MYOSTATIN INHIBITION FOR ENHANCING MUSCLE AND/OR IMPROVING

MUSCLE FUNCTION

[0001] This invention was developed with partial government support under grant number NS052530 from the National Institutes of Health and Project A.L.S. The government may have certain rights in the invention.

Field of the Invention

[0002] The present invention relates to methods for inhibiting myostatin, a regulator of muscle mass, for enhancing muscle (including inducing hypertrophy and/or hyperplasia) as well as improving muscle function (including decreasing atrophy and/or increasing endurance, force and/or strength). Some of the methods involve delivering genes to cells using gene delivery or other delivery techniques known in the art in order to inhibit myostatin. Examples of genes to be delivered are genes encoding proteins such as Follistatin, Follistatin-related gene-1 (FLRG-I), growth differentiation factor associated protein- 1 (GASP-I) and myostatin precursor propeptide. The genes can be delivered using, for example, a recombinant Adeno-associated virus (rAAV), lentivirus or equine-associated virus capable of infecting the cells. Following introduction, the genes are expressed in the cell body of the infected cell and the encoded proteins are secreted systemically. In other methods of the invention, expression of proteins such as activin lib and myostatin is inhibited by oligonucleotide techniques to effect muscle enhancement and/or improve muscle function. All the methods have applications in the treatment of musculoskeletal and neurodegenerative disorders among others, as well as enhancing muscle in livestock.

Background

[0003] Molecular advances have provided greater understanding of skeletal muscle diseases, such as muscular dystrophy (MD), beginning with the discovery of the dystrophin gene and its gene product. There are nine types of MD, each a genetic degenerative disease primarily affecting voluntary muscles. Duchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. The progressive muscle weakness and degeneration usually lead to loss of ambulation and wheelchair dependency in the early teens. Death occurs anytime after age 18 due to respiratory infection usually complicated by cardiac failure. The disease is caused by mutations in the dystrophin gene, which encodes a large (427 kDa) cytoskeletal protein in

both skeletal and cardiac muscle. The dystrophin gene is the largest gene identified to date. It shows one of the highest sporadic mutation rates, with 1 of every 10,000 germ cells showing cle novo mutations. Thus, the high incidence (1/3500), de novo mutations, early morbidity and fatality, and the lack of effective treatment require urgency in the search for novel therapeutics. Since DMD was described more than 140 years ago, the life-span of the DMD patient has only been marginally prolonged and the quality of life may not have changed significantly improved despite tremendous advances in medicine. Few treatments have been added to the current repertoire. In the muscular dystrophies, only corticosteroids have altered the natural history of disease and indeed the mechanism of action of corticosteroids in DMD remains unknown. In fact, the known myopathic effects of steroids might predict a deleterious effect but instead there is a paradoxical response resulting in an increase in muscle mass contrasting with the effects in normal muscle. Unfortunately the benefit of steroids comes at a high cost in terms of side effects (bone loss, cataracts, delayed puberty, weight gain and hypertension) providing compelling reasons to find other approaches.

[0004] Amyotrophic Lateral Sclerosis (ALS) is another disease that results in loss of muscle and/or muscle function. First characterized by Charcot in 1869, it is a prevalent, adult-onset neurodegenerative disease affecting nearly 5 out of 100,000 individuals. ALS occurs when specific nerve cells in the brain and spinal cord that control voluntary movement gradually degenerate. Within two to five years after clinical onset, the loss of these motor neurons leads to progressive atrophy of skeletal muscles, which results in loss of muscular function resulting in paralysis, speech deficits, and death due to respiratory failure. The genetic defects that cause or predispose ALS onset are unknown, although missense mutations in the SOD-I gene occurs in approximately 10% of familial ALS cases, of which up to 20% have mutations in the gene encoding Cu/Zn superoxide dismutase (SODl), located on chromosome 21. SOD-I normally functions in the regulation of oxidative stress by conversion of free radical superoxide anions to hydrogen peroxide and molecular oxygen. To date, over 90 mutations have been identified spanning all exons of the SOD-I gene. Some of these mutations have been used to generate lines of transgenic mice expressing mutant human SOD-I to model the progressive motor neuron disease and pathogenesis of ALS.

[0005] Another musculoskeletal disorder, inclusion body myositis (IBM), was originally named by Yunis and Samaha in 1971 to describe a patient with a chronic inflammatory myopathy who had intranuclear and intracytoplasmic tubular filaments within muscle fibers on electron microscopy. Many clinical and pathologic studies over more than three decades

have supported this condition as a disorder distinct from other idiopathic inflammatory myopathies. Although incidence and prevalence statistics need further refinement, it is unequivocally the most common acquired muscle disease occurring after age of 50, with an estimated prevalence at 4-9:1,000,000. More men are affected than women by a ratio of greater than 2:1. Typically IBM is a sporadic disorder with insidious onset, and distinctive clinical and histopathological features (sIBM). Inflammation is prominent, helping to distinguish it from the group of inherited disorders (hIBM). These include autosomal recessive and dominant conditions with pathologic features resembling sIBM without inflammatory infiltration. Perhaps the best characterized hIBM is associated with mutations of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) [6].

[0006] The clinical features of sIBM include an average time of about 6 years from symptom onset to diagnosis. Difficulty with ambulation and frequent falls can be attributed to weak knee extensor muscles. Early involvement of the quadriceps and forearm flexor muscle compartment is typical. This is accompanied by a scooped-out appearance of the medial aspect of the forearms and thin, atrophic quadriceps muscles. Quadriceps atrophy and weakness is directly related to loss of safe ambulation. Frequent, sudden falls occur in three- fourths of cases and are the prime reason for wheelchair use. Only a minority are totally wheelchair dependent (-15%). Mean time between symptom onset and wheelchair use ranged from 13±8 (6-32) years. Clinical dysphagia is common with estimates reaching as high as 66%. Finger flexor weakness can be detected early in the course of illness. About 40% of patients have facial muscle weakness. In the only prospective study of decline of muscle strength, the investigators documented a 4% decline in 6 months (0.66% per month) based on quantitative myometry using maximal isometric contraction testing. A similar rate of decline was also found in another study using manual muscle testing. Natural history studies document a fairly consistent, slow rate of decline in most patients with sIBM.

[0007] Therapy for sIBM has focused on the underlying inflammatory response with either immunosuppressive or immunomodulating drags. Uncontrolled trials with corticosteroids, cyclophosphamide, chlorambucil, azathioprine and cyclosporin, and methotrexate fail to show convincing evidence of benefit. An initial pilot study of IVIG in IBM showed encouraging results but a subsequent randomized controlled trial with IVIG was negative. A prospective, double-blind, placebo-controlled, 6 month trial of weekly IM injections of 30 μg of β-interferon-la showed no benefit. A recently published open-label trial using etanercept showed increased hand grip strength without any other functional benefits. Considering the

complexity of pathogenic factors, it may be sometime before an effective treatment can target both autoimmune and myodegenerative factors. Attention has shifted to growth modulatory approaches. In a 3 month, randomized, placebo-controlled, crossover study of oxandrolone, a synthetic anabolic steroid, a borderline significant improvement in overall strength was reported, as measured by quantitative myometry. The study was limited by small sample size and short duration of treatment. Exercise is another means of growth modulation and this has been tried in inflammatory muscle diseases. An increase in isometric torque and an improvement in functional activities was reported in five patients with sIBM following a 12- week isotonic training program for the knee extensors and flexors and the elbow and wrist flexors. The small sample size disallows any conclusion but exercise had no deleterious effects based on muscle biopsies performed pre- and post-exercise.

[0008] Research has shown that skeletal muscle utilizes a regulatory mechanism to control tissue mass. In a screen for novel members of the transforming growth factor-β (TGF-β) superfamily of growth and differentiation factors, myostatin [previously called growth and differentiation factor-8 (GDF-8)] was identified and has subsequently been shown to be a negative regulator of muscle formation. Myostatin is expressed in the myotome compartment of developing somites at E9.5 with expression continuing throughout adulthood, predominantly in skeletal muscles and adipose tissue. Myostatin is synthesized in a precursor form that undergoes two proteolytic processing events to remove the N-terminal signal sequence and the C-terminal fragment, which possess receptor-binding activity. Following proteolytic processing, the propeptide and the disulfide linked C-terminal dimer remain bound noncovalently in a latent complex. Myostatin can be activated by dissociation of the propeptide after proteolytic cleavage by a metalloproteinase of the bone morphogenic (BMP)/tolloid family. The dissociated C-terminal fragment is thus the biologically active species. For a review of the biosynthesis and signaling pathway of myostatin, see Lee, Ann Rev Cell Dev Biol, 20: 61-86 (2004).

[0009] Myostatin is conserved among species, especially in its C-terminal fragment which is identical across human, rat, murine, porcine, turkey and chicken species. Mutations within myostatin have been shown to be linked to the double muscling phenotype in cattle [Grobet et al, Nat Genet, 17: 71-74 (1997); Kambadur et al, Genome Res, 7:910-915 (1997); and McPherron and Lee, Proc Natl Acad Sci USA, 94: 12457- 12461 (1997)] and gross muscle hypertrophy in human subjects [Schuelke et al, NEngJMed, 350: 2682-2688 (2004)]. Forced muscle atrophy has even been achieved with recombinant myostatin administration or

over-expression of myostatin [Zimmers et al, Science, 296(5572): 1486-1488 (2002)]. Histology of muscles from myostatin null mice shows increased muscle mass resulting from hyperplasia and hypertrophy of the muscle with less fat and connective tissues.

[0010] The hypothesis that it may be beneficial to block, remove, or reduce myostatin to promote regeneration and reduce fibrosis in MD has been explored in animal studies. Wagner et al, Ann Neurol, 52: 832-836 (2002) describes data obtained from crossing myostatin null mutant mice with mdx mice (which are models for dystrophin deficiency) showing that mdx mice lacking myostatin were stronger and more muscular than their mdx counterparts. In addition, Bogdanovich et al, Nature, 420: 418-421 (2002) report that when a neutralizing antibody to myostatin was administered to 4 week old mdx mice by intraperitoneal injection, an increase in body weight, muscle mass, muscle size and absolute muscle strength along with a significant decrease in muscle degeneration and concentrations of serum creatine kinase was observed. Similarly, Whittemore et al, Biochem Biophys Res Commun, 300: 965-971 (2003) describes that myostatin neutralizing antibodies increase muscle mass in adult mice. Tobin and Celeste, Curr Opin Pharma, 5: 328-332 (2005) reviews the myostatin pathway as well as studies testing the effects of reducing myostatin expression/activity.

[0011] Another review article, Wagner, Curr Opin Rheumatol, 17: 720-724 (2005), lists various therapeutic approaches of inhibiting myostatin that have been considered for treating human disease. For example, Wyeth has developed a humanized, anti-myostatin antibody called MYO-029 for clinical trials for treatment of muscular dystrophy in adult patients. The review article states the antibody or similar agent will hopefully be tested in other indications such as inflammatory myopathies, cachexia and sarcopenia. The author also notes that a number of endogenous inhibitors of myostatin, including the myostatin propeptide, follistatin, FLRG and GASP-I could be modified for use as therapeutic agents. The review refers to two articles describing the effects of modified propeptide on muscle in mice, Wolfman et al, Proc Natl Acad Sci US, 100: 15842-15846 (2003) and Bogdanovich et al, FASEB J 19: 543- 549 (2004).

[0012] The Wagner review article states that there is significant data that follistatin is an in vivo inhibitor of myostatin and refers to the results of studies described in Lee and McPherron, Proc Natl Acad Sci USA, 98{\β): 9306-9311 (2001) and Amthor et al, Developmental Biol, 270: 19-30 (2004) to support that statement. Follistatin is a secreted protein that inhibits the activity of TGF-β family members such as GDF-1 1 /BMP-1 1.

Follistatin-344 is a follistatin precursor that undergoes peptide cleavage to form the circulating Follistatin-315 isoform which includes a C-terminal acidic region. It circulates with myostatin propeptide in a complex that includes two other proteins, follistatin related gene (FLRG) and GDF associated serum protein (GASP-I). Follistatin-317 is another follistatin precursor that undergoes peptide cleavage to form the membrane-bound Follistatin- 288 isoform. The DNA and amino acid sequences of the follistatin-344 precursor are respectively set out in SEQ ID NOs: 3 and 4. The Follistatin-288 isoform, which lacks a C- terminal acidic region, exhibits strong affinity for heparin-sulfate-proteoglycans, is a potent suppressor of pituitary follicle stimulating hormone, is found in the follicular fluid of the ovary, and demonstrates high affinity for the granulose cells of the ovary. The testis also produce Follistatin-288. The DNA and amino acid sequences of the follistatin-317 precursor are respectively set out in SEQ ID NOs: 5 and 6. Lack of follistatin results in reduced muscle mass at birth.

[0013] In the experiments described in the Lee and McPherron article, follistatin was over- expressed in transgenic mice. The mice showed increased muscling resulting from a combination of hyperplasia (increased muscle fiber number) and hypertrophy (increased muscle fiber diameter). The article proposes that follistatin binds the C-terminal dimer of myostatin and, in turn, inhibits the ability of myostatin to bind to activin type II receptors. Transgenic mice expressing high levels of myostatin propepetide or a dominant-negative form of activin type II receptor (Act RIIB) were also shown to exhibit increased muscle mass in the article.

[0014] The Amthor et al. article is stated to report that follistatin directly binds myostatin with high affinity, is co-expressed with myostatin in somites and prevents myostatin- mediated inhibition of limb muscle development in chick embryos. Indicating that the inhibitory effects of follistatin are not specific to myostatin evening in regard to muscle growth, the Wagner review article alternatively indicates that FLRG and GASP-I, which bind to and inhibit circulating myostatin, may prove to be specific inhibitors of myostatin for therapeutic use. FLRG is a protein that exhibits homology to a 10-cysteine repeat in follistatin. Hill et al, J Biol Chem, 277(43): 40735-40741 reports that FLRG binds circulating myostatin in vivo.

[0015] Yet another review article addressing the regulation of muscle mass by myostatin and clinical implications is Lee, Annu Rev Cell Dev Biol, 20: 61-86 (2404).

[0016] Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). The nucleotide sequence of the AAV serotype 2 (AA V2) genome is presented in Srivastava et al., J Virol, 45: 555-564 (1983) as corrected by Ruffing et al, J Gen Virol, 75: 3385-3392 (1994). Cώ-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, pi 9, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and pi 9), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VPl, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0017J AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65 ⁰ C for several hours),

making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0018] Multiple studies have demonstrated long-term (> 1.5 years) recombinant AAV- mediated protein expression in muscle. See, Clark et al, Hum Gene Ther, 8: 659-669 (1997); Kessler et al, Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996); and Xiao et al, J Virol, 70: 8098-8108 (1996). See also, Chao et al, MoI Ther, 2:619-623 (2000) and Chao et al, MoI Ther, 4:2X1-222 (2001). Moreover, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of trans gene products in the systemic circulation following intramuscular injection as described in Herzog et al, Proc Natl Acad Sci USA, 94: 5804-5809 (1997) and Murphy et al, Proc Natl Acad Sci USA, 94: 13921- 13926 (1997). Moreover, Lewis et al, J Virol, 76: 8769-8775 (2002) demonstrated that skeletal myofibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics.

Brief Summary of the Invention

[0019] The present invention provides methods and materials useful for muscle enhancement and/or improving muscle function. The methods of the invention involve delivering inhibitors of myostatin, a regulator of muscle mass, to muscle cells. Use of the methods and materials is indicated, for example, for treating musculoskeletal diseases/disorders and neurodegenerative diseases/disorders in which muscle is adversely affected as well as treating sarcopenia, cachexia, obesity, Type II diabetes, Pompe disease and lysosomal storage disorders. The methods and materials are also useful, for example, for muscle enhancement in livestock including, but not limited to, cattle, pigs and fowl.

[0020] The terms "inhibitor of myostatin" and "myostatin inhibitor" are intended to be interchangeable herein. The myostatin inhibitor may be a protein or may be an oligonucleotide (RNA or DNA). The terms "muscle enhancement" and "enhancing muscle" are also intended to be interchangeable herein and include, but are not limited to, inducement of hyperplasia (increased muscle fiber number), inducement of hypertrophy (increased muscle fiber diameter) or both. "Enhanced muscle performance" includes, but is not limited to, one or more of decreased atrophy, increased muscle endurance, increased muscle force and increased muscle strength.

[0021] Myostatin inhibitor proteins according to the invention may be peptides or polypeptides. The proteins may inhibit myostatin by binding myostatin [McPherron et al, Nature, 357(6628): 83-90 (1997)]or by binding the myostatin receptor activin lib [McPherron et al, Nat. Genet., 22(3): 260-264 (1999)]. Examples of proteins that inhibit myostatin by binding to myostatin are myostatin propeptide, follistatin [Shimasaki et al., U.S. Patent No. 5,041,538], other follistatin-like proteins (U.S. Patent Nos. 5,942,420; 6,410,232; 6,537,966; and 6,953,662), FLRG (SEQ ID NO: 8, the corresponding nucleotide sequence is SEQ ID NO: 7) [Hill et al, J. Biol. Chem., 277(43): 40735-40741 (2002)]and GASP-I(SEQ ID NO: 10, corresponding nucleotide sequence is SEQ ID NO: 9) [Hill et al., MoI Endocrinol, 17: 1144-1154 (2003)] . Proteins that are myostatin inhibitors according to the invention may be protein fragments or may be chimeric (i.e. , fusion) proteins.

[0022] Myostatin inhibitor oligonucleotides of the invention may be antisense oligonucleotides [Eckstein, Antisense Nucleic Acid Drug Dev., 10: 117-121 (2000); Crooke, Methods Enzymol, 313: 3-45 (2000); Guvakova et al, J. Biol. Chem., 270: 2620-2627 (1995); Manoharan, Biochim. Biophys. Acta, 1489: 117-130 (1999); Baker et al., J. Biol. Chem., 272: 11994-12000 (1997); Kurreck, Eur. J. Biochem., 270: 1628-1644 (2003); Sierakowska et al, Proc. Natl. Acad. ScL USA, 93: 12840-12844 (1996); Marwick, J. Am. Med. Assoc. 280: 871 (1998); Tomita and Morishita, Curr. Pharm. Des., 10: 797-803 (2004); Gleave and Monia, Nat. Rev. Cancer, 5: 468-479 (2005) and Patil, AAPSJ. 7: E61-E77 (2005], triplex oligonucleotides [Francois et al, Nucleic Acids Res., 16: 11431-11440 (1988) and Moser and Dervan, Science, 238: 645-650 (1987)], ribozymes/deoxyribozymes(DNAzymes) [Kruger et al, Tetrahymena. Cell, 31: 147-157 (1982); Uhlenbeck, Nature, 328: 596-600 (1987); Sigurdsson and Eckstein, Trends Biotechnol, 13 286-289 (1995); Kumar et al, Gene Ther., 12: 1486-1493 (2005); Breaker and Joyce, Chem. Biol, 1: 223-229 (1994); Khachigian, Curr. Pharm. Biotechnol., 5: 337- 339 (2004); Khachigian, Biochem. Pharmacol, 68: 1023-1025 (2004) and Trulzsch and Wood, J. Neurochem., 88: 257-265 (2004)], small-interfering RNAs/RNAi [Fire et al, Nature, 391: 806-811 (1998); Montgomery et al, Proc. Natl Acad. ScL U.S.A., 95: 15502- 15507 (1998); Cullen, Nat. Immunol, 3: 597-599 (2002); Hannon, Nature, 418: 244-251 (2002); Bernstein et al, Nature, 409: 363-366 (2001); Nykanen et al, Cell, 107: 309-321 (2001); Gilmore et al, J. Drug Target., 12: 315-340 (2004); Reynolds et al, Nat. Biotechnol, 22: 326-330 (2004); Soutschek et al, Nature, 432173-178 (2004); Ralph et al, Nat. Med., 11: 429-433 (2005); Xia et al, Nat. Med., 70816-820 (2004) and Miller et al, Nucleic Acids Res.,

32: 661-668 (2004)], aptamers [Ellington and Szostak, Nature, 346: 818-822 (1990); Doudna et al, Proc. Natl. Acad. Sci. U.S.A., 92: 2355-2359 (1995); Tuerk and Gold, Science, 249: 505-510 (1990); White et al, MoI Ther., 4: 567-573 (2001); Rusconi et al, Nature, 419: 90- 94 (2002); Nimjee et al, MoI. Ther., 14: 408-415 (2006); Gragoudas et al, N. Engl. J. Med., 351: 3805-2816 (2004); Vinores, Curr. Opin. MoI Ther., 5673-679 (2003) and Kourlas and Schiller et al, Clin. Ther., 28 36-44 (2006)] or decoy oligonucleotides [Morishita et al, Proc. Natl Acad. Sci. U.S.A., 92: 5855-5859 (1995); Alexander et al, J. Am. Med. Assoc, 294: 2446-2454 (2005); Mann and Dzau, J. Clin. Invest., 106: 1071-1075 (2000) and Nimjee et al, Annu. Rev. Med., 56: 555-583 (2005). The myostatin inhibitor oligonucleotides inhibit the expression of myostatin or expression of its receptor activin lib. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to methods of designing, making and using oligonucleotides.

[0023] Some embodiments of the invention exploit the unique properties of AAV to deliver polynucleotides encoding myostatin inhibitors to muscle cells. Other embodiments of the invention utilize other vectors (for example, other viral vectors such as adenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus, pox viruses, herpes virus, polio virus, sindbis virus and vaccinia viruses) to deliver polynucleotides encoding myostatin inhibitors.

Detailed Description of the Invention

[0024] In one aspect, the invention provides rAAV genomes. The rAAV genomes comprise one or more AAV ITRs flanking a polynucleotide encoding one or more myostatin inhibitors. If the polynucleotide encodes one or more myostatin inhibitor proteins the polynucleotide is operatively linked to transcriptional control DNA, specifically promoter DNA and polyadenylation signal sequence DNA that are functional in target cells to form a gene cassette. The gene cassette may also include intron sequences to facilitate processing of the RNA transcript when expressed in mammalian cells. Alternatively, the polynucleotide in the rAAV genome be a myostatin inhibitor RNA or may encode one or more myostatin inhibitor RNAs. The myostatin inhibitor RNAs may be antisense RNAS, ribozymes, small interfering RNAs (RNAi) or aptamers that inhibit expression of myostatin or its receptor activin lib. For example, an antisense RNA complementary to the translation initiation site of myostatin or activin lib may be encoded by the rAAV genome. As another example, an RNA

that binds to the myostatin or activin lib double-stranded DNA may be encoded that prevents DNA unwinding and transcription. As yet another example, commercial providers such as Ambion Inc. (Austin, TX), Darmacon Inc. (Lafayette, CO), InvivoGen (San Diego, CA), and Molecular Research Laboratories, LLC (Hemdon, VA) generate custom siRNA molecules. In addition, commercially kits are available to produce custom siRNA molecules, such as SILENCER™ siRNA Construction Kit (Ambion Inc., Austin, TX) or psiRNA System (InvivoGen, San Diego, CA).

[0025] The rAAV genomes of the invention lack AAV rep and cap DNA. AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV-I, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-IO and AAV-11.The nucleotide sequences of the genomes of the AAV serotypes are known in the art. For example, the complete genome of AAV-I is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC 001401 and Srivastava et ai, J. Virol., 45: 555-564 {1983); the complete genome of AAV-3 is _. provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV -9 genome is provided in Gao et ah, J. Virol, 78: 6381-6388 (2004); the AAV-10 genome is provided in MoI Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004)..

[0026] In another aspect, the invention provides DNA plasmids comprising rAAV genomes of the invention. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, El -deleted adenovirus or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-I, AAV-2, AAV-3,

AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.

[0027] A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al, 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells.

[0028] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129). Various approaches are described in Ratschin et al., MoI. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81 :6466 (1984); Tratschin et al., MoI. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol, 62:1963 (1988); and Lebkowski et al., 1988 MoI. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Patent No. 5,173,414; WO 95/13365 and corresponding U.S. Patent No. 5,658.776 ; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/1 1764; Perrin et al. (1995) Vaccine 13:1244- 1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Patent. No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent. No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to rAAV production.

[0029] The invention thus provides packaging cells that produce infectious rAAV. In one embodiment packaging cells may be stably transformed cancer cells such as HeLa cells, 293

cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells such as low passage 293 cells (human fetal kidney cells transformed with El of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

[0030] In another aspect, the invention provides rAAV (i.e., infectious encapsidated rAAV particles) comprising a rAAV genome of the invention. Embodiments include, but are not limited to, the exemplified rAAV named "rAAV follistatin-344" and "rAAV MCK follistatin-344." The rAAV follistatin-344 genome includes in sequence an AA V2 ITR, the CMV promoter, an intron from the human β-globin gene, the follistatin DNA set out in SEQ ID NO: 11, a polyadenylation signal sequence from the human β-globin gene and another AA V2 ITR. The rAAV MCK follistatin-344 genome includes in sequence an AA V2 ITR, a truncated human muscle creatine kinase (MCK) promoter, a chimeric intron, the follistatin DNA set out in SEQ ID No: 11, the SV40 late polyadenylation signal and another AA V2 ITR. The chimeric intron is composed of the 5' donor site from the first intron of the human β-globin gene and the branchpoint and 3' splice acceptor site from the intron that is between the leader and the body of an immunoglobulin gene heavy chain variable region. The sequences of the donor and acceptor sites, along with the branchpoint site, have been changed to match the consensus sequences for splicing. The SV40 polyadenylation signal is used for efficient transcription termination. Both genomes lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genome.

[0031] The rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example, Clark et ah, Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods MoI. Med., 69 427- 443 (2002); U.S. Patent No. 6,566,118 and WO 98/09657.

[0032] In another embodiment, the invention contemplates compositions comprising rAAV of the present invention. These compositions may be used to enhance muscle and/or improve muscle function. In one embodiment, compositions of the invention comprise a rAAV encoding a myostatin inhibitor of interest. In other embodiments, compositions of the present invention may include two or more rAAV encoding different myostatin inhibitors of interest.

[0033] Compositions of the invention comprise rAAV in a pharmaceutically acceptable carrier. The compositions may also comprise other ingredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-formig counterions such as sodium; and/or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).

[0034] Titers of rAAV to be administered in methods of the invention will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of rAAV may range from about 1x10 ⁶ , about 1x10 ⁷ , about 1x10 ⁸ , about 1x10 ⁹ , about IxIO ¹⁰ , about IxIO ¹¹ , about IxIO ¹² , about IxIO ¹³ to about lxl0 ¹⁴ or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg).

[0035] Methods of transducing a target cell with rAAV, in vivo or in vitro, are contemplated by the invention. The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a rAAV of the invention to an animal (including a human being) in need thereof. If the dose is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development of a disorder/disease, the administration is therapeutic. In embodiments of the invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease state being treated,that slows or prevents progression to a disorder/disease state,that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival. Examples of disease states contemplated for treatment with methods of the invention are musculoskeletal diseases/disorders [for example, muscular dystrophies or inclusion body myositis (IBM)], neurodegenerative diseases/disorders in which muscle is adversely affected (for example, Amyotrophic Lateral

Sclerosis multiple sclerosis and spinal muscular atrophy), sarcopenia, cachexia, obesity, Type II diabetes, Pompe disease and lysosomal storage disorders.

[0036] Combination therapies are also contemplated by the invention. Combination as used herein includes both simultaneous treatment or sequential treatments. Combinations of methods of the invention with standard medical treatments (e.g., corticosteroids for muscular dystrophies) are specifically contemplated, as are combinations with novel therapies. For example, for treatment of Muscular Dystrophies, methods of the invention may be combined with follistatin administration, followed by simultaneous or concomitant treatment to correct the genetic disorder. Correcting a genetic disorder may involve, for example, replacing sarcoglycans in sarcoglycan deficiency, correcting or replacing dystrophin in disorders such as Duchenne's Muscular Dystrophy, treating ALS patients with IGF-I or mutant SODl interference strategies). Given that in disorder contemplated for treatment by the present invention, significant amount of muscle is lost, the prevention or rescue of muscle will give a substrate (preserved or regenerated muscle) for subsequent gene correction. In this respect, it may be conceivable to inhibit myostatin to enhance muscle, increase muscle size, and then provide the secondary treatment. Such secondary treatments for Muscular Dystrophy may be IGF-I, interfering RNA approaches, exon-skipping, calpain inhibition, dystrophin upregulation, and dystroglycan expression. Further, there may be additions to myostatin inhibition approaches to enhance the muscle boosting effects. For example, addition of IGF- 1 or other trophic factors or muscle precursor injections could be performed. Myostatin inhibition in concert with muscle precursor cells (satellite cells, stem cells) may allow more of these cells to be incorporated into the tissue.

[0037] Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. Route(s) of administration and serotype(s) of AAV components of rAAV (in particular, the AAV ITRs and capsid protein) of the invention may be chosen and/or matched by those skilled in the art taking into account the infection and/or disease state being treated and the target cells/tissue(s) that are to express the myostatin inhibitor protein(s).

[0038] In particular, actual administration of rAAV of the present invention may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal. Administration according to the invention includes, but is not limited to, injection into muscle, the bloodstream and/or directly into the liver. Simply

resuspending a rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV). Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as muscle. See, for example, WO 02/053703, the disclosure of which is incorporated by reference herein. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations to be delivered to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the invention. The rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0039] For purposes of intramuscular injection, solutions in an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV 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 hydroxpropylcellulose. A dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

[0040] 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 easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions 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. The prevention

of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include 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.

[0041] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0042] Transduction with rAAV may also be carried out in vitro. In one embodiment, desired target muscle cells are removed from the subject, transduced with rAAV and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used where those cells will not generate an inappropriate immune response in the subject.

[0043] Suitable methods for the transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and/or PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, and the composition introduced into the subject by various techniques, such as by intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle, using e.g., a catheter.

[0044] Transduction of cells with rAAV of the invention results in sustained expression of myostatin inhibitors. The present invention thus provides methods of administering/delivering rAAV which express myostatin inhibitors to an animal, preferably a human being. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or more rAAV of the present invention. Transduction may be carried out with gene

cassettes comprising tissue specific control elements. For example, one embodiment of the invention provides methods of transducing muscle cells and muscle tissues directed by muscle specific control elements, including, but not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family [See Weintraub et al. Science, 251: 161-166 (1991)], the myocyte- specific enhancer binding factor MEF-2 [Cserjesi and Olson, MoI Cell Biol 11: 4854-4862 (1991)], control elements derived from the human skeletal actin gene [Muscat et al, MoI Cell Biol, 7: 4089-4099 (1987)], the cardiac actin gene, muscle creatine kinase sequence elements [See Johnson et al, MoI Cell Biol, 9:3393-3399 (1989)] and the murine creatine kinase enhancer (mCK) element, control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene and the slow-twitch troponin I gene: hypozia-inducible nuclear factors [Semenza et al, Proc Natl Acad Sci USA, 88: 5680-5684 (1991)], steroid-inducible elements and promoters including the glucocorticoid response element (GRE) [See Mader and White, Proc. Natl. Acad. Sci. USA 90: 5603-5607 (1993)], and other control elements.

[0045] Muscle tissue is an attractive target for in vivo gene delivery and gene therapy, because it is not a vital organ and is easy to access. The invention contemplates sustained expression of biologically active myostatin inhibitor proteins from transduced myo fibers.

[0046] By "muscle cell" or "muscle tissue" is meant a cell or group of cells derived from muscle of any kind (for example, skeletal muscle and smooth muscle, e.g. from the digestive tract, urinary bladder, blood vessels or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomyoblasts. Since muscle tissue is readily accessible to the circulatory system, a protein produced and secreted by muscle cells and tissue in vivo will logically enter the bloodstream for systemic delivery, thereby providing sustained, therapeutic levels of protein secretion from muscle.

[0047] The term "transduction" is used to refer to the administration/delivery of myostatin inhibitor DNA to a recipient cell either in vivo or in vitro, via a replication-deficient rAAV of the invention resulting in expression of a functional myostatin inhibitor by the recipient cell.

[0048] Thus, the invention provides methods of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV that encode inhibitors of myostatin to a patient in need thereof.

Brief Description of the Drawing

Figure 1 demonstrates that AAV Follistatin induces gross body mass increase in mice.

Figure 2 depicts the dose response of AAV-Follistatin on motor function tests in mice.

Figure 3 shows that AAV-Follistatin induces muscle mass increase in mice.

Figure 4 depicts Succinate Dehydrogenase (SDH) staining on tibialis anterior and triceps muscles in mice.

Figures 5-7 respectively represent the dark, intermediate and light tibialis anterior (TA) muscle fiber diameters that result from various doses of follistatin administration.

Figures 8-10 respectively represent the dark, intermediate and light tricep muscle fiber diameters that result from various doses of follistatin administration.

Figure 11 demonstrates that follistatin treatment attenuates the levels of serum creatine kinase.

Figure 12 depicts histological examination of diaphragms at 1OX from mdx animals stained with Masson's Trichrome.

Figure 13 depicts histological examination of diaphragms at 2OX from mdx animals stained with Masson's Trichrome.

Figure 14 depicts histological examination of diaphragms at 4OX from mdx animals stained with Masson's Trichrome.

Figure 15 shows that follistatin induces hypertrophy in diaphragm muscle fibers.

Figure 16 shows that follistatin-treated animals demonstrate higher numbers of revertant fibers in gastrocnemius.

Figure 17 depicts studies done to assess muscle function of 8-week old animals that were forced to exercise daily on a treadmill for 12 weeks.

Figure 18 demonstrates that myostatin inhibitor proteins can increase muscle mass and strength in wild type C57BL/6 mice.

Figure 19 demonstrates that a single injection of AAVl-FS can increase grip strength in aged mdx mice.

Figure 20 depicts levels of FS in circulating plasma following AAV-CMV-FS administration to non-human primates.

Figure 21 depicts an analysis of fiber size showing muscle hypertrophy in MCK-FS injected quads compared to non-injected quads.

Examples of the Invention

[0049] Examples 1 and 2 below respectively describe the effect of a rAAV-delivered myostatin inhibitor in animal models of Limb-girdle muscular dystrophy Type 2D and Amyotrophic Lateral Sclerosis. Example 3 describes the effect of a rAAV-delivered myostatin inhibitor in the mdx animal model of muscular dystrophy. Example 4 describes a clinical protocol for treatment of inclusion body myositis with a rAAV-delivered myostatin inhibitor. Example 5 describes the effect of a rAAV-delivered myostatin inhibitor in wild type C57B1/6 mice. Example 6 describes the effect of a rAAV-delivered myostatin inhibitor in older mdx animals. Example 7 describes the effect of a rAAV-delivered myostatin inhibitor in non-human primates.

Example 1

[0050] Limb-girdle muscular dystrophy Type 2D (LGMD2D) is a debilitating disease of children and young adults. Caused by a mutation in any one of at least fifteen different genes, some types of LGMD2D are autosomal dominant and others are autosomal recessive. LGMD2D progresses slowly, resulting in weakness and wasting first of the muscles around the shoulders and hips (limb girdles) and sometimes later the muscles involved in cardiopulmonary function. To date, there is no proven treatment to delay the disease progression. Alpha-sarcoglycan knock-out mice are animal models for LGMD2D [Duclos et al, J. Cell Biol, 142(6): 1461-1471 (1993)]. Experiments examining the effect of delivering follistatin using a rAAV are described below.

[0051] An AAV serotype 1 recombinant virus was constructed to encode a human follistatin DNA under the control of the strong human cytomegalovirus promoter. The rAAV was designated "rAAV follistatin UCSD". The human follistatin DNA (SEQ ID NO: 1) was a kind gift from Dr. Shumazaki (University of California-San Diego, La Jolla, CA). The amino acid sequence encoded by the DNA is set out in SEQ ID NO: 2. The DNA was subcloned into the EcoRI site of Bluescript containing a novel 5' Sfi I site and 3' Pme I site. The cDNA was then subcloned directionally into these sites of an AAV2-ITR-containing vector under the control of the human cytomegalovirus promoter [Kaspar et al, MoI Ther, 5: 50-56 (2002)]. Recombinant AAV-I vectors were produced by triple transfection using calcium phosphate in human embryonic kidney carcinoma 293 cells (HEK-293). Briefly, a plasmid containing the Rep from serotype 2 and capsid from serotype 1 [Rabinowitz et al, J. Virol, 76: 791- 801 (2002)] along with a helper adenoviral plasmid (Stratagene, Palo Alto,

CA) was used. Transfected HEK293 cells were harvested 48 hours after transfection and resuspended in a small volume of 20 niM Tris (pH 8.0), 1 mM MgC12 and 150 mM NaCl (T2 OMIN 150). Cells were frozen and thawed four times and incubated with Benzonase (35 u/ml) for 30 minutes at 37°C. Cell debris were pelleted by centrifugation at 3,000 rpm at 40 ⁰ C for 15 minutes in a Beckmann GS-6R centrifuge. The cleared lysate was purified by a CsCl gradient purification process followed by anion exchange chromatography (POROS HQ-50). A contract manufacturing company (Virapur LLC, San Diego, CA) was used for some virus preparations and titers confirmed. Titer was determined by QPCR techniques and titers were 3 x 10 ¹² DNase Resistant Particles (DRP)/ml.

[0052] To test myostatin inhibition in vivo, 4-8 week old male alpha-Sarcoglycan deficient mice were injected with a total of 1 X 10 ¹⁰ DNase Resistant Particles of the rAAV follistatin UCSD or AAV-GFP (green fluorescent protein; control). The animals had been tested for baseline motor function at 3 weeks of age.

[0053] Gross body mass increases were seen in mice following administration of AAV- follistatin. (Figure 1).

[0054] Motor functional tests included the accelerating Rotarod test and hindlimb and forelimb grip strength measurements (Columbus Instruments, Columbus, OH) (Figure 2). Measurements were performed weekly (same day and time of each week). Each weekly session consisted of three trials on the elevated accelerating rotarod beginning at 4 rpm/min. The time each mouse remained on the rod was registered automatically. Grip strength meter testing was performed by allowing the animals to grasp a platform followed by pulling the animal until it release the platform, the force measurement recorded in four separate trials.

[0055] These motor function tests showed improvements in muscle strength in AAV- follistatin treated mice.

[0056] At 120 days, gross muscle evaluation and analysis of muscle weight, fiber number and cross-sectional area (CSA) on HE sections were performed as well as a follistatin ELISA assay. See Example 2 for procedures.

[0057] Increased serum follistatin levels accompanied by a local and remote increase in muscle mass were observed in rAAV-follistatin UCSD treated animals (p<0.05) (Figure 3). Muscle hyperplasia was observed in comparison to controls. The number of central nucleated fibers was increased in AAV-follistatin treated animals indicating enhanced muscle regeneration. In addition, succinate dehydrogenase (SDH) staining was done on tibialis

anterior (TA) muscle histological sections from control, treated, and wild type mice. SDH is a marker for oxidative, mitochondrial metabolism and classifies myofibers into oxidative or glycolytic fibers. This data supports a therapeutic effect of AAV-follistatin treatment in MD. This approach represents a clinically applicable gene delivery method to enhance muscle mass and function in LGMD2D with potential for treatment for other forms of MD.

Example 2

[0058] Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig's disease in the United States, is an adult onset disease that causes degeneration of motor neurons. The loss of motor neurons results in stiffness, atrophy of muscles, severe weakness of limb muscles, inability to walk, speak or swallow, and death two to five years after failure of the muscles that control respirations. Ten percent of ALS is familial and one fifth of these familial cases are caused by mutations in superoxide dismustase 1 (SODl). Transgenic mice and rats expressing a mutant human SODl transgene develop an ALS phenotype [Gurney et ah, Science, 264: 1772-1775 (1994)]. Experiments examining effect of delivering follistatin using a rAAV are described below.

[0059] To test the ability for follistatin to reverse the myostatin-dependent inhibition on myoblast proliferation, C2C12 myoblasts were incubated with a growth inhibitory dose of myostatin (3 μg/ml) in the presence or absence of conditioned media from 293 cells infected with rAAV follistatin UCSD (described in Example 1) or conditioned media from cells infected with rAAV encoding Red Fluorescent Protein (RFP). The virus was produced as described for the AAV-follistatin in Example 1 except virus was collected 72 hours post- transfection and processed on cesium chloride gradients as previously described (Kaspar et ah, 2002) C2C12 myoblasts were grown in DMEM (Invitrogen, Temecula, CA) containing 10% FBS (Invitrogen, Temecula, CA). Cell proliferation assays were conducted in a 96-well Nunc Microtiter plates and seeded at 1000 cells per well. After attachment, myostatin (R&D Systems) was added at a concentration of 3 μg/ml in the presence or absence of conditioned media. Conditioned media was created by infecting a well of 12 well dish with either 109 viral particles of AAV-Red Fluorescent Protein (RFP) or AAV-Follistatin (FS) and collecting the media 48 hours after infection. Three-days following myostatin inhibition, myoblast proliferation was assayed by the MTT assay. MTT proliferation assay was performed using a commercially available kit (Pierce Technologies). Data was collected in quadruplicate and

read on a microplate reader set for absorbance at 570 nM. Results were presented as means and standard errors.

[0060] Myoblasts cultured in growth media steadily increased in cell number compared to myostatin-treated cultures, in which a significant decrease in proliferation was seen. Follistatin-conditioned media reversed the inhibition of proliferation by myostatin, whereas control RFP conditioned media did not.

[0061] To directly assess whether increased muscle proliferation affected disease course in a mouse model of inherited ALS caused by mutation in SODl, rAAV follistatin UCSD or AAV-GFP (green fluorescent protein; control) (1 x 10 ¹⁰ viral genomes per injection) were injected bilaterally via intramuscular delivery into the hindlimb quadriceps and tibialis muscles of 16 mice each at 40 days of age (equal distribution of male and female animals).

[0062] Mice were observed daily for survival. Survival analysis was performed by Kaplan-Meier analysis which generates a χ2 value to test for significance. The Kaplan-Meier test was performed using the log-rank test equivalent to the Mantel-Haenszel test. In addition, two tailed p values were calculated. When comparing survival curves, median survival times were calculated with a 95% confidence interval. All other statistical tests not involved in survival analysis were performed by multi-way analysis of variance followed by a Bonferroni post hoc analysis of means differences between groups (GraphPad Prizm Software, San Diego, CA). Testing of motor function using a rotarod device (Columbus Instruments, Columbus, OH) began at 35-40 days of age. Each weekly session consisted of three trials on the elevated accelerating rotarod beginning at 5 rpm/min. The time each mouse remained on the rod was registered. Grip Strength measurements for forelimb and hindlimbs were tested weekly using a grip strength meter (Columbus Instruments, Columbus, OH). Each weekly session consisted of 4 tests per animal per limb. To determine mortality in a reliable and humane fashion, we used an artificial end point, defined by the inability of mice to right themselves 30 seconds after being placed on their sides. The moribund mice were scored as "dead" and were euthanized, and tissues were collected.

[0063] Both sets of animals reached end stage disease at -126 days. Despite this, follistatin treated muscles showed gross changes including widespread increased muscle mass in compared to the GFP treated animals. Easily seen from visual inspection, wet weights of multiple muscles (n=10-15 animals each), including the tibialis anterior, gastrocnemius, medial quadriceps and triceps muscles were significantly (p<0.05) after injection of AAV-

follistatin. Increased muscle mass was not limited to the hindlimb muscles injected. Using a commercially available ELISA assay (R & D Systems) specific for human follistatin, circulating blood levels of follistatin were found to be significantly elevated (>10 ng/ml) at 100 days of age of AAV-follistatin treated animals, but not AAV-GFP animals, indicating that follistatin was acting in a paracrine manner on all skeletal muscles. Data was collected in triplicate for each animal and presented as means with standard error.

[0064] To determine whether the muscle weight increase was due to hyperplasia, hypertrophy and/or muscle sparing in the ALS animals, skeletal muscle hypertrophy and myofϊber number were investigated. Muscles were embedded in Optimal Cutting Temperature Compound (OCT) and snap frozen in liquid nitrogen cooled isopentane. Transverse 10 μm sections were cut through the middle of the muscle and sections stained with H & E and trichrome stains. The sections (4 sections each animal) were photographed on a Zeiss Axiovert microscope connected to a Zeiss micrometer on a Dell Workstation. The total area of the muscle cross section was calculated and individual myo fibers were counted and diameters measured. Graphs of total fiber numbers per section and a percentage of fiber diameters were plotted (Figures 5-10).

[0065] This revealed that at end stage (126-127 days) approximately 2 fold more myo fibers were present in the gastrocnemius compared to GFP-treated animals (n=8 animals) (pO.Ol). Follistatin treated muscles were also slightly hypertrophic based on measurements of myo fiber diameters in the quadriceps and gastrocnemius muscles compared to AAV-GFP treated animals. Moreover, serum creatine kinase levels were decreased in AAV-follistatin treated animals versus AAV-GFP treated animals (Figure 11).

[0066] Increased muscle mass, increased myo fiber number, and hypertrophy of muscle fibers translated, as expected, into increased strength in the follistatin-treated animals. By 75 days of age, follistatin-treated animals showed a 40% increase in hindlimb strength compared with GFP treated littermates, remaining comparable to the non-transgenic animals at 75 days. Strength in the forelimbs was also increased from 60-120 days of age or end-stage. The increased strength did not afford these mice better performance with other measures, including a rotarod test, presumably reflecting that strength is only one measure of performance in this particular test. Despite maintenance of muscle strength and increased muscle mass even through end stage, no statistically significant increase in survival (defined by paralysis so severe that the animal was unable to right itself within 30 seconds) was seen

in the follistatin-treated mice versus untreated or AAV GFP treated cohorts (AAV-GFP 126, AAV-Follistatin 130; p value 0.06, Chi Square 3.504).

[0067] Enhancement of muscle mass and prevention of muscle atrophy by inhibition of myostatin with virally delivered follistatin yielded the expected increase in muscle mass and delayed atrophy. It is contemplated that this result will translate into therapeutic benefit in human patients treated with a myostatin inhibitor of the invention.

Example 3

[0068] Another AAV serotype 1 recombinant virus was constructed to encode a human follistatin DNA under the control of the strong human cytomegalovirus promoter. The rAAV was designated "rAAV follistatin-344". The DNA and amino acid sequences of the follistatin encoded are respectively set out in SEQ ID NOs: 11 and 12. The rAAV follistatin- 344 was administered to mdx mice (Bogdanovich et al, supra) which are an animal model of MD.

[0069] Eighteen three- week old mice were injected with 1 x 10 ¹⁰ drp/limb of the rAAV while fifteen three-week old mice received 1 x 10 ⁹ drp/limb. Twelve other mice received 1 x 10 ¹⁰ drp/limb AAV-GFP. Results of the treatment are presented in Figures 1 through 17.

[0070] Treatment with rAAV follistatin-344 resulted in muscle enhancement and improved muscle function in the treated animals and it is contemplated that these results will translate into therapeutic benefit in human MD patients treated with a myostatin inhibitor of the invention.

Example 4

[0071] Inclusion body myositis (IBM) is an inflammatory muscle disease characterized by progressive muscle weakness and wasting. The disorder is similar to another inflammatory myopathy called polymyositis. IBM is often the diagnosis for cases of polymyositis that are unresponsive to therapy, but IBM has its own distinctive features. The onset of muscle weakness in IBM is generally gradual (over months or years). Falling and tripping are usually the first noticeable symptoms. For some patients the disorder begins with weakness in the hands causing difficulty with gripping, pinching, and buttoning. IBM occurs more frequently in men than in women and affects both the proximal (closest to the center of the body) and

distal (farthest from the center of the body) muscles. There may be weakness of the wrist and finger muscles and atrophy of the quadricep muscles in the legs. Atrophy or shrinking of the forearms is also characteristic. Difficulty swallowing (dysphagia) occurs in approximately half of IBM cases. Symptoms of the disease usually begin after the age of 50, although the disease can occur earlier. IBM is generally resistant to all therapies, and its rate of progression appears to be unaffected by any currently available treatments.

[0072] It is contemplated that muscle enhancement and particularly quadriceps muscle enhancement will benefit patients suffering from sporadic IBM (sIBM). Enhancement of muscle and/or improved muscle function resulting from delivering follistatin using a rAAV may be confirmed as described below where methods of treatment of mdx mice and sIBM human patients are set out.

Treatment of mdx mice

[0073] C57B1/10 (wild-type) and mdx animals (Bogdanovich et al, supra) receive bilateral injections using a 26 gauge Hamilton needle of rAAV follistatin-344 into the quadriceps at 3 weeks of age (n=8 animals/dosage/vector). Four doses are tested, 1 x 10 ^π , 1 x 10 ¹⁰ , 1 x 10 ⁹ , 1 x 10 ⁸ viral particles per quadriceps. One group of animals (n=8) will be injected with AAV.CMV.GFP (rAAV comprising DNA encoding green fluorescent protein linked to the CMV promoter) as a control using the high dosage; 1 x 10 ^π viral particles. Tacrolimus and MMF were given daily to animals 1 week prior to viral delivery and for 8 weeks following viral gene delivery. Doses were based on comparable schedules to patient dosing (0.2 mg/kg tacrolimus and 25 mg/kg MMF).

[0074] Levels of follistatin were measured monthly. Blood was collected by retro-orbital eye bleeds (routinely performed in our lab) and serum separated in serum-separator tubes. A commercially available ELISA assay kit specific for human follistatin is available (R&D Systems) and may be used to detect follistatin. Animals were sacrificed at 4 months of age. The quadriceps, tibialis anterior, extensor digitorum longus muscles, gastrocnemius, soleus, plantaris, diaphragm and biceps femoris muscles were dissected from each side of the animals. Muscles were mounted and snap frozen in liquid nitrogen-cooled isopentane, and cross-sectioned at 8-12 μm on a cryostat at -18-2O ⁰ C followed by histopathology stains (trichrome and H&E). To distinguish between hypertrophy and hyperplasia, morphometric examination of the muscles was performed using systematic sampling of unbiased counting frames using a semi-automatic stereology system and morphometric analysis will be

performed using Metamorph. Pathologic irregularities including fibrosis, inflammation and cytoplasmic bodies were noted (Figures 12-14). Muscle hypertrophy was noted in those animals receiving follistatin treatment (Figure 15), and they also produce higher numbers of revertant fibers in gastrocnemius muscle (Figure 16).

[0075] Exercise studies done on 8-week old mdx animals that were treated with AAV- follistatin were compared to non-exercised animals as well as wild type non-exercised animals. The results show that administration of AAV-follistatin mice could perform better than both the non-exercised mdx and non-exercised wild type mice in endurance as well as accelerating RotaRod tests (Figure 17).

[0076] rAAVl -follistatin virus is produced in 293 cells by calcium phosphate transient transfection using the AAV vector plasmid containing the AAVl inverted terminal repeats and using pXRl helper plasmid providing rep and cap serotype 1 in trans, and pHelper- Adenoviral plasmid [Xiao and Samulski, J. Virol, 72(3): 2224-2232 (1998)]. Three days following transfection, cells are harvested, freeze thawed and purified by cesium chloride density gradient ultracentrifugation. Fractions containing virus are dialyzed against Hepes buffered saline. Determination of viral titer was determined using real time quantitative PCR as well as functional testing of transgene expression by limiting dilutions of virus on 293 cells using immunohistochemistry.

[0077] Statistical Analysis: Statistical analysis was performed by multi-way ANOVA followed by a Bonferroni post-hoc analysis of means differences between groups (InStat and GraphPad Software).

Treatment of sIBM Human Patients

[0078] Patients receive an immunosuppression regimen of tacrolimus and MMF during the study period. One group of patients is injected in the quadriceps with rAAV follistatin-344 and the other group receives sham injections, Since the patients in the trial will receive an immunosuppression regimen that could influence their course, at minimum a separate group receiving tacrolimus and MMF should be treated to distinguish the effects of the drug regimen from gene transfer.

[0079] Six sIBM patients will be included based on the following criteria. Subjects must fulfill the following characteristic features of sIBM a) duration of illness > 6 months, b) age of onset > 30 yrs; c) quadriceps muscle strength must be in the MRC grade 4 range; d) muscle biopsy proven diagnosis of IBM: (including mononuclear cell inflammation,

vacuolated muscle fibers and either intracellular amyloid deposits or 15-18nm tubulofilaments by EM). Sexually active patients must also be willing to practice a reliable method of contraception during the study. Exclusion Criteria will be active viral infection, concomitant illness or requirement for chronic drug treatment that in the opinion of the PI creates unnecessary risks for gene transfer or conflicts with immunosuppressive regimen and pregnant women

[0080] Dose escalation study of rAAVl carrying the human follistatin gene under control of the CMV promoter (rAAV follistatin-344) given as a multiple intramuscular injections to the quadriceps muscles on both sides. The planned dose for cohort 1 (three patients) will be 3 x 10 ¹² [in six divided quadriceps muscle injections of 2.5 x 10 ⁿ viral genomes (vg)] followed by cohort 2 receiving 3 x 10 ¹³ (in six divided quadriceps muscle injections of 2.5 x 10 ¹² vg). Patients will be injected at six week intervals. Patients will receive tacrolimus (0.2 mg) and mycophenolate mofetil (I g BID) daily starting one week prior to gene transfer and drug will continue for at least six months post gene transfer. Patients may opt to continue the drug depending on the discretion of the principal investigator. At minimum a separate, parallel group of IBM patients will receive immunosuppression using tacrolimus and MMF will be compared to the gene transfer cohort.

[0081] Baseline measures to be taken prior to injection (day -7 to day -1) are quantitative muscle testing (Maximum voluntary isometric contractions or MVICT) to measure quadriceps (knee extensors) muscle strength and hand strength will be assessed with a grip dynamometer. MVICT on additional muscles including elbow flexion and extension and knee flexion are also obtained. These will be the muscles used to evaluate strength following gene transfer. Functional testing will include time to climb four standard steps, time to walk 30 feet, and time to get up from a chair. Baseline laboratory work will include CBC, platelets, blood urea nitrogen (BUN), GGT, bilirubin, alkaline phosphatase, creatinine, amylase, serum protein electrophoresis, protime, and PTT, neutralizing antibodies to AAV and western blot detection of antibodies to follistatin. Patients will require hepatitis screening, chest x-ray, echocardiogram, and EKG. Females of childbearing potential will have a pregnancy test. Adult males will be asked to provide a semen sample.

[0082] Patients will return to a designated monitored bed following gene transfer and vital signs will be obtained hourly for four hours following the injection and then every 4 hours for 3 days prior to discharge. Patients will return for follow up visits on days 7, 14, 30, 60, and 90. Muscle biopsies will be performed on both quadriceps muscles on day 90. Blood will be

taken for neutralizing antibody to AAVl, and antibody to follistatin at each visit. In addition, ELISpots will be performed using capsid peptides to establish T cell responses to rAAVl and follistatin. On days 14, 30, 60, and 90, a more comprehensive battery of tests will include GGT, bilirubin, BUN, alkaline phosphatase, creatinine, CBC, platelets, amylase, cholesterol, triglycerides. Semen samples will be requested on days 60 and 90. Muscle will be studied using multiple serial sections. For leukocyte markers (CD45, CD3, CD4, CD8, MAC 387). Muscle will also be examined for histological appearance using modified trichrome, H & E, oxidative enzymes, and ATPase. Muscle sections will also be pooled for PCR analysis for viral DNA. Patients will be followed for every six months for two years.

[0083] Muscle strength based on MVICT of each individual muscle including quadriceps, knee extensors, elbow extensors, and elbow flexors and hand grip dynamometer will be performed prior to gene transfer and at six months (completion of study). The best of three measures at each of these time points will be used for analysis. An intermediate time point will be obtained at 3 months but used only to evaluate as a safety measure in case of adverse effects of the gene transfer. Muscle biopsy will be performed on both quadriceps post gene transfer. This muscle will not undergo biopsy pre-gene transfer for a variety of reasons. First, it will be too invasive to do biopsies on both quadriceps muscles before and after. The post gene transfer biopsy at six months will be necessary to establish the size of individual muscle fibers and degree of inflammation. Fiber size distribution histograms (# / mm2) will be obtained from 5 randomly selected areas of the removed muscle (one central and four peripheral). In addition, fiber type histograms will be analyzed separately to determine if there is a preferential effect. Muscle will also be analyzed for number and distribution of T and B cells and macrophages, number of internal nuclei, number of muscle fibers undergoing necrosis, and number of regenerating muscle fibers. Vector titers in the muscle will be determined in the tissue by quantitative PCR.

[0084] Outcome variables will include changes from baseline to 6 months with a two-way analysis of variance model with 24-week change from baseline as the dependent variable and treatment group as the independent variable. An F test will be performed for significance of the difference in the adjusted treatment group means, and a 95% CI for the difference will be constructed. This analysis will apply mainly to MVICT but will also be applied to variables in the muscle biopsies between treatment groups.

Example 5

[0085] To demonstrate the effect of AAV delivery of various myostatin inhibitors in vivo, mice were treated with AAVl according to the present invention. Briefly, 1 x 10 ⁿ AAVl viral particles were administered per animal. Recombinant AAV administered were: rAAV follistatin-344, rAAV encoding FLRG, rAAV encoding GASP-I, or rAAV encoding GFP. The DNA and amino acid sequences of FLRG are respectively set out in SEQ ID NOs: 7 and 8 while the DNA and amino acid sequences of GASP-I are respectively set out in SEQ ID NOs: 9 and 10. The rAAV were administered bilaterally into the quadriceps and tibialis anterior muscles of 4-week-old wild-type C57B1/6 mice. Results are presented in Figure 18.

[0086] All animals treated with the myostatin inhibitors demonstrated an increase in body mass with an observable gross enhancement of muscles when analyzed at 725-days of age compared to GFP-treated controls (Figure 18a,b). Evaluation of individual muscle weights showed an increase in muscle mass for all myostatin inhibitor-treated animals, with the greatest increase in FS-treated animals compared to GFP controls. The increased muscle mass was found in the injected hindlimb muscles as well as remote muscles to the injection site, such as the triceps. Thus, these inhibitors were secreted into the circulation from the site of muscle injection, enhancing skeletal muscle mass at remote sites (Figure 18c). The enlarged muscle mass was accompanied by functional improvement demonstrated by an increase in hindlimb grip strength (Figure 18d). There was no effect on heart mass or histological appearance of cardiomyocytes, indicating that myostatin inhibition was selective to skeletal muscle tissue (data not shown). No change in reproductive capacity in mice treated with rAAV follistatin-344 was found. Furthermore, no histological/pathological alterations in the gonadal tissue of rAAV follistatin-344-treated mice was seen compared to controls (data not shown).

[0087] The results discussed above demonstrate that administration of AAV encoding myostatin inhibitors according to the invention enhances muscle and improves muscle function in vivo.

Example 6

[0088] The ability of rAAV follistain-344 to increase muscle strength in older mdx animals was also examined.

[0089] In experiments similar to those described in Example 3 and/or 4, at 210 days of age it was found that high dose (1 x 10 ¹ ' AAVl particles) FS administration increased muscle strength versus administration of AAVl-GFP in control animals -60 days post administration (see Figure 19). These results demonstrated that myostatin inhibition by FS treatment was beneficial in aged mdx animals that had undergone multiple rounds of muscle degeneration and regeneration.

[0090] Translation to a clinical parallel suggests that AAV-mediated FS gene therapy is indicated for the older DMD patient independent of replacing a missing gene and is indicated in combination therapy similar to that demonstrated for IGF-I and mini-dystrophin gene replacement [Abmayr et al, MoI Ther., 72:441-450 (2005)].

Example 7

[0091] The effects of rAAV-follistatin-344 were studied in non-human primates using cynomologous macaques.

[0092] The treatment included delivery of the follistatin sequences either under the control of the CMV promoter (rAAV-follistatin-344) or the mouse creatine kinase (MCK) promoter, to test the efficacy of a stronger (CMV) promoter versus a muscle specific promoter. The rAAV comprising the MCK promoter are designated "rAAV MCK follistatin-344" herein. Animals were injected into the quadriceps of one of their hindlimbs with 1 x 10 ¹³ viral genomes in a total volume of 1.5 ml spread over three injections per quadriceps muscle in a volume of 0.5 ml per injection. Studies conducted to date have been accompanied by an immunosuppression regimen of Tacrolimus (1.5 mg/kg body weight) and microphenylate (50 mg/kg body weight) administered daily 1 week prior to vector delivery and daily post-vector injection. Animals have been followed bi-weekly for any evidence of toxicity, immune response and levels of Follistatin. No adverse events have been noted in any of the studies conducted to date, that includes a total of 6 animals (3 animals with CMV-, 3 animals with MCK-Follistatin). All animals have had detectable levels of Follistatin detected in the plasma, with the greatest amounts found in the CMV-Follistatin treated animals (Figure 20).

[0093] A biopsy of muscle from the rAAV MCK follistatin-344 treated animals demonstrated a significant increase in the diameter of myo fibers in the injected muscles indicating that the lower expressed MCK-Follistatin was having biological effect to induce muscle hypertrophy in treated animals at 14-weeks post-injection (Figure 21).

[0094] While the present invention has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.

[0095] All documents referred to in this application are hereby incorporated by reference in their entirety.