WO1997048806A1 - Therapie genique pour le traitement de l'obesite - Google Patents

Therapie genique pour le traitement de l'obesite Download PDF

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WO1997048806A1
WO1997048806A1 PCT/US1997/010371 US9710371W WO9748806A1 WO 1997048806 A1 WO1997048806 A1 WO 1997048806A1 US 9710371 W US9710371 W US 9710371W WO 9748806 A1 WO9748806 A1 WO 9748806A1
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Prior art keywords
leptin
mice
gene
adenovirus
vector
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PCT/US1997/010371
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English (en)
Inventor
Manal A. Morsy
Ming Cheng Gu
Jing Zhao
C. Thomas Caskey
Stefan Kochanet
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Merck & Co., Inc.
Baylor College Of Medicine
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Priority claimed from GBGB9615788.8A external-priority patent/GB9615788D0/en
Application filed by Merck & Co., Inc., Baylor College Of Medicine filed Critical Merck & Co., Inc.
Priority to US09/202,684 priority Critical patent/US6630346B1/en
Priority to AU34887/97A priority patent/AU3488797A/en
Priority to EP97931187A priority patent/EP0954579A1/fr
Priority to JP50319398A priority patent/JP2002514904A/ja
Publication of WO1997048806A1 publication Critical patent/WO1997048806A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; 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)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • This invention relates to methods of gene therapy by vector-assisted delivery of a peptide hormone, and to transgenic non- human mammals so produced. This invention also related to gene therapy for obesity. This invention also relates to vectors useful in this gene therapy.
  • peptides or proteins include leptin, insulin, calcitonin, erythropoietin, (EPO), growth hormone, interferons, interleukin-2, hemophilia factors, vascular endothelial growth factors such as VEGF, granuiocyte-macrophage colony stimulating factor, alpha 1 anti-trypsin, and others.
  • EPO erythropoietin
  • growth hormone interferons
  • interleukin-2 hemophilia factors
  • vascular endothelial growth factors such as VEGF, granuiocyte-macrophage colony stimulating factor, alpha 1 anti-trypsin, and others.
  • VEGF vascular endothelial growth factors
  • granuiocyte-macrophage colony stimulating factor alpha 1 anti-trypsin
  • Leptin is a protein expressed by the ob gene. Leptin is secreted by adipose tissue and appears to be both a satiety factor and a regulator of metabolism (Levin et al., 1996 Proc. Natl Acad. Sci. USA 93:1726-1730). Both the mouse gene and its human homologue have recently been identified and sequenced (Zhang et al., 1994 Nature (London) 372:425-431.)
  • mice which are homozygous for the ob gene are obese, perhaps due to leptin deficiency.
  • oblob mice are given daily injections of recombinant protein, their food intake was markedly inhibited and they experienced a reduction in body weight and fat.
  • lean mice daily injections of leptin lead to modest decreases of food intake and body weight.
  • body fat The results for body fat have been confirmatory to the effect of leptin on fat metabolism. (Pelleymounter et al., 1995, Science 269:540-543; Halaas et al., 1995 Science 269: 543-546; and Campfield et al, 1995 Science 269:546-549).
  • Obesity in humans is a major disorder associated with mortality, and may result from a number of causes, and at least some may be due to an insufficient amount of leptin produced or resistance. Since leptin is a protein, and vulnerable to breakdown and inactivation by the gastrointestinal system, it cannot be delivered orally. It would be desirable to develop a therapy for leptin delivery for obese patients whose obesity is due, at least in part, to a paucity of leptin or resistance to the sustained peripheral levels.
  • This invention relates to gene therapy wherein a viral vector is used to deliver a protein or peptide which is also a hormone.
  • One aspect of this invention involves a method of treating a condition which is caused at least in part by an insufficient production of or resistance to a peptide or protein hormone in a mammal comprising administration of a viral vector comprising a gene encoding the peptide or protein hormone. It has been found, in accordance with this invention, that the amount of protein or peptide hormone needed to produce a biological result is much lower when the protein or peptide hormone is made by the mammal receiving the gene therapy, as compared to the amount needed to produce the same biological result when the peptide or protein hormone is delivered to the mammal by, e.g.
  • muscular injection or intravenous administration Further, the mammal which is expressing the gene which has been introduced by gene therapy does not develop antibodies to the peptide or protein hormone. In contrast, a mammal which receives the same peptide or protein hormone by injection or intravenous administration, may develop antibodies to the hormone.
  • one aspect of this invention relates to a method of treating a condition caused, at least in part by an insufficient production of a peptide or protein hormone in a mammal comprising administration of a viral vector comprising a gene encoding the peptide or protein hormone wherein the gene encodes a hormone selected from the group consisting of: leptin, insulin, calcitonin, erythropoietin, growth hormone, interferons, interleukin-2, a hemophilia factor, a vascular endothelial growth factor, granulocyte-macrophage colony stimulating factor, and alpha 1 anti-trypsin.
  • This invention is also related to gene therapy for obesity.
  • One aspect of this invention involves a method of treating obesity, lowering serum glucose levels or lowering serum insulin levels in a mammal in need of such therapy comprising delivering a gene encoding an obesity regulating gene to said mammal; and allowing sufficient time to pass for transcription and translation of the obesity regulating gene.
  • Some types of obesity are caused by an insufficient amount of leptin.
  • the individual may also experience elevated serum glucose and/or insulin levels.
  • another aspect of this invention is a method of treating obesity, elevated serum glucose levels, or elevated insulin levels comprising delivering a gene encoding leptin to a mammal wherein transcription and translation of the gene occurs in vivo.
  • the mammal may produce functional leptin, but at lower levels than required; alternatively the animal may have a complete inability to produce leptin; or in yet another alternative, the animal produces a mutated form of leptin which either functions less efficiently than native leptin or does not function at all.
  • Non-human mammals particularly rodents such as mice and rats which have received peptide or protein hormone transgenes and which express the peptide or protein hormone make up another aspect of this invention.
  • mice which have received the leptin gene transgenically and which express leptin form another aspect of this invention.
  • Mice may be oblob, Obi? or homozygous wild-type for the ob gene prior to receipt of the leptin gene. Progeny of these mammals make up yet another aspect of this invention.
  • viral vectors for the delivery of peptide or protein hormone genes to be used in gene therapy.
  • adenoviral vectors are provided, including those with deletions in all viral protein coding sequences, which are less immunogenic than previous vectors.
  • Still further aspects of this invention include mammalian cells transformed with vectors of this invention.
  • Another aspect of this invention is a method of determining whether a compound has the ability to modulate leptin activity in vivo comprising administering the compound to a transgenic animal of this invention and monitoring the animal's reaction to the compound.
  • leptin agonists, antagonists and mimetics may be identified, or if a compound shows leptin modulating activity in in vitro assays, it can be determined if this activity is retained in vivo.
  • FIGURE 1 is a photograph of two oblob mice.
  • the mouse on the right is from a control group.
  • the mouse on the left received gene therapy in accordance with the pilot study (Example 3) of this invention.
  • Figure 2 is a graph showing the body weight changes of mice treated with recombinant human leptin protein as described in the Example 3 pilot study. Injections of human recombinant leptin were given daily IP, at 1 mg/gm body weight. Arrows indicate bleeding points.
  • Figure 3 is a graph showing body weight changes of mice of the pilot study treated with adenovirus carrying a reporter gene ( ⁇ - galactosidase), used as a control.
  • Figure 4 is a graph showing body weight changes of mice of the pilot study treated with adenovirus carrying the human leptin gene.
  • Figure 5 is a graph showing the percent of body weight changes for all groups of mice of the pilot study.
  • Figure 6A (left graph) is a graph showing the amount of human leptin found in the plasma of mice treated with adenovirus containing the leptin gene as described in Example 3.
  • Figure 6B (right graph) shows results for mice in the pilot study treated with five daily injections of recombinant human leptin.
  • Figure 7A shows the amount of insulin and leptin in plasma of mice of the pilot study treated with adenovirus containing the leptin gene.
  • Figure 7B shows the amount of insulin and leptin in plasma of mice of the pilot study treated with recombinant human leptin injections.
  • Figure 8 shows glucose levels of the mice of the pilot study treated with either recombinant leptin, reporter gene or adenovirus containing the leptin gene.
  • Figure 9 compares body weight changes in the mice of the pilot study which received human leptin gene (Figure 9A), with mice of the expanded study (Example 5) which received mouse leptin gene (Figure 9B) and with mice of the expanded study which received human leptin gene (Figure 9C).
  • Figure 10 shows the body weight changes in oblob mice which were used as controls.
  • Figure 10A is the pilot study mice;
  • Figure 10B shows the expanded study mice which received iv. injections of dialysis buffer, and
  • Figure IOC shows the expanded study mice which received the control adenoviral injection.
  • the arrowhead shows the first day that injections occurred. All controls gained weight.
  • Figures 11A-C show the results of IP injections of human leptin.
  • Figure 11 A is the pilot study mice.
  • Figure 1 IB shows mice in the expanded study which received control injections, and
  • Figure 11C shows mice in the expanded study which received leptin injections.
  • Figure 12 is a graph summarizing percent body weight changes for all the treatment groups of the expanded study.
  • Figure 13 is a graph summarizing the mean body weight changes for the oblob mice in the expanded study.
  • Figures 14A-B summarize the levels of human leptin found in the plasma of mice in the expanded study.
  • Figure 14A shows mice which received adenovirus carrying human leptin gene
  • Figure 14B shows mice which received human leptin IP injections.
  • Figures 15A-B summarizes findings relating to the injection of recombinant leptin in the expanded study.
  • Figure 15A shows serum levels of human or mouse leptin and weight for animals receiving leptin genes.
  • Figure 15B shows leptin levels and weight for animals receiving leptin injections, and notes the antibody response.
  • Figure 16 is a graph illustrating glucose levels of animals in the expanded study.
  • Figure 17A shows glucose levels in adenovirus -treated mice in the expanded study and Figure 17B shows the same in mice receiving protein injections.
  • Figure 18 shows food intake in relation to weight loss for the expanded study.
  • Figure 19 shows body weight changes in treated lean mice relative to day 0 for the expanded study.
  • Figure 20 shows the food intake relative to control for mice treated in the expanded study with adenovirus containing leptin or recombinant leptin.
  • Figure 21 A shows leptin levels and weight changes of lean mice treated in the expanded study with adenovirus carrying leptin genes.
  • Figure 21 B shows leptin levels and weight for lean mice receiving recombinant leptin IP.
  • Figure 22 shows glucose levels in lean ⁇ Obi?) mice in the expanded study.
  • Figure 23 shows anti-adenovirus antibody levels and anti- leptin antibody levels measured after day 15 in the expanded study.
  • Figure 24 shows anti-adenovirus antibody levels and anti- leptin antibody levels measured after day 15 in the expanded study.
  • Figure 25A illustrates the HD-Leptin vector.
  • the DNA composite fragments of HD-leptin inserted into pBluescript 11KS are (left to right): the left end terminus of Ad5, composed of the inverted
  • -t - terminal repeat sequences and the packaging signal ⁇ (nucleotides (nt.) 1-440, solid arrow), the Pmel-EclXI 16054 bp fragment of HPRT (nt.1799- 17853 of HUMHPRTB, shaded bar), the leptin expression cassette, composed of the HCMV promoter, the murine leptin cDNA (500 bp) and the bovine growth hormone poly A tail (open bar), the Hindm 9063 bp fragment of C346 cosmid (nt. 12421-21484 of HUMDXS455A, shaded bar) and the right end terminus of Ad5, composed of the ITR sequence (nt. 35818-35935).
  • the ITRs are flanked by unique Pmel restriction site used to liberate the vector from the plasmid backbone prior to the initial transfection into 293-cre4 cells for viral propagation and rescue.
  • Figure 25B illustrates detection of leptin protein expression mediated by the HD-leptin virus in vitro using a Western blot with polyclonal antibodies. Details are provided in Example 6.
  • Figures 26 A-F show effects of HD-leptin and Ad-leptin in lean mice.
  • solid circle is HD-leptin
  • circle with cross is Ad-leptin
  • open circle is AD- ⁇ -gal
  • open triangle is dialysis buffer.
  • Figure 26A shows serum leptin levels collected 2-3 times weekly (mean + SEM).
  • Figure 26B shows weight (mean ⁇ SEM) measured daily.
  • Figure 26C shows food intake measured daily.
  • Figure 26D is a Northern Blot of RNA extracted from liver of Ad-leptin at 2, 4, and 8 weeks.
  • Figure 26E is serum glucose measured in all animal groups (mean + SEM).
  • Figure 26F is insulin levels measured in all animal groups (mean ⁇ SEM).
  • Figures 27 A-F show effects of HD-leptin and Ad-leptin in oblob mice.
  • solid circle is HD-leptin; circle with cross is Ad-leptin; open circle is AD- ⁇ -gal; open triangle is dialysis buffer; as asterisk is lean control values plotted for relative comparison.
  • Figure 27A shows serum leptin levels collected 2-3 times weekly (mean + SEM).
  • Figure 27B shows weight (mean + SEM) measured daily.
  • Figure 27C shows food intake measured daily.
  • Figure 27D is a Northern Blot of RNA extracted from liver of Ad-leptin and HD-leptin at 1 , 2, 4, and 8 weeks.
  • Figure 27E is serum glucose measured in all animal groups (mean + SEM).
  • Figure 27F is insulin levels measured in all animal groups (mean ⁇ SEM).
  • Figure 28 shows phenotypic correction of HD-leptin treated oblob mice. From left to right, representative oblob mouse treated with HD-leptin at day 54 post treatment next to a littermate treated with Ad- leptin 54 days post treatment. Lean control mouse and untreated oblob mouse are provided for comparison.
  • “Native” a gene or protein is native if it naturally occurs in a given organism.
  • Transgene a gene which has been introduced into a mammal or its ancestor using a viral vector.
  • Transgene construct or “expression cassette” means a transgene and associated DNA, such as promoter(s) enhancer(s), and terminal sequences needed for control of transcription and translation of the transgene.
  • Leptin gene a gene from any mammal which encodes a native leptin, or a derivative thereof.
  • a “derivative” is a modified leptin molecule which retains at least 80% of the biological activity of native leptin.
  • Protein or peptide hormone any hormone which is in the form of a protein or peptide, whether or not post-translationally modified. Examples include: leptin, insulin, calcitonin, EPO, growth hormone, interferon, EL-2, vascular endothelial growth factors such as VEGF, GMCSF and alpha 1 anti-trypsin.
  • Olesity regulating gene a gene whose gene product is involved in the regulation of obesity in a mammal, including genes encoding leptin, leptin receptors, neuropeptide Y, and the like.
  • lean means the animal expresses normal levels of leptin and genotypically are homozygous normal for the leptin gene. "Obi?" means the animal expresses leptin, but it is unknown if the animal is homozygous normal or a carrier of the leptin gene defect.
  • IP intraperitoneal injection
  • IM intramuscular injection
  • Ad means adenovirus
  • Ad-leptin means an adenovirus vector carrying a leptin transgene.
  • HD helper-dependent virus system
  • HD-leptin means a helper dependent virus carrying a leptin transgene.
  • wild-type or wt refers to the non-mutated form of a gene. With reference to the ob gene, a mouse with a wild-type phenotype is lean.
  • recombinant peptide hormones have been administered to mammals (including man) suffering from conditions caused at least in part by an insufficiency of the hormone.
  • mammals including man
  • insufficiency of the hormone examples include replacement of congenital (e.g. hemophilias) or acquired (e.g. erythropoetin) deficiencies.
  • Enhanced expression of loci that might provide a therapeutic benefit e.g. LDL receptor or apolipoprotein A-I
  • loci that might provide a therapeutic benefit
  • tissue-specific and physiologic regulation e.g. LDL receptor or apolipoprotein A-I
  • these conditions include diabetes, alpha 1-anti-trypsin deficiency, etc.
  • one problem with such therapy is the inconvenience and associated expense of an injection or iv administration.
  • the recipient may develop antibodies to the administered hormone.
  • Recombinant leptin has been administered to animals who exhibiting an obese phenotype, and a daily injection has been shown to decrease body weight.
  • Injections are not a particularly convenient method of treatment, particularly for long-term treatments.
  • the half-life of leptin is short, so the duration of a single treatment was found to be only about 24 hours, after which the animals were observed to re-gain weight.
  • a peptide or protein hormone which is expressed in vivo is more advantageous than administration of the recombinant form of that hormone; its effects last longer, and most surprisingly, is up to 20 fold more potent than recombinant peptide hormone administered by injection. Further, if the recipient mammal endogenously produces the hormone, no immune response with respect to the transgenic hormone is observed.
  • This invention utilizes the leptin gene delivered to oblob mice as a model for peptide hormones in general.
  • Leptin was chosen because its nucleic acid and amino acid sequences were known, and its effects on obese oblob mice are visually apparent as well as biochemically apparent. Applicants are not aware of any reason why their findings for leptin are not generally applicable to all peptide or protein hormones, and intend for this invention to be construed broadly.
  • nucleotide sequence of leptin Since the amino acid and nucleotide sequence of leptin is known, it is well within the skill of one of the ordinary artisan to construct a nucleotide sequence which encodes a desired mutant form of leptin. These can be used to study structure and function relationships involved in leptin binding and signaling in the transgenic animal model.
  • the gene which encodes the leptin should also contain at least one element which allows for expression of the gene when introduced into the host cell environment.
  • These sequences include, but are not limited to promoters, response elements, and enhancer elements.
  • promoters are chosen which are regulatable; i.e. are inducible rather than constitutive.
  • Particular examples of such promoters include: the Gene SwitchTM mifepristone inducible gene regulation system commercially available from Gene Medicine; the "two component gene regulation system” commercially available from Ariad, regulatable tet, P-450, and constitutive promoters such as EF-1 alpha, SR-alpha, CMV, albumin and the like.
  • the heterologous leptin gene may be delivered to the organism using a vector or other delivery vehicle.
  • DNA delivery vehicles can include viral vectors such as adenoviruses, adeno-associated viruses, helper dependent adenoviruses, and retroviral vectors. See, for example: Chu et al, 1994 Gene Ther. 1 :292-299; Couture et al, 1994 Hum. Gene Ther. 5:667-277; and Eiverhand et al, 1995 Gene Ther. 2:336-343.
  • Non-viral vectors which are also suitable include DNA-lipid complexes, for example liposome-mediated or ligand/poly-L-Lysine conjugates, such as asialoglyco-protein-mediated delivery systems.
  • a viral vector is chosen as the delivery vehicle it may be one which is capable of integrating into the host genome, so that the gene can be expressed permanently, but this is not required. In cases where the vector does not integrate into the host genome, the expression of the gene may be transient rather than permanent.
  • One vector which is suitable for transient expression of the ob gene is an adenovirus which has a deletion in the El gene.
  • Such vectors are known, as taught in the aforementioned WO 95/00655 and Mitani et al, 1995 publications. These viruses preferentially infect hepatocytes, where they persist for approximately 3-4 weeks after the initial infection. While in the hepatocytes, these viruses can express the heterologous gene.
  • an adenovirus vector is preferably administered as an injection at a dose range of from about 5.0 x 10 to about 10 x 10 ⁇ plaque forming units (PFU) per gram body weight.
  • PFU plaque forming units
  • PFU plaque forming units
  • helper dependent viral vector be utilized.
  • Ad vectors are currently among the most efficient gene transfer vehicles for both in vitro and in vivo delivery, but the utilization of first generation Ad for many gene therapy applications is limited due to the transient nature of transgene expression obtained by these vectors.
  • Several factors have been shown to contribute to and modulate the duration of Ad-mediated gene expression as well as the immunogenicity of these vectors, including "leaky" viral protein expression and the transgene delivered.
  • the development of Ad vectors, deleted in all viral protein coding sequences, offers the prospects of a potentially safer, less immunogenic vector with an insert capacity of up to approximately 37 kb. This vector requires supplementation of viral regulatory and structural proteins in trans for packaging and rescue, thus helper dependent (HD).
  • HD helper dependent
  • Ad vectors deleted in all viral protein coding sequences, has resulted in a less immunogenic vector with an insert capacity of up to approximately 37 kb.
  • This class of vectors requires supplementation of viral regulatory and structural proteins in trans for packaging and rescue, and are thus termed "Helper Dependent” (HD).
  • HD Helper Dependent
  • the HD vector containing a leptin transgene construct comprises the Ad5 inverted terminal repeats (ITR) and packaging signal sequences, a leptin transgene construct, and "sniffer" DNA.
  • Sniffer DNA is human genomic DNA sequences or other non-transcribed DNA sequences used to increase the vector insert size to at least approximately 28 kb.
  • the sniffer DNA is a segment of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene.
  • HPRT human hypoxanthine-guanine phosphoribosyltransferase
  • pSTK120 the sniffer DNA is intronic HPRT which also contains a matrix association region (MAR).
  • MAR matrix association region
  • a specific embodiment of this invention is vector pSTK120 comprises a first segment of adenovirus a first segment of sniffer DNA, a transgene DNA, construct a second segment of sniffer DNA, and a second segment of adenovirus DNA.
  • the adenovirus DNA is preferably adenovirus type 5 DNA sequences, and together comprise adenovirus inverted terminal repeats (ITRs) that comprise the viral origin of replication and packaging signals.
  • the non-viral DNA can contain up to 38 kb.
  • An important aspect of the vector is that no adenoviral proteins are expressed in the host cell.
  • the first segment of adenovirus type 5 DNA preferably comprises at least about nucleotides 1-440, although more may be present.
  • the second segment of adenovirus type 5 DNA preferably comprises at least about nucleotides 35818-35935.
  • helper virus system containing a modified El deleted vector with lox sites flanking the packaging signals (Ad LC ⁇ cluc) and a 293 cell line expressing Cre recombinase such as 293-C/W is preferred.
  • Ad LC ⁇ cluc modified El deleted vector with lox sites flanking the packaging signals
  • 293 cell line expressing Cre recombinase such as 293-C/W
  • Animals which transiently or permanently express a peptide or protein hormone such as the ob gene product are valuable research tools. For example, they can be used to monitor the effects of decreasing amounts of leptin, or the effect of various exogenously supplied substances (such as hormones or putative leptin receptor agonists and antagonists) in an environment of decreasing leptin availability.
  • this invention is specifically directed to gene therapy for humans.
  • an adenovirus or helper dependent adenovirus containing a transgenic leptin gene is administered to mice which are obese (oblob).
  • the leptin gene or a derivative
  • the gene which is from the same species as the host is used.
  • mice dbldb mice (obese, but unresponsive to leptin injections due to a receptor defecit), and lean mice (wild-type phenotype, genotype Oblob or OblOb ).
  • Bodv-weight Figure 2 illustrates the body weight changes for the oblob mice of Example 3 ("pilot study") receiving 1 mg/gm body weight human recombinant leptin protein injections daily, compared to untreated controls. Animals receiving leptin were injected for five consecutive days, shown by the darkened symbols on the graph.
  • Leptin The amount of human leptin in plasma was measured in the pilot study animals which received injections of human recombinant leptin and those which received the vector carrying the leptin gene. Those receiving the recombinant protein were noted to have leptin levels which were approximately 20-fold higher than the amount of leptin found in control (lean, wild type) animals; peak amounts of 399.8 ⁇ 40.91 ng/ml. Those receiving the leptin gene had levels of leptin in their plasma which was within the normal range found in a wild-type mouse (17.52 ⁇ 4.66 ng/ml). In both groups of animals of the pilot study, weight gain was synchronized with the fall of human leptin detected in the plasma. This is illustrated in Figure 6.
  • Insulin The amount of insulin in the plasma was measured in the pilot study animals receiving recombinant protein and those which received the gene therapy. This is illustrated in Figure 7. In both
  • Example 5 An expanded study was also conducted, and is detailed in Example 5. This differed from the pilot study in that somewhat older animals were used, additional investigations were performed, and wild- type lean mice were also treated. The observations are set forth below.
  • Weight loss The oblob mice which were treated with adenovirus carrying the human leptin gene lost weight, (18.61 % over a 10-12 day period) as did the mice in the pilot study, as is shown in Figure 9. Weight loss occurred within 24 hours, with 9.17% loss from day 1-5 post treatment. Weight gain was restored within 10-20 days post-treatment. On the other hand, mice receiving control treatments continued to gain weight throughout the experiment, as did their counterparts in the pilot study, as shown in Figure 10. The oblob mice receiving iv injections of adenovirus carrying the mouse leptin gene lost more weight than did mice receiving iv injections of adenovirus carrying the human gene. The pattern of weight loss and time of weight gain were substantially the same as those
  • mice receiving IP injections of recombinant leptin protein lost weight within 24 hours, and plateaued at day 3 of the daily injection, as illustrated in Figure 11. After day 1-5 post-treatment, loss was 4.7%, significantly less than that of groups receiving gene therapy. Control animals gained weight throughout the experiment, as can be seen in Figures 1 IB and 1 IC.
  • IP leptin led to a response similar to that seen in the pilot study.
  • a sharp drop in weight in the first three days was observed which was substantially identical to that observed for the gene-treated animals. This was followed by a moderation in weight reduction in the IP protein treated animals, and a bifurcation in weight reduction slopes for both treatments.
  • the adenoviral-mediated effect was transient, and weight gain was observed at day 11-12 post treatment for the iv. injected animals and day 8 post treatment for the IM injected animals. While not wishing to be bound by theory, it appears that this is due to an immune response to the adenovirus and or adenoviral genes, and not due to an immune response to the leptin produced.
  • Leptin levels Levels of leptin in blood plasma were studied in each of the mice groups, and is illustrated in Figure 14. Those which were treated with the adenovirus carrying the human leptin gene (Figure 14A) had levels which were within the normal range found in wild-type mice (averaging 12.5 ng/ml). In the group treated with recombinant protein (Figure 14B), levels of human leptin exceeded wild-type by about 20-fold; for mouse leptin, concentration exceeded wild type by about 10-fold ( Figure 15A). Weight gain was observed to be synchronized with the fall of leptin levels in plasma.
  • Glucose levels The glucose levels dropped within 6-9 days post-treatment in mice receiving adenovirus carrying the leptin gene and receiving recombinant injected leptin. (See Figures 16, 17A and 17B). No change in glucose levels were observed in any control treated mice. The only treated group whose glucose levels reached that of normal (lean) mice were the mice receiving adenovirus carrying the human leptin gene, and the normal glucose levels was sustained for at least two weeks. Mice receiving recombinant leptin injections sustained reduced glucose levels for less than one week.
  • Food intake An attempt was made to measure food intake, and is shown in Figure 18. Accurate measurements in the group receiving adenovirus carrying a leptin gene and in the group receiving human recombinant leptin was not possible after about one week, as the animals became hyperactive and spilled food out of their containers. In the group receiving adenovirus carrying the human leptin gene, food suppression was 76.6 % + 6.75, whereas in the group receiving recombinant human leptin injections, suppression was 43.8 % + 4.8. Comparisons with lean mice: Various treatments were tried on lean, "normal" mice whose genotype was (Ob/?). The changes in body weight are shown in Figure 19.
  • the gene therapy treatment response of the lean mice resembled that of the oblob mice in that the effect was transient.
  • Table 2 presents the percent weight change in lean mice:
  • Food intake for treated mice relative to controls was measured and is presented in Figure 20.
  • food intake was suppressed by about 50%.
  • food intake was suppressed by about 30%.
  • mice were treated with a single tail vein administration of
  • 1-2 x 10 particles of either HD-leptin, adenovims carrying the leptin gene (Ad-leptin) or control virus In the lean mice, treatment with Ad-leptin resulted in a transient increase in serum leptin levels and weight loss for only 7-10 days (Figs. 26A and 26B). In contrast, treatment with HD-leptin resulted in persistent high serum leptin levels (6-10 fold over background) and approximately a 20% weight loss for at least 2 months. Weight loss in HD-leptin treated mice was associated with satiety that persisted over a longer period (2-3 weeks) as opposed to those treated with Ad-leptin (5-7 days) (Fig. 26C).
  • the HD-leptin was also found to be more effective in obese (oblob) mice than the Ad-leptin vector used in the previous-described pilot and extended studies.
  • serum levels of leptin increased only transiently during the first 1-4 days of treatment after which the levels declined and returned to baseline within 10 days post injection (Fig. 27A).
  • Fig. 27B serum levels of leptin levels resulted in transient body weight loss of only approximately 25% and mice began gaining weight within 2 weeks of treatment.
  • Fig. 27B In contrast, in the oblob HD-leptin treated mice, increasing serum leptin levels were observed up to approximately 15 days post-treatment, after which the levels gradually dropped to baseline over the subsequent 25 days.
  • Leptin specific antibodies were detected in sera of Ad-leptin and HD-leptin oblob treated mice, and it was essential to determine whether the drop observed in serum leptin levels was due to interference of the antibodies with the ELISA assay utilized to measure leptin or if the drop was due to loss of gene expression. Leptin gene expression was examined by total RNA northern blot analysis, in Ad- leptin oblob treated mice expression was transient and RNA levels were beyond the sensitivity level of detection at 1 week post treatment, where as in HD-leptin treated mice gene expression was detected up to 4 weeks post- injection but was undetectable at 8 weeks (Fig. 27C). Serum glucose and insulin levels dropped during the first 1 -
  • transgene immunogenicity plays a central role in loss of gene expression, which may explain the transient effect of HD-leptin seen in the oblob and not in the lean mice.
  • the leptin model used in these studies provided an excellent tool and was invaluable with regards to the specificity and sensitivity of the numerous parameters used to indirectly and directly follow relative changes in gene expression.
  • the differences between the longevity of expression mediated by the HD deleted vector and the transient effect observed by others may reflect differences in the size of the recombinant virus used as it pertains to its stability and efficient packaging which have been characterized.
  • sniffer DNA may play a role in improving stability of gene expression.
  • the HD-vector system of this invention thus reflects a significant advance over previous Ad vectors with regards to vector capacity and reduced immunogenicity in relation to viral protein expression, they have and thus wide application in gene therapy.
  • Another aspect of this invention is a method of permanently expressing a transgenic peptide or protein hormone gene in vivo by administering the gene to a mammal using a helper dependent adenoviral vector, wherein the mammal also endogenously expresses a non-transgenic version of the peptide or protein hormone gene.
  • a helper dependent adenoviral vector wherein the mammal also endogenously expresses a non-transgenic version of the peptide or protein hormone gene.
  • transgenic leptin and endogenously produced leptin were immunologically indistinguishable.
  • the immunological system treats transgenic leptin as if it were a foreign protein, and mounts an antibody attack.
  • leptin or other hormones
  • Human obesity is generally not the result of a double recessive mutation in which no leptin is produced.
  • the amount of leptin produced is abnormally low.
  • gene therapy with a helper dependent adenovirus carrying a leptin gene would provide a way of permanently correcting the leptin level.
  • the helper-dependent adenovirus system can be use to permanently deliver other protein or peptide hormone genes to a human patient, provided that patient endogenously expresses that hormone (even at a low level).
  • This gene therapy can be used to raise protein or peptide hormone levels from below normal to a normal range. Examples include: insulin, calcitonin, erythropoietin, growth hormone, interferons, interleukin 2, hemophilia factors, VEGF, GMCSF, and alpha 1 anti-trypsin.
  • Two PCR cDNA amplification fragments were obtained from Jefferson University (generated by cloning both variants from a Clontech phage human hypothalamic library): one coding for the human leptin and one for the human leptin variant with glutamine, (Zhang et al, 1994, Nature 372:425; Considine et al, 1995 J. Clin. Invest. 95:2986). Both PCR fragments were amplified for cloning purposes. Two primers were designed and ordered from GIBCO BRL Custom Primers: Forward primer: ATG CAT TGG GGA ACC CTG TG (SEQ.ED.NO:l )
  • Reverse primer TCA GCA CCC AGG GCT GAG GT (SEQ.ID.NO:2)
  • the primers were used to re-amplify the cDNA as follows:
  • each primer (0.3 ⁇ g/ ⁇ l stock), 2 ⁇ l dNTP (10 ⁇ M, Pharmacia), 10 ⁇ l 10 X PCR Buffer (Buffer 2 from Expand Long Template PCR System Kit, Boehringer Mannheim), 2 ⁇ l Taq polymerase (Perkin Elmer), 3 ⁇ l template DNA and 18 ⁇ l water.
  • PCR cycling conditions were as follows: Mixture was incubated at 94°C initially (without the addition of the Taq enzyme) for 1 -2 minute, Taq was then added to each tube and the cycling program was initiated, 20 cycles of 94°C for 30 seconds, 45 °C for 45 seconds and 72°C for 1 minute. At the end of the 20 rounds of amplification the samples were incubated at 72°C for 7 minutes.
  • the expected fragment size in each case was 501 and 504, respectively.
  • the PCR amplified fragments were cloned into pCR- Script SK(+) plasmid (Stratagene) and several selected bacterial colonies were grown, plasmids extracted and sequenced to verify correct sequence of both cloned.
  • adenoviral vectors used in this study are essentially the same as those described in Morsey et al, 1993 J. Clin. Invest. 92: 1580-86, which is hereby incorporated by reference, except
  • the three shuttle vectors from the previous example are used in rescue replication of the deficient El deleted adenoviral vectors.
  • 293 cells, commercially available from Microbix, passage 27-30 were set up one day ahead of transfection in 60 mm dishes, and were about 70-80% confluent at the time of use.
  • Plaques were identified, and plugged out of the agarose overlay using a sterile glass Pasteur pipette. Each plugged plaque was resuspended in 100-500 ⁇ l of PBS (with calcium and magnesium) in 10% glycerol, frozen at -80°C and thawed (1 -3 times). The thawed plaque was then used to infect a 90% confluent 6 cm plate of 293 cells to expand the isolated virus.
  • PBS with calcium and magnesium
  • CPE cytopathic effects
  • mice were fed milled Purina Chow 5008 starting from day of arrival or day after arrival. Food consumption was also measured. After approximately 4 days on milled chow, the mice were weighed and bled for determination of plasma levels of glucose and insulin. Injections were started 8 days after the initiation of base line measurements but before injections, mice were weighed and blood samples were obtained from all study mice for determination of plasma glucose and insulin. Leptin level in plasma were also measured. Mice were housed 5 per cage and fed milled Purina Chow
  • mice were injected as follows: A. oblob mouse groups: iv injections. 5 mice/group
  • Group 1 received 2.75 x 10 8 / gm wt of AdHCMV-hob-BGHP A
  • Group 2 received 2.75 x 10 ⁇ / gm wt of AdHCMV- ⁇ gal reporter
  • Group 3 received 500 ml dialysis buffer in tail vein.
  • Group 4 received 1 mg/kg wt active leptin daily IP injections for
  • mice/group B. dbldb mouse groups: iv injections. 5 mice/group
  • Group 1 received 2.75 x 10 8 / gm wt of AdHCMV-hob-BGHP A (in 500 ⁇ l dialysis buffer) in the tail vein.
  • Group 2 received 2.75 x 10 ⁇ / gm wt of AdHCMV- ⁇ gal reporter (in 500 ⁇ l dialysis buffer) in tail vein.
  • Group 3 received 1 mg/kg wt active leptin daily IP injections for 5 days.
  • Adenovirus vectors are made similarly to those described in Examples 2-3, except that the leptin receptor gene replaces the leptin gene.
  • Mice which are dbldb are used in place of the oblob mice. Results for the dbldb mice are similar to those observed with the oblob mice reported herein. After injection, glucose levels fall, insulin levels fall and the mice loose weight. No effect is observed in control mice and in oblob mice injected with vector carrying a leptin receptor gene.
  • Plasmid pSTK Construction of plasmid pSTK was as follows using standard techniques, known to those of ordinary skill in the art. Bluescript KSII (Stratagene) DNA was cleaved with EcoRV. A double- stranded oligodeoxynucleotide with the restriction sites Ascl-Avrll- Fsel-PacI was generated by annealing the single-stranded oligodeoxynucleotides #17302 and #17303:
  • the oligodeoxynucleotide was inserted into the EcoRV side of Bluescript KSII using T4 DNA ligase (NEB). The resulting plasmid was called STK2. STK2 was cleaved with BstXI and the BstXI site was made blunt ended using T4-DNA Polymerase (Pharmacia). A double- stranded deoxyoligonucleotide with the restriction sites Swal-Pmel- SNaBI was generated by annealing the single-stranded oligodeoxynucleotides #17300 and #17301 :
  • the oligodeoxynucleotide was inserted into the blunt-ended BstXI site using T4-DNA ligase.
  • the resulting plasmid was called STK3.
  • the left terminus of Adenovirus type 5 was amplified by
  • STK3 was cleaved with Pmel and Hindm and the Pmel/Hindm cleaved PCR product described above was insewrted using T4-DNA ligase.
  • the resulting plasmid was called STK3-23531/24147.
  • STK31 was cleaved with EcoO109I The EcoO109I site was made blunt-ended using the Klenow fragment of DNA Polymerase I (Pharmacia).
  • Plasmid STK3-23531/24147 was cleaved with SnaBI and EcoRV. The resulting SnaBI-EcoRV fragment containing the right terminus of Adenovirus type 5 was inserted into the EcoO109I site of STK31.
  • the resulting plasmid was called STK42.
  • a cosmid containing part of the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene (U72D8) was cleaved with EclXI and Pmel.
  • EclXI cleaves at bp 1799 and Pmel at bp 17853 of the sequences which is deposited in the GenBank database (Locus: Human HPRT gene [HUMHPRTB]; gb:humhprtb).
  • STK3 was cleaved with Pmel and EclXI.
  • the 16054 bp EclXI/Pmel HPRT fragment from the HPRT gene containing cosmid was inserted into the Pmel and EclXI sites of STK3.
  • the resulting plasmid was called STK55.
  • STK42 was cleaved with HincII.
  • STK55 was cleaved with
  • the Sail site was made blunt-ended using the Klenow fragment of DNA Polymerase I.
  • the resulting fragment that contained the 16054 bp EclXI/Pmel HPRT fragment was inserted into the HincII site of STK42.
  • the resulting plasmid was called STK68.
  • STK68 was cleaved with Ascl.
  • Ascl was made blunt-ended using the Klenow fragment of DNA Polymerase I.
  • the cosmid C346 (Andersson et al. 1995 DNA Seq. 5: 219-223, which is hereby incorporated by reference) was cleaved with Hindm and the ends were made blunt-ended using the Klenow fragment of DNA Polymerase I.
  • Plates were infected with 1 ml lysate and 1 ml media (MEM- ⁇ supplemented with 10% serum 1 X pen/strep, 1 X L-glutamine and sodium bicarbonate), followed 24 hours later with 0.1 -1 X 10 ⁇ pfu of the helper virus. After CPE (approximately 4-5 days), lysate was collected and used to infect 10 cm plates of 293-cre4 cells, the same process was followed, infection in 10 cm plates used 2 ml lysate, 8 ml media and 1-5 X 10 ⁇ helper virus (added 24 hours post lysate infection).
  • CPE collection was followed and lysate was used to infect 15 cm plates of 293-cre4 cells (approximately 1 X 10? cells). 5 ml cells plus 15 ml media was used with 0.1-1 X 10? pfu helper was added 24 hours post- lysate collection. After CPE collected lysate was used to infect twenty 15 cm plates of 293 cre4 cells, this step (passage 5) is the first passage used for cesium banding and virus purification.
  • Cesium banding and virus purification was identical to the process used to purify the first generation virus. Supernatant from passage 5 was also used to further propagate the virus. Occasionally two cesium bands were observed, the lower band was the helper dependent virus. Virus was dialyzed and as was the case with the first generation, titered on regular 293 cells to determine the level of contaminating helper viruses which are capable of plaque formation. The helper dependent virus is not capable of forming plaques; therefore to estimate levels of rescued virus, optical density (OD) readings were made. It is estimated that the infective particles are approximately 10 to 100-fold lower that the estimated particle number measured by optical density. Also, comparisons between the viral vectors described in
  • Examples 1-5 and the helper dependent leptin gene expression were used to estimate particle number. Yield was approximately 8 x 10 12
  • COS cells were infected with 10 ⁇ l of HD-leptin or with Ad-leptin at a moi of l0 or l5. Cells were washed 30 minutes post-infection and serum-free media was added. 100 ⁇ l aliquots of media were collected from infected plates at 24, 30, 48 and 54 hours post-treatment, and compared by western blot analysis for leptin protein levels, using a polyclonal anti -leptin antibody (Santa Cruz Biotech) (See Figure 25B). Leptin was detected as a single band at approximately 16 kD.
  • the HD- leptin mediated expression was equivalent to the 15 moi infected plates, and based on the pfu titer of Ad-leptin, the estimated titer was approximately 1-2 x 10 /ml with a particle to infectious unit ratio of approximately 1 :100.
  • Oblob C57BL/J6-ob/ob
  • litter mates homozygous normal lean mice, age and sex matched (females) were purchased from Jackson Laboratories (Bar Harbor, ME) for the use in this study.
  • mice were free of all common murine pathogens. Eight to twelve weeks old mice (oblob approximately 70 gm and lean approximately 28 gm) were re-distributed based on equal representation of weight and caged in groups of 5 on day 0, immediately preceding treatment. After a series of baseline blood samples were obtained by tail incision from conscious mice, animals were divided into 4 groups and treated by tail vein injection of a single 100 ⁇ l aliquot of 1-2 x 10 particles of HD- leptin, Ad-leptin, Ad- ⁇ gal (control) or dialysis buffer (control). Body weight and food intake were measured daily and blood was collected 2-3 times weekly, pre- and post-treatment. Animals were killed by carbon dioxide inhalation and organs removed for immunohistochemistry and RNA analysis.

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Abstract

La thérapie génique peut traiter l'obésité chez les mammifères. On administre un gène de régulation de l'obésité à un mammifère. De préférence, le gène code la leptine ou un récepteur de la leptine. La protéine qui est administrée et exprimée in vivo est plus efficace que la protéine qui est injectée dans l'animal.
PCT/US1997/010371 1996-06-20 1997-06-20 Therapie genique pour le traitement de l'obesite WO1997048806A1 (fr)

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US6620413B1 (en) 1995-12-27 2003-09-16 Genentech, Inc. OB protein-polymer chimeras
US7074397B1 (en) 1996-01-08 2006-07-11 Genentech, Inc. Method for enhancing proliferation or differentiation of a cell using ob protein
US6541604B1 (en) 1996-01-08 2003-04-01 Genentech, Inc. Leptin receptor having a WSX motif
EP0921820A4 (fr) * 1996-06-20 2003-01-15 Merck & Co Inc Therapie genique de l'obesite
EP0921820A1 (fr) * 1996-06-20 1999-06-16 Merck & Co., Inc. Therapie genique de l'obesite
WO1999064577A1 (fr) * 1998-06-09 1999-12-16 Merck & Co., Inc. Nouveaux vecteurs adenoviraux utilises en therapie genique
JP2002521496A (ja) * 1998-07-28 2002-07-16 フラームス・インテルウニフェルシタイル・インスティチュート・フォール・ビオテヒノロヒー レプチン介在性遺伝子誘導
EP1151091A4 (fr) * 1999-02-04 2002-09-04 Merck & Co Inc Amelioration du systeme vecteur dependant d'un assistant pour la therapie genique
EP1151091A1 (fr) * 1999-02-04 2001-11-07 Merck & Co., Inc. Amelioration du systeme vecteur dependant d'un assistant pour la therapie genique
EP1889914A1 (fr) * 1999-02-19 2008-02-20 Engene, Inc. Compositions de thérapie génétique pour le diabète
US7132277B1 (en) 2000-01-31 2006-11-07 Merck & Co., Inc. Helper dependent vector system for gene therapy
WO2001094605A3 (fr) * 2000-06-09 2002-12-05 Univ Florida Compositions a base de vecteur viral associe a l'adenovirus et leurs utilisations therapeutiques
WO2001094605A2 (fr) * 2000-06-09 2001-12-13 University Of Florida Research Foundation, Inc. Compositions a base de vecteur viral associe a l'adenovirus et leurs utilisations therapeutiques
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