Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/18—Growth factors; Growth regulators
  • A61K38/1816—Erythropoietin [EPO]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
  • A61K38/212—IFN-alpha
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/4846—Factor VII (3.4.21.21); Factor IX (3.4.21.22); Factor Xa (3.4.21.6); Factor XI (3.4.21.27); Factor XII (3.4.21.38)
• • 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
  • 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
  • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
  • C12N2750/00011—Details
  • C12N2750/14011—Parvoviridae
  • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
  • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• Adeno-associated viruses were discovered as satellite viruses present in preparations of adenovirus.
• AAV isolates human, simian and avian
• AAV isolates cause no apparent disease and are not associated with cancer or any other adverse effects.
• Several studies have shown AAV to have a protective effect on carcinogenesis mediated by other viruses, such as tumorigenic strains of adenovirus and bovine papilloma virus.
• Recombinant AAV vectors have been used to express gene products in vitro (usually tissue cultures) and to express gene products in animals, see, for example, U.S. Pat. No. 5,193,941 and WO 9413788, usually in the lungs or oral mucosa, the normal sites of AAV infection, but also in the central nervous system and in cardiac tissue.
• Transduction with recombinant AAV vectors has been accomplished with more direct routes of administration, such as, intraperitoneal injection, intratracheal injection and direct endobronchial instillation in animals, see, for example, Flotte and Carter, Gene Therapy 2:357-362 (1995).
• Those methods of delivering rAAV were focussed on situating the recombinant AAV vector at a site closest to the lung as the experiments were directed to treating patients with cystic fibrosis.
• intraperitoneal, intratracheal and endobronchial injection generally are undesirable clinical techniques for routine use in humans, for example, due to the dangers of producing infection and adhesions, see Goodman and Gil an, "The Pharmacological Basis of Therapeutics" pp. 1-32 (8th ed. , 1993); see also “Remington's Pharmaceutical Sciences” p. 1686 (18th ed., 1990).
• One goal of gene therapy is to provide long-term, therapeutically effective levels of expression of a particular gene of interest in or adjacent to the tissues where such gene expression is therapeutically beneficial.
• There are many challenges to the development of effective gene therapy including obtaining expression of the gene of interest in the targeted tissues, which requires at least a promoter in the recombinant AAV vector that operates in the targeted tissues, and a mode of administration that delivers the recombinant AAV vector to the appropriate tissues.
• the instant invention provides new methods of administering recombinant AAV vectors for effective, long-term expression of a gene of interest in animals, including humans.
• Provided herein are methods of subcutaneously injecting recombinant AAV vectors containing a gene of interest encoding any diffusible polypeptide, ribozyme, nucleic acid or antisense oligonucleotide for gene therapy.
• the instant invention also provides for a topical administration means of rAAV to a host.
• the methods of the instant invention provide significant advantages, including the ability to deliver any suitable gene of interest via subcutaneous injection or topical means and obtain high level, long-term expression in regions approximate or distant from the site of administration.
• the administration routes of the instant invention provide a safe, effective and non-invasive procedure for the administration of recombinant AAV vectors as described herein.
• the methods and recombinant AAV vectors described herein provide a significant development in the field of recombinant AAV vector gene therapy.
• FIG. 1 is a schematic representation of the rAAV-MFG-human-Factor IX vector.
• ITR AAV inverted terminal repeat
• MFG murine Moloney virus long terminal repeat
• MuLV IVS mRNA splice donor/splice acceptor
• Human FIX human Factor IX cDNA
• bGH pA bovine growth hormone polyadenylation site.
• FIG. 2 is a schematic representation of the rAA -MD-human-interferon vector.
• ITR AAV inverted terminal repeat
• CMV Cytomegalovirus immediate early promoter
• /3-gb IVS jS-globin mRNA splice donor/splice acceptor
• Human IFN human interferon cDNA
• j3-gb pA 0-globin polyadenylation site.
• FIG. 3 is a schematic representation of the rAAV-MD-mouse erythropoietin vector.
• ITR AAV inverted terminal repeat
• CMV Cytomegalovirus immediate early promoter
• / 8-gb IVS 3-globin mRNA splice donor/splice acceptor
• Mouse EPO mouse erythropoietin cDNA
• jS-gb pA ⁇ -globin polyadenylation site.
• FIG. 4 is a schematic representation of the rAAV-CMV-LacZ vector.
• ITR AAV inverted terminal
• CMV Cytomegalovirus immediate early promoter
• SV40 IVS SV40 mRNA splice donor/splice acceptor
• FIG. 5 is a graph showing long-term expression of human Factor IX in immunocompetent mice that were injected subcutaneously with 5.9 x 10 10 particles of rAAV-MFG-hFactor IX.
• FIG. 6 is a graph showing long-term expression of human Factor IX in immunocompetent mice that were injected subcutaneously with 5.9 x 10 9 particles of rAAV-MFG-hFactor IX.
• FIG. 7 is a graph showing long-term expression of human Factor IX in immunocompetent mice that were injected subcutaneously with 5.9 x 10 8 particles of rAAV-MFG-hFactor IX.
• FIGS. 8A and 8B are two charts showing anti-human Factor IX antibody levels and anti-AAV antibody levels, respectively, in animals subcutaneously injected with a single injection of particles of rAAV-MFG-hFactor IX.
• FIG. 8A depicts antibody titers for anti-FIX activity.
• FIG. 8B shows antibody titers for anti-AAV activity.
• FIGS. 9A and 9B depict long-term expression of human Factor IX in immunocompetent (BALB/c) and immunocompromised (NIH-3) mice after subcutaneous injection with recombinant AAV-MFG-human Factor IX (each injection contained 5.9 x IO 10 , 5.9 x IO 9 or 5.9 x 10* particles) .
• FIG. 10 illustrates the anti-human Factor IX antibody levels of the animals described in FIG. 9.
• FIGS. 11A and 11B depict long-term expression of human interferon in immunocompetent (BALB/c) and immunocompromised (NIH-3) mice after subcutaneous injection with recombinant AAV-MD-interferon (at 8.5 x 10 10 , 8.5 x 10 9 or 8.5 x 10* particles).
• FIG. 12 is a chart illustrating expression of mouse EPO in BALB/c mice following subcutaneous injection of rAAV-MD-mouse-EPO ( 8 . 9 x 10 9 or 8 . 9 x 10 10 particles) .
• the expression of mouse EPO was measured by monitoring the hematoc it.
• FIG. 13 Epo protein levels at day 105 following SC injection of rAAV-MD-Epo. The protein levels were measured by RIA.
• FIGS. 14A and 14B depict the hematocrit of BALB/c mice after subcutaneous or intramuscular injection of rAAV-MD-mouse-Erythropoietin.
• FIG. 15 Epo protein levels at day 180 following subcutaneous or intramuscular injection of rAAV-MD- Epo. Protein levels were measured by RIA.
• FIG. 16 is a chart illustrating human interferon levels in the serum of BALB/c mice after intravenous injection of AAV-MD-human IFN.
• FIG. 17 is a chart illustrating human Factor IX levels in the serum of BALB/c mice after intramuscular injection of rAAV-MFG-F9.
• FIG. 18 ⁇ -galactosidase expression was not detected in the panniculus carnosus of uninfected animals.
• FIG. 19 ⁇ -galactosidase expression in the panniculus carnosus following subcutaneous injection of rAAV-CMV-LacZ.
• FIG. 20 is a diagram of pSSV9 MD-2 AAV expression vector .
• FIG. 21 is a diagram of the SSV9 MD2-mEPO AAV vector .
• FIG. 22 is a diagram of the SSV9 MD2-human alpha
• FIG. 23 is a diagram of the SSV9-MFG-ShuF9 vector .
• FIG. 24 is a diagram of the MFG-S-hFIX vector.
• DESCRIPTION OF SPECIFIC EMBODIMENTS Provided herein are methods of subcutaneously injecting or topically applying recombinant AAV vectors containing a foreign gene of interest encoding any diffusible polypeptide, ribozyme, nucleic acid or antisense polynucleotide for gene therapy.
• the methods of the instant invention provide significant advantages, including the ability to deliver any diffusible gene of interest via subcutaneous injection or topical application to obtain high level, long-term expression in regions proximal or distal from the site of administration.
• human Factor IX was expressed in high levels in approximately 2/3 of the immunocompetent mice subcutaneously injected with a recombinant AAV vector containing the human Factor IX gene.
• the immunocompetent mice that expressed human Factor IX was expressed in high levels in approximately 2/3 of the immunocompetent mice subcutaneously injected with a recombinant AAV vector containing the human Factor IX gene.
• the immunocompetent mice that expressed human Factor IX was expressed in high levels in approximately 2/3 of the immunocompetent mice subcutaneously injected with a recombinant AAV vector containing the human Factor IX gene.
• AAV vectors containing the human interferon gene or the mouse erythropoietin gene also resulted in high serum levels of both gene products, lasting at least 175 days and 35 days after subcutaneous injection, respectively.
• Subcutaneous injection or topical application for gene therapy is a simple, safe and non-invasive method of administering the recombinant AAV vectors, which is much preferred to other, more invasive methods of administration described previously (e.g., intraperitoneal, intrathecal and endobronchial) .
• AAV vectors also provides the advantages of ease of handling and administration (e.g. self administration) .
• Subcutaneous injection of recombinant AAV vector particles enables immunocompetent animals to express high levels of the gene of interest over a long term, whereas other routes of delivery of certain recombinant AAV vector particles do not result in comparable levels of expression, compare FIGS. 12, 16 and 17.
• Expression of the gene of interest in immunocompromised animals was found to be dose-dependent and consistent. Expression was found to increase within the time periods tested.
• delivery of a syngeneic gene, like mouse erythropoietin injected via a recombinant AAV vector in immunocompetent mice, resulted in dose-dependent, high level and consistent expression, see FIG. 12.
• the recombinant vectors of the instant invention comprise the following components: (1) a promoter derived from a non-AAV source, (2) an mRNA splice donor/splice acceptor, (3) a gene of interest, for example, that encodes a therapeutic polypeptide or a therapeutically effective portion thereof, (4) a polyadenylation site and (5) two AAV inverted terminal repeats flanking components (l)-(4) above.
• the gene of interest can be any polynucleotide that encodes a diffusible polypeptide, i.e. a polypeptide that can diffuse from the site of administration to effect systemic delivery, or a diffusible nucleic acid.
• diffusible polypeptides that may be useful for gene therapy include insulin, Factor VIII and any cytokines, including interferons (IFN- ⁇ , IFN-jS and IFN-7) , interleukins, GM-CSF (granulocyte-macrophage colony-stimulating factor) , M-CSF (macrophage colony-stimulating factor) , tumor necrosis factors and growth factors (TGF-jS (transforming growth factor-0) and PDGF (platelet-derived growth factor) ) .
• IFN- ⁇ interferons
• IFN-jS interferons
• IFN- ⁇ interferons
• IFN- ⁇ interleukins
• GM-CSF granulocyte-macrophage colony-stimulating factor
• M-CSF macrophage colony-stimulating factor
• TGF-jS transforming growth factor-0
• PDGF platelet-derived growth factor
• any diffusible polypeptides of therapeutic interest can be used to express, via subcutaneous injection or topical administration, any diffusible polypeptides of therapeutic interest.
• the gene of interest also may be an antisense polynucleotide or ribozyme.
• the promoter can be any of the many characterized viral (e.g., thymidine kinase and Rous sarcoma virus) or cellular promoters (e.g. neural-specific enolase, vimentin and fibronectin) .
• the accompanying regulatory signals also can be used for expression of transgenes in the instant vector backbone.
• Regulated gene expression from an inducible promoter e.g. metallothionein etc.
• CMV expression cassettes also can be used for controlled gene expression.
• Recombinant AAV vector particles encoding human Factor IX (huFIX) or human ⁇ -interferon (huIFN) were prepared and injected under the skin of immunocompetent BALB/c mice (10 8 to 10 10 particles in a 300 ⁇ l volume of HBSS (Hank's Balanced Salt Solution)). The presence of the secreted proteins was monitored in serum samples collected over time after a single subcutaneous injection. Using either vector, a durable systemic release of the human proteins was observed reproducibly.
• levels of 5-50 ng/ml huFIX and 2-7 ng/ml huIFN were attained over a period of several weeks. The levels remained constant or increased slightly until the latest available time point. The situation typically was observed in 2 or 3 animals within experimental groups of 5.
• Recombinant AAV vector particles encoding mouse erythropoietin also were prepared and injected subcutaneously as described above in immunocompetent BALB/c mice.
• the gene of interest is syngeneic with the host.
• Expression of mouse erythropoietin was monitored in serum samples collected over time. Over the time period monitored, expression of mouse erythropoietin was assayed by hematocrit and high level expression was observed.
• Subcutaneous delivery is a very attractive mode for in vivo gene delivery because the skin is not a vital organ and is very easy to access. If a disease is caused by a defective gene product which is required to be produced and/or secreted, such as in hemophilia, diabetes and Gaucher's disease etc., subcutaneous delivery is a good candidate mode of delivering the gene product.
• Actual delivery of the recombinant AAV vectors is accomplished by using any physical method that will transport the AAV recombinant vector into the subcutaneous region of a host.
• AAV vector means both a bare recombinant AAV vector and recombinant AAV vector polynucleotides packaged into viral coat proteins, as is well known for AAV administration.
• compositions can be prepared as injectable formulations or as topical formulations to be delivered subcutaneously or by transdermal transport, including implantable subcutaneous pumps (known by those of skill in the art and described in, for example, U.S. Pat. No. 5,474,552. Numerous formulations for subcutaneous injection and transdermal transport have been developed and can be used in the practice of the instant invention.
• the vectors can be used with any pharmaceutically acceptable carrier for ease of administration and handling.
• solutions in an adjuvant such as sesame or peanut oil, or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions.
• aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose.
• Solutions of the AAV vector as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
• a dispersion of AAV viral particles also can be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils.
• the preparations Under ordinary conditions of storage and use, the preparations contain a preservative to prevent the growth of microorganisms.
• the sterile aqueous media employed all are obtainable readily by standard techniques well known to those skilled in the art.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that the final preparation can be administered by the device means selected.
• the pharmaceutical must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) , suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.
• a coating such as lecithin
• surfactants for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
• isotonic agents for example, sugars or sodium chloride.
• Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.
• Sterile injectable solutions are prepared by incorporating the AAV vector in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other inert ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and the freeze drying technique which yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
• dilute sterile, aqueous solutions for purposes of topical administration, dilute sterile, aqueous solutions, usually in about 0.1% to 5%, or greater as needed, concentration, otherwise similar to the above parenteral solutions, are prepared in containers suitable for delivery by a transdermal patch, and can include known carriers, such as pharmaceutical grade dimethylsulfoxide (DMSO) .
• DMSO dimethylsulfoxide
• topical preparations such as ointments, creams, washes, drops, sprays and the like can be exercised as known in the art for preparing such forms for use at the dermal surface or as drops and sprays for instillation, for example in the eye or nose.
• topical is meant to indicate any local or particular readily accessible locale of the host, for application of the invention of interest.
• ointments and creams to the surface of the skin, drops or ointment for instillation, for example, in the ear or nose, sprays or mists for instillation in the nose or mouth, suppositories and the like are contemplated to fall within the scope of the instant invention.
• Topical administration may be coupled with slight abrasion of the skin to enable ready access past the keratinized epidermis to the der is, underlying connective tissue, muscle, tissue spaces and the circulatory system.
• the therapeutic compounds of the instant invention may be administered to a mammal alone or in combination with pharmaceutically acceptable carriers.
• pharmaceutically acceptable carriers As noted above, the relative proportions of active ingredient and carrier are determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
• the rAAV particles can be encapsulated in microvessels, such as liposomes, microcapsules, microbeads and the like.
• Standard pharmaceutic manufacturing methods can be used to make the microcapsules and microbeads.
• standard methods for making liposomes and other such lipid membrane structures encircling an aqueous core can be made practicing methods known in the art.
• the dosage of the instant therapeutic agents which will be most suitable for prophylaxis or treatment will vary with the form of administration, the particular recombinant AAV vector chosen and the physiological characteristics of the particular patient under treatment. Generally, small dosages will be used initially and, if necessary, will be increased by small increments until the optimum effect under the circumstances is reached. Exemplary dosages for humans includes within the range of IO 8 up to approximately 5 x IO 14 particles in a total volume of up to about 3-10 ml, subcutaneously injected in aliquots into different sites on the human body, including the thighs, arms and back.
• AAV has in the past been shown to have a broad host range and now has been demonstrated to be operable via subcutaneous injection, there are no known limits on the hosts in which the herein described methods of delivery can take place, particularly in mammals, birds, fish and reptiles, especially domesticated mammals and birds, such as cattle, sheep, pigs, horses, dogs, cats chickens and turkeys. Both human and veterinary uses are preferred.
• the gene being expressed can be either a DNA segment encoding a protein, with whatever control elements (e.g., promoters, operators) are desired by the user, or a non-coding DNA segment, the transcription of which produces all or part of some RNA-containing molecule, such as a ribozyme or an anti-sense molecule. Since the spirit of the instant invention is directed to a route of delivery and to the vector rather than to the material being delivered, there are no limitations on the foreign DNA (non-AAV DNA) being delivered by the vector.
• the gene need not be limited to those strictly useful in the subcutaneous or topical region of administration since the host vascular system will deliver the gene product to other parts of the body. Also, the AAV particles themselves may enter the circulatory system and be transported to sites distal from the point of administration.
• Adenovirus vectors can infect the non-dividing cells and therefore, can be delivered directly into the mature tissues, such as muscle.
• the transgenes delivered by adenovirus vectors are not useful to maintain long term expression. Since adenovirus vectors retain most of the viral genes, those vectors pose potential problems, i.e. safety. The expression of those genes can cause the immune system to destroy the cells containing the vectors (see, for example, Yang et al. 1994, Proc. Natl Acad. Sci. 91:4407-4411) . Thus, an adenovirus vector cannot be delivered repeatedly. Also, because adenovirus is not an integration virus, the DNA eventually will be diluted or degraded in the cells.
• retrovirus vectors achieve stable integration into the host chromosomes, the use thereof is restricted because currently-used vectors only infect dividing cells whereas a large majority of target cells are non-dividing.
• Adeno-associated virus vectors have certain advantages over the above-mentioned vector systems. First, like adenovirus, AAV efficiently can infect non-dividing cells. Second, all the AAV viral genes are eliminated in the vector. Since the viral gene expression-induced immune reaction is no longer a concern, AAV vectors are safer than adenovirus vectors. Third, AAV is an integration virus by nature and integration into the host chromosome will maintain the transgene in the cells. Fourth, AAV is an extremely stable virus which is resistant to many detergents, pH changes and heat (stable at 56 °C for more than an hour) . AAV also can be lyophilized and redissolved without losing significant activity. Finally, AAV causes no known diseases or pathogenic symptoms in humans. Therefore, it is a very promising delivery vehicle for gene therapy.
• EXAMPLE 1 RECOMBINANT AAV VECTOR: rAAV-MFG-HUMAN- FACTOR IX.
• the recombinant AAV vector for expressing human Factor IX contains an MFG promoter, which is derived from the murine Moloney virus long terminal repeat, the entire human Factor IX cDNA and bGH pA, which is the bovine growth hormone polyadenylation site, see FIG. l.
• MFG promoter which is derived from the murine Moloney virus long terminal repeat, the entire human Factor IX cDNA and bGH pA, which is the bovine growth hormone polyadenylation site, see FIG. l.
• SSV9-MFG-ShF9 huFIX
• the recombinant AAV vector for expressing human interferon contains an MD promoter, which is derived from the Cytomegalovirus immediate early promoter, the entire human interferon cDNA and a ⁇ -globin polyadenylation site.
• Plasmid SSV9-MD AAV (pSSV9/MD-2) (FIG. 20) was constructed by subcloning a 2.35 kbp Xbal fragment from pMDG (Naldini et al. (1996) Science 272:263-267).
• PSSV922 MD-2 contains the Cytomegalovirus (CMV) immediate early gene promoter/enhancer (nucleotide positions 195-993), exon 2 and 3, intervening sequence 2 (IVS2) (nucleotide position 1036-1561) and a polyadenylation signal (nucleotide positions 1573-2355) derived from the human j8-globin gene.
• CMV Cytomegalovirus
• IVS2 intervening sequence 2
• polyadenylation signal (nucleotide positions 1573-2355) derived from the human j8-globin gene.
• the fragment was modified by PCR to contain restriction sites for Pmll, EcoRl and Bglll at nucleotide positions 1562, 1567, and 1573, respectively, for subcloning of selected transgenes.
• pSSV9-MD-hu alpha interferon (huAIFN) (FIG. 22) was constructed by insertion of the huIFN sequence between the Pmll and Bglll cloning sites of pSSV9-MD2 (FIG. 20) .
• the huIFN cDNA also was generated by PCR using multiple overlapping oligonucleotides (FIG. 22) .
• EPO contains an MD promoter, derived from the cytomegalovirus immediate early promoter, the entire mouse erythropoietin cDNA and a /3-globin polyadenylation site (FIG. 3) .
• pSSV9-MD2-murine erythropoietin (muEPO) (FIG. 21) was constructed by insertion of the muEPO cDNA between the Pmll and Bglll cloning sites of pSSV9-MD-2 (FIG. 20) . In that construct, the muEPO gene is flanked by the CMV promoter, /3-globin intron and the /3-globin poly (A) .
• the 579 base pair muEPO coding region was generated by PCR using multiple overlapping oligonucleotides (Dillion & Rosen (1990) Biotechniques 9:298-299) and contained compatible Pmll and Bglll sites for subcloning into the vector backbone.
• the rAAV-CMV-LacZ construct see FIG. 4, is known as pdx-31-LacZ and is described in McCown et al. (1996) Brain Res. 713:99-107.
• the recombinant AAV vectors were packaged as described by R. Snyder et al., "Production of recombinant adeno-associated viral vectors," in Current Protocols in Human Genetics, pp. 12.1.1- 12.1.24, N. Dracopoli et al., eds. (John Wiley & Sons, New York, 1996) .
• Recombinant AAV-MFG-Human Factor IX was administered subcutaneously as a single injection of either 5.9 x 10 10 , 5.9 x 10 9 or 5.9 x 10 8 particles in a volume of 300 microliters of Hanks' Balanced Salt Solution (HBSS) on the dorsal side of BALB/c mice (each dose of recombinant AAV vector was administered to a group of five mice) .
• Mice were bled using heparin-coated capillary tubes. Blood was collected into EDTA-coated tubes at the indicated number of days after subcutaneous injection depicted in FIGS. 5, 6 and 7, and stored frozen.
• human Factor IX The expression of human Factor IX was determined by ELISA using monoclonal or polyclonal antibodies for human Factor IX (monoclonal anti-factor IX antibody from Boehringer Mannheim, Cat. # 1199 277, rabbit anti-human factor IX from DAKO, Cat. # A300 and Asserachrom® IX:Ag test kit, Cat. # 00564) . Individual animals expressing human Factor IX at detectable levels are identified by number: 100, 108, 202, 204, 208 and 306.
• FIGS. 8A and 8B Animal numbers correspond to the numbers indicated in FIG. 5, 6 and 7. Animal
• 400 is a control animal injected with HBSS.
• the presence of antibodies to the AAV capsid proteins was determined by an ELISA as follows: 1 x 10 10 functional rAAV-LacZ purified virions (-5 x 10 12 total particles) in 100 ⁇ l were used to coat the wells of 96-well plates for 16 hours. The unbound virions were washed from the plate, the plate was blocked and incubated with 200 ⁇ l of 1:50, 1:800 and 1:600 dilutions of mouse serum for 2 hours. The plate was developed using an anti-mouse horseradish peroxidase-conjugated antibody and 1 mg/ml O-phenylenediamine dihydrochloride substrate (Sigma) in 7.5% H 2 0 2 . Titers were calculated by determining the serum dilution at which the OD value was half-maximal.
• Titers of antibody of hFIX were determined by an ELISA on selected days postinjection. Animals that expressed detectable levels of hFIX for the duration of the experiment are indicated by the plus (+) sign.
• EXAMPLE 8 COMPARISON OF LONG-TERM GENE EXPRESSION IN IMMUNOCOMPETENT AND IMMUNOCOMPROMISED MICE.
• rAAV-MFG-human Factor IX was administered subcutaneously once as an injection of either 5.9 x 10 10 , 5.9 x 10 9 or 5.9 x 10 8 particles in a volume of 300 microliters of HBSS on the dorsal side of BALB/c or NIH-3 mice (each dose of vector was administered to a group of five mice) . Mice were bled using heparin-coated capillary tubes. The blood was collected into EDTA-coated tubes at the time points indicated in FIGS. 9A and 9B and stored frozen.
• human Factor IX The expression of human Factor IX was determined by ELISA, and the results are shown in FIGS. 9A and 9B. Individual BALB/c animals expressing human Factor IX at detectable levels are identified by number: 103, 104 and 204. The expression of human Factor IX for immunocompetent mice correlated positively to the amount of recombinant AAV vector administered.
• EXAMPLE 10 LONG-TERM EXPRESSION OF HUMAN INTERFERON IN IMMUNOCOMPETENT AND IMMUNOCOMPROMISED MICE.
• rAAV-MD-human interferon was administered subcutaneously once as a single injection of either 8.5 x 10 10 , 8.5 x 10 9 or 8.5 x 10 8 particles in a volume of 300 microliters of HBSS on the dorsal side of BALB/c or NIH-3 mice (each dose of vector was administered to a group of five mice) . Mice were bled using heparin-coated capillary tubes. Blood was collected into EDTA-coated tubes at the time points indicated in FIGS. 11A and 11B and stored frozen. The expression of human interferon was determined by ELISA.
• EXAMPLE 11 EXPRESSION OF MOUSE ERYTHROPOIETIN IN IMMUNOCOMPETENT BALB/c MICE.
• rAAV-MD-mouse-erythropoietin was administered subcutaneously once as a single injection of either 8.9 x 10 10 or 8.9 x 10 9 particles in a volume of 100 microliters of HBSS on the dorsal side of BALB/c mice (each dose of vector was administered to a group of six or eight mice) . Mice were bled at the time points indicated on FIG. 12 and the hematocrit was determined.
• Epo protein levels were determined at day 105 for mice in FIG. 12. Epo protein levels were determined by radioimmunoassay (RIA), see FIG. 13.
• EXAMPLE 13 COMPARISON BETWEEN INTRAMUSCULAR AND SUBCUTANEOUS EXPRESSION OF HOUSE ERYTHROPOIETIN IN IMMUNOCOMPETENT MICE.
• rAAV-MD-mouse erythropoietin was administered subcutaneously once as a single injection of either 8.9 x IO 10 , 8.9 x IO 9 or 8.9 x IO 8 particles in a volume of 100 microliters of HBSS on the dorsal side of BALB/c mice or intramuscularly as a single injection of either 8.9 x 10 10 , 8.9 x 10 9 or 8.9 x 10 8 particles in a volume of 30 microliters of HBSS to the quadriceps of BALB/c mice (each dose of vector was administered to a group of three mice) . Mice were bled at the time points indicated in FIGS. 14A and 14B and the hematocrit was measured.
• Epo protein levels were determined at day 180 for mice in FIGS. 14A and 14B. Epo protein levels were determined by RIA, see FIG. 15.
• EXAMPLE IS. EXPRESSION OF HUMAN INTERFERON ADMINISTERED INTRAVENOUSLY IN IMMUNOCOMPETENT MICE.
• rAAV-MD-human interferon (8.5 x 10 10 particles) was administered intravenously as a single injection in a volume of 100 microliters of HBSS into the tail vein of BALB/c (the dose of vector was administered to a group of five mice) . Mice were bled using heparin-coated capillary tubes. Blood was collected into EDTA-coated tubes at the indicated times and stored frozen. The expression of human interferon was determined by ELISA (Endogen) and the results are shown in FIG . 16.
• EXAMPLE 16 EXPRESSION OF HUMAN FACTOR IX ADMINISTERED TO THE MUSCLE IN IMMUNOCOMPETENT MICE. rAAV-MFG-human Factor IX (2.5 x 10 10 particles) was administered intramuscularly as a single injection in a volume of 50 microliters of Hanks' Balanced Salt
• mice the dose of vector was administered to a group of five mice. Mice were bled using heparin-coated capillary tubes. The blood was collected into
• Recombinant AAV-CMV-LacZ (6.9 x 10 7 functional particles) was administered subcutaneously as a single injection in a volume of 300 ⁇ l PBS on the dorsal side of four BALB/c mice.
• Two weeks and six weeks after injection two mice from the rAAV-CMV-LacZ group and one mouse from the PBS control group were euthanized and skin samples, muscle samples, inguinal lymph nodes, inguinal fat pads and spleen were harvested for histology.
• the skin samples were obtained by raising the skin at the injection site with forceps, forming a tent as described for injection and cutting around the base of the tent of skin.
• the muscle samples were collected just lateral on both sides of the dorsal midline in the lumbar region. Inguinal lymph nodes and fat pads were collected from both the left and right side of each mouse.
• tissue samples were flash frozen, cut into 10 ⁇ m sections and collected onto glass slides. The sections were fixed with 0.5% glutaraldehyde for 10 min., washed with PBS and stained with X-gal (5-bromo-4-chloro-3-indolyl- ⁇ - D-galactopyranoside) using standard procedures. The sections then were washed with PBS and counterstained with nuclear fast red.
• ⁇ -galactosidase protein activity was detected in the skin tissue, such as, in the panniculus carnosus, a skeletal muscle layer located in the skin, see FIG. 19. ⁇ -galactosidase activity was not present in non-injected animals, see FIG. 18.

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