Link:  Pharm/Biotech Resources

Title:  Adiponectin gene therapy

United States Patent:  6,967,018

Issued:  November 22, 2005

Inventors:  Zolotukhin; Sergei (Gainesville, FL); Tennant; Michael D. (Seattle, WA)

Assignee:  Applied Genetic Technologies Corporation (Alachua, FL)

Appl. No.:  341972

Filed:  January 13, 2003

Adiponectin cDNA was cloned into AAV serotypes 1, 2, and 5-based expression vectors. Virions containing these vectors were administered to the livers of rat subjects via portal vein injection. A single injection of 6×10¹¹ virions of the vector caused a sustained and statistically significant reduction in body weight of the treated animals compared to the control animals. This occurred in the absence of side effects. Compared to control animals, the subject rats also exhibited reduced adipose tissue mass, reduced appetite, improved insulin sensitivity, and improved glucose tolerance.

SUMMARY OF THE INVENTION

The invention relates to methods and compositions for modulating adiponectin nucleic acid and/or protein levels in a subject. In the experiments described below, adiponectin cDNA was cloned into AAV serotypes 1, 2, and 5-based expression vectors (i.e., rAAV-Acrp30). Virions containing these vectors were administered to the livers of rat subjects via portal vein injection. A single injection of 6×10¹¹ virions of the vector caused a sustained and statistically significant reduction in body weight of the treated animals compared to the control animals. This occurred in the absence of side effects. Compared to control animals, the subject rats also exhibited reduced adipose tissue mass, reduced appetite, improved insulin sensitivity, and improved glucose tolerance.

Accordingly, the invention features a nucleic acid including a first AAV terminal repeat (TR); a second AAV TR; and interposed between the first and second AAV TRs, a nucleotide sequence that encodes at least a portion of an adiponectin protein that has at least one functional activity of native adiponectin. The nucleotide sequence can be one derived from a mammal such as a mouse, rat, or human. It can also be one that encodes a full-length adiponectin protein or a fragment thereof (e.g., the active globular domain of adiponectin). The TRs can be derived from a number of different serotypes. For example, one of the TRs can be derived from AAV serotypes 1, 2, or 5.

In variations of the invention, the nucleotide sequence can further include an expression control sequence. The expression control sequence can be one that effects tissue-specific (e.g., liver-specific or muscle specific) expression of the nucleotide sequence. It can be, for example, a chicken β-actin promoter or a cytomegalovirus enhancer operably linked to the nucleotide sequence. The nucleic acid can be included within a cell and/or included within an AAV virion.

In another aspect, the invention features a method of modulating adiponectin protein levels in a subject. This method includes the step of administering a nucleic acid of the invention into the subject. In variations of the methods of the invention, the modulation of adiponectin protein levels results in a reduction of weight gain, an increase in insulin sensitivity, an increase in glucose tolerance, and/or a reduction of appetite in the subject. In the method of the invention, the nucleic acid can be administered to the subject by intravenous or intramuscular injection.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions for modulating adiponectin protein and nucleic acid levels in a subject. In the experiments described below, adiponectin nucleic acids and proteins were delivered to rats using a rAAV vector. Compared to control rats, the subject rats exhibited reduced adipose tissue mass, reduced appetite, improved insulin sensitivity, and improved glucose tolerance.

The below described preferred embodiments illustrate adaptations of these compositions and methods. Nonetheless, from the description of these embodiments, other aspects of the invention can be made and/or practiced based on the description provided below.

Biological Methods

Methods involving conventional molecular biology techniques are described herein. Such techniques are generally known in the art and are described in detail in methodology treatises such as Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22:1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc., 103:3185, 1981. Chemical synthesis of nucleic acids can be performed, for example, on commercial automated oligonucleotide synthesizers. Immunological methods (e.g., preparation of antigen-specific antibodies, immunoprecipitation, and immunoblotting) are described, e.g., in Current Protocols in Immunology, ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992. Conventional methods of gene transfer and gene therapy are described in, e.g., Gene Therapy: Principles and Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins, Humana Press, 1997; and Retro-vectors for Human Gene Therapy, ed. C. P. Hodgson, Springer Verlag, 1996.

AAV Vectors

The invention utilizes rAAV vectors and virions for delivering an adiponectin-encoding nucleic acid to a subject. AAV is an attractive vector system for human gene therapy because it is non-pathogenic for humans, it has a high frequency of integration, and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells both in tissue culture and in whole animals. Muzyczka, Curr. Top. Microbiol. Immunol., 158:97-129, 1992.

AAV is a helper-dependent parvovirus in that it requires coinfection with another virus (either adenovirus [Ad] or a member of the herpes virus family) to undergo a productive infection in cultured cells. Muzyczka, N., Curr. Top. Microbiol. Immunol., 158:97-129, 1992. In the absence of coinfection with helper virus, AAV establishes a latent state by insertion of its genome into human chromosome 19, where it resides in a latent state as a provirus. Kotin et al., Proc. Natl. Acad. Sci. USA, 87:2211-2215, 1990; Samulski et al., EMBO J. 10:3941-3950, 1991. When a cell carrying an AAV provirus is superinfected with a helper virus, the AAV genome undergoes rescue and proceeds through a normal productive infection. Samulski et al., Cell, 33:135-143, 1983; McLaughlin et al., J. Virol., 62:1963-1973, 1988; Kotin et al., Proc. Natl. Acad. Sci. USA, 87:2211-2215, 1990; Muzyczka, N., Curr. Top. Microbiol. Immunol., 158:97-129, 1992.

Recent studies have demonstrated AAV to be a potentially useful vector for gene delivery. LaFace et al., Viology., 162:483-486, 1998; Zhou et al., Exp. Hematol. (NY), 21:928-933, 1993; Flotte et al., PNAS 90:10613-10617, 1993; and Walsh et al., Blood 84:1492-1500, 1994. Recombinant AAV vectors have been used successfully for in vitro and in vivo transduction of marker genes (Kaplitt et al., Nature Genetics, 8:148-154, 1994; Lebkowski et al., Mol. Cell. Biol. 8:3988-3996, 1988; Samulski et al., J. Virol., 63:3822-3828, 1989; Shelling, A. N., and Smith, M. G., Gene Therapy, 1:165-169, 1994; Yoder et al., Blood, 82: suppl. 1:347A, 1994; Zhou et al., J. Exp. Med., 179:1867-1875, 1994; Hermonat, P. L. and Muzyczka, N., Proc. Nalt. Acad. Sci. USA., 81:6466-6470, 1984; Tratschin et al., Mol. Cell. Biol., 4:2072-2081, 1984; McLaughlin et al., J. Virol., 62:1963-1973, 1988) as well as genes involved in human diseases (Flotte et al., Am. J. Respir. Cell Mol. Biol., 7:349-356, 1992; Luo et al., Blood, 82:suppl. 1,303A, 1994; Ohi et al., Gene, 89L:27914 282, 1990; Walsh et al., PNAS 89:7257-7261, 1992; Wei et al., Gene Therapy, 1:261-268, 1994). Recently, an AAV vector has been approved for phase I human trials for the treatment of cystic fibrosis.

Typically, rAAV virus (virions) is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV TRs (McLaughlin et al., J. Virol., 62:1963-1973, 1988; Samulski et al., J. Virol., 63:3822-3828, 1989) and an expression vector containing the WT AAV coding sequences without the TRs, for example pIM45. McCarty et al., J. Virol., 65:2936-2945, 1991. The cells are also infected or transfected with Ad or plasmids carrying the Ad genes required for AAV helper function. rAAV virus (virion) stocks made in this fashion are contaminated with Ad which must be physically separated from the rAAV particles (for example, by cesium chloride density centrifugation). Alternatively, Ad vectors containing the AAV coding regions or cell lines containing the AAV coding regions and some or all of the Ad helper genes could be used. Yang et al., J. Virol., 68:4847-4856, 1994; Clark et al., Human Gene Therapy, 6:1329-1341, 1995. Cell lines carrying the rAAV DNA as an integrated provirus can also be used (Flotte et al., Gene Therapy, 2:29-37, 1995) in the production of infectious virions.

An rAAV vector of the invention is a recombinant AAV-derived nucleic acid sequence that includes at least those AAV sequences required in cis for replication and packaging (e.g., functional TRs) of the virus. In some applications, rAAV vectors contain a non-AAV nucleic acid. Non-AAV nucleic acids include, for example, marker or reporter genes (e.g., a nucleic acid encoding green fluorescent protein). Non-AAV nucleic acids also include, for example, therapeutic genes (e.g., an adiponectin gene). Examples of useful rAAV vectors are those that have one or more AAV WT genes deleted in whole or in part, but retain functional flanking ITR sequences. Other useful rAAV vectors include those that contain rep and cap genes. rAAV vectors can be derived from any AAV serotype, including 1, 2, 3, 4, 5, 6, and 7.

AAV Serotypes 1, 2 and 5

The rAAV vectors and virions of the invention may be derived from any of several AAV serotypes. To develop optimized vectors for the delivery of therapeutic genes including adiponectin, rAAV vectors derived from AAV serotypes other than type 2 have been constructed. A new generation of rAAV vectors, based on different serotypes, with altered biodistribution and level of transgene product, has emerged as an alternative and more efficacious platform for gene delivery. Serotype-based AAV vectors have been shown to mediate transgene expression up to several logs higher compared to AAV-2. Halbert et al., J Virol, 75:6615-24, 2001; Chao et al., Mol Ther, 2:619-23, 2000; Duan et al., J Virol, 75:7662-71 2001; Zabner et al., J Virol, 74:3852-8, 2000. Research suggests that rAAV-1 vectors exhibit higher transgene expression in muscle compared to rAAV-2 vectors, and that rAAV-5 vectors exhibit higher transgene expression in hepatocytes (liver) compared to rAAV-2 vectors.

Because the site of action of adiponectin has yet to be determined (although early research suggests that adiponectin may act in either the liver or muscle), an adiponectin expression cassette has been packaged into three different capsid serotypes: 1, 2 and 5. This set of vectors may be tailored to a particular route of administration for the most efficacious expression of adiponectin in target tissues, namely liver and muscle. Accordingly, the increased transduction capabilities of AAV vectors 1 and 5 may be exploited to achieve higher expression of adiponectin in muscle and liver, respectively. Techniques involving nucleic acids and capsids of different AAV serotypes are known in the art and are described in Halbert et al., J. Virol., 74:1524-1532, 2000; Halbert et al., J. Virol., 75:6615-6624, 2001; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001.

Pseudotyped Vectors

Another aspect of the invention relates to the administration of pseudotyped rAAV-Acrp30 vectors to a subject for controlling weight gain, glucose tolerance and insulin sensitivity, as well as appetite. Pseudotyped rAAV virions contain an rAAV vector derived from a particular serotype that is encapsidated within a capsid containing proteins of another serotype. Vectors of the invention include AAV2 vectors pseudotyped with a capsid gene derived from an AAV serotype other than 2 (e.g., AAV1, AAV3, AAV4, AAV5, AAV6 or AAV7 capsids). For example, particularly preferred vectors of the invention are AAV2 vectors encoding Acrp30 pseudotyped with a capsid gene derived from AAV serotypes 1 or 5. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art and are described in Duan et al., J. Virol., 75:7662-7671, 2001; Halbert et al., J. Virol., 74:1524-1532, 2000; and Zolotukhin et al., Methods, 28:158-167, 2002.

rAAV Mutants

The invention provides methods for modulating adiponectin protein levels (e.g., for regulating weight gain, insulin sensitivity and glucose tolerance) by administering rAAV-Acrp30 vector-containing virions that have mutations within the virion capsid. For example, suitable rAAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types (e.g., hepatocytes). The construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol., 74:8635-45, 2000. Pseudotyped rAAV virions that have mutations within the capsid may also be produced and purified according to methods of the invention. Techniques involving nucleic acids and viruses of different AAV serotypes are known in the art and are described in Halbert et al., J. Virol., 74:1524-1532, 2000; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001. Other rAAV virions that can be used in methods of the invention include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See Soong et al., Nat. Genet., 25:436-439, 2000; and Kolman and Stemmer Nat. Biotechnol., 19:423-428, 2001.

Nucleic Acids

The invention provides nucleic acids (polynucleotides) that include (1) a first AAV TR, (2) a second AAV TR, and interposed between the first and second AAV TRs, (3) a nucleotide sequence that encodes at least a portion of an adiponectin protein that has at least one functional activity of a native adiponectin ("adiponectin-encoding nucleotide sequence").

The AAV TR sequences that are contained within the nucleic acid can be derived from any AAV serotype (e.g., 1, 2, 3, 4, 5, 6 and 7) or can be derived from more than one serotype. For use in a vector, the first and second TRs should include at least the minimum portions of a WT or engineered TR that are necessary for packaging and replication.

The adiponectin-encoding nucleotide sequence can take many different forms. For example, the sequence may be a native mammalian adiponectin nucleotide sequence such as the mouse or human adiponectin nucleotide sequences deposited with Genbank as accession nos. AF304466/NM009605 and AF304467, respectively. Other native mammalian adiponectin nucleotide sequences that may be used within the invention include rat (accession nos. NM_—144744, AY033885), canine (accession no. AF417206), and rhesus macaque (accession no. AF404407) nucleotide sequences.

The adiponectin-encoding nucleotide sequence may also be a non-native coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as does a native mammalian adiponectin nucleotide sequence. Other adiponectin-encoding nucleotide sequences within the invention are those that encode fragments, analogs and derivatives of a native adiponectin protein. Such variants may be, e.g., a naturally occurring allelic variant of a native adiponectin-encoding nucleic acid, a homolog of a native adiponectin-encoding nucleic acid, or a non-naturally occurring variant of native adiponectin-encoding nucleic acid. These variants have a nucleotide sequence that differs from native adiponectin-encoding nucleic acid in one or more bases. For example, the nucleotide sequence of such variants can feature a deletion, addition, or substitution of one or more nucleotides of a native adiponectin encoding nucleic acid. Nucleic acid insertions are preferably of about 1 to 10 contiguous nucleotides, and deletions are preferably of about 1 to 30 contiguous nucleotides.

In some applications, variant adiponectin-encoding nucleotide sequences encode polypeptides that substantially maintain an adiponectin protein functional activity. For other applications, such variants encode polypeptides that lack or feature a significant reduction in an adiponectin protein functional activity. Where it is desired to retain a functional activity of native adiponectin protein, preferred variant nucleotide sequences feature silent or conservative nucleotide changes.

In other applications, variant adiponectin polypeptides displaying substantial changes in one or more functional activities of native adiponectin protein can be generated by making nucleotide substitutions that cause less than conservative changes in the encoded polypeptide. Examples of such nucleotide substitutions are those that cause changes in (a) the structure of the polypeptide backbone; (b) the charge or hydrophobicity of the polypeptide; or (c) the bulk of an amino acid side chain. Nucleotide substitutions generally expected to produce the greatest changes in protein properties are those that cause non-conservative changes in codons. Examples of codon changes that are likely to cause major changes in protein structure are those that cause substitution of (a) a hydrophilic residue, e.g., serine or threonine, for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histadine, for (or by) an electronegative residue, e.g., glutamine or aspartine; or (d) a residue having a bulky side chain, e.g., phenylalanine, for (or by) one not having a side chain, e.g., glycine.

Naturally occurring allelic variants of a native adiponectin-encoding nucleic acid within the invention are nucleotide sequences isolated from a mammalian subject (e.g., human, mouse, rat, dog, and macaque) that have at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with a native adiponectin-encoding nucleic acid, and encode polypeptides having as at least one functional activity in common with a native adiponectin protein. Homologs of a native adiponectin-encoding nucleic acid within the invention are nucleotide sequences isolated from other species that have at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with a native adiponectin-encoding nucleic acid, and encode polypeptides having at least one functional activity in common with a native adiponectin protein.

Non-naturally occurring adiponectin-encoding nucleic acid variants are nucleotide sequences that do not occur in nature (e.g., are made by the hand of man), have at least 75% (e.g., 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%) sequence identity with a native adiponectin-encoding nucleic acid, and encode polypeptides having at least one functional activity in common with a native adiponectin protein. Examples of non-naturally occurring adiponectin nucleotide sequences are those that encode a fragment of an adiponectin protein, those that hybridize to a native adiponectin-encoding nucleic acid or a complement of a native adiponectin-encoding nucleic acid under stringent conditions, those that share at least 65% sequence identity with a native adiponectin-encoding nucleic acid or a complement of a native adiponectin encoding nucleic acid, and those that encode an adiponectin fusion protein.

Nucleotide sequences encoding fragments of adiponectin protein are those that encode, e.g., 2, 5, 10, 25, 50, 100, 150, 200, or more amino acid residues of an adiponectin protein. A particularly useful fragment or portion of an adiponectin protein is the active globular domain of adiponectin (e.g., amino acids 110-247 or 104-247 of murine adiponectin). See Philipp et al., J. Biol. Chem. 270:26746-26749, 1995; and Fruebis et al., PNAS 98:2005-2010, 2001. The adiponectin-encoding nucleotide sequence can also be one that encodes an adiponectin fusion protein. Such a sequence can be made by ligating a first polynucleotide encoding an adiponectin protein fused in frame with a second polynucleotide encoding another protein (e.g., one that encodes a detectable label).

Expression Control Sequences

In addition to the AAV TRs and the adiponectin-encoding nucleotide sequence, the nucleic acids of the invention can also include one or more expression control sequences operatively linked to the adiponectin-encoding nucleotide sequence. Numerous such sequences are known. Those to be included in the nucleic acids of the invention can be selected based on their known function in other applications. Examples of expression control sequences include promoters, insulators, silencers, enhancers, initiation sites, termination signals, and polyA tails.

To achieve appropriate levels of adiponectin proteins, any of a number of promoters suitable for use in the selected host cell may be employed. For example, constitutive promoters of different strengths can be used to express adiponectin proteins.

Expression vectors and plasmids in accordance with the present invention may include one or more constitutive promoters, such as viral promoters or promoters from mammalian genes that are generally active in promoting transcription. Examples of constitutive viral promoters include the Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A and CMV promoters. Examples of constitutive mammalian promoters include various housekeeping gene promoters, as exemplified by the β-actin promoter.

Inducible promoters and/or regulatory elements may also be contemplated for use with the nucleic acids of the invention. Examples of suitable inducible promoters include promoters from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone-inducible genes, such as the estrogen gene promoter. Another example of an inducible promoter is the tetVP16 promoter that is responsive to tetracycline. The sterol regulatory-binding protein (SREBP-1c) promoter is another inducible promoter useful within the invention (Deng et al., Biochem. Biphys. Res. Commun 290:256-262, 2002; Kim et al., J. Clin. Invest. 101:1-9, 1998; and Azzout-Marniche et al., Biochem. J. 350 Pt 2:389-393, 2000). Expression of the SREBP-1c gene is induced by nutritional stimuli, in particular by insulin and glucose.

A rAAV vector has been constructed that encodes mouse Acrp30 under the control of a rat SREPB-1c promoter. The presence of this vector in a mammalian subject is expected to up-regulate the expression of adiponectin by hyperinsulinemia induced by overnutrition (a condition known to be associated with low serum adiponectin levels).

Tissue-specific promoters and/or regulatory elements are useful in certain embodiments of the invention. Examples of such promoters that may be used with the expression vectors of the invention include promoters from the transphyretin, α1-antitrypsin, plasminogen activator inhibitor type 1 (PAI-1), apolipoprotein AI and LDL receptor genes, specific for liver cells; and the utrophin promoter A and human dystrophin muscle specific promoters, specific for muscle cells.

The promoter used to express the adiponectin gene is not critical to the present invention. In the examples given, the human chicken β-actin promoter has been used (Miyazaki et al., Gene, 79:269-77, 1989) which results in the sustained, high-level expression of the foreign gene. However, the use of other promoters (such as viral, mammalian or cellular promoters) which are well known in the art, is also suitable to achieve expression of the adiponectin gene. Preferably the promoter will direct expression of an adiponectin-encoding nucleic acid in an amount sufficient to reduce body weight gain and appetite as well as increase insulin sensitivity in a subject.

Modulating Adiponectin Protein Levels in a Subject

The invention provides compositions and methods for modulating adiponectin protein levels in a subject using an rAAV vector. The method includes the step of administering to the subject a nucleic acid containing a first AAV TR, a second AAV TR, and a nucleotide sequence that encodes at least a portion of an adiponectin protein that has at least one functional activity of native adiponectin ("an AAV-adiponectin vector"). The subject can be animal into which a nucleic acid of the invention can be administered. In general, animals that typically express adiponectin such as mammals (e.g., human beings, dogs, cats, pigs, sheep, mice, rats, rabbits, cattle, goats, etc.) are suitable subjects.

Administration of rAAV-Acrp30 Compositions

Any suitable method for administering a nucleic acid to a subject may be used to administer an AAV-adiponectin nucleic acid, vector, or virion to the subject. For example, an nucleic acid, vector, or virion can be administered to a subject by parenteral administration (e.g., intravenous and/or intramuscular injection). An AAV-adiponectin nucleic acid, vector, or virion can be delivered to a particular site by known methods. For example, to deliver an AAV-adiponectin nucleic acid, vector, or virion to the liver (a preferred target), intravenous injection of the portal vein may be performed. Intracranial injection or injection into peripheral (non-CNS) sites may also be used in certain applications. The effectiveness of particular protocols can be assessed using conventional clinical assays, e.g., examining the subject's visceral fat pad, its ability to normalize circulating glucose levels, and measuring the sensitivity of the subject's hepatocytes to insulin.

The nucleic acids, vectors, and virions of the present invention can be administered to a subject by ex vivo delivery, where cells not contained within a subject (e.g., cells isolated from a mammalian subject such as hepatocytes) are transduced with AAV-adiponectin nucleic acid or vector or infected with a rAAV-adiponectin virion in vitro, and the cells are then introduced into the subject (e.g., transduced isolated cells are delivered to the liver). In one example of a suitable ex vivo protocol, liver cells (e.g., hepatocytes) may be harvested from the subject and transduced with AAV-adiponectin nucleic acid or vector or infected with a rAAV-adiponectin virion in vitro. These genetically modified cells may then be transplanted back into the subject. Modified liver cells may be reintroduced into the subject's liver by any suitable delivery route (e.g., intravenous delivery to the portal vein). Microencapsulation of cells transduced with AAV-adiponectin nucleic acid or vector or infected with a rAAV-adiponectin virion in vitro modified ex vivo is another technique that may be used within the invention. Delivery of an adiponectin-encoding nucleic acid may also involve methods of ex vivo gene transfer using stem cells and progenitor cells. Such methods involve the isolation and expansion of selected stem or progenitor cells, introduction of a therapeutic gene into the cells ex vivo, and return of the genetically modified cells to the host. Autologous and allogeneic cell transplantation may be used according to the invention.

Parenteral administration of vectors or virions by injection can be performed, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the vectors or virions may be in powder form (e.g., lyophilized) for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

To facilitate delivery of the vectors or virions to a subject, the vectors or virions of the invention can be mixed with a carrier or excipient. Suitable carriers and diluents can be selected on the basis of mode and route of administration and standard pharmaceutical practice. Carriers and excipients that might be used include saline (especially sterilized, pyrogen-free saline) saline buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. USP grade carriers and excipients are particularly preferred for delivery of vectors or virions to human subjects. A description of exemplary pharmaceutically acceptable carriers and diluents, pharmaceutical formulations, and methods for making such formulations can be found in, for example, Remington's Pharmaceutical Sciences (Remington: The Science and Practice of Pharmacy, 19th ed., A. R. Gennaro (ed), Mack Publishing Co., N.J., 1995).

Effective Doses

An effective amount is an amount which is capable of producing a desirable result in a treated subject (e.g., reduction of body weight gain, reduction of appetite, and increase of insulin sensitivity). As is well known in the medical and veterinary arts, dosage for any one mammal depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, time and route of administration, general health, and other drugs being administered concurrently. It is expected that a composition of rAAV-Acrp30 may be administered intravenously in the portal vasculature or intramuscularly in a dosage range of about 1×10 to 1×10¹³ viral particles. Although these doses are based on experiments with small mammals, one of skill in the art, without undue experimentation, could determine appropriate doses for use in human subjects by employing known principles of pharmacology. Dosage treatment may be a single dose schedule or a multiple dose schedule.
 

Claim 1 of 18 Claims

1. A method for increasing adiponectin protein levels in a mammal, comprising injecting a mammal in need thereof, a recombinant adeno-associated (rAAV) expression vector comprising a nucleic acid sequence that encodes full-length adiponectin polypeptide operably linked to an expression control sequence.

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