Adeno-associated virus vector delivery of micro-dystrophin to treat muscular dystrophy

Abstract

To provide adeno-associated virus vector delivery of micro-dystrophin to treat muscular dystrophy.SOLUTION: The invention provides recombinant AAV vectors comprising a miniaturized human micro-dystrophin gene, and methods of using the recombinant vectors to reduce or prevent fibrosis in subjects suffering from muscular dystrophy. The present invention is directed to gene therapy methods that directly reduce the three primary components of connective tissue (collagen 1, collagen 3 and fibronectin) by delivering the microRNA miR29. In this system, the miR29 binds to the 3' UTR of the collagen and fibronectin gene to down-regulate expression. The invention is directed to gene therapy vectors, e.g., AAV, expressing the guide strand of the microRNA miR29, and a method of delivering miR29 to the muscle to reduce and / or prevent fibrosis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 323,163, filed April 15, 2016, and U.S. Provisional Application No. 62 / 473,253, filed March 17, 2017, both of which are incorporated by reference in their entireties. Technical Field

[0002] The present invention provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, that express a miniaturized human micro-dystrophin gene, and methods of using these vectors to reduce and prevent fibrosis in subjects suffering from muscular dystrophy. The present invention also provides combination gene therapies that protect muscle fibers from damage and increase muscle strength. [Background technology]

[0003] The importance of muscle mass and strength for daily activities such as movement and breathing, as well as for whole-body metabolism, is unquestionable. Impairments in muscle function result in muscular dystrophies (MDs), characterized by muscle weakness and wasting and severely impacting quality of life. The most well-characterized MDs result from mutations in genes encoding members of the dystrophin-associated protein complex (DAPC). These MDs result from membrane fragility associated with loss of sarcolemma-cytoskeleton tethering by DAPCs. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle diseases, affecting 1 in 5,000 newborn boys.

[0004] This application includes two translational approaches to developing a treatment for DMD. Fibrotic infiltration is severe in DMD and poses a significant obstacle to any potential therapy. It is also important to consider that gene replacement alone is hampered by the severity of fibrosis already present in very young children with DMD. Indeed, muscle biopsies at the usual age of diagnosis, 4-5 years of age, demonstrate highly significant levels of fibrosis.

[0005] DMD is caused by mutations in the DMD gene, which result in reduced mRNA and the absence of dystrophin, a 427-kD sarcolemmal protein associated with the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51(6):919-28, 1987). DAPC consists of multiple proteins in the sarcolemma that form structural links between the extracellular matrix (ECM) and the cytoskeleton via the actin-binding protein dystrophin and the laminin-binding protein α-dystroglycan. These structural links act to stabilize the sarcolemma during contraction and protect it from contraction-induced damage. With dystrophin loss, membrane fragility leads to sarcolemmal rupture and calcium influx, triggering calcium-activated proteases and segmental fiber necrosis (Straub et al., Curr Opin. Neurol. 10(2):168-75, 1997). This uncontrolled cycle of muscle degeneration and regeneration ultimately depletes the muscle stem cell population (Sacco et al., Cell, 2010. 143(7): p. 1059-71; Wallace et al., Annu Rev Physiol, 2009. 71: p. 37-57), resulting in progressive muscle weakness, endomysial inflammation, and fibrotic scar formation.

[0006] Without membrane stabilization from dystrophin or microdystrophin, DMD exhibits an uncontrolled cycle of tissue damage and repair, ultimately resulting in connective tissue proliferation replacing lost muscle fibers with fibrotic scar tissue. Fibrosis is characterized by the excessive deposition of ECM matrix proteins, including collagen and elastin. ECM proteins are primarily produced from cytokines, such as TGFβ, released by activated fibroblasts in response to stress and inflammation. While the primary pathological hallmark of DMD is muscle fiber degeneration and necrosis, fibrosis as a pathological consequence has an equal impact. Excessive production of fibrotic tissue limits muscle regeneration and contributes to the progressive muscle weakness of DMD patients. In one study, the presence of fibrosis in initial DMD muscle biopsies was highly correlated with poor motor outcomes at 10-year follow-up (Desguerre et al., J Neuropathol Exp Neurol, 2009. 68(7):762-7). These results point to fibrosis as a major cause of DMD muscle dysfunction and emphasize the need to develop therapies to reduce fibrotic tissue. Most antifibrotic therapies tested in mdx mice act to block fibrotic cytokine signaling by inhibiting the TGFβ pathway. MicroRNAs (miRNAs) are ~22-nucleotide single-stranded RNAs that mediate gene silencing at the post-transcriptional level by pairing with bases within the 3'UTR of mRNAs, inhibiting translation, or promoting mRNA degradation. A 7-bp seed sequence at the 5' end of the miRNA targets the miRNA, and additional recognition is provided by the remainder of the target sequence and its secondary structure. miRNAs play an important role in muscle disease pathology and exhibit expression profiles that are uniquely dependent on the type of muscular dystrophy in question (Eisenberg et al., Proc Natl Acad Sci USA, 2007. 104(43):p.17016-21). Increasing evidence suggests that miRNAs are involved in fibrotic processes in many organs, including the heart, liver, kidney, and lung (Jiang et al., Proc Natl Acad Sci USA, 2007.104(43):p.17016-21). Recently, downregulation of miR-29 has been shown to contribute to cardiac fibrosis (Cacchiarelli et al., Cell Metab, 2010.12(4):p.341-51), and reduced expression of miR-29 has been genetically associated with muscle in human DMD patients (Eisenberg et al. Proc Natl Acad Sci USA, 2007.104(43):p.17016-2). The miR-29 family consists of three family members expressed from two bicistronic miRNA clusters. MiR-29a is coexpressed with miR-29b (miR-29b-1), and miR-29c is coexpressed with a second copy of miR-29b (miR-29b-2). The miR-29 family shares a conserved seed sequence, with miR-29a and miR-29b each differing by a single base from miR-29c. Furthermore, electroporation of miR-29 plasmids (cluster of miR-29a and miR-29b-1) into mdx mouse muscle reduced the expression levels of ECM components, collagen, and elastin, and significantly reduced collagen deposition in muscle sections within 25 days of treatment (Cacchiarelli et al., Cell Metab, 2010, 12(4):341-51).

[0007] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length, including 145-nucleotide inverted terminal repeats (ITRs). There are several serotypes of AAV. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the nucleotide sequence of the AAV serotype 2 (AAV2) genome is described in Ruffing et al. The present invention is presented in Srivastava et al., J Virol, 45:555-564 (1983), as modified by Srivastava et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077, 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_001862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos. 7,282,199 and 7,790,449 regarding AAV-8), and the AAV-9 genome is described in Gao et al. The AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The AAVrh74 serotype is described in Rodino-Klapac et al. J. Trans. Med. 5:45 (2007). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19), linked by differential splicing of a single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The 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 encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation 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).

[0008] AAV has 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 in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, enabling the potential for targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the lifespan of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA within a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making cryopreservation of AAV less important. AAV can be lyophilized. Finally, AAV-infected cells do not tolerate superinfection.

[0009] 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., Mol Ther, 2:619-623 (2000); and Chao et al., Mol Ther, 4:217-222 (2001). Furthermore, because muscle is highly vascularized, recombinant AAV transduction resulted in the appearance of transgene products in the systemic circulation after intramuscular injection, as described by 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). Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle fibers possess the cellular factors necessary for proper antibody glycosylation, folding, and secretion, indicating that muscle can stably express secreted protein therapeutics. Both gene restoration and reduction of fibrosis are necessary for functional improvement in patients with DMD and other muscular dystrophies. There is a need for methods to reduce fibrosis that can be repaired by gene restoration for more effective treatment of DMD and other muscular dystrophies. miR29 is a potential gene regulator and an ideal candidate for reducing muscle fibrosis. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Hoffman et al., Cell 51(6):919-28,1987 Summary of the Invention [Means for solving the problem]

[0011] The present invention is directed to a gene therapy that directly reduces three major components of connective tissue (collagen 1, collagen 3, and fibronectin) by delivering the microRNA miR29. In this system, miR29 binds to the 3'UTRs of the collagen and fibronectin genes and downregulates their expression. The present invention is directed to gene therapy vectors, e.g., AAVs, that express the guide strand of the microRNA miR29, as well as methods for delivering miR29 to muscle to reduce and / or prevent fibrosis.

[0012] Additionally, the present invention provides a combination therapy and approach for reducing and preventing fibrosis using a gene therapy vector that delivers miR-29, which suppresses fibrosis, along with miR-dystrophin, which addresses the genetic defect observed in DMD. As shown in Examples 5-7, the combination treatment resulted in greater reduction in fibrosis, increased muscle size, and increased muscle strength.

[0013] In one embodiment, the present invention provides an rAAV vector that expresses miR-29. For example, the rAAV vector includes a polynucleotide sequence that expresses miR29c, such as a miR-29c target guide strand of SEQ ID NO: 3, a miR-29c guide strand of SEQ ID NO: 4, and a nucleotide sequence that includes native miR-30 and a stem-loop (SEQ ID NO: 5). An exemplary polynucleotide sequence that includes the miR-29c cDNA in the miR-30 backbone is shown as SEQ ID NO: 2 (FIG. 1).

[0014] An exemplary rAAV of the invention is pAAV.CMV.Mir29C comprising the nucleotide sequence of SEQ ID NO: 1, wherein the CMV promoter spans nucleotides 120-526, the EF1a intron spans nucleotides 927-1087 and nucleotides 1380-1854, the guide strand of miR-29c spans nucleotides 1257-1284, the shRNA-miR29-c with primary seed sequence spans nucleotides 1088-1375, and the polyA sequence spans nucleotides 1896-2091. In one embodiment, the rAAV vector of the invention is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0015] Another exemplary rAAV of the invention is pAAV.MHC.Mir29C comprising the nucleotide sequence of SEQ ID NO: 12, wherein the MCK enhancer spans nucleotides 190-395, the MHC promoter spans nucleotides 396-753, the EF1a intron spans nucleotides 1155-1315 and nucleotides 1609-2083, the guide strand of miR-29c spans nucleotides 1487-1512, shRNA-miR29-c with the primary seed sequence spans nucleotides 1316-1608, and a polyA sequence spans nucleotides 2094-2146. In one embodiment, the rAAV vector of the invention is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0016] In another embodiment, the rAAV vectors of the invention can be operably linked to a muscle-specific regulatory element, such as a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor MEF, a muscle creatine kinase (MCK), a tMCK (truncated MCK), a myosin heavy chain (MHC), a C5-12 (synthetic promoter), a mouse creatine kinase enhancer element, a fast skeletal troponin C gene element, a slow cardiac troponin C gene element, a slow troponin I gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a glucocorticoid response element (GRE).

[0017] For example, any of the rAAV vectors of the invention is operably linked to a muscle-specific regulatory element comprising the MCK enhancer nucleotide sequence of SEQ ID NO:10 and / or the MCK promoter sequence of SEQ ID NO:11.

[0018] The present invention also provides pharmaceutical compositions (or sometimes simply referred to herein as "compositions") comprising any of the rAAV vectors of the present invention.

[0019] In another embodiment, the invention provides a method for producing rAAV vector particles, comprising culturing cells transfected with any of the rAAV vectors of the invention and recovering the rAAV particles from the supernatant of the transfected cells. The invention also provides viral particles comprising any of the recombinant AAV vectors of the invention.

[0020] In another embodiment, the present invention provides a method for reducing fibrosis in a subject in need thereof, comprising administering any rAAV vector of the present invention that expresses a therapeutically effective amount of miR-29.For example, any rAAV of the present invention is administered to a subject suffering from muscular dystrophy to reduce fibrosis, particularly to reduce the fibrosis in the subject's skeletal muscle or cardiac muscle.These methods can further comprise administering a rAAV vector that expresses microdystrophin.

[0021] "Fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and abnormal repair processes in tissues after injury, including skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas. Deposited ECM components include fibronectin and collagen, e.g., collagen 1, collagen 2, or collagen 3.

[0022] In another embodiment, the present invention provides a method for preventing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of any recombinant AAV vector of the present invention expressing miR-29.For example, any rAAV of the present invention is administered to a subject suffering from muscular dystrophy to prevent fibrosis, for example, the rAAV of the present invention expressing miR-29 is administered before fibrosis is observed in the subject.Furthermore, the rAAV of the present invention expressing miR-29 is administered to a subject at risk of developing fibrosis, such as a subject suffering from or diagnosed with muscular dystrophy, such as DMD.The rAAV of the present invention is administered to a subject suffering from muscular dystrophy to prevent new fibrosis in these subjects.These methods can further comprise administering a rAAV vector expressing microdystrophin.

[0023] The present invention also provides methods for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of any of the rAAV vectors of the invention expressing miR-29. These methods can further comprise administering an rAAV vector expressing microdystrophin. The terms "combination therapy" and "combination treatment" refer to the administration of an rAAV vector of the invention expressing miR-29 and an rAAV vector expressing micro-dystrophin.

[0024] In any of the methods of the present invention, the subject may be suffering from a muscular dystrophy, such as DMD, Becker muscular dystrophy, or any other dystrophin-associated muscular dystrophy. In addition, in any of the methods of the present invention, the subject may be suffering from a dystrophinopathy.

[0025] In another embodiment, the present invention provides a recombinant AAV vector comprising a nucleotide sequence encoding a micro-dystrophin protein. The present invention provides an rAAV vector comprising: a) a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO:7 and encoding a functional micro-dystrophin protein; b) the nucleotide sequence of SEQ ID NO:7; or c) the nucleotide sequence of SEQ ID NO:9.

[0026] An exemplary rAAV expressing microdystrophin of the invention is pAAV.mck.microdystrophin, which comprises the nucleotide sequence of SEQ ID NO:9 and is shown in Figures 10 and 11. This rAAV vector comprises an MCK promoter, a chimeric intron sequence, the coding sequence of the microdystrophin gene, polyA, ampicillin resistance, and a pGEX plasmid backbone with a pBR322 origin of replication. In one aspect, the recombinant AAV vector of the invention is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh.74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0027] The present invention provides rAAV vectors encoding microdystrophin proteins that are at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, or 99% sequence identical to, for example, SEQ ID NO: 8, where the protein retains microdystrophin activity. The microdystrophin protein provides stability to muscle membrane during muscle contraction, e.g., microdystrophin acts as a shock absorber during muscle contraction.

[0028] The present invention provides microdystrophin-expressing rAAV vectors comprising a nucleotide sequence that has, for example, at least 65%, at least 70%, at least 75%, at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more typically at least 90%, 91%, 92%, 93%, or 94%, and even more typically at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:7, and encodes a functional microdystrophin protein.

[0029] The present invention provides a microdystrophin-expressing rAAV vector comprising a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO: 7 or its complement and encodes a functional microdystrophin protein.

[0030] The term "stringent" refers to conditions generally understood in the art as stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide, or other denaturing agents) can also be used, but the rate of hybridization will be affected. In instances where deoxyoligonucleotide hybridization is a concern, additional exemplary stringent hybridization conditions include washing in 6x SSC 0.05% sodium pyrophosphate at 37°C (14-base oligo), 48°C (17-base oligo), 55°C (20-base oligo), and 60°C (23-base oligo).

[0031] Other agents may be included in the hybridization and wash buffers to reduce nonspecific and / or background hybridization. Examples include 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate, NaDodSO4 (SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or other non-complementary DNA), and dextran sulfate; however, other suitable agents may also be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are typically performed at pH 6.8-7.4, although under typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one of skill in the art to accommodate these variables and allow DNAs with different sequence relatedness to form hybrids.

[0032] In another embodiment, the microdystrophin-expressing rAAV vector comprises the coding sequence of the microdystrophin gene operably linked to a muscle-specific regulatory element, such as a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor (MEF), a muscle creatine kinase (MCK), a tMCK (truncated MCK), a myosin heavy chain (MHC), a C5-12 (synthetic promoter), a mouse creatine kinase enhancer element, a fast skeletal troponin C gene element, a slow cardiac troponin C gene element, a slow troponin I gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a glucocorticoid response element (GRE).

[0033] Additionally, the present invention provides a micro-dystrophin-expressing rAAV vector comprising a muscle-specific regulatory element comprising the nucleotide sequence of SEQ ID NO:10 or SEQ ID NO:11.

[0034] The present invention also provides pharmaceutical compositions (or sometimes simply referred to herein as "compositions") comprising any of the rAAV vectors of the present invention.

[0035] In another embodiment, the invention provides a method for producing rAAV vector particles, comprising culturing cells transfected with any of the rAAV vectors of the invention and recovering the rAAV particles from the supernatant of the transfected cells. The invention also provides viral particles comprising any of the recombinant AAV vectors of the invention.

[0036] The present invention also provides a method for producing a functional micro-dystrophin protein, comprising infecting a host cell with a recombinant AAV vector expressing the micro-dystrophin of the present invention, and expressing the functional micro-dystrophin protein in the host cell.

[0037] In another embodiment, the present invention provides a method for reducing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of any of the rAAV vectors of the present invention that express micro-dystrophin. For example, any of the rAAV vectors of the present invention is administered to a subject suffering from muscular dystrophy or dystrophinopathy to reduce fibrosis, particularly in the subject's skeletal or cardiac muscle.

[0038] In another embodiment, the present invention provides a method for preventing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of any recombinant AAV vector of the present invention that expresses micro-dystrophin.For example, any rAAV of the present invention is administered to a subject suffering from muscular dystrophy or dystrophinopathy to prevent fibrosis, for example, the rAAV of the present invention that expresses micro-dystrophin is administered before fibrosis is observed in the subject.In addition, the rAAV of the present invention that expresses micro-dystrophin is administered to a subject at risk of developing fibrosis, such as a subject suffering from or diagnosed with dystrophinopathy or muscular dystrophy, for example, DMD or Becker muscular dystrophy.The rAAV of the present invention is administered to a subject suffering from dystrophinopathy or dystrophinopathy muscular dystrophy to prevent new fibrosis in these subjects.

[0039] The present invention also provides a method for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy or a dystrophinopathy, comprising administering a therapeutically effective amount of any of the rAAV vectors of the present invention expressing miR-29.

[0040] Any of the aforementioned methods comprising administering an rAAV vector of the present invention expressing miR-29c can further comprise administering any of the microdystrophin-expressing rAAV vectors described herein. The terms "combination therapy" and "combination treatment" refer to the administration of an rAAV vector of the present invention expressing miR-29c and an rAAV vector expressing microdystrophin.

[0041] In the method of administering an rAAV vector expressing miR-29 and an rAAV vector expressing a micro-dystrophin protein, these rAAV vectors may be administered simultaneously, or sequentially, with the rAAV vector expressing miR29 administered immediately before the rAAV expressing the micro-dystrophin protein, or sequentially, with the rAAV vector expressing miR29 administered immediately after the rAAV expressing the micro-dystrophin protein. Alternatively, the method of the present invention may be carried out in which the AAV vector expressing the micro-dystrophin protein is administered within about 1 to 5 hours, 5 to 12 hours, 12 to 15 hours, or 15 to 24 hours after administration of the rAAV expressing miR-29, or in which the AAV vector expressing the micro-dystrophin protein is administered within about 1 to 5 hours, 5 to 12 hours, 12 to 15 hours, or 15 to 24 hours before administration of the rAAV expressing miR-29c. Alternatively, the method of the invention is carried out in which an AAV vector expressing a micro-dystrophin protein is administered within about 1, 6, 12, or 24 hours after administration of an rAAV expressing miR-29, or in which an AAV vector expressing a micro-dystrophin protein is administered within about 1, 6, 12, or 24 hours before administration of an rAAV expressing miR-29c.

[0042] The present invention contemplates administering any of the AAV vectors of the invention to a patient diagnosed with a dystrophinopathy or muscular dystrophy, such as DMD or Becker muscular dystrophy, before fibrosis is observed in the subject, or before muscle weakness is experienced in the subject, or before muscle mass is lost in the subject.

[0043] The present invention also contemplates administering any of the rAAVs of the present invention to subjects with a dystrophinopathy or muscular dystrophy, such as DMD or Becker muscular dystrophy, who have already developed fibrosis, to prevent new fibrosis in these subjects. The present invention also provides for administering any of the rAAVs of the present invention to patients with muscular dystrophy who have already experienced muscle weakness or muscle mass loss to protect their muscles from further damage.

[0044] In any of the methods of the invention, the rAAV vector is administered by intramuscular or intravenous injection.

[0045] Additionally, in any of the methods of the invention, the rAAV vector is administered systemically, e.g., the rAAV vector or composition is administered parenterally by injection, infusion, or implantation.

[0046] In another embodiment, the present invention provides a composition for reducing fibrosis in a subject in need thereof, comprising either a miR29-expressing rAAV vector or a micro-dystrophin-expressing rAAV vector, or comprising both a miR-29-expressing rAAV vector and a micro-dystrophin-expressing rAAV vector. Furthermore, the present invention provides a composition for preventing fibrosis in a patient suffering from a dystrophinopathy or muscular dystrophy, such as DMD or Becker muscular dystrophy, comprising either a miR29-expressing recombinant AAV vector or a micro-dystrophin-expressing rAAV vector, or comprising both a miR-29-expressing rAAV vector and a micro-dystrophin-expressing rAAV vector.

[0047] The present invention also provides compositions for increasing muscle strength and / or muscle mass in a subject suffering from a dystrophinopathy or muscular dystrophy, such as DMD or Becker muscular dystrophy, comprising any of the rAAV vectors of the present invention that express miR29 or any of the rAAV vectors that express a microdystrophin protein, or comprising both an rAAV vector that expresses miR-29 and an rAAV vector that expresses a microdystrophin protein.

[0048] In further embodiments, the present invention provides compositions for the treatment of dystrophinopathy or muscular dystrophy, such as DMD or Becker muscular dystrophy, comprising any of the rAAV vectors of the present invention expressing miR29 or any of the rAAV vectors expressing microdystrophin protein, or comprising both an rAAV vector expressing miR-29 and an rAAV vector expressing microdystrophin protein.

[0049] The compositions of the present invention are formulated for intramuscular injection or intravenous injection.The compositions of the present invention are also formulated for systemic administration, such as parenteral administration by injection, infusion or implantation.In addition, any of the compositions are formulated for administration to the subject suffering from dystrophinopathy or muscular dystrophy, such as DMD, Becker's dystrophy or any other dystrophin-related muscular dystrophy.

[0050] In a further embodiment, the present invention provides use of either an rAAV vector of the present invention expressing miR29 or an rAAV vector expressing microdystrophin, or both an rAAV vector expressing miR-29 and an rAAV vector expressing microdystrophin, for the preparation of a medicament for reducing fibrosis in a subject in need thereof. For example, the subject is suffering from a dystrophinopathy or muscular dystrophy, such as DMD, Becker muscular dystrophy, or any other dystrophin-associated muscular dystrophy, and is in need of treatment.

[0051] In another embodiment, the present invention provides the use of any of the rAAV vectors of the present invention expressing miR29 or any of the rAAV vectors expressing microdystrophin, or both an rAAV vector expressing miR-29 and an rAAV vector expressing microdystrophin, for the preparation of a medicament for preventing fibrosis in a subject suffering from muscular dystrophy. Additionally, the present invention provides the use of any of the recombinant AAV vectors of the present invention expressing miR29 or any of the rAAV vectors expressing microdystrophin, or both an rAAV vector expressing miR-29 and an rAAV vector expressing microdystrophin, for the preparation of a medicament for increasing muscle strength and / or muscle mass in a subject suffering from a dystrophinopathy or muscular dystrophy, such as DMD or Becker muscular dystrophy.

[0052] The present invention contemplates the use of any of the AAV vectors of the invention for the preparation of a medicament for administration to a patient diagnosed with DMD before fibrosis is observed in the subject, or before muscle weakness occurs in the subject, or before muscle mass is lost in the subject.

[0053] The present invention also contemplates the use of any of the AAV vectors of the present invention to prepare a medicament for administration to subjects with muscular dystrophy who have already developed fibrosis to prevent new fibrosis in these subjects. The present invention also provides for the administration of any of the rAAV vectors of the present invention to patients with muscular dystrophy who already have muscle weakness or loss of muscle mass to protect their muscles from further damage.

[0054] The present invention also provides the use of either an rAAV vector of the present invention expressing miR296 or an rAAV vector expressing microdystrophin, or both an rAAV vector expressing miR-29 and an rAAV vector expressing microdystrophin, for the preparation of a medicament for the treatment of muscular dystrophy.

[0055] In any of the uses of the present invention, the medicament is formulated for intramuscular injection. Furthermore, any of the medicaments can be prepared for administration to a subject suffering from muscular dystrophy, such as DMD or any other dystrophin-related muscular dystrophy.

[0056] Furthermore, any of the medicaments of the present invention may be a combination therapy in which an rAAV vector expressing miR-29 and an rAAV vector expressing micro-dystrophin are administered simultaneously, or sequentially with an rAAV vector expressing miR29 administered immediately before an rAAV vector expressing micro-dystrophin, or sequentially with an rAAV vector expressing miR29 administered immediately after an rAAV vector expressing micro-dystrophin. Alternatively, the medicament comprises administration of an AAV vector expressing micro-dystrophin administered within about 1-5 hours after administration of an rAAV expressing miR-29c, or the medicament comprises an AAV vector expressing micro-dystrophin administered within about 1-5 hours before administration of an rAAV expressing miR-29c. The present invention provides, for example, the following items. (Item 1) a) a nucleotide sequence having at least 85% identity to the nucleotide sequence of SEQ ID NO: 7 and encoding a functional micro-dystrophin protein; b) the nucleotide sequence of SEQ ID NO: 7, or c) A recombinant AAV vector comprising the nucleotide sequence of SEQ ID NO: 9. (Item 2) 2. The recombinant AAV vector of item 1, wherein the vector is of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. (Item 3) 3. The recombinant AAV vector of item 1 or 2, wherein the polynucleotide sequence is operably linked to a muscle-specific regulatory element. (Item 4) 4. The recombinant AAV vector of item 3, wherein the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a muscle cell-specific enhancer-binding factor mef, a muscle creatine kinase (MCK), a truncated MCK (tMCK), a myosin heavy chain (MHC), C5-12, a mouse creatine kinase enhancer element, a fast skeletal troponin C gene element, a slow cardiac troponin C gene element, a slow troponin I gene element, a hypoxia-inducible nuclear factor, a steroid-inducible element, or a glucocorticoid response element (gre). (Item 5) 5. The recombinant AAV vector of item 3 or 4, wherein the muscle-specific regulatory element comprises the nucleotide sequence of SEQ ID NO: 10 or SEQ ID NO: 11. (Item 6) A composition comprising the recombinant AAV vector of any one of items 1 to 5. (Item 7) A method for treating muscular dystrophy or dystrophinopathy, comprising administering a therapeutically effective amount of the recombinant AAV vector according to any one of items 1 to 5 or the composition according to item 6. (Item 8) A method for reducing or preventing fibrosis in a subject suffering from muscular dystrophy or a dystrophinopathy, comprising administering a therapeutically effective amount of the recombinant AAV vector of any one of Items 1 to 5 or the composition of Item 6. (Item 9) A method for increasing muscle strength or muscle mass in a subject suffering from muscular dystrophy or dystrophinopathy, comprising administering a therapeutically effective amount of the recombinant AAV vector of any one of Items 1 to 5 or the composition of Item 6. (Item 10) 10. The method of any one of items 7 to 9, wherein the recombinant AAV vector or the composition is administered by intramuscular injection, intravenous injection, parenteral administration, or systemic administration. (Item 11) 11. The method of any one of items 7 to 10, wherein the recombinant AAV is administered before fibrosis is observed in the subject, before muscle strength is lost in the subject, or before muscle mass is lost in the subject. (Item 12) 12. The method according to any one of items 7 to 11, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. (Item 13) A composition comprising the recombinant AAV vector of any one of items 1 to 5 for the treatment of muscular dystrophy. (Item 14) A composition comprising the recombinant AAV vector of any one of items 1 to 5 for reducing or preventing fibrosis in a subject suffering from muscular dystrophy. (Item 15) A composition comprising the recombinant AAV vector of any one of items 1 to 5 for increasing muscle strength in a subject suffering from muscular dystrophy. (Item 16) 16. The composition of any one of items 13 to 15, wherein the composition is administered before fibrosis is observed in the subject, or before muscle strength is lost in the subject, or before muscle mass is lost in the subject. (Item 17) 17. The composition of any one of items 13 to 16, wherein the subject suffers from Duchenne muscular dystrophy or Becker muscular dystrophy. (Item 18) Use of a recombinant AAV vector according to any one of items 1 to 5 or a composition according to item 6 for the preparation of a medicament for the treatment of muscular dystrophy. (Item 19) Use of a recombinant AAV vector according to any one of items 1 to 5 or a composition according to item 6 for the preparation of a medicament for reducing or preventing fibrosis in a subject suffering from muscular dystrophy. (Item 20) Use of a recombinant AAV vector according to any one of items 1 to 5 or a composition according to item 6 for the preparation of a medicament for increasing muscle strength or muscle mass in a subject suffering from muscular dystrophy. (Item 21) 19. The use of any one of items 16 to 18, wherein the agent is administered before fibrosis is observed in the subject, or before muscle strength is lost in the subject, or before muscle mass is lost in the subject. (Item 22) 22. The use according to any one of items 18 to 21, wherein the subject suffers from Duchenne muscular dystrophy or Becker muscular dystrophy. (Item 23) 23. The composition or use according to any one of items 13 to 22, wherein the composition or medicament is formulated for intramuscular administration, intravenous injection, parenteral administration, or systemic administration. (Item 21) A method for producing a functional micro-dystrophin protein, comprising infecting a host cell with the recombinant AAV vector according to any one of items 1 to 5, and expressing the functional micro-dystrophin protein in the host cell. [Brief explanation of the drawings]

[0057] [Figure 1] 1 provides a schematic representation of the nucleotide sequence of miR-29c in the rAAV vector scAAVCrh.74.CMV.miR29c and the native miR-30 backbone and predicted hairpin structure. [Figure 2A]We show that injection of miR-29c into muscle reduces collagen throughout the muscle and restores miR-29c expression. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 3A] Figure 1 shows that injection of miR-29c improves absolute muscle strength (Panel A) and specific muscle strength (Panel B) but does not protect against contraction-induced damage (Panel C). [Figure 3B] Same as above. [Figure 3C] Same as above. [Figure 4A] The number of myofibers expressing microdystrophin, which measures the efficacy of transgene delivery, is shown. [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 5A] Figure 1 shows that co-delivery of miR-29c with miR-dystrophin reduces collagen expression (Panel A) and fibrosis-induced dystrophin expression. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6A] Figure 1 shows that intramuscular injection of miR-29c / microdystrophin inhibits extracellular matrix in mdx / utrn+ / - mice, as measured by collagen 1 alpha (Panel A), collagen 3 alpha (Panel B), fibronectin (Panel C), and TGF-β (Panel D). [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 7A] 1 demonstrates that intramuscular injection of miR-29c increased absolute force in muscle (Panel A), normalized specific force (Panel B), and provided additional protection from contraction-induced injury (Panel C). [Figure 7B] Same as above. [Figure 7C] Same as above. [Figure 8]Figure 1 shows that the miR-29c / μ-dys combination increases muscle size in treated mice at 3 months of age. Sections of treated and untreated mdx / utrn+ / - gastrocnemius muscles stained with picrosirius red, which stains collagen, are shown. Areas of fibrosis are pink, while intact muscle is green. At the macroscopic level, the miR-29c / μ-dys combination reduces fibrosis and increases total cross-sectional area. [Figure 9A] Treatment with miR-29c co-delivered with microdystrophin increased muscle hypertrophy and hyperplasia, as shown by increased total weight of injected gastrocnemius muscles (Panel A), increased mean fiber size (Panel B), increased muscle cross-sectional area (Panel D, uninjected: 24.6 vs. miR-29c: 26.3 vs. microdys: 26.6 vs. microdys / miR-29c: 33.1), and increased myofiber number (Panel E) compared with either injected muscle alone, although the number of myofibers per unit area was unaffected (Panel F). Panel C compares mdx / utrn+ / - controls with miR-29c / μ-dys-treated mdx / utrn+ / -, with an average diameter increased from 25.96 to 30.97 μm. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Same as above. [Figure 10A]This shows that early treatment with AAV.miR-29c / microdystrophin combination therapy is more effective in reducing fibrosis and ECM expression. Panel A shows picrosirius red staining of wild-type, uninjected, and AAV.miR-29c, AAV.microdystrophin, and AAV.miR-29c / AAV.microdystrophin-injected mice at 4-5 weeks of age, harvested 12 weeks after injection. Panel B provides quantification of picrosirius red staining, showing that the combination-treated muscles had a 51.1% reduction in collagen compared to uninjected GAS muscles. Panel C demonstrates that qRT-PCR confirms increased miR-29c transcript levels in the treated cohort. Semiquantitative qRT-PCR shows significant reductions in collagen I and III (panels d and e), fbn (panel f), and TGF-β1 (panel g) levels in AAV.miR-29c / AAV.microdystrophin-treated muscle compared with the contralateral limb and monotherapy, respectively. Error bars, SEM, n = 5 (scAAVrh.74.CMV.miR-29c), n = 5 (scAAVrh.74.CMV.miR-29c / ssAAVrh.74.MCK.microdystrophin), n = 6 (ssAAVrh.74.MCK.microdystrophin), n = 9 (mdx / utrn+ / − mice). One-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. [Figure 10F] Same as above. [Figure 10G] Same as above. [Figure 11]We demonstrate that early combination therapy restores force and protects against contraction-induced damage. Measurements of absolute (Panel A) and normalized specific force (Panel B) after tetanic contractions of GAS muscles injected with all three treatments were significantly increased compared to untreated mdx / utrn+ / - muscles (Panel C). Muscles were then assessed for loss of force after repeated eccentric contractions. Only mice treated with miR-29c / microdystrophin combination and microdystrophin monotherapy showed protection from force loss compared to untreated mdx / utrn+ / - muscles (blue). Two-way ANOVA demonstrates significance in the decay curves. Error bars, SEM of n = 5 (rAAVrh.74.CMV.miR-29c), n = 6 (rAAVrh.74.CMV.miR-29c / rAAVrh.74.MCK.microdystrophin), n = 5 (rAAVrh74.MCK.microdystrophin), and n = 15 (mdx / utrn+ / - mice). One-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). [Figure 12] This figure shows that miR-29c / microdystrophin combination treatment increases muscle size in treated mice at 1 month of age. Treated and untreated mdx / utrn+ / - GAS muscles were sectioned and stained with picrosirius red, which stains collagen. Areas of fibrosis are pink, while intact muscle is green. At the macroscopic level, the miR-29c / microdystrophin combination reduces fibrosis and increases total cross-sectional area. [Figure 13A]This shows that early treatment (4-5 weeks) with AAV.MCK.miR-29c / microdystrophin combination therapy is more effective in reducing fibrosis and ECM expression. Panel A provides picrosirius red staining of uninjected and AAV.MCK.miR-29c / AAV.MCK.microdystrophin-injected mice at 4-5 weeks of age, harvested 12 weeks after injection. Original magnification, 20x. Panel B provides quantification of picrosirius red staining, showing that combination-treated muscles had a 50.9% reduction in collagen compared to untreated GAS muscles. Panel C provides qRT-PCR confirming increased miR-29c transcript levels in the treated cohort. Semiquantitative qRT-PCR shows a significant decrease in collagen 1A (Col1A, panel D) and collagen 3A (Col3A, panel E), fibronectin (Fbn, panel F), and Tgfβ1 (panel G) levels in AAV.MCK.miR-29c / AAV.microdystrophin-treated muscles compared to the contralateral limb (*p<0.05, ****p<0.0001). [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 13D] Same as above. [Figure 13E] Same as above. [Figure 13F] Same as above. [Figure 13G] Same as above. [Figure 14A]Late treatment (treatment at 12 weeks) with AAV.MCK.miR-29c / microdystrophin combination therapy is effective in reducing fibrosis and ECM expression. Panel A provides picrosirius red staining of untreated, AAV.MCK.miR-29c, and AAV.MCK.miR-29c / AAV.microdystrophin muscles 12 weeks after injection. Original magnification, 20x. Panel B provides quantification of picrosirius red staining, showing that combination-treated muscles had a 30.3% reduction in collagen compared to untreated GAS muscles. Panel C provides qRT-PCR confirming increased miR-29c transcript levels in the treated cohort. Semiquantitative qRT-PCR demonstrated significant decreases in collagen 1A (Col1A, panel D), collagen 3A (Col3A, panel E), fibronectin (Fbn, panel F), and Tgfβ1 (panel G) levels in AAV.miR-29c / AAV.miR-dystrophin-treated muscles compared to the contralateral limb. One-way ANOVA. All data represent the mean ± SEM. (**p<0.01, ****p<0.0001). [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 14D] Same as above. [Figure 14E] Same as above. [Figure 14F] Same as above. [Figure 14G] Same as above. [Figure 15A] (C) The results demonstrate that early combination therapy (treatment at 4–5 weeks) restored force and protected against contraction-induced damage. Measurements of absolute (Panel A) and normalized specific force (Panel B) after tetanic contractions of GAS muscles injected with MCK.miR-29c and microdystrophin were significantly increased compared to untreated mdx / utrn+ / − muscles. (D) Muscles were then assessed for loss of force after repeated eccentric contractions. Mice treated with miR-29c / microdystrophin combination and microdystrophin monotherapy showed protection from force loss compared to untreated mdx / utrn+ / − muscles (red). Two-way ANOVA. All data represent mean ± SEM (****P<0.0001). [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 16A] These results demonstrate that late combination therapy restored force and protected against contraction-induced injury. Measurements of absolute (Panel A) and normalized specific force (Panel B) after tetanic contractions of GAS muscles injected with rAAV expressing rAAV expressing rAAV.MCK.miR-29c and microdystrophin were significantly increased compared to untreated mdx / utrn+ / − muscles. In Panel C, muscles were then assessed for loss of force after repeated eccentric contractions. Mice co-treated with rAAV expressing rAAV expressing rAAV.MCK.miR-29c / microdystrophin showed protection from force loss compared to untreated mdx / utrn+ / − muscles (red). Two-way ANOVA. All data represent the mean ± SEM (**p<0.01, ****P<0.0001). [Figure 16B] Same as above. [Figure 16C] Same as above. [Figure 17A] The combined treatment increases muscle hypertrophy 3 months after injection. Panel A shows rAAV. MCK.miR-29c co-delivered with rAAV expressing microdystrophin did not increase the total weight of injected GAS. Panel B demonstrates that rAAV.MCK.miR-29c / microdystrophin-expressing rAAV combined treatment induced an increase in mean fiber size. Comparing mdx / utrn+ / - controls with miR-29c / microdystrophin-treated mdx / utrn+ / - mice, the mean diameter increased from 28.96 to 36.03 μm. Panel C shows that combined delivery resulted in a shift toward wild-type fiber size distribution. Panel D shows that the number of myofibers per mm2 in miR-29c / microdystrophin combined treatment was significantly lower than in untreated mice and wild-type mice (***p<0.01, ****p<0.0001). [Figure 17B] Same as above. [Figure 17C] Same as above. [Figure 17D] Same as above. [Figure 18A]The nucleic acid sequence of an exemplary rAAV vector (SEQ ID NO:1 pAAV.CMV.Mir29C) is provided, which contains the mature guide strand of miR-29c (nucleotides 1257-1284) and the native miR-30 backbone (nucleotides 1088-1375). The construct also contains a CMV promoter (nucleotides 120-526), two EF1a introns at nucleotides 927-1087 and 1380-1854, and polA at nucleotides 1896-2091. [Figure 18B] Same as above. [Figure 19] 1 provides a schematic diagram of the rAAV vector pAAV.MCK.micro-dystrophin. [Figure 20A] The nucleic acid sequence of an exemplary rAAV vector expressing microdystrophin (SEQ ID NO: 9, pAAV.MCK.microdystrophin) is provided. [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D] Same as above. [Figure 21A] 1 provides the nucleotide sequence of human microdystrophin nucleotide sequence (SEQ ID NO: 7). [Figure 21B] Same as above. [Figure 21C] Same as above. [Figure 22A] The nucleotide sequence of an exemplary rAAV vector (SEQ ID NO: 12, pAAV.MCK.Mir29C) is provided, which contains the mature guide strand of miR-29c (nucleotides 1487-1512) and the native miR-30 backbone (nucleotides 1088-1375). The construct also contains the MCK enhancer (nucleotides 190-395), the MCK promoter (nucleotides 396-753), two EF1a introns at nucleotides 1155-1315 and 1609-2083, and polA at nucleotides 2094-2148. [Figure 22B] Same as above. [Figure 22C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0058] The present invention provides gene therapy vectors, e.g., rAAV vectors, that overexpress the miR-29 microRNA, and methods for reducing and preventing fibrosis in patients with muscular dystrophy. The present invention also provides combination gene therapy methods, including administering a gene therapy vector expressing miR-29 in combination with a gene therapy vector expressing the micro-dystrophin deleted in DMD patients.

[0059] Muscle biopsies taken at the earliest age of diagnosis of DMD reveal significant connective tissue proliferation. Muscle fibrosis is detrimental in multiple ways. It reduces the normal transport of endomysial nutrients across the connective tissue barrier, reduces blood flow, deprives muscles of blood-borne nutrients, and functionally contributes to early loss of ambulation due to limb contractures. Over time, the treatment challenge multiplies as a result of significant fibrosis in the muscle, which can be observed in muscle biopsies comparing connective tissue proliferation at successive time points. This process continues to worsen, causing loss of ambulation and accelerating loss of control, especially in wheelchair-dependent patients.

[0060] Without a parallel approach to alleviate fibrosis, it is unlikely that the benefits of exon skipping, stop codon readthrough, or gene replacement therapy can be fully realized. Even small molecule or protein replacement strategies are likely to fail without an approach to alleviate muscle fibrosis. Previous studies in aged mdx mice with pre-existing fibrosis treated with AAV or microdystrophin showed that complete functional recovery could not be achieved (Human Molecular Genetics 22, 4929-4937 (2013)). Progression of DMD cardiomyopathy is also known to be accompanied by scar formation and fibrosis in the ventricular wall. MicroRNA delivery is particularly innovative due to its lack of immune barriers and relatively easy delivery. MicroRNAs are small (~200 bp) and therefore can be packaged into AAV along with therapeutic cassettes that correct or bypass gene defects.

[0061] As used herein, the term "AAV" is a general abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. Currently, there are 13 characterized serotypes of AAV. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, New York. However, since it is well known that the various serotypes are very closely related, both structurally and functionally, even at the genetic level, it is fully expected that these same principles will be applicable to additional AAV serotypes. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R.P.Tattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes, and they all possess related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by extensive cross-hybridization between genotypes along the length of the genome and heteroduplex analysis, which reveals the presence of similar self-annealing segments at the ends corresponding to the "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0062] As used herein, "AAV vector" refers to one or more polynucleotides of interest (or transgenes) flanked by AAV interterminal repeats (ITRs), which can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0063] An "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a viral particle consisting of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, production of an AAV vector particle necessarily includes production of an AAV vector, since such a vector is contained within the AAV vector particle.

[0064] AAV The recombinant AAV genome of the present invention comprises a nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The generation of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote skeletal muscle-specific expression, AAV1, AAV6, AAV8, or AAVrh.74 can be used.

[0065] The DNA plasmids of the present invention comprise the rAAV genome of the present invention. The DNA plasmids are introduced into cells permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for incorporation of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles in which the AAV genome is packaged, the rep and cap genes, and helper virus functions are provided to the cell are standard in the art. rAAV production requires that the following components be present in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and the helper virus functions. The AAV rep and cap genes can be of any AAV serotype from which a recombinant virus can be derived, and can be from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The generation of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0066] The method for generating packaging cells involves creating a cell line that stably expresses all components necessary for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the cell's genome. The AAV genome has been introduced into a bacterial plasmid by techniques such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or 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 advantage of this method is that the cells are selectable and are suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus rather than a plasmid to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0067] The 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., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Ratschin et al., Mol. Cell. Biol. 5:3251 (1985), McLaughlin et al., J. Virol., 62:1963 (1988), and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828), U.S. Pat. No. 5,173,414, WO95 / 13365 and corresponding U.S. Pat. No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. (1995) Vaccine 13:1244-1250, Paul et al. 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 incorporated herein by reference in their entireties, with particular emphasis on the portions of the documents relating to rAAV production.

[0068] Thus, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (an allogeneic 293 strain). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), 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).

[0069] The recombinant AAV of the invention (i.e., infectious, encapsidated rAAV particles) comprise a rAAV genome. In an exemplary embodiment, the genomes of both rAAVs lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome. Examples of rAAVs that can be constructed to contain the nucleic acid molecules of the invention are described in International Patent Application No. PCT / US2012 / 047999 (WO2013 / 016352), which is incorporated herein by reference in its entirety.

[0070] rAAV can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO 98 / 09657.

[0071] In another embodiment, the present invention contemplates a composition comprising the rAAV of the present invention. The composition of the present invention comprises an rAAV and a pharmaceutically acceptable carrier. The composition may also contain other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to recipients and are preferably inert at the dosages and concentrations used, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight polypeptides, 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-forming counterions such as sodium; and / or non-ionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

[0072] The titer of the rAAV administered in the methods of the invention will vary depending, for example, on the particular rAAV, the mode of administration, the therapeutic goal, the individual being targeted, and the cell type(s), and can be determined by standard methods in the art. The titer of the rAAV is approximately 1 x 10 per mL. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 Dosages may be expressed in units of viral genomes (vg).

[0073] Methods for transducing target cells with rAAV in vivo or in vitro are contemplated by the present invention. In vivo methods include administering an effective dose or effective multiple doses of a composition comprising an rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of the present invention, an "effective dose" is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to the disorder / disease state, slows or prevents progression of the disorder / disease state, reduces the extent of the disease, causes disease remission (partial or complete), and / or prolongs survival. An example of a disease contemplated for prevention or treatment by the methods of the present invention is FSHD.

[0074] Combination therapies are also contemplated by the present invention. As used herein, combination therapy includes simultaneous and sequential therapy. Combination of the methods of the present invention with standard medical treatments (e.g., corticosteroids), including combinations with novel therapies, is specifically contemplated.

[0075] Administration of an effective dose of the composition may be by any route standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or intravaginal. The administration route(s) and serotype(s) of the AAV components (specifically, AAV ITRs and capsid proteins) of the rAAV of the present invention may be selected and / or adapted by one skilled in the art, taking into consideration the infectious disease and / or disease state to be treated and the target cell / tissue(s) expressing miR-29 miRNA and / or microdystrophin.

[0076] The present invention provides for local and systemic administration of effective doses of the rAAV and compositions of the present invention, including the combination therapies of the present invention. For example, systemic administration is administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration by injection, infusion, or implantation.

[0077] Specifically, the actual administration of the rAAV of the present invention can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, intramuscular injection, injection into the bloodstream, and / or direct injection into the liver. Simply resuspending 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 limitations on the carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the usual practice with rAAV). The capsid protein of rAAV can be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations delivered to muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in practicing the present invention. The rAAV may be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0078] The dose of rAAV administered in the methods disclosed herein will vary depending, for example, on the particular rAAV, mode of administration, therapeutic goal, targeted individual, and cell type(s), and can be determined by standard methods in the art. The titer of each rAAV administered can be in the range of about 1 x 10, about 1 x 10, about 1 x 10, about 1 x 10, about 1 x 10, about 1 x 10, about 1 x 10, about 1 x 10, about 1 x 10, about 1 x 10, or about 1 x 10 or more DNase-resistant particles (DRP) per ml. Dosages are measured in units of viral genomes (vg) (i.e., 1 x 10, respectively). 7 vg, 1x10 8 vg, 1x10 9 vg, 1x10 10 vg, 1x10 11 vg, 1x10 12 vg, 1x10 13 vg, 1x1014 vg, 1×10 15 Dosages may be expressed in units of viral genomes (vg) per kilogram (kg) of body weight (i.e., 1x10 10 vg / kg, 1x10 11 vg / kg, 1x10 12 vg / kg, 1x10 13 vg / kg, 1x10 14 vg / kg, 1 × 10 15 AAV titration may be expressed as a percentage of the total antibody titer (vg / kg). Methods for titrating AAV are described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

[0079] Specifically, the actual administration of the rAAV of the present invention can be achieved by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, intramuscular injection, injection into the bloodstream, and / or direct injection into the liver. Simply resuspending 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 limitations on the carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in the usual practice with rAAV). The capsid protein of rAAV can be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations delivered to muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in practicing the present invention. The rAAV may be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0080] For intramuscular injection, solutions in adjuvants such as sesame or peanut oil, or in aqueous propylene glycol, as well as sterile aqueous solutions, can be used. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV as the free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. rAAV dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media employed can be readily obtained by standard techniques well known to those skilled in the art.

[0081] Pharmaceutical carriers, diluents, or excipients 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 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 action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. 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 dispersions, and by the use of surfactants. Protection against 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 is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0082] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent with various other ingredients as listed above, as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above. For the preparation of sterile powders for sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient plus any additional desired ingredients from the previously sterile-filtered solution.

[0083] Transduction with rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from a subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used if they do not generate an inappropriate immune response in the subject.

[0084] Suitable methods for transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with muscle cells in an appropriate medium and screening for cells carrying the DNA of interest using conventional techniques such as Southern blot and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, which can be introduced into the subject by a variety of techniques, such as intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth myocardium, for example, using a catheter.

[0085] Transduction of cells with the rAAVs of the present invention results in sustained expression of miR-29 or microdystrophin. Accordingly, the present invention provides methods for administering / delivering rAAVs expressing miR-29 and / or microdystrophin to animals, preferably humans. These methods involve transducing tissues (including, but not limited to, tissues such as muscle, organs such as the liver and brain, and glands such as salivary glands) with one or more rAAVs of the present invention. Transduction can be performed with gene cassettes containing tissue-specific regulatory elements. For example, one embodiment of the present invention may be used in combination with, but is not limited to, the actin and myosin gene families, such as those from the myoD gene family [see Weintraub et al., Science, 251:761-766 (1991)], the muscle cell-specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol., 11:4854-4862 (1991)], the human skeletal muscle actin gene [Muscat et al., Mol. Cell. Biol., 7:4089-4099 (1987)], the cardiac actin gene, and the muscle creatine kinase sequence element [Johnson et al. al., Mol. Cell. Biol., 9:3393-3399 (1989)], and regulatory elements derived from the mouse creatine kinase enhancer (mCK) element, regulatory elements derived from the fast skeletal troponin C gene, the slow cardiac troponin C gene, and the slow troponin I gene: hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)), steroid-inducible elements, and promoters containing glucocorticoid response elements (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993)), as well as other regulatory elements.

[0086] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The present invention contemplates sustained expression of miRNA from transduced myofibrils.

[0087] By "muscle cell" or "muscle tissue" is meant a cell or group of cells derived from any type of muscle (e.g., skeletal and smooth muscle, e.g., from the gastrointestinal tract, bladder, blood vessels, or heart tissue). Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[0088] The term "transduction" is used to refer to the administration / delivery of the miiR29 guide strand or micro-dystrophin coding region to a recipient cell either in vivo or in vitro via a replication-deficient rAAV of the present invention, resulting in expression of miR29 or micro-dystrophin by the recipient cell.

[0089] Thus, the present invention provides methods of administering an effective dose (or doses administered essentially simultaneously or doses given at an interval) of an rAAV encoding miR29 and / or micro-dystrophin to a patient in need thereof. [Example]

[0090] Example 1 Validation of Duchenne muscular dystrophy model The mdx mouse provides a convenient, yet imperfect, animal model for studying DMD pathogenesis. This model, a cross between mdx mice and a heterozygous knockout of the utrophin gene (mdx:utrn+ / -), exhibits increased fibrosis and more closely mimics human DMD pathology. Mdx mice harbor a nonsense mutation in exon 23 of DMD, resulting in a relatively mild phenotype and a nearly normal lifespan. By 3 weeks of age, the diaphragm and limb muscles of mdx mice develop signs of endomysial inflammation. These symptoms subside in limb muscles after the mice reach adulthood, whereas inflammation in diaphragm muscles continues to gradually worsen. In mdx mice lacking telomerase, muscular dystrophy progressively worsens with age, and mdx mice lacking utrophin (DKO) have a phenotype more characteristic of human DMD, with early-onset muscle weakness, severe fibrosis, and early death. Utrophin, an autosomal paralog of dystrophin, shares a high degree of sequence homology that may compensate for the lack of dystrophin in double KO (dystrophin + utrophin) mdx mice, resulting in a severe phenotype with early death. Early death in DKO mice abrogates the progression of inflammation and fibrosis, but mdx:utrn + / - Mice present a model with similarities to human disease, exhibiting a significant degree of fibrosis and a longer survival time than DKO, and offer a better model for proposed translational studies. + / - The use of mice has been identified as an ideal model for studying fibrosis in the context of DMD. In this study, increased fibrosis, as measured by Sirius Red staining, was accompanied by increased collagen transcript levels and decreased mir29c levels.

[0091] Example 2 Delivery of miR29 to DMD mice reduces fibrosis Preliminary studies have been conducted in human DMD patients and mdx / utrn + / -We demonstrate a significant increase in Sirius red staining for collagen and a decrease in miR-29c levels in mice. Gene delivery of miR-29 using muscle-specific AAV vectors is potentially safe and efficient. To generate the rAAV vector referred to herein as rAAVrh.74.CMV.miR29c, the 22-nucleotide miR29c sequence (target strand SEQ ID NO: 3 and guide strand SEQ ID NO: 4) was cloned into a miR-30 scaffold driven by a CMV promoter. The expression cassette (SEQ ID NO: 2) was cloned into a self-complementary AAV plasmid and packaged using AAVrh.74, a serotype known to express well in muscle. miR-29c cDNA was synthesized using custom primers containing the miR-30c target (sense) strand, miR-30 stem-loop, and miR-29c guide (antisense) strand in the miR-30 backbone. Three bases in the miR-29c sequence were modified. This sequence was then cloned into a plasmid containing self-complementary AAV ITRs driven by a CMV promoter and polyA sequence.

[0092] As shown in Figure 1, the pAAV.CMV.miR29C plasmid contains the miR-30 stem-loop backbone of the miR-30 gene, flanked by AAV2 inverted terminal repeats (ITRs). It is this sequence that is encapsidated into AAVrh.74 virions. Furthermore, several nucleotides within the miR-29c target sequence were modified to mimic Watson-Crick pairing at this site, as in shRNA-miR(luc). According to the shRNA-luc design, the hairpin must be perfectly complementary throughout its entire length. Additionally, the more modifications made to the passenger strand, the greater the likelihood of eliminating endogenous mechanisms regulating miR-29 processing that can recognize the miRNA through the stem. The 19th base of the guide strand was modified to cytosine to mimic the nucleotide preceding the cleavage site in the native miR-29c sequence, and the corresponding base on the other strand was modified to preserve pairing.

[0093] Gene therapy vector scrAAVrh.74.CMV.miR29c (1 × 10 11 vg) to 3-month-old mdx / utrn + / - The quadriceps muscles of young mdx / utrn mice were injected. The quadriceps muscles were analyzed 3 months after injection by Sirius Red staining and analyzed using NIH ImageJ software as described by Nevo et al. (PloS One, 6: e18049 (2011)). MiR29c, collagen, and elastin levels were quantified by RT-PCR. + / - Delivery of miR-29c to 6-month-old mdx / utrn mice + / - In the quadriceps muscles of mice (3 months after injection), mir-29c levels were significantly increased and Sirius red staining was significantly decreased. As assessed by RT-PCR, there was a decrease in collagen and elastin levels in the treated muscles.

[0094] mdx / utrn + / - The demonstration of increased fibrosis and decreased miR29 expression in mice and dystrophin-deficient patients validates the mouse model as representative of human disease. Early results using AAV-delivered miR29 as an antifibrotic therapy suggest significant beneficial effects on Sirius red staining and reduced collagen and elastin levels, which are important drivers of fibrosis.

[0095] Example 3 Injection of miR-29c reduces collagen and restores miR-29c To determine whether rAAVrh.74.CMV.MiR-29c can attenuate fibrosis, 12-week-old mdx / utrn +￥- Mice received 5x10 injections into the left gastrocnemius (GAS) muscle. 11Mice were intramuscularly injected with rAAVrh.74.CMV.MiR-29c (vg). Mice were analyzed 12 weeks after injection. Picrosirius red staining revealed a significant decrease in collagen staining throughout the GAS muscle (Figure 2a) compared to untreated contralateral mdx / utrn+ / - GAS muscle. Quantification of picrosirius red staining indicates that treated muscles had an 18.3% decrease in collagen compared to untreated muscles (treated -23.3% ± 1.3 vs. untreated -29.5% ± 0.7) (Figure 2b). To confirm miR-29c overexpression in treated muscles, 24-week-old WT, miR-29c-treated, and mdx / utrn mice were analyzed. + / - Total RNA was extracted from the GAS muscle of mice and subjected to quantitative reverse transcription PCR (qRT-PCR) analysis for miR-29c expression. The results showed that miR-29c was significantly increased in the GAS muscle of treated mice compared with untreated mice (Figure 2d).

[0096] Example 4 MiR-29c improves absolute and specific muscle strength but does not protect against contraction-induced injury Since fibrosis can affect muscle function, we hypothesized that reducing fibrosis by increasing MiR-29c expression could protect mdx / utrn from contraction-induced damage. + / - We wanted to test whether rAAVrh.74.CMV.MiR-29c-treated mdx / utrn mice could protect their muscles and increase overall strength. + / - The functional properties of the gastrocnemius muscle from mice were evaluated. Twelve weeks after injection, the GAS was isolated and in vivo force measurements were performed.

[0097] The GAS procedure followed the protocol outlined by Hakim et al. (Methods Mol Biol. 709:75-89, 2011) for analyzing transverse abdominis muscle physiology, but was adapted for the GAS. Briefly, mice were anesthetized with a ketamine / xylazine mixture. The skin on the hind limbs was removed to expose the GAS muscle and Achilles tendon. The distal tendon was excised, and a 4-0 suture was tied around the tendon in a double square knot as close to the muscle as possible. A second double square knot was then made immediately adjacent to the first knot, and the tendon was then severed. The exposed muscle was constantly moistened with saline. The mouse was then transferred to a thermo-controlled platform maintained at 37°C. The knee was secured to the platform with a needle passed through the patellar tendon, the tendon was sutured to the level arm of a force transducer (Aurora Scientific, Aurora, ON, Canada), and the foot was secured with tape. GAS muscle contraction was induced by stimulating the sciatic nerve via a bipolar platinum electrode. Once the muscle was stabilized, the optimal length was determined by incrementally stretching the muscle until maximum contractile force was achieved. After a 3-minute rest period, the GAS was stimulated at 50, 100, 150, and 200 Hz, with a 1-minute rest period between each stimulation, to determine maximum tetanic force. Muscle length was measured. After a 5-minute rest, the sensitivity of the GAS muscle to contraction-induced damage was assessed. After a 500-ms stimulation, the muscle lengthened by 10% of its optimal length. This consisted of stimulating the muscle for 700 ms at 150 Hz. After stimulation, the muscle returned to its optimal length. This cycle was repeated every minute for a total of five cycles. Specific force was calculated by dividing the maximum tetanic force by the GAS muscle cross-sectional area. After the eccentric contraction, the mice were then euthanized, and the GAS muscles were excised, weighed, and frozen for analysis.

[0098] Each GAS was subjected to a series of repeated eccentric contractions. Comparing the force ratio of each contraction to the first contraction revealed that after the fifth contraction, the untreated muscle had decayed to 0.56 ± 0.05 compared to 0.50 ± 0.04 in the treated group (P < 0.0001). The treated group showed a slightly reduced degree of protection compared to WT controls, which had decayed to 0.92 ± 0.02 (Fig. 3c). These data indicate that reducing fibrosis by increasing miR-29c expression increases both absolute and specific force but does not significantly protect muscles from contraction-induced damage.

[0099] rAAVrh.74.MiR-29c-treated GAS muscles were significantly higher than untreated mdx / utrn + / - showed a significant improvement in absolute force when compared to the GAS muscle (rAAV.miR-29c-2227 ± 161.7 vs mdx / utrn + / - Untreated -1722 ± 145.7, Figure 3a), and normalized specific force in rAAVrh.74.miR-29c-treated GAS muscle specifically improved when compared with untreated GAS muscle (rAAV.miR-29c -204.7 ± 11.7 vs. mdx / utrn + / - Untreated -151.6 ± 14.5, Figure 3b). When compared to wild-type controls, titers were still significantly reduced (rAAV.miR-29c -204.7 ± 11.7 vs. wild-type -312.0 ± 34.1).

[0100] Example 5 Co-delivery with micro-dystrophin further reduces fibrosis To determine whether a combined miR-29c / microdystrophin gene therapy approach would be more beneficial in reducing fibrosis, 12-week-old mdx / utrn +￥- Mice received 5x10 injections into the left gastrocnemius muscle. 11 Three-month-old mdx / utrn mice received intramuscular injections of 1000 mg of rAAVrh.74.CMV.MiR-29c. The following gene therapy vectors were administered: scAAVrh.74.CMV.miR-29c alone, co-delivery with rAAVrh.74.MCK.microdystrophin, and rAAVrh.74.MCK.microdystrophin alone. + / -Mice were administered by intramuscular injection (IM) into the left gastrocnemius (GAS) muscle of a DMD mouse model.

[0101] The pAAV.MCK.microdystrophin plasmid contains a human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs), as shown in Figure 10. It is this sequence that is encapsidated into AAVrh.74 virions. The pAAV.MCK.microdystrophin plasmid was constructed by inserting an MCK expression cassette driving a codon-optimized human microdystrophin cDNA sequence into an AAV cloning vector, as described by Rodino-Klapac et al. (Mol Ther. 2010 Jan;18(1):109-17). The MCK promoter / enhancer sequence, used to drive muscle-specific gene expression, consists of the mouse MCK core enhancer (206 bp) fused to the 351 bp MCK core promoter (proximal). The core promoter is followed by 53 bp of endogenous mouse MCK Exon 1 (untranslated) for efficient transcription initiation, followed by the SV40 late 16S / 19S splice signal (97 bp) and a small 5' UTR (61 bp). The intron and 5' UTR are derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette contains a consensus Kozak sequence immediately before the ATG start and a small 53 bp synthetic poly(A) signal for mRNA termination. The human microdystrophin cassette contains the (R4-R23 / Δ71-78) domain. Complementary DNA was codon-optimized for human use and synthesized by GenScript (Piscataway, NJ).

[0102] Mice were analyzed 12 and 24 weeks after injection. The efficacy of transgene delivery was assessed using the number of myofibers expressing microdystrophin, ensuring similar levels of microdystrophin expression in each group. Microdystrophin expression was not found to differ between cohorts treated with microdystrophin alone (71.85 ± 2.25%) compared with miR-29c / microdystrophin combination therapy (75.03 ± 1.91%) (Figure 4).

[0103] GAS muscles were analyzed 12 months after injection to assess collagen accumulation by Sirius red staining and subsequent quantification using ImageJ. Further results included miR-29c and collagen transcript levels, force measurements in GAS muscles, fiber diameter measurements, and Western blot analysis of proteins involved in muscle regeneration (MyoD, myogenin). The amount of fibrosis was analyzed by picrosirius red staining, revealing a significant decrease in collagen staining throughout GAS muscles in all treatment groups compared to untreated contralateral mdx / utrn+ / - GAS muscles or microdystrophin alone (Figure 5a). Quantification of picrosirius red staining indicated that the combination-treated muscles had a 40.8% decrease in collagen compared to untreated muscles (treated -17.47% ± 0.75 vs. untreated -29.5% ± 0.7) (Figure 5b). To confirm the expression of miR-29c, qRT-PCR was performed in GAS muscle, and all treatment groups had an increase in miR-29c compared to untreated muscle (Figure 5c).

[0104] Similar to DMD tissue, mdx / utrn + / -A significant decrease in miR-29c levels in muscle was observed, correlating with an increase in fibrosis measured by picrosirius red staining. After 3 months of treatment with scAAV.miR-29c alone, there was a significant decrease in fibrosis in GAS muscle (treated -23.5% ± 1.3 vs. untreated -27.8% ± 0.6). When co-delivered with miR-dystrophin, a further decrease in collagen (41%) was observed by picrosirius red staining (combined treatment: 17.47% ± 0.75 vs. untreated: 29.5% ± 0.7) (p<0.0001) (Figure 5b). To confirm miR-29c expression, qRT-PCR was performed in GAS muscle, and all treatment groups had an increase in miR-29c compared to untreated muscle (Figure 5b).

[0105] At 24 weeks after injection, results were similar to those observed at 12 weeks after injection: there was a 47% decrease in collagen stained by picrosirius red compared to untreated muscle (combination therapy: 16.5 ± 1.23 vs. untreated: 31.07 ± 0.93, p < 0.0001) and a concomitant increase in miR-29c transcript levels.

[0106] To further verify the collagen reduction observed by picrosirius red staining, qRT-PCR was performed on muscle to quantify transcript levels of Col1A, Col3A, and another ECM component, fibronectin (Fbn). While qRT-PCR analysis detected a decrease in Col1A and Col3A after each treatment, only the cohort treated with both miR-29c and microdystrophin showed a significant decrease (Figures 6a and 6b). Analysis revealed that Fbn was significantly reduced only in the combined treatment cohort (Figure 6c).

[0107] TGF-β1 has previously been shown to be upregulated in dystrophic muscle and is likely involved in initiating the fibrosis cascade. TGF-β1 is a known pro-fibrotic cytokine that downregulates miR-29c and is responsible for the conversion of myoblasts to myofibroblasts, accompanied by increased collagen and muscle fiber formation. qRT-PCR analysis shows that the combination-treated muscles had significantly lower levels of TGF-β1 compared with uninjected muscles and either monotherapy (Figure 6d). Six months after injection, the combination-treated muscles continued to show reduced Col1A, Col3A, Fbn, and TGF-β1 levels, whereas only a slight decrease in Col1A mRNA levels was observed in the miR-29 and microdystrophin-only group.

[0108] Increases in specific and absolute force were observed in muscles treated with miR-29c alone compared to untreated limbs, and when combined with miR-29c, resulted in absolute and specific forces that were not significantly different from wild-type. Also, a significant increase in gastrocnemius muscle weight was observed in the combined treated muscles.

[0109] Initial results using rAAV.miR-29c as an antifibrotic therapy suggest beneficial effects in reducing collagen levels, a major cause of fibrosis. Furthermore, when combined with microdystrophin to improve membrane stability, miR29 upregulation normalized muscle strength.

[0110] Example 6 Further increase in absolute force and additional protection from contraction-induced damage Given that miR-29-treated muscles showed modest but significant increases in absolute and specific force, we investigated the effect of combined miR-29c overexpression and microdystrophin gene replacement on muscle function. Twelve weeks after injection, GAS were isolated and in vivo force measurements were performed. The rAAVrh.74.MiR-29c vector and rAAV, as described above in Example 2, were used.

[0111] GAS muscles treated with rAAVrh.74.MiR-29c and rAAV expressing MiR-29c in combination therapy were significantly higher than untreated mdx / utrn + / - showed a significant improvement in absolute force when compared to the GAS muscle (combined treatment -3582.4 ± 79.4 nM vs mdx / utrn + / - Untreated, −1722 ± 145.7 nM vs. wild-type, −3005 ± 167.3 nM) (Figure 7), and normalized specific force in rAAVrh.74.miR-29c / miR-29c-treated GAS muscles compared with untreated GAS muscles (combination-treated mice, −244.2 ± 6.6 nM / mm 2 vs. mdx / utrn + / - Untreated -151.6±14.5nM / mm 2 vs. 312.0±34.1nM / mm 2 ) (Figure 7). Both absolute and specific forces were not significantly different from the wild-type control.

[0112] Each GAS was subjected to a series of repeated eccentric contractions. Comparing the force ratio of each contraction to the first contraction revealed that after the fifth contraction, untreated muscles were attenuated to 0.54 ± 0.06 compared with 0.66 ± 0.04 with combined treatment (P ≤ 0.0001). This can be attributed to microdystrophin, as microdystrophin alone also attenuated the force to 0.66 ± 0.04. The treated group was still significantly lower than the wild-type group, attenuated to 0.92 ± 0.02 (Figure 7c). Similar findings were observed 24 weeks after injection. These data indicate that fibrosis reduction and gene replacement significantly protect muscles from contraction-induced damage, increasing both absolute and specific force.

[0113] Example 7 Combination therapy increases muscle hypertrophy and hyperplasia MiR-29c co-delivered with micro-dystrophin increased the total weight of the injected gastrocnemius muscle compared to either injection alone at 3 months of age (Figures 8 and 9a). To investigate the cause of the increased muscle mass, we measured muscle fiber diameter. The miR-29c / μ-dys combination treatment showed an increase in average fiber size. + / -Controls were treated with miR-29c / μ-dys and mdx / utrn + / - Compared to untreated, the mean diameter increased from 25.96 to 30.97 μm (Figure 9b). Co-delivery resulted in a shift toward wild-type fiber size distribution (Figure 9c). While mean fiber size increased, this did not account for the ~30% increase in total muscle weight. Total muscle cross-sectional area was also measured. Gastrocnemius muscles from all groups were subjected to full-slide scans, and total area was measured. Muscles co-treated with Microdis / miR-29c had a significant increase in cross-sectional area compared to untreated and either monotherapy (uninjected: 24.6 vs. miR-29c: 26.3 vs. Microdis: 26.6 vs. Microdis / miR-29c: 33.1) (Figure 8, Figure 9d).

[0114] miR-29c has been reported to play a role in the myoD / Pax7 / myogenin pathway, and we hypothesized that miR-29c may influence the regeneration and activation of satellite cells (muscle stem cells) that differentiate into the myogenic lineage. To test this, we counted the total number of myofibers from full-slide scan images. The number of myofibers increased after miR-29c / μ-dys combination treatment (Figure 9e). Finally, given that myofiber diameters in mdx / utrn+ / − mice differed, with many small-diameter fibers and some hypertrophied fibers, we determined whether the number of fibers per unit area (cells / mm2) was affected by treatment. The number of fibers per unit area (cells / mm2) was unchanged from wild-type after miR-29c / μ-dys combination treatment (Figure 9f).

[0115] Example 8 Early combination treatment prevents fibrosis Considering the potential importance of combinatorial miR-29c and microdystrophin as a preventive therapy for DMD, younger mdx / utrn + / -Cohorts of mice were treated at 4 weeks of age. Using the same paradigm as the other groups described herein, the following treatments were compared for efficacy in preventing fibrosis following intramuscular injection of GAS: scAAVrh.74.CMV.miR-29c alone at the same dose, ssAAVrh74.MCK.microdystrophin + scAAVrh74.CMV.miR-29c combination therapy, or ssAAVrh74.MCK.microdystrophin alone. Mice were necropsied 12 weeks after injection. Untreated contralateral mdx / utrn + / - A significant decrease in collagen staining throughout GAS muscle was observed in all treated groups compared to GAS muscle (Figure 10A). Quantification of picrosirius red staining showed that muscles treated with miR-29c / miR-29c had a 51% decrease in collagen compared to untreated muscle (treated -11.32% ± 1.18 vs. untreated -23.15% ± 0.90) (p<0.0001) (Figure 10). pRT-PCR confirmed the decrease in Col1A, Col3A, Fbn, and TGF-β1 after combinatorial therapy (Figures 10D and 10E).

[0116] Example 9 Early combination therapy restores force better than late treatment and protects against contraction-induced injury In vivo force measurements were performed on GAS in mice treated early with the combination therapy as described in Example 8. 4-week-old mdx / utrn + / - Combination therapy with miR-29c / microdystrophin significantly improved the survival of untreated mdx / utrn mice. + / - Compared to wild-type mice, absolute force showed significant improvement with no difference from wild-type (combined treatment: 2908 ± 129.5 mN vs. untreated: 1639.4 ± 116.9 mN vs. wild-type: 3369.73 ± 154.1 mN). Specific force also normalized to wild-type levels after combinatorial therapy (combined treatment 338.9 ± 22.34 mN / mm vs. untreated 184.3 ± 13.42 mN / mm). 2 vs. WT 364.3±7.79mN / mm 2 ) (Figures 11A and B and 12).

[0117] Next, each GAS muscle underwent a series of repeated eccentric contractions. Comparing the force ratio of each contraction by the fifth contraction, untreated muscles were attenuated to 0.53 ± 0.04 compared to 0.82 ± 0.04 for the combined treatment (P < 0.0001). The combined treatment group was slightly lower than the wild-type group, attenuated to 0.93 ± 0.01, but the difference was not significant (Figure 11C). These data indicate that fibrosis reduction and gene replacement significantly protect muscles from contraction-induced damage, increasing both absolute and specific force.

[0118] These experiments suggest that gene replacement should begin in the neonatal period. Efforts are clearly moving toward identifying DMD and other muscular dystrophies in the neonatal period. The Ohio Newborn Screening Study demonstrates the feasibility of identifying DMD in newborns using CK 7 Neurol. as a biomarker (>2000 U / L) with DNA confirmation on the same dried blood spot (Mendell et al., Ann. Neurol. 71:304-313, 2012). This methodology is now being expanded in the United States (PPMD May 16, 2016: Next Steps with Newborn Screening) and other countries, notably the United Kingdom (UK National Screening Committee) and China (Perkin Elmer™ has begun screening in China).

[0119] miR-29 has also shown promise as a therapeutic modality for cardiac, pulmonary, and liver fibrosis. Myocardial infarction in mice and humans is associated with miR-29 downregulation. Rooij et al. (Proc. Natl. Acad. Sci. USA 105:13027-13032, 2008) demonstrated that exposing fibroblasts to a miR-29b mimic reduced collagen transcripts, providing a pathway for clinical transformation of cardiac fibrosis. Subsequent studies demonstrated that attenuation of fibrosis could be achieved using the Sleeping Beauty (SB) transposon system-based delivery of miR-29b in a bleomycin-induced pulmonary fibrosis mouse model. 14 Currently, a miR-29b mimic is undergoing clinical Phase 1 Safety-Tolerance topical transdermal study in healthy volunteers (miRagen Therapeutics™ MRG-201). Compared to miR-29 oligonucleotide delivery, which requires repeated administration related to the half-life of the oligonucleotide, AAV gene therapy could potentially provide a route for single-delivery gene transfer.

[0120] Example 10 Treatment with muscle-specific expression of miR-29 and microdystrophin reduced fibrosis and ECM expression An AAV vector containing the miR29c sequence and the muscle-specific promoter MCK was also generated and tested as a combination therapy with an AAV vector expressing microdystrophin. To generate the rAAV vector referred to herein as rAAV.MCK.miR29c, the 22-nucleotide miR29c sequence (target strand SEQ ID NO: 3 and guide strand SEQ ID NO: 4) was cloned into the miR-30 scaffold (SEQ ID NO: 11) driven by the MCK promoter. The expression cassette (SEQ ID NO: 12) was cloned into a single-stranded AAV plasmid and packaged using AAVrh74, a serotype known to express well in muscle. miR-29c cDNA was synthesized using custom primers containing the miR-30c target (sense) strand, miR-30 stem-loop, and miR-29c guide (antisense) strand in the miR-30 scaffold. Three bases in the miR-29c sequence were modified. This sequence was then cloned into a plasmid containing a single AAV ITR driven by the MCK promoter and polyA sequence.

[0121] The pAAV.MCK.miR29C plasmid contains the miR-30 stem-loop backbone of the miR-30 gene, flanked by AAV2 inverted terminal repeats (ITRs). This sequence is encapsidated into AAVrh74 virions. Furthermore, several nucleotides within the miR-29c target sequence were modified to mimic Watson-Crick pairing at this site, as in shRNA-miR(luc). According to the shRNA-luc design, the hairpin must be perfectly complementary throughout its entire length. Additionally, more variation in the passenger strand potentially eliminates endogenous mechanisms regulating miR-29 processing that can recognize the miRNA through the stem. The 19th base of the guide strand was modified to cytosine to mimic the nucleotide preceding the cleavage site in the native miR-29c sequence, and the corresponding base on the other strand was altered to preserve pairing.

[0122] Early treatment with AAV.MCK.miR-29c / microdystrophin combination therapy was more effective in reducing fibrosis and ECM expression. +￥- Mice were inoculated with 5x10 punctures into the left gastrocnemius muscle as described in Example 5. 11 Each mouse received an intramuscular injection of rAAVrh.74.MCK.miR-29c and rAAVrh74.MCK.micro-dystrophin (vg). Muscles were harvested 12 weeks after injection. Picrosirius red staining of muscles harvested from uninjected and mice injected with the rAAV.MCK.miR-29c / rAAV.MCK.micro-dystrophin combination therapy showed that the combination-treated muscles had a 50.9% reduction in collagen compared to untreated GAS muscles (see Figures 13a and 13b), and qRT-PCR confirmed increased miR-29c transcript levels in the treated cohort (Figure 13c). Semiquantitative qRT-PCR showed significant decreases in collagen 1A and collagen 3A (Figures 13d, e), fibronectin (Figure 13f), and Tgfβ1 (Figure 13g) levels in AAV.MCK.miR-29c / AAV.microdystrophin-treated muscles compared to contralateral limb therapy (*p<0.05, ****p<0.0001). Late-stage treatment with AAV.MCK.miR-29c / microdystrophin combination therapy is effective in reducing fibrosis and ECM expression. Three-month-old mdx / utrn +￥- Mice were inoculated with 5x10 punctures into the left gastrocnemius muscle as described in Example 5. 11Each rat received an intramuscular injection of rAAVrh.74.MCK.miR-29c and rAAVrh.74.MCK.micro-dystrophin. Muscles were harvested 12 weeks after injection. Picrosirius red staining of untreated, AAV.MCK.miR-29c, and AAV.MCK.miR-29c / AAV.micro-dystrophin-treated muscles showed a 30.3% reduction in collagen compared to untreated GAS muscle (see Figures 14a and 14b). qRT-PCR confirmed increased miR-29c transcript levels in the treated cohort (Figure 14c). Semiquantitative qRT-PCR showed significant decreases in collagen 1A and collagen 3A (Figures 14d and 14e), fibronectin (Figure 14f), and Tgfβ1 (Figure 14g) levels in AAV.miR-29c / AAV.micro-dystrophin-treated muscles compared to the contralateral limb. One-way ANOVA. All data represent mean ± SEM. (**p<0.01, ****p<0.0001).

[0123] Example 11 Early combination therapy restores force better than late treatment and protects against contraction-induced injury In vivo force measurements were performed on GAS of mice treated early with muscle-specific expression of miR-29 and microdystrophin as described in Examples 8 and 9. 4-week-old mdx / utrn + / - Combination therapy with rAAV.MCK.miR-29c / and rAAV expressing microdystrophin improved the survival of untreated mdx / utrn mice. + / - Compared with wild-type mice, the combination therapy showed a significant improvement in absolute force, with no difference from wild-type (Figure 15a). Specific force also normalized to wild-type levels after combination therapy (Figure 15b).

[0124] Muscles were then assessed for loss of force after repeated eccentric contractions as described in Example 9. rAAV.miR-29c / rAAV.MCK.microdystrophin combination treatment and rAAV.MCK.microdystrophin mono-treated mice showed similar results to untreated mdx / utrn mice. +￥-showed protection from loss of force compared to muscle (Fig. 15c).

[0125] 12-week-old mdx / utrn + / - In mice, combined treatment with rAAV.MCK.miR-29c / and rAAV expressing microdystrophin restored force and protected against contraction-induced injury. Measurements of absolute force (Fig. 16a) and normalized specific force (Fig. 16b) after tetanic contractions of GAS muscles injected with rAAV.MCK.miR-29c and rAAV expressing microdystrophin were significantly greater than those of untreated mdx / utrn mice. + / - The force loss was significantly increased compared to untreated mdx / utrn muscles. Muscles were then evaluated for loss of force after repeated eccentric contractions as described in Example 9. MCK.miR-29c / microdystrophin co-treated mice showed a significant increase in force loss compared to untreated mdx / utrn muscles. +￥- These data demonstrate that fibrosis reduction and gene replacement significantly protects muscle from contraction-induced damage, increasing both absolute and specific force.

[0126] Example 12 Early combination treatment increases muscle hypertrophy and hyperplasia Co-delivery of rAAV.MCK.miR-29 with rAAV expressing microdystrophin did not increase the total weight of injected gastrocnemius muscles compared to either injected alone 3 months after injection (Fig. 17a). Myofiber diameter was also measured. miR-29c / microdystrophin combination treatment showed an increase in mean fiber size. mdx / utrn + / - Controls were treated with miR-29c / microdystrophin and mdx / utrn + / - The mean diameter increased from 28.96 to 36.03 μm compared with the control group (Fig. 17b). Co-delivery resulted in a shift to wild-type fiber size distribution (Fig. 17c). 2 The number of muscle fibers per muscle was significantly lower than in untreated mice and wild-type mice (Fig. 17d, ***p<0.01, ****p<0.0001).

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Claims

[Claim 1] The method or recombinant AAV vector described in the specification.

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