WO1998049350A1 - In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes - Google Patents
In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes Download PDFInfo
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- WO1998049350A1 WO1998049350A1 PCT/US1998/008834 US9808834W WO9849350A1 WO 1998049350 A1 WO1998049350 A1 WO 1998049350A1 US 9808834 W US9808834 W US 9808834W WO 9849350 A1 WO9849350 A1 WO 9849350A1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
- A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
- A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
Definitions
- the invention concerns methods and compositions for the use of recombinagenic oligonucleobases in vivo for the correction of disease causing genetic defects.
- the invention is particularly suited for the treatment of genetic defects of the type where the correction of the genetic defect in the hepatocytes of the subject will ameliorate the disease.
- CMV Chimeric Mutational Vectors
- 5,565,350 (the '350 patent) described duplex CMV for the introduction of genetic changes in eukaryotic cells.
- Examples in a Ustilago maydis gene and in the murine ras gene were reported.
- the latter example was designed to introduce a transforming mutation into the ras gene so that the successful mutation of the ras gene in NIH 3T3 cells would cause the growth of a colony of cells ("transformation").
- the '350 patent reported that the maximum rate of transformation of NIH 3T3 was less than 0.1 %, i.e., about 100 transformants per 10 6 cells exposed to the ras duplex CMV. In the Ustilago maydis system the rate of transformants was about 600 per 10 6 .
- a chimeric vector designed to introduce a mutation into a human bcl-2 gene was described in Kmiec, E.B., February 1996, Seminars in Oncology 23, 188.
- a duplex CMV designed to repair the mutation in codon 12 of K-ras was described in Kmiec, E.B., December 1995, Advanced Drug Delivery Reviews 17, 333- 40.
- the duplex CMV was tested in Capan 2, a cell line derived from a human pancreatic adenocarcinoma, using LIPOFECTINTM to introduce the duplex CMV into the Capan 2 cells. Twenty four hours after the duplex CMV were introduced, the cells were harvested and genomic DNA was extracted; a fragment containing codon 12 of K- ras was amplified by PCR and the rate of conversion estimated by hybridization with allele specific probes. The rate of repair was reported to be approximately 18%.
- a duplex CMV designed to repair a mutation in the gene encoding liver/bone/kidney type alkaline phosphatase was reported in Yoon, K., et al., March 1996, Proc. Natl. Acad. Sci. 93, 2071.
- the alkaline phosphatase gene was transiently introduced into CHO cells by a plasmid. Six hours later the duplex CMV was introduced. The plasmid was recovered at 24 hours after introduction of the duplex CMV and analyzed. The results showed that approximately 30 to 38% of the alkaline phosphatase genes were repaired by the duplex CMV.
- WO 97/4141 1 by E.B. Kmiec, A. Cole-Strauss and K. Yoon, and the publication Cole-Strauss, A., et al., Sept 1996, SCIENCE 273, 1386 disclose duplex CMV that are used in the treatment of genetic diseases of hematopoietic cells, e.g., Sickle Cell Disease, Thalassemia and Gaucher Disease.
- WO 97/48714 and counterpart United States Patent No. 5,731 ,181 by E.B. Kmiec describes duplex CMV having non-natural nucleotides for use in specific, site-directed mutagenesis.
- Kmiec and his colleagues concerned cells that are mitotically active, i.e., proliferating cells, at the time they are exposed to CMV.
- Kmiec and colleagues used a CMV/liposomal macromolecular carrier complex in which the CMV were mixed with a pre-formed liposome or lipid vesicle. In such a complex the CMV are believed to adhere to the surface of the liposome.
- Branched chain polyethylenimine has been used as a carrier to introduce nucleic acids into eukaryotic cells both in vivo and in vitro. Boussif, O., et al., 1995, Proc. Natl. Acad. Sci. 92, 7297; Abdallah, B. et al., 1996, Human Gene Therapy 7, 1947. Boletta, A., et al., 1997, 8, 1243-1251 .
- the in vitro use o galactosylated polyethylenimine to introduce DNA into cultured HepG2 hepatocarcinoma cell lines is reported by Zanta, et al., October 1 , 1997, Bioconjugate Chemistry 8, 839-844.
- Branched chain polyethylenimines contain secondary and tertiary amino groups having a broad range of pK's and, consequently these polyethylenimines have a substantial buffering capacity at a pH where polylysine has little or no capacity, i.e., less than about 8. Tang, M.K., & Szoka, F.C., 1997, Gene Therapy 4, 823-832.
- Hposomes or lipid vesicles to introduce DNA encoding a foreign protein into cells has been described.
- the most frequently used techniques adhere the DNA to the surface of a positively charged liposome, rather than encapsulating the DNA, although encapsulated DNA techniques were known.
- United States Patent Nos. 4,235,871 and 4,394,448 are relevant. The field is reviewed by Smith, J.G., et al., 1993, Biochim. Biophy. Acta 1154, 327-340 and Staubinger, R.M., et al., 1987, Methods in Enzymology 185, 512.
- DOTAP a cationic lipid in a liposome to transfect hepatic cells in vivo
- Fabrega A.J., et al., 1996, Transplantation 62, 1866-1871 .
- the use of cationic lipid-containing iiposomes to transfect a variety of cells of adult mice is described in Zhu, N., et al., 1993, Science 261, 209.
- the use of phosphatidylserine containing lipids to form DNA encapsulating Iiposomes for transfection is described in Fraley, R., et al., 1981 , Biochemistry 20, 6978-87.
- United States Patent Nos. 5,166,320 and 5,635,383 disclose the transfection of hepatocytes by forming a complex of a DNA, a polycationic macromolecular carrier and a ligand for the asialoglycoprotein receptor.
- the macromolecular carrier was polylysine.
- lactosylcerebroside containing liposome to transfect a hepatocyte in vivo is described by Nandi, P.K., et al., 1986, J.
- the present invention concerns compositions comprising a recombinagenic oligonucleobase and methods of their use to introduce specific genetic alterations in the DNA of the cells of a subject individual, a process termed "transmutation.”
- a recombinagenic oligonucleobase is a nucleobase polymer that contains fewer than 60 bases of the sequence of the gene that is the target of the transmutation.
- the invention can be used with any recombinagenic oligonucleobase now known or to be developed.
- the recombinagenic nucleobase is a Chimeric Mutational Vector (CMV) and the process of using CMV to effect transmutation is termed "Chimeraplasty.”
- CMV Chimeric Mutational Vector
- Chrimeraplasty the process of using CMV to effect transmutation.
- the compositions and methods of the invention can be administered to the subject individual to effect the genetic alteration in vivo.
- the present invention depends in part on the unexpected discovery that CMV are effective to transmutate non-proliferating, i.e., non-mitotic, cells.
- the invention further depends upon the unexpected discovery that CMV complexed with a macromolecular carrier to which is attached a ligand for a cell-surface receptor that is internalized through clathrin-coated pits into endosomes are suitable for chimeraplasty (a "clathrin-coated pit receptor").
- the invention provides a composition comprising a CMV and a macromolecular carrier selected from the group consisting of: an aqueous- cored lipid vesicle of between 25 nm and 400 nm diameter, wherein the aqueous core contains the CMV; a lipid nanosphere of between 25 nm and 400 nm diameter, having a lipid core, wherein the lipid core contains a lipophilic salt of the CMV; and a polycationic salt of the CMV.
- polycations for such salts include polyethylenimine, polylysine and histone HI .
- the poiycation is a branched chain polyethylenimine (PEI) salt having a mass average molecular weight greater than 500 daltons and less than 1.3 Md.
- PEI branched chain polyethylenimine
- the carrier can be optionally substituted with a ligand for a cellular receptor that is internalized through a clathrin-coated pit into an endosome.
- the ligand is a lactosyl moiety and the receptor is the asialoglycoprotein receptor of the hepatocyte.
- Other receptors include the receptors for transferrin, insulin, glucagon, EGF and low density lipoproteins (LDL) and the ligand can be a fragment of the natural ligand that binds the receptor.
- the ligand can be any compound that binds specifically to the receptor, whether related to a naturally occurring ligand or otherwise.
- composition of the CMV/macromolecular carrier/I igand further comprises a pharmaceutically acceptable aqueous carrier, such as a buffered saline solution.
- a pharmaceutically acceptable aqueous carrier such as a buffered saline solution.
- cell culture medium which can be supplemented with serum.
- a further embodiment of the invention comprises the administration of the above described complex to a human subject having a disease-causing genetic condition.
- the complex can be administered parenterally and in a preferred embodiment the complex is administered directly to the organ that is affected by the disease-causing genetic condition.
- the invention comprises the use of the CMV/macromolecular carrier/I igand complex for chimeraplasty in a subject individual and in cell culture.
- An oligonucleobase is a polymer of nucleobases, which polymer can hybridize by Watson-Crick base pairing to a DNA having the complementary sequence.
- Nucleobases comprise a base, which is a purine, pyrimidine, or a derivative or analog thereof.
- Nucleobases include peptide nucleobases. the subunits of peptide nucleic acids, and morpholine nucleobases as well as nucleosides and nucleotides.
- Nucleosides are nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2'-deoxyriboside.
- Nucleosides can be linked by one of several linkage moieties, which may or may not contain a phosphorus. Nucleosides that are linked by unsubstituted phosphodiester linkages are termed nucleotides.
- a oligonucleobase chain has a single 5' and 3' terminus, which are the ultimate nucleobases of the polymer.
- a particular oligonucleobase chain can contain nucleobases of all types.
- An oligonucleobase compound is a compound comprising one or more oligonucleobase chains that are complementary and hybridized by Watson-Crick base pairing. Nucleobases are either deoxyribo-type or ribo-type. Ribo- tvpe nucleobases are pentosefuranosyl containing nucleobases wherein the 2' carbon is a methylene substituted with a hydroxyl, alkyloxy or halogen.
- Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases and include all nucleobases that do not contain a pentosefuranosyl moiety.
- An oligonucleobase strand generically includes both oligonucleobase chains and segments or regions of oligonucleobase chains.
- An oligonucleobase strand has a 3' end and a 5' end. When a oligonucleobase strand is coextensive with a chain, the 3' and 5' ends of the strand are also 3' and 5' termini of the chain.
- a region is a portion of an oligonucleobase, the sequence of which is derived from some particular source, e.g., a CMV having a region of at least 15 nucleotides having the sequence of a fragment of the human ⁇ -globin gene.
- a segment is a portion of a CMV having some characteristic structural feature.
- a given segment or a given region can contain both 2'-deoxynucleotides and ribonucleotides.
- a ribo- tvpe segment or a 2 '-deoxyribo-type segment contain only ribo-type and 2'-deoxyribo- type nucleobases, respectively.
- the Chimeric Mutational Vectors are comprised of polymers of purines and pyrimidines that hybridize, i.e., form Watson-Crick base pairs of purines and pyrimidines, to DNA having the appropriate sequence.
- Each CMV is divided into a first and a second strand of at least 15 bases each that are complementary to each other and can be, but need not be, covalently linked.
- the subunits of the oligonucleobase polymers are termed nucleobases. Nucleobases contain a base, which is either a purine or a pyrimidine or analog or derivative thereof. There are two types of nucleobases.
- Ribo-type nucleobases are ribonucleosides having a 2'-hydroxyl, substituted hydroxyl or 2'-halo-substituted ribose. All nucleobases other than ribo-type nucleobases are deoxyribo-type nucleobases.
- the sequence of the first and second strands consists of at least two regions that are homologous to the target gene and one or more regions (the "mutator regions") that differ from the target gene and introduce the genetic change into the target gene.
- the mutator region is directly adjacent to homologous regions in both the 3' and 5' directions.
- each mutator region is adjacent in both the 3' and 5' directions to homologous regions of at least three bases.
- each mutator region is flanked in both the 3' and 5' directions by ribo-type oligonucleobase segments of at least three bases, which segments need not be adjacent to the mutator region.
- flanking ribo-type nucleobase segments need not be directly adjacent to the mutator region, i.e, a portion of the homologous region comprising deoxyribo-type nucleobases can intervene.
- the total length of all homologous regions is preferably at least 16 bases and is more preferably from about 20 nucleotides to about 60 nucleotides in length. If the CMV contains two homologous regions separated by a mutator region, the homologous regions can more preferably be each between 8 and 1 5 bases long and most preferably be 10 bases long.
- At least two homologous regions of the first strand are comprised of at least three contiguous ribo-type nucleobases which are Watson-Crick paired to deoxyribo-type nucleobases of the second strand. In a preferred embodiment there are between 9 and 25 ribo-type nucleobases and more preferably 20 ribo-type nucleobases in the first strand, which are Watson-Crick paired to deoxyribo-type nucleobases of the second strand. In one embodiment there are no ribo-type nucleobases in the second strand.
- the mutator region of the first strand consists of deoxyribo-type nucleobases and is flanked by deoxyribo-type nucleobases.
- the mutator region can be comprised of ribo-type nucleobases of the first strand and deoxyribo-type nucleobases of the second strand.
- the mutator region consists of 20 or fewer bases, more preferably 6 or fewer bases and most preferably 3 or fewer bases.
- the mutator region can be of a length different than the length of the sequence that separates the regions of the target gene homology with the homologous regions of the CMV so that an insertion or deletion of the target gene results.
- the CMV is used to introduce a deletion in the target gene there is no base identifiable as within the mutator region. Rather, the mutation is effected by the juxtaposition of the two homologous regions that are separated in the target gene.
- the length of the mutator region of a CMV that introduces a deletion in the target gene is deemed to be the length of the deletion.
- the mutator region is a deletion of from 6 to 1 bases or more preferably from 3 to 1 bases.
- Multiple separated mutations can be introduced by a single CMV, in which case there are multiple mutator regions in the same CMV.
- multiple CMV can be used simultaneously to introduce multiple genetic changes in a single gene or, altematively to introduce genetic changes in multiple genes of the same cell.
- the mutator region is also termed the heterologous region.
- the CMV is a single oligonucleobase chain of between 20 and 200 nucleobases and preferably between 40 and 100 nucleobases.
- the CMV comprises a first and a second oligonucleobase chain, each of between 20 and 100 bases; wherein the first chain comprises the first strand and the second chain comprises the second strand.
- the first and second chains can be linked covalently by other than nucleobases or, alternatively, can be associated only by Watson-Crick base pairings.
- Covalent linkage of the first and second strands can be made by oligo-alkanediols such as polyethyleneglycol, poly-1 ,3-propanediol or poly-1 ,4-butanediol.
- oligo-alkanediols such as polyethyleneglycol, poly-1 ,3-propanediol or poly-1 ,4-butanediol.
- the invention can be practiced using CMV comprising deoxynucleotides or deoxynucleosides and 2'-0 substituted ribonucleotides or ribonucleosides.
- Suitable substituents include the substituents taught by the Kmiec Application, C,. 6 alkane.
- Alternative substituents include the substituents taught by U.S. Patent No. 5,334,71 1 (Sproat) and the substituents taught by patent publications EP 629 387 and EP 679657 (collectively, the Martin Applications), which are hereby incorporated by reference.
- 2' fluoro, chloro or bromo derivative of a ribonucleotide or a ribonucleotide having a substituted 2'-0 as described in the Martin Applications or Sproat is termed a "2 '-Substituted Ribonucleotide.”
- Particular preferred embodiments of 2'-Substituted Ribonucleotides are 2'-fluoro, 2'-methoxy, 2'-propyloxy, 2'-allyloxy, 2'-hydroxylethyloxy, 2'-methoxyethyloxy, 2'-fluoropropyloxy and 2'- trifluoropropyloxy substituted ribonucleotides.
- 2'- Substituted Ribonucleotides are 2'-fluoro, 2'-methoxy, 2'-methoxyethyloxy, and 2'- allyloxy substituted nucleotides.
- 2'-Substituted Ribonucleosides are defined analogously. Particular preferred embodiments of 2'-Substituted Ribonucleosides are 2'-fluoro, 2'-methoxy, 2'-propyloxy, 2'-allyloxy, 2'-hydroxylethyloxy, 2'-methoxyethyloxy, 2'-fluoropropyloxy and 2'- trifluoropropyloxy substituted ribonucleotides. In a more preferred embodiments of 2'- Substituted Ribonucleosides are 2'-fluoro, 2'-methoxy, 2'-methoxyethyloxy, and 2'- allyloxy substituted nucleotides.
- nuclease resistant ribonucleoside encompasses 2 '-Substituted Ribonucleosides, including 2 '-Substituted Ribonucleotides and also all 2'-hydroxyl ribonucleosides other than ribonucleotides.
- the CMV preferably includes at least three and more preferably six nuclease resistant ribonucleosides. In one preferred embodiment the CMV contains no nuclease sensitive ribonucleosides. In an alternative preferred embodiment, every other ribonucleoside is nuclease resistant. Certain 2'-blocking groups can be more readily synthesized for purines or pyrimidines. In one embodiment of the CMV only the ribonucleoside purines or only the ribonucleoside pyrimdines are nuclease resistant.
- the CMV is still further characterized by containing at least three nuclease resistant ribo-type nucleobases.
- all ribo-type nucleobases are nuclease resistant.
- the prior art formulations of CMV and a macromolecular carrier are of limited utility for in vivo use because of their low capacity for CMV and because the CMV is not protected from extracellular enzymes.
- the invention provides three alternative macromolecular carriers that overcome the limitations of the prior art.
- the carriers are polyethylenimine (PEI), aqueous-cored lipid vesicles, which are also termed unilamellar Iiposomes and lipid nanospheres.
- Each of the carriers can be further provided with a ligand that is complementary to a cell-surface protein of the target cell. Such ligands are useful to increase both the amount and specificity of the uptake of CMV into the targeted cell.
- the target cell is a hepatocyte and the ligand is a galactose saccharide or lactose disaccharide that binds to the asialoglycoprotein receptor.
- the invention can be practiced using any polycation that is non-toxic when administered to cells in vitro or to subjects in vivo.
- suitable examples include polybasic amino acids such as polylysine, polyarginine, basic proteins such as histone H1 , and synthetic polymers such as the branched-chain polyethylenimine: (-NHCH 2 CH 2 -) X [-N(CH 2 CH 2 NH 2 )CH 2 CH 2 -] Y .
- the invention can be practiced with any branched chain polyethylenimine (PEI) having an average molecular weight of greater than about 500 daltons, preferably greater than between about 10 Kd and more preferably about 25 Kd (mass average molecular weight determined by light scattering)
- PEI branched chain polyethylenimine
- the upper limit of suitability is determined by the toxicity and solubility of the PEI. Toxicity and insolubility of molecular weights greater than about 1 .3 Md makes such PEI material less suitable.
- the use of high molecular weight PEI as a carrier to transfect a cell with DNA is described in Boussif, O. et al., 1995, Proc. Natl. Acad. Sci. 92, 7297, which is hereby incorporated by reference.
- PEI solutions can be prepared according to the procedure of Boussif et al.
- the CMV carrier complex is formed by mixing an aqueous solution of CMV and a neutral aqueous solution of PEI at a ratio of between 9 and 4 PEI nitrogens per CMV phosphate. In a preferred embodiment the ratio is 6.
- the complex can be formed, for example, by mixing a 10 mM solution of PEI, at pH 7.0 in 0.15 M NaCl with CMV to form a final CMV concentration of between 100 and 500 nM.
- a ligand for a clathrin-coated pit receptor can be attached to the polycation or to a fraction of the polycations.
- the ligand is a saccharide or disaccharide that binds to the asialoglycoprotein receptor, such as lactose, galactose, or N-acetylgalactosamine. Any technique can be used to attach the ligands.
- the optimal ratio of ligand to polyethylene subunit can be determined by fluorescently labeling the CMV and injecting fluorescent CMV/molecular carrier/I igand complexes directly into the tissue of interest and determining the extent of fluorescent uptake according to the method of Kren et al., 1997, Hepatology 25, 1462-1468.
- Good results can be obtained using a 1 :1 mixture of lactosylated PEI having a ratio of 0.4-0.8 lactosyl moieties per nitrogen and unmodified PEI.
- the mixture is used in a ratio of between 4 and 9 PEI nitrogens per CMV phosphate.
- a preferred ratio of oligonucleotide phosphate to nitrogen is 1 :6.
- Good results can be obtained with PEIs having a mass average molecular weight of 25 Kd and 800 Kd which are commercially available from Aldrich Chemical Co., Catalog No. 40,872-7 and 18,197-8, respectively.
- the polycationic carrier can be a basic protein such as histone H 1 , which can be substituted with a ligand for a clathrin-coated pit receptor.
- histone H 1 a basic protein
- a 1 :1 (w/w) mixture of histone and CMV can be used to practice the intention.
- Lipid nanospheres can be constructed using semi-purified lipid biological preparations, e.g., soybean oil (Sigma Chem. Co.) egg phosphatidyl choline (EPC) (Avanti Polar Lipids).
- soybean oil Sigma Chem. Co.
- EPC egg phosphatidyl choline
- DOPC dioleoyl phosphatidylcholine
- DOPE di
- lipids that can be used in the invention can be found in Gao, X. and Huany, L., 1995, Gene Therapy 2, 710.
- Saccharide ligands can be added in the form of saccharide cerebrosides, e.g., lactosylcerebroside or galactocerebroside (Avanti Polar Lipids).
- the particular choice of lipid is not critical.
- Hydrogenated EPC or lysolecithin can be used in place of EPC.
- DPPC dipalmitoyl phosphatidylcholine
- Lipid nanospheres can be constructed by the following process.
- a methanol or chloroform methanol solution of phospholipids is added to a small test tube and the solvent removed by a nitrogen stream to leave a lipid film.
- a lipophilic salt of CMV is formed by mixing an aqueous saline solution of CMV with an ethanolic solution of a cationic lipid. Good results can be obtained when the cationic species are in about a 4 fold molar excess relative to the CMV anions (phosphates).
- the lipophilic CMV salt solution is added to the lipid film, vortexed gently followed by the addition of an amount of neutral lipid equal weight to the phospholipids.
- the concentration of CMV can be up to about 3% (w/w) of the total amount of lipid.
- the emulsion is sonicated at 4°C for about 1 hour until the formation of a milky suspension with no obvious signs of separation.
- the suspension is extruded through polycarbonate filters until a final diameter of about 50 nm is achieved.
- the target cell is a reticuloendothelial cell
- the preferred diameter of the lipid nanospheres is about 100-200 nm.
- the CMV-carrying lipid nanospheres can then be washed and placed into a pharmaceutically acceptable carrier or tissue culture medium.
- the capacity of lipid nanospheres is about 2.5 mg CMV/ 500 ⁇ l of a nanosphere suspension.
- a lipid film is formed by placing a chloroform methanol solution of lipid in a tube and removing the solvent by a nitrogen stream.
- An aqueous saline solution of CMV is added such that the amount of CMV is between 20% and 50% (w/w) of the amount of lipid, and the amount of aqueous solvent is about 80% (w/w) of the amount of lipid in the final mixture.
- the liposome-containing liquid is forced through successively finer polycarbonate filter membranes until a final diameter of about 50 nm is achieved.
- the passage through the successively finer polycarbonate filter results in the conversion of polylamellar Iiposomes into unilamellar Iiposomes, i.e., vesicles.
- the target cell is a reticuloendothelial cell
- the preferred diameter of the lipid nanospheres is about 100-200 nm.
- the CMV-carrying lipid nanospheres can then be washed and placed into a pharmaceutically acceptable carrier or tissue culture medium.
- the CMV are entrapped in the aqueous core of the vesicles. About 50% of the added CMV is entrapped.
- a variation of the basic procedure comprises the formation of an aqueous solution containing a PEI/CMV condensate at a ratio of about 4 PEI imines per CMV phosphate.
- the condensate can be particularly useful when the Iiposomes are positively charged, i.e., the lipid vesicle contains a concentration of cations of cationic lipids such as DOTAP, DOTMA or DOSPER, greater than the concentration of anions of anionic lipids such as DOPS.
- the capacity of lipid vesicles is about 150 ⁇ g CMV per 500 ⁇ l of a lipid vesicle suspension.
- the lipid vesicles contain a mixture of the anionic phospholipid, DOPS, and a neutral lipid such as DOPE or DOPC.
- negatively charged phospholipids that can be used to make lipid vesicles include dioleoyl phosphatidic acid (DOPA) and dioleoyl phosphatidyl glycerol (DOPG).
- DOPA dioleoyl phosphatidic acid
- DOPG dioleoyl phosphatidyl glycerol
- the neutral lipid is DOPC and the ratio of DOPS:DOPC is between 2:1 and 1 :2 and is preferably about 1 :1.
- the ratio of negatively charged to neutral lipid should be greater than 1 :9 because the presence of less than 10% charged lipid results in instability of the lipid vesicles because of vesicle fusion.
- a particular lipid vesicle formulation can be tested by using the formulation to transfect a target cell population with a plasmid of about 5.0 kb in length that expresses some readily detectable product in the transfected target cell.
- Lipid vesicles can be used to transfect a cell with the plasmid if the plasmid is condensed with PEI at an imine:phosphate ratio of about 9-4:1.
- the capacity of the lipid vesicle formulation to transfect a cell with a plasmid is indicative of the formulation's capacity to introduce a CMV into a cell and effect a transmutation.
- Certain lipids, particularly the polycationic lipids can be toxic to certain cell lines and primary cell cultures. The formulation of the lipid vesicles should be adjusted to avoid such toxic lipids.
- Ligands for clathrin-coated pit receptors can be introduced into the lipid vescicles by a variety of means. Cerebrosides, such as lactocerebroside or galactocerebroside can be intorduced into the lipid mixture and are incorporated into the vesicle to produce a ligand for the asialoglycoprotein receptor.
- the lipid vesicle further comprise a integral membrane protein that inserts itself into the lipid bi layer of the vesicle.
- the protein is a fusigenic (F-protein) from the virus altematively termed Sendai Virus or Hemagglutinating Virus of Japan (HVJ).
- F-protein fusigenic
- HVJ Hemagglutinating Virus of Japan
- the invention can be used to correct any disease-causing mutation, in which the mutation results in the change of one or more nucleotides or in the insertion or deletion of from one to about 30 nucleotides. In a preferred embodiment the deletion or insertion is of from one to about six nucleotides.
- the disease-causing mutation is corrected by administering a CMV containing the sequence of the wild type gene that is homologous to the locus of the mutation.
- the CMV is constructed so that there are regions of homology with the mutant DNA sequence flanking the heterologous region, i.e, the region of the CMV that contains the portion of the wild-type sequence that is absent from the mutant.
- the heterologous region of the CMV is considered to be the point which is homologous to the site of the insertion. Accordingly, the length of the heterologous region of the CMV is deemed to be the length of the insertion in the mutant sequence.
- the sequence of the CMV is determined by the location of the mutation, however, the sequence of the mutation is not important. Rather, the sequence of a CMV is always the sequence of the wild type gene or a desired related sequence. In each of the sequences that follow the heterologous region is underlined.
- a first embodiment of the invention is a CMV that can be used to correct the mutation that causes the von Willebrand's Disease.
- a CMV to correct this mutation contains the sequence 5'-CTC GGA GAG C CCC CTC GCA-3' (SEQ ID No. 1), the sequence of a mixed ribo-deoxyribo oligonucleobase having the same sequence of bases or a sequence that differs by the substitution of the thymine by uracil, or the sequence of the complement thereof.
- the tissue in which the von Willebrand's factor gene needs to be corrected is the vascular endothelium.
- a further embodiment of the invention is a CMV that can be used to correct the mutation that causes Hemophilia B, which is an A ⁇ C substitution at nt 1234 of the human coagulation Factor IX gene.
- CMV to correct this mutation contains the sequence 5'-CAA GGA GAT AGT GGG GGA C-3' (SEQ ID No. 2), the sequence of a mixed ribo-deoxyribo oligonucleobase having the same sequence of bases or a sequence that differs by the substitution of the thymine by uracil, or the sequence of the complement thereof.
- the invention can be used to correct other mutations in the human coagulation Factor IX gene, the sequence of which is given in Kurachi, K., et al., 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 6461-6464, which is hereby incorporated by reference.
- the tissue in which the factor IX gene needs to be corrected is the hepatocellular liver.
- a further embodiment of the invention is a CMV that can be used to correct the Z-mutation that causes ⁇ 1-antitrypsin deficiency.
- the Z mutation is a G ⁇ A substitution located at nt 1 145 of the human ⁇ 1-antitrypsin gene.
- CMV to correct this mutation contains the sequence 5'-ACC ATC GAC GAG AAA GGG A-3' (SEQ ID No. 3), the sequence of a mixed ribo-deoxyribo oligonucleobase having the same sequence of bases or a sequence that differs by the substitution of the thymine by uracil, or the sequence of the complement thereof.
- the invention can be used to correct other mutations in the ⁇ l-antitrypsin gene, the sequence of which is given in Long, G.L., et al., 1984, Biochemistry 23, 4828-4837, which is hereby incorporated by reference.
- the tissue in which the ⁇ l-antitrypsin gene needs to be corrected is the hepatocellular liver.
- a further embodiment of the invention is a CMV that can be used to correct a mutation in the low density lipoprotein receptor (LDLR) that causes familial hypercholesterolemia (FH).
- LDLR low density lipoprotein receptor
- FH familial hypercholesterolemia
- Surveys of the more than 105 point mutations or insertions or deletions of 25 nt or fewer that cause FH can be found in Hobbs, H.H., et al., 1992, Hum. Mutat. 1, 445- 466 and Leren, T.P., et al., Hum. Genet. 95, 671-676, which are hereby incorporated by reference in their entirety.
- the complete sequence of the human LDLR cDNA is published in Yamamoto, T., et al., 1984, CELL 39, 27-38.
- the tissue in which the LDLR can be corrected to obtain amelioration of FH is the hepatocellular liver.
- a further embodiment of the invention is a CMV that can be used to correct a mutation in the glucocerebrosidase gene that causes Gaucher Disease.
- the structure of CMV that can be used to correct a Gaucher Disease mutation can be found in commonly licensed U.S. application Serial No. 08/640,51 7.
- the tissue in which the glucocerebrosidase mutation can be corrected to obtain amelioration of Gaucher Disease is the reticuloendothelial (Kupffer Cell) liver.
- a further embodiment of the invention is a CMV that can be used to correct a mutation in the glucose-6-phosphatase (G-6-P) gene that causes type 1 Glycogen Storage Disease (GSD).
- G-6-P glucose-6-phosphatase
- SCIENCE 262, 580 which is hereby incorporated by reference.
- the two most common mutations that cause type 1 GSD are C-T at nt 326, C ⁇ T at nt 1 1 18, and an insertion of TA at nt 459, as described in Lei, K.-J., et al., J. Clin. Investigation 95, 234- 240, which is hereby incorporated by reference.
- CMV to correct the two most common mutations contain the sequence 5'-TTT GGA CAG CGT CCA TAC T-3' (SEQ ID No. 4), or 5'-TGC CTC GCC CAG GTC CTG G-3' (SEQ ID No. 5), the sequence of a mixed ribo-deoxyribo oligonucleobase having the same sequence of bases or a sequence that differs by the substitution of the thymine by uracil, or the sequence of the complement thereof.
- a further embodiment of the invention is a CMV that can be used to correct a mutation in the Ornithine Transcarbamylase (OTC) gene an X-linked gene that catalyzes the condensation of ornithine and carbamyl phosphate to yield citruline and phosphate.
- OTC Ornithine Transcarbamylase
- the complete sequence of the human OTC cDNA is given in Horwich, A.L., et al., 1984, Science 224, 1068, which is hereby incorporated by reference.
- the structure of OTC gene and a review of the structure of identified mutants is reviewed in Tuchman, M., 1992, Human Mutation 2, 174.
- a further embodiment of the invention is a CMV that can be used to correct a mutation in the human UDP-glucuronosyltransferase gene that causes Crigler-Najjar syndrome.
- the sequence of the human UDP-glucuronosyltransferase gene is given in Bosma, P.J., et al., 1992, Hepatology, 15, 941-7, which is hereby incorporated by reference.
- the tissue in which the UDP-glucuronosyltransferase gene can be corrected to obtain amelioration of Crigler-Najjar syndrome is the hepatocellular liver.
- a further embodiment of the invention is a CMV that can be used to correct a mutation in a galactose-1 -phosphate uridyltransferase gene that cause galactosemia.
- the sequence of the human galactose-1 -phosphate uridyltransferase gene is described in Flack, J.E., et al., 1990 Mol. Biol. Med. 7, 365, and the molecular biology and population genetics of galactosemia are described in Reichert, J.K.V., et al., 1991 , Proc. Natl. Acad. Sci. 88, 2633-37 and Reichert, J.K.V., et al., 1991 , Am. J. Hum.
- the most common mutation that causes galactosemia is Q ⁇ R at amino acid 188.
- the CMV to correct this mutation contains the sequence 5'-CC CAC TGC CAG GTA TGG GC-3' (SEQ ID No. 6), the sequence of a mixed ribo-deoxyribo oligonucleobase having the same sequence of bases or a sequence that differs by the substitution of the thymine by uracil, or the sequence of the complement thereof.
- a further embodiment of the invention is a CMV that can be used to correct a mutation in the phenylalanine hydroxylase (PAH, phenylalanine 4-monooxygenase, EC 1.14.16.1 ) that cause phenylketonuria (PKU) or hyperphenylalaninemia.
- PAH phenylalanine hydroxylase
- PKU phenylketonuria
- the molecular and population genetics of phenylketonuria are described in Woo, SLC, 1989, Biochemistry 28, 1-7, the sequence of human PAH is described in Kowk, S.C.M., et al., 1985, Biochemistry 28, 556-561 , which are hereby incorporated by reference. Further examples of PKU-causing mutations can be found in Sworniczak, B., et al., 1992, Hum. Mutat. 1, 138-146.
- the present invention can be used to make beneficial alterations in "normal" genes, where the identity of such beneficial alteration has been identified. For example, as many as 1 in 3,000 persons have serum cholesterol levels of 100 mg/dl or less because such individuals are heterozygous for a truncated lipoprotein apo B gene. Kane, J..P. and Havel, R.J., in THE METABOLIC BASIS OF INHERITED DISEASE, VOL. II (1995, McGraw Hill, New York) p. 1866.
- a "disease-causing mutation” includes examples such as the apo B gene, wherein the "normal” gene is associated with a disease, i.e., atherosclerosis, and a rare allele, in heterozygous form, is protective from that disease. Accordingly, in these circumstances, the statistically most prevalent gene should be regarded as the “disease- causing gene” and the gene having the beneficial alteration should be regarded as the "wild-type gene.”
- the CMV of the invention can be parenterally administered directly to the target organ at a dose of between 50 and 250 ⁇ g/gm.
- the target organ is the liver muscle or kidney
- the CMV/macromolecular carrier complex can be injected directly into the organ.
- the target organ is the liver, intravenous injection into the hepatic or portal veins of a liver, having temporarily obstructed circulation can be used.
- the CMV/macromolecular complex can further comprise a hepatic targeting ligand, such as a lactosyl or galactosyl saccharide, which allows for administration of the CMV/macromolecular complex intravenously into the general circulation.
- the target organ is the lung or a tissue thereof, e.g., the bronchiolar epithelium CMV/macromolecular complex can be administered by aerosol.
- Small particle aerosol delivery of liposomal/DNA complexes is described in Schwarz L.A., et al., 1996, Human Gene Therapy 7, 731-741.
- the target organ is the vascular endothelium, as for example in von Willebrand's Disease
- the CMV/macromolecular complex can be delivered directly into the systemic circulation.
- Other organs can be targeted by use of Iiposomes that are provided with ligands that enable the liposome to be extravasated through the endothelial cells of the circulatory system.
- therapeutic effects can be obtained by correcting the genes of about 1 % of the cells of the affected tissue.
- treatments with CMV can be repeatedly performed to obtain an increased therapeutic effect.
- EPC Egg phosphatidylcholine
- DOTAP and galactocerebroside
- Gc galactocerebroside
- soybean oil Sigma Chemical Co.
- DOGS ® dioctadecyldiamidoglycyl spermine
- EPC, DOTAP and Gc were previously dissolved at defined concentrations in chloroform or anhydrous methanol and stored in small glass vials in desiccated containers at -20°C until use.
- EPC (40-45 mg), DOTAP (200 ⁇ g) and Gc (43 ⁇ g) solutions were aliquoted into a small 10 x 75 mm borosilicate tube and solvents removed under a stream of nitrogen.
- CMV were diluted in 0.15 M NaCI (-80-125 ⁇ g/250-300 ⁇ l); DOGS (as a 10 mg/ml solution in ethanol) was diluted into 250-300 ⁇ l 0.1 5 M NaCI at 3-5 times the weight of added CMV.
- the two solutions were mildly vortexed to mix contents and then CMV solution was added slowly to the DOGS solution.
- the contents were mixed by gentle tapping and inverting the tube a few times.
- the DOGS-complex solution was added to the dried lipids followed by soybean oil (40-45 mg), the mixture was vortexed on high for a few seconds and bath sonicated in a FS-15 (Fisher Scientific) bath sonicator for -1 hr in a 4°C temperature controlled room. Occasionally, the tube was removed from the bath and vortexed.
- FS-15 Fisher Scientific
- DOPC Dioleoyl phosphatidylcholine
- DOPS dioleoyl phosphatidylserine
- Gc galactocerebroside
- lactosylcerebroside (Avanti Polar Lipids).
- DOPS, DOPC and Gc at a molar ratio of 1 :1 :0.16 were dissolved in chloroform:methanol (1 :1 v/v) and then dried under a stream of nitrogen to obtain a uniform lipid film.
- the CMV were diluted in 500 ⁇ l of 0.15 M NaCI (approximately 100-250 ⁇ g/500 ⁇ l). The solution was added to the lipid film at room temperature. Lipids were dispersed entirely by alternate mild vortexing and warming (in a water bath at 37-42°C).
- lipid vesicles were stored at 4°C until use. Under these conditions the lipid vesicles were stable for at least one month. The final product can be lyophilized.
- DOPC Dioleoyl phosphatidylcholine
- DOPE dioleoyl phosphatidylethanolamine
- Gc galactocerebroside
- lactosylcerebroside (Avanti Polar Lipids).
- DOPC, DOPE and Gc (1 :1 :0.16 molar ratio) or DOPGGc (1 :0.08) were dissolved in chloroform:methanol (1 :1 v/v) and then dried under a stream of nitrogen to obtain a uniform lipid film.
- the oligonucleotides (or chimeric molecules) were diluted in 500 ⁇ l of 0.15 M NaCI (approximately 100-250 ⁇ g/500 ⁇ l). The solution was added to the lipid film at room temperature. Lipids were dispersed entirely by alternate mild vortexing and warming (in a water bath at 37-42°C).
- lipid vesicles were stored at 4°C until use. The size of the lipid vesicles of the preparation was stable for about 5 days.
- DOPC Dioleoyl phosphatidylcholine
- DOTAP dioleoyl trimethylammonium propane
- Gc galactocerebroside
- lactosylcerebroside (Avanti Polar Lipids).
- PEI Polyethylenimine (PEI) (M.W. 800 Kd), Fluka Chemicals.
- DOPC, DOTAP and Gc (6:1 :0.56 molar ratio) (500 ⁇ g total lipid) were dissolved in chlorofo ⁇ rumethanol (1 :1 v/v) and then dried under a stream of nitrogen to obtain a uniform lipid film.
- PEI was diluted to a concentration of 45 mg/100 ml using water. pH of the solution was adjusted to -7.6 using HCI. This PEI stock solution was prepared fresh each time and was equivalent to approximately 50 nmol amine/ ⁇ l.
- CMV were diluted into 0.15 M NaCI at a concentration of ⁇ 125 ⁇ g in 250 ⁇ l.
- PEI was further diluted into 250 ⁇ l 0.15 M NaCI so that approximately 4 moles of PEI amine were present per mole of oligonucleotide/chimeric phosphate.
- PEI solution was added dropwise to the CMV solution (both at room temperature) and vortexed for 5-10 minutes.
- the PEI-complex solution was then added to the lipid film and the lipids dispersed as described above. After a uniform milky suspension was formed, it was extruded through a series of polycarbonate membranes (pore sizes 0.8, 0.4, 0.2, 0.1 and 0.05 ⁇ m) using a Liposofast ® mini-extruder. Extrusion was done 5 to 7 times through each pore size.
- PEI 25 kDa was purchased from Aldrich Chemical (Milwaukee, Wl). PEI (800 kDa) was purchased from Fluka chemicals (Ronkonkoma, NY, USA) . Lactosylation of the PEI was carried out by modification of a previously described method for the conjugation of oligosaccharides to proteins. Briefly, 3 to 5 ml of PEI (0.1 to 1.2 M monomer) in ammonium acetate (0.2 M) or Tris buffer (0.2 M) (pH 7.6) solution was incubated with 7 to 8 mg of sodium cyanoborohydride (Sigma Chemical Co., St. Louis, MO) and approximately 30 mg of lactose monohydrate (Sigma Chemical Co., St. Louis, MO).
- Reaction was carried out in polypropylene tubes, tightly capped in a 37°C shaking water bath. After 10 days the reaction mixture was dialyzed against distilled water (500 ml) for 48 h with 1 to 2 changes of water. The purified complex was sterile filtered through 0.2 ⁇ m filter and stored at 4°C. The amount of sugar (as galactose) associated with PEI was determined by the phenol-sulphuric acid method.
- the number of moles of free amine (primary -(- secondary) in the lactosylated PEI was determined as follows: a standard curve was set up using a 0.02M stock solution of PEI; several aliquots of the stock were diluted to 1 ml using deionized water in glass tubes, then 50 ⁇ l of Ninhydrin reagent (Sigma Chemical Co., St. Louis, Mo) was added to each tube and vortexed vigorously for 10 sec. Color development was allowed to proceed at room temperature for 10 to 12 min. and then O.D. was read (within 4 minutes) at 485 nm on a Beckman DU-64 spectrophotometer.
- Lactosylated-PEI (L-PEI) complexes were prepared as follows: an equivalent of 3 mmol of amine as L-PEI and 3 mmol of amine as PEI, per mmol of RNA/DNA phosphate, were mixed together and diluted in 0.15M NaCI as required; the mixture was added dropwise to a solution of the chimeric and vortexed for 5 min.
- Fetal bovine serum was obtained from Atlanta Biologicals, Inc. (Atlanta, GA).
- the terminal transferase, fluorescein-12-dUTP, ExpandTM high fidelity PCR system, dNTPs and high pure PCR template preparation kit were obtained from Boehringer Mannheim Corp. (Indianapolis, IN).
- ReflectionTM NEF-496 autoradiography film and ReflectionTM NEF-491 intensifying screens were from DuPont NEN ® Research Products (Boston, MA).
- Polyethylenimine (PEI) 800 kDa was obtained from Fluka Chemical Corp. (Ronkonkoma, NY).
- the [ ⁇ - 32 P]ATP was obtained from ICN Biochemicals, Inc. (Costa Mesa, CA).
- pCRTM2.1 was obtained from Invitrogen (San Diego, CA).
- OPTIMEMTM Dulbecco's modified Eagle's medium, William's E medium and oligonucleotides 365-A and 365-C were from Life Technologies, Inc. (Gaithersburg, MD).
- Spin filters of 30,000 mol wt cutoff were purchased from Millipore Corp. (Bedford, MA).
- Dil and SlowFadeTM antifade mounting medium were obtained from Molecular Probes, Inc. (Eugene, OR).
- T4 polynucleotide kinase was purchased from New England Biolabs, Inc. (Beverly, MA).
- MSI MagnaGraph membrane was purchased from Micron Separations, Inc. (Westboro, MA).
- the primers used for PCR amplification were obtained from Oligos Etc., Inc. (Wilsonville, OR). Tetramethylammonium chloride was purchased from Sigma Chemical Company (St. Louis, MO). All other chemicals were molecular biology or reagent grade and purchased from Aldrich Chemical Company (Milwaukee, Wl), Curtin Matheson Scientific, Inc. (Eden Prairie, MN), and Fisher Scientific (Itasca, IL).
- RNA/DNA oligonucleotides HIXF, RIXF and RIXR were synthesized.
- the CMV were prepared with DNA and 2'-0-methyl RNA phosphoramidite nucleoside monomers on an ABI 394 synthesizer.
- the DNA phosphoramidite exocyclic amine groups were protected with benzoyl (adenosine and cytidine) and isobutyryl (guanosine).
- the protective groups on the 2'-0-methyl RNA phosphoramidites were phenoxyacetyl for adenosine, isobutyryl for cytidine, and dimethylformamide for guanosine.
- the base protecting groups were removed following synthesis by heating in ethanol/concentrated ammonium hydroxide for 20 h at 55°C.
- the crude oligonucleotides were electrophoresed on 15% polyacrylamide gels containing 7 M urea, and the DNA visualized using UV shadowing.
- the chimeric molecules were eluted from the gel slices, concentrated by precipitation and desalted using G-25 spin columns. Greater than 95% of the purified oligonucleotides were full length.
- RIXR The structure of the RIXR, RIXF and HIXR CMV is as follows
- Uppercase letters are deoxyribonucleotides, lower case letters are 2'OMe- ribonucleotides.
- the nucleotide of the heterologous region is underlined.
- HuH-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% (vol/vol) heat inactivated fetal bovine serum in a humidified C0 2 atmosphere at 37°C. Twenty four hours prior to transfection 1 x 10 5 cells were plated per 35 mm culture dish. At the time of transfection, the cells were rinsed twice with OPTIMEMTM media and transfections were performed in 1 ml of the same media. Eighteen hours after transfection, 2 ml of Dulbecco's modified Eagle's medium containing 20% (vol/vol) heat inactivated fetal bovine serum was added to each 35 mm dish and the cells maintained for an additional 30 h prior to harvesting for DNA isolation.
- HuH-7 vehicle control transfections utilized the same amount of PEI as was used in the HulXF transfections, but substituted an equal volume of 10 mM Tris-HCI pH 7.6 for the oligonucleotides.
- Primary rat hepatocytes were isolated from 250 g male Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN) by a two step collagenase perfusion as previously described (Fan et al., Oncogene 12:1909-1919, 1996, which is hereby incorporated by reference) and plated on PrimariaTM plates at a density of 4 x 10 5 cells per 35 mm dish.
- the cultures were maintained in William's E medium supplemented with 10% heat inactivated FBS, 26 mM sodium bicarbonate, 23 mM HEPES, 0.01 U/ml insulin, 2 mM L-glutamine, 10 nM dexamethasone, 5.5 mM glucose, 100 U/ml penicillin and 100 U/ml streptomycin. Twenty four hours after plating, the hepatocytes were washed twice with the same medium and 1 ml of fresh medium added and the cells transfected using PEI/chimeric oligonucleotide complexes at the identical concentrations as for the HuH-7 cells. After 18 h, an additional 2 ml of the medium was added and the cells harvested 6 or 30 h later.
- Chimeric duplexes were 3' end- labeled using terminal transferase and fluorescein-12-dUTP according to the manufacturer's recommendation, and were then mixed with unlabeled oligonucleotides at a 2:3 ratio.
- Transfections were performed as described above and after 24 h the cells were fixed in phosphate buffered saline, pH 7.4, containing 4% paraformaldehyde (wt/vol) for 10 min at room temperature. Following fixation, the cells were counterstained using a 5 ⁇ M solution of Dil in 0.32 M sucrose for 10 min according to the manufacturer's recommendation.
- the cells were coversliped using SlowFadeTM antifade mounting medium in phosphate buffered saline and examined using a MRC1000 confocal microscope (BioRad, Inc., Hercules, CA).
- the caudate lobes of liver in situ were injected with fluorescently-labeled chimerics as described above and harvested 24 h post-injection.
- the lobes were bisected longitudinally, embedded using OCT and frozen.
- Cryosections were cut - 10 ⁇ m thick, fixed for 10 min at room temperature using phosphate buffered saline, pH 7.4, containing 4% paraformaldehyde (wt/vol). Following fixation, the cells were counterstained using a 5 ⁇ M solution of Dil in 0.32 M sucrose for 10 min according to the manufacturer's recommendation. After washing with 0.32 M sucrose and then phosphate buffered saline, pH 7.4, the sections were coversliped using SlowFadeTM antifade mounting medium and examined using a MRC1000 confocal microscope (BioRad, Inc.). The collection series for the fixed cells and sectioned tissue were made at 1 ⁇ m steps to establish the presence of the chimeric in the nucleus.
- DNA isolation and cloning The cells were harvested by scrapping 24 and 48 h after transfection. Genomic DNA larger than 100-150 base pairs was isolated using the high pure PCR template preparation kit according to the manufacturer's recommendation. PCR amplification of a 317-nt fragment of the eighth exon in the human liver IX gene was performed with 500 ng of the isolated DNA.
- the primers used were 5'-CATTGCTGACAAGGAATACACGAAC-3' (SEQ ID No. 14) and 5'-ATTTGCCTTTCATTGCACACTCTTC-3' (SEQ ID No. 15) corresponding to nucleotides 1008-1032 and 1300-1324, respectively, of the human factor IX cDNA.
- Primers were annealed at 58°C for 20 sec, elongation was for 45 sec at 72°C and denaturation proceeded for 45 sec at 94°C.
- the sample was amplified for 30 cycles using Expand Hi-fidelityTM polymerase.
- PCR amplification of a 374-nt fragment of the rat factor IX gene was performed with 500 ng of the isolated DNA from either the primary hepatocytes or liver caudate lobe.
- the primers used were 5'- ATTGCCTTGCTGGAACTGGATAAC-3' (SEQ ID No. 16) and 5'-TTGCCTTTCATTGCACATTCTTCAC-3' (SEQ ID No.
- probes were 32 P-end-labeled using [ ⁇ - 32 ]ATP ( > 7,000 Ci/mmol) and T4 polynucleotide kinase according to the manufacturer's recommendations.
- Hybridizations were preformed at 37°C in 2X sodium chloride sodium citrate containing 1 % SDS, 5X Denhardt's and 200 ⁇ g/ml denatured sonicated fish sperm DNA.
- the filters were rinsed in 1 X sodium chloride phosphate EDTA, 0.5% SDS and then washed at 54°C for 1 h in 50 mM Tris-HCl, pH 8.0 containing 3 M tetramethylammonium chloride, 2 mM EDTA, pH 8.0, 0.1 % SDS.
- Autoradiography was performed with NEN ® Reflection film at -70°C using an intensifying screen. Plasmid DNA was prepared from colonies identified as hybridizing with 365-A or 365-C using Qiagen minprep kit (Chatsworth, CA) and subjected to automatic sequencing using the mp13 reverse primer on an ABI 370A sequencer (Perkin-Elmer, Corp., Foster City, CA).
- Chimeric oligonucleotides were fluorescein-labeled and used to determine whether direct injection into the caudate lobe of the liver was feasible. The results indicated that the hepatocytes adjacent to the injection site within the caudate lobe showed uptake of the fluorescently-labeled chimeric molecules similar to that observed in isolated primary hepatocytes and HuH-7 cells. Although some punctate material was present in the cytoplasm, the labeled material was detected primarily in the nucleus. In fact, only nuclear labeling was observed in hepatocytes farthest from the injection site.
- the unlabeled PEI/RIXF chimeric complexes and vehicle controls were injected directly into the caudate lobe using the same protocol and the animals sacrificed 24 and 48 h post-injection.
- Liver DNA was isolated as described in Methods, subjected to PCR amplification of a 374 nt sequence spanning the targeted nt exchange site. Following subcloning and transformation of Escherichia coli with the PCR amplified material, duplicate filter lifts of the transformed colonies were performed. The filters were hybridized with 32 -labeled 17-mer oligonucleotides specific for either 365-A (wild-type) or 365-C (factor IX mutation) and processed post-hybridization as described in Methods.
- Rats which received direct hepatic injection of the RIXF chimeric molecules exhibited a A-C conversion frequency of -1 % at both 24 and 48 h.
- the vehicle controls showed no hybridization with the 365-C probe.
- Colonies that hybridized with the 365-C probe from the RIXF treated animals were cultured, the plasmid DNA isolated and subjected to sequencing to confirm the A ⁇ C conversion.
- the ends of the amplified 374-nt fragment correspond exactly with the primers and the only nucleotide change observed was an A-C at the targeted exchange site.
- CMV complexed to a mixture of lactosylated-PEI and PEI was prepared using the RIXR oligonucleotide as described in Section 6.1.5 above.
- a CMV directed to the complementary strand of the same region of the factor IX was also constructed (RIXR C ).
- the nuclear localization of the fluorescently-labeled chimeric molecules indicated efficient transfection in the isolated rat hepatocytes.
- the cultured hepatocytes were then transfected with the unlabeled chimeric molecules factor RIXR C and RIXR at comparable concentrations using 800 kDa PEI as the carrier. Additionally, vehicle control transfections were performed simultaneously. Forty eight hours after transfection, the cells were harvested and the DNA isolated and processed for hybridization as described in Section 6.1 .5.
- the A ⁇ C targeted nucleotide conversion at Ser 365 was determined by hybridization of duplicate colony lifts of the PCR-amplified and cloned 374-nt stretch of exon 8 of the factor IX gene (Sarkar, B., Koeberl, D. D. & Somer, S. S., "Direct Sequencing of the activation peptide and the catalytic domain of the factor IX gene in six species," Genomics, 6, 133-143, 1990.)
- the 1 7 mer oligonucleotide probes used to distinguish between the wild-type 365-A (5'- AAGGAGATAGTGGGGGA-3') (SEQ ID No. 20) or converted 365-C (5'- AAGGAGATCGTGGGGGA-3') (SEQ ID No. 21) corresponded to nucleotides 710 through 726 of the cDNA sequence.
- the overall frequency of conversion of the targeted nucleotide was calculated by dividing the number of clones hybridizing with the 365-C oligonucleotide by the total number of clones hybridizing with both oligonucleotide probes.
- the results are summarized in Table 1 for RIXR C .
- a ⁇ C conversion at Ser 365 was observed only in primary hepatocytes transfected with the RIXR or RIXR C . Similar conversion frequencies were observed in hepatocytes transfected with RIXR or RIXR C .
- the fluorescein-labeled oligonucleotides were also used to determine cellular uptake of the chimeric molecules after direct injection into the caudate lobe of the liver.
- some punctate material was present in the cytoplasm of the hepatocytes, the labeled material was primarily present in the nucleus. In fact, only nuclear labeling was observed in those areas farthest from the injection site.
- the unlabeled RIXR chimeric oligonucleotides and vehicle controls were then administered in vivo by tail vein injection of the 25 kDa PEI and liver tissue harvested 5 days post-injection. Liver DNA was isolated and subjected to PCR amplification of a 374-nt sequence spanning the targeted nucleotide exchange site, using the same primers as those used with the primary hepatocytes. Following subcloning and transformation of E. coli with the PCR-amplified material, duplicate filter lifts of the transformed colonies were done.
- the filters were hybridized with the same 32 P-labeled 17-mer oligonucleotides specific for either 365-A (wild-type) or 365-C (mutant) and processed post-hybridization.
- Rats treated with 100 ⁇ g of the RIXR chimeric oligonucleotides exhibited an A ⁇ C conversion frequency ranging from 13.9% to 18.9%, while those that received a total of 350 ⁇ g in two injections showed 40% conversion. In contrast, the vehicle controls showed no hybridization with the 365-C probe.
- RT-PCR hybridization of isolated RNA indicated A ⁇ C conversion frequencies of 26.4% to 28.4% in the high dose livers.
- the APTT for vehicle-treated rats ranged from 89.7% to 181.9% of control values (131.84% + 32.89%), while the APTT for the oligonucleotide-treated animals ranged from 48.9% to 61.7% (53.8% ⁇ 4.8%).
- the factor IX activity of duplicate samples was determined from a log-log standard curve that was constructed from the APTT results for dilution (1 :10 to 1 :80) of pooled plasma from 12 normal male rats, 6-8 weeks old.
- APTT activated partial thromboplastin time
- RNA isolation and cloning The cells were harvested by scrapping 48 h after transfection. Genomic DNA larger than 100-150 base pairs was isolated using the high pure PCR template preparation kit (Boehringer Mannheim, Corp., Indianapolis, IN). RNA was isolated using RNAzolTM B (Tel-Test, Inc., Friendswood, TX), according to the manufacturer's protocol. PCR amplification of a 374-nt fragment of the rat factor IX gene was performed with 300 ng of the isolated DNA from either the primary hepatocytes or liver tissue. The primers were designed as 5'- ATTGCCTTGCTGGAACTGGATAAAC-3' (SEQ ID No.
- PCR amplification products from both the hepatocytes and intact liver factor IX genes were subcloned into the TA cloning vector pCRTM2.1 (Invitrogen, San Diego, CA), and the ligated material used to transform frozen competent E. coli.
- pCRTM2.1 Invitrogen, San Diego, CA
- 300 ng of control DNA was incubated with 0.5, 1.0 and 1.5 ⁇ g of the oligonucleotide prior to the PCR-amplification reaction. Additionally, 1.0 ⁇ g of the chimeric alone was used as the "template" for the PCR amplification.
- RT-PCR amplification was done utilizing the TitianTM one tube RT-PCR system (Boehringer Mannheim, Corp.) According to the manufacturer's protocol using the same primers as those used for the DNA PCR amplification.
- the RNA samples were treated with RQ1 DNase free RNase (Promega Corp., Madison, Wl) and RT-PCR negative controls of RNased RNA samples were performed in parallel with the RT-PCR reaction.
- Each of the PCR reactions were ligated into the same TA cloning vector and transformed into frozen competent E. coli.
- the probes were 32 P-end-labeled using ( ⁇ - 2 P) ATP ( > 7,000 Ci/mmol) and T4 polynucleotide kinase (New England Biolabs, Inc., Beverly MA). Hybridizations were performed at 37°C in 2X sodium chloride sodium citrate containing 1 % SDS, 5X Denhardt's and 200 ⁇ g/ml denatured sonicated fish sperm DNA.
- the filters were rinsed in I X sodium chloride sodium phosphate EDTA, 0.5% SDS and then washed at 54°C for 1 h in 50 mM Tris-HCl, pH 8.0 containing 3 M tetramethylammonium chloride, 2 mM EDTA, pH 8.0, 0.1 % SDS (Melchior, W. B. & Von Hippel, P. H. "Alteration of the relative stability of dA.dT and dG.dC base pairs in DNA,” Proc. Natl. Acad. Sci. USA, 70, 298-302, 1973.). Autoradiography was performed with NEN s Reflection film at -70°C using an intensifying screen.
- Plasmid DNA was prepared from colonies identified as hybridizing with 365-A or 365-C using Qiagen miniprep kit (Chatsworth, CA) and subjected to automatic sequencing using the mp13 forward and reverse primers as well as a gene specific primer, 5'GTTGACCGAGCCACATGCCTTAG-3' (SEQ ID No. 26) corresponding to nucleotides 616 to 638 of the rat factor IX cDNA using an ABI 370A sequencer (Perkin-Elmer, Corp., Foster City, CA).
- the Chapel Hill strain of dogs which has a (G ⁇ A) 1477 mutation that results in hemophilia in the animals, was used to obtain primary cultured hepatocytes.
- a CMV designed to correct the mutation is given below.
- Mutant rats with hyperbilirubinemia termed Gunn rats, have a single nucleotide deletion in the gene encoding bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (UGT1A 1). Roy Chowdhury, J., et al., 1991 , J. Biol. Chem. 266, 18294. Human patients with Crigler-Najjar syndrome type I also have mutations of the UGT1A 1 gene, resulting in life-long hyperbilirubinemia and consequent brain damage. Bosma, P.J., et al., 1992, FASEB J.
- CN3 (mut ⁇ T) (SEQ ID No . 27)
- Gunn rat primary cultured hepatocytes were treated with 150 nM CN3 according to the above protocol except that the carrier was either the negatively charged glycosylated lipid vesicles of section 6.2.2 or a lactosylated-PEI carrier at a ratio of oligonucleotide phosphate to imine of 1 :4 .
- the results were 8.5% conversion with the negatively charged liposome and 3.6% conversion with lactosylated-PEI carrier.
- a Gunn rat was injected on five successive days with 1 mg/Kg of CN3 complexed with 25 kDa Lac-PEI as above. Twenty five days after the final injection the serum bilirubin had declined from 6.2 mg/dl to 3.5 mg/dl and remained at that level for a further 25 days.
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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EP98920930A EP0979311A1 (en) | 1997-04-30 | 1998-04-30 | $i(IN VIVO) USE OF RECOMBINAGENIC OLIGONUCLEOBASES TO CORRECT GENETIC LESIONS IN HEPATOCYTES |
AU73654/98A AU749410B2 (en) | 1997-04-30 | 1998-04-30 | In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes |
JP54742998A JP2002511851A (en) | 1997-04-30 | 1998-04-30 | Use of recombinant oligonucleobases in vivo to correct hepatocyte genetic lesions in vivo |
NZ500694A NZ500694A (en) | 1997-04-30 | 1998-04-30 | Use of chimeric mutational vector (CMV), a macromolecule and a ligand for cell surface receptor adhesion molecule (clatherin) in gene therapy; the CMV vector derived from REC2 homologous pairing with Ustilago maydis |
CA002288209A CA2288209A1 (en) | 1997-04-30 | 1998-04-30 | In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes |
US09/108,006 US6524613B1 (en) | 1997-04-30 | 1998-06-30 | Hepatocellular chimeraplasty |
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US4528897P | 1997-04-30 | 1997-04-30 | |
US60/045,288 | 1997-04-30 | ||
US5483797P | 1997-08-05 | 1997-08-05 | |
US60/054,837 | 1997-08-05 | ||
US6499697P | 1997-11-10 | 1997-11-10 | |
US60/064,996 | 1997-11-10 |
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US09/108,006 Continuation-In-Part US6524613B1 (en) | 1997-04-30 | 1998-06-30 | Hepatocellular chimeraplasty |
Publications (3)
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WO1998049350A1 true WO1998049350A1 (en) | 1998-11-05 |
WO1998049350B1 WO1998049350B1 (en) | 1999-01-07 |
WO1998049350A8 WO1998049350A8 (en) | 2000-05-25 |
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PCT/US1998/008834 WO1998049350A1 (en) | 1997-04-30 | 1998-04-30 | In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes |
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EP (1) | EP0979311A1 (en) |
JP (1) | JP2002511851A (en) |
KR (1) | KR20010020375A (en) |
CN (1) | CN1268186A (en) |
AU (1) | AU749410B2 (en) |
CA (1) | CA2288209A1 (en) |
NZ (1) | NZ500694A (en) |
WO (1) | WO1998049350A1 (en) |
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Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8148164B2 (en) | 2003-06-20 | 2012-04-03 | Roche Diagnostics Operations, Inc. | System and method for determining the concentration of an analyte in a sample fluid |
US20210018492A1 (en) * | 2018-03-13 | 2021-01-21 | Corning Incorporated | High density 3d hepatocyte spheroid platform for drug adme studies |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5264618A (en) * | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
WO1994004032A1 (en) * | 1992-08-21 | 1994-03-03 | The Regents Of The University Of California | Composition and method for altering dna sequences by homologous recombination |
WO1994012650A2 (en) * | 1992-12-03 | 1994-06-09 | Transkaryotic Therapies, Inc. | Activating expression of an amplifying endogenous gene by homologous recombination |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5166320A (en) * | 1987-04-22 | 1992-11-24 | University Of Connecticut | Carrier system and method for the introduction of genes into mammalian cells |
-
1998
- 1998-04-30 AU AU73654/98A patent/AU749410B2/en not_active Ceased
- 1998-04-30 CN CN98806642A patent/CN1268186A/en active Pending
- 1998-04-30 KR KR1019997009997A patent/KR20010020375A/en not_active Application Discontinuation
- 1998-04-30 NZ NZ500694A patent/NZ500694A/en unknown
- 1998-04-30 JP JP54742998A patent/JP2002511851A/en active Pending
- 1998-04-30 CA CA002288209A patent/CA2288209A1/en not_active Abandoned
- 1998-04-30 WO PCT/US1998/008834 patent/WO1998049350A1/en not_active Application Discontinuation
- 1998-04-30 EP EP98920930A patent/EP0979311A1/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5264618A (en) * | 1990-04-19 | 1993-11-23 | Vical, Inc. | Cationic lipids for intracellular delivery of biologically active molecules |
WO1994004032A1 (en) * | 1992-08-21 | 1994-03-03 | The Regents Of The University Of California | Composition and method for altering dna sequences by homologous recombination |
WO1994012650A2 (en) * | 1992-12-03 | 1994-06-09 | Transkaryotic Therapies, Inc. | Activating expression of an amplifying endogenous gene by homologous recombination |
Non-Patent Citations (1)
Title |
---|
CAPECCHI M. R.: "ALTERING THE GENOME BY HOMOLOGOUS RECOMBINATION.", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 244., 16 June 1989 (1989-06-16), US, pages 1288 - 1292., XP002910193, ISSN: 0036-8075, DOI: 10.1126/science.2660260 * |
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US8680202B2 (en) | 2002-09-06 | 2014-03-25 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
US8399431B2 (en) | 2002-09-06 | 2013-03-19 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
US8389499B2 (en) | 2002-09-06 | 2013-03-05 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
US8475781B2 (en) | 2002-09-06 | 2013-07-02 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
US8580242B2 (en) | 2002-09-06 | 2013-11-12 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
US8609081B2 (en) | 2002-09-06 | 2013-12-17 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
US8603454B2 (en) | 2002-09-06 | 2013-12-10 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
US8580244B2 (en) | 2002-09-06 | 2013-11-12 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
US8580243B2 (en) | 2002-09-06 | 2013-11-12 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
US8252276B2 (en) | 2002-09-06 | 2012-08-28 | Cerulean Pharma Inc. | Cyclodextrin-based polymers for therapeutics delivery |
EP2003205A2 (en) | 2004-12-28 | 2008-12-17 | Pioneer Hi-Bred International, Inc. | Improved grain quality through altered expression of seed proteins |
EP2338905A2 (en) | 2005-02-23 | 2011-06-29 | North Carolina State University | Alteration of tobacco alkaloid content through modification of specific cytochrome p450 genes |
EP2261362A2 (en) | 2005-05-25 | 2010-12-15 | Pioneer Hi-Bred International Inc. | Methods for improving crop plant architecture and yield |
EP2261361A2 (en) | 2005-05-25 | 2010-12-15 | Pioneer Hi-Bred International Inc. | Methods for improving crop plant architecture and yield |
EP1764091A2 (en) * | 2005-09-15 | 2007-03-21 | Novosom AG | Improvements in or relating to amphoteric liposomes |
WO2007031333A2 (en) * | 2005-09-15 | 2007-03-22 | Novosom Ag | Improvements in or relating to amphoteric liposomes |
EP1764091A3 (en) * | 2005-09-15 | 2007-06-13 | Novosom AG | Improvements in or relating to amphoteric liposomes |
US9066867B2 (en) | 2005-09-15 | 2015-06-30 | Marina Biotech, Inc. | Amphoteric liposomes |
US9737484B2 (en) | 2005-09-15 | 2017-08-22 | Marina Biotech, Inc. | Amphoteric liposomes |
AU2006291429B2 (en) * | 2005-09-15 | 2013-04-04 | Biontech Delivery Technologies Gmbh | Improvements in or relating to amphoteric liposomes |
WO2007031333A3 (en) * | 2005-09-15 | 2007-07-19 | Novosom Ag | Improvements in or relating to amphoteric liposomes |
EP2112223A2 (en) | 2005-11-10 | 2009-10-28 | Pioneer Hi-Bred International Inc. | DOF (DNA binding with one finger) sequences and method of use |
WO2007059077A2 (en) | 2005-11-14 | 2007-05-24 | E.I.Du Pont De Nemours And Company | Compositions and methods for altering alpha- and beta-tocotrienol content |
WO2007079161A2 (en) | 2005-12-30 | 2007-07-12 | Pioneer Hi-Bred International, Inc. | Udp-xylose synthases (uxs) polynucleotides, polypeptides, and uses thereof |
EP2465341A1 (en) | 2006-01-12 | 2012-06-20 | Cibus Europe B.V. | EPSPS Mutants |
EP2465340A1 (en) | 2006-01-12 | 2012-06-20 | Cibus Europe B.V. | EPSPS mutants |
US8268622B2 (en) | 2006-01-12 | 2012-09-18 | Cibus Us Llc | EPSPS mutants |
EP2923563A2 (en) | 2006-01-12 | 2015-09-30 | Cibus Europe B.V. | EPSPS mutants |
EP2730587A2 (en) | 2006-02-09 | 2014-05-14 | Pioneer Hi-Bred International, Inc. | Genes for enhancing nitrogen utilization efficiency in crop plants |
EP2251349A1 (en) | 2006-04-19 | 2010-11-17 | Pioneer Hi-Bred International, Inc. | Isolated polynucleotide molecules corresponding to mutant and wild-type alleles of the maize D9 gene and methods of use |
US9796938B2 (en) | 2006-06-28 | 2017-10-24 | Nucelis Inc. | Fatty acid blends and uses therefor |
US8361173B2 (en) | 2006-06-28 | 2013-01-29 | Nucelis Inc. | Fatty acid blends and uses therefor |
EP2679687A2 (en) | 2006-06-28 | 2014-01-01 | Nucelis Inc. | Fatty acid blends and uses therefor |
US8029579B2 (en) | 2006-06-28 | 2011-10-04 | COH Inc. | Fatty acid blends and uses therefor |
US9610360B2 (en) | 2007-01-24 | 2017-04-04 | Ceruliean Pharma Inc. | Polymer drug conjugates with tether groups for controlled drug delivery |
US8497365B2 (en) | 2007-01-24 | 2013-07-30 | Mark E. Davis | Cyclodextrin-based polymers for therapeutics delivery |
WO2008145629A2 (en) | 2007-05-25 | 2008-12-04 | Cropdesign N.V. | Yield enhancement in plants by modulation of maize alfins |
WO2009046334A1 (en) | 2007-10-05 | 2009-04-09 | Cibus Llc | Mutated acetohydroxyacid synthase genes in brassica |
EP3584325A1 (en) | 2007-10-05 | 2019-12-25 | Cibus Europe B.V. | Mutated acetohydroxyacid synthase genes in brassica |
EP2573177A1 (en) | 2007-11-12 | 2013-03-27 | North Carolina State University | Alteration of tobacco alkaloid content through modification of specific cytochrome P450 genes |
WO2009067580A2 (en) | 2007-11-20 | 2009-05-28 | Pioneer Hi-Bred International, Inc. | Maize ethylene signaling genes and modulation of same for improved stress tolerance in plants |
EP3260549A1 (en) | 2008-05-23 | 2017-12-27 | Nucelis LLC | Production of squalene using yeast |
US10100334B2 (en) | 2008-05-23 | 2018-10-16 | Nucelis Inc. | Production of squalene using yeast |
EP3441470A1 (en) | 2008-09-26 | 2019-02-13 | BASF Agrochemical Products, B.V. | Herbicide-resistant ahas-mutants and methods of use |
EP2821490A2 (en) | 2008-10-30 | 2015-01-07 | Pioneer Hi-Bred International Inc. | Manipulation of glutamine synthetases (GS) to improve nitrogen use efficiency and grain yield in higher plants |
WO2010065867A1 (en) | 2008-12-04 | 2010-06-10 | Pioneer Hi-Bred International, Inc. | Methods and compositions for enhanced yield by targeted expression of knotted1 |
WO2010083328A2 (en) | 2009-01-14 | 2010-07-22 | The Salk Institute For Biological Studies | Methods for screening and compounds that protect against amyloid diseases |
WO2010101818A1 (en) | 2009-03-02 | 2010-09-10 | Pioneer Hi-Bred International, Inc. | Nac transcriptional activators involved in abiotic stress tolerance |
WO2010120862A1 (en) | 2009-04-14 | 2010-10-21 | Pioneer Hi-Bred International, Inc. | Modulation of acc synthase improves plant yield under low nitrogen conditions |
EP2666781A1 (en) | 2009-05-04 | 2013-11-27 | Pioneer Hi-Bred International, Inc. | Yield enhancement in plants by modulation of AP2 transcription factor |
WO2011011273A1 (en) | 2009-07-24 | 2011-01-27 | Pioneer Hi-Bred International, Inc. | The use of dimerization domain component stacks to modulate plant architecture |
WO2011017492A2 (en) | 2009-08-05 | 2011-02-10 | Pioneer Hi-Bred International, Inc. | Novel eto1 genes and use of same for reduced ethylene and improved stress tolerance in plants |
WO2011041796A1 (en) | 2009-10-02 | 2011-04-07 | Pioneer Hi-Bred International, Inc. | Down-regulation of acc synthase for improved plant performance |
US11952577B2 (en) | 2009-11-23 | 2024-04-09 | Nucelis Llc | Yarrowia lipolytica yeast strains expressing an HMG-CoA reductase having increased activity or expression |
WO2011063350A2 (en) | 2009-11-23 | 2011-05-26 | Cibus Oils, Llc | Methods and compositions for producing squalene using yeast |
US8470568B2 (en) | 2009-11-23 | 2013-06-25 | Nucelis Inc. | Methods and compositions for producing squalene using yeast |
EP3219815A2 (en) | 2009-11-23 | 2017-09-20 | Nucelis Inc. | Methods and compositions for producing squalene using yeast |
WO2011085062A1 (en) | 2010-01-06 | 2011-07-14 | Pioneer Hi-Bred International, Inc. | Identification of diurnal rhythms in photosynthetic and non-photosynthetic tissues from zea mays and use in improving crop plants |
WO2011094205A1 (en) | 2010-01-26 | 2011-08-04 | Pioneer Hi-Bred International, Inc. | Hppd-inhibitor herbicide tolerance |
WO2011094199A1 (en) | 2010-01-26 | 2011-08-04 | Pioneer Hi-Bred International, Inc. | Polynucleotide and polypeptide sequences associated with herbicide tolerance |
WO2011139431A1 (en) | 2010-05-06 | 2011-11-10 | Pioneer Hi-Bred International, Inc. | Maize acc synthase 3 gene and protein and uses thereof |
US11111500B2 (en) | 2010-08-03 | 2021-09-07 | Cibus Us Llc | Mutated protoporphyrinogen IX oxidase (PPX) genes |
EP3441469A1 (en) | 2010-08-03 | 2019-02-13 | Cibus US LLC | Mutated protoporphyrinogen ix oxidase (ppx) genes |
WO2012018862A2 (en) | 2010-08-03 | 2012-02-09 | Cibus Us Llc | Mutated protoporphyrinogen ix oxidase (ppx) genes |
WO2012021785A1 (en) | 2010-08-13 | 2012-02-16 | Pioneer Hi-Bred International, Inc. | Compositions and methods comprising sequences having hydroxyphenylpyruvate dioxygenase (hppd) activity |
WO2012028673A1 (en) | 2010-09-02 | 2012-03-08 | Ludwig-Maximilians-Universitaet Muenchen | Spontaneous organogenesis in plants |
EP2426204A1 (en) | 2010-09-02 | 2012-03-07 | Ludwig-Maximilians-Universität München | Spontaneous nodule organogenesis in plants |
US9254313B2 (en) | 2010-11-08 | 2016-02-09 | Amicus Therapeutics, Inc. | Variant, recombinant beta-glucocerebrosidase proteins with increased stability and increased retained catalytic activity |
US9821038B2 (en) | 2010-11-08 | 2017-11-21 | Amicus Therapeutics, Inc. | Variant, recombinant beta-glucocerebrosidase proteins with increased stability and increased retained catalytic activity |
US9566316B2 (en) | 2010-11-08 | 2017-02-14 | Amicus Therapeutics, Inc. | Variant, recombinant beta-glucocerebrosidase proteins with increased stability and increased retained catalytic activity |
US8962564B2 (en) | 2010-11-08 | 2015-02-24 | Amicus Therapeutics, Inc. | Variant, recombinant beta-glucocerebrosidase proteins with increased stability and increased retained catalytic activity |
WO2012148835A1 (en) | 2011-04-29 | 2012-11-01 | Pioneer Hi-Bred International, Inc. | Down-regulation of a homeodomain-leucine zipper i-class homeobox gene for improved plant performance |
WO2012174139A2 (en) | 2011-06-14 | 2012-12-20 | Synthon Biopharmaceuticals B.V. | Compositions and methods for making and b ioc ont aining auxotrophic transgenic plants |
WO2013066805A1 (en) | 2011-10-31 | 2013-05-10 | Pioneer Hi-Bred International, Inc. | Improving plant drought tolerance, nitrogen use efficiency and yield |
WO2013138309A1 (en) | 2012-03-13 | 2013-09-19 | Pioneer Hi-Bred International, Inc. | Genetic reduction of male fertility in plants |
WO2013138358A1 (en) | 2012-03-13 | 2013-09-19 | Pioneer Hi-Bred International, Inc. | Genetic reduction of male fertility in plants |
WO2013177400A1 (en) | 2012-05-24 | 2013-11-28 | Iowa State University Research Foundation, Inc. | Arabidopsis nonhost resistance gene(s) and use thereof to engineer disease resistant plants |
US11464871B2 (en) | 2012-10-02 | 2022-10-11 | Novartis Ag | Methods and systems for polymer precipitation and generation of particles |
WO2014100525A2 (en) | 2012-12-21 | 2014-06-26 | Pioneer Hi-Bred International, Inc. | Compositions and methods for auxin-analog conjugation |
WO2014164014A1 (en) | 2013-03-11 | 2014-10-09 | Pioneer Hi-Bred International, Inc. | Genes for improving nutrient uptake and abiotic stress tolerance in plants |
WO2014164961A2 (en) | 2013-03-12 | 2014-10-09 | Pioneer Hi-Bred International, Inc. | Manipulation of dominant male sterility |
WO2014164116A1 (en) | 2013-03-13 | 2014-10-09 | Pioneer Hi-Bred International, Inc. | Functional expression of bacterial major facilitator superfamily (sfm) gene in maize to improve agronomic traits and grain yield |
WO2014164074A1 (en) | 2013-03-13 | 2014-10-09 | Pioneer Hi-Bred International, Inc. | Enhanced nitrate uptake and nitrate translocation by over-expressing maize functional low-affinity nitrate transporters in transgenic maize |
US9790515B2 (en) | 2013-03-14 | 2017-10-17 | Cibus Us Llc | Mutated allene oxide synthase 2 (AOS2) genes |
WO2014153178A2 (en) | 2013-03-14 | 2014-09-25 | Cibus Us Llc | Mutated allene oxide synthase 2 (aos2) genes |
WO2014160122A1 (en) | 2013-03-14 | 2014-10-02 | Pioneer Hi-Bred International, Inc. | Maize stress related transcription factor 18 and uses thereof |
WO2014143996A2 (en) | 2013-03-15 | 2014-09-18 | Pioneer Hi-Bred International, Inc. | Compositions and methods of use of acc oxidase polynucleotides and polypeptides |
US10954522B2 (en) | 2013-03-15 | 2021-03-23 | Cibus Us Llc | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
WO2014144987A2 (en) | 2013-03-15 | 2014-09-18 | Cibus Us Llc | Methods and compositions for increasing efficiency of increased efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
US11761011B2 (en) | 2013-03-15 | 2023-09-19 | Cibus Us Llc | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
EP4136963A1 (en) | 2013-03-15 | 2023-02-22 | Cibus US LLC | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
WO2014152507A2 (en) | 2013-03-15 | 2014-09-25 | Pioneer Hi-Bred International, Inc. | Modulation of acc deaminase expression |
US10287594B2 (en) | 2013-03-15 | 2019-05-14 | Cibus Us Llc | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
EP3527068A1 (en) | 2013-03-15 | 2019-08-21 | Cibus US LLC | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
US11434494B2 (en) | 2013-03-15 | 2022-09-06 | Cibus Us Llc | Targeted gene modification using oligonucleotide-mediated gene repair |
US11421240B2 (en) | 2013-03-15 | 2022-08-23 | Cibus Us Llc | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
US9957515B2 (en) | 2013-03-15 | 2018-05-01 | Cibus Us Llc | Methods and compositions for targeted gene modification |
US11459579B2 (en) | 2013-07-09 | 2022-10-04 | Board Of Trustees Of Michigan State University | Transgenic plants produced with a K-domain, and methods and expression cassettes related thereto |
US10570409B2 (en) | 2013-07-09 | 2020-02-25 | Board Of Trustees Of Michigan State University | Transgenic plants produced with a K-domain, and methods and expression cassettes related thereto |
WO2015006105A1 (en) | 2013-07-09 | 2015-01-15 | Board Of Trustees Of Michigan State University | Transgenic plants produced with a k-domain, and methods and expression cassettes related thereto |
EP4357452A2 (en) | 2014-03-14 | 2024-04-24 | Cibus US LLC | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
WO2015139008A1 (en) | 2014-03-14 | 2015-09-17 | Cibus Us Llc | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
US10435700B2 (en) | 2014-10-06 | 2019-10-08 | Altria Client Services Llc | Genetic control of axillary bud growth in tobacco plants |
US11236349B2 (en) | 2014-10-06 | 2022-02-01 | Altria Client Services Llc | Genetic control of axillary bud growth in tobacco plants |
WO2016100309A1 (en) | 2014-12-16 | 2016-06-23 | Pioneer Hi-Bred International, Inc. | Restoration of male fertility in wheat |
US11542515B2 (en) | 2016-02-09 | 2023-01-03 | Cibus Us Llc | Methods and compositions for increasing efficiency of targeted gene modification using oligonucleotide-mediated gene repair |
US10731173B2 (en) | 2016-03-11 | 2020-08-04 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having reduced or eliminated suckers |
WO2017156535A1 (en) | 2016-03-11 | 2017-09-14 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having reduced or eliminated suckers |
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US10647989B2 (en) | 2016-10-07 | 2020-05-12 | Altria Client Services Llc | Composition and methods for producing tobacco plants and products having reduced tobacco-specific nitrosamines (TSNAs) |
US11634724B2 (en) | 2016-10-07 | 2023-04-25 | Altria Client Services Llc | Composition and methods for producing tobacco plants and products having reduced tobacco-specific nitrosamines (TSNAs) |
WO2018067985A1 (en) | 2016-10-07 | 2018-04-12 | Altria Client Services Llc | Composition and methods for producing tobacco plants and products having reduced tobacco-specific nitrosamines (tsnas) |
WO2018140214A1 (en) | 2017-01-24 | 2018-08-02 | Pioneer Hi-Bred International, Inc. | Nematicidal protein from pseudomonas |
WO2019051232A1 (en) | 2017-09-11 | 2019-03-14 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having reduced or eliminated suckers |
US11879130B2 (en) | 2017-09-11 | 2024-01-23 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having reduced or eliminated suckers |
US11104912B2 (en) | 2017-09-11 | 2021-08-31 | Altria Client Services Llc | Compositions and methods for producing tobacco plants and products having reduced or eliminated suckers |
US11359208B2 (en) | 2018-01-09 | 2022-06-14 | Cibus Us Llc | Shatterproof genes and mutations |
EP4062774A1 (en) | 2018-04-03 | 2022-09-28 | Altria Client Services LLC | Composition and methods for producing tobacco plants and products having increased phenylalanine and reduced tobacco-specific nitrosamines (tsnas) |
WO2019195237A1 (en) | 2018-04-03 | 2019-10-10 | Altria Client Services Llc | Composition and methods for producing tobacco plants and products having increased phenylalanine and reduced tobacco-specific nitrosamines (tsnas) |
US11377665B2 (en) | 2018-04-03 | 2022-07-05 | Altria Client Services Llc | Composition and methods for producing tobacco plants and products having increased phenylalanine and reduced tobacco-specific nitrosamines (TSNAs) |
WO2020154466A1 (en) | 2019-01-24 | 2020-07-30 | Altria Client Services Llc | Tobacco plants comprising reduced nicotine and reduced tobacco specific nitrosamines |
WO2023012342A1 (en) | 2021-08-06 | 2023-02-09 | Kws Vegetables B.V. | Durable downy mildew resistance in spinach |
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EP0979311A1 (en) | 2000-02-16 |
KR20010020375A (en) | 2001-03-15 |
CN1268186A (en) | 2000-09-27 |
CA2288209A1 (en) | 1998-11-05 |
AU7365498A (en) | 1998-11-24 |
JP2002511851A (en) | 2002-04-16 |
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WO1998049350A8 (en) | 2000-05-25 |
AU749410B2 (en) | 2002-06-27 |
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