrowhead Pharmaceuticals, Inc. | RNAi agents and compositions for inhibiting expression of apolipoprotein C-III (APOC3) |
US11279934B2 (en) | 2014-04-28 | 2022-03-22 | Phio Pharmaceuticals Corp. | Methods for treating cancer using nucleic acids targeting MDM2 or MYCN |
WO2023237873A1 (en) | 2022-06-07 | 2023-12-14 | The University Court Of The University Of Glasgow | Targets for cancer therapy |
Families Citing this family (455)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6605712B1 (en) | 1990-12-20 | 2003-08-12 | Arch Development Corporation | Gene transcription and ionizing radiation: methods and compositions |
US20040266706A1 (en) * | 2002-11-05 | 2004-12-30 | Muthiah Manoharan | Cross-linked oligomeric compounds and their use in gene modulation |
US20040203024A1 (en) * | 1996-06-06 | 2004-10-14 | Baker Brenda F. | Modified oligonucleotides for use in RNA interference |
US20050042647A1 (en) * | 1996-06-06 | 2005-02-24 | Baker Brenda F. | Phosphorous-linked oligomeric compounds and their use in gene modulation |
US20050119470A1 (en) * | 1996-06-06 | 2005-06-02 | Muthiah Manoharan | Conjugated oligomeric compounds and their use in gene modulation |
US20040171031A1 (en) * | 1996-06-06 | 2004-09-02 | Baker Brenda F. | Sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation |
US20050053976A1 (en) * | 1996-06-06 | 2005-03-10 | Baker Brenda F. | Chimeric oligomeric compounds and their use in gene modulation |
US20040171028A1 (en) * | 1996-06-06 | 2004-09-02 | Baker Brenda F. | Phosphorous-linked oligomeric compounds and their use in gene modulation |
US20050118605A9 (en) * | 1996-06-06 | 2005-06-02 | Baker Brenda F. | Oligomeric compounds having modified bases for binding to adenine and guanine and their use in gene modulation |
US20040171032A1 (en) * | 1996-06-06 | 2004-09-02 | Baker Brenda F. | Non-phosphorous-linked oligomeric compounds and their use in gene modulation |
US20040161844A1 (en) * | 1996-06-06 | 2004-08-19 | Baker Brenda F. | Sugar and backbone-surrogate-containing oligomeric compounds and compositions for use in gene modulation |
US20040171030A1 (en) * | 1996-06-06 | 2004-09-02 | Baker Brenda F. | Oligomeric compounds having modified bases for binding to cytosine and uracil or thymine and their use in gene modulation |
US20040161777A1 (en) * | 1996-06-06 | 2004-08-19 | Baker Brenda F. | Modified oligonucleotides for use in RNA interference |
US6586661B1 (en) | 1997-06-12 | 2003-07-01 | North Carolina State University | Regulation of quinolate phosphoribosyl transferase expression by transformation with a tobacco quinolate phosphoribosyl transferase nucleic acid |
US7601494B2 (en) | 1999-03-17 | 2009-10-13 | The University Of North Carolina At Chapel Hill | Method of screening candidate compounds for susceptibility to biliary excretion |
WO2000063364A2 (en) * | 1999-04-21 | 2000-10-26 | American Home Products Corporation | Methods and compositions for inhibiting the function of polynucleotide sequences |
US20040138168A1 (en) * | 1999-04-21 | 2004-07-15 | Wyeth | Methods and compositions for inhibiting the function of polynucleotide sequences |
US20030181412A1 (en) * | 1999-12-21 | 2003-09-25 | Ingeneus Corporation | Method for modifying transcription and/or translation in an organism for therapeutic, prophylactic and/or analytic uses |
WO2003070918A2 (en) | 2002-02-20 | 2003-08-28 | Ribozyme Pharmaceuticals, Incorporated | Rna interference by modified short interfering nucleic acid |
US8202979B2 (en) | 2002-02-20 | 2012-06-19 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid |
US20030084471A1 (en) * | 2000-03-16 | 2003-05-01 | David Beach | Methods and compositions for RNA interference |
US8202846B2 (en) | 2000-03-16 | 2012-06-19 | Cold Spring Harbor Laboratory | Methods and compositions for RNA interference |
JP2003526367A (en) * | 2000-03-16 | 2003-09-09 | ジェネティカ インコーポレイテッド | RNA interference method and RNA interference composition |
DK2360253T3 (en) * | 2000-03-30 | 2014-06-16 | Whitehead Biomedical Inst | Process for the preparation of knockdown cells or knockdown organisms by RNA sequence specific mediators of RNA interference and applications thereof |
US20030190635A1 (en) | 2002-02-20 | 2003-10-09 | Mcswiggen James A. | RNA interference mediated treatment of Alzheimer's disease using short interfering RNA |
US20020165192A1 (en) | 2000-09-19 | 2002-11-07 | Kerr William G. | Control of NK cell function and survival by modulation of ship activity |
US7575924B2 (en) | 2000-11-13 | 2009-08-18 | Research Development Foundation | Methods and compositions relating to improved lentiviral vectors and their applications |
EP1229134A3 (en) | 2001-01-31 | 2004-01-28 | Nucleonics, Inc | Use of post-transcriptional gene silencing for identifying nucleic acid sequences that modulate the function of a cell |
WO2002066638A1 (en) * | 2001-02-22 | 2002-08-29 | Gencom Corporation | Recombinant gene containing inverted repeat sequence and utilization thereof |
US8034791B2 (en) | 2001-04-06 | 2011-10-11 | The University Of Chicago | Activation of Egr-1 promoter by DNA damaging chemotherapeutics |
US20050148530A1 (en) | 2002-02-20 | 2005-07-07 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA) |
US20030175950A1 (en) * | 2001-05-29 | 2003-09-18 | Mcswiggen James A. | RNA interference mediated inhibition of HIV gene expression using short interfering RNA |
US7109165B2 (en) | 2001-05-18 | 2006-09-19 | Sirna Therapeutics, Inc. | Conjugates and compositions for cellular delivery |
US9994853B2 (en) | 2001-05-18 | 2018-06-12 | Sirna Therapeutics, Inc. | Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference |
EP1409506B1 (en) | 2001-07-23 | 2012-05-09 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for rnai mediated inhibition of gene expression in mammals |
US10590418B2 (en) | 2001-07-23 | 2020-03-17 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for RNAi mediated inhibition of gene expression in mammals |
WO2003012054A2 (en) | 2001-08-02 | 2003-02-13 | Institut Clayton De La Recherche | Methods and compositions relating to improved lentiviral vector production systems |
EP1446503A2 (en) | 2001-09-27 | 2004-08-18 | University Of Delaware | Composition and methods for enhancing oligonucleotide-directed nucleic acid sequence alteration |
KR20040054699A (en) | 2001-10-02 | 2004-06-25 | 엥스띠뛰 끌레이톤 드 라 러쉐르쉬 | Methods and compositions relating to restricted expression lentiviral vectors and their applications |
JP2003144141A (en) * | 2001-11-14 | 2003-05-20 | Gencom Co | ES CELL HAVING ENHANCED RNAi EFFECT |
AU2002354121A1 (en) * | 2001-11-28 | 2003-06-10 | Toudai Tlo, Ltd. | siRNA Expression System and Method for Producing Functional Gene Knockdown Cell Using the Same |
WO2003048371A2 (en) * | 2001-12-07 | 2003-06-12 | University Of Liverpool | Dna vaccine |
US7294504B1 (en) | 2001-12-27 | 2007-11-13 | Allele Biotechnology & Pharmaceuticals, Inc. | Methods and compositions for DNA mediated gene silencing |
JP2005532787A (en) * | 2002-02-06 | 2005-11-04 | エクセリクシス・インコーポレイテッド | MINR as a modifier of insulin receptor signaling and methods of use thereof |
ATE519774T1 (en) * | 2002-02-20 | 2011-08-15 | Sirna Therapeutics Inc | RNA DISRUPTION-MEDIATED INHIBITION OF HEPATITIS C VIRUS (HCV) GENE EXPRESSION WITH SHORT INTERFERING NUCLEIC ACID (SINA) |
EP1495041A4 (en) * | 2002-02-20 | 2006-02-01 | Sirna Therapeutics Inc | RNA INTERFERENCE MEDIATED INHIBITION OF G72 AND D-AMINO ACID OXIDASE (DAAO) GENE EXPRESSION USING SHORT INTERFERING NUCLEIC ACID (siNA) |
US9657294B2 (en) | 2002-02-20 | 2017-05-23 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA) |
AU2003207708A1 (en) | 2002-02-20 | 2003-09-09 | Sirna Therapeutics, Inc. | Rna interference mediated inhibition of map kinase genes |
US9181551B2 (en) | 2002-02-20 | 2015-11-10 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA) |
AU2003207099A1 (en) * | 2002-02-22 | 2003-09-09 | Otsuka Pharmaceutical Co., Ltd. | Polynucleotide for target gene |
EP1572923A4 (en) * | 2002-03-06 | 2007-10-31 | Rigel Pharmaceuticals Inc | Novel method for delivery and intracellular synthesis of sirna molecules |
US7357928B2 (en) | 2002-04-08 | 2008-04-15 | University Of Louisville Research Foundation, Inc. | Method for the diagnosis and prognosis of malignant diseases |
US7541150B2 (en) | 2002-04-08 | 2009-06-02 | University Of Louisville Research Foundation, Inc | Method for the diagnosis and prognosis of malignant diseases |
WO2003095652A2 (en) * | 2002-05-08 | 2003-11-20 | Xantos Biomedicine Ag | Expression constructs for producing double-stranded (ds) rna and the use thereof |
JP2006506961A (en) | 2002-05-23 | 2006-03-02 | セプティア, インコーポレイテッド | Regulation of PTP1B signal transduction by RNA interference |
JP4171256B2 (en) * | 2002-07-18 | 2008-10-22 | 三菱化学株式会社 | Method for producing non-human mammal having RNAi phenotype using papillomavirus vector |
WO2004018676A2 (en) * | 2002-08-21 | 2004-03-04 | The University Of British Columbia | Rnai probes targeting cancer-related proteins |
AU2003268531A1 (en) | 2002-09-06 | 2004-03-29 | University Of South Florida | Materials and methods for treatment of allergic diseases |
US20080260744A1 (en) | 2002-09-09 | 2008-10-23 | Omeros Corporation | G protein coupled receptors and uses thereof |
EP1575518A4 (en) | 2002-10-10 | 2007-08-22 | Wyeth Corp | Compositions, organisms and methodologies employing a novel human kinase |
DE60325494D1 (en) * | 2002-10-17 | 2009-02-05 | Taconicartemis Gmbh | SIRNA-CONTROLLED GENE EXPRESSION SUPPRESSION IN TRANSGENETIC ANIMALS |
EP1554385A2 (en) | 2002-10-24 | 2005-07-20 | Wyeth | Calcineurin-like human phoshphoesterase |
WO2004041889A2 (en) * | 2002-11-05 | 2004-05-21 | Isis Pharmaceuticals, Inc. | Polycyclic sugar surrogate-containing oligomeric compounds and compositions for use in gene modulation |
AU2003287464A1 (en) * | 2002-11-05 | 2004-06-03 | Isis Pharmaceuticals, Inc. | 2'-fluoro substituted oligomeric compounds and compositions for use in gene modulations |
US9150605B2 (en) * | 2002-11-05 | 2015-10-06 | Isis Pharmaceuticals, Inc. | Compositions comprising alternating 2′-modified nucleosides for use in gene modulation |
US9150606B2 (en) * | 2002-11-05 | 2015-10-06 | Isis Pharmaceuticals, Inc. | Compositions comprising alternating 2'-modified nucleosides for use in gene modulation |
US9719094B2 (en) | 2002-11-14 | 2017-08-01 | Thermo Fisher Scientific Inc. | RNAi targeting SEC61G |
US9839649B2 (en) | 2002-11-14 | 2017-12-12 | Thermo Fisher Scientific Inc. | Methods and compositions for selecting siRNA of improved functionality |
US9879266B2 (en) | 2002-11-14 | 2018-01-30 | Thermo Fisher Scientific Inc. | Methods and compositions for selecting siRNA of improved functionality |
WO2006006948A2 (en) | 2002-11-14 | 2006-01-19 | Dharmacon, Inc. | METHODS AND COMPOSITIONS FOR SELECTING siRNA OF IMPROVED FUNCTIONALITY |
US8163896B1 (en) | 2002-11-14 | 2012-04-24 | Rosetta Genomics Ltd. | Bioinformatically detectable group of novel regulatory genes and uses thereof |
US9771586B2 (en) | 2002-11-14 | 2017-09-26 | Thermo Fisher Scientific Inc. | RNAi targeting ZNF205 |
US9719092B2 (en) | 2002-11-14 | 2017-08-01 | Thermo Fisher Scientific Inc. | RNAi targeting CNTD2 |
US7250496B2 (en) | 2002-11-14 | 2007-07-31 | Rosetta Genomics Ltd. | Bioinformatically detectable group of novel regulatory genes and uses thereof |
US9228186B2 (en) | 2002-11-14 | 2016-01-05 | Thermo Fisher Scientific Inc. | Methods and compositions for selecting siRNA of improved functionality |
US7655785B1 (en) | 2002-11-14 | 2010-02-02 | Rosetta Genomics Ltd. | Bioinformatically detectable group of novel regulatory oligonucleotides and uses thereof |
US10011836B2 (en) | 2002-11-14 | 2018-07-03 | Thermo Fisher Scientific Inc. | Methods and compositions for selecting siRNA of improved functionality |
JP2006514554A (en) | 2002-11-21 | 2006-05-11 | ワイス | Diagnosis of renal cell carcinoma and other solid tumors |
US20130130231A1 (en) | 2002-11-26 | 2013-05-23 | Isaac Bentwich | Bioinformatically detectable group of novel viral regulatory genes and uses thereof |
US7217807B2 (en) | 2002-11-26 | 2007-05-15 | Rosetta Genomics Ltd | Bioinformatically detectable group of novel HIV regulatory genes and uses thereof |
US7696334B1 (en) | 2002-12-05 | 2010-04-13 | Rosetta Genomics, Ltd. | Bioinformatically detectable human herpesvirus 5 regulatory gene |
WO2004050831A2 (en) | 2002-11-27 | 2004-06-17 | Wyeth | Compositions, organisms and methodologies employing a novel human kinase |
US20040248299A1 (en) * | 2002-12-27 | 2004-12-09 | Sumedha Jayasena | RNA interference |
GB0306715D0 (en) * | 2003-03-24 | 2003-04-30 | Novartis Ag | Organic compounds |
US8158420B2 (en) | 2003-04-04 | 2012-04-17 | The Trustees Of Columbia University In The City Of New York | Methods for inhibiting the differentation of proliferative telencephalic cells in vitro by addition of ATF5 |
WO2004101756A2 (en) | 2003-05-09 | 2004-11-25 | Diadexus, Inc. | Ovr110 antibody compositions and methods of use |
WO2005000403A2 (en) | 2003-05-19 | 2005-01-06 | The Trustees Of Columbia University In The City Of New York | Compositions and methods for treating and preventing heart tissue degeneration, and uses thereof |
JP4505749B2 (en) | 2003-05-30 | 2010-07-21 | 日本新薬株式会社 | Oligo double-stranded RNA that suppresses expression of Bcl-2 and pharmaceutical composition containing the same |
EP2241572A3 (en) | 2003-06-03 | 2011-04-06 | Eli Lilly And Company | Modulation of survivin expression |
US20060241072A1 (en) * | 2003-06-20 | 2006-10-26 | Isis Pharmaceuticals, Inc. | Oligomeric compounds for use in gene modulation |
AU2004257167B2 (en) * | 2003-07-03 | 2012-03-29 | The Trustees Of The University Of Pennsylvania | Inhibition of Syk kinase expression |
US7683036B2 (en) * | 2003-07-31 | 2010-03-23 | Regulus Therapeutics Inc. | Oligomeric compounds and compositions for use in modulation of small non-coding RNAs |
US7888497B2 (en) | 2003-08-13 | 2011-02-15 | Rosetta Genomics Ltd. | Bioinformatically detectable group of novel regulatory oligonucleotides and uses thereof |
WO2005040388A2 (en) * | 2003-08-22 | 2005-05-06 | Nucleonics Inc. | Eukariotic expression systems for expression of inhibitory rna in multiple intracellular compartments |
JP2007505887A (en) * | 2003-09-16 | 2007-03-15 | ファルマシア・コーポレーション | PACE4 inhibitor for the treatment of arthritis |
US7553822B2 (en) | 2003-10-30 | 2009-06-30 | The United States Of America As Represented By The Department Of Health And Human Services | Compositions and methods for inhibiting translation of a Mect1-MAML2 chimeric gene |
WO2005081714A2 (en) * | 2003-11-21 | 2005-09-09 | Revivicor, Inc. | Use of interfering rna in the production of transgenic animals |
WO2005068630A1 (en) * | 2003-12-16 | 2005-07-28 | National Institute Of Advanced Industrial Science And Technology | Double-stranded rna for interference |
JP2007520221A (en) | 2004-01-23 | 2007-07-26 | ニュー・イングランド・バイオラブズ・インコーポレイティッド | Composition and production method of short double-stranded RNA using mutant RNase |
CA2554212A1 (en) | 2004-02-10 | 2005-08-25 | Sirna Therapeutics, Inc. | Rna interference mediated inhibition of gene expression using multifunctional short interfering nucleic acid (multifunctional sina) |
US20060019914A1 (en) | 2004-02-11 | 2006-01-26 | University Of Tennessee Research Foundation | Inhibition of tumor growth and invasion by anti-matrix metalloproteinase DNAzymes |
CA2559853A1 (en) | 2004-02-17 | 2005-10-13 | University Of South Florida | Materials and methods for treatment of inflammatory and cell proliferation disorders |
AU2005231692B2 (en) | 2004-03-26 | 2011-01-27 | Curis, Inc. | RNA interference modulators of Hedgehog signaling and uses thereof |
CA2557532A1 (en) | 2004-04-23 | 2005-11-10 | Angela M. Christiano | Inhibition of hairless protein mrna |
EP1784501B1 (en) | 2004-05-14 | 2015-11-18 | Rosetta Genomics Ltd | VIRAL AND VIRUS ASSOCIATED MicroRNAS AND USES THEREOF |
US7687616B1 (en) | 2004-05-14 | 2010-03-30 | Rosetta Genomics Ltd | Small molecules modulating activity of micro RNA oligonucleotides and micro RNA targets and uses thereof |
DE102004025881A1 (en) | 2004-05-19 | 2006-01-05 | Beiersdorf Ag | Oligoribonucleotides for influencing hair growth |
US10508277B2 (en) | 2004-05-24 | 2019-12-17 | Sirna Therapeutics, Inc. | Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference |
US7795419B2 (en) | 2004-05-26 | 2010-09-14 | Rosetta Genomics Ltd. | Viral and viral associated miRNAs and uses thereof |
EP1765416A4 (en) * | 2004-06-03 | 2010-03-24 | Isis Pharmaceuticals Inc | Double strand compositions comprising differentially modified strands for use in gene modulation |
EP1773375A1 (en) | 2004-07-14 | 2007-04-18 | University of Utah Research Foundation | Netrin-related compositions and uses |
WO2007001324A2 (en) | 2004-07-23 | 2007-01-04 | The University Of North Carolina At Chapel Hill | Methods and materials for determining pain sensitivity and predicting and treating related disorders |
US7582741B2 (en) * | 2004-07-26 | 2009-09-01 | University Of Massachusetts | Conditional disruption of dicer1 in cell lines and non-human mammals |
EP1789070B1 (en) | 2004-08-03 | 2012-10-24 | Biogen Idec MA Inc. | Taj in neuronal function |
US20060057590A1 (en) * | 2004-09-14 | 2006-03-16 | Azeddine Si-Ammour | RNA probes |
ATE487484T1 (en) | 2004-09-18 | 2010-11-15 | Univ Maryland | THERAPEUTIC AGENTS FOR TARGETING THE NC CA ATP CHANNEL AND METHOD OF USE THEREOF |
US10583094B2 (en) | 2004-09-18 | 2020-03-10 | University Of Maryland | Therapeutic methods that target the NCCA-ATP channel |
MX2007003795A (en) | 2004-09-28 | 2007-07-11 | Quark Biotech Inc | Oligoribonucleotides and methods of use thereof for treatment of alopecia, acute renal failure and other diseases. |
WO2006042112A2 (en) | 2004-10-05 | 2006-04-20 | California Institute Of Technology | Aptamer regulated nucleic acids and uses thereof |
SG158921A1 (en) * | 2004-10-27 | 2010-02-26 | Schering Corp | Compositions and methods for short interfering nucleic acid inhibition of nav1.8 |
US7517870B2 (en) | 2004-12-03 | 2009-04-14 | Fondazione Telethon | Use of compounds that interfere with the hedgehog signaling pathway for the manufacture of a medicament for preventing, inhibiting, and/or reversing ocular diseases related with ocular neovascularization |
US7361752B2 (en) * | 2004-12-14 | 2008-04-22 | Alnylam Pharmaceuticals, Inc. | RNAi modulation of MLL-AF4 and uses thereof |
US8137907B2 (en) | 2005-01-03 | 2012-03-20 | Cold Spring Harbor Laboratory | Orthotopic and genetically tractable non-human animal model for liver cancer and the uses thereof |
JP2008526883A (en) | 2005-01-07 | 2008-07-24 | ディアデクサス インコーポレーテッド | Ovr110 antibody compositions and methods of use |
CA2593238C (en) | 2005-01-07 | 2014-11-18 | State Of Oregon Acting By And Through The State Board Of Higher Education On Behalf Of Oregon State University | Method to trigger rna interference |
US20060178334A1 (en) | 2005-02-04 | 2006-08-10 | City Of Hope | Double-stranded and single-stranded RNA molecules with 5 ' triphosphates and their use for inducing interferon |
RS59399B1 (en) | 2005-03-23 | 2019-11-29 | Genmab As | Antibodies against cd38 for treatment of multiple myeloma |
ES2440953T3 (en) | 2005-03-31 | 2014-01-31 | The General Hospital Corporation | Modulation of HGF / HGFR activity to treat lymphedema |
KR20060119412A (en) * | 2005-05-20 | 2006-11-24 | 아주대학교산학협력단 | Sirna for inhibiting il-6 expression and composition containing them |
US20070044164A1 (en) | 2005-05-31 | 2007-02-22 | Cold Spring Harbor Laboratory | Methods for producing microRNAs |
US8703769B2 (en) | 2005-07-15 | 2014-04-22 | The University Of North Carolina At Chapel Hill | Use of EGFR inhibitors to prevent or treat obesity |
US20070036773A1 (en) * | 2005-08-09 | 2007-02-15 | City Of Hope | Generation and application of universal T cells for B-ALL |
CA2620387C (en) | 2005-09-20 | 2018-09-18 | Basf Plant Science Gmbh | Methods for controlling gene expression using ta-sirna |
WO2007035744A1 (en) | 2005-09-20 | 2007-03-29 | Osi Pharmaceuticals, Inc. | Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors |
EP1965814A4 (en) | 2005-09-26 | 2012-01-11 | Univ Columbia | Side population cells in cardiac repair |
JP2007104969A (en) * | 2005-10-13 | 2007-04-26 | Bio Think Tank Co Ltd | NUCLEIC ACID FOR PRODUCING SHORT HAIRPIN RNA (shRNA) PRECURSOR, AND UTILIZATION THEREOF |
CA3046754A1 (en) | 2005-11-29 | 2007-06-07 | Cambridge Enterprise Limited | Markers for breast cancer including refsnp_id 3817198 |
CA2637254A1 (en) * | 2006-01-17 | 2007-07-26 | Biolex Therapeutics, Inc. | Compositions and methods for humanization and optimization of n-glycans in plants |
US20090060921A1 (en) * | 2006-01-17 | 2009-03-05 | Biolex Therapeutics, Inc. | Glycan-optimized anti-cd20 antibodies |
TWI428143B (en) | 2006-01-18 | 2014-03-01 | Gen Hospital Corp | Methods of increasing lymphatic function |
US7825099B2 (en) | 2006-01-20 | 2010-11-02 | Quark Pharmaceuticals, Inc. | Treatment or prevention of oto-pathologies by inhibition of pro-apoptotic genes |
NL2000439C2 (en) | 2006-01-20 | 2009-03-16 | Quark Biotech | Therapeutic applications of inhibitors of RTP801. |
EP1981902B1 (en) | 2006-01-27 | 2015-07-29 | Biogen MA Inc. | Nogo receptor antagonists |
US9150882B2 (en) | 2006-01-31 | 2015-10-06 | The Board Of Trustees Of The Leland Stanford Junior University | Self-complementary parvoviral vectors, and methods for making and using the same |
US9044461B2 (en) | 2006-04-07 | 2015-06-02 | The Research Foundation Of State University Of New York | Transcobalamin receptor polypeptides, nucleic acids, and modulators thereof, and related methods of use in modulating cell growth and treating cancer and cobalamin deficiency |
EP2010226B1 (en) | 2006-04-07 | 2014-01-15 | The Research Foundation of State University of New York | Transcobalamin receptor polypeptides, nucleic acids, and modulators thereof, and related methods of use in modulating cell growth and treating cancer and cobalamin deficiency |
CA2656990A1 (en) | 2006-04-28 | 2007-11-08 | University Of South Florida | Materials and methods for reducing inflammation by inhibition of the atrial natriuretic peptide receptor |
EP1886685A1 (en) | 2006-08-11 | 2008-02-13 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods, uses and compositions for modulating replication of hcv through the farnesoid x receptor (fxr) activation or inhibition |
WO2008058291A2 (en) * | 2006-11-09 | 2008-05-15 | California Institute Of Technology | Modular aptamer-regulated ribozymes |
WO2008067283A2 (en) | 2006-11-27 | 2008-06-05 | Diadexus, Inc. | Ovr110 antibody compositions and methods of use |
US20080199475A1 (en) | 2006-11-27 | 2008-08-21 | Patrys Limited | Novel glycosylated peptide target in neoplastic cells |
WO2008089103A2 (en) | 2007-01-12 | 2008-07-24 | University Of Maryland, Baltimore | Targeting ncca-atp channel for organ protection following ischemic episode |
CN101641010A (en) | 2007-01-26 | 2010-02-03 | 路易斯维尔大学研究基金会公司 | Be used as the modification of the allochthon component of vaccine |
CA2676613A1 (en) * | 2007-01-29 | 2008-08-07 | Wyeth | Immunophilin ligands and methods for modulating immunophilin and calcium channel activity |
DK2129680T3 (en) | 2007-03-21 | 2015-08-10 | Brookhaven Science Ass Llc | COMBINED hairpin ANTISENSE COMPOSITIONS AND METHODS FOR MODULATING EXPRESSION OF |
CN101986785A (en) | 2007-05-11 | 2011-03-16 | 托马斯杰弗逊大学 | Methods of treatment and prevention of neurodegenerative diseases and disorders |
US9029383B2 (en) | 2007-05-11 | 2015-05-12 | The Trustees Of The University Of Pennsylvania | Methods of treatment of skin ulcers |
WO2009002832A2 (en) | 2007-06-22 | 2008-12-31 | University Of Maryland, Baltimore | Inhibitors of ncca-atp channels for therapy |
ES2474176T3 (en) | 2007-06-27 | 2014-07-08 | Quark Pharmaceuticals, Inc. | Compositions and methods to inhibit the expression of pro-apoptotic genes |
US20090082217A1 (en) * | 2007-07-16 | 2009-03-26 | California Institute Of Technology | Selection of nucleic acid-based sensor domains within nucleic acid switch platform |
US20120165387A1 (en) | 2007-08-28 | 2012-06-28 | Smolke Christina D | General composition framework for ligand-controlled RNA regulatory systems |
US8367815B2 (en) * | 2007-08-28 | 2013-02-05 | California Institute Of Technology | Modular polynucleotides for ligand-controlled regulatory systems |
US8865667B2 (en) | 2007-09-12 | 2014-10-21 | California Institute Of Technology | Higher-order cellular information processing devices |
CN101815521B (en) | 2007-10-03 | 2014-12-10 | 夸克制药公司 | Novel siRNA structures |
US7968525B1 (en) | 2007-12-03 | 2011-06-28 | University Of Florida Research Foundation, Inc. | Use of RNA interference to validate new termiticide target sites and a method of termite control |
US9029524B2 (en) * | 2007-12-10 | 2015-05-12 | California Institute Of Technology | Signal activated RNA interference |
US8614311B2 (en) | 2007-12-12 | 2013-12-24 | Quark Pharmaceuticals, Inc. | RTP801L siRNA compounds and methods of use thereof |
DK2245464T3 (en) | 2008-01-25 | 2017-02-20 | Multivir Inc | P53 BIOMARKETS |
WO2009117513A2 (en) * | 2008-03-21 | 2009-09-24 | The Regents Of The University Of California | Modified dicer polypeptide and methods of use thereof |
US8278287B2 (en) | 2008-04-15 | 2012-10-02 | Quark Pharmaceuticals Inc. | siRNA compounds for inhibiting NRF2 |
WO2009147684A2 (en) | 2008-06-06 | 2009-12-10 | Quark Pharmaceuticals, Inc. | Compositions and methods for treatment of ear disorders |
NZ590605A (en) | 2008-07-09 | 2012-11-30 | Biogen Idec Inc | Compositions comprising antibodies to lingo or fragments thereof |
WO2010033560A2 (en) | 2008-09-16 | 2010-03-25 | University Of Maryland, Baltimore | Sur1 inhibitors for therapy |
EP2349235A1 (en) | 2008-11-07 | 2011-08-03 | Triact Therapeutics, Inc. | Use of catecholic butane derivatives in cancer therapy |
EP2201982A1 (en) | 2008-12-24 | 2010-06-30 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Histamine H4 receptor antagonists for the treatment of vestibular disorders |
US8329882B2 (en) | 2009-02-18 | 2012-12-11 | California Institute Of Technology | Genetic control of mammalian cells with synthetic RNA regulatory systems |
US20120189641A1 (en) | 2009-02-25 | 2012-07-26 | OSI Pharmaceuticals, LLC | Combination anti-cancer therapy |
JP2012519170A (en) | 2009-02-26 | 2012-08-23 | オーエスアイ・ファーマシューティカルズ,エルエルシー | INSITU method for monitoring EMT status of tumor cells in vivo |
WO2010099138A2 (en) | 2009-02-27 | 2010-09-02 | Osi Pharmaceuticals, Inc. | Methods for the identification of agents that inhibit mesenchymal-like tumor cells or their formation |
WO2010099364A2 (en) | 2009-02-27 | 2010-09-02 | Osi Pharmaceuticals, Inc. | Methods for the identification of agents that inhibit mesenchymal-like tumor cells or their formation |
EP2401614A1 (en) | 2009-02-27 | 2012-01-04 | OSI Pharmaceuticals, LLC | Methods for the identification of agents that inhibit mesenchymal-like tumor cells or their formation |
US8748493B2 (en) | 2009-03-20 | 2014-06-10 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Inhibitors of cathepsin S for prevention or treatment of obesity-associated disorders |
US9145555B2 (en) | 2009-04-02 | 2015-09-29 | California Institute Of Technology | Integrated—ligand-responsive microRNAs |
WO2010115874A1 (en) | 2009-04-07 | 2010-10-14 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the treatment and the diagnosis ofpulmonary arterial hypertension |
SI2432467T1 (en) | 2009-05-20 | 2018-06-29 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Serotonin 5-ht3 receptor antagonists for use in the treatment of lesional vestibular disorders |
ES2432618T3 (en) | 2009-05-20 | 2013-12-04 | Inserm (Institut National De La Santé Et De La Recherche Medicale) | Serotonin 5-HT3 receptor antagonists for use in the treatment or prevention of a pathology of the inner ear with vestibular deficit |
EP2445334A1 (en) | 2009-06-26 | 2012-05-02 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Non human animal models for increased retinal vascular permeability |
US9409983B2 (en) | 2009-07-23 | 2016-08-09 | The Board Of Trustess Of The University Of Illinois | Methods and compositions involving PBEF inhibitors for lung inflammation conditions and diseases |
US20120258093A1 (en) | 2009-08-20 | 2012-10-11 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Vla-4 as a biomarker for prognosis and target for therapy in duchenne muscular dystrophy |
US20120219543A1 (en) | 2009-10-20 | 2012-08-30 | Raphael Scharfmann | Methods and pharmaceutical compositions for the treatment of disorders of glucose homeostasis |
WO2011054916A1 (en) | 2009-11-06 | 2011-05-12 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical composition for the treatment of atherosclerosis |
WO2011062997A2 (en) | 2009-11-17 | 2011-05-26 | Musc Foundation For Research Development | Human monoclonal antibodies to human nucleolin |
TW201124160A (en) | 2009-11-26 | 2011-07-16 | Quark Pharmaceuticals Inc | SiRNA compounds comprising terminal substitutions |
KR101168726B1 (en) | 2009-11-30 | 2012-07-30 | 한국생명공학연구원 | Pharmaceutical composition for treating cancer |
US20120283190A1 (en) | 2009-12-09 | 2012-11-08 | Institut National de la Santé et de la Recherche Medicale (INSERM) | Endothelin inhibitors for the treatment of rapidly progressive glomerulonephritis |
US8293718B2 (en) | 2009-12-18 | 2012-10-23 | Novartis Ag | Organic compositions to treat HSF1-related diseases |
WO2011080261A1 (en) | 2009-12-28 | 2011-07-07 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for improved cardiomyogenic differentiation of pluripotent cells |
US9109227B2 (en) | 2010-01-05 | 2015-08-18 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | FLT3 receptor antagonists for the treatment or the prevention of pain disorders |
WO2011084193A1 (en) | 2010-01-07 | 2011-07-14 | Quark Pharmaceuticals, Inc. | Oligonucleotide compounds comprising non-nucleotide overhangs |
PT2523661T (en) | 2010-01-15 | 2017-06-21 | Inserm (Institut Nat De La Santé Et De La Rech Médicale) | Nkcc inhibitors for the treatment of autism |
WO2016187217A2 (en) | 2015-05-18 | 2016-11-24 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for treating aging-associated impairments |
CN102770767A (en) | 2010-02-10 | 2012-11-07 | 诺瓦提斯公司 | Methods and compounds for muscle growth |
CA2783665A1 (en) | 2010-03-03 | 2011-09-09 | OSI Pharmaceuticals, LLC | Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors |
US20110275644A1 (en) | 2010-03-03 | 2011-11-10 | Buck Elizabeth A | Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors |
WO2011118739A1 (en) | 2010-03-26 | 2011-09-29 | 協和発酵キリン株式会社 | Novel antibody having modification site introduced therein, and antibody fragment |
EP2561077B1 (en) | 2010-04-23 | 2016-04-13 | Arrowhead Research Corporation | ORGANIC COMPOSITIONS TO TREAT BETA-ENaC-RELATED DISEASES |
US8993532B2 (en) | 2010-04-23 | 2015-03-31 | Cold Spring Harbor Laboratory | Structurally designed shRNAs |
EP2977084B1 (en) | 2010-05-10 | 2017-07-05 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for the treatment of fluid accumulation in and/ or under the retina |
WO2011157798A1 (en) | 2010-06-16 | 2011-12-22 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for stimulating reepithelialisation during wound healing |
US9095591B2 (en) | 2010-06-28 | 2015-08-04 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Pharmaceutical composition for use in the treatment of glaucoma |
WO2012010696A1 (en) | 2010-07-23 | 2012-01-26 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for cancer management targeting co-029 |
WO2012019991A1 (en) | 2010-08-09 | 2012-02-16 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of hiv-1 infections |
WO2012028703A1 (en) | 2010-09-02 | 2012-03-08 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for the prognosis of the progression of cancer |
EP2621511A1 (en) | 2010-09-28 | 2013-08-07 | INSERM - Institut National de la Santé et de la Recherche Médicale | Methods and pharmaceutical compositions for the treatment of bone density related diseases |
ES2606140T3 (en) | 2010-10-01 | 2017-03-22 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods to predict progression and treat chronic kidney disease in a patient |
US9260471B2 (en) | 2010-10-29 | 2016-02-16 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using short interfering nucleic acids (siNA) |
WO2012072681A1 (en) | 2010-12-01 | 2012-06-07 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Diagnostic and treatment of chronic heart failure |
EP2646555B1 (en) | 2010-12-03 | 2015-04-22 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Pharmaceutical compositions for the treatment of heart failure |
WO2012107589A1 (en) | 2011-02-11 | 2012-08-16 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment and prevention of hcv infections |
US20120214830A1 (en) | 2011-02-22 | 2012-08-23 | OSI Pharmaceuticals, LLC | Biological markers predictive of anti-cancer response to insulin-like growth factor-1 receptor kinase inhibitors in hepatocellular carcinoma |
WO2012120130A1 (en) | 2011-03-09 | 2012-09-13 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods to characterize patients suffering from hemolysis |
US10184942B2 (en) | 2011-03-17 | 2019-01-22 | University Of South Florida | Natriuretic peptide receptor as a biomarker for diagnosis and prognosis of cancer |
WO2012129145A1 (en) | 2011-03-18 | 2012-09-27 | OSI Pharmaceuticals, LLC | Nscle combination therapy |
US9217156B2 (en) | 2011-04-13 | 2015-12-22 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Non human animal model for ulcerative colitis and its main complications |
EP2702173A1 (en) | 2011-04-25 | 2014-03-05 | OSI Pharmaceuticals, LLC | Use of emt gene signatures in cancer drug discovery, diagnostics, and treatment |
EP2702145A1 (en) | 2011-04-28 | 2014-03-05 | Institut National de la Sante et de la Recherche Medicale (INSERM) | Methods for preparing accessory cells and uses thereof for preparing activated nk cells |
EP2714037B1 (en) | 2011-05-25 | 2016-07-13 | Université Paris Descartes | Erk inhibitors for use in treating spinal muscular atrophy |
WO2012163848A1 (en) | 2011-05-27 | 2012-12-06 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of crohn's disease |
EP2714094B1 (en) | 2011-06-02 | 2016-02-24 | The University of Louisville Research Foundation, Inc. | Anti-nucleolin agent-conjugated nanoparticles |
WO2012175711A1 (en) | 2011-06-24 | 2012-12-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for predicting the responsiveness of a patient affected with an osteosarcoma to a chemotherapy |
US20140227293A1 (en) | 2011-06-30 | 2014-08-14 | Trustees Of Boston University | Method for controlling tumor growth, angiogenesis and metastasis using immunoglobulin containing and proline rich receptor-1 (igpr-1) |
US9120858B2 (en) | 2011-07-22 | 2015-09-01 | The Research Foundation Of State University Of New York | Antibodies to the B12-transcobalamin receptor |
EP2737083A1 (en) | 2011-07-27 | 2014-06-04 | INSERM (Institut National de la Santé et de la Recherche Scientifique) | Methods for diagnosing and treating myhre syndrome |
WO2013024022A1 (en) | 2011-08-12 | 2013-02-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for treatment of pulmonary hypertension |
EP2748192B2 (en) | 2011-08-23 | 2022-04-20 | Foundation Medicine, Inc. | Kif5b-ret fusion molecules and uses thereof |
KR20140057356A (en) | 2011-08-31 | 2014-05-12 | 제넨테크, 인크. | Diagnostic markers |
EP2751272A2 (en) | 2011-09-02 | 2014-07-09 | Novartis AG | Organic compositions to treat hsf1-related diseases |
US10040853B2 (en) | 2011-09-09 | 2018-08-07 | Fred Hutchinson Cancer Research Center | Methods and compositions involving NKG2D inhibitors and cancer |
WO2013055530A1 (en) | 2011-09-30 | 2013-04-18 | Genentech, Inc. | Diagnostic methylation markers of epithelial or mesenchymal phenotype and response to egfr kinase inhibitor in tumours or tumour cells |
JP2014528959A (en) | 2011-10-03 | 2014-10-30 | 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 the treatment of Th2-mediated diseases |
WO2013053919A2 (en) | 2011-10-14 | 2013-04-18 | Inserm | Biomarkers of renal disorders |
EP2768971A1 (en) | 2011-10-20 | 2014-08-27 | Institut National de la Sante et de la Recherche Medicale (INSERM) | Methods for the detection and the treatment of cardiac remodeling |
WO2013068836A1 (en) | 2011-11-07 | 2013-05-16 | INSERM (Institut National de la Santé et de la Recherche Médicale) | A ddr1 antagonist or an inhibitor of ddr1 gene expression for use in the prevention or treatment of crescentic glomerulonephritis |
US20140328864A1 (en) | 2011-11-22 | 2014-11-06 | Inserm (Institut National De La Sente Et De La Recherche Medicale) | Methods and pharmaceutical compositions for reducing airway hyperresponse |
CN104302768A (en) | 2012-01-09 | 2015-01-21 | 诺华股份有限公司 | Rnai agents to treat beta-catenin related diseases |
WO2013113762A1 (en) | 2012-01-31 | 2013-08-08 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and kits for predicting the risk of having a cutaneous melanoma in a subject |
WO2013121034A1 (en) | 2012-02-17 | 2013-08-22 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for reducing adipose tissue inflammation |
CA2860994A1 (en) | 2012-03-16 | 2013-09-19 | Klaus P. Hoeflich | Methods of treating melanoma with pak1 inhibitors |
WO2013152252A1 (en) | 2012-04-06 | 2013-10-10 | OSI Pharmaceuticals, LLC | Combination anti-cancer therapy |
EP3272868A1 (en) | 2012-05-02 | 2018-01-24 | Arrowhead Pharmaceuticals, Inc. | Organic compositions to treat kras-related diseases |
EP2844668A1 (en) | 2012-05-03 | 2015-03-11 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method and pharmaceutical composition for use in the treatment and diagnotic of anemia of inflammation |
JP2015522528A (en) | 2012-05-09 | 2015-08-06 | 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 | Method and pharmaceutical composition for preventing or treating chronic obstructive pulmonary disease |
WO2013173364A2 (en) | 2012-05-14 | 2013-11-21 | Biogen Idec Ma Inc. | Lingo-2 antagonists for treatment of conditions involving motor neurons |
WO2013171296A1 (en) | 2012-05-16 | 2013-11-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Diagnostic and treatment of sarcoidosis |
US20150140010A1 (en) | 2012-05-22 | 2015-05-21 | Inserm 9Institut National De La Sante Et De La R- Echerche Medicale) | Methods for diagnosing and treating focal segmental glomerulosclerosis |
PT2858647T (en) | 2012-06-08 | 2018-10-03 | Sensorion | H4 receptor inhibitors for treating tinnitus |
WO2014006025A2 (en) | 2012-07-02 | 2014-01-09 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Marker of pathogenicity in salmonella |
WO2014013005A1 (en) | 2012-07-18 | 2014-01-23 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for preventing and treating chronic kidney disease (ckd) |
CA3163776A1 (en) | 2012-08-03 | 2014-02-06 | Foundation Medicine, Inc. | Human papilloma virus as predictor of cancer prognosis |
WO2014045126A2 (en) | 2012-09-18 | 2014-03-27 | Uti Limited Partnership | Treatment of pain by inhibition of usp5 de-ubiquitinase |
WO2014053871A1 (en) | 2012-10-04 | 2014-04-10 | INSERM (Institut National de la Santé et de la Recherche Médicale) | A method for screening a compound capable of inhibiting the notch1 transcriptional activity |
KR20210063443A (en) | 2012-10-09 | 2021-06-01 | 바이오젠 엠에이 인코포레이티드 | Combination therapies and uses for treatment of demyelinating disorders |
EP2906589A1 (en) | 2012-10-10 | 2015-08-19 | INSERM - Institut National de la Santé et de la Recherche Médicale | Methods and pharmaceutical compositions for treatment of gastrointestinal stromal tumors |
EP2911655A1 (en) | 2012-10-24 | 2015-09-02 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Tpl2 kinase inhibitors for preventing or treating diabetes and for promoting -cell survival |
WO2014064192A1 (en) | 2012-10-26 | 2014-05-01 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method and pharmaceutical composition for use in the treatment and prediction of myocardial infraction |
EP2911681A1 (en) | 2012-10-26 | 2015-09-02 | Institut National de la Santé et de la Recherche Médicale (INSERM) | Lyve-1 antagonists for preventing or treating a pathological condition associated with lymphangiogenesis |
WO2014068070A1 (en) | 2012-10-31 | 2014-05-08 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for preventing antiphospholipid syndrome (aps) |
CA2890207A1 (en) | 2012-11-05 | 2014-05-08 | Foundation Medicine, Inc. | Novel ntrk1 fusion molecules and uses thereof |
EP2917347B1 (en) | 2012-11-08 | 2019-01-02 | Institut National de la Sante et de la Recherche Medicale (INSERM) | Methods and pharmaceutical compositions for the treatment of bone metastases |
EP2732815A1 (en) | 2012-11-16 | 2014-05-21 | Neurochlore | Modulators of intracellular chloride concentration for treating fragile X syndrome |
EP2945652B1 (en) | 2013-01-18 | 2021-07-07 | Foundation Medicine, Inc. | Methods of treating cholangiocarcinoma |
EP2951589A1 (en) | 2013-02-01 | 2015-12-09 | INSERM - Institut National de la Santé et de la Recherche Médicale | Methods for predicting and preventing metastasis in triple negative breast cancers |
WO2014122199A1 (en) | 2013-02-06 | 2014-08-14 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for treatment of chronic intestinal pseudo-obstruction |
WO2014128127A1 (en) | 2013-02-19 | 2014-08-28 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for treatment of prostate cancer |
AU2014223548A1 (en) | 2013-02-26 | 2015-10-15 | Triact Therapeutics, Inc. | Cancer therapy |
EP2961843A2 (en) | 2013-02-28 | 2016-01-06 | Arrowhead Research Corporation | Organic compositions to treat epas1-related diseases |
CN105143470B (en) | 2013-02-28 | 2020-06-09 | 德克萨斯大学系统董事会 | Methods for classifying cancer as susceptible to TMEPAI-directed therapy and treating the cancer |
WO2014140243A1 (en) | 2013-03-15 | 2014-09-18 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method and pharmaceutical composition for use in the treatment and prediction of myocardial infarction |
WO2014147246A1 (en) | 2013-03-21 | 2014-09-25 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method and pharmaceutical composition for use in the treatment of chronic liver diseases associated with a low hepcidin expression |
WO2014170712A1 (en) | 2013-04-15 | 2014-10-23 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Rac-1 inhibitors or pi3k inhibitors for preventing intestinal barrier dysfunction |
CA2909434A1 (en) | 2013-04-18 | 2014-10-23 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Methods and pharmaceutical compositions for inhibiting lymphocyte proliferation in a subject in need thereof |
US10155001B2 (en) | 2013-06-14 | 2018-12-18 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | RAC1 inhibitors for inducing bronchodilation |
WO2015001053A1 (en) | 2013-07-03 | 2015-01-08 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the screening of substances that may be useful for the prevention and treatment of infections by enterobacteriaceae family |
EP3022303B1 (en) | 2013-07-17 | 2023-11-01 | Foundation Medicine, Inc. | Methods of treating urothelial carcinomas |
EP3044593A4 (en) | 2013-09-09 | 2017-05-17 | Triact Therapeutics, Inc. | Cancer therapy |
US10016424B2 (en) | 2013-09-16 | 2018-07-10 | INSERM (Institut National de la Santé et de la Recherche Médicale | Method and pharmaceutical composition for use in the treatment of epilepsy |
EP3052133A2 (en) | 2013-10-03 | 2016-08-10 | INSERM - Institut National de la Santé et de la Recherche Médicale | Methods and pharmaceutical compositions for modulating autophagy in a subject in need thereof |
WO2015051135A2 (en) | 2013-10-04 | 2015-04-09 | Novartis Ag | Organic compositions to treat hepcidin-related diseases |
US20170002064A1 (en) | 2013-11-08 | 2017-01-05 | The Board Of Regents Of The University Of Texas System | Vh4 antibodies against gray matter neuron and astrocyte |
JP6490077B2 (en) | 2013-12-20 | 2019-03-27 | フォンダツィオーネ・イスティトゥート・イタリアーノ・ディ・テクノロジャFondazione Istituto Italiano Di Tecnologia | Modulator of intracellular chloride concentration for treating Down's syndrome |
WO2015140351A1 (en) | 2014-03-21 | 2015-09-24 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for enhancing myelination |
EP3131569B1 (en) | 2014-04-16 | 2020-05-27 | Philippe Rouet | Apoo for use in a method for treating cancer and various pathophysiological situations |
ES2718042T3 (en) | 2014-07-09 | 2019-06-27 | Inst Nat Sante Rech Med | Methods and compositions for the treatment of neuropathic pain |
EP3169337A1 (en) | 2014-07-17 | 2017-05-24 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for treating neuromuscular junction-related diseases |
WO2016025629A1 (en) | 2014-08-12 | 2016-02-18 | The Regents Of The University Of California | Molecular composition for enhancing and rejuvenating maintenance and repair of mammalian tissues |
EP3194581A4 (en) | 2014-09-15 | 2018-04-25 | Children's Medical Center Corporation | Methods and compositions to increase somatic cell nuclear transfer (scnt) efficiency by removing histone h3-lysine trimethylation |
NZ730319A (en) | 2014-09-19 | 2024-03-22 | Memorial Sloan Kettering Cancer Center | Methods for treating brain metastasis |
WO2016046414A2 (en) | 2014-09-26 | 2016-03-31 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Cdc25a inhibitor for the treatment of drug resistant cancer or for the prevention of tumor relapse |
WO2016061144A1 (en) | 2014-10-14 | 2016-04-21 | The Regents Of The University Of California | Use of cdk9 and brd4 inhibitors to inhibit inflammation |
WO2016059220A1 (en) | 2014-10-16 | 2016-04-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Tcr-activating agents for use in the treatment of t-all |
EP3009147A1 (en) | 2014-10-16 | 2016-04-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for treating resistant glioblastoma |
WO2016066608A1 (en) | 2014-10-28 | 2016-05-06 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for treatment of pulmonary cell senescence and peripheral aging |
WO2016066671A1 (en) | 2014-10-29 | 2016-05-06 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for treating resistant cancers using progastrin inhibitors |
JP2018504400A (en) | 2015-01-08 | 2018-02-15 | バイオジェン・エムエイ・インコーポレイテッドBiogen MA Inc. | LINGO-1 antagonist and use for treatment of demyelinating disorders |
WO2016128523A1 (en) | 2015-02-12 | 2016-08-18 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for predicting the responsiveness of a patient affected with malignant hematological disease to chemotherapy treatment and methods of treatment of such disease |
WO2016131944A1 (en) | 2015-02-20 | 2016-08-25 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New method for treating cardiovascular diseases |
CA2978449A1 (en) | 2015-03-02 | 2016-09-09 | 180 Therapeutics Lp | Method of treating a localized fibrotic disorder using an il-33 antagonist |
WO2016139331A1 (en) | 2015-03-05 | 2016-09-09 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of melanoma |
WO2016142427A1 (en) | 2015-03-10 | 2016-09-15 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method ank kit for reprogramming somatic cells |
US11203753B2 (en) | 2015-03-13 | 2021-12-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Hepcidin antagonists for use in the treatment of inflammation |
EP3078378B1 (en) | 2015-04-08 | 2020-06-24 | Vaiomer | Use of factor xa inhibitors for regulating glycemia |
WO2016166110A1 (en) | 2015-04-13 | 2016-10-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for treatment of haemorrhagic diseases |
WO2016170027A1 (en) | 2015-04-22 | 2016-10-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of th17 mediated diseases |
WO2016170382A1 (en) | 2015-04-23 | 2016-10-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Pharmaceutical compositions comprising a bradykinin 2 receptor antagonist for prevention or treatment of impaired skin wound healing |
EP3291839A1 (en) | 2015-05-05 | 2018-03-14 | The University of Louisville Research Foundation, Inc. | Anti-nucleolin agent-conjugated nanoparticles as radio-sensitizers and mri and/or x-ray contrast agents |
WO2016185026A1 (en) | 2015-05-20 | 2016-11-24 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical composition for modulation polarization and activation of macrophages |
WO2016189091A1 (en) | 2015-05-26 | 2016-12-01 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions (ntsr1 inhibitors) for the treatment of hepatocellular carcinomas |
WO2017029391A1 (en) | 2015-08-20 | 2017-02-23 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New method for treating cancer |
WO2017067944A1 (en) | 2015-10-19 | 2017-04-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for predicting the survival time of subjects suffering from triple negative breast cancer |
US20180327806A1 (en) * | 2015-11-04 | 2018-11-15 | The Broad Institute, Inc. | Multiplex high-resolution detection of micro-organism strains, related kits, diagnostics methods and screening assays |
WO2017085566A1 (en) | 2015-11-20 | 2017-05-26 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for increase/induction of immune responses |
WO2017093354A1 (en) | 2015-11-30 | 2017-06-08 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Nmdar antagonists for the treatment of diseases associated with angiogenesis |
US20180353486A1 (en) | 2015-12-01 | 2018-12-13 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Methods and pharmaceutical compositions for the treatment of darier disease |
AU2016364855B2 (en) | 2015-12-03 | 2019-08-29 | Les Laboratoires Servier | MAT2A inhibitors for treating MTAP null cancer |
WO2017129558A1 (en) | 2016-01-25 | 2017-08-03 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for predicting or treating myelopoiesis-driven cardiometabolic diseases and sepsis |
US11725247B2 (en) | 2016-02-29 | 2023-08-15 | Foundation Medicine, Inc. | Methods of treating cancer |
EP3423568A4 (en) | 2016-03-04 | 2019-11-13 | University Of Louisville Research Foundation, Inc. | Methods and compositions for ex vivo expansion of very small embryonic-like stem cells (vsels) |
PL3430404T3 (en) | 2016-03-15 | 2022-05-02 | Institut National De La Santé Et De La Recherche Médicale (Inserm) | Early and non invasive method for assessing a subject's risk of having pancreatic ductal adenocarcinoma and methods of treatement of such disease |
WO2017158396A1 (en) | 2016-03-16 | 2017-09-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Cytidine deaminase inhibitors for the treatment of pancreatic cancer |
EP3433615A1 (en) | 2016-03-21 | 2019-01-30 | Institut National de la Sante et de la Recherche Medicale (INSERM) | Methods for diagnosis and treatment of solar lentigo |
US10639384B2 (en) | 2016-03-23 | 2020-05-05 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Targeting the neuronal calcium sensor 1 for treating wolfram syndrome |
WO2017182834A1 (en) | 2016-04-19 | 2017-10-26 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New method for treating resistant glioblastoma |
EP3464357A1 (en) | 2016-05-24 | 2019-04-10 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of pulmonary bacterial infections |
US10829563B2 (en) | 2016-06-16 | 2020-11-10 | INSERM (Institute National de la Santé et de la Recherche Médicale) | Method of screening a candidate compound for activity as an elastase 2A (ELA2A) inhibitor |
MX2019000327A (en) | 2016-07-13 | 2019-04-11 | Biogen Ma Inc | Dosage regimens of lingo-1 antagonists and uses for treatment of demyelinating disorders. |
AU2017298385A1 (en) | 2016-07-19 | 2019-03-07 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Oncolytic viruses targeting STAT3 |
WO2018019843A1 (en) | 2016-07-26 | 2018-02-01 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Antagonist of mineralocorticoid receptor for the treatment of osteoarthritis |
EP3491387A1 (en) | 2016-07-28 | 2019-06-05 | Institut National de la Sante et de la Recherche Medicale (INSERM) | Methods of treatement of cancer disease by targetting tumor associated macrophage |
EP3493670A1 (en) | 2016-08-05 | 2019-06-12 | Institut National de la Sante et de la Recherche Medicale (INSERM) | Methods and compositions for the preservation of organs |
US20190242908A1 (en) | 2016-09-08 | 2019-08-08 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for diagnosing and treating nephrotic syndrome |
WO2018055023A1 (en) | 2016-09-22 | 2018-03-29 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of lung cancer |
WO2018069232A1 (en) | 2016-10-10 | 2018-04-19 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for predicting the risk of having cardiac hypertrophy |
WO2018078083A1 (en) | 2016-10-28 | 2018-05-03 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New method for treating multiple myeloma |
EP3318277A1 (en) | 2016-11-04 | 2018-05-09 | Institut du Cerveau et de la Moelle Epiniere-ICM | Inhibitors of glucosylceramide synthase for the treatment of motor neuron diseases |
EP3538102A4 (en) | 2016-11-10 | 2020-06-24 | Memorial Sloan-Kettering Cancer Center | Inhibition of kmt2d for the treatment of cancer |
EP3538140A1 (en) | 2016-11-14 | 2019-09-18 | Institut National de la Sante et de la Recherche Medicale (INSERM) | Methods and pharmaceutical compositions for modulating stem cells proliferation or differentiation |
WO2018115083A1 (en) | 2016-12-21 | 2018-06-28 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method of treatment of gut diseases such as irritable bowel syndrome (ibs) |
US20210403511A1 (en) * | 2017-01-06 | 2021-12-30 | Pillargo, Inc. | Nucleic acids and methods for genome editing |
WO2018138106A1 (en) | 2017-01-27 | 2018-08-02 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of heart failure |
WO2018141753A1 (en) | 2017-01-31 | 2018-08-09 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for treating squamous cell carcinomas |
WO2018167283A1 (en) | 2017-03-17 | 2018-09-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the diagnosis and treatment of pancreatic ductal adenocarcinoma associated neural remodeling |
WO2018185516A1 (en) | 2017-04-05 | 2018-10-11 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for treating cardiovascular toxicity induced by anti-cancer therapy |
US20200088732A1 (en) | 2017-04-13 | 2020-03-19 | INSERM (Institut National de la Santé et de la Recherche Mèdicale) | Methods for the diagnosis and treatment of pancreatic ductal adenocarcinoma |
WO2018209288A1 (en) | 2017-05-12 | 2018-11-15 | Massachusetts Institute Of Technology | Argonaute protein-double stranded rna complexes and uses related thereto |
JP2020519665A (en) | 2017-05-17 | 2020-07-02 | アンセルム(アンスティテュト ナショナル ド ラ サンテ エ ド ラ ルシェルシュ メディカル) | FLT3 inhibitors for improving opioid pain management |
EP3412288A1 (en) | 2017-06-08 | 2018-12-12 | Galderma Research & Development | Vegf inhibitors for use for preventing and/or treating acne |
WO2018234370A1 (en) | 2017-06-20 | 2018-12-27 | Institut Curie | Immune cells defective for suv39h1 |
CN111032025A (en) | 2017-06-20 | 2020-04-17 | 居里研究所 | Use of an inhibitor of SUV39H1 histone methyltransferase in the combination therapy of cancer |
WO2018234538A1 (en) | 2017-06-23 | 2018-12-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Hepcidin antagonist or agonist for use in the treatment of dysregulation of mo and/or mn metabolism |
WO2019014398A1 (en) | 2017-07-11 | 2019-01-17 | Actym Therapeutics, Inc. | Engineered immunostimulatory bacterial strains and uses thereof |
WO2019012030A1 (en) | 2017-07-13 | 2019-01-17 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Dhodh inhibitor and chk1 inhibitor for treating cancer |
US20200237864A1 (en) | 2017-10-10 | 2020-07-30 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for treating fibrotic interstitial lung disease |
WO2019072885A1 (en) | 2017-10-11 | 2019-04-18 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Magnetic nanoparticles for the treatment of cancer |
CA3079865C (en) | 2017-10-26 | 2023-09-12 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Methods and pharmaceutical compositions for treating tubulin carboxypeptidases associated diseases |
US11618884B2 (en) | 2017-11-14 | 2023-04-04 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Regulatory T cells genetically modified for the lymphotoxin alpha gene and uses thereof |
US20200352913A1 (en) | 2017-11-23 | 2020-11-12 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New method for treating dengue virus infection |
WO2019108835A1 (en) | 2017-11-29 | 2019-06-06 | The Trustees Of Columbia University In The City Of New York | Delta-2-tubulin as a biomarker and therapeutic target for peripheral neuropathy |
WO2019106126A1 (en) | 2017-12-01 | 2019-06-06 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Mdm2 modulators for the diagnosis and treatment of liposarcoma |
WO2019121872A1 (en) | 2017-12-20 | 2019-06-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the diagnosis and treatment of liver cancer |
WO2019158512A1 (en) | 2018-02-13 | 2019-08-22 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the prognosis and the treatment of glioblastoma |
EP3762105A1 (en) | 2018-03-06 | 2021-01-13 | Institut Curie | Inhibitor of setdb1 histone methyltransferase for use in cancer combination therapy |
EP3775206A1 (en) | 2018-03-28 | 2021-02-17 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for treating cancer |
WO2019207066A1 (en) | 2018-04-26 | 2019-10-31 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for the treatment of sjögren's syndrome |
WO2019211370A1 (en) | 2018-05-03 | 2019-11-07 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for treating cancer |
WO2019211369A1 (en) | 2018-05-03 | 2019-11-07 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for treating cancer |
WO2019234099A1 (en) | 2018-06-06 | 2019-12-12 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for diagnosing, predicting the outcome and treating a patient suffering from heart failure with preserved ejection fraction |
WO2019234221A1 (en) | 2018-06-08 | 2019-12-12 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for stratification and treatment of a patient suffering from chronic lymphocytic leukemia |
CA3106143A1 (en) | 2018-07-11 | 2020-01-16 | Actym Therapeutics, Inc. | Engineered immunostimulatory bacterial strains and uses thereof |
WO2020016160A1 (en) | 2018-07-16 | 2020-01-23 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method to treat neurological diseases |
US20210290633A1 (en) | 2018-07-19 | 2021-09-23 | INSERM (Insstitut National de la Santé et de la Recherche Médicale) | Combination for treating cancer |
WO2020047161A2 (en) | 2018-08-28 | 2020-03-05 | Actym Therapeutics, Inc. | Engineered immunostimulatory bacterial strains and uses thereof |
JP7444858B2 (en) | 2018-09-05 | 2024-03-06 | 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 compositions for treating asthma and allergic diseases |
US20210340240A1 (en) | 2018-10-18 | 2021-11-04 | INSERM (Institut National de la Santé et de la Recherche Médicale | Combination of a big-h3 antagonist and an immune checkpoint inhibitor for the treatment of solid tumor |
EP3873455A1 (en) | 2018-10-31 | 2021-09-08 | Institut National de la Santé et de la Recherche Médicale (INSERM) | Method for treating t-helper type 2 mediated disease |
US11733242B2 (en) | 2018-10-31 | 2023-08-22 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Biomarkers and methods of use for radiation-induced lung injury |
EP3650040A1 (en) | 2018-11-07 | 2020-05-13 | Galderma Research & Development | Vegf inhibitors for use for preventing and/or treating atopic dermatitis |
KR20200071198A (en) | 2018-12-10 | 2020-06-19 | 네오이뮨텍, 인코퍼레이티드 | Development of new adoptive T cell immunotherapy by modification of Nrf2 expression |
AU2020215271A1 (en) | 2019-02-01 | 2021-07-15 | Universität Basel | Calcineurin inhibitor resistant immune cells for use in adoptive cell transfer therapy |
US20220117911A1 (en) | 2019-02-04 | 2022-04-21 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for modulating blood-brain barrier |
WO2020169707A1 (en) | 2019-02-21 | 2020-08-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Foxo1 inhibitor for use in the treatment of latent virus infection |
WO2020178193A1 (en) | 2019-03-01 | 2020-09-10 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method of treatment of sarcoidosis |
WO2020183011A1 (en) | 2019-03-14 | 2020-09-17 | Institut Curie | Htr1d inhibitors and uses thereof in the treatment of cancer |
WO2020193740A1 (en) | 2019-03-28 | 2020-10-01 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New strategy for treating pancreatic cancer |
WO2020208082A1 (en) | 2019-04-09 | 2020-10-15 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for treating cmv related diseases |
JP2022531841A (en) | 2019-04-19 | 2022-07-12 | ソルボンヌ ウニベルシテ | P16INK4a inhibitor for the prevention or treatment of Huntington's disease |
EP3969472A1 (en) | 2019-05-16 | 2022-03-23 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method to treat type 2 inflammation or mast-cell dependent disease |
WO2020249769A1 (en) | 2019-06-14 | 2020-12-17 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for treating ocular diseases related to mitochondrial dna maintenance |
WO2021001539A1 (en) | 2019-07-04 | 2021-01-07 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New strategy to detect and treat eosinophilic fasciitis |
EP3997225A1 (en) | 2019-07-10 | 2022-05-18 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for the treatment of epilepsy |
EP4013466A1 (en) | 2019-08-14 | 2022-06-22 | Vanarix SA | Method for in vitro production of hyaline cartilage tissue |
WO2021044012A1 (en) | 2019-09-05 | 2021-03-11 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method of treatment and pronostic of acute myeloid leukemia |
US20220290151A1 (en) | 2019-09-27 | 2022-09-15 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Use of müllerian inhibiting substance inhibitors for treating cancer |
US20230016983A1 (en) | 2019-11-19 | 2023-01-19 | lNSERM (INSTITUT NATIONAL DE LA SANTÉ ET DE LA RECHERCHE MÉDICALE) | Antisense oligonucleotides and thier use for the treatment of cancer |
WO2021105384A1 (en) | 2019-11-27 | 2021-06-03 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Targeting the nls region of nupr1 protein to treat cancer |
WO2021105391A1 (en) | 2019-11-27 | 2021-06-03 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Combination comprising nupr1 inhibitors to treat cancer |
US20230070181A1 (en) | 2020-02-05 | 2023-03-09 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods of treatment of cancer disease by targeting an epigenetic factor |
WO2021224401A1 (en) | 2020-05-07 | 2021-11-11 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for determining a reference range of β-galactose exposure platelet |
WO2021233962A1 (en) | 2020-05-19 | 2021-11-25 | Institut Curie | Methods for the diagnosis and treatment of cytokine release syndrome |
EP3919062A1 (en) | 2020-06-02 | 2021-12-08 | Institut Gustave-Roussy | Modulators of purinergic receptors and related immune checkpoint for treating acute respiratory distress syndrom |
EP4157288A2 (en) | 2020-06-02 | 2023-04-05 | Institut Gustave-Roussy | Modulators of purinergic receptors and related immune checkpoint for treating acute respiratory distress syndrom |
EP4161583A1 (en) | 2020-06-05 | 2023-04-12 | Institut National de la Santé et de la Recherche Médicale (INSERM) | Methods and pharmaceutical compositions for treating ocular diseases |
JP2023528663A (en) | 2020-06-09 | 2023-07-05 | ジェネトン | Treatment of Hereditary Dilated Cardiomyopathy |
EP4162049A1 (en) | 2020-06-09 | 2023-04-12 | Genethon | Cilp-1 inhibitors for use in the treatment of dilated cardiomyopathies |
EP4162047A1 (en) | 2020-06-09 | 2023-04-12 | Roche Innovation Center Copenhagen A/S | Guanosine analogues for use in therapeutic polynucleotides |
EP4168006A1 (en) | 2020-06-18 | 2023-04-26 | Institut National de la Santé et de la Recherche Médicale (INSERM) | New strategy for treating pancreatic cancer |
US20230305023A1 (en) | 2020-06-25 | 2023-09-28 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods of treatment and diagnostic of pathological conditions associated with intense stress |
US20230266325A1 (en) | 2020-06-30 | 2023-08-24 | Lunglife Ai, Inc. | Methods for detecting lung cancer |
BR112023000428A2 (en) | 2020-07-10 | 2023-03-14 | Inst Nat Sante Rech Med | METHODS AND COMPOSITIONS TO TREAT EPILEPSY |
WO2022018667A1 (en) | 2020-07-24 | 2022-01-27 | Pfizer Inc. | Combination therapies using cdk2 and cdc25a inhibitors |
AU2021316727A1 (en) | 2020-07-30 | 2023-03-02 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Immune cells defective for SOCS1 |
WO2022049273A1 (en) | 2020-09-07 | 2022-03-10 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods of treatment of inflammatory bowel diseases |
EP4291898A1 (en) | 2021-02-12 | 2023-12-20 | Institut National de la Santé et de la Recherche Médicale (INSERM) | Method for prognosis and treating a patient suffering from cancer |
WO2022218998A1 (en) | 2021-04-13 | 2022-10-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for treating hepatitis b and d virus infection |
EP4322938A1 (en) | 2021-04-14 | 2024-02-21 | Institut National de la Santé et de la Recherche Médicale (INSERM) | New method to improve nk cells cytotoxicity |
WO2022219076A1 (en) | 2021-04-14 | 2022-10-20 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New method to improve the anti-tumoral activity of macrophages |
TW202305125A (en) | 2021-04-30 | 2023-02-01 | 美商凱立凡爾免疫治療股份有限公司 | Oncolytic viruses for modified mhc expression |
EP4337769A1 (en) | 2021-05-10 | 2024-03-20 | SQZ Biotechnologies Company | Methods for delivering genome editing molecules to the nucleus or cytosol of a cell and uses thereof |
WO2022251644A1 (en) | 2021-05-28 | 2022-12-01 | Lyell Immunopharma, Inc. | Nr4a3-deficient immune cells and uses thereof |
WO2022256437A1 (en) | 2021-06-02 | 2022-12-08 | Lyell Immunopharma, Inc. | Nr4a3-deficient immune cells and uses thereof |
WO2022253910A1 (en) | 2021-06-02 | 2022-12-08 | INSERM (Institut National de la Santé et de la Recherche Médicale) | A new method to treat an inflammatory skin disease |
WO2022258755A1 (en) | 2021-06-09 | 2022-12-15 | Universität Duisburg-Essen | Method for immortalising vesicle-secreting cells |
WO2023012165A1 (en) | 2021-08-02 | 2023-02-09 | Universite De Montpellier | Compositions and methods for treating cmt1a or cmt1e diseases with rnai molecules targeting pmp22 |
WO2023012343A1 (en) | 2021-08-06 | 2023-02-09 | Institut Du Cancer De Montpellier | Methods for the treatment of cancer |
WO2023041744A1 (en) | 2021-09-17 | 2023-03-23 | Institut Curie | Bet inhibitors for treating pab1 deficient cancer |
WO2023041805A1 (en) | 2021-09-20 | 2023-03-23 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for improving the efficacy of hdac inhibitor therapy and predicting the response to treatment with hdac inhibitor |
WO2023057484A1 (en) | 2021-10-06 | 2023-04-13 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods for predicting and improving the efficacy of mcl-1 inhibitor therapy |
WO2023064924A1 (en) | 2021-10-14 | 2023-04-20 | Codiak Biosciences, Inc. | Modified producer cells for extracellular vesicle production |
WO2023073099A1 (en) | 2021-10-28 | 2023-05-04 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method to improve phagocytosis |
WO2023078906A1 (en) | 2021-11-03 | 2023-05-11 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for treating acute myeloid leukemia |
WO2023078900A1 (en) | 2021-11-03 | 2023-05-11 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and compositions for treating triple negative breast cancer (tnbc) |
WO2023089032A1 (en) | 2021-11-19 | 2023-05-25 | Institut Curie | Methods for the treatment of hrd cancer and brca-associated cancer |
WO2023089159A1 (en) | 2021-11-22 | 2023-05-25 | INSERM (Institut National de la Santé et de la Recherche Médicale) | New strategy targeting stroma/tumor cell crosstalk to treat a cancer |
WO2023099763A1 (en) | 2021-12-03 | 2023-06-08 | Institut Curie | Sirt6 inhibitors for use in treating resistant hrd cancer |
WO2023111173A1 (en) | 2021-12-16 | 2023-06-22 | INSERM (Institut National de la Santé et de la Recherche Médicale) | An ezh2 degrader or inhibitor for use in the treatment of resistant acute myeloid leukemia |
WO2023225665A1 (en) | 2022-05-19 | 2023-11-23 | Lyell Immunopharma, Inc. | Polynucleotides targeting nr4a3 and uses thereof |
WO2023230531A1 (en) | 2022-05-24 | 2023-11-30 | Lunglife Ai, Inc. | Methods for detecting circulating genetically abnormal cells |
WO2024017990A1 (en) | 2022-07-21 | 2024-01-25 | Institut National de la Santé et de la Recherche Médicale | Methods and compositions for treating chronic pain disorders |
WO2024028476A1 (en) | 2022-08-05 | 2024-02-08 | Institut National de la Santé et de la Recherche Médicale | Methods for the treatment of th2-mediated diseases |
WO2024037910A1 (en) | 2022-08-17 | 2024-02-22 | Institut National de la Santé et de la Recherche Médicale | Syk inhibitors for use in the treatment of cancer |
WO2024047110A1 (en) | 2022-08-31 | 2024-03-07 | Institut National de la Santé et de la Recherche Médicale | Method to generate more efficient car-t cells |
WO2024052503A1 (en) | 2022-09-08 | 2024-03-14 | Institut National de la Santé et de la Recherche Médicale | Antibodies having specificity to ltbp2 and uses thereof |
WO2024056659A1 (en) | 2022-09-13 | 2024-03-21 | Institut National de la Santé et de la Recherche Médicale | Method for treating prostate cancer and other epithelial cancers |
WO2024064958A1 (en) | 2022-09-23 | 2024-03-28 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells |
WO2024077174A1 (en) | 2022-10-05 | 2024-04-11 | Lyell Immunopharma, Inc. | Methods for culturing nr4a-deficient cells |
WO2024074713A1 (en) | 2022-10-07 | 2024-04-11 | Institut National de la Santé et de la Recherche Médicale | Method to generate improving car-t cells |
Citations (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6130092A (en) * | 1994-07-04 | 2000-10-10 | Max-Planck Gesellschaft Zur Forderung Der Wissenschaften E.V. | Ribozyme gene library and method for making |
US6326193B1 (en) * | 1999-11-05 | 2001-12-04 | Cambria Biosciences, Llc | Insect control agent |
US20020086356A1 (en) * | 2000-03-30 | 2002-07-04 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US20020114784A1 (en) * | 1999-01-28 | 2002-08-22 | Medical College Of Georgia Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA |
US6506559B1 (en) * | 1997-12-23 | 2003-01-14 | Carnegie Institute Of Washington | Genetic inhibition by double-stranded RNA |
US20030084471A1 (en) * | 2000-03-16 | 2003-05-01 | David Beach | Methods and compositions for RNA interference |
US6573099B2 (en) * | 1998-03-20 | 2003-06-03 | Benitec Australia, Ltd. | Genetic constructs for delaying or repressing the expression of a target gene |
US20040018999A1 (en) * | 2000-03-16 | 2004-01-29 | David Beach | Methods and compositions for RNA interference |
US20050164210A1 (en) * | 2004-01-23 | 2005-07-28 | Vivek Mittal | Regulated polymerase III expression systems and related methods |
US20050197315A1 (en) * | 2001-11-28 | 2005-09-08 | Toudai Tlo, Ltd. | siRNA expression system and method for producing functional gene knock-down cell using the system |
Family Cites Families (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5246921A (en) * | 1990-06-26 | 1993-09-21 | The Wistar Institute Of Anatomy And Biology | Method for treating leukemias |
NZ255028A (en) | 1992-07-02 | 1997-03-24 | Hybridon Inc | Antisense oligonucleotides resistant to nucleolytic degradation |
US5624803A (en) | 1993-10-14 | 1997-04-29 | The Regents Of The University Of California | In vivo oligonucleotide generator, and methods of testing the binding affinity of triplex forming oligonucleotides derived therefrom |
US6107027A (en) | 1994-12-14 | 2000-08-22 | University Of Washington | Ribozymes for treating hepatitis C |
US5814500A (en) | 1996-10-31 | 1998-09-29 | The Johns Hopkins University School Of Medicine | Delivery construct for antisense nucleic acids and methods of use |
AU5927598A (en) | 1997-01-23 | 1998-08-18 | Immusol Incorporated | Gene functional analysis and discovery using randomized or target-specific ribozyme gene vector libraries |
GB9803351D0 (en) | 1998-02-17 | 1998-04-15 | Oxford Biomedica Ltd | Anti-viral vectors |
KR101085210B1 (en) | 1998-03-20 | 2011-11-21 | 커먼웰쓰 사이언티픽 앤드 인더스트리얼 리서치 오가니제이션 | Control of gene expression |
GB9827152D0 (en) | 1998-07-03 | 1999-02-03 | Devgen Nv | Characterisation of gene function using double stranded rna inhibition |
DE19956568A1 (en) | 1999-01-30 | 2000-08-17 | Roland Kreutzer | Method and medicament for inhibiting the expression of a given gene |
US5998148A (en) | 1999-04-08 | 1999-12-07 | Isis Pharmaceuticals Inc. | Antisense modulation of microtubule-associated protein 4 expression |
WO2000063364A2 (en) | 1999-04-21 | 2000-10-26 | American Home Products Corporation | Methods and compositions for inhibiting the function of polynucleotide sequences |
AU1086501A (en) * | 1999-10-15 | 2001-04-30 | Carnegie Institution Of Washington | Rna interference pathway genes as tools for targeted genetic interference |
GB9927444D0 (en) | 1999-11-19 | 2000-01-19 | Cancer Res Campaign Tech | Inhibiting gene expression |
DE10100586C1 (en) | 2001-01-09 | 2002-04-11 | Ribopharma Ag | Inhibiting gene expression in cells, useful for e.g. treating tumors, by introducing double-stranded complementary oligoRNA having unpaired terminal bases |
GB9930691D0 (en) | 1999-12-24 | 2000-02-16 | Devgen Nv | Improvements relating to double-stranded RNA inhibition |
US20050229272A1 (en) | 1999-12-30 | 2005-10-13 | Monica Driscoll | Compositions and methods for gene silencing |
BRPI0115814B8 (en) | 2000-12-01 | 2021-05-25 | Europaeisches Laboratorium Fuer Molekularbiologie Embl | double-stranded RNA molecules, their method of preparation and pharmaceutical composition comprising them |
JP2004532616A (en) | 2000-12-28 | 2004-10-28 | ジョンソン・アンド・ジョンソン・リサーチ・ピー・ティー・ワイ・リミテッド | Double-stranded RNA-mediated gene suppression |
GB0104948D0 (en) | 2001-02-28 | 2001-04-18 | Novartis Res Foundation | Novel methods |
BR0211111A (en) | 2001-07-12 | 2004-06-22 | Univ Massachusetts | Isolated nucleic acid molecule, vector, host cell, transgene, engineered rna precursor, non-human transgenic animal, and method of inducing ribonucleic acid interference from a target gene in a cell |
EP1409506B1 (en) | 2001-07-23 | 2012-05-09 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for rnai mediated inhibition of gene expression in mammals |
US20030198627A1 (en) | 2001-09-01 | 2003-10-23 | Gert-Jan Arts | siRNA knockout assay method and constructs |
AU2002354121A1 (en) | 2001-11-28 | 2003-06-10 | Toudai Tlo, Ltd. | siRNA Expression System and Method for Producing Functional Gene Knockdown Cell Using the Same |
GB0130955D0 (en) | 2001-12-24 | 2002-02-13 | Cancer Res Ventures | Expression system |
CA2499188A1 (en) | 2002-09-27 | 2004-04-08 | Cold Spring Harbor Laboratory | Cell-based rna interference and related methods and compositions |
-
2001
- 2001-03-16 JP JP2001567320A patent/JP2003526367A/en active Pending
- 2001-03-16 AU AU2001245793A patent/AU2001245793A1/en not_active Abandoned
- 2001-03-16 WO PCT/US2001/008435 patent/WO2001068836A2/en not_active Application Discontinuation
- 2001-03-16 CA CA002403397A patent/CA2403397A1/en not_active Abandoned
- 2001-03-16 EP EP01918752A patent/EP1272630A2/en not_active Ceased
- 2001-03-16 IL IL15178101A patent/IL151781A0/en unknown
- 2001-05-16 US US09/858,862 patent/US7732417B2/en not_active Expired - Fee Related
- 2001-05-24 US US09/866,557 patent/US20020162126A1/en not_active Abandoned
-
2003
- 2003-01-24 US US10/350,798 patent/US20040086884A1/en not_active Abandoned
-
2008
- 2008-05-16 US US12/152,837 patent/US8383599B2/en not_active Expired - Lifetime
Patent Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6130092A (en) * | 1994-07-04 | 2000-10-10 | Max-Planck Gesellschaft Zur Forderung Der Wissenschaften E.V. | Ribozyme gene library and method for making |
US20030051263A1 (en) * | 1997-12-23 | 2003-03-13 | The Carnegie Institution Of Washington | Genetic inhibition by double-stranded RNA |
US20030055020A1 (en) * | 1997-12-23 | 2003-03-20 | The Carnegie Institution Of Washington | Genetic inhibition by double-stranded RNA |
US20030056235A1 (en) * | 1997-12-23 | 2003-03-20 | The Carnegie Institution Of Washington | Genetic inhibition by double-stranded RNA |
US6506559B1 (en) * | 1997-12-23 | 2003-01-14 | Carnegie Institute Of Washington | Genetic inhibition by double-stranded RNA |
US6573099B2 (en) * | 1998-03-20 | 2003-06-03 | Benitec Australia, Ltd. | Genetic constructs for delaying or repressing the expression of a target gene |
US20020114784A1 (en) * | 1999-01-28 | 2002-08-22 | Medical College Of Georgia Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA |
US6326193B1 (en) * | 1999-11-05 | 2001-12-04 | Cambria Biosciences, Llc | Insect control agent |
US20030084471A1 (en) * | 2000-03-16 | 2003-05-01 | David Beach | Methods and compositions for RNA interference |
US20040018999A1 (en) * | 2000-03-16 | 2004-01-29 | David Beach | Methods and compositions for RNA interference |
US20040086884A1 (en) * | 2000-03-16 | 2004-05-06 | Genetica, Inc. | Methods and compositions for RNA interference |
US20020086356A1 (en) * | 2000-03-30 | 2002-07-04 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US20050197315A1 (en) * | 2001-11-28 | 2005-09-08 | Toudai Tlo, Ltd. | siRNA expression system and method for producing functional gene knock-down cell using the system |
US20050164210A1 (en) * | 2004-01-23 | 2005-07-28 | Vivek Mittal | Regulated polymerase III expression systems and related methods |
Cited By (349)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040171156A1 (en) * | 1995-06-07 | 2004-09-02 | Invitrogen Corporation | Recombinational cloning using nucleic acids having recombination sites |
US20030068799A1 (en) * | 1995-06-07 | 2003-04-10 | Invitrogen Corporation | Recombinational cloning using engineered recombination sites |
US7282326B2 (en) | 1995-06-07 | 2007-10-16 | Invitrogen Corporation | Recombinational cloning using engineered recombination sites |
US20040063207A1 (en) * | 1995-06-07 | 2004-04-01 | Invitrogen Corporation | Recombinational cloning using nucleic acids having recombination sites |
US7223576B2 (en) | 1995-06-07 | 2007-05-29 | Invitrogen Corporation | Recombinational cloning using engineered recombination sites |
US20030100110A1 (en) * | 1995-06-07 | 2003-05-29 | James L. Hartley | Recombinational cloning using engineered recombination sites |
US7304130B2 (en) | 1995-06-07 | 2007-12-04 | Invitrogen Corporation | Recombinational cloning using engineered recombination sites |
US9096636B2 (en) | 1996-06-06 | 2015-08-04 | Isis Pharmaceuticals, Inc. | Chimeric oligomeric compounds and their use in gene modulation |
US7695902B2 (en) | 1996-06-06 | 2010-04-13 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides and ribonucleases for cleaving RNA |
US7812149B2 (en) | 1996-06-06 | 2010-10-12 | Isis Pharmaceuticals, Inc. | 2′-Fluoro substituted oligomeric compounds and compositions for use in gene modulations |
US20090215880A1 (en) * | 1999-01-28 | 2009-08-27 | Med. College Of Georgia Research Institute, Inc. | Composition and Method for IN VIVO and IN VITRO Attenuation of Gene Expression Using Double Stranded RNA |
US20090156520A1 (en) * | 1999-01-28 | 2009-06-18 | Med. College Of Georgia Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA |
US8148345B2 (en) | 1999-01-28 | 2012-04-03 | Georgia Health Sciences University Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA |
US20040147475A1 (en) * | 1999-01-28 | 2004-07-29 | Medical College Of Georgia Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA |
US7888325B2 (en) | 1999-01-28 | 2011-02-15 | Medical College Of Georgia Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA |
US20020114784A1 (en) * | 1999-01-28 | 2002-08-22 | Medical College Of Georgia Research Institute, Inc. | Composition and method for in vivo and in vitro attenuation of gene expression using double stranded RNA |
US9902955B2 (en) | 1999-01-30 | 2018-02-27 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US20080261303A1 (en) * | 1999-01-30 | 2008-10-23 | Roland Kreutzer | Method and medicament for inhibiting the expression of a given gene |
US8183362B2 (en) | 1999-01-30 | 2012-05-22 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US20040053875A1 (en) * | 1999-01-30 | 2004-03-18 | Ribopharma Ag | Method and medicament for inhibiting the expression of a given gene |
US8202980B2 (en) | 1999-01-30 | 2012-06-19 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US20040072779A1 (en) * | 1999-01-30 | 2004-04-15 | Ribopharma Ag | Method and medicament for inhibiting the expression of a given gene |
US8119608B2 (en) | 1999-01-30 | 2012-02-21 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US20040102408A1 (en) * | 1999-01-30 | 2004-05-27 | Ribopharma Ag | Method and medicament for inhibiting the expression of a given gene |
US8114851B2 (en) | 1999-01-30 | 2012-02-14 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US9133454B2 (en) | 1999-01-30 | 2015-09-15 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US20080182981A1 (en) * | 1999-01-30 | 2008-07-31 | Roland Kreutzer | Method and medicament for inhibiting the expression of a given gene |
US8168776B2 (en) | 1999-01-30 | 2012-05-01 | Alnylam Pharmaceuticals, Inc. | Method for making a 21 nucleotide double stranded RNA chemically linked at one end |
US8114981B2 (en) | 1999-01-30 | 2012-02-14 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US20080171861A1 (en) * | 1999-01-30 | 2008-07-17 | Roland Kreutzer | Method and medicament for inhibiting the expression of a given gene |
US8101742B2 (en) | 1999-01-30 | 2012-01-24 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US8101584B2 (en) | 1999-01-30 | 2012-01-24 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US20080166800A1 (en) * | 1999-01-30 | 2008-07-10 | Roland Kreutzer | Method and medicament for inhibiting the expression of a given gene |
US8729037B2 (en) | 1999-01-30 | 2014-05-20 | Alnylam Pharmaceuticals, Inc. | Method and medicament for inhibiting the expression of a given gene |
US20050100907A1 (en) * | 1999-01-30 | 2005-05-12 | Ribopharma, Ag | Method and medicament for inhibiting the expression of a given gene |
US8883988B2 (en) | 1999-03-02 | 2014-11-11 | Life Technologies Corporation | Compositions for use in recombinational cloning of nucleic acids |
US8241896B2 (en) | 1999-03-02 | 2012-08-14 | Life Technologies Corporation | Compositions for use in recombinational cloning of nucelic acids |
US7670823B1 (en) | 1999-03-02 | 2010-03-02 | Life Technologies Corp. | Compositions for use in recombinational cloning of nucleic acids |
US20040221337A1 (en) * | 1999-10-27 | 2004-11-04 | Baulcombe David C. | Gene silencing |
US20080312176A1 (en) * | 1999-10-27 | 2008-12-18 | David Charles Baulcombe | Gene silencing |
US20090288182A1 (en) * | 1999-10-27 | 2009-11-19 | David Charles Baulcombe | Gene silencing |
US20090286254A1 (en) * | 1999-10-27 | 2009-11-19 | David Charles Baulcombe | Gene silencing |
US8779236B2 (en) | 1999-10-27 | 2014-07-15 | Plant Bioscience Limited | Gene silencing |
US20050102710A1 (en) * | 1999-10-27 | 2005-05-12 | Plant Bioscience Limited | Cells and animals produced by gene silencing |
US20050102709A1 (en) * | 1999-10-27 | 2005-05-12 | Plant Bioscience Limited | RNA molecules and vectors for gene silencing |
US20060168669A1 (en) * | 1999-10-27 | 2006-07-27 | Baulcombe David C | Gene silencing |
US8097710B2 (en) | 1999-10-27 | 2012-01-17 | Plant Bioscience Limited | Gene silencing |
US7704688B2 (en) | 1999-10-27 | 2010-04-27 | Plant Bioscience Limited | Methods of detecting silencing mammalian cells |
US8759102B2 (en) | 1999-10-27 | 2014-06-24 | Plant Bioscience Limited | Short RNA producing gene silencing in cells |
US8258285B2 (en) | 1999-10-27 | 2012-09-04 | Plant Bioscience Limited | RNA molecules and vectors for gene silencing |
US8263569B2 (en) | 1999-10-27 | 2012-09-11 | Plant Biosciences Limited | Gene silencing |
US8299235B2 (en) | 1999-10-27 | 2012-10-30 | Plant Bioscience Limited | RNA molecules and vectors for gene silencing |
US8349607B2 (en) | 1999-10-27 | 2013-01-08 | Plant Bioscience Limited | Gene silencing |
US20040175703A1 (en) * | 1999-11-24 | 2004-09-09 | Ribopharma Ag | Compositions and methods for inhibiting expression of a target gene |
US7829693B2 (en) | 1999-11-24 | 2010-11-09 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a target gene |
US7393632B2 (en) | 1999-12-10 | 2008-07-01 | Invitrogen Corp. | Use of multiple recombination sites with unique specificity in recombinational cloning |
US20020007051A1 (en) * | 1999-12-10 | 2002-01-17 | David Cheo | Use of multiple recombination sites with unique specificity in recombinational cloning |
US20070003962A1 (en) * | 2000-03-30 | 2007-01-04 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US8420391B2 (en) | 2000-03-30 | 2013-04-16 | University Of Massachusetts | RNA sequence-specific mediators of RNA interference |
US10472625B2 (en) | 2000-03-30 | 2019-11-12 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA sequence-specific mediators of RNA interference |
US20020086356A1 (en) * | 2000-03-30 | 2002-07-04 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US9012138B2 (en) | 2000-03-30 | 2015-04-21 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA sequence-specific mediators of RNA interference |
US20090186843A1 (en) * | 2000-03-30 | 2009-07-23 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US9012621B2 (en) | 2000-03-30 | 2015-04-21 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA sequence-specific mediators of RNA interference |
US8790922B2 (en) | 2000-03-30 | 2014-07-29 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA sequence-specific mediators of RNA interference |
US8394628B2 (en) | 2000-03-30 | 2013-03-12 | University Of Massachusetts | RNA sequence-specific mediators of RNA interference |
US9193753B2 (en) | 2000-03-30 | 2015-11-24 | University Of Massachusetts | RNA sequence-specific mediators of RNA interference |
US20070003960A1 (en) * | 2000-03-30 | 2007-01-04 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US8552171B2 (en) | 2000-03-30 | 2013-10-08 | University Of Massachusetts | RNA sequence-specific mediators of RNA interference |
US20070003963A1 (en) * | 2000-03-30 | 2007-01-04 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US8632997B2 (en) | 2000-03-30 | 2014-01-21 | University Of Massachusetts | RNA sequence-specific mediators of RNA interference |
US20070003961A1 (en) * | 2000-03-30 | 2007-01-04 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US20080132461A1 (en) * | 2000-03-30 | 2008-06-05 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US20030108923A1 (en) * | 2000-03-30 | 2003-06-12 | Whitehead Institute For Biomedical Research | RNA sequence-specific mediators of RNA interference |
US8742092B2 (en) | 2000-03-30 | 2014-06-03 | University Of Massachusetts | RNA sequence-specific mediators of RNA interference |
US20030186233A1 (en) * | 2000-05-21 | 2003-10-02 | Invitrogen Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites |
US9309520B2 (en) | 2000-08-21 | 2016-04-12 | Life Technologies Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites |
US8765930B2 (en) | 2000-12-01 | 2014-07-01 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US20110020234A1 (en) * | 2000-12-01 | 2011-01-27 | Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. | Rna interference mediating small rna molecules |
US8796016B2 (en) | 2000-12-01 | 2014-08-05 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US8993745B2 (en) | 2000-12-01 | 2015-03-31 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US8933044B2 (en) | 2000-12-01 | 2015-01-13 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US8778902B2 (en) | 2000-12-01 | 2014-07-15 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US8362231B2 (en) | 2000-12-01 | 2013-01-29 | Max-Planck-Gesellschaft zur Föderung der Wissenschaften E.V. | RNA interference mediating small RNA molecules |
US10633656B2 (en) | 2000-12-01 | 2020-04-28 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | RNA interference mediating small RNA molecules |
US20050026278A1 (en) * | 2000-12-01 | 2005-02-03 | Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. | RNA interference mediating small RNA molecules |
US20040259247A1 (en) * | 2000-12-01 | 2004-12-23 | Thomas Tuschl | Rna interference mediating small rna molecules |
US8372968B2 (en) | 2000-12-01 | 2013-02-12 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US8853384B2 (en) | 2000-12-01 | 2014-10-07 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US8329463B2 (en) | 2000-12-01 | 2012-12-11 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US8895721B2 (en) | 2000-12-01 | 2014-11-25 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US20110112283A1 (en) * | 2000-12-01 | 2011-05-12 | Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. | Rna interference mediating small rna molecules |
US8445237B2 (en) | 2000-12-01 | 2013-05-21 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US20110054159A1 (en) * | 2000-12-01 | 2011-03-03 | Maxplanck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. | Rna interference mediating small rna molecules |
US8895718B2 (en) | 2000-12-01 | 2014-11-25 | MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. | RNA interference mediating small RNA molecules |
US8945884B2 (en) | 2000-12-11 | 2015-02-03 | Life Technologies Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiplerecognition sites |
US8030066B2 (en) | 2000-12-11 | 2011-10-04 | Life Technologies Corporation | Methods and compositions for synthesis of nucleic acid molecules using multiple recognition sites |
US20090053808A1 (en) * | 2001-01-09 | 2009-02-26 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting the expression of anti-apoptopic genes |
US7868160B2 (en) | 2001-01-09 | 2011-01-11 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US7767802B2 (en) | 2001-01-09 | 2010-08-03 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US7423142B2 (en) | 2001-01-09 | 2008-09-09 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US9587240B2 (en) | 2001-01-09 | 2017-03-07 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a target gene |
US20040001811A1 (en) * | 2001-01-09 | 2004-01-01 | Ribopharma Ag | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US9074213B2 (en) | 2001-01-09 | 2015-07-07 | Alnylam Pharmacuticals, Inc. | Compositions and methods for inhibiting expression of a target gene |
US20050176667A1 (en) * | 2001-01-09 | 2005-08-11 | Alnylam Europe Ag | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US7473525B2 (en) | 2001-01-09 | 2009-01-06 | Alnylam Europe Ag | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US20060084621A1 (en) * | 2001-01-09 | 2006-04-20 | Hans-Peter Vornlocher | Compositions and methods for inhibiting expression of anti-apoptotic genes |
US20030124555A1 (en) * | 2001-05-21 | 2003-07-03 | Invitrogen Corporation | Compositions and methods for use in isolation of nucleic acid molecules |
US7893036B2 (en) | 2001-07-12 | 2011-02-22 | University Of Massachusetts | In vivo production of small interfering RNAs that mediate gene silencing |
US20110207224A1 (en) * | 2001-07-12 | 2011-08-25 | University Of Massachusetts | In vivo production of small interfering rnas that mediate gene silencing |
US20100234448A1 (en) * | 2001-07-12 | 2010-09-16 | University Of Massachusetts | In vivo production of small interfering rnas that mediate gene silencing |
US8530438B2 (en) | 2001-07-12 | 2013-09-10 | University Of Massachusetts | Vivo production of small interfering RNAs that mediate gene silencing |
US10731155B2 (en) | 2001-07-12 | 2020-08-04 | University Of Massachusetts | In vivo production of small interfering RNAs that mediate gene silencing |
US9850487B2 (en) | 2001-07-12 | 2017-12-26 | University Of Massachusetts | In vivo production of small interfering RNAs that mediate gene silencing |
US8232260B2 (en) | 2001-07-12 | 2012-07-31 | University Of Massachusetts | In vivo production of small interfering RNAs that mediate gene silencing |
US8557785B2 (en) | 2001-07-12 | 2013-10-15 | University Of Massachusetts | In vivo production of small interfering RNAS that mediate gene silencing |
US9175287B2 (en) | 2001-07-12 | 2015-11-03 | University Of Massachusetts | In vivo production of small interfering RNAs that mediate gene silencing |
US20080200420A1 (en) * | 2001-07-12 | 2008-08-21 | Zamore Phillip D | In vivo production of small interfering RNAs that mediate gene silencing |
US20060252035A1 (en) * | 2001-08-06 | 2006-11-09 | Lori Friedman | Sphks as modifiers of the p53 pathway and methods of use |
US20080241131A1 (en) * | 2001-08-06 | 2008-10-02 | Exelixis Inc. | SPHKS as modifiers of the p53 pathway and methods of use |
US7348314B2 (en) | 2001-10-12 | 2008-03-25 | Alnylam Europe Ag | Compositions and methods for inhibiting viral replication |
US7763590B2 (en) | 2001-10-12 | 2010-07-27 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of a mutant gene |
US20050074757A1 (en) * | 2001-10-12 | 2005-04-07 | Ribopharma Ag | Compositions and methods for inhibiting expression of a mutant gene |
US7745418B2 (en) | 2001-10-12 | 2010-06-29 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting viral replication |
US20040126791A1 (en) * | 2001-10-26 | 2004-07-01 | Ribopharma Ag | Compositions and methods for treating trail-resistant cancer cells |
US20040002077A1 (en) * | 2001-11-28 | 2004-01-01 | Center For Advanced Science And Technology Incubation, Ltd. | siRNA expression system and method for producing functional gene knock-down cell using the system |
US20050048647A1 (en) * | 2001-11-28 | 2005-03-03 | Kazunari Taira | Sirna expression system and process for producing functional gene knockdown cell or the like using the same |
US20030190654A1 (en) * | 2002-01-22 | 2003-10-09 | Ribopharma | Double-stranded RNA (dsRNA) and method of use for inhibiting expression of a fusion gene |
US7846907B2 (en) | 2002-01-22 | 2010-12-07 | Alnylam Pharmaceuticals, Inc. | Double-stranded RNA (dsRNA) and method of use for inhibiting expression of a fusion gene |
US7196184B2 (en) | 2002-01-22 | 2007-03-27 | Alnylam Europe Ag | Double-stranded RNA (DSRNA) and method of use for inhibiting expression of the AML-1/MTG8 fusion gene |
US8524680B2 (en) | 2002-02-01 | 2013-09-03 | Applied Biosystems, Llc | High potency siRNAS for reducing the expression of target genes |
US10196640B1 (en) | 2002-02-01 | 2019-02-05 | Life Technologies Corporation | Oligonucleotide compositions with enhanced efficiency |
US8815821B2 (en) | 2002-02-01 | 2014-08-26 | Life Technologies Corporation | Double-stranded oligonucleotides |
US10036025B2 (en) | 2002-02-01 | 2018-07-31 | Life Technologies Corporation | Oligonucleotide compositions with enhanced efficiency |
US20030166282A1 (en) * | 2002-02-01 | 2003-09-04 | David Brown | High potency siRNAS for reducing the expression of target genes |
US9592250B2 (en) | 2002-02-01 | 2017-03-14 | Life Technologies Corporation | Double-stranded oligonucleotides |
US9777275B2 (en) | 2002-02-01 | 2017-10-03 | Life Technologies Corporation | Oligonucleotide compositions with enhanced efficiency |
US10626398B2 (en) | 2002-02-01 | 2020-04-21 | Life Technologies Corporation | Oligonucleotide compositions with enhanced efficiency |
US9796978B1 (en) | 2002-02-01 | 2017-10-24 | Life Technologies Corporation | Oligonucleotide compositions with enhanced efficiency |
US10106793B2 (en) | 2002-02-01 | 2018-10-23 | Life Technologies Corporation | Double-stranded oligonucleotides |
US20030169748A1 (en) * | 2002-03-11 | 2003-09-11 | Weyman Raphael J. | Stackable network units with resiliency facility |
US8202845B2 (en) | 2002-04-18 | 2012-06-19 | Acuity Pharmaceuticals, Inc. | Means and methods for the specific modulation of target genes in the CNS and the eye and methods for their identification |
US8946180B2 (en) | 2002-04-18 | 2015-02-03 | Opko Pharmaceuticals, Llc | Means and methods for the specific modulation of target genes in the CNS and the eye and methods for their identification |
US20060003915A1 (en) * | 2002-04-18 | 2006-01-05 | Karina Drumm | Means and methods for the specific modulation of target genes in the cns and the eye and methods for their identification |
US20110021605A1 (en) * | 2002-04-18 | 2011-01-27 | Schulte Ralf Wilhelm | Means and methods for the specific inhibition of genes in cells and tissue of the cns and/or eye |
US8299042B2 (en) * | 2002-04-26 | 2012-10-30 | Alnylam Pharmaceuticals, Inc. | Methods and compositions for silencing genes without inducing toxicity |
US20090137514A1 (en) * | 2002-04-26 | 2009-05-28 | Nucleonics, Inc. | Methods and compositions for silencing genes without inducing toxicity |
US20030224432A1 (en) * | 2002-05-03 | 2003-12-04 | Jason Myers | Methods and compositions for use in preparing siRNAs |
US20090286287A1 (en) * | 2002-05-03 | 2009-11-19 | Jason Myers | METHODS AND COMPOSITIONS FOR USE IN PREPARRING siRNAs |
US7556944B2 (en) * | 2002-05-03 | 2009-07-07 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for use in preparing siRNAs |
US8119610B2 (en) * | 2002-05-31 | 2012-02-21 | The Regents Of The University Of California | Method for efficient RNA interference in mammalian cells |
US20040014113A1 (en) * | 2002-05-31 | 2004-01-22 | The Regents Of The University Of California | Method for efficient RNA interference in mammalian cells |
AU2003253618B2 (en) * | 2002-05-31 | 2007-11-15 | The Regents Of The University Of California | Method for efficient RNA interference in mammalian cells |
US20040248094A1 (en) * | 2002-06-12 | 2004-12-09 | Ford Lance P. | Methods and compositions relating to labeled RNA molecules that reduce gene expression |
US20040033602A1 (en) * | 2002-06-12 | 2004-02-19 | Ambion, Inc. | Methods and compositions relating to polypeptides with RNase III domains that mediate RNA interference |
US20100009856A1 (en) * | 2002-06-21 | 2010-01-14 | Sinogenomax Sompany LTD. | Randomized dna libraries and double-stranded rna libraries, use and method of production thereof |
US20040086911A1 (en) * | 2002-06-24 | 2004-05-06 | Baylor College Of Medicine | Inhibition of gene expression in vertebrates using double-stranded RNA (RNAi) |
US20170067050A1 (en) * | 2002-07-10 | 2017-03-09 | Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. | Rna-interference by single-stranded rna molecules |
US10023865B2 (en) * | 2002-07-10 | 2018-07-17 | Max-Planck-Gesellschaft Zur Förderung De Wissenschaften E.V. | RNA-interference by single-stranded RNA molecules |
US20090104259A1 (en) * | 2002-07-24 | 2009-04-23 | The Trustees Of The University Of Pennsylvania | Compositions and methods for sirna inhibition of angiogenesis |
US9150863B2 (en) | 2002-07-24 | 2015-10-06 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiogenesis |
US20040018176A1 (en) * | 2002-07-24 | 2004-01-29 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiogenesis |
US7345027B2 (en) | 2002-07-24 | 2008-03-18 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiogenesis |
US7674895B2 (en) | 2002-07-24 | 2010-03-09 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiogenesis |
US20080188437A1 (en) * | 2002-07-24 | 2008-08-07 | The Trustees Of The University Of Pennsylvania | Compositions and Methods for siRNA Inhibition of Angiogenesis |
US7148342B2 (en) | 2002-07-24 | 2006-12-12 | The Trustees Of The University Of Pennyslvania | Compositions and methods for sirna inhibition of angiogenesis |
US8546345B2 (en) | 2002-07-24 | 2013-10-01 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiogenesis |
US20060286073A1 (en) * | 2002-07-24 | 2006-12-21 | Tolentino Michael J | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS |
US20070149471A1 (en) * | 2002-07-24 | 2007-06-28 | Reich Samuel J | Compositions and methods for siRNA inhibition of angiogenesis |
US8541384B2 (en) | 2002-07-24 | 2013-09-24 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiogenesis |
US8946403B2 (en) | 2002-07-24 | 2015-02-03 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiogenesis |
US20060292120A1 (en) * | 2002-07-24 | 2006-12-28 | Tolentino Michael J | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS |
US20070003523A1 (en) * | 2002-07-24 | 2007-01-04 | Tolentino Michael J | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS |
US7750143B2 (en) | 2002-07-24 | 2010-07-06 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiogenesis |
US20070037761A1 (en) * | 2002-07-24 | 2007-02-15 | Tolentino Michael J | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS |
US20070037760A1 (en) * | 2002-07-24 | 2007-02-15 | Tolentino Michael J | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS |
US20070037762A1 (en) * | 2002-07-24 | 2007-02-15 | Tolentino Michael J | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS |
US20060211637A1 (en) * | 2002-08-06 | 2006-09-21 | Intradigm Corporation | Methods of down regulating target gene expression in vivo by introduction of interfering rna |
US8729036B2 (en) | 2002-08-07 | 2014-05-20 | University Of Massachusetts | Compositions for RNA interference and methods of use thereof |
US9611472B2 (en) | 2002-08-07 | 2017-04-04 | University Of Massachusetts | Compositions for RNA interference and methods of use thereof |
US20040203145A1 (en) * | 2002-08-07 | 2004-10-14 | University Of Massachusetts | Compositions for RNA interference and methods of use thereof |
WO2004015062A2 (en) | 2002-08-12 | 2004-02-19 | New England Biolabs, Inc. | Methods and compositions relating to gene silencing |
US20040038278A1 (en) * | 2002-08-12 | 2004-02-26 | George Tzertzinis | Methods and compositions relating to gene silencing |
US20080206835A1 (en) * | 2002-08-12 | 2008-08-28 | New England Biolabs, Inc. | Methods and Compositions Relating to Gene Silencing |
US7700758B2 (en) * | 2002-08-12 | 2010-04-20 | New England Biolabs, Inc. | Methods and compositions relating to gene silencing |
EP2806025A1 (en) | 2002-09-05 | 2014-11-26 | California Institute of Technology | Use of zinc finger nucleases to stimulate gene targeting |
US20040242518A1 (en) * | 2002-09-28 | 2004-12-02 | Massachusetts Institute Of Technology | Influenza therapeutic |
US20090106852A1 (en) * | 2002-09-28 | 2009-04-23 | Massachusetts Institute Of Technology | Influenza Therapeutic |
US7521431B2 (en) | 2002-11-01 | 2009-04-21 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of HIF-1 alpha |
US7645744B2 (en) | 2002-11-01 | 2010-01-12 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of HIF-1 alpha |
US8236775B2 (en) | 2002-11-01 | 2012-08-07 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of HIF-1 α |
US20100136101A1 (en) * | 2002-11-01 | 2010-06-03 | The Trustees Of The University Of Pennsylvania | Compositions and methods for sirna inhibition of hif-1 alpha |
US8604183B2 (en) | 2002-11-05 | 2013-12-10 | Isis Pharmaceuticals, Inc. | Compositions comprising alternating 2′-modified nucleosides for use in gene modulation |
US20050059028A1 (en) * | 2002-11-18 | 2005-03-17 | Genospectra, Inc. | RNAi-based sensors, caged interfering RNAs, and methods of use thereof |
US20050282203A1 (en) * | 2002-11-18 | 2005-12-22 | Genospectra, Inc. | Caged RNAs and methods of use thereof |
EP1583836A2 (en) * | 2002-12-11 | 2005-10-12 | University of Massachusetts | METHOD OF INTRODUCING siRNA INTO ADIPOCYTES |
US20050014264A1 (en) * | 2002-12-11 | 2005-01-20 | University Of Massachusetts | Method of introducing siRNA into adipocytes |
EP1583836A4 (en) * | 2002-12-11 | 2006-12-27 | Univ Massachusetts | METHOD OF INTRODUCING siRNA INTO ADIPOCYTES |
US20110092571A1 (en) * | 2003-01-16 | 2011-04-21 | The Trustees Of The University Of Pennsylvania | Compositions and methods for sirna inhibition of icam-1 |
US7847090B2 (en) | 2003-01-16 | 2010-12-07 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of ICAM-1 |
US20090104260A1 (en) * | 2003-01-16 | 2009-04-23 | The Trustees Of The University Of Pennsylvania | Compositions and methods for sirna inhibition of icam-1 |
US8193163B2 (en) | 2003-01-16 | 2012-06-05 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of ICAM-1 |
US20050260756A1 (en) * | 2003-01-28 | 2005-11-24 | Troy Carol M | Complex for facilitating delivery of dsRNA into a cell and uses thereof |
US20040248174A1 (en) * | 2003-04-18 | 2004-12-09 | Thetrustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiopoietin 1and 2 and their receptor Tie2 |
US7994305B2 (en) | 2003-04-18 | 2011-08-09 | The Trustees Of The University Of Pennsylvania | Compositions and methods for siRNA inhibition of angiopoietin 1 and 2 and their receptor Tie2 |
WO2005019410A3 (en) * | 2003-04-25 | 2005-12-08 | Intradigm Corp | Rnai agents for anti-sars coronavirus therapy |
US8722340B2 (en) | 2003-05-29 | 2014-05-13 | Board Of Regents, The University Of Texas System | JAB1 as a prognostic marker and a therapeutic target for human cancer |
US20050069918A1 (en) * | 2003-05-29 | 2005-03-31 | Francois Claret | JAB1 as a prognostic marker and a therapeutic target for human cancer |
US20050059044A1 (en) * | 2003-06-03 | 2005-03-17 | Graham Michael Wayne | Double-stranded nucleic acid |
US7790691B2 (en) | 2003-06-20 | 2010-09-07 | Isis Pharmaceuticals, Inc. | Double stranded compositions comprising a 3′-endo modified strand for use in gene modulation |
US20070015722A1 (en) * | 2003-06-20 | 2007-01-18 | Kraynack Brian A | Double stranded compositions comprising a 3'-endo modified strand for use in gene modulation |
WO2005016504A2 (en) | 2003-06-23 | 2005-02-24 | Pioneer Hi-Bred International, Inc. | Disruption of acc synthase genes to delay senescence in plants |
US20070105113A1 (en) * | 2003-08-14 | 2007-05-10 | Takara Bio Inc. | Methods of degrading dsrna and synthesizing rna |
US20050112763A1 (en) * | 2003-08-21 | 2005-05-26 | Cold Spring Harbor Laboratory | RNAI-based modification of heterochromatin |
US20070218524A1 (en) * | 2003-09-30 | 2007-09-20 | Jun Tomono | Polypeptide Having Rnase III Activity |
US7962316B2 (en) | 2003-10-27 | 2011-06-14 | Merck Sharp & Dohme Corp. | Method of designing siRNAs for gene silencing |
US20080234941A1 (en) * | 2003-10-27 | 2008-09-25 | Jackson Aimee L | Method of Designing Sirnas for Gene Silencing |
US8457902B2 (en) | 2003-10-27 | 2013-06-04 | Merck Sharp & Dohme Corp. | Method for selecting SIRNAs from a plurality of SIRNAs for gene silencing |
US20070275919A1 (en) * | 2003-11-04 | 2007-11-29 | Sergei Gryaznov | Rna Amidates and Thioamidates for Rnai |
US9822360B2 (en) | 2003-11-04 | 2017-11-21 | Geron Corporation | RNA amidates and thioamidates for RNAi |
US10655127B2 (en) | 2003-11-04 | 2020-05-19 | Geron Corporation | RNA amidates and thioamidates for RNAi |
US9133233B2 (en) | 2003-11-04 | 2015-09-15 | Geron Corporation | RNA amidates and thioamidates for RNAi |
US8304189B2 (en) | 2003-12-01 | 2012-11-06 | Life Technologies Corporation | Nucleic acid molecules containing recombination sites and methods of using the same |
US9534252B2 (en) | 2003-12-01 | 2017-01-03 | Life Technologies Corporation | Nucleic acid molecules containing recombination sites and methods of using the same |
US20090155268A1 (en) * | 2004-03-05 | 2009-06-18 | Vegenics Limited | Growth Factor Binding Constructs Materials and Methods |
US8691967B2 (en) | 2004-03-05 | 2014-04-08 | Benitec, Inc. | Multiple promoter expression cassettes for simultaneous delivery of RNAi agents |
EP2363483A2 (en) | 2004-03-05 | 2011-09-07 | Benitec, Inc. | Multiple promoter expression cassettes for simultaneous delivery of RNAi agents |
US7422741B2 (en) | 2004-03-05 | 2008-09-09 | Vegenics Limited | VEGFR-3 fusion proteins |
US20110003378A1 (en) * | 2004-03-05 | 2011-01-06 | Benitec, Inc. | MULTIPLE PROMOTER EXPRESSION CASSETTES FOR SIMULTANEOUS DELIVERY OF RNAi AGENTS |
US7855178B2 (en) | 2004-03-05 | 2010-12-21 | Vegenics Limited | Growth factor binding constructs materials and methods |
US8283461B2 (en) | 2004-03-05 | 2012-10-09 | Benitec, Inc. | Multiple promoter expression cassettes for simultaneous delivery of RNAi agents |
US8569474B2 (en) | 2004-03-09 | 2013-10-29 | Isis Pharmaceuticals, Inc. | Double stranded constructs comprising one or more short strands hybridized to a longer strand |
US8063198B2 (en) | 2004-04-05 | 2011-11-22 | Alnylam Pharmaceuticals, Inc. | Processes and reagents for desilylation of oligonucleotides |
US8058448B2 (en) | 2004-04-05 | 2011-11-15 | Alnylam Pharmaceuticals, Inc. | Processes and reagents for sulfurization of oligonucleotides |
US8431693B2 (en) | 2004-04-05 | 2013-04-30 | Alnylam Pharmaceuticals, Inc. | Process for desilylation of oligonucleotides |
US8470988B2 (en) | 2004-04-27 | 2013-06-25 | Alnylam Pharmaceuticals, Inc. | Single-stranded and double-stranded oligonucleotides comprising a 2-arylpropyl moiety |
US7626014B2 (en) | 2004-04-27 | 2009-12-01 | Alnylam Pharmaceuticals | Single-stranded and double-stranded oligonucleotides comprising a 2-arylpropyl moiety |
US20100197899A1 (en) * | 2004-04-27 | 2010-08-05 | Alnylam Pharmaceuticals, Inc. | Single-stranded and double-stranded oligonucleotides comprising a 2-arylpropyl moiety |
US7674778B2 (en) | 2004-04-30 | 2010-03-09 | Alnylam Pharmaceuticals | Oligonucleotides comprising a conjugate group linked through a C5-modified pyrimidine |
US20090048192A1 (en) * | 2004-06-03 | 2009-02-19 | Isis Pharmaceuticals, Inc. | Double Strand Compositions Comprising Differentially Modified Strands for Use in Gene Modulation |
US8394947B2 (en) | 2004-06-03 | 2013-03-12 | Isis Pharmaceuticals, Inc. | Positionally modified siRNA constructs |
US7615618B2 (en) | 2004-06-30 | 2009-11-10 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a non-phosphate backbone linkage |
US7723512B2 (en) | 2004-06-30 | 2010-05-25 | Alnylam Pharmaceuticals | Oligonucleotides comprising a non-phosphate backbone linkage |
US8013136B2 (en) | 2004-06-30 | 2011-09-06 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a non-phosphate backbone linkage |
US7579451B2 (en) | 2004-07-21 | 2009-08-25 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a modified or non-natural nucleobase |
US7772387B2 (en) | 2004-07-21 | 2010-08-10 | Alnylam Pharmaceuticals | Oligonucleotides comprising a modified or non-natural nucleobase |
US7632932B2 (en) | 2004-08-04 | 2009-12-15 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a ligand tethered to a modified or non-natural nucleobase |
US7893224B2 (en) | 2004-08-04 | 2011-02-22 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a ligand tethered to a modified or non-natural nucleobase |
US7884086B2 (en) | 2004-09-08 | 2011-02-08 | Isis Pharmaceuticals, Inc. | Conjugates for use in hepatocyte free uptake assays |
US20070036740A1 (en) * | 2004-10-06 | 2007-02-15 | Reed Kenneth C | Modulation of hair growth |
US20060115455A1 (en) * | 2004-10-22 | 2006-06-01 | Reed Kenneth C | Therapeutic RNAi agents for treating psoriasis |
US7622454B2 (en) | 2004-12-23 | 2009-11-24 | Alcon, Inc. | RNAi inhibition of CTGF for treatment of ocular disorders |
US7838507B2 (en) | 2004-12-23 | 2010-11-23 | Alcon, Inc. | RNAi inhibition of CTGF for treatment of ocular disorders |
US20060142228A1 (en) * | 2004-12-23 | 2006-06-29 | Ambion, Inc. | Methods and compositions concerning siRNA's as mediators of RNA interference |
US8058255B2 (en) | 2004-12-23 | 2011-11-15 | Applied Biosystems, Llc | Methods and compositions concerning siRNA's as mediators of RNA interference |
US20060166919A1 (en) * | 2004-12-23 | 2006-07-27 | Alcon, Inc. | RNAi inhibition of CTGF for treatment of ocular disorders |
US20060172961A1 (en) * | 2004-12-23 | 2006-08-03 | Alcon, Inc. | RNAi inhibition of serum amyloid a for treatment of glaucoma |
US20110054008A1 (en) * | 2004-12-23 | 2011-03-03 | Alcon, Inc. | RNAi Inhibition of Serum Amyloid A For Treatment of Glaucoma |
US20100159591A1 (en) * | 2004-12-23 | 2010-06-24 | Life Technologies Corporation | METHODS AND COMPOSITIONS CONCERNING siRNA'S AS MEDIATORS OF RNA INTERFERENCE |
US20100152279A1 (en) * | 2004-12-23 | 2010-06-17 | Alcon, Inc. | RNAi Inhibition of Serum Amyloid A For Treatment of Glaucoma |
US20090023671A1 (en) * | 2005-01-06 | 2009-01-22 | Brashears Sarah J | Rnai Agents for Maintenance of Stem Cells |
US20090326044A1 (en) * | 2005-02-01 | 2009-12-31 | Alcon Research, Ltd. | RNAi-Mediated Inhibition of Ocular Targets |
US20060172963A1 (en) * | 2005-02-01 | 2006-08-03 | Alcon, Inc. | RNAi-mediated inhibition of ocular hypertension targets |
US20060172965A1 (en) * | 2005-02-01 | 2006-08-03 | Alcon, Inc. | RNAi-mediated inhibition of ocular targets |
US7592324B2 (en) | 2005-02-01 | 2009-09-22 | Alcon, Inc. | RNAi-mediated inhibition of ocular targets |
EP2172549A2 (en) | 2005-02-03 | 2010-04-07 | Benitec, Inc. | RNAi expression constructs |
US20070025969A1 (en) * | 2005-02-03 | 2007-02-01 | Roelvink Petrus W | RNAi expression constructs |
US7803611B2 (en) | 2005-02-03 | 2010-09-28 | Benitec, Inc. | RNAi expression constructs |
US8993530B2 (en) | 2005-02-03 | 2015-03-31 | Benitec, Inc. | RNAi expression constructs |
US8076471B2 (en) | 2005-02-03 | 2011-12-13 | Benitec, Inc. | RNAi expression constructs |
US8173617B2 (en) | 2005-03-11 | 2012-05-08 | Novartis Ag | RNAi-mediated inhibition of frizzled related protein-1 for treatment of glaucoma |
US9040494B2 (en) | 2005-03-11 | 2015-05-26 | Novartis Ag | RNAi-mediated inhibition of frizzled related protein-1 for treatment of glaucoma |
US9550994B2 (en) | 2005-03-11 | 2017-01-24 | Arrowhead Pharmaceuticals, Inc. | RNAI-mediated inhibition of frizzled related protein-1 for treatment of glaucoma |
US20110190381A1 (en) * | 2005-03-11 | 2011-08-04 | Alcon Inc. | Rnai-mediated inhibition of frizzled related protein-1 for treatment of glaucoma |
US20060223773A1 (en) * | 2005-03-11 | 2006-10-05 | Alcon, Inc. | RNAi-mediated inhibition of Frizzled Related Protein-1 for treatment of glaucoma |
US7947660B2 (en) | 2005-03-11 | 2011-05-24 | Alcon, Inc. | RNAi-mediated inhibition of frizzled related protein-1 for treatment of glaucoma |
US20090203055A1 (en) * | 2005-04-18 | 2009-08-13 | Massachusetts Institute Of Technology | Compositions and methods for RNA interference with sialidase expression and uses thereof |
US20070081982A1 (en) * | 2005-04-28 | 2007-04-12 | Elisabeth Evertsz | Multiple RNAi expression cassettes for simultaneous delivery of RNAi agents related to heterozygotic expression patterns |
US20090209628A1 (en) * | 2005-04-28 | 2009-08-20 | Benitec Limited | MULTIPLE RNAi EXPRESSION CASSETTES FOR SIMULTANEOUS DELIVERY OF RNAi AGENTS RELATED TO HETEROZYGOTIC EXPRESSION PATTERNS |
US20090175871A1 (en) * | 2005-11-25 | 2009-07-09 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method for demonstrating presence or absence of markers associated with the presence and/or the chemosensitivity of tumors |
EP2292739A1 (en) | 2006-03-24 | 2011-03-09 | Institut National De La Recherche Agronomique | Method for preparing differentiated avian cells and genes involved in the maintenance of pluripotency |
EP2548578A1 (en) | 2006-05-17 | 2013-01-23 | The Ludwig Institute for Cancer Research | Targeting VEGF-B regulation of fatty acid transporters to modulate human diseases |
EP2548579A1 (en) | 2006-05-17 | 2013-01-23 | The Ludwig Institute for Cancer Research | Targeting VEGF-B regulation of fatty acid transporters to modulate human diseases |
US20080152654A1 (en) * | 2006-06-12 | 2008-06-26 | Exegenics, Inc., D/B/A Opko Health, Inc. | COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS |
US20110143400A1 (en) * | 2006-09-08 | 2011-06-16 | Opko Ophthalmics, Llc | Sirna and methods of manufacture |
US7872118B2 (en) | 2006-09-08 | 2011-01-18 | Opko Ophthalmics, Llc | siRNA and methods of manufacture |
US20090061487A1 (en) * | 2006-09-08 | 2009-03-05 | Samuel Jotham Reich | Sirna and methods of manufacture |
US8084422B2 (en) | 2007-04-04 | 2011-12-27 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Method of treating insulin resistance with a selective inhibitor of CB2 receptor activity |
US10131904B2 (en) | 2008-02-11 | 2018-11-20 | Rxi Pharmaceuticals Corporation | Modified RNAi polynucleotides and uses thereof |
US10633654B2 (en) | 2008-02-11 | 2020-04-28 | Phio Pharmaceuticals Corp. | Modified RNAi polynucleotides and uses thereof |
US9029119B2 (en) * | 2008-04-29 | 2015-05-12 | Eberhard-Karls-Universitaet Tuebingen Universitaetsklinikum | Composition for the cultivation of sophisticated bacteria |
US20110070627A1 (en) * | 2008-04-29 | 2011-03-24 | Eberhard-Karls-Universitaet Tuebingen Universitaetsklinikum | Composition for the cultivation of sophisticated bacteria |
US8815818B2 (en) | 2008-07-18 | 2014-08-26 | Rxi Pharmaceuticals Corporation | Phagocytic cell delivery of RNAI |
US9175289B2 (en) | 2008-09-22 | 2015-11-03 | Rxi Pharmaceuticals Corporation | Reduced size self-delivering RNAi compounds |
US10774330B2 (en) | 2008-09-22 | 2020-09-15 | Phio Pharmaceuticals Corp. | Reduced size self-delivering RNAI compounds |
US10815485B2 (en) | 2008-09-22 | 2020-10-27 | Phio Pharmaceuticals Corp. | RNA interference in skin indications |
US10876119B2 (en) | 2008-09-22 | 2020-12-29 | Phio Pharmaceuticals Corp. | Reduced size self-delivering RNAI compounds |
US8664189B2 (en) | 2008-09-22 | 2014-03-04 | Rxi Pharmaceuticals Corporation | RNA interference in skin indications |
US9303259B2 (en) | 2008-09-22 | 2016-04-05 | Rxi Pharmaceuticals Corporation | RNA interference in skin indications |
US8796443B2 (en) | 2008-09-22 | 2014-08-05 | Rxi Pharmaceuticals Corporation | Reduced size self-delivering RNAi compounds |
US9938530B2 (en) | 2008-09-22 | 2018-04-10 | Rxi Pharmaceuticals Corporation | RNA interference in skin indications |
US10138485B2 (en) | 2008-09-22 | 2018-11-27 | Rxi Pharmaceuticals Corporation | Neutral nanotransporters |
US10041073B2 (en) | 2008-09-22 | 2018-08-07 | Rxi Pharmaceuticals Corporation | Reduced size self-delivering RNAi compounds |
US11396654B2 (en) | 2008-09-22 | 2022-07-26 | Phio Pharmaceuticals Corp. | Neutral nanotransporters |
US11254940B2 (en) | 2008-11-19 | 2022-02-22 | Phio Pharmaceuticals Corp. | Inhibition of MAP4K4 through RNAi |
US9074211B2 (en) | 2008-11-19 | 2015-07-07 | Rxi Pharmaceuticals Corporation | Inhibition of MAP4K4 through RNAI |
US8470792B2 (en) | 2008-12-04 | 2013-06-25 | Opko Pharmaceuticals, Llc. | Compositions and methods for selective inhibition of VEGF |
US10167471B2 (en) | 2009-01-05 | 2019-01-01 | Rxi Pharmaceuticals Corporation | Inhibition of PCSK9 through RNAI |
US9493774B2 (en) | 2009-01-05 | 2016-11-15 | Rxi Pharmaceuticals Corporation | Inhibition of PCSK9 through RNAi |
US9745574B2 (en) | 2009-02-04 | 2017-08-29 | Rxi Pharmaceuticals Corporation | RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality |
US10479992B2 (en) | 2009-02-04 | 2019-11-19 | Phio Pharmaceuticals Corp. | RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality |
US11667915B2 (en) | 2009-02-04 | 2023-06-06 | Phio Pharmaceuticals Corp. | RNA duplexes with single stranded phosphorothioate nucleotide regions for additional functionality |
WO2011004003A1 (en) | 2009-07-10 | 2011-01-13 | Schmuelling Thomas | Disruption of ckx3 and at least one other ckx gene in a plant or plant cell leads to improved traits |
US20130180001A1 (en) * | 2009-11-30 | 2013-07-11 | Jean-Philippe Vielle-Calzada | Plants that reproduce via unreduced gametes |
US10240149B2 (en) | 2010-03-24 | 2019-03-26 | Phio Pharmaceuticals Corp. | Reduced size self-delivering RNAi compounds |
US10662430B2 (en) | 2010-03-24 | 2020-05-26 | Phio Pharmaceuticals Corp. | RNA interference in ocular indications |
US9095504B2 (en) | 2010-03-24 | 2015-08-04 | Rxi Pharmaceuticals Corporation | RNA interference in ocular indications |
US10184124B2 (en) | 2010-03-24 | 2019-01-22 | Phio Pharmaceuticals Corp. | RNA interference in ocular indications |
US9340786B2 (en) | 2010-03-24 | 2016-05-17 | Rxi Pharmaceuticals Corporation | RNA interference in dermal and fibrotic indications |
US11584933B2 (en) | 2010-03-24 | 2023-02-21 | Phio Pharmaceuticals Corp. | RNA interference in ocular indications |
US10913948B2 (en) | 2010-03-24 | 2021-02-09 | Phio Pharmaceuticals Corp. | RNA interference in dermal and fibrotic indications |
US11118178B2 (en) | 2010-03-24 | 2021-09-14 | Phio Pharmaceuticals Corp. | Reduced size self-delivering RNAI compounds |
US9080171B2 (en) | 2010-03-24 | 2015-07-14 | RXi Parmaceuticals Corporation | Reduced size self-delivering RNAi compounds |
US9963702B2 (en) | 2010-03-24 | 2018-05-08 | Rxi Pharmaceuticals Corporation | RNA interference in dermal and fibrotic indications |
US20130197207A1 (en) * | 2010-06-01 | 2013-08-01 | Universidty of Kentucky Research Foundation | Method of inhibiting alu rna and therapeutic uses thereof |
US8809517B2 (en) * | 2010-06-01 | 2014-08-19 | University Of Kentucky Research Foundation | Method of inhibiting Alu RNA and therapeutic uses thereof |
US20130160156A1 (en) * | 2010-06-08 | 2013-06-20 | Devgen Private Limited | Method for down-regulating gene expression in fungi |
EP2423304A1 (en) | 2010-08-30 | 2012-02-29 | IMBA-Institut für Molekulare Biotechnologie GmbH | Use of a RNA ligase |
WO2012028606A1 (en) | 2010-08-30 | 2012-03-08 | Imba - Institut Für Molekulare Biotechnologie Gmbh | Use of a hspc117 molecule as rna ligase |
EP2433644A1 (en) | 2010-09-22 | 2012-03-28 | IMBA-Institut für Molekulare Biotechnologie GmbH | Breast cancer therapeutics |
WO2012038504A2 (en) | 2010-09-22 | 2012-03-29 | Imba - Institut Für Molekulare Biotechnologie Gmbh | Breast cancer therapeutics |
EP3124610A1 (en) | 2010-10-28 | 2017-02-01 | Benitec Biopharma Limited | Hbv treatment |
WO2012084715A1 (en) | 2010-12-20 | 2012-06-28 | Schmuelling Thomas | Disruption of ahp6 gene leads to plants with improved seed yield |
WO2013160291A2 (en) | 2012-04-23 | 2013-10-31 | Imba - Institut Für Molekulare Biotechnologie Gmbh | Archease as rna ligase complex member |
EP2700949A1 (en) | 2012-08-24 | 2014-02-26 | IMG Institut für medizinische Genomforschung Planungsgesellschaft M.B.H. | Use of biliverdin reductase proteins as cancer marker |
US10934550B2 (en) | 2013-12-02 | 2021-03-02 | Phio Pharmaceuticals Corp. | Immunotherapy of cancer |
EP3693384A1 (en) | 2014-03-11 | 2020-08-12 | Cellectis | Method for generating t-cells compatible for allogenic transplantation |
US11279934B2 (en) | 2014-04-28 | 2022-03-22 | Phio Pharmaceuticals Corp. | Methods for treating cancer using nucleic acids targeting MDM2 or MYCN |
US10240153B2 (en) | 2014-07-16 | 2019-03-26 | Arrowhead Pharmaceuticals, Inc. | Organic compositions to treat APOC3-related diseases |
US11066667B2 (en) | 2014-07-16 | 2021-07-20 | Arrowhead Pharmaceuticals, Inc. | Organic compositions to treat APOC3-related diseases |
US10900039B2 (en) | 2014-09-05 | 2021-01-26 | Phio Pharmaceuticals Corp. | Methods for treating aging and skin disorders using nucleic acids targeting Tyr or MMP1 |
US11926828B2 (en) | 2014-09-05 | 2024-03-12 | Phio Pharmaceuticals Corp. | Methods for treating aging and skin disorders using nucleic acids targeting TYR or MMP1 |
WO2016099918A1 (en) | 2014-12-17 | 2016-06-23 | Pioneer Hi Bred International Inc | Modulation of yep6 gene expression to increase yield and other related traits in plants |
US11001845B2 (en) | 2015-07-06 | 2021-05-11 | Phio Pharmaceuticals Corp. | Nucleic acid molecules targeting superoxide dismutase 1 (SOD1) |
US10808247B2 (en) | 2015-07-06 | 2020-10-20 | Phio Pharmaceuticals Corp. | Methods for treating neurological disorders using a synergistic small molecule and nucleic acids therapeutic approach |
US11021707B2 (en) | 2015-10-19 | 2021-06-01 | Phio Pharmaceuticals Corp. | Reduced size self-delivering nucleic acid compounds targeting long non-coding RNA |
WO2018229251A1 (en) | 2017-06-16 | 2018-12-20 | Imba - Institut Für Molekulare Biotechnologie Gmbh | Blood vessel organoid, methods of producing and using said organoids |
US11214801B2 (en) | 2017-09-11 | 2022-01-04 | Arrowhead Pharmaceuticals, Inc. | RNAi agents and compositions for inhibiting expression of apolipoprotein C-III (APOC3) |
WO2023237873A1 (en) | 2022-06-07 | 2023-12-14 | The University Court Of The University Of Glasgow | Targets for cancer therapy |
Also Published As
Publication number | Publication date |
---|---|
US20040086884A1 (en) | 2004-05-06 |
WO2001068836A2 (en) | 2001-09-20 |
US20090193531A1 (en) | 2009-07-30 |
AU2001245793A1 (en) | 2001-09-24 |
US7732417B2 (en) | 2010-06-08 |
IL151781A0 (en) | 2003-04-10 |
CA2403397A1 (en) | 2001-09-20 |
WO2001068836A3 (en) | 2002-03-14 |
EP1272630A2 (en) | 2003-01-08 |
JP2003526367A (en) | 2003-09-09 |
US8383599B2 (en) | 2013-02-26 |
US20040018999A1 (en) | 2004-01-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7732417B2 (en) | Methods and compositions for RNA interference using recombinant Dicer and Argonaut | |
US20030084471A1 (en) | Methods and compositions for RNA interference | |
US8829264B2 (en) | Methods and compositions for RNA interference | |
US8742092B2 (en) | RNA sequence-specific mediators of RNA interference | |
AU2007202668A1 (en) | Methods and compositions for RNA interference | |
EP1728862A1 (en) | Methods and compositions for RNA interference | |
AU2008246266B2 (en) | Methods and compositions for RNA interference | |
AU2012261651B2 (en) | Methods and compositions for RNA interference | |
AU2003210621A1 (en) | Methods and compositions for RNA interference |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF Free format text: CONFIRMATORY LICENSE;ASSIGNOR:COLD SPRING HARBOR LABORATORY;REEL/FRAME:020906/0815 Effective date: 20011121 |
|
AS | Assignment |
Owner name: COLD SPRING HARBOR LABORATORY, NEW YORK Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:GENETICA INCORPORATED;REEL/FRAME:021251/0021 Effective date: 20040107 Owner name: COLD SPRING HARBOR LABORATORY,NEW YORK Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:GENETICA INCORPORATED;REEL/FRAME:021251/0021 Effective date: 20040107 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |