Adeno-associated viral vector delivery of muscle-specific microdystrophin to treat muscular dystrophies

Abstract

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 express micro-dystrophin in skeletal muscles, including the diaphragm and cardiac muscle, to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis in subjects suffering from muscular dystrophy. In one embodiment, the present invention provides an rAAV vector comprising a nucleotide sequence of a muscle-specific regulatory element and a nucleotide sequence encoding a micro-dystrophin protein. [Selected Figure] Figure 1

Description

[Technical field]

[0001] This invention was made with Government support under Grant No. NS055958 awarded by the National Institutes of Health / National Institute of Neurological Disorders and Stroke. The Government has certain rights in the invention. This application claims priority to U.S. Provisional Patent Application No. 62 / 473,148, filed March 17, 2017, which is incorporated by reference in its entirety.

[0002] Incorporation by Reference of Electronically Submitted Materials This application contains, as a separate part of this disclosure, a sequence listing in computer readable form, identified as follows, which is incorporated by reference in its entirety: Filename: 51475_Seqlisting.txt; Size: 29,519 bytes; Created; March 13, 2018.

[0003] 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 express micro-dystrophin in skeletal muscles, including the diaphragm and cardiac muscle, to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis in subjects suffering from muscular dystrophy. [Background technology]

[0004] The importance of muscle mass and strength for daily activities such as locomotion and respiration, as well as whole-body metabolism, is clear. Deficits in muscle function give rise to muscular dystrophies (MDs), characterized by muscle weakness and wasting, severely impacting quality of life. The best-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 sarcolemmal-cytoskeleton anchoring by DAPC. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle diseases, affecting 1 in 5000 newborn males.

[0005] DMD is caused by mutations in the DMD gene that result in reduced mRNA and absence of dystrophin, a 427 kD sarcolemmal protein that binds to the dystrophin-associated protein complex (DAPC) (Hoffman et al., Cell 51(6):919-28, 1987). The DAPC is composed of multiple proteins in the sarcolemma and forms structural links with the extracellular matrix (ECM) and the cytoskeleton via the actin-binding protein dystrophin and the laminin-binding protein alpha-dystroglycan. These structural links act to stabilize the muscle cell membrane during contraction and protect it from contraction-induced damage. With loss of dystrophin, membrane fragility leads to sarcolemmal avulsion 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 eventually exhausts 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), leading to progressive muscle weakness, endomysitis, and fibrotic scarring.

[0006] Without membrane stabilization by dystrophin or microdystrophin, DMD exhibits an uncontrollable cycle of tissue damage and repair, eventually replacing lost muscle fibers with fibrous scar tissue through proliferation of connective tissue. Fibrosis is characterized by the excessive deposition of ECM matrix proteins, including collagen and elastin. ECM proteins are primarily generated from cytokines, such as TGFβ, released by activated fibroblasts in response to stress and inflammation. Although the primary pathological hallmark of DMD is degeneration and necrosis of muscle fibers, fibrosis as a pathological consequence has an equal impact. Overproduction of fibrous tissue limits muscle regeneration and contributes to the progressive muscle weakness of DMD patients. In one study, the presence of fibrosis in early DMD muscle biopsies was highly correlated with poor motor outcome at 10-year follow-up (Desguerre et al., J Neuropathol Exp Neurol, 2009.68(7):p.762-7). These results indicate that fibrosis is a major cause of DMD muscle dysfunction and highlight the need for early intervention before overt fibrosis.

[0007] Adeno-associated virus (AAV) is a replication-defective parvovirus, whose single-stranded DNA genome is about 4.7 kb long, including an inverted terminal repeat (ITR) of 145 nucleotides. 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 presented in Srivastava et al., J Virol, 45:555-564 (1983), as amended by Ruffing et al., J Gen Virol, 75:3385-3392 (1994). As other examples, the complete genome of AAV-1 is presented in GenBank Accession No. NC_002077, the complete genome of AAV-3 is presented in GenBank Accession No. NC_1829, the complete genome of AAV-4 is presented in GenBank Accession No. NC_001829, the AAV-5 genome is presented in GenBank Accession No. AF085716, the complete genome of AAV-6 is presented in GenBank Accession No. NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are presented 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. al., J. Virol., 78:6381-6388 (2004), the AAV-10 genome is presented in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is presented in Virology, 330(2):375-383 (2004). The cloning of the AAVrh.74 serotype is described in Rodino-Klapac., et al. Journal of translational medicine 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 (named p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (e.g., at nucleotides 2107 and 2227 in AAV2), generate four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins have multiple enzymatic properties that are ultimately involved in the replication of 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 associated 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 characteristics 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 non-cytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and non-dividing cells and persist essentially for the life of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in 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, some or all of the internal approximately 4.3 kb of the genome (encoding the replication and structural capsid protein, 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 resist superinfection.

[0009] Several 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 muscles are highly vascularized, recombinant AAV transduction results 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 the correct glycosylation, folding, and secretion of antibodies, indicating that muscle can stably express secreted protein therapeutics. Genetic correction at the early stages of the disease is necessary to improve function in patients suffering from DMD and other muscular dystrophies. Treatments are needed that increase muscle strength and protect against muscle damage in patients suffering from DMD. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Hoffman et al.,Cell 51(6):919-28,1987 [Non-Patent Document 2] Straub et al.,Curr Opin.Neurol.10(2):168-75,1997 [Non-Patent Document 3] Sacco et al.,Cell,2010.143(7):p.1059-71 [Non-Patent Document 4] Wallace et al.,Annu Rev Physiol,2009.71:p.37-57 Summary of the Invention [Means for solving the problem]

[0011] The present invention is directed to gene therapy vectors, e.g., AAV, expressing the micro-dystrophin gene for skeletal muscles, including the diaphragm and cardiac muscle, to protect muscle fibers from damage, increase muscle strength, and reduce and / or prevent fibrosis.

[0012] The present invention provides treatments and approaches to increase muscle strength and / or increase muscle mass by delivering micro-dystrophin using gene therapy vectors to address the genetic deficiencies observed in DMD. As shown in Example 2, treatment with micro-dystrophin gene therapy resulted in greater muscle strength in vivo. Furthermore, intramuscular and systemic delivery of micro-dystrophin gene therapy demonstrated delivery of dystrophin to muscle in vivo in mouse models.

[0013] In one embodiment, the present invention provides an rAAV vector comprising a nucleotide sequence of a muscle-specific control element and a nucleotide sequence encoding a micro-dystrophin protein. For example, the nucleotide sequence encodes a functional micro-dystrophin protein, where the nucleotide 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%, 99%, or 100% sequence identity to SEQ ID NO: 1, and the protein retains micro-dystrophin activity. The micro-dystrophin protein provides stability to muscle membranes during muscle contraction, for example, micro-dystrophin functions as a shock absorber during muscle contraction.

[0014] The present invention also provides rAAV vectors encoding functional microdystrophin proteins, wherein the nucleotide sequence comprises a nucleotide sequence that hybridizes under stringent conditions to the nucleic acid sequence of SEQ ID NO:1, or complements thereof.

[0015] In one embodiment, the rAAV vector is a non-replicating, recombinant adeno-associated virus (AAV) called rAAVrh74.MHCK7.microdystrophin. The vector genome contains the minimal elements required for gene expression, including the AAV2 inverted terminal repeat (ITR) under the control of the MHCK7 promoter / enhancer, microdystrophin, SV40 intron (SD / SA), and synthetic polyadenylation (polyA) signal. A schematic diagram of the vector genome and expression cassette is shown in Figure 1. The AAVrh74 serotype can be used to achieve efficient gene transfer in skeletal and cardiac muscles after IV administration.

[0016] The term "stringent" is used to refer to conditions that are generally understood in the art as stringent. Hybridization stringency is determined primarily by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015M sodium chloride, 0.0015M sodium citrate at 65-68°C or 0.015M sodium chloride, 0.0015M 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, NY1989). 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. Where deoxyoligonucleotide hybridization is concerned, examples of additional stringent hybridization conditions include washing in 6×SSC, 0.05% sodium pyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).

[0017] Other agents can be included in the hybridization and washing 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, although other suitable agents can be used. The concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4, although under typical ionic strength conditions, the rate of hybridization is largely independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach, Ch. 4, IRL Press Limited (Oxford, England). Hybridization conditions can be adjusted by one of skill in the art to take these variables into account, allowing DNAs of different sequence similarity to form hybrids.

[0018] The term "muscle-specific control element" refers to a nucleotide sequence that regulates the expression of a coding sequence specific for expression in muscle tissue. These control elements include enhancers and promoters. The present invention provides constructs that include the muscle-specific control elements MCKH7 promoter, MCK promoter and MCK enhancer.

[0019] The term "operably linked" refers to an arrangement of regulatory element nucleotide sequences, such as a promoter nucleotide sequence, that confers expression of said nucleotide sequence by said regulatory element.

[0020] In one aspect, the invention provides an rAAV vector, wherein the muscle-specific control element is a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor (MEF), a muscle creatine kinase (MCK), a truncated MCK (tMCK), a myosin heavy chain (MHC), a hybrid alpha-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7), C5-12, a mouse creatine kinase enhancer factor, 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).

[0021] For example, the muscle-specific control element is SEQ ID NO:2 of the MHCK7 promoter nucleotide sequence, or the muscle-specific control element is SEQ ID NO:4 of the MCK nucleotide sequence. Furthermore, in any of the rAAV vectors of the present invention, the muscle-specific control element nucleotide sequence, e.g., the MHCK7 or MCK nucleotide sequence, is operably linked to a nucleotide sequence encoding a microdystrophin protein. For example, the MHCK7 promoter nucleotide sequence (SEQ ID NO:2) is operably linked to a human microdystrophin coding sequence (SEQ ID NO:1) as depicted in FIG. 1 or the construct (SEQ ID NO:3) presented in FIG. 10. In another example, the MCK promoter (SEQ ID NO:4) is operably linked to a human microdystrophin coding sequence (SEQ ID NO:1) as depicted in FIG. 7 or the construct (SEQ ID NO:5) presented in FIG. 11. In another embodiment, the present invention provides a rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2. The present invention also provides a rAAV vector comprising the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:4.

[0022] In a further aspect, the invention provides an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 5. For example, the rAAVrh74.MHCK7.microdystrophin vector comprises the nucleotide sequence of SEQ ID NO: 3 and is shown in Figure 10. This rAAV vector comprises a pGEX plasmid backbone with an MHCK7 promoter, a chimeric intron sequence, a coding sequence for the human microdystrophin gene, polyA, ampicillin resistance, and a pBR322 origin of replication.

[0023] The present invention provides an rAAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 3. The rAAV vector is of the AAV serotype AAVrh.74.

[0024] The present invention also provides an rAAV vector comprising the nucleotide sequence of the pAAV.MHCK7.microdystrophin construct of SEQ ID NO: 3. This rAAV vector is of the AAV serotype AAVrh.74.

[0025] The rAAV vectors of the invention can be any AAV serotype, such as serotypes AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13.

[0026] The present invention also provides pharmaceutical compositions (or, as often referred to herein simply as "compositions") comprising any of the rAAV vectors of the present invention.

[0027] 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 culture supernatant of the transfected cells. The invention also provides viral particles comprising any of the recombinant AAV vectors of the invention.

[0028] The invention provides a method for treating muscular dystrophy comprising administering a therapeutically effective amount of any of the recombinant AAV vectors of the invention expressing human microdystrophin.

[0029] The present invention provides a method of treating muscular dystrophy, comprising administering a recombinant AAV vector comprising a therapeutically effective amount of the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2.

[0030] The present invention also provides a method of treating muscular dystrophy, comprising administering a therapeutically effective amount of a recombinant AAV vector comprising the nucleotide sequence of the pAAV.MHCK7.microdystrophin construct of SEQ ID NO:3.

[0031] "Fibrosis" refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and the abnormal repair process of tissues upon injury, including skeletal muscle, cardiac muscle, liver, lung, kidney, and pancreas. Deposited ECM components include fibronectin and collagens, such as collagen 1, collagen 2, or collagen 3.

[0032] The present invention also provides a method for reducing or preventing fibrosis in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of any of the recombinant AAV vectors of the present invention.

[0033] 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.For example, any of the rAAVs of the present invention can be administered to a subject suffering from muscular dystrophy to prevent fibrosis, for example, an rAAV expressing the human microdystrophin protein of the present invention is administered before fibrosis is observed in the subject.In addition, an rAAV expressing the human microdystrophin gene of the present invention can be administered to a subject at risk of developing fibrosis, such as those suffering from or diagnosed with muscular dystrophy, e.g., DMD.The rAAV of the present invention can be administered to a subject suffering from muscular dystrophy to prevent de novo fibrosis in these subjects.

[0034] The present invention contemplates administering any of the AAV vectors of the present invention before fibrosis is observed in a subject.Furthermore, the rAAV of the present invention can be administered to subjects at risk of developing fibrosis, such as those who suffer from or are diagnosed with muscular dystrophy, e.g., DMD.The rAAV of the present invention can be administered to subjects who suffer from muscular dystrophy and have already developed fibrosis, to prevent new fibrosis in these subjects.

[0035] 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 human micro-dystrophin-expressing rAAV vectors of the present invention. These methods can further comprise administering a micro-dystrophin-expressing rAAV.

[0036] The present invention contemplates administering any of the AAV vectors of the invention to a patient diagnosed with DMD before fibrosis is observed in the subject, or before muscle strength is lost, or before muscle mass is lost.

[0037] The invention also contemplates administering the AAV of the invention to subjects suffering from muscular dystrophies who have already developed fibrosis to prevent new fibrosis in these subjects or to reduce fibrosis in these patients. The invention also provides for administering any of the rAAV of the invention to patients suffering from muscular dystrophies who already have muscle weakness or loss of muscle mass to protect the muscle from further damage.

[0038] In any of the methods of the invention, the subject may be suffering from a muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy.

[0039] In another embodiment, the rAAV vector expressing the microdystrophin protein comprises a coding sequence of the microdystrophin gene operably linked to a muscle-specific control element other than MHCK7 or MCK. For example, the muscle-specific control element is a human skeletal actin gene element, a cardiac actin gene element, a muscle cell-specific enhancer-binding factor MEF, a truncated MCK (tMCK), a myosin heavy chain (MHC), a C5-12 (synthetic promoter), a mouse creatine kinase enhancer factor, 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).

[0040] In any of the methods of the invention, the rAAV vector or composition can be administered by intramuscular or intravenous injection.

[0041] Additionally, in any of the methods of the invention, the rAAV vector or composition can be administered systemically, for example, the rAAV vector or composition can be administered parenterally by injection, infusion, or implantation.

[0042] In another embodiment, the invention provides a composition comprising any of the rAAV vectors of the invention for reducing fibrosis in a subject in need thereof.

[0043] Furthermore, the present invention provides a composition comprising any of the recombinant AAV vectors of the present invention for preventing fibrosis in a patient suffering from muscular dystrophy.

[0044] The present invention provides a composition comprising any of the recombinant AAV vectors of the present invention for treating muscular dystrophy.

[0045] The present invention provides a composition comprising a recombinant AAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 for the treatment of muscular dystrophy.

[0046] The present invention provides a composition comprising a recombinant AAV vector comprising the pAAV.MHCK7.microdystrophin construct comprising the nucleotide sequence of SEQ ID NO:3 for the treatment of muscular dystrophy.

[0047] The present invention also provides compositions comprising any of the rAAV vectors of the present invention for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy. In a further embodiment, the present invention provides compositions comprising any of the rAAV vectors of the present invention for the treatment of muscular dystrophy.

[0048] The compositions of the invention can be formulated for intramuscular or intravenous injection. The compositions of the invention are also formulated for systemic administration, such as parenteral administration by injection, infusion, or implantation.

[0049] Additionally, any of the compositions can be formulated for administration to a subject suffering from a muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy.

[0050] In a further embodiment, the present invention provides the use of any of the rAAV vectors of the present invention for the preparation of a medicament for reducing fibrosis in a subject in need thereof.For example, the subject in need thereof may suffer from a muscular dystrophy, such as DMD or any other dystrophin-related muscular dystrophy.

[0051] In another embodiment, the present invention provides the use of an rAAV vector of the present invention for the preparation of a medicament for preventing fibrosis in a subject suffering from muscular dystrophy.

[0052] Furthermore, the present invention provides the use of a recombinant AAV vector of the present invention in the preparation of a medicament for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy.

[0053] The present invention also provides the use of an rAAV vector of the present invention for the preparation of a medicament for the treatment of muscular dystrophy.

[0054] The present invention provides the use of a recombinant AAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO: 1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO: 2 for the preparation of a medicament for the treatment of muscular dystrophy.

[0055] The present invention provides the use of a recombinant AAV vector comprising the nucleotide sequence of the pAAV.MHCK7.microdystrophin construct of SEQ ID NO:3 for the treatment of muscular dystrophy.

[0056] In any of the uses of the present invention, the agent may be formulated for intramuscular or intravenous injection. Additionally, in any of the uses of the present invention, the agent is formulated for systemic administration, such as parenteral administration by injection, infusion, or implantation.

[0057] Any of the agents can be prepared for administration to a subject suffering from a muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy. The present invention provides, for example, the following items: (Item 1) A recombinant AAV vector comprising a nucleotide sequence of a muscle-specific control element and a nucleotide sequence encoding a micro-dystrophin protein. (Item 2) The nucleotide sequence encoding the micro-dystrophin protein is a) a nucleotide sequence that is at least 85% identical to the nucleotide sequence of SEQ ID NO:1 and that encodes a functional micro-dystrophin protein; or b) The recombinant AAV vector according to item 1, comprising the nucleotide sequence of SEQ ID NO:1. (Item 3) 3. The recombinant AAV vector of item 1 or 2, wherein the muscle-specific regulatory element is a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer-binding factor (MEF), a muscle creatine kinase (MCK), a truncated MCK (tMCK), a myosin heavy chain (MHC), a hybrid alpha-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7), C5-12, a mouse creatine kinase enhancer factor, 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 4) 4. The recombinant AAV vector according to any one of items 1 to 3, wherein the muscle-specific control element is muscle creatine kinase (MCK) or hybrid alpha-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7). (Item 5) 5. The recombinant AAV vector according to any one of items 1 to 4, wherein the muscle-specific control element is a muscle creatine kinase (MCK) comprising the nucleotide sequence of SEQ ID NO: 4, or a hybrid alpha-myosin heavy chain enhancer / MCK enhancer promoter (MHCK7) comprising the nucleotide sequence of SEQ ID NO: 2. (Item 6) 6. The recombinant AAV vector according to any one of items 1 to 5, comprising the nucleotide sequences of SEQ ID NOs: 1 and 2. (Item 7) 7. The recombinant AAV vector according to any one of items 1 to 6, comprising the nucleotide sequence of SEQ ID NO: 3. (Item 8) A recombinant AAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2. (Item 9) A recombinant AAV vector comprising the nucleotide sequence of the pAAV.MHCK7.micro-dystrophin construct of SEQ ID NO:3. (Item 10) 6. The recombinant AAV vector according to any one of items 1 to 5, comprising the nucleotide sequences of SEQ ID NOs: 1 and 4. (Item 11) 11. The recombinant AAV vector of any one of items 1 to 5 or 10, comprising the nucleotide sequence of SEQ ID NO:5. (Item 12) 12. The recombinant AAV vector according to any one of items 1 to 11, wherein the vector is of the serotype AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. (Item 13) 13. The recombinant AAV vector of any one of claims 1 to 12, wherein the nucleotide sequence of the muscle-specific control element is operably linked to a microdystrophin nucleotide sequence. (Item 14) A composition comprising the recombinant AAV vector according to any one of items 1 to 13 and a pharma- ceutically acceptable carrier. (Item 15) A method for increasing muscle strength or muscle mass in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of a recombinant AAV vector described in any one of items 1 to 13 or a composition described in item 14. (Item 16) A method for reducing or preventing fibrosis in a subject suffering from muscular dystrophy, comprising administering a therapeutically effective amount of a recombinant AAV vector described in any one of items 1 to 13 or a composition described in item 14. (Item 17) A method for treating muscular dystrophy, comprising administering a therapeutically effective amount of a recombinant AAV vector described in any one of items 1 to 13 or a composition described in item 14. (Item 18) A method for treating muscular dystrophy, comprising administering a therapeutically effective amount of a recombinant AAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2. (Item 19) A method for treating muscular dystrophy, comprising administering a therapeutically effective amount of a recombinant AAV vector comprising the nucleotide sequence of the pAAV.MHCK7.microdystrophin construct of SEQ ID NO:3. (Item 20) 20. The method according to any one of items 15 to 19, wherein the muscular dystrophy is Duchenne muscular dystrophy. (Item 21) 20. The method of any one of items 15 to 19, wherein the recombinant AAV vector or the composition is administered by intramuscular or intravenous injection. (Item 22) 20. The method of any one of items 15 to 19, wherein the recombinant AAV vector or the composition is administered systemically. (Item 23) 23. The method of item 22, wherein the recombinant AAV vector or the composition is administered parenterally by injection, infusion, or implantation. (Item 24) 14. A composition comprising a recombinant AAV vector according to any one of items 1 to 13 for increasing muscle strength or muscle mass in a subject suffering from muscular dystrophy. (Item 25) A composition comprising a recombinant AAV vector according to any one of items 1 to 13 for the treatment of muscular dystrophy. (Item 26) A composition comprising a recombinant AAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter sequence of SEQ ID NO:2 for the treatment of muscular dystrophy. (Item 27) A composition comprising a recombinant AAV vector comprising the pAAV.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO:3 for the treatment of muscular dystrophy. (Item 28) 28. The composition according to any one of items 24 to 27, wherein the muscular dystrophy is Duchenne muscular dystrophy. (Item 29) 29. The composition according to any one of items 24 to 28, formulated for intramuscular or intravenous injection. (Item 30) 29. The method of any one of items 24 to 28, wherein the recombinant AAV vector or the composition is administered systemically. (Item 31) 31. The method of item 30, wherein the recombinant AAV vector or composition is administered parenterally by injection, infusion, or implantation. (Item 32) Use of a recombinant AAV vector according to any one of items 1 to 13 or a composition according to item 14 for the preparation of a medicament for increasing muscle strength or muscle mass in a subject suffering from muscular dystrophy. (Item 33) Use of a recombinant AAV vector according to any one of items 1 to 13 or a composition according to item 14 for the preparation of a medicament for the treatment of muscular dystrophy. (Item 34) Use of a recombinant AAV vector according to any one of items 1 to 13 or a composition according to item 14 for the preparation of a medicament for reducing or preventing fibrosis in a subject suffering from muscular dystrophy. (Item 35) 2. Use of a recombinant AAV vector comprising the human microdystrophin nucleotide sequence of SEQ ID NO:1 and the MHCK7 promoter nucleotide sequence of SEQ ID NO:2 for the preparation of a medicament for the treatment of muscular dystrophy. (Item 36) Use of a recombinant AAV vector comprising the nucleotide sequence of the pAAV.MHCK7.microdystrophin construct of SEQ ID NO:3 for the treatment of muscular dystrophy. (Item 37) 37. The use according to any one of items 32 to 36, wherein the muscular dystrophy is Duchenne muscular dystrophy. (Item 38) 38. The use according to any one of items 32 to 37, wherein the medicament is formulated for intramuscular or intravenous administration. (Item 39) 38. The use according to any one of items 32 to 37, wherein the medicament is formulated for systemic delivery. (Item 40) 40. The use according to item 39, wherein the medicament is formulated for parenteral administration by injection, infusion or implantation. [Brief description of the drawings]

[0058] [Figure 1] The pAAV.MHCK7.microdystrophin construct is illustrated. In this construct, the cDNA expression cassette is flanked by AAV2 inverted terminal repeats (ITRs). The construct features an in-frame rod deletion (R4 to R23) that maintains hinges 1, 2, and 4 (H1, H2, and H4) and a cysteine-rich domain to produce a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) is driven by the MHCK7 promoter (795 bp). The intron and 5'UTR are derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette had a consensus Kozak immediately prior to the ATG start and a small 53 bp synthetic polyA signal for mRNA termination. The human microdystrophin cassette contained the (R4-R23 / Δ71-78) domain as previously described by Harper et al. (Nature Medicine 8, 253-261 (2002)). [Diagram 2] Demonstrate the expression of dystrophin protein after intramuscular delivery of AAVrh74.MHCK7 construct. 1x10vg (n=5 per group) was injected into the tibialis anterior muscle of mdx mice. After 6 weeks, muscle was harvested and stained for dystrophin expression with dystrophin N-terminal antibody and hematoxylin and eosin staining. [Figure 3A]Quantification of skeletal muscle force measurements and microdystrophin expression following intramuscular injection of the AAVrh74.MHCK7 construct. (A) The tibialis anterior muscles of mdx mice were injected with the AAVrh74.MHCK7 construct at 1x10vg (n=5). Six weeks later, tibialis anterior muscles were harvested and in vivo force measurements were performed. Treated cohorts demonstrated significantly greater force generation than untreated mdx controls. [Figure 3B] Quantification of skeletal muscle force measurements and microdystrophin expression following intramuscular injection of the AAVrh74.MHCK7 construct. (A) The tibialis anterior muscles of mdx mice were injected with the AAVrh74.MHCK7 construct at 1x10vg (n=5). Six weeks later, tibialis anterior muscles were harvested and in vivo force measurements were performed. Treated cohorts demonstrated significantly greater force generation than untreated mdx controls. [Figure 3C] Quantification of skeletal muscle force measurements and microdystrophin expression following intramuscular injection of the AAVrh74.MHCK7 construct. (A) The tibialis anterior muscles of mdx mice were injected with the AAVrh74.MHCK7 construct at 1x10vg (n=5). Six weeks later, tibialis anterior muscles were harvested and in vivo force measurements were performed. Treated cohorts demonstrated significantly greater force generation than untreated mdx controls. [Figure 4A] Demonstrating widespread transduction of skeletal, diaphragm, and cardiac muscle fibers following systemic administration of the AAVrh.74.MHCK7.micro-dys construct. (A) Mdx mice were treated systemically with 6x1012vg (2x1014vg / kg) AAVrh.74.MHCK7.micro-dystrophin via the tail vein at 6 weeks of age, following 12 weeks of treatment. (B) Micro-dystrophin staining shows quantification of the percentage of myofibers expressing micro-dystrophin in each tissue. (C) Specific force measured in the diaphragm at low and high (clinical) doses. No significant differences were seen at the low dose, but there was a marked improvement at the high dose. [Figure 4B]Demonstrating widespread transduction of skeletal, diaphragm, and cardiac muscle fibers following systemic administration of the AAVrh.74.MHCK7.micro-dys construct. (A) Mdx mice were treated systemically with 6x1012vg (2x1014vg / kg) AAVrh.74.MHCK7.micro-dystrophin via the tail vein at 6 weeks of age, following 12 weeks of treatment. (B) Micro-dystrophin staining shows quantification of the percentage of myofibers expressing micro-dystrophin in each tissue. (C) Specific force measured in the diaphragm at low and high (clinical) doses. No significant differences were seen at the low dose, but there was a marked improvement at the high dose. [Figure 4C] Demonstrating widespread transduction of skeletal, diaphragm, and cardiac muscle fibers following systemic administration of the AAVrh.74.MHCK7.micro-dys construct. (A) Mdx mice were treated systemically with 6x1012vg (2x1014vg / kg) AAVrh.74.MHCK7.micro-dystrophin via the tail vein at 6 weeks of age, following 12 weeks of treatment. (B) Micro-dystrophin staining shows quantification of the percentage of myofibers expressing micro-dystrophin in each tissue. (C) Specific force measured in the diaphragm at low and high (clinical) doses. No significant differences were seen at the low dose, but there was a marked improvement at the high dose. [Diagram 5] Figure 1 shows dystrophin protein expression following systemic delivery of the AAVrh.74.MHCK7.microdystrophin construct. Mdx mice (n=5) were treated systemically with 6x1012 vg of AAVrh.74.MHCK7.microdystrophin via the tail vein starting at 6 weeks of age. After 12 weeks of treatment, all muscles were harvested and stained for restoration of dystrophin and DAPC components (showing beta-sarcoglycan). [Figure 6A]Demonstrating toxicity / safety of AAVrh.74.MHCK7. Hematoxylin and eosin (H&E) staining was performed on the following muscle tissues to analyze toxicity: tibialis anterior (TA), gastrocnemius (GAS), quadriceps (QD), psoas (PSO), triceps (TRI), and diaphragm (DIA) (Figure 6A). No toxicity was observed. As an indication of efficacy, the number of muscle fibers with centrally located nuclei (CN) was quantified (Figure 6B). A decrease in CN demonstrates therapeutic efficacy, as CN indicates cycles of muscle degeneration and regeneration. (Figure 6C) Demonstrating that the total number of fibers is unchanged by treatment. Creatine kinase amounts are shown in (D) and demonstrate improvement at higher doses. An independent t-test was used to identify differences (p<0.05) and data are reported as mean ± SEM. [Figure 6B] Demonstrating toxicity / safety of AAVrh.74.MHCK7. Hematoxylin and eosin (H&E) staining was performed on the following muscle tissues to analyze toxicity: tibialis anterior (TA), gastrocnemius (GAS), quadriceps (QD), psoas (PSO), triceps (TRI), and diaphragm (DIA) (Figure 6A). No toxicity was observed. As an indication of efficacy, the number of muscle fibers with centrally located nuclei (CN) was quantified (Figure 6B). A decrease in CN demonstrates therapeutic efficacy, as CN indicates cycles of muscle degeneration and regeneration. (Figure 6C) Demonstrating that the total number of fibers is unchanged by treatment. Creatine kinase amounts are shown in (D) and demonstrate improvement at higher doses. An independent t-test was used to identify differences (p<0.05) and data are reported as mean ± SEM. [Figure 6C]Demonstrating toxicity / safety of AAVrh.74.MHCK7. Hematoxylin and eosin (H&E) staining was performed on the following muscle tissues to analyze toxicity: tibialis anterior (TA), gastrocnemius (GAS), quadriceps (QD), psoas (PSO), triceps (TRI), and diaphragm (DIA) (Figure 6A). No toxicity was observed. As an indication of efficacy, the number of muscle fibers with centrally located nuclei (CN) was quantified (Figure 6B). A decrease in CN demonstrates therapeutic efficacy, as CN indicates cycles of muscle degeneration and regeneration. (Figure 6C) Demonstrating that the total number of fibers is unchanged by treatment. Creatine kinase amounts are shown in (D) and demonstrate improvement at higher doses. An independent t-test was used to identify differences (p<0.05) and data are reported as mean ± SEM. [Figure 6D] Demonstrating toxicity / safety of AAVrh.74.MHCK7. Hematoxylin and eosin (H&E) staining was performed on the following muscle tissues to analyze toxicity: tibialis anterior (TA), gastrocnemius (GAS), quadriceps (QD), psoas (PSO), triceps (TRI), and diaphragm (DIA) (Figure 6A). No toxicity was observed. As an indication of efficacy, the number of muscle fibers with centrally located nuclei (CN) was quantified (Figure 6B). A decrease in CN demonstrates therapeutic efficacy, as CN indicates cycles of muscle degeneration and regeneration. (Figure 6C) Demonstrating that the total number of fibers is unchanged by treatment. Creatine kinase amounts are shown in (D) and demonstrate improvement at higher doses. An independent t-test was used to identify differences (p<0.05) and data are reported as mean ± SEM. [Figure 7] 1 shows the pAAV.MCK.micro-dystrophin plasmid construct. [Figure 8] Results of rAAVrh74.MCK micro-dystrophin (human) potency assay are provided. tibialis anterior muscles of mdx mice were injected with 3x109, 3x1010, or 1x1011 vg (n=3 per group). After 4 weeks, muscles were harvested and stained for dystrophin expression with N-terminal Dys3 antibody. There was a linear correlation between expression and dose, with very little expression (no effect level) at 3x109 vg and 89% expression at 1x1011 vg. [Figure 9A] Human micro-dystrophin has been shown to improve force generation and protection from damage due to abnormal contractions. (A) Immunostaining of dystrophin protein in the extensor digitorum longus (EDL) and TA shows expression in mdx muscle fibers after rAAVrh.74-MCK-micro-dystrophin (human) injection via the femoral artery. Mock-infected muscles were stained in an identical manner and exposure times were matched. (B) rAAVrh.74-MCK-micro-dystrophin significantly increased normalized specific force compared to mock-treated mdx muscles (P<0.05 vs. mdx). (C) mdx muscles infected with rAAVrh.74-MCK-Micro-dys (human) were compared to mock-infected contralateral mdx EDL muscles and WT (WT C57Bl / 10) EDL muscles for force reduction during repetitive eccentric contractions at 12 weeks post-gene transfer. rAAVrh.74-MCK-micro-dystrophin (Micro-dys) treatment significantly protected against loss of force compared to mock-treated mdx muscles (P<0.001 vs. mdx). Error bars are SEM. [Figure 9B]Human micro-dystrophin has been shown to improve force generation and protection from damage due to abnormal contractions. (A) Immunostaining of dystrophin protein in the extensor digitorum longus (EDL) and TA shows expression in mdx muscle fibers after rAAVrh.74-MCK-micro-dystrophin (human) injection via the femoral artery. Mock-infected muscles were stained in an identical manner and exposure times were matched. (B) rAAVrh.74-MCK-micro-dystrophin significantly increased normalized specific force compared to mock-treated mdx muscles (P<0.05 vs. mdx). (C) mdx muscles infected with rAAVrh.74-MCK-Micro-dys (human) were compared to mock-infected contralateral mdx EDL muscles and WT (WT C57Bl / 10) EDL muscles for force reduction during repetitive eccentric contractions at 12 weeks post-gene transfer. rAAVrh.74-MCK-micro-dystrophin (Micro-dys) treatment significantly protected against loss of force compared to mock-treated mdx muscles (P<0.001 vs. mdx). Error bars are SEM. [Figure 9C] Human micro-dystrophin has been shown to improve force generation and protection from damage due to abnormal contractions. (A) Immunostaining of dystrophin protein in the extensor digitorum longus (EDL) and TA shows expression in mdx muscle fibers after rAAVrh.74-MCK-micro-dystrophin (human) injection via the femoral artery. Mock-infected muscles were stained in an identical manner and exposure times were matched. (B) rAAVrh.74-MCK-micro-dystrophin significantly increased normalized specific force compared to mock-treated mdx muscles (P<0.05 vs. mdx). (C) mdx muscles infected with rAAVrh.74-MCK-Micro-dys (human) were compared to mock-infected contralateral mdx EDL muscles and WT (WT C57Bl / 10) EDL muscles for force reduction during repetitive eccentric contractions at 12 weeks post-gene transfer. rAAVrh.74-MCK-micro-dystrophin (Micro-dys) treatment significantly protected against loss of force compared to mock-treated mdx muscles (P<0.001 vs. mdx). Error bars are SEM. [Figure 10-1] The nucleic acid sequence (SEQ ID NO:3, rAAVrh74.MHCK7.microdystrophin) is presented. [Figure 10-2] The nucleic acid sequence (SEQ ID NO:3, rAAVrh74.MHCK7.microdystrophin) is presented. [Figure 10-3] The nucleic acid sequence (SEQ ID NO:3, rAAVrh74.MHCK7.microdystrophin) is presented. [Figure 10-4] The nucleic acid sequence (SEQ ID NO:3, rAAVrh74.MHCK7.microdystrophin) is presented. [Figure 11-1] The nucleic acid sequence (SEQ ID NO:5) rAAVrh74.MCK.microdystrophin is presented. [Figure 11-2] The nucleic acid sequence (SEQ ID NO:5) rAAVrh74.MCK.microdystrophin is presented. [Figure 11-3] The nucleic acid sequence (SEQ ID NO:5) rAAVrh74.MCK.microdystrophin is presented. [Figure 11-4] The nucleic acid sequence (SEQ ID NO:5) rAAVrh74.MCK.microdystrophin is presented. [Figure 12A] presents the immune response to systemic delivery of AAVrh74.MHCK7.microdystrophin to non-human primates. (A) ELISpot responses to AAV capsid and microdystrophin peptide pools. ConA is a positive control and DMSO is a negative control. There were three pools for AAVrh74 and four peptide pools specific for microdystrophin. (B) ELISA positive titers of circulating neutralizing antibodies to vector capsid. Serum was isolated from primates every two weeks and analyzed for antibody titers. The reported titers correspond to the last dilution with a ratio of responses ≥2. [Figure 12B]presents the immune response to systemic delivery of AAVrh74.MHCK7.microdystrophin to non-human primates. (A) ELISpot responses to AAV capsid and microdystrophin peptide pools. ConA is a positive control and DMSO is a negative control. There were three pools for AAVrh74 and four peptide pools specific for microdystrophin. (B) ELISA positive titers of circulating neutralizing antibodies to vector capsid. Serum was isolated from primates every two weeks and analyzed for antibody titers. The reported titers correspond to the last dilution with a ratio of responses ≥2. [Figure 13A] We demonstrate systemic delivery of AAVrh74.MHCK7.micro-dystrophin in rhesus macaques. Anti-FLAG immunofluorescence staining of the left lateral muscle demonstrated robust micro-dystrophin expression. [Figure 13B] We demonstrate systemic delivery of AAVrh74.MHCK7.micro-dystrophin in rhesus macaques. Anti-FLAG immunofluorescence staining of the left lateral muscle demonstrated robust micro-dystrophin expression. [Figure 14] Demonstrating the effect of systemic treatment with rAAVrh74.MHCK7.micro-dystrophin on transgene expression. Immunofluorescent staining of micro-dystrophin using an N-terminal dystrophin antibody in the heart, diaphragm, psoas, and tibialis anterior (TA) muscles demonstrates robust expression in animals 3 months post-injection treated with medium dose (6e12vg; 2e14vg / kg) and high dose (1.2e13vg; 6e14vg / kg). 20x images are shown. [Figure 15] Demonstrating the effect of systemic treatment with rAAVrh74.MHCK7.micro-dystrophin on transgene expression. Immunofluorescent staining of micro-dystrophin using an N-terminal dystrophin antibody in gastrocnemius, quadriceps, triceps, and gluteus muscles demonstrates robust expression in animals 3 months post-injection treated with the mid dose (6e12vg; 2e14vg / kg) and the highest dose (1.2e13vg; 6e14vg / kg). Images at 20x magnification are shown. [Figure 16]Demonstrating the effect of systemic treatment with rAAVrh74.MHCK7.microdystrophin on muscle pathology. (A) H&E staining of diaphragm, tibialis anterior, gastrocnemius, and quadriceps muscles from C57BL / 6 WT, mdx, and rAAVrh74.MHCK7.microdystrophin-treated mice (medium dose 2e14vg / kg; high dose -6e14vg / kg), (B) quantification of mean fiber size demonstrated normalization of fiber size across all tissues. ****p<0.001, one-way ANOVA; data are reported as mean ± SEM. 20x images are shown. [Figure 17] Demonstrating the effect of systemic treatment with rAAVrh74.MHCK7.microdystrophin on muscle pathology. (A) H&E staining of triceps, gluteus, and psoas muscles from C57BL / 6 WT, mdx, and rAAVrh74.MHCK7.microdystrophin-treated mice (medium dose 2e14vg / kg; high dose -6e14vg / kg), (B) quantification of average fiber size demonstrated larger fibers in a dose-dependent manner. ****p<0.001, one-way ANOVA; data are reported as mean ± SEM. 20x images are shown. [Figure 18] 13 demonstrates the effect of systemic treatment with rAAVrh74.MHCK7.microdystrophin on central nucleation. Dose escalation shows a decrease in central nucleation in all skeletal muscles and diaphragm. Two-way ANOVA was used to determine differences (p<0.05). Data are reported as mean±SEM. [Figure 19] 13 demonstrates the effect of systemic treatment with rAAVrh74.MHCK7.microdystrophin on collagen deposition. Dose escalation shows a decrease in diaphragm collagen accumulation (%). *p<0.05, one-way ANOVA; data are reported as mean±SEM. 20x images are shown. [Figure 20] Correction of diaphragm force deficits is shown. After 3 or 6 months of treatment, diaphragm strips were harvested to measure specific force (normalized to cross-sectional area). Treatment restored force to WT levels. *p<0.05. One-way ANOVA was used to determine differences from mdx-LR mice. [Figure 21] Correction of force deficits in the TA. (A) After 3-6 months of treatment, TA muscles were harvested (both left and right) to measure specific force (normalized to TA weight). Treatment restored force to WT levels. (B) After a rigorous protocol of eccentric contractions, treatment rescued TA muscles from fatigue. *p<0.05. One-way ANOVA was used to determine differences from mdx-LR mice. [Figure 22] Presenting the distribution of average vg copies in various tissues from three mdx mice following IV delivery of rAAVrh74.MHCK7.microdystrophin. [Figure 23] Serum chemistry of mice systemically injected with ssAAVrh74.MHCK7.microdystrophin and age-matched controls was analyzed by an independent CRO (Charles River Laboratories) showing normal values ​​for all chemicals analyzed. The only abnormal values ​​were elevated AST and ALT seen in MDX vehicle-treated animals [MDX-LR (lactated Ringer's solution)] which normalized with treatment. AST and ALT are known to be elevated in DMD. ALT=Alanine aminotransferase, ALP / K=Alkaline phosphatase, AST=Aspartate aminotransferase, BUN=Blood urea nitrogen, B / C=Blood / creatinine ratio, CREAT=Creatine, GLU=Glucose, TP=Total protein, TBIL=Total bilirubin, DBIL=Direct bilirubin. [Figure 24] FIG. 13 presents muscle and organ biodistribution western blots of mdx mice systemically injected with rAAVrh74.MHCK7.micro-dystrophin. [Diagram 25] 1 presents the pNLREP2-Caprh74 AAV helper plasmid map. [Figure 26] Presents the Ad Helper plasmid pHELP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] The present invention provides gene therapeutic vectors, e.g., rAAV vectors, that overexpress human microdystrophin, as well as methods for reducing and preventing fibrosis in patients with muscular dystrophies. 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 passage of endomysial nutrients through the connective tissue barrier, reduces blood flow, deprives muscles of vascular-derived nutrients, and functionally contributes to early loss of ambulation due to limb contractures. Over time, the challenge of treatment increases as a result of significant fibrosis of the muscle. This can be observed in muscle biopsies comparing connective tissue proliferation at successive time points. This process continues to worsen, resulting in loss of ambulation and accelerating loss of control, especially in wheelchair-dependent patients.

[0060] Without early treatment, including a parallel approach to reduce fibrosis, the benefits of exon skipping, stop codon read-through, or gene replacement therapy are unlikely to be fully achieved. Even small molecule or protein replacement strategies may fail without an approach to reduce muscle fibrosis. Previous studies in aged mdx mice with pre-existing fibrosis treated with AAV.microdystrophin showed that full functional recovery could not be achieved (Liu, M., et al., Mol Ther 11, 245-256 (2005)). The progression of DMD cardiomyopathy is also known to be accompanied by scarring and fibrosis of the ventricular wall.

[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 where certain functions are provided by a co-infecting helper virus. Currently, there are 13 serotypes of AAV that have been characterized. 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, it is well known that various serotypes are very closely related, both structurally and functionally, even at the genetic level, so 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. Puttson, 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 have three 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) that are flanked by AAV terminal repeats (ITRs) that can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector that encodes and expresses the rep and cap gene products.

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

[0064] AAV The recombinant AAV genome of the present invention comprises the nucleic acid molecule of the present invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA of the rAAV genome can be derived from any AAV serotype that can derive recombinant virus, including but not limited to AAV serotypes AAVrh.74, 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 production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV mutants are also contemplated, such as rAAV with capsid mutations. 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. AAV1, AAV6, AAV8 or AAVrh.74 can be used to promote skeletal muscle-specific expression.

[0065] The DNA plasmid of the present invention comprises the rAAV genome of the present invention. The DNA plasmid is introduced into a cell that is permissive for infection by a helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) of AAV to assemble the rAAV genome into an infectious viral particle. The technology of producing rAAV particles is standard in the art, in which the AAV genome to be packaged, the rep and cap genes, and the helper virus functions are provided to the cell. The production of rAAV requires that the following components are 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 may be derived from any AAV serotype from which a recombinant virus may be derived, or may be derived from an AAV serotype different from the rAAV genomic ITRs, including, but not limited to, 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 to generate packaging cells is to create a cell line that stably expresses all the components required for the production of AAV particles. 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 genome of the cell. The AAV genome has been introduced into a bacterial plasmid by methods such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), the addition of a synthetic linker containing a restriction endonuclease cleavage site (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 plasmids 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 include Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81: 6466 (1984), Tratschin et al., Mo 1. Cell. Biol. al., J. Virol., 62:1963 (1988), and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988), Samulski et al. al., J. Virol., 63: 3822-3828 (1989), 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. Vaccine 13: 1244-1250 (1995), Paul et al. al. Human Gene Therapy 4:609-615 (1993), Clark et al. Gene Therapy 3:1124-1132 (1996), 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] Therefore, the present invention provides a packaging cell that produces infectious rAAV.In one embodiment, the packaging cell can be a stably transformed cancer cell, such as HeLa cell, 293 cell, and PerC.6 cell (similar 293 line).In another embodiment, the packaging cell is a cell that is not a transformed cancer cell, such as low-passage 293 cell (human fetal kidney cell transformed with adenovirus E1), MRC-5 cell (human fetal fibroblast), WI-38 cell (human fetal fibroblast), Vero cell (monkey kidney cell), and FRhL-2 cell (rhesus fetal lung cell).

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

[0070] In an exemplary embodiment, a recombinant AAV vector of the invention is produced by a triple transfection method (Xiao et al., J Virol 72, 2224-2232 (1998) using the AAV vector plasmids pAAV.MHCK7.microdystrophin, pNLRep2-Caprh74, and pHelp, where pAAV contains a microdystrophin gene expression cassette flanked by AAV2 inverted terminal repeats (ITRs). It is this sequence that is encapsidated into the AAVrh74 virion. The plasmid contains the microdystrophin sequence and the core promoter elements of the MHCK7 enhancer and muscle-specific promoter that drive gene expression. The expression cassette also contains an SV40 intron (SD / SA) to promote high levels of gene expression and uses the bovine growth hormone polyadenylation signal for efficient transcription termination.

[0071] pNLREP2-Caprh74 is an AAV helper plasmid encoding the four wild-type AAV2 rep proteins and three wild-type AAV VP capsid proteins from serotype rh74. A schematic diagram of the pNLREP2-Caprh74 plasmid is shown in Figure 25.

[0072] The pHELP adenovirus helper plasmid is 11,635 bp and was obtained from Applied Viromics. This plasmid contains regions of the adenovirus genome important for AAV replication, namely E2A, E4ORF6, and VA RNA (adenovirus E1 function is provided by 293 cells). The adenovirus sequences present in this plasmid are only about 40% of the adenovirus genome and do not contain cis elements important for replication such as the adenovirus terminal repeats. Therefore, infectious adenovirus is not expected to be generated from such a production system. A schematic diagram of the pHELP plasmid is shown in Figure 26.

[0073] rAAV can be purified by standard methods in the art, such as by 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 WO98 / 09657.

[0074] In another embodiment, the present invention contemplates a composition comprising the rAAV of the present invention. The composition of the present invention comprises rAAV and a pharma- ceutically acceptable carrier. The composition may also comprise other components, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are non-toxic to recipients, preferably inert at the dosages and concentrations employed, and include buffers and surfactants such as pluronics.

[0075] The titer of the rAAV administered in the methods of the invention will vary depending, for example, on the particular rAAV, the method of administration, the therapeutic goal, the individual, and the cell type(s) being targeted, and can be determined by standard methods in the art. The titer of the rAAV is approximately 1×10 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 , about 1×10 14 Dosages may range from 1000 mg to 1500 mg of DNase resistant particles (DRP) or more. Dosages may be expressed in units of viral genomes (vg).

[0076] Methods of 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 doses of a composition comprising the rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered before the onset of the disorder / disease, the administration is prophylactic. If the dose is administered after the onset of the disorder / disease, the administration is therapeutic. In an embodiment 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 to be treated, slows or prevents progression to the disorder / disease state, reduces the extent of the disease, causes remission (partial or complete) of the disease, and / or prolongs survival. An example of a disease contemplated for prevention or treatment by the method of the present invention is DMD.

[0077] Combination therapy is also contemplated by the present invention. As used herein, combination includes both simultaneous and sequential treatment. Combination of the method of the present invention with standard medical treatment (e.g., corticosteroids) is specifically contemplated, as well as combination with novel treatments.

[0078] Administration of an effective dose of the composition may be by routes standard in the art, including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal, or vaginal. The administration route(s) and serotype(s) of the AAV components (particularly the AAV ITRs and capsid proteins) of the rAAV of the present invention may be selected and / or adapted by the skilled artisan taking into account the disease state to be infected and / or treated and the target cell / tissue(s) expressing micro-dystrophin protein.

[0079] The present invention provides for local and systemic administration of effective doses of the rAAV and compositions of the present invention.For example, systemic administration refers to administration into the circulatory system so that the whole body is affected.Systemic administration includes enteral administration, such as absorption through the digestive tract, and parenteral administration by injection, infusion or implantation.

[0080] In particular, the actual administration of the rAAV of the present invention can be accomplished 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, injection into muscle and injection into the bloodstream. Simply resuspending rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for expression in muscle tissue, and there are no known limitations on the carrier or other components that can be co-administered with rAAV (compositions that degrade DNA should be avoided in the usual manner involving rAAV). The capsid protein of rAAV may be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or as a local formulation that is delivered to muscle by transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been developed to date and can be used in the practice of the present invention. The rAAV can be used with any pharma- ceutically acceptable carrier to facilitate administration and handling.

[0081] The dose of rAAV administered in the methods disclosed herein will vary depending, for example, on the particular rAAV, the method of administration, the therapeutic goal, the individual, and the cell type(s) being targeted, and can be determined by standard methods in the art. The titer of each rAAV administered 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 , about 1×10 14 or about 1 × 10 15 Dosages may range from 1×10 to 1×10 DNase resistant particles (DRP) or more. Dosages may be expressed in units of viral genomes (vg) (i.e., 1×10 7 vg, 1×10 8 vg, 1×10 9 vg, 1×1010 vg, 1×10 11 vg, 1×10 12 vg, 1×10 13 vg, 1×10 14 vg, 1×10 15 Dosages may be expressed in units of viral genomes (vg) per kilogram (kg) of body weight (i.e., 1×10 10 vg / kg, 1×10 11 vg / kg, 1×10 12 vg / kg, 1×10 13 vg / kg, 1×10 14 vg / kg, 1×10 15 Methods for titrating AAV are described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

[0082] In particular, the actual administration of the rAAV of the present invention can be accomplished 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, injection into muscle and injection into the bloodstream. Simply resuspending rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for expression in muscle tissue, and there are no known limitations on the carrier or other components that can be co-administered with rAAV (compositions that degrade DNA should be avoided in the usual manner involving rAAV). The capsid protein of rAAV may be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, WO02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or as a local formulation that is delivered to muscle by transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been developed to date and can be used in the practice of the present invention. The rAAV can be used with any pharma- ceutically acceptable carrier to facilitate administration and handling.

[0083] For the purpose of 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 necessary, and the liquid diluent is first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains an acidic phosphate group) or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions of rAAV 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 connection, all sterile aqueous media employed are readily available by standard techniques well known to those skilled in the art.

[0084] 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 must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating agent, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include 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.

[0085] Sterile injectable solution is prepared by incorporating the required amount of rAAV into a suitable solvent, and then sterilizing by filtration, as necessary with various other components listed above.Generally, dispersion is prepared by mixing sterilized active ingredient into a sterile vehicle that contains basic dispersion medium and other required components listed above.For the preparation of sterile powder for the preparation of sterile injectable solution, the preferred method of preparation is vacuum drying and freeze-drying technology, which produces powder of active ingredient plus any additional desired components from their solution that has been previously sterilized and filtered.

[0086] Transduction with rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from the subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used if they do not generate an inappropriate immune response in the subject.

[0087] 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 a suitable medium and screening for cells with 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, and the composition can be introduced into a subject by a variety of techniques, such as by intramuscular, intravenous, subcutaneous, and intraperitoneal injection, or by injection into smooth and cardiac muscles, for example, using a catheter.

[0088] Transduction of cells with the rAAV of the present invention results in sustained expression of micro-dystrophin protein. Thus, the present invention provides methods for administering / delivering rAAV expressing micro-dystrophin protein to animals, preferably humans. These methods include transducing one or more rAAV of the present invention into tissues, including but not limited to tissues such as muscle, organs such as liver and brain, and glands such as salivary glands. Transduction may be performed using gene cassettes that contain tissue-specific control elements. For example, one embodiment of the present invention utilizes regulatory elements derived from the actin and myosin gene families, such as the myoD gene family (see Weintraub et al., Science, 251:761-766 (1991)), muscle cell specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11:4854-4862 (1991)), the human skeletal actin gene (Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)), the cardiac actin gene, muscle creatine kinase sequence elements (see Johnson et al., Mol Cell Biol, 9:3393-3399 (1989)), and the mouse creatine kinase enhancer (mCK) element, the fast skeletal troponin C gene, the slow cardiac troponin C gene element, and the slow troponin I gene, hypoxia-inducible nuclear factor (Semenza et al., Mol Cell Biol, 1997; The present invention provides methods for transducing muscle cells and muscle tissue induced by muscle-specific control elements, including, but not limited to, control elements derived from IL-16 (E. et al., Proc Natl Acad Sci USA, 88:5680-5684 (1991)), steroid-inducible elements and promoters including glucocorticoid response elements (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), and other control elements.

[0089] Because muscle tissue is a non-vital organ and easily accessible, it is an attractive target for in vivo DNA delivery. The present invention contemplates sustained expression of micro-dystrophin from transduced muscle fibers.

[0090] 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 derived from the digestive tract, bladder, blood vessels, or heart tissue). Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[0091] The term "transduction" is used to refer to the administration / delivery of the coding region for microdystrophin to a recipient cell, either in vivo or in vitro, via a replication-deficient rAAV of the present invention, resulting in expression of microdystrophin by the recipient cell.

[0092] Thus, the present invention provides methods of administering an effective dose (or essentially simultaneous or spaced doses) of a rAAV encoding micro-dystrophin to a patient in need thereof. EXAMPLES

[0093] Example 1 Generation of pAAV.MHCK7.micro-dystrophin construct The pAAV.MHCK7.microdystrophin plasmid contains a human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see Figure 1). The microdystrophin construct features an in-frame rod deletion (R4-R23) that maintains hinges 1, 2, and 4, as well as the cysteine-rich domain to produce a 138 kDa protein. Expression of the microdystrophin protein (3579 bp) was driven by the MHCK7 promoter (795 bp). The plasmid was constructed by removing the MCK promoter from the pAAV.MCK.microdystrophin plasmid and inserting the MHCK7 promoter. After the core promoter, 53 bp of endogenous mouse MCK exon 1 (untranslated) are present 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 had a consensus Kozak immediately before the ATG start and a small 53 bp synthetic polyA signal for mRNA termination. The human microdystrophin cassette contained the (R4~R23 / Δ71~78) domain as previously described by Harper et al. (Nature Medicine 8,253-261(2002)). Complementary DNA was codon-optimized for human use and synthesized by GenScript (Piscataway, NJ) (Mol Ther 18,109-117(2010)). The only viral sequences included in this vector were the AAV2 terminal inverted sequences required for both viral DNA replication and packaging. The microdystrophin cassette has a small 53 bp synthetic polyA signal for mRNA termination.

[0094] Previous studies have demonstrated expression in the heart using the MHCK7 promoter (Salva et al. Mol Ther 15, 320-329 (2007)) and AAVrh74 achieved expression in skeletal muscle, diaphragm, and cardiac muscle (Sondergaard et al. Annals of clinical and Transl Neurology 2, 256-270 (2015)). The sequences of the constructs in Figure 1 were encapsidated into AAVrh.74 virions. A molecular clone of the AAVrh.74 serotype was cloned from a lymph node of a rhesus macaque and is described in Rodino-Klapac et al. Journal of Translational medicine 5, 45 (2007). Table 1 shows the molecular characteristics of plasmid pAAV.MHCK7.microdystrophin (SEQ ID NO:3). [Table 1]

[0095] Example 2 Intramuscular expression studies using rAAV.MHCK7.micro-dystrophin Expression studies were performed with a human microdystrophin construct (rAAVrh74.MHCK7.microdystrophin; described in Example 1) by intramuscular injection. mdx mice (a spontaneous Dmd mouse that does not express dystrophin) were mdx 1 × 10 11 vg cassette (n=5 per group). Six weeks later, muscles were harvested and stained for dystrophin expression with an N-terminal antibody to dystrophin (Dys3) and hematoxylin and eosin (HE) staining. Figure 2 shows that dystrophin expression increased at 1×10 compared to untreated muscle. 11 Figure 1 shows diffuse gene expression and a decrease in centrally located nuclei at a dose of 1000 vg. Additionally, a decrease in central nucleation accompanied by an increase in average fibers / frame was observed after treatment with the microdystrophin construct. Expression levels of the rAAVrh74.MHCK7.microdystrophin construct were quantified to be approximately 73%.

[0096] In addition to measuring micro-dystrophin localization and expression levels, we measured skeletal muscle force following intramuscular injection of the cassette. Intramuscular expression of the pAAV.MHCK7.micro-dystrophin construct significantly increased absolute and specific force production compared to untreated controls (Figures 3A and 3B, respectively).

[0097] Example 3 Systemic delivery of rAAVrh.74.MHCK7.microdystrophin into mdx mice Cohorts of mdx mice were injected with 2 × 10 12 vg(8×10 13 vg / kg) or high dose (planned clinical dose) 6 × 10 12 vg(2×10 14 Mice were injected via the tail vein with either rAAVrh.74.MHCK7.micro-dystrophin at 6 × 10 ng / kg or 1 × 10 ng / kg. After 12 weeks of treatment, all muscles were harvested and stained for restoration of dystrophin and DAPC components. Systemically injected (tail vein) mice showed high levels of staining for dystrophin throughout all muscles. Figure 4A shows that 6 × 10 12 vg(2×10 14 Figure 4B shows the quantification of the percentage of myofibers expressing microdystrophin in each tissue. Finally, functional improvement of the diaphragm was tested (Figure 4C). No significant differences were observed at low doses, but there was a marked improvement at high doses. Importantly, Figure 5 shows that other components of DAPCs were fully restored after microdystrophin delivery. Shown is beta-sarcoglycan (B-SG).

[0098] The toxicology / safety of AAVrh.74.MHCK7.microdystrophin was assessed by administering the vector by intravenous (iv) injection into the tail vein of mdx mice according to Table 2. There was no evidence of toxicity in any of the muscle tissues analyzed, including: tibialis anterior (TA), gastrocnemius (GAS), quadriceps (QD), psoas (PSO), triceps brachii (TRI), and diaphragm (DIA) (Figures 6A and 6B). The number of centrally located nuclei was significantly higher at the high dose of 6 × 10 12 vg(2×10 14 vg / kg). Historically, central nucleation in skeletal muscle of untreated age-matched mdx mice averages approximately 80%. Finally, preliminary data from a small sample size (n=3) demonstrates a decrease in CK release levels (U / L) in serum of high dose (D)-treated mice. Differences were identified using independent t-tests (p<0.05) and data are reported as mean ± SEM. [Table 2]

[0099] Example 4 Generation of pAAV.MCK.micro-dystrophin construct The pAAV.MCK.microdystrophin plasmid was constructed by inserting an MCK expression cassette driving a codon-optimized human microdystrophin cDNA sequence into the AAV cloning vector psub201 (Samulski et al., J. Virol. 61(10):3096-3101). A muscle-specific regulatory element was included in the construct to drive muscle-specific gene expression. This regulatory element consisted of the mouse MCK core enhancer (206 bp) fused to the 351 bp MCK core promoter (proximal). After the core promoter, the construct contained 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 were derived from the plasmid pCMVβ (Clontech). The microdystrophin cassette has a consensus Kozak immediately before the ATG start and a small 53 bp synthetic polyA signal for mRNA termination. The human microdystrophin cassette contains the (R4~R23 / Δ71~78) domain as previously described by Harper et al. Nat. Med. 8(3):253-61, 2002.

[0100] The pAAV.MCK.microdystrophin plasmid contained a human microdystrophin cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs) (see FIG. 7). This sequence was encapsidated into AAVrh.74 virions. A molecular clone of the AAVrh.74 serotype was cloned from a lymph node of a rhesus macaque and described in Rodino-Klapac et al. Journal of Tran.Med. 45 (2007).

[0101] Example 5 Potency and dosage analysis using rAAV.MCK.micro-dystrophin Expression studies were performed with a human microdystrophin construct (rAAV.MCK.microdystrophin; described in Example 1) by intramuscular injection. mdx mice (a spontaneous Dmd mouse that does not express dystrophin) were used. mdx 3 × 10 9 , 3×10 10 , or 1 × 10 11 vg cassette (n=3 per group). After 4 weeks, muscles were harvested and stained for dystrophin expression using an antibody specific for N-terminal Dys3 as well as with hematoxylin and eosin (HE) staining. 9 Very little expression (no effect level) in vg and 1 × 10 11 There is a linear correlation between expression and dose with 89% expression in vg.

[0102] Example 6 Vascular delivery of rAAV.MCK.micro-dystrophin into mdx mice Using the isolated limb perfusion model (Rodino-Klapac et al., J. Trans. Med. 5(45):1-11, 2007), mdx mice (n=10) were administered 1×10 11 rAAVrh.74.MCK.microdystrophin vg was injected via the femoral artery and analysis of the results was performed. Three months after gene transfer, lower limb muscles were harvested and efficacy studies showed significant improvements in both force and resistance to eccentric contraction-induced injury (Figure 9).

[0103] Immunostaining of dystrophin protein in extensor digitorum longus (EDL) and TA muscles shows expression in mdx muscle fibers after rAAVrh.74-MCK-microdystrophin treatment via the femoral artery (Figure 9A). Mock-infected muscles were stained in an identical manner and exposure times were matched. Figure 9B demonstrates that rAAVrh.74-MCK-microdystrophin significantly increased normalized specific force compared to mock-treated mdx muscles (P<0.05 vs. mdx). Furthermore, mdx muscles infected with rAAVrh.74-MCK-microdystrophin (human) were compared to mock-infected contralateral mdx EDL muscles (blue) and wild-type (WT C57Bl / 10) EDL muscles for force reduction during repetitive eccentric contractions at 12 weeks post-gene transfer (Figure 9C). rAAVrh.74-MCK-micro-dystrophin (Micro-dys) treatment was found to significantly protect against loss of force compared to mock-treated mdx muscles (P<0.001 vs. mdx).

[0104] Example 7 Primate Research To translate the preclinical findings in mice to a clinical paradigm, non-human primates (NHPs) were administered systemically 2 × 10 of AAVrh74.MHCK7.microdystrophin.FLAG to assess safety and efficacy in future clinical trials. 14 The effects of a total dose of 1000 mg / kg delivered intravenously via the cephalic vein were studied in non-human primates. This dose was proportional (based on the animal's body weight) to the systemic dose given to mice and was 100% of the intermediate dose given to mice (6.0 × 10 12 vg total dose).

[0105] Immunological studies, including baseline chemical and enzyme-linked immunosorbent spot assay (ELISpot) analyses, were performed to measure T cells against AAVrh.74 capsid and microdystrophin, as well as anti-AAV antibody titers. Three peptide pools were used for the AAVrh.74 capsid protein (Genemed Synthesis, San Antonio, TX), each containing 34–36 peptides, 18 amino acids long and overlapping by 11 residues. Four peptide pools containing the microdystrophin.FLAG protein (Genemed Synthesis), each 18 amino acids long and overlapping by 11 residues, were used. Concanavalin A (ConA) (Sigma, 1 μg / mL) was used as a positive control, and 0.25% dimethyl sulfoxide (DMSO) was used as a negative control. These studies were repeated every 2 weeks throughout the study. Three months after treatment, animals were euthanized to obtain a complete tissue necropsy. Immunologic assays showed no unexpected responses against the capsid or transgene by ELISpot (FIG. 12A), nor unexpected antibody responses against the AAVrh74 capsid by ELISA (FIG. 12B).

[0106] Additionally, as shown in Table 3 below, a complete blood count and chemistry panel revealed slightly elevated liver enzymes, which normalized back to baseline without the need for intervention or treatment. [Table 3]

[0107] There were no other unexpected chemical values ​​during the study period. Finally, a complete analysis of all skeletal muscles showed widespread expression in myofibers by immunofluorescence staining with a FLAG-specific antibody and Western blot detection using a mouse monoclonal antibody against dystrophin (Figure 14A, B).

[0108] Data demonstrate that systemic delivery of AAVrh74.MHCK7.microdystrophin.FLAG establishes safety and efficacy with widespread expression throughout all skeletal muscles in non-human primates.

[0109] Example 8 Preclinical studies demonstrating efficacy A preclinical study was conducted to demonstrate the efficacy of systemic delivery of rAAVrh74.MHCK7.microdystrophin in treating skeletal and cardiac defects in mdx mice. The AAVrh74 vector containing the codon-optimized human microdystrophin transgene driven by the muscle- and cardiac-specific promoter MHCK7 described in Example 1 was used in this study.

[0110] Systemic injection of rAAVrh74.MHCK7.micro-dystrophin via the tail vein of mdx (dystrophin null) mice was used for a dose-response study. Results of this study demonstrated that systemic injection into mdx mice was effective in normalizing histological and functional outcomes measured in the limbs and diaphragm in a dose-dependent manner. Furthermore, no significant vector-associated toxicity was reported after formal histopathology review by a board-certified veterinary pathologist.

[0111] Vectors for this study were created by the Viral Vector Core at Nationwide Children's Hospital utilizing a triple transfection method in HEK293 cells under research grade conditions. Post-production vector characterization included titer determination by qPCR with supercoiled standards, endotoxin level measurements (EU / mL) and sterility assessment. Produced vectors were analyzed by SDS-PAGE to verify consistency of banding patterns with expected rAAV. Vectors were created using a plasmid containing the micro-dystrophin construct, a muscle-specific MHCK7 promoter driving expression, a consensus Kozak sequence (CCACC), an SV40 chimeric intron, and a synthetic polyadenylation site (53bp) (Error! Reference source not found). The micro-dystrophin expression cassette was cloned between AAV2 ITRs packaged into the AAVrh74 vector to enhance transduction of skeletal and cardiac tissues.

[0112] Efficacy determination of the rAAVrh74.MHCK7.microdystrophin test samples was accomplished by intramuscular injection of the vector into mdx mice, with wild type mice serving as a positive control and injection of sterile lactated Ringer's solution into mdx mice serving as a negative control. [Table 4]

[0113] Animals as shown in Table 4 were administered tail vein injections for systemic delivery at the ages indicated (4-5 weeks of age). To ensure accurate dosing with intramuscular injections, animals were briefly anesthetized by isoflurane inhalation. The dose was administered by direct injection into the tibialis anterior muscle of the lower hind leg. Anesthesia was not required for accurate dosing with systemic delivery. The dose was administered by the vasculature through the tail vein. Care was taken to place the entire vector dose precisely in the blood vessel. After dosing was administered, animals were placed on a heating pad until locomotor activity was restored, after which they were returned to their cages. Observations of each animal were performed weekly for the entire duration of the study.

[0114] Mice were overdosed with a ketamine / xylazine mixture (200 mg / kg / 20 mg / kg) at the appropriate ages as described in Table 4. Blood was collected by cardiac puncture, whole blood was sent for complete blood count (CBC) analysis, and serum was stored at -80°C until analyzed by Charles Rivers Laboratory. Tissues were then collected and sent for analysis by an independent veterinary histopathologist and in-house analysis.

[0115] rAAVrh74.MHCK7.microdystrophin at 1 × 10 11 When delivered intramuscularly to dystrophin-null mice at a total dose of vg, approximately 70% of dystrophin was expressed in the injected TA muscle. Immunofluorescence imaging of vector-treated mice confirmed expression of the microdystrophin gene.

[0116] Restoration of dystrophin expression following systemic treatment with rAAVrh74.MHCK7.microdystrophin Potency determination of the rAAVrh74.MHCK7.micro-dystrophin test samples was performed using low, medium, and high doses (2.0 x 10 12 vg total dose: 6.0 × 10 12 vg total dose: 1.2 x 10 13 In a dose-escalation study, mdx mice (genotype: C57BL / 10ScSn-DMD mdx This was achieved by administering systemic injections of 100 mg / J (100 mg / kg / day) to mice. Mice were injected at 4–5 weeks of age, and complete necropsies were performed at both 3 and 6 months post-injection. Based on the average animal weight per group, these doses were 8 × 10 13 vg / kg, 2×10 14 vg / kg and 6 × 10 14 vg / kg. Injection of an equivalent volume of lactated Ringer's solution into C57BL / 6 mice served as a negative control. Injection of an equivalent volume of lactated Ringer's solution into C57BL / 6 mice served as a positive control. Safety was assessed using a dose of 6.0 × 10 12The dose of 1000 mg / kg total dose was determined by systemic injection into WT mice (referred to as WT TX-medium dose group). Immunofluorescence staining of skeletal muscles, tibialis anterior (TA), gastrocnemius (GAS), quadriceps (QUAD), gluteus maximus (GLUT), psoas, triceps (TRI), diaphragm (DIA), and heart, was performed to determine dystrophin restoration and confirm the efficacy of the rAAVrh74.MHCK7.microdystrophin viral vector.

[0117] For analysis, skeletal muscles (TA, QUAD, GLUT, TRI) were extracted along with the heart and diaphragm. Organs were also harvested for toxicology and biodistribution studies. Expression of the micro-dystrophin transgene remained high after 3-6 months of treatment. This was accompanied by improved muscle histopathology and improved function, without adverse effects in off-target organs.

[0118] Reversal of the dystrophic phenotype in rAAVrh74.MHCK7.microdystrophin-systemically treated mdx mice Hematoxylin & Eosin (H&E) staining of skeletal muscle, diaphragm, and heart was performed for each dose of 2 × 10 mice with euthanasia at 12 weeks post-injection. 12 vg total dose (low dose; n = 1), 6 × 10 12 Total dose of vg (medium dose; n = 8) 1.2 × 10 13 We assessed reversal and amelioration of dystrophic pathology following systemic injection of rAAVrh74.MHCK7.microdystrophin at a total dose of 10 vg (high dose; n = 8). At 24 weeks post-injection, 12 A second cohort of animals treated with 100 mg / kg total dose was evaluated for reversal and amelioration of dystrophin pathology (n=5).

[0119] Immunofluorescence staining for human micro-dystrophin protein was used to determine micro-dystrophin transgene expression in both the left and right sides of six skeletal muscles (TA, GAS, QUAD, GLUT, psoas, TRI), as well as the diaphragm and heart, in all dystrophin null mice injected with the micro-dystrophin vector. This determined the restoration of dystrophin and was performed in mice injected with 2 × 10 12 vg total dose (low dose; n = 2), 6 × 10 12 Total dose of vg (medium dose; n = 8) 1.2 × 10 13 This was performed to ensure efficacy of the rAAVrh74.MHCK7.microdystrophin viral vector at a total dose of .vg (high dose; n=8).

[0120] To assess expression and transduction efficiency, images from all three dose cohorts and both the left and right sides of each muscle were utilized for quantification. Four 20x images of each muscle were taken and the percentage of microdystrophin positive fibers in each image was determined to obtain the average percent transduction for each muscle. Figures 14 and 15 show the mean percent transduction for each muscle after the medium dose (6×10 12 vg;2×10 14 vg / kg) and high dose (1.2 × 10 13 vg;6×10 14 Representative images of mice treated with 100 mg / kg of lactated Ringer's solution are shown. Age-matched dystrophin null mice injected with lactated Ringer's solution were included as negative controls, and wild-type mice injected with lactated Ringer's solution were included as positive controls. Hearts showed >75% in all animals analyzed.

[0121] Muscles from untreated animals showed extensive myopathy, including focal areas of fatty infiltration, central nucleation, fibrosis, and necrosis. H&E staining in Error!Reference source not found. 16 and Error!Reference source not found. 17 demonstrated this dystrophic phenotype in dystrophin null mice compared with normal WT mice, as well as the increased risk of myopathy after a moderate dose (6 × 10 12 vg;2×10 14 vg / kg) or high dose (1.2 × 10 13 vg;6×10 14The results show improvement in muscle pathology following treatment with 16 and 17 vg / kg. Quantification of histological parameters revealed a reduction in central nucleation (Figure 18) and normalization of mean fiber diameter (Error! Reference source not found. 16 and 17) in all muscles in a dose-dependent manner in treated mice. Sirius Red staining showed a reduction in collagen deposition in the diaphragm in both the mid and high dose cohorts compared to the untreated (mdx LR) cohort (Figure 19).

[0122] Functional assessment of systemic treatment with rAAVrh74.MHCK7.micro-dystrophin To determine whether microdystrophin gene transfer provides functional strength benefits to diseased muscles, we assessed the functional properties of both the diaphragm and tibialis anterior muscles in mdx mice, WT mice, and mice treated with vector at three dose levels. Dose escalation included a low dose (8 × 10 13 vg / kg), medium dose (2×10 14 vg / kg), and high dose (6 × 10 14 vg / kg) was included. 12 In animals systemically injected with rAAVrh74.MHCK7.microdystrophin at a vg total dose (medium dose), functional assessment of systemic treatment with rAAVrh74.MHCK7.microdystrophin was utilized 24 weeks post-injection using ex vivo assessment of reduction in specific force and force output following eccentric contractions of the TA. Additionally, specific force output at the diaphragm was assessed in the same animals.

[0123] As outlined in the figures above, histopathology indicated a more normalized environment with improvements in central nucleation, collagen deposition, and fiber size at the mid and high doses. Tail vein delivery of rAAVrh74.MHCK7.microdystrophin resulted in a gradual improvement in specific force output in the diaphragm (176.9 mN / mm in the mid dose group). 2 vs. 227.78mN / mm in the high dose group 2 ) and the long-term treatment cohort received injections (medium dose 2 × 10 14 vg / kg) in mice 6 months after the treatment, there was no long-term deviation in diaphragm specific force output (176.9 mN / mm 2194.9mN / mm 2 )(Error! Reference source not found. 20).

[0124] Furthermore, functional deficits in the tibialis anterior muscle of mdx mice were observed compared to WT mice. Mdx mice showed a 50% reduction in force output (171.3 mN / mm) compared to WT mice. 2 291.65mN / mm 2 ) and a greater loss of force following eccentric contractions (32% loss in mdx vs. 5% loss in WT). Systemic delivery of mid-dose levels of rAAVrh74.MHCK7.micro-dystrophin resulted in recovery of 65.5% dystrophin and improved specific force output to 235.4 mN / mm2 in tibialis anterior muscles, protecting the muscle from the damaging effects of repeated eccentric contractions with only a 25% decrease in force (Figure 21). The WT mid-dose group represents a wild-type treated cohort to demonstrate a lack of toxicity following vector treatment and maintenance of functional outcome measures.

[0125] overview After initial demonstration of biopotency by intramuscular injection, equivalent or increased restoration of microdystrophin was achieved by vascular delivery while transducing skeletal muscle, diaphragm, and heart. Efficacy demonstrated dose-dependent reversal of dystrophic function with reduced inflammation, fewer degenerated fibers, and improved functional recovery by protecting against eccentric contractions in the tibialis anterior and diaphragm. Functional benefits of the vector included a gradual improvement to wild-type levels in force production in the diaphragm and TA.

[0126] Example 9 Toxicity and biodistribution of systemic treatment with rAAVrh74.MHCK7.microdystrophin Organs and tissues from mdx mice given systemic injections of rAAVrh74.MHCK7.microdystrophin were collected for real-time quantitative PCR to detect specific sequences in the vector DNA. Proteins extracted from all collected organs and tissues were analyzed by Western blot to detect microdystrophin in off-target organs.

[0127] The test sample was administered intravenously at a low dose (2 × 10 12 vg;8×10 13 vg / kg), medium dose (6×10 12 vg;8×10 14 vg / kg), and high dose (1.2 × 10 13 vg;6×10 14 The vector was given at three dose levels: 0.01 mg / kg (vg / kg). To evaluate the safety of the vector, H&E staining was performed on frozen sections of muscle tissue and all major organs taken from the same cohort of mice described above. Also included were organs and muscles from C57BL6 WT mice treated systemically with the medium dose of vector. Lactated Ringer's solution-treated mdx and WT mice were also included in the histopathology analysis. These sections were formally examined for toxicity by an independent board-certified veterinary pathologist, and no adverse effects were detected in any samples from any mice. The results are summarized below.

[0128] Group details and study design are shown in 4 below. [Table 5]

[0129] Histopathological examination of vector-transduced tissues Intravenous injection of rAAVrh74.MHCK7.microdystrophin did not induce any microscopic changes in muscle fibers in any of the skeletal muscles examined. Furthermore, no treatment-related pathology was found in any of the tissues evaluated histologically. Any changes noted were seen in both treated and control mice and were considered incidental findings. Collectively, these data indicate that the test sample was well tolerated by the test subjects. Furthermore, compared to reference specimens from age-matched untreated mdx mice, administration of rAAVrh74.MHCK7.microdystrophin reduced muscle fiber atrophy in treated mdx mice, thus indicating that the test sample can ameliorate the degree of myopathy associated with mdx deficiency.

[0130] In addition to investigating disease-bearing mdx mice treated systemically with the vector, rAAVrh74.MHCK7.microdystrophin was administered at a minimal effective dose (MED) of 6 × 10, the dose established in the above studies in mdx mice. 12 Total dose of vg (2 × 10 14 The same dose of 100 mg / kg of 1000 mg ...

[0131] Vector genome biodistribution A real-time quantitative PCR assay (qPCR) was used to examine the presence of test sample-specific DNA sequences. Biodistribution analysis was performed on tissue samples collected from three vector-treated mdx animals per dose level. A positive signal was 100 or more single-stranded DNA copies per μg of genomic DNA detected. Tissues were collected at necropsy using vector-specific primer probe sets specific for sequences in the MHCK7 promoter. Figure 22 and Table 6 below show the vector genome copies detected in each tissue sample from rAAVrh74.MHCK7.microdystrophin-injected mice. [Table 6]

[0132] The rAAVrh74.MHCK7.microdystrophin transcript was detected at various levels in all tissues collected. As expected, the highest levels were found in skeletal muscle and heart. The lowest levels were detected in gonads, lung, kidney, and spleen. These data indicate that the test samples were efficiently delivered to all examined tissues of vector-treated mice.

[0133] As the qPCR results above show, intravenous delivery of rAAVrh74.MHCK7.microdystrophin resulted in varying levels of vector transcript distribution in most tissues, with the highest levels occurring in liver, heart, and quadriceps (medium dose), as well as liver, heart, and gastrocnemius (high dose). Thus, the purpose of this part of the study was to determine protein expression of the human microdystrophin transgene in these tissues and ensure functionality of the muscle-specific MHCK7 promoter. Western blotting was used to detect microdystrophin expression in tissue samples.

[0134] Protein expression and biodistribution of the vector was also assessed using qPCR and Western blotting (Figure 23), and these data show normal levels of vector in off-site organs and minimal detection of micro-dystrophin protein in livers treated with the high dose. These results correlated with the absence of toxicity as determined by a liver pathologist. Additionally, serum chemistries were analyzed by an independent CRO (Charles River Laboratories), showing normal values ​​across all chemistries analyzed. There were three abnormal values ​​for the liver enzyme AST, two in the mdx-LR group and one in the mid-dose group (Figure 23). A subset of animals underwent creatine kinase analysis (CK), however samples were analyzed before and after physiological evaluation. Analysis of serum corroborates the lack of toxicity following delivery of test samples.

[0135] Micro-dystrophin protein expression was observed in variable amounts in all skeletal muscle and heart samples (Figure 24). However, minimal protein was detected in the high dose cohort in liver. This is believed to be a benign finding, and its presence in liver may be due to expression in hepatic smooth muscle. Importantly, there were no adverse histopathological effects as reported by an independent pathologist in liver.

[0136] overview Histopathological examination concluded that the mdx-LR cohort exhibited widespread myopathy affecting all seven skeletal muscles evaluated and the right ventricular wall of the heart. Major findings of histopathological review included prominent widespread myofiber atrophy (30-75% of normal myofiber size), minimal to mild mononuclear cell inflammation, increased interstitial spaces, and increased cytoplasmic mineral deposition. The diaphragm showed the most prominent changes of mononuclear cell infiltration and myofiber atrophy. The heart showed small foci of minimal mononuclear cell accumulation in the ventricular myocardium. Myopathy in all skeletal tissues and the heart was significantly reduced in the vector-treated cohort. The reduction in histopathological findings was dose-dependent, with substantially less degeneration and inflammation in the high-dose group. There were no adverse effects from vector treatment with rAAVrh74.MHCK7.micro-dystrophin, as demonstrated in the WT-treated and vector-treated mdx cohorts. There were incidental findings in the liver and lungs of mdx and WT mice where the mice exhibited mild vacuolization of hepatocyte cytoplasm, regardless of treatment. Thus, the test samples were safe and effective, and the protective effect was dose-dependent. References 1.Hoffman, EP, Brown, RH, Jr. & Kunkel, LMDystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51, 919-928 (1987). 2. Straub, V. & Campbell, KPMuscular dystrophies and the dystrophin-glycoprotein complex. Curr Opin Neurol 10, 168-175 (1997). 3.Sacco, A., et al.Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx / mTR mice.Cell 143,1059-1071(2010). 4.Wallace,G.Q.&McNally,E.M.Mechanisms of muscle degeneration, regeneration,and repair in the muscular dystrophies.Annu Rev Physiol 71,37-57(2009). 5.Zhou,L.& Lu,H.Targeting fibrosis in Duchenne muscular dystrophy.J Neuropathol Exp Neurol 69,771-776(2010). 6.Desguerre,I.,et al.Endomysial fibrosis in Duchenne muscular dystrophy:a marker of poor outcome associated with macrophage alternative activation.J Neuropathol Exp Neurol 68,762-773(2009). 7.DiPrimio,N.,McPhee,S.W.&Samulski,R.J.Adeno-associated virus for the treatment of muscle diseases:toward clinical trials.Curr Opin Mol Ther 12,553-560(2010). 8.Mendell,J.R.,et al.Sustained alpha-sarcoglycan gene expression after gene transfer in limb-girdle muscular dystrophy,type 2D.Ann Neurol 68,629-638(2010). 9.Mendell,J.R.,et al.Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins.Ann Neurol 66,290-297(2009). 10.Mendell,J.R.,et al.A phase 1 / 2a follistatin gene therapy trial for becker muscular dystrophy.Molecular therapy:the journal of the American Society of Gene Therapy 23,192-201(2015). 11.Carnwath,J.W.&Shotton,D.M.Muscular dystrophy in the mdx mouse: histopathology of the soleus and extensor digitorum longus muscles.J Neurol Sci 80,39-54(1987). 12.Coulton,G.R.,Morgan,J.E.,Partridge,T.A.&Sloper,J.C.The mdx mouse skeletal muscle myopathy:I.A histological,morphometric and biochemical investigation.Neuropathol Appl Neurobiol 14,53-70(1988). 13.Cullen,M.J.&Jaros,E.Ultrastructure of the skeletal muscle in the X chromosome-linked dystrophic(mdx)mouse.Comparison with Duchenne muscular dystrophy.Acta Neuropathol 77,69-81(1988). 14.Dupont-Versteegden,E.E.&McCarter,R.J.Differential expression of muscular dystrophy in diaphragm versus hindlimb muscles of mdx mice.Muscle Nerve 15,1105-1110(1992). 15.Stedman,H.H.,et al.The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.Nature 352,536-539(1991). 16.Deconinck,A.E.,et al.Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy.Cell 90,717-727(1997). 17.Grady,R.M.,et al.Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy.Cell 90,729-738(1997). 18.Love,D.R.,et al.An autosomal transcript in skeletal muscle with homology to dystrophin.Nature 339,55-58(1989). 19.Tinsley,J.M.,et al.Primary structure of dystrophin-related protein.Nature 360,591-593(1992). 20.Tinsley,J.,et al.Expression of full-length utrophin prevents muscular dystrophy in mdx mice.Nat Med 4,1441-1444(1998). 21.Squire,S.,et al.Prevention of pathology in mdx mice by expression of utrophin:analysis using an inducible transgenic expression system.Hum Mol Genet 11,3333-3344(2002). 22.Rafael,J.A.,Tinsley,J.M.,Potter,A.C.,Deconinck,A.E.&Davies,K.E.Skeletal muscle-specific expression of a utrophin transgene rescues utrophin-dystrophin deficient mice.Nat Genet 19,79-82(1998). 23.Zhou,L.,et al.Haploinsufficiency of utrophin gene worsens skeletal muscle inflammation and fibrosis in mdx mice.J Neurol Sci 264,106-111(2008). 24.Gutpell,K.M.,Hrinivich,W.T.&Hoffman,L.M.Skeletal Muscle Fibrosis in the mdx / utrn+ / -Mouse Validates Its Suitability as a Murine Model of Duchenne Muscular Dystrophy.PloS one 10,e0117306(2015). 25.Rodino-Klapac,L.R.,et al.Micro-dystrophin and follistatin co-delivery restores muscle function in aged DMD model.Human molecular genetics 22,4929-4937(2013). 26.Nevo,Y.,et al.The Ras antagonist,farnesylthiosalicylic acid(FTS),decreases fibrosis and improves muscle strength in dy / dy mouse model of muscular dystrophy.PloS one 6,e18049(2011). 27.Rodino-Klapac,L.R.,et al.A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy.J Transl Med 5,45(2007). 28.Mulieri,L.A.,Hasenfuss,G.,Ittleman,F.,Blanchard,E.M.&Alpert,N.R.Protection of human left ventricular myocardium from cutting injury with 2,3-butanedione monoxime.Circ Res 65,1441-1449(1989). 29.Rodino-Klapac,L.R.,et al.Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery.Molecular therapy:the journal of the American Society of Gene Therapy 18,109-117(2010). 30.Grose,W.E.,et al.Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer.PloS one 7,e39233(2012). 31.Liu,M.,et al.Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury.Mol Ther 11,245-256(2005). 32.Harper,S.Q.,et al.Modular flexibility of dystrophin:implications for gene therapy of Duchenne muscular dystrophy.Nature medicine 8,253-261(2002). 33.Rodino-Klapac,L.R.,et al.Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery.Mol Ther 18,109-117(2010). 34.Salva,M.Z.,et al.Design of tissue-specific regulatory cassettes for high-level rAAV-mediated expression in skeletal and cardiac muscle.Mol Ther 15,320-329(2007). 35.Sondergaard,P.C.,et al.AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models.Annals of clinical and translational neurology 2,256-270(2015). 36.De,BP,et al.High levels of persistent expression of alpha1-antitrypsin mediated by the nonhuman primate serotype rh.10 adeno-associated virus despite preexisting immunity to common human adeno-associated viruses.Mol Ther 13,67-76(2006). 37.Rodino-Klapac,LR,et al.A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy.Journal of translational medicine 5,45(2007). 38.Bulfield et al.,X chromosome-linked muscular dystrophy(mdx) in the mouse.Proc Natl Acad Sci US A.1984;81(4):1189-1192. 39.Sicinski et al.,The molecular basis of muscular dystrophy in the mdx mouse:a point mutation.Science.1989 30;244(4912):1578-80 Sequence Listing <110> Nationwide Children's Hospital Research Institute <120> Muscle-specific adeno-associated viral vector delivery Microdystrophins for treating muscular dystrophies <130> 28335 / 51475A PCT <150> US62 / 473,148 <151> 2017-03-17<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <160> 5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <170> PatentInバージョン3.5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 3579<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Homo sapiens<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atgctgtggt gggaggaggt ggaggattgt tatgaaaggg aggacgtgca gaagaagact 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tttaccaagt gggtgaacgc tcagttcagc aaatttggga agcagcacat cgagaatctg 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttttccgacc tgcaggatgg gagacggctg ctggatctgc tggaaggact gactggccag 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aagctgccca aagagaaggg gagcactagg gtgcacgccc tgaacaacgt gaacaaagct 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgagagtgc tgcagaacaa caacgtggat ctggtgaata ttggcagtac tgatatcgtg 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gacgggaacc acaaactgac actgggcctg atctggaaca ttattctgca ctggcaggtg 360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaaaatgtga tgaagaacat catggccggg ctgcagcaga ccaattccga gaagatcctg 420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgtcttggg tgcggcagag cacccgcaac tatccccagg tgaacgtgat taacttcact 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acatcctgga gcgaccgggct ggccctgaat gctctgattc acagccacag gcctgatctg 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttcgactgga atagcgtggt gtgccagcag tctgccacac agcgcctgga acatgccttc 600<h2 style=";text-align:left;direction:ltr"> aatatcgctc ggtaccagct ggggatcgaa aaactgctgg acccagagga tgtggacact 660 acatacccag ataaaaagtc tattctgatg tacattacta gcctgttcca ggtgctgcca 720 cagcaggtgt ctattgaagc cattcaggag gtggaaatgc tgccccgccc ccccaaagtg 780 actaaagagg agcattttca gctgcatcat cagatgcatt acagccagca gattaccgtg 840 agcctggctc agggatatga gcgcaccagt agtccaaaac cacggttcaa gtcctacgct 900 tatacccagg ctgcctacgt gacaactagc gaccctacta gatccccctt tccatcccag 960 cacctggagg ccccagagga caagagcttt gggtccagcc tgatggaaag cgaggtgaat 1020 ctggatcggt accagacagc cctggaggag gtgctgagct ggctgctgag tgctgaagac 1080 acactgcagg cccagggcga aatttccaat gacgtggaag tggtgaagga tcagttccac 1140 acacacgagg gctatatgat ggacctgaca gctcaccagg ggcgcgtggg caatatcctg 1200 1260 1320 aagcagtcca acctgcaccg ggtgctgatg gacctgcaga accagaaact gaaagagctg 1380 aacgactggc tgacaaagac tgaggaacgc acaaggaaga tggaggagga gccactggga 1440 cccgacctgg aggatctgaa gagacaggtg cagcagcata aggtgctgca ggaggatctg 1500 gaacaggagc aggtgcgggt gaactccctg acacatatgg tggtggtggt ggacgaatct 1560 agtggagatc acgccaccgc cgccctggag gaacagctga aggtgctggg ggaccggtgg 1620 gccaacattt gccggtggac cgaggacagg tgggtgctgc tgcaggacat cctgctgaaa 1680 tggcagaggc tgaccgagga gcagtgtctg tttagtgctt ggctgagcga gaaagaggac 1740 gccgtgaaca agatccacac aaccggcttt aaggatcaga acgaaatgct gtctagcctg 1800 cagaaactgg ctgtgctgaa ggccgatctg gagaaaaaga agcagagcat gggcaaactg 1860 tatagcctga aacaggacct gctgagcacc ctgaagaaca agagcgtgac ccagaagaca 1920 gaagcctggc tggataactt tgcccgctgc tgggacaacc tggtgcagaa actggagaaa 1980 agtacagctc agatctctca ggctgtgacc acaacccagc ctagcctgac ccagacaacc 2040 gtgatggaaa ccgtgaccac cgtgacaacc cgcgaacaga tcctggtgaa acatgcccag 2100 gaagagctgc cacctccacc tccccagaag aagagaaccc tggagcggct gcaggagctg 2160 caggaagcca ctgacgaact ggacctgaag ctgaggcagg ccgaagtgat taaggggtct 2220 tggcagcctg tgggcgatct gctgattgat tccctgcagg accacctgga aaaggtgaag 2280 gctctgagag gcgaaattgc tccactgaag gagaacgtga gtcatgtgaa cgatctggct 2340 agacagctga caacactggg catccagctg agcccataca atctgagcac actggaggac 2400 ctgaatacca ggtggaagct gctgcaggtg gctgtggaag accgggtgcg gcagctgcat 2460 gaggcccatc gcgacttcgg accagccagc cagcactttc tgagcacatc cgtgcagggg 2520 ccctgggaga gggccatttc tcccaacaag gtgccctact atattaatca cgagacccag 2580 accacttgtt gggaccatcc caagatgaca gaactgtacc agtccctggc cgatctgaac 2640 aacgtgaggt ttagcgctta cagaaccgct atgaagctga gacggctgca gaaggccctg 2700 tgcctggatc tgctgtccct gtccgccgcc tgcgatgccc tggatcagca taatctgaag 2760 cagaacgatc agccaatgga tatcctgcag atcatcaact gcctgaccac tatctacgac 2820 aggctggagc aggagcacaa caacctggtg aacgtgcctc tgtgcgtgga tatgtgcctg 2880 aactggctgc tgaacgtgta tgacactggg cgcaccggcc ggatcagagt gctgagtttt 2940 aaaactggga ttatctccct gtgtaaggcc cacctggagg acaagtcag gtacctgttc 3000 aagcaggtgg ctagtagcac tggattttgt gaccagcgcc gcctgggact gctgctgcat 3060 gatagtatcc agattcctag acagctggga gaggtggcta gtttcggagg atctaacatc 3120 gaacccagcg tgcgcagctg tttccagttt gccaataaca aacctgaaat cgaggctgct 3180 ctgttcctgg attggatgcg cctggaacca cagagcatgg tgtggctgcc tgtgctgcac 3240 agagtggctg ccgccgaaac tgccaagcac caggctaaat gcaacatctg caaggaatgt 3300 cccattatcg gctttcgcta caggagtctg aaacatttta actacgatat ttgccagagc 3360 3420 tgcaccccaa ctacatctgg cgaagatgtg cgcgattttg ccaaggtgct gaagaataag 3480 tttcggacta agaggtactt cgccaagcac ccccgcatgg ggtatctgcc agtgcagaca 3540 gtgctggaag gagacaatat ggagaccgat acaatgtga 3579 <210> 2 <211> 810 <212> DNA <213> Adeno-associated virus <400> 2 gtttaaacaa gcttgcatgt ctaagctaga cccttcagat taaaaataac tgaggtaagg 60 gcctgggtag gggaggtggt gtgagacgct cctgtctctc ctctatctgc ccatcggccc 120 tttggggagg aggaatgtgc ccaaggacta aaaaaaggcc atggagccag aggggcgagg 180 gcaacagacc tttcatgggc aaaccttggg gccctgctgt ctagcatgcc ccactacggg 240 tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct ggttataatt 300 aacccagaca tgtggctgcc cccccccccc caacacctgc tgcctctaaa aataaccctg 360 tccctggtgg atcccctgca tgcgaagatc ttcgaacaag gctgtggggg actgagggca 420 ggctgtaaca ggcttggggg ccagggctta tacgtgcctg ggactcccaa agtattactg 480 ttccatgttc ccggcgaagg gccagctgtc ccccgccagc tagactcagc acttagttta 540 ggaaccagtg agcaagtcag cccttggggc agcccataca aggccatggg gctgggcaag 600 ctgcacgcct gggtccgggg tgggcacggt gcccgggcaa cgagctgaaa gctcatctgc 660 tctcaggggc ccctccctgg ggacagcccc tcctggctag tcacaccctg taggctcctc 720 tatataaccc aggggcacag gggctgccct cattctacca ccacctccac agcacagaca 780 gacactcagg agccagccag cggcgcgccc 810 <210> 3 <211> 8611 <212> DNA <213> Adeno-associated virus <400> 3 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcgcg 60 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc 120 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt 180 ccttgtagtt aatgattaac ccgccatgct aattatctac gtagccatgt ctagagttta 240 aacaagcttg catgtctaag ctagaccctt cagattaaaa ataactgagg taagggcctg 300 ggtaggggag gtggtgtgag acgctcctgt ctctcctcta tctgcccatc ggccctttgg 360 ggaggaggaa tgtgcccaag gactaaaaaa aggccatgga gccagagggg cgaggggcaac 420 agacctttca tgggcaacc ttggggccct gctgtctagc atgccccact acgggtcttag 480 gctgcccatg taaggaggca aggctgggg acacccgaga tgctggtta taattaaccc 540 agacatgtgg ctgccccccc ccccaaca cctgctgcct ctaaaaataa ccctgctccct 600 ggtggatccc ctgcatgcga agatcttcga acaggctgt gggggactga gggcaggctg 660 taacaggctt gggggccagg gcttatacgt gcctgggact cccaaagtat tactgttcca 720 tgttcccggc gaagggccag ctgtcccccg ccagctagac tcagcactta gtttaggaac 780 cagtgagcaa gtcagccctt ggggcagccc atacaaggcc atggggctgg gcaagctgca 840 cgcctgggtc cggggtgggc acggtgcccg ggcaacgagc tgaagctca tctgctca 900 ggggcccctc cctggggaca gcccctctg gctagtcaca ccctgtaggc tcctctatat 960 aacccagggg cacaggggct gcctcattc taccaccc tccacagcac agacagacac 1020 tcaggagcca gccagcggcg cgcccaggta agtttagtct tttgtctt tattcaggt 1080 cccggatccg gtggtggtgc aaatcaaaga actgctcctc agtggatgtt gcctttactt 1140 ctaggcctgt acggaagtgt tacttctgct ctaaaagctg cggaattgta cccgcggccg 1200 ccaccatgct gtggtgggag gaggtggagg attgttatga aagggaggac gtgcagaaga 1260 agacttttac caagtgggtg aacgctcagt tcagcaaatt tgggaagcag cacatcgaga 1320 atctgttttc cgacctgcag gatgggagac ggctgctgga tctgctggaa ggactgactg 1380 gccagaagct gcccaaagag aaggggagca ctagggtgca cgccctgaac aacgtgaaca 1440 aagctctgag agtgctgcag aacaacaacg tggatctggt gaatattggc agtactgata 1500 tcgtggacgg gaaccacaaa ctgacactgg gcctgatctg gaacattatt ctgcactggc 1560 aggtgaaaaa tgtgatgaag aacatcatgg ccgggctgca gcagaccaat tccgagaaga 1620 tcctgctgtc ttgggtgcgg cagagcaccc gcaactatcc ccaggtgaac gtgattaact 1680 tcactacatc ctggagcgac gggctggccc tgaatgctct gattcacagc cacaggcctg 1740 atctgttcga ctggaatagc gtggtgtgcc agcagtctgc cacacagcgc ctggaacatg 1800 ccttcaat cgctcggtac cagctgggga tcgaaaact gctggaccca gaggatgtgg 1860 acactacata cccagataaa aagtctattc tgatgtacat tactacctg ttccaggtgc 1920 tgccacagca gtgtctatt gaagccattc aggaggtgga aatgctgccc cgccccccca 1980 aagtgactaa agaggagcat ttcagctgc atcatcagat gcattacagc cagcagatta 2040 ccgtgagcct ggctcaggga tatgagcgca ccagtagtcc aaaaccacgg ttcaagctcct 2100 acgcttatac ccaggctgcc tacgtgacaa ctagcgaccc tactagatcc ccctttccat 2160 cccagcacct ggaggcccca gaggacaga gctttgggtc cagcctgatg gaaagcgagg 2220 tgaatctgga tcggtaccag acagccctgg aggaggtgct gagctggctg ctgagtgctg 2280 aagacacact gcaggcccag ggcgaattt ccaatgacgt ggaagtggtg aaggatcagt 2340 tccacacaca cgaggctat atgatggacc tgacagctca ccaggggcgc gtggggcaata 2400 tcctgcagct gggctctaaa ctgatcggca ccggaaact gagtgaggac gaggaacag 2460 aagtgcagga gcagatgaac ctgctgaaca gccgctggga gtgtctgaga gtggctagta 2520 tggagaagca gtccaacctg caccgggtgc tgatggacct gcagaaccag aaactgaaag 2580 agctgaacga ctggctgaca aagactgagg aacgcacaag gaagatggag gaggccac 2640 tgggacccga cctggaggat ctgaagagac aggtgcagca gcataaggtg ctgcaggagg 2700 atctggaaca ggagcaggtg cgggtgaact ccctgacaca tatggtggtg gtggtggacg 2760 aatctagtgg agatcacgcc accgccgccc tggaggaaca gctgaaggtg ctgggggacc 2820 ggtgggccaa catttgccgg tggaccgagg acaggtgggt gctgctgcag gacatcctgc 2880 tgaaatggca gaggctgacc gaggagcagt gtctgtttag tgcttggctg agcgagaaag 2940 aggacgccgt gaacaagatc cacacaaccg gctttaagga tcagaacgaa atgctgtcta 3000 gcctgcagaa actggctgtg ctgaaggccg atctggagaa aaagaagcag agcatgggca 3060 aactgtatag cctgaaacag gacctgctga gcaccctgaa gaacaagagc gtgacccaga 3120 agacagaagc ctggctggat aactttgccc gctgctggga caacctggtg cagaaactgg 3180 agaaaagtac agctcagatc tctcaggctg tgaccacaac ccagcctagc ctgacccaga 3240 caaccgtgat ggaaaccgtg accaccgtga caacccgcga acagatcctg gtgaaacatg cccaggaga gctgccacct ccacctcccc aaccctggag cggctgcagg 3360 agctgcagga agccactgac gaactggacc tgaagctgag gcaggccga gtgattaagg ggtcttggca gcctgtgggc gatctgctga ttgattccct gcaggaccac ctggaaaagg 3480 tgaaggctct gagaggcga attgctccac tgaaggaga cgtgagtcat gtgaacgatc tggctagaca gctgacaaca ctgggcatcc agctgagccc attackcaatctg agcacactgg aggacctgaa taccaggtgg aagctgctgc aggtggctgt ggaagaccgg gtgcggcagc tgcatgaggc ccatcgcgac ttcggaccag ccagccagca ttttctgagc acatccgtgc 3720 aggggccctg ggagagggcc atttctccca acaaggtgcc ctactatatt aatcacgaga cccagaccac ttgttgggac catcccaaga tgacagaact gtaccagtcc ctggccgatc tgaacaacgt gaggtttagc gcttacagaa ccgctatgaa gctgagacgg ctgcagaagg ccctgtgcct ggatctgctg tccctgtccg ccgcctgcga tgccctggat cagcataatc 3960 tgaagcagaa cgatcagcca atggatatcc tgcagatcat caactgcctg accactatct 4020 acgacaggct ggagcaggag cacaacaacc tggtgaacgt gcctctgtgc gtggatatgt 4080 gcctgaactg gctgctgaac gtgtatgaca ctgggcgcac cggccggatc agagtgctga 4140 gttttaaaac tgggattatc tccctgtgta aggcccacct ggaggacaag tacaggtacc 4200 tgttcaagca ggtggctagt agcactggat tttgtgacca gcgccgcctg ggactgctgc 4260 tgcatgatag tatccagatt cctagacagc tgggagaggt ggctagtttc ggaggatcta 4320 acatcgaacc cagcgtgcgc agctgtttcc agtttgccaa taacaaacct gaaatcgagg 4380 ctgctctgtt cctggattgg atgcgcctgg aaccacagag catggtgtgg ctgcctgtgc 4440 tgcacagagt ggctgccgcc gaaactgcca agcaccaggc taaatgcaac atctgcaagg 4500 aatgtcccat tatcggcttt cgctacagga gtctgaaaca ttttaactac gatatttgcc 4560 agagctgctt cttttccgga agagtggcca aaggacacaa gatgcactac cctatggtgg 4620 aatattgcac cccaactaca tctggcgaag atgtgcgcga ttttgccaag gtgctgaaga 4680 ataagtttcg gactaagagg tacttcgcca agcaccccg catggggtat ctgccagtgc 4740 agacagtgct ggaaggagac aatatggaga ccgatacaat gtgagcggcc gcaataaaag 4800 atctttattt tcattagatc tgtgtgttgg ttttttgtgt gtctagagca tggctacgta 4860 gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc 4920 actccctc tgcgcgctg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc 4980 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gccagctggc gtaatagcga 5040 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggaagtt 5100 ccagacgatt gagcgtcaaa atgtaggtat ttccatgagc gttttcctg ttgcaatggc 5160 tggcggtaat attgttctgg atattaccag caaggccgat agtttgagtt cttctactca 5220 ggcaagtgat gttattacta atcaaagaag tattgcgaca acggttaatt tgcgtgatgg 5280 acagactctt ttactcggtg gcctcactga ttataaaaac acttctcagg attctggcgt 5340 accgtcctg tctaaaatcc cttaatcgg cctcctgttt agctcccgct ctgattctaa 5400 cgaggaaagc acgttatacg tgctcgtcaa agcaaccata gtacgcgccc tgtagcggcg 5460 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 5520 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 5580 gtcaagctct aaatcgggg ctccctttag ggttccgatt tagtgattta cggcacctcg 5640 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 5700 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 5760 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 5820 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 5880 tattaacgtt tacaattttaa atatttgctt atacaatctt cctgtttttg gggcttttct 5940 gattatcaac cggggtacat atgattgaca tgctagtttt acgattaccg ttcatcgatt 6000 ctcttgtttg ctccagactc tcaggcaatg acctgatagc ctttgtagag acctctcaaa 6060 aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc atattgatgg 6120 tgatttgact gtctccggcc tttctcaccc gtttgaatct ttacctacac attactcagg 6180 cattgcattt aaaatatatg agggttctaa aaatttttat ccttgcgttg aaataaaggc 6240 ttctcccgca aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg 6300 ctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga 6360 tgttggaagt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 6420 atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac 6480 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 6540 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 6600 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata 6660 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 6720 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 6780 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 6840 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 6900 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 6960 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 7020 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 7080 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 7140 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 7200 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 7260 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 7320 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 7380 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 7440 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 7500 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 7560 aagccctccc gtatcgtagt tatctacacg acggggagtc agcaactat ggatgaacga 7620 atagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 7680 gtttactcat atatacttta gattgattta aaacttcatt tttaattta aaggatctag 7740 gtgaagatcc ttttgataa tctcatgacc aaaatcctt aacgtgagtt tcgttccac 7800 tgagcgtcag accccgtaga aagatcaa ggatctctt gagatcctttt ttttctgcgc 7860 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggttg tttgccggat 7920 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 7980 actgtccttc tagtgtagcc gtagttaggc caccactca agaactctgt agcaccgcgt 8040 acataccctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 8100 cttaccggt tggactcaag acgatagtta ccggatagg cgcagcggtc gggctgaacg 8160 gggggttcgt gcacagcc cagcttggag cgaacgacct acacgact gagataccta 8220 cagcgtgagc tatgagaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 8280 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 8340 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 8400 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 8460 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat 8520 aaccgtatta ccgggtttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 8580 agcgagtcag tgagcgacca agcggaagag c 8611 <210> 4 <211> 564 <212> DNA <213> Adeno-associated virus <400> 4 cagccactat gggtctaggc tgcccatgta aggaggcaag gcctggggac acccgagatg 60 cctggttata attaacccag acatgtggct gctccccccc cccaacacct gctgcctgag 120 cctcaccccc accccggtgc ctgggtctta ggctctgtac accatggagg agaagctcgc 180 tctaaaaata accctgtccc tggtgggctg tgggggactg agggcaggct gtaacaggct 240 tgggggccag ggcttatacg tgcctgggac tcccaaagta ttactgttcc atgttcccgg 300 cgaagggcca gctgtccccc gccagctaga ctcagcactt agtttaggaa ccagtgagca 360 agtcagccct tggggcagcc catacaaggc catggggctg ggcaagctgc acgcctgggt 420 ccggggtggg cacggtgccc gggcaacgag ctgaaagctc atctgctctc aggggcccct 480 ccctggggac agcccctcct ggctagtcac accctgtagg ctcctctata taacccaggg 540 gcacaggggc tgcccccggg tcac 564 <210> 5 <211> 8409 <212> DNA <213> Adeno-associated virus <400> 5 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcgcg 60 ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc 120 cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt 180 ccttgtagtt aatgattaac ccgccatgct aattatctac gtagccatgt ctagacagcc 240 actatgggtc taggctgccc atgtaaggag gcaaggcctg gggacacccg agatgcctgg 300 ttataattaa cccagacatg tggctgctcc ccccccccaa cacctgctgc ctgagcctca 360 cccccacccc ggtgcctggg tcttaggctc tgtacaccat ggaggagaag ctcgctctaa 420 aaataaccct gtccctggtg ggctgtgggg gactgagggc aggctgtaac aggcttgggg 480 gccagggctt atacgtgcct gggactccca aagtattact gttccatgtt cccggcgaag 540 ggccagctgt cccccgccag ctagactcag cacttagttt aggaaccagt gagcaagtca 600 gcccttgggg cagcccatac aaggccatgg ggctgggcaa gctgcacgcc tgggtccggg 660 gtgggcacgg tgcccgggca acgagctgaa agctcatctg ctctcagggg cccctccctg 720 gggacagccc ctcctggcta gtcacaccct gtaggctcct ctatataacc caggggcaca 780 ggggctgccc ccgggtcacc accacctcca cagcacagac agacactcag gagccagcca 840 gccaggtaag tttagtcttt ttgtctttta tttcaggtcc cggatccggt ggtggtgcaa 900 atcaaagaac tgctcctcag tggatgttgc ctttacttct aggcctgtac ggaagtgtta 960 cttctgctct aaaagctgcg gaattgtacc cgcggccgcc accatgctgt ggtgggagga 1020 ggtggaggat tgttatgaaa gggaggacgt gcagaagaag acttttacca agtgggtgaa 1080 cgctcagttc agcaaatttg ggaagcagca catcgagaat ctgttttccg acctgcagga 1140 tgggagacgg ctgctggatc tgctggaagg actgactggc cagaagctgc ccaaagagaa 1200 ggggagcact agggtgcacg ccctgaacaa cgtgaacaaa gctctgagag tgctgcagaa 1260 caacaacgtg gatctggtga atattggcag tactgatatc gtggacggga accacaaact 1320 gacactgggc ctgatctgga acattattct gcactggcag gtgaaaaatg tgatgaagaa 1380 catcatggcc gggctgcagc agaccaattc cgagaagatc ctgctgtctt gggtgcggca 1440 gagcacccgc aactatcccc aggtgaacgt gattaacttc actacatcct ggagcgacgg 1500 gctggccctg aatgctctga ttcacagcca caggcctgat ctgttcgact ggaatagcgt 1560 ggtgtgccag cagtctgcca cacagcgcct ggaacatgcc ttcaatatcg ctcggtacca 1620 gctggggatc gaaaaactgc tggacccaga ggatgtggac actacatacc shitaaaa 1680 gtctattctg atgtacatta ctagcctgtt ccaggtgctg ccacagcagg tgtctattga 1740 agccattcag gaggtggaaa tgctgccccg cccccccaaa gtgactaaag aggagcattt 1800 tcagctgcat catcagatgc attacagcca gcagattacc gtgagcctgg ctcagggata tgagcgcacc agtagtcca aaccacggtt caagtcctac gcttataccc aggctgccta cgtgacaact agcgacccta ctagatcccc ctttccatcc cgcacctgg aggccccaga ggacaagagc tttgggtcca gcctgatgga aagcgaggtg aatctggatc ggtaccagac agccctggag gaggtgctga gctggctgct gagtgctga gacacactgc aggcccaggg cgaaatttcc aatgacgtgg aagtggtgaa ggatcagttc cacacacacg agggctatat gatggacctg acagctcacc aggggcgcgt gggcaatatc ctgcagctgg gctctaaact gatcggcacc gggaaactga gtgaggacga ggaaacagaa gtgcaggagc agatgaacct gctgaacagc cgctggggagt gtctgagagt ggctagtatg gagaagcagt ccaacctgca ccgggtgctg atggacctgc agaaccagaa actgaaagag ctgaacgact ggctgacaaa rich cgcacaagga rich rich ggagccactg ggacccgacc tggaggatct gaagagacag gtgcagcagc ataaggtgct gcaggaggat ctggaacagg agcaggtgcg ggtgaactcc ctgacacata tggtggtggt ggtggacgaa tctagtggag atcacgccac 2580 cgccgccctg gaggaacagc tgaggtgct gggggaccgg tgggccaca ttgccggtg 2640 gaccgaggac aggtgggtgc tgctgcagga catcctgctg aaatggcaga ggctgaccga 2700 ggagcagtgt ctgtttagtg cttggctgag cgagaaagg gacgccgtga acaagatcca 2760 cacaacggc tttaggatc agaacgaat gctgtctagc ctgcagaac tggctgtgct 2820 gaaggccgat ctggagaaaa agaagcagag catgggcaaa ctgtatagcc tgaacagga 2880 cctgctgagc accctgaaga acagagcgt gacccagaag acagaagccct ggctggataa 2940 ctttgcccgc tgctgggaca acctgtgca gaactggag aaagtacag ctcagatctc 3000 tcaggctgtg accacaaccc agcctagcct gacccagaca accgtgatgg aaccgtgac 3060 caccgtgaca acccgcgac agatcctggt gaaacatgcc caggaagagc tgccacctcc 3120 acctccccag agagagaa ccctggagcg gctgcaggag ctgcaggaag ccactgacga 3180 actggacctg aagctgaggc aggccgaagt gattagggg tcttggcagc ctgtggggcga 3240 tctgctgatt gattccctgc aggaccacct ggaaaaggtg aaggctctga gaggcgaaat 3300 tgctccactg aaggaacg tgagtcatgt gaacgatctg gctagacagc tgacaacact 3360 gggcatccag ctgagcccat aaatctgag caactggag gacctgaata ccaggtggaa 3420 gctgctgcag gtggctgtgg aagaccgggt gcggcagctg catgaggccc atcgcgactt 3480 cggaccagcc agccagcact ttctgagcac atccgtgcag gggccctggg agagggccat 3540 ttctcccaac aaggtgccct actatattaa tcacgagacc cagaccactt gttgggacca 3600 tcccaagatg agaactgt accagtccct ggccgatctg aacaacgtga ggtttagcgc 3660 ttacagaacc gctatgaagc tgagacggct gcagaaggcc ctgtgcctgg atctgctgtc 3720 cctgtccgcc gcctgcgatg ccctggatca gcataatctg aagcagaacg atcagccaat 3780 ggatatcctg cagatcatca actgcctgac cactatctac gacaggctgg agcaggagca 3840 caacaacctg gtgaacgtgc ctctgtgcgt ggatatgtgc ctgaactggc tgctgaacgt 3900 gtatgacact gggcgcaccg gccggatcag agtgctgagt tttaaaactg ggattatctc 3960 4020 cactggattt tgtgaccagc gccgcctggg actgctgctg catgatagta tccagattcc 4080 tagacagctg ggagaggtgg ctagtttcgg aggatctaac atcgaaccca gcgtgcgcag 4140 ctgtttccag tttgccaata aaaacctga aatcgaggct gctctgttcc tggattggat 4200 gcgcctggaa ccacagagca tggtgtggct gcctgtgctg cacagagtgg ctgccgccga 4260 aactgccaag caccaggcta aatgcaacat ctgcaaggaa tgtcccatta tcggctttcg 4320 ctacaggagt ctgaaacatt ttaactacga tatttgccag agctgcttct tttccggaag 4380 4440 tggcgaagat gtgcgcgatt ttgccaaggt gctgaagaat aagtttcgga ctaagaggta 4500 cttcgccaag cacccccgca tggggtatct gccagtgcag acagtgctgg aaggacaa 4560 tatggagacc gatacaatgt gagcggccgc aataaaagat ctttattttc attagatctg 4620 tgtgttggtt ttttgtgtgt ctagagcatg gctacgtaga tagtagcat ggcgggtaa 4680 tcattaacta caaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 4740 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 4800 cagtgagcga gcgagcgcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct 4860 tcccaacagt tgcgcagcct gaatggcgaa tggaagttcc agacgattga gcgtcaaaat 4920 gtaggtattt ccatgagcgt ttttcctgtt gcaatggctg gcggtaatat tgttctggat 4980 attaccagca aggccgatag tttgagttct tctactcagg caagtgatgt tattactataat 5040 ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc 5100 ctcactgatt ataaaaac ttctcaggat tctggcgtac cgttcctgtc taaaatccct 5160 ttaatcggcc tcctgtttag ctcccgctct gattctaacg aggaaagcac gttatacgtg 5220 ctcgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 5280 ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 5340 cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcggggggct 5400 ccctttaggg ttccgattta gtgatttacg gcacctcgac cccaaaaaac ttgattaggg 5460 tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 5520 gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 5580 ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 5640 gctgatttaa caaaaattta acgcgaattt taacaaata ttaacgttta caatttaaat 5700 atttgcttat acaatcttcc tgtttttgg gcttttctga ttatcaaccg gggtacatat 5760 gattgacatg ctagttttac gattaccgtt catcgattct cttgtttgct ccagactctc 5820 aggcaatgac ctgatagcct ttgtagagac ctctcaaaaa tagctaccct ctccggcatg 5880 aatttatcag ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt 5940 tctcaccccgt ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag 6000 ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag 6060 ggtcataatg tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat 6120 tttgctaatt ctttgccttg cctgtatgat ttatggatg ttggaagttc ctgatgcggt 6180 attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 6240 tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 6300 cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 6360 gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 6420 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 6480 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt attttctaa atacattcaa 6540 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 6600 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 6660 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 6720 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagtttc 6780 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 6840 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 6900 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 6960 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 7020 cgatcggagg accgaaggag ctaaccgcttt tttgcacaa catgggggat catgtaactc 7080 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 7140 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 7200 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 7260 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 7320 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 7380 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 7440 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 7500 ttgattaaa acttcattttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 7560 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 7620 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 7680 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 7740 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 7800 agttaggcca ccacttcaag aactctgtag caccgcgtac atacctcgct ctgctaatcc 7860 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 7920 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 7980 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 8040 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 8100 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 8160 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 8220 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 8280 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gggtttgagt 8340 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaccaag 8400 cggaagagc 8409

Claims

1. 1. A recombinant AAVrh74 vector comprising, in 5' to 3' direction, an inverted terminal repeat (ITR), an MHCK7 muscle-specific regulatory element, a chimeric intron sequence, a nucleotide sequence of SEQ ID NO:1, a synthetic polyA signal set forth at nucleotides 4787 to 4842 of SEQ ID NO:3, and an ITR.

2. The recombinant AAVrh74 vector described in claim 1, wherein the chimeric intron sequence is shown in nucleotides 1046 to 1195 of SEQ ID NO:

3.

3. 3. A composition comprising the recombinant AAVrh74 vector of claim 1 or 2 and a pharma- ceutically acceptable carrier.

4. A composition comprising a recombinant AAVrh74 vector according to claim 1 or 2, or a composition according to claim 3, for use in treating muscular dystrophy in a subject in need thereof.

5. A composition comprising a recombinant AAVrh74 vector according to claim 1 or 2, or a composition according to claim 3, for reducing or preventing fibrosis in a subject suffering from muscular dystrophy.

6. The composition of claim 4 or 5, wherein the subject is suffering from Duchenne muscular dystrophy.

7. The composition according to any one of claims 4 to 6, characterized in that the composition is administered by intramuscular or intravenous injection.

8. The composition according to any one of claims 4 to 6, characterized in that the composition is administered systemically.

9. 9. The composition of claim 8, wherein the composition is administered by injection, infusion, or implantation.

10. 13. Use of a recombinant AAVrh74 vector according to claim 1 or 2 or a composition according to claim 3 for the manufacture of a medicament for treating muscular dystrophy in a subject in need thereof.

11. 13. Use of a recombinant AAVrh74 vector according to claim 1 or 2 or a composition according to claim 3 for the manufacture of a medicament for reducing or preventing fibrosis in a subject suffering from muscular dystrophy.

12. 12. The use according to claim 10 or 11, wherein the subject suffers from Duchenne muscular dystrophy.

13. The use according to any one of claims 10 to 12, wherein the medicament is formulated for intramuscular or intravenous injection.

14. The use according to any one of claims 10 to 12, wherein the medicament is formulated for systemic administration.

15. 15. The use according to claim 14, wherein the medicament is formulated for parenteral administration by injection, infusion, or implantation.

Images