RECOMBINANT VECTOR AAVRH74, COMPOSITION AND USES THEREOF

Abstract

The present invention provides gene therapy vectors, such as adeno-associated virus (aav) vectors, that express a miniaturized human microdystrophin gene and a method of using these vectors to express microdystrophin in skeletal muscles, including the diaphragm and cardiac muscle, and to protect muscle fibers from injury, increase muscle strength, and reduce and / or prevent fibrosis in subjects suffering from muscular dystrophy.

Description

Descriptive Report of the Invention Patent for RECOMBINANT AAVRH74 VECTOR, COMPOSITION AND USES THEREOF.

[001] The present invention was made with government support under grant number NS055958 granted by the National Institutes of Health / National Institute of Neurological Disorders and Stroke. The government has certain rights to the invention.

[002] This application claims priority for the Patent Application Provisional US 62 / 473,148 filed on March 17, 2017, which is incorporated herein by reference in its entirety. INCORPORATION FOR REFERENCE PURPOSES OF MATERIAL SUBMITTED ELECTRONICALLY

[003] This application contains, as a separate part of the disclosure, a Sequence Listing in machine-readable format, which is incorporated by reference in its entirety and identified as follows: File name: 51475_Seqlisting.txt; Size: 29,519 bytes, created on: March 13, 2018. FIELD OF THE INVENTION

[004] The present invention provides gene therapy vectors, such as adeno-associated virus (AAV) vectors, that express a miniaturized human microdystrophin gene and a method of using these vectors to express microdystrophin in skeletal muscles, including the diaphragm and cardiac muscle, and to protect muscle fibers from injury, increase muscle strength, and reduce and / or prevent fibrosis in subjects suffering from muscular dystrophy. BACKGROUND

[005] The importance of muscle mass and strength for daily activities, such as locomotion and breathing, and for the metabolism of the entire body is unequivocal. Deficits in muscle function produce dis Petition 870210023177, dated 11 / 03 / 2021, p. 6 / 74 2 / 62 Muscular atrophies (MDs) are characterized by muscle weakness and wasting and have serious impacts on 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 sarcolemma-cytoskeleton constriction by DAPC. Duchenne muscular dystrophy (DMD) is one of the most devastating muscle diseases, affecting 1 in 5,000 newborn males.

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

[007] Without membrane stabilization by dystrophin or a microdystrophin, DMD will manifest uncontrolled cycles of tissue damage, and repair will replace lost muscle fibers with fibrotic scar tissue through connective tissue proliferation. The fi Petition 870210023177, dated 11 / 03 / 2021, page 7 / 74 3 / 62 Fibrosis is characterized by excessive deposits of ECM matrix proteins, including collagen and elastin. ECM proteins are primarily produced from cytokines, such as TGFe, which are released by activated fibroblasts responding to stress and inflammation. Although the main pathological feature of DMD is myofiber degeneration and necrosis, fibrosis as a pathological consequence has equal repercussions. The overproduction of fibrotic tissue restricts muscle regeneration and contributes to progressive muscle weakness in patients with DMD. In one study, the presence of fibrosis in early DMD muscle biopsies was highly correlated with unsatisfactory motor outcome at 10-year follow-up (Desguerre et al., J Neuropathol Exp Neurol, 2009. 68(7): p. 762-767). These results indicate fibrosis as a major contributor to DMD muscle dysfunction and highlight the need for early intervention before overt fibrosis.

[008] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long, including 145-nucleotide inverted-terminal repeats (ITRs). There are multiple AAV serotypes. The nucleotide sequences of the AAV serotype genomes 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 corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994). As other examples, the complete AAV-1 genome is provided in GenBank accession number NC_002077; The complete genome of AAV-3 is provided in GenBank accession number NC_1829; the complete genome of AAV-4 is provided in GenBank accession number NC_001829; the genome of AAV-5 is provided in GenBank accession number AF085716; the complete genome of AAV-6 is provided in GenBank accession number NC_001862; at least portions of AAV genomes Petition 870210023177, dated 11 / 03 / 2021, p. 8 / 74 4 / 62 and AAV-8 are provided in GenBank accession numbers AX753246 and AX753249, respectively (see also US Patents 7,282,199 and 7,790,449 with respect to AAV-8); the genome of AAV-9 is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the genome of AAV-10 is provided in Mol. Ther., 13(1): 67-76 (2006); and the genome of AAV-11 is provided in Virology, 330(2): 375-383 (2004). 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), capsidation / packaging, and host cell chromosome integration are contained within ITRs. Three AAV promoters (named p5, p19, and p40 for their relative locations on the map) drive the expression of the two internal open reading frames of AAV that encode rep and cap genes.The two rep promoters (p5 and p19), coupled to differential splicing of the single AAV intron (e.g., at AAV2 nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. Rep proteins have multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensual translational initiation sites are responsible for the production of the three related capsid proteins. A single consensual polyadenylation site is located at position 95 on the AAV genome map. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97 to 129 (1992).

[009] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cultured cells is non-cytopathic, and natural infection of humans and other animals is silent. Petition 870210023177, dated 11 / 03 / 2021, page 9 / 74 5 / 62 sa and asymptomatic. Furthermore, AAV infects various mammalian cells, allowing for the possibility of targeting many different tissues in vivo. Additionally, AAV slowly transduces dividing and non-dividing cells and can persist essentially for the lifespan of these cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids, enabling the construction of recombinant genomes. Furthermore, because the signals that direct AAV replication, encapsulation, and genome integration are contained in the ITRs of the AAV genome, some or all of the interior, approximately 4.3 kb of the genome (which encodes replication and structural capsid proteins, rep-cap), can be replaced by foreign DNA, such as a genetic cassette containing a promoter, DNA of interest, and a polyadenylation signal. The rep and cap proteins can be trans-delivered.Another significant feature of AAV is that it is an extremely stable and healthy virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), which makes cold preservation of AAV less critical. AAV can even be lyophilized. Finally, cells infected with AAV are not resistant to superinfection.

[0010] 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, since muscle is highly vascularized, recombinant AAV transduction resulted in the appearance of transgenic products in the systemic circulation after intramuscular injection, as described in Herzog et al., Proc Natl Acad Sci USA, 94: 5804-5809 (1997) and Murphy. Petition 870210023177, dated 11 / 03 / 2021, page 10 / 74 6 / 62 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 glycosylation, folding, and correct antibody secretion, indicating that muscle is capable of stable expression of secreted protein therapy.

[0011] Functional improvement in patients suffering from DMD and other muscular dystrophies requires genetic restoration at an early stage of the disease. There is a need for treatments that increase muscle strength and protect against muscle injuries in patients suffering from DMD. SUMMARY OF THE INVENTION

[0012] The present invention relates to gene therapy vectors, for example, AAV, which express the microdystrophin gene in skeletal muscles, including the diaphragm and cardiac muscle, to protect muscle fibers from injury, increase muscle strength, and reduce and / or prevent fibrosis.

[0013] The invention provides therapies and approaches to increase muscle strength and / or increase muscle mass using gene therapy vectors to deliver microdystrophin to treat the genetic defect observed in DMD. As shown in Example 2, treatment with microdystrophin gene therapy resulted in greater muscle strength in vivo. Furthermore, intramuscular and systemic administration of microdystrophin gene therapy showed dystrophin delivery to muscles in in vivo mouse models.

[0014] In one embodiment, the invention provides an rAAV vector comprising a muscle-specific control element nucleotide sequence and a nucleotide sequence encoding the microdystrophin protein. For example, the nucleotide sequence encoding Petition 870210023177, dated 11 / 03 / 2021, page 11 / 74 7 / 62 ca a functional microdystrophin protein, wherein the nucleotide is, 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, wherein the protein maintains microdystrophin activity. The microdystrophin protein provides stability to the muscle membrane during muscle contraction, for example, the microdystrophin acts as a buffer during muscle contraction.

[0015] The invention also provides rAAV vectors in which the nucleotide sequence encodes a functional microdystrophin protein comprising a nucleotide sequence that hybridizes under stringent conditions with the nucleic acid sequence of SEQ ID NO: 1, or its complements, and encodes a functional microdystrophin protein.

[0016] In one embodiment, the rAAV vector is a non-replicating recombinant adeno-associated virus (AAV), designated rAAVrh74.MHCK7.microdystrophin. This vector genome contains the minimum elements necessary for gene expression, including AAV2 inverted terminal repeats (ITR), microdystrophin, SV40 intron (SD / SA), and synthetic polyadenylation signal (Poly A), all under the control of the MHCK7 promoter / enhancer. The vector genome and expression cassette scheme is shown in Figure 1. The AAVrh74 serotype can be employed to achieve efficient gene transfer in skeletal and cardiac muscle after IV administration.

[0017] The term stringent is used to refer to conditions that are normally understood in the art as stringent. Hybridization stringency is primarily determined by temperature, ionic strength, and the concentration of denaturing agents, such Petition 870210023177, dated 11 / 03 / 2021, page 12 / 74 8 / 62 as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65 to 68 °C or 0.015 M sodium chloride, 0.0015 M sodium citrate and 50% formamide at 42 °C. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY 1989). More stringent conditions (such as higher temperature, lower ionic strength, higher formamide content or other denaturing agent) can also be used; however, the hybridization rate will be affected. In cases related to the hybridization of deoxyoligonucleotides, additional exemplary stringent hybridization conditions include washing in 6x SSC 0.05% sodium pyrophosphate at 37 °C (for 14-base oligonucleotides), 48 °C (for 17-base oligonucleotides), 55 °C (for 20-base oligonucleotides), and 60 °C (for 23-base oligonucleotides).

[0018] Other agents may be included in hybridization and washing buffers with the aim of reducing non-specific and / or background hybridization. Examples are 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 may also be used. The concentration and types of these additives may be altered without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are generally carried out at pH 6.8 to 7.4; however, under typical ionic strength conditions, the rate of hybridization is almost pH-independent. See Anderson et al., Nucleic Acid Hybridization: A Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).The hybridization conditions can be adjusted by someone skilled in the art in order to... Petition 870210023177, dated 11 / 03 / 2021, page 13 / 74 9 / 62 accommodate these variables and allow DNAs with different sequence relationships to form hybrids.

[0019] The term muscle-specific control element refers to a nucleotide sequence that regulates the expression of a coding sequence that is specific for expression in muscle tissue. These control elements include enhancers and promoters. The invention provides constructs comprising the MCKH7 promoter, the MCK promoter, and the MCK enhancer of muscle-specific control elements.

[0020] The term operationally linked refers to the positioning of the nucleotide sequence of the regulatory element, for example, promoter nucleotide sequence, to confer expression of said nucleotide sequence by said regulatory element.

[0021] In one aspect, the invention provides an rAAV vector, wherein the muscle-specific control element is a human skeletal actin gene element, cardiac actin gene element, myocyte-specific enhancer-binding factor (MEF), muscle creatine kinase (MCK), truncated MCK (tMCK), myosin heavy chain (MHC), MCK enhancer / hybrid α-myosin heavy chain enhancer (MHCK7), C5-12, murine creatine kinase enhancer element, fast-twitch troponin c gene skeletal element, slow-twitch cardiac troponin c gene element, slow-twitch troponin i gene element, hypoxia-inducible nuclear factors, steroid-inducible element or glucocorticoid response (GRE) element.

[0022] For example, the muscle-specific control element is the MHCK7 promoter nucleotide sequence SEQ ID NO: 2 or the muscle-specific control element is the MCK nucleotide sequence SEQ ID NO: 4. Furthermore, in either of the rAAV vectors of the invention, the nucleotide sequence of the control element Petition 870210023177, dated 11 / 03 / 2021, page 14 / 74 10 / 62 specific to the muscle, for example, the nucleotide sequence of MHCK7 or MCK, is operationally linked to the nucleotide sequence encoding the microdystrophin protein. For example, the nucleotide sequence of the MHCK7 promoter (SEQ ID NO: 2) is operationally linked to the coding sequence of human microdystrophin (SEQ ID NO: 1), as established in the construct provided in Figure 1 or Figure 10 (SEQ ID NO: 3). In another example, the MCK promoter (SEQ ID NO: 4) is operationally linked to the coding sequence of human microdystrophin (SEQ ID NO: 1), as established in the construct provided in Figure 7 or Figure 11 (SEQ ID NO: 5). In another aspect, the invention provides an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2. The invention also provides an rAAV vector comprising the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 4.

[0023] 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 rAAV vector rh74.MHCK7.microdystrophin comprises the nucleotide sequence of SEQ ID NO: 3 and shown in Figure 10. This rAAV vector comprises the MHCK7 promoter, a chimeric intron sequence, the coding sequence of the human microdystrophin gene, polyA, ampicillin resistance, and the pGEX plasmid main structure originating from or replicating pBR322.

[0024] The invention provides 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: 3. This rAAV vector is the AAV serotype AAVrh.74.

[0025] The invention also provides a recombinant AAV vector comprising the pAAV.MHCK7.microdystrophin construct nucleotide sequence of SEQ ID NO: 3. This rAAV vector is the Petition 870210023177, dated 11 / 03 / 2021, p. 15 / 74 11 / 62 serotype AAV AAVrh.74.

[0026] The rAAV vectors of the invention can be any AAV serotype, such as serotype AAVrh.74, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or AAV13.

[0027] The invention also provides pharmaceutical compositions (or sometimes simply referred to herein as compositions) comprising any of the rAAV vectors of the invention.

[0028] In another embodiment, the invention provides methods for producing an rAAV vector particle comprising culturing a cell that has been transfected with any rAAV vector of the invention and recovering rAAV particles from the supernatant of the transfected cells. The invention also provides viral particles comprising any of the recombinant AAV vectors of the invention.

[0029] The invention provides methods for treating muscular dystrophy comprising administering a therapeutically effective amount of any of the recombinant AAV vectors of the invention expressing human microdystrophin.

[0030] The invention provides methods 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.

[0031] The invention also provides methods 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.

[0032] Fibrosis refers to the excessive or unregulated deposition of extracellular matrix (ECM) components and repair processes. Petition 870210023177, dated 11 / 03 / 2021, page 16 / 74 12 / 62 abnormalities in tissues following injury, including skeletal muscle, heart muscle, liver, lung, kidney, and pancreas. The deposited ECM components include fibronectin and collagen, for example, collagen 1, collagen 2, or collagen 3.

[0033] The invention also provides methods of reducing or preventing fibrosis in a subject suffering from muscular dystrophy comprising administering a therapeutically effective amount of any recombinant AAV vector of the invention.

[0034] In another embodiment, the invention provides methods for preventing fibrosis in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant AAV vector of the invention. For example, any of the rAAVs of the invention can be administered to subjects suffering from muscular dystrophy to prevent fibrosis, for example, the rAAV of the invention expressing a human microdystrophin protein administered before fibrosis is observed in the subject. Furthermore, the rAAV of the invention expressing a human microdystrophin gene can be administered to a subject at risk of developing fibrosis, such as those suffering from or diagnosed with muscular dystrophy, for example, DMD. The rAAV of the invention can be administered to the subject suffering from muscular dystrophy in order to prevent further fibrosis in these subjects.

[0035] The invention contemplates the administration of any of the AAV vectors of the invention before fibrosis is observed in the subject. Furthermore, the rAAV of the invention can be administered to a subject at risk of developing fibrosis, such as those suffering from or diagnosed with muscular dystrophy, for example, DMD. The rAAV of the invention can be administered to a subject suffering from muscular dystrophy who has already developed fibrosis in order to prevent further fibrosis in these individuals. Petition 870210023177, dated 11 / 03 / 2021, page 17 / 74 13 / 62

[0036] The 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 an rAAV vector of the invention expressing a human microdystrophin gene. These methods may further comprise the step of administering an rAAV expressing microdystrophin.

[0037] The invention contemplates administering any of the AAV vectors of the invention to patients diagnosed with DMD before fibrosis is observed in the subject or before reduction in muscle strength or before reduction in muscle mass.

[0038] The invention also contemplates administering an AAV of the invention to a subject suffering from muscular dystrophy who has already developed fibrosis, in order to prevent further fibrosis in these individuals or to reduce fibrosis in these patients. The invention also provides for the administration of any of the rAAVs of the invention to a patient suffering from muscular dystrophy who already has reduced muscle strength or reduced muscle mass, in order to protect the muscle against further injury.

[0039] In any of the methods of the invention, the subject may be suffering from muscular dystrophy, such as DMD or any other muscular dystrophy associated with dystrophin.

[0040] In another aspect, an rAAV vector expressing the microdystrophin protein comprises the microdystrophin gene coding sequence operationally linked to a muscle-specific control element other than MHCK7 or MCK. For example, where the muscle-specific control element is the human skeletal actin gene element, the cardiac actin gene element, the myocyte-specific enhancer-binding factor MEF, tMCK (truncated MCK), myosin heavy chain (MHC), C5-12 Petition 870210023177, dated 11 / 03 / 2021, page 18 / 74 14 / 62 (synthetic promoter), murine creatine kinase potentiating element, fast-twitch skeletal troponin gene element C, slow-twitch cardiac troponin gene element, slow-twitch troponin gene element I, hypoxia-inducible nuclear factors, steroid-inducible element or glucocorticoid response element (GRE).

[0041] In either of the methods of the invention, the rAAV vector or composition can be administered by intramuscular injection or intravenous injection.

[0042] Furthermore, 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.

[0043] In another embodiment, the invention provides a composition comprising any of the rAAV vectors of the invention to reduce fibrosis in an individual in need thereof.

[0044] In addition, the invention provides a composition comprising any of the recombinant AAV vectors of the invention to prevent fibrosis in a patient suffering from muscular dystrophy.

[0045] The invention provides compositions comprising any of the recombinant AAV vectors of the invention for the treatment of muscular dystrophy.

[0046] The invention provides compositions 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.

[0047] The invention provides a composition comprising a recombinant AAV vector comprising the pAAV.MHCK7.microdystrophin construct comprising the nucleotide sequence SEQ ID NO: 3 for the treatment of muscular dystrophy. Petition 870210023177, dated 11 / 03 / 2021, page 19 / 74 15 / 62

[0048] The invention also provides compositions comprising any of the rAAV vectors of the invention for increasing muscle strength and / or muscle mass in a subject suffering from muscular dystrophy. In another embodiment, the invention provides compositions comprising any of the rAAV vectors of the invention for the treatment of muscular dystrophy.

[0049] The compositions of the invention can be formulated for intramuscular injection or intravenous injection. The composition of the invention is also formulated for systemic administration, such as parenteral administration by injection, infusion or implantation.

[0050] In addition, any of the compositions can be formulated for administration to a subject suffering from muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy.

[0051] In another embodiment, the invention provides the use of any of the rAAV vectors of the invention for the preparation of a medicament to reduce fibrosis in a subject in need thereof. For example, the subject in need may be suffering from muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy.

[0052] In another embodiment, the invention provides the use of an rAAV vector of the invention for the preparation of a medicament to prevent fibrosis in a subject suffering from muscular dystrophy.

[0053] In addition, the invention provides the use of a recombinant AAV vector of the invention for the preparation of a medicament to increase muscle strength and / or muscle mass in a subject suffering from muscular dystrophy.

[0054] The invention also provides the use of the rAAV vectors of the invention for the preparation of a medicament for the treatment of muscular dystrophy. Petition 870210023177, dated 11 / 03 / 2021, page 20 / 74 16 / 62

[0055] The 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.

[0056] The 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.

[0057] In any of the uses of the invention, the medicament can be formulated for intramuscular injection or intravenous injection. Furthermore, in any of the uses of the invention, the medicament can be formulated for systemic administration, such as parenteral administration by injection, infusion or implantation.

[0058] Any of the medications can be prepared for administration to a subject suffering from muscular dystrophy, such as DMD or any other dystrophin-associated muscular dystrophy. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 illustrates the pAAV.MHCK7.microdystrophin construct. In this construct, the cDNA expression cassette is flanked by inverted terminal repeat sequences of AAV2 (ITR). The construct is characterized by a stem deletion in the structure (R4-R23), while hinges 1, 2, and 4 (Hi, H2, and H4) and the cysteine-rich domain continue to produce a 138 kDa protein. The expression of the microdystrophin protein (3579 bp) is guided by a MHCK7 promoter (795 bp). The intron and 5' UTR are derived from the pCMVB plasmid (Clontech). The microdystrophin cassette had a Kozak consensus immediately ahead of the ATG start and a small 53 bp synthetic polyA signal for mRNA termination. The human microdystrophin cassette contained the domains (R4-R23 / Δ71-78), as described. Petition 870210023177, dated 11 / 03 / 2021, page 21 / 74 17 / 62 previously by Harper et al. (Nature Medicine 8, 253-261 (2002)).

[0060] Figure 2 demonstrates dystrophin protein expression after intramuscular delivery of the AAVrh74.MHCK7 construct. The anterior tibial muscle of mdx mice was injected with 1 x 1011vg (n=5 per group). Six weeks later, the muscles were harvested and stained for dystrophin expression with an N-terminal antibody for dystrophin staining and hematoxylin and eosin.

[0061] Figures 3A to 3C provide measurements of skeletal muscle strength and quantification of microdystrophin expression after intramuscular injection of the AAVrh74.MHCK7 construct. (A) The tibialis anterior muscle of mdx mice was injected with 1 x 1011vg (n=5) of the AAVrh74.MHCK7 construct. Six weeks later, the tibialis anterior muscles were harvested and subjected to in vivo strength measurements. The dosed cohort had significantly greater force output than the untreated mdx controls.

[0062] Figures 4A to 4C demonstrate generalized transduction of skeletal, diaphragmatic, and cardiac muscle fibers after systemic administration of the construct AAVrh.74.MHCK7.micro-dys. (A) mdx mice were systemically treated at 6 weeks of age via the tail vein with 6 x 10¹²vg (2 x 10¹⁴vg / kg) of AAVrh.74.MHCK7.microdystrophin after 12 weeks of treatment. (B) Microdystrophin staining demonstrates the quantification of the percentage of muscle fibers expressing microdystrophin in each tissue. (C) Shows the specific force measured in the diaphragm at the low and high doses (clinically planned). No significant difference was observed at low doses; however, there was a significant improvement at the high dose.

[0063] Figure 5 demonstrates the expression of dystrophin protein after systemic administration of the AAVrh.74.MHCK7.microdystrophin construct. mdx mice (n=5) were systemically treated with Petition 870210023177, dated 11 / 03 / 2021, page 22 / 74 18 / 62 from 6 weeks of age via tail vein with 6 x 1012vg of AAVrh.74.MHCK7.microdystrophin. After 12 weeks of treatment, all muscles were harvested and stained for dystrophin and restoration of DAPC components (beta-sarcoglycan shown).

[0064] Figures 6A to 6D demonstrate the toxicology / 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 indicator of efficacy, the number of muscle fibers with centrally positioned nuclei (CN) was quantified (Figure 6B). CNs are indicative of muscle degeneration and regeneration cycles, and thus, a reduction in CN demonstrates the effect of treatment. (Figure 6C) shows that the total number of fibers remains unchanged with treatment. The amount of creatine kinase is provided in (D), which shows improvement at high doses. Independent t-tests were used to locate differences (p<0.05); The data are reported as mean ± SEM.

[0065] Figure 7 illustrates the pAAV plasmid construct. MCK.microdystrophin.

[0066] Figure 8 presents the results of a potency assay of a rAAVrh74.MCK.microdystrophin (human). 3 x 10⁹, 3 x 10¹⁰ or 1 x 10¹¹vg (n=3 per group) was injected into the tibialis anterior muscle of mdx mice. Four weeks later, the muscles were harvested and stained for dystrophin expression with the N-terminal Dys3 antibody. There was a linear correlation between expression and dose with very little expression (no effect level) at 3 x 10⁹vg and 89% expression at 1 x 10¹¹vg.

[0067] Figures 9A to 9C demonstrate that human microdystrophin improves force generation and protection against injury-induced movements. Petition 870210023177, dated 11 / 03 / 2021, page 23 / 74 19 / 62 by eccentric contraction. (A) Immunostaining of dystrophin protein in extensor digitorum longus (EDL) and TA shows expression in mdx myofibers after injection of rAAVrh.74-MCK-microdystrophin (human) via the femoral artery. Cell transfection-infected muscle stained identically and exposures coincide over time. (B) rAAVrh.74-MCK-microdystrophin significantly increased normalized specific strength compared to cell transfection-treated mdx muscles (P<0.05 vs. mdx). (C) mdx muscles infected with rAAVrh.74-MCK-Micro-dys (human) were compared with contralateral mdx EDL muscles infected by sham and WT EDL muscles (WT C57Bl / 10) for strength loss during repetitive eccentric contractions 12 weeks after gene transfer. Treatment with rAAVrh.74-MCK-microdystrophin (Micro-dys) significantly protected against strength loss compared with cell transfection-treated mdx muscles (P< 0.001 vs.mdx). Errors are SEMs.

[0068] Figure 10 provides the nucleic acid sequence (SEQ ID NO: 3 rAAVrh74.MHCK7.microdystrophin).

[0069] Figure 11 provides the nucleic acid sequence (SEQ ID NO: 5) rAAVrh74.MHCK7.microdystrophin.

[0070] Figures 12A to 12B provide the immune response to systemic administration of AAVrh74.MHCK7.microdystrophin in the non-human primate. (A) ELISpot response to microdystrophin and AAV capsid peptide clusters. ConA is the positive control and DMSO is the negative control. There were three clusters for AAVrh74 and four clusters of microdystrophin-specific peptides. (B) Positive ELISA titers of circulating neutralizing antibodies to the vector capsid. Serum was isolated from primates biweekly and analyzed for antibody titer. The reported titer corresponds to the last dilution at which the response ratio... Petition 870210023177, 11 / 03 / 2021, p. 24 / 74 20 / 62 ta is >2.

[0071] Figures 13A and B demonstrate systemic delivery in rhesus monkeys with AAVrh74.MHCK7.microdystrophin. Anti-FLAG immunofluorescence staining in muscles on the left side demonstrated robust microdystrophin expression.

[0072] Figure 14 demonstrates the effect of systemic treatment with rAAVrh74.MHCK7.microdystrophin on transgenic expression. Immunofluorescence staining for microdystrophin using an N-terminal dystrophin antibody in the heart, diaphragm, psoas, and tibialis anterior (TA) demonstrates robust expression at medium doses (6 and 12 vg; 2 and 14 vg / kg) and high doses (1, 2 and 13 vg; 6 and 14 vg / kg) of animals treated 3 months after injection. 20x images are shown.

[0073] Figure 15 demonstrates the effect of systemic treatment with rAAVrh74.MHCK7.microdystrophin on transgenic expression. Immunofluorescence staining for microdystrophin using an N-terminal dystrophin antibody in the gastrocnemius, quadriceps, triceps, and gluteus muscles demonstrates robust expression at intermediate doses (6 and 12 vg; 2 and 14 vg / kg) and at the highest dose (1, 2, and 13 vg; 6 and 14 vg / kg) in animals 3 months post-injection. 20x magnification images are shown.

[0074] Figure 16 demonstrates the effect of systemic treatment with rAAVrh74.MHCK7.microdystrophin on muscle pathology. (A) H&E staining of diaphragm, tibialis anterior, gastrocnemius, and quadriceps muscles of mice treated with C57BL / 6 WT, mdx, and rAAVrh74.MHCK7.microdystrophin (average dose 2 and 14 vg / kg; high dose 6 and 14 vg / kg), (B) Quantification of mean fiber size demonstrated a normalization of fiber size in all tissues. ****p<0.001, one-way ANOVA; data are reported as mean ± SEM. 20x images are shown.

[0075] Figure 17 demonstrates the effect of systemic treatment Petition 870210023177, dated 11 / 03 / 2021, page 25 / 74 21 / 62 with rAAVrh74.MHCK7.microdystrophin in muscle pathology. (A) H&E staining of triceps, gluteus, and psoas muscle from mice treated with C57BL / 6 WT, mdx, and rAAVrh74.MHCK7.microdystrophin (medium dose - 2 and 14 vg / kg; high dose - 6 and 14 vg / kg), (B) quantification of mean fiber size demonstrated larger fibers in a dose-dependent manner. ****p<0.001, one-way ANOVA; data are reported as mean ± SEM. Images are shown 20x.

[0076] Figure 18 demonstrates the effect of systemic treatment with rAAVrh74.MHCK7.microdystrophin on central nucleation. The dose scale illustrates reductions in central nucleation in all skeletal muscles and the diaphragm. Two-way ANOVA was used to locate differences (p<0.05). Data are reported as mean ± SEM.

[0077] Figure 19 demonstrates the effect of systemic treatment with rAAVrh74.MHCK7.microdystrophin on collagen deposition. Increasing dose illustrates reductions in collagen accumulation (%) in the diaphragm. *p<0.05, one-way ANOVA; data are reported as mean ± SEM. 20x magnification images are shown.

[0078] Figure 20 demonstrates the correction of strength deficits in the diaphragm. After 3 or 6 months of treatment, strips of diaphragm muscle were harvested to measure specific strength (normalized to the cross-sectional area). Treatment restored strength to TP levels. *p<0.05. One-way ANOVA was used to determine differences in mdx-LR mice.

[0079] Figure 21 demonstrates the correction of strength deficits in TA. (A) After 3 to 6 months of treatment, TA muscles were harvested (left and right) to measure specific strength (normalized with TA weight). Treatment restored strength to WT levels. (B) Treatment rescued TA muscles from fatigue after a rigorous eccentric contraction protocol. *p<0.05. One-way ANOVA Petition 870210023177, dated 11 / 03 / 2021, p. 26 / 74 The 22 / 62 ratio was used to determine the differences in mdx-LR mice.

[0080] Figure 22 provides the distribution of mean vg copies in various tissues from three mdx mice after IV administration of rAAVrh74.MHCK7.microdystrophin.

[0081] Figure 23. Serum chemistry for mice systemically injected with ssAAVrh74.MHCK7.microdystrophin and serum chemistry from age-matched control groups were analyzed by an independent CRO (Charles River Laboratories), which indicate normal values ​​in all chemistry analyzed. The only abnormal values ​​were elevated AST and ALT observed in animals treated with MDX vehicle [MDX-LR (Ringer's lactate)], 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.

[0082] Figure 24 provides biodistribution western blots in the muscles and organs of mdx mice systemically injected with rAAVrh74.MHCK7.microdystrophin.

[0083] Figure 25 provides the auxiliary plasmid map pNLREP2-Caprh74 AAV.

[0084] Figure 26 provides the Ad Helper pHELP plasmid. DETAILED DESCRIPTION

[0085] The present invention provides gene therapy vectors, for example, rAAV vectors, microdystrophin overexpression, and methods for reducing and preventing fibrosis in patients with muscular dystrophy. Muscle biopsies performed at the earliest age of DMD diagnosis reveal prominent tissue proliferation. Petition 870210023177, dated 11 / 03 / 2021, page 27 / 74 23 / 62 connective tissue. Muscle fibrosis is detrimental in several ways. It reduces the normal transit of endomysial nutrients through connective tissue barriers, reduces blood flow, deprives muscles of nutritional constituents of vascular origin, and functionally contributes to early loss of ambulation due to limb contractures. Over time, treatment challenges multiply as a result of accentuated fibrosis in the muscle. This can be observed in muscle biopsies comparing the proliferation of connective tissue at successive time points. The process continues to worsen, leading to loss of ambulation and accelerating out of control, especially in wheelchair-dependent patients.

[0086] Without early treatment, including a parallel approach to reduce fibrosis, the benefits of exon skipping, stop codon reading, or gene replacement therapies are unlikely to be fully achieved. Even small molecule or protein replacement strategies are likely to fail without an approach to reduce muscle fibrosis. Previous work in aged mdx mice with existing fibrosis treated with AAV.microdystrofin demonstrated that complete functional restoration was not achieved (Liu, M., et al., Mol Ther 11, 245-256 (2005)). It is also known that the progression of DMD cardiomyopathy is accompanied by scarring and fibrosis in the ventricular wall.

[0087] As used in this document, the term AAV is a standardized 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, thirteen AAV serotypes have been characterized. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Volume 1, pages 169 to 228 and Berns, 1990, Virology, pages Petition 870210023177, dated 11 / 03 / 2021, page 28 / 74 24 / 62 1,743 to 1,764, Raven Press, (New York). However, it is expected that these same principles will be applicable to additional AAV serotypes, since it is well known that the various serotypes are closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pages 165 to 174 of Parvoviruses and Human Disease, J.R. Pattison, 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 all have three capsid proteins like those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-cooking segments in the terminal that corresponds to inverted terminal repeat sequences (ITRs).The similar inefficiency patterns also suggest that replication functions in each serotype are under similar regulatory control.

[0088] An AAV vector, as used herein, refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). These AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

[0089] An AAV virion or AAV viral particle or AAV vector particle refers to a viral particle composed of at least one AAV capsid protein and an encapsulated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome as a transgene to be delivered to a cell of Petition 870210023177, dated 11 / 03 / 2021, page 29 / 74 25 / 62 mammal), the same typically refers to an AAV vector particle or simply an AAV vector. Thus, the production of an AAV vector particle necessarily includes the production of an AAV vector, as a vector contained within an AAV vector particle. AAV

[0090] The recombinant AAV genomes of the invention comprise a nucleic acid molecule of the invention and one or more AAV ITRs flanking a nucleic acid molecule. The AAV DNA in the rAAV genomes can be of any AAV serotype for which a recombinant virus can be derived, 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, AAV12 and AAV-13. The production of pseudotyped rAAV is disclosed, for example, in document no. WO 01 / 83692. Other types of rAAV variants, for example, rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900 to 1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of several AAV serotypes are known in the art. To promote specific skeletal muscle expression, AAV1, AAV6, AAV8, AAV8 or AAVrh.74 can be used.

[0091] The DNA plasmids of the invention comprise rAAV genomes of the invention. The DNA plasmids are transferred into cells permitted for infection with an AAV helper virus (e.g., adenovirus, adenovirus with E1 deleted, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. The techniques for producing rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell, are standard in the art. Petition 870210023177, dated 11 / 03 / 2021, page 30 / 74 26 / 62 rAAV production requires the following components to be present within a single cell (denoted herein as a packaging cell): an rAAV genome, AAV rep and cap genes separate from (i.e., not within) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus may be derived, and may be from an AAV serotype different from the ITR of the rAAV genome, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAV is disclosed, for example, in document no. WO 01 / 83692, which is incorporated by reference in its entirety herein.

[0092] One method of generating a packaging cell is to create a cell line that stably expresses all the components necessary for the production of AAV particles. For example, a plasmid (or multiple plasmids) comprising an rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separated from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tail production (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or by direct end ligation (Senapathy and Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus, such as adenovirus.The advantages of this method are that the cells are selectable and suitable for large-scale production of rAAV. Petition 870210023177, dated 11 / 03 / 2021, page 31 / 74 27 / 62 Other examples of suitable methods employ adenoviruses or baculoviruses instead of plasmids to introduce rAAV genomes and / or rep and cap genes into packaging cells.

[0093] 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). Several approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., Mol. Cell. Biol., 7: 349 (1988). Samulski et al., J. Virol., 63:3822 to 3828 (1989); Patent No. US 5,173,414; WO 95 / 13365 and corresponding US Patent No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. Vaccine 13: 1244-1250 (1995); Paul et al. Human Gene Therapy 4: 609-615 (1993); Clark et al. Gene Therapy 3: 1124-1132 (1996); Patent No. US 5,786,211; Patent document No. US 5,871,982; and US Patent No. 6,258,595. The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on those sections of the documents relating to the production of rAAV.

[0094] The invention thus provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (a 293 cognate cell line). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells). Petition 870210023177, dated 11 / 03 / 2021, page 32 / 74 28 / 62 cells transformed with E1 adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (fetal rhesus lung cells).

[0095] The recombinant AAV (i.e., infectious encapsulated rAAV particles) of the invention comprises an rAAV genome. In exemplary embodiments, the genomes of both rAAVs lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genomes. Examples of rAAVs that can be constructed to comprise the nucleic acid molecules of the invention are presented in International Patent Application No. PCT / US2012 / 047999 (WO 2013 / 016352) incorporated by reference herein in its entirety.

[0096] In an exemplary embodiment, the recombinant AAV vector of the invention is produced by the triple transfection method (Xiao et al., J Virol 72, 2224-2232 (1998)) using the AAV vector plasmids pAAV.MHCK7.microdystrophin, pNLRep2-Caprh74 and pHelp. pAAV contains the microdystrophin gene expression cassette flanked by AAV2 inverted-terminal repeat (ITR) sequences. It is this sequence that is encapsulated in the AAVrh74 virions. The plasmid contains the microdystrophin sequence and the core promoter and muscle-specific promoter MHCK7 enhancer elements to drive gene expression. The expression cassette also contains an SV40 intron (SD / SA) to promote high-level gene expression, and the bovine growth hormone polyadenylation signal is used. for efficient transcription completion.

[0097] pNLREP2-Caprh74 is an AAV helper plasmid that encodes the 4 wild-type AAV2 rep proteins and the 3 wild-type AAV capsid VP proteins of rh74 serotype. A map Petition 870210023177, dated 11 / 03 / 2021, page 33 / 74 29 / 62 Schematic of the pNLREP2-Caprh74 plasmid is shown in Figure Error! Reference source not found.25.

[0098] The pHELP adenovirus helper plasmid is 11,635 bp long and was obtained from Applied Viromics. The plasmid contains the adenovirus genome regions that are important for AAV replication, namely E2A, E4ORF6, and VA RNA (adenovirus E1 functions are provided by 293 cells). The adenovirus sequences present in this plasmid represent only ~40% of the adenovirus genome and do not contain the cis elements critical for replication, such as adenovirus terminal repeats. Therefore, no infectious adenovirus is expected to be generated from such a production system. A schematic map of the pHELP plasmid is shown in Figure 26.

[0099] rAAV can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art and include methods disclosed, for example, in Clark et al., Hum. Gene Ther., 10(6):1031 to 1039 (1999); Schenpp and Clark, Methods Mol. Med., 69:427 to 443 (2002); US Patent No. 6,566,118 and WO No. 98 / 09657.

[00100] In another embodiment, the invention contemplates compositions comprising rAAV of the present invention. The compositions of the invention comprise rAAV in a pharmaceutically acceptable carrier. The compositions may also comprise other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are non-toxic to receptors and are preferably inert at the dosages and concentrations employed and include buffers and surfactants, such as pluronic acid.

[00101] The rAAV titers to be administered in the methods of the invention will vary depending, for example, on the particular rAAV, the Petition 870210023177, dated 11 / 03 / 2021, p. 34 / 74 30 / 62 The method of administration, the treatment objective, the individual, and the type (or types) of target cell, can be determined by standard methods in the technique. rAAV titers can range from approximately 1x10⁶, approximately 1x10⁷, approximately 1x10⁸, approximately 1x10⁹, approximately 1x10¹⁰, approximately 1x10¹¹, approximately 1x10¹², approximately 1x10¹³ to approximately 1x10¹⁴ or more DNAase-resistant particles (DRPs) per ml. Dosages can also be expressed in viral genome units (vg).

[00102] Methods for transducing a target cell with rAAV, in vivo or in vitro, are contemplated by the invention. In vivo methods comprise the step of administering an effective dose, or multiple effective doses, of a composition comprising an rAAV of the invention to an animal (including a human) in need thereof. If the dose is administered before the development of a disorder / disease, the administration is prophylactic. If the dose is administered after the development of a disorder / disease, the administration is therapeutic. In embodiments of the invention, an effective dose is a dose that relieves (eliminates or reduces) at least one symptom associated with the disorder / disease state to be treated, that diminishes or prevents progression to a disorder / disease state, that slows or prevents progression from a disorder / disease state, that diminishes the extent of the disease, that results in remission (partial or total) of the disease, and / or that prolongs survival.An example of a disease contemplated for prevention or treatment with methods of the invention is DMD.

[00103] Combination therapies are also contemplated by the invention. Combination, as used in this document, includes simultaneous treatment and sequential treatments. Combinations of methods of the invention with standard medical treatments (e.g., corticosteroids) are specifically contemplated, as well as Petition 870210023177, dated 11 / 03 / 2021, page 35 / 74 31 / 62 combinations with innovative therapies.

[00104] Administration of an effective dose of the compositions may be by standard routes in the art including, but not limited to, intramuscular, parenteral, intravenous, oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular, rectal or vaginal. The route(s) of administration and the serotype(s) of AAV components of the rAAV (in particular, the AAV ITRs and the capsid protein) of the invention may be chosen and / or matched by those skilled in the art taking into account the state of the infection and / or disease to be treated and the target cells / tissue(s) that should express the microdystrophin protein.

[00105] The invention provides local and systemic administration of an effective dose of rAAV and compositions of the invention. For example, systemic administration is administration into the circulatory system so that the whole body is affected. Systemic administration includes enteric administration, such as absorption through the gastrointestinal tract, and parenteral administration via injection, infusion, or implantation.

[00106] In particular, the actual administration of rAAV of the present invention can be carried out using any physical method that will transport the recombinant rAAV vector into the target tissue of an animal. Administration according to the invention includes, but is not limited to, injection into the muscle and injection into the bloodstream. It has been demonstrated that simple resuspension of an rAAV in phosphate-buffered saline solution is sufficient to provide a useful vehicle for muscle tissue expression, and there are no known restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid proteins of an rAAV can be modified so that the rAAV is directed... [Petition 870210023177, dated 11 / 03 / 2021, page 36 / 74] 32 / 62 targeted to a particular tissue of interest, such as muscle. See, for example, document no. WO 02 / 053703, the description of which is incorporated herein by reference. The pharmaceutical compositions can be prepared as injectable formulations or as topical formulations for delivery to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the invention. The rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[00107] The dose of rAAV to be administered in the methods disclosed in this document will vary depending, for example, on the specific rAAV, mode of administration, treatment objective, individual and type (or types) of target cell, and can be determined by standard methods of the technique. The titers of each rAAV can vary from about 1x10⁶, about 1x10⁷, about 1x10⁸, about 1x10⁹, about 1x10¹⁰, about 1x10¹¹, about 1x10¹², about 1x10¹³ to about 1x10¹⁴ or more DNase-resistant particles (DRP) per ml. Doses can also be expressed in viral genome units (vg) (i.e., 1x10⁷ vg, 1x10⁸ vg, 1x10⁹ vg, 1x10¹⁰ vg, 1x10¹¹ vg, 1x10¹² vg, 1x10¹³ vg, 1x10¹⁴ vg, 1x10¹⁵ respectively). Doses can also be expressed in viral genome units (vg) per kilogram (kg) of body weight (i.e., 1x10¹⁰ vg / kg, 1x10¹¹ vg / kg, 1x10¹² vg / kg, 1x10¹³ vg / kg, 1x10¹³ vg / kg, 1x10¹⁴ vg / kg, 1x10¹⁵ vg / kg, respectively).Methods for AAV titration are described in Clark et al., Hum. Gene Ther., 10: 1031 to 1039 (1999).

[00108] In particular, the actual administration of rAAV of the present invention can be carried out using any physical method that will transport the recombinant rAAV vector into the target tissue of an animal. Administration according to the invention includes, but is not limited to Petition 870210023177, dated 11 / 03 / 2021, p. 37 / 74 33 / 62 tion, injection into the muscle and injection into the bloodstream. It has been demonstrated that simple resuspension of an rAAV in phosphate-buffered saline solution is sufficient to provide a useful vehicle for muscle tissue expression, and there are no known restrictions on the carriers or other components that may be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid proteins of an rAAV can be modified so that the rAAV is targeted to a particular target tissue of interest, such as muscle. See, for example, document no. WO 02 / 053703, the description of which is incorporated herein by reference. Pharmaceutical compositions may be prepared as injectable formulations or as topical formulations for delivery to muscles by transdermal transport.Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in the practice of the invention. rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[00109] For intramuscular injection purposes, solutions in an adjuvant, such as sesame or peanut oil, or in aqueous propylene glycol, as well as sterile aqueous solutions, may be used. Such aqueous solutions may be buffered, if desired, and the liquid diluent made isotonic first with saline or glucose solution. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. A dispersion of rAAV may also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils. Under normal storage and use conditions, these preparations contain a preservative to prevent the growth of Petition 870210023177, dated 11 / 03 / 2021, page 38 / 74 34 / 62 microorganisms. In this sense, the sterile aqueous media employed are all readily obtained by conventional techniques well known to those skilled in the art.

[00110] Suitable pharmaceutical carriers, diluents or excipients for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the dosage form must be sterile and flowable until easily syringeable. It must be stable under manufacturing and storage conditions and protected against contaminating actions by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. Appropriate flowability may be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants.The prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and similar substances. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be obtained by using agents that delay absorption, for example, aluminum monostearate and gelatin.

[00111] Sterile injectable solutions are prepared by incorporating rAAV in the required quantity into the appropriate solvent with various other ingredients listed above, as needed, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing the basic dispersion medium and the other ingredients. Petition 870210023177, dated 11 / 03 / 2021, page 39 / 74 35 / 62 required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation methods are vacuum drying and the lyophilization technique, which yields a powder of the active ingredient plus any desired additional ingredient from the solution previously sterilized by filtration.

[00112] Transduction with rAAV can also be performed in vitro. In one modality, the desired target muscle cells are removed from the subject, subjected to rAAV transduction, and reintroduced into the subject. Alternatively, syngenetic or xenogeneic muscle cells can be used when these cells do not generate an inappropriate immune response in the subject.

[00113] Suitable methods for transducing and reintroducing transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with muscle cells, for example, in an appropriate medium, and screening for cells harboring the DNA of interest using conventional techniques such as Southern blots and / or PCR, or using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, and the composition introduced into the subject by various techniques such as intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into smooth and cardiac muscle using, for example, a catheter.

[00114] Transduction of cells with rAAV of the invention results in the continuous expression of the microdystrophin protein. The present invention thus provides methods of administering / delivering rAAV expressing the microdystrophin protein to an animal, preferably a human. These methods include transducing tissues (including, but not limited to, tissues such as muscle, organs such as liver and brain, and glands such as salivary glands) with one or Petition 870210023177, dated 11 / 03 / 2021, page 40 / 74 36 / 62 plus rAAV of the present invention. Transduction can be performed with genetic cassettes comprising tissue-specific control elements. For example, one embodiment of the invention provides methods of transducing muscle cells and muscle tissues directed by muscle-specific control elements, including, but not limited to, those derived from the actin and myosin gene families, such as the myoD gene family [see Weintraub et al., Science, 251: 761 to 766 (1991)], the myocyte-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11: 4854 to 4862 (1991)), control elements derived from the human skeletal actin gene (Muscat et al., Mol Cell Biol, 7: 4089 to 4099 (1987)), the cardiac actin gene, muscle creatine kinase sequence elements (See Johnson et al., Mol Cell Biol, 9: 3393 to 3.399 (1989)) and the murine creatine kinase (mCK) enhancing element, control elements derived from the fast-twitch skeletal troponin C gene, the slow-twitch cardiac troponin C gene, and the slow-twitch troponin I gene: hypoxia-inducible nuclear factors (Semenza et al., Proc Natl Acad Sci USA, 88: 5680-5684 (1991)), promoters and steroid-inducible elements, including the glucocorticoid response element (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA 90: 5603-5607 (1993)), and other control elements.

[00115] Muscle tissue is an attractive target for in vivo DNA delivery, as it is not a vital organ and is easily accessible. The invention contemplates the continuous expression of microdystrophin from transduction-treated muscle fibers.

[00116] A muscle cell or muscle tissue is understood to be a cell or group of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, for example, of the digestive tract, urinary bladder, blood vessels or cardiac tissue). Such Petition 870210023177, dated 11 / 03 / 2021, page 41 / 74 37 / 62 muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[00117] The term transduction is used to refer to the administration / delivery of microdystrophin to a recipient cell, in vivo or in vitro, by means of a replication-deficient rAAV of the invention resulting in the expression of microdystrophin by the recipient cell.

[00118] Thus, the invention provides methods for administering an effective dose (or doses, administered essentially simultaneously or doses administered at intervals) of microdystrophin-encoding rAAV to a patient in need thereof. EXAMPLES EXAMPLE 1 GENERATION OF THE PAAV.MHCK7.MICRODYSTROPHIN CONSTRUCT

[00119] The pAAV.MHCK7.microdystrophin plasmid contains a human microdystrophin cDNA expression cassette, flanked by AAV2 inverted-terminal repeat (ITR) sequences (see Figure 1). The microdystrophin construct was characterized by a deletion in structure (R4-R23), while hinges 1, 2, and 4 and the cysteine-rich domain continue to produce a 138 kDa protein. The expression of the microdystrophin protein (3,579 bp) was guided by an MHCK7 promoter (795 bp). The plasmid was constructed from the pAAV.MCK.microdystrophin plasmid by removing the MCK promoter and inserting the MHCK7 promoter. Following the main promoter, the endogenous mouse MCK exon 1 of 53 bp (untranslated) is present for efficient transcription initiation, followed by late 16S / 19S SVS splice signals (97 bp) and a small 5'UTR (61 bp). The intron and 5'UTR are derived from the pCMVβ plasmid (Clontech).The microdystrophin cassette had a Kozak consensus signal immediately ahead of the ATG start and a small 53 bp synthetic polyA signal for mRNA termination. The microdystrophin cassette. Petition 870210023177, dated 11 / 03 / 2021, page 42 / 74 Human 38 / 62 contained the domains (R4-R23 / A71-78), 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) (Moi Ther 18, 109-117 (2010)). The only viral sequences included in this vector were the inverted terminal repeats of AAV2, necessary for viral DNA replication and packaging. The microdystrophin cassette has a small 53 bp synthetic polyA signal for mRNA termination.

[00120] Previous studies validated cardiac expression using the MHCK7 promoter (Salva et al. Moi Ther 15, 320-329 (2007)) and AAVrh74 achieving expression in skeletal muscle, diaphragm, and cardiac tissue (Sondergaard et al. Annals of Clinical and Transi Neurology 2, 256-270 (2015)). The construct sequence in Figure 1 was encapsulated in AAVrh.74 virions. The molecular clone of the AAVrh.74 serotype was cloned from a rhesus monkey lymph node and is described in Rodino-Klapac et al. Journal of Translational Medicine 5, 45 (2007). Table 1 shows the molecular characteristics of the pAAV.MHCK7.microdystrophin plasmid (SEQ ID NO: 3). TABLE 1. MOLECULAR CHARACTERISTICS OF PAAV.MHCK7.MICRODYSTROPHIN PLASMID TYPE START END NAME DESCRIPTION REGION 7 116 5' ITR Jungle-type AAV2 inverted-terminal repeat REGION 236 1036 MHCK7 Mouse myosin heavy chain complex muscle box creatine kinase fusion enhancer / promoter REGION 1046 1195 Chimeric intron 5' donor site of human β-globin gene and branch site and 3' splice receptor site of IgG heavy chain variable region GENE 1206 4786 huDys cDNA Human microdystrophin cDNA REGION 4787 4842 PolyA Synthetic PolyA REGION 4.933 5.042 3' ITR Wild-type AAV2 inverted-terminal repeat GENE 6.808 7.668 AmpR β-lactamase gene REGION 7.823 8.442 O ri Replication plasmid origin EXAMPLE 2 Intramuscular expression studies using RAAV.MHCK7.MICRODYSTROPHIN

[00121] The expression studies were conducted with the construct Petition 870210023177, dated 11 / 03 / 2021, page 43 / 74 39 / 62 to human microdystrophin (rAAVrh74.MHCK7.microdystrophin; described in Example 1) by intramuscular injection. 1 x 1011vg of the cassette (n=5 per group) was injected into the tibialis anterior muscle of mdx mice (spontaneous Dmdmdx mutant mice that do not express dystrophin). Six weeks later, the muscles were harvested and stained for dystrophin (Dys3) expression with an N-terminal antibody for dystrophin staining and hematoxylin and eosin (HE). Figure 2 shows the diffuse gene expression and reduction in centrally located nuclei with a dose of 1 x 1011vg compared to untreated muscle. In addition, a decrease in central nucleation was observed, with an increase, on average, in fibers / structure after treatment with the microdystrophin construct. Expression levels of the rAAVrh74.MHCK7.microdystrophin construct were quantified at approximately 73%.

[00122] In addition to measuring microdystrophin localization and expression levels, skeletal muscle strength was measured after intramuscular injection of the cassette. Intramuscular expression of the pAAV.MHCK7.microdystrophin construct resulted in significantly greater absolute and specific force output compared to untreated controls (Figures 3A and 3B, respectively). EXAMPLE 3 Systemic delivery of RAAVRH.74.MHCK7.MICRODYSTROPHIN in MDX mice

[00123] 2 x 1012vg (8 x 1013vg / kg) or a high dose (planned clinical dose) of 6 x 1012vg (2 x 1014vg / kg) of rAAVrh.74.MHCK7.microdystrophin was injected via the tail vein into cohorts of 6-week-old mdx mice. After 12 weeks of treatment, all muscles were harvested and stained for dystrophin and restoration of DAPC components. Mice injected systemically (tail vein) showed high levels of staining. Petition 870210023177, dated 11 / 03 / 2021, page 44 / 74 40 / 62 dystrophin in all muscles. Figure 4A represents the generalized transduction of skeletal, diaphragmatic, and cardiac muscle fibers after a systemic dose of 6 x 10¹²vg (2 x 10¹⁴vg / kg). Figure 4B shows the quantification of the percentage of muscle fibers expressing microdystrophin in each tissue. Finally, the diaphragm was tested for functional improvement (Figure 4C). No significant difference was observed at low doses; however, there was significant improvement at the high dose. It is important to note that Figure 5 demonstrates that other components of the DAPC were completely restored after microdystrophin delivery. Beta-sarcoglycan (B-SG) is shown.

[00124] The toxicology / safety of AAVrh.74.MHCK7.microdystrophin is evaluated by administering the vector via 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 (TRI), and Diaphragm (DIA) (Figures 6A and 6B). The number of centrally placed nuclei was decreased with the elevated dose 6 x 1012vg (2 x 1014vg / kg). Historically, central nucleation of skeletal muscles in untreated age-matched mdx mice averages ~80%. Finally, preliminary data from a small sample size (n=3) demonstrate a reduced level of CK release (U / L) in the serum of mice treated with high doses (D). Independent t-tests were used to locate differences (p<0.05); data are reported as mean ± SEM. Petition 870210023177, dated 11 / 03 / 2021, page 45 / 74 41 / 62 TABLE 2. SUMMARY OF TOXICOLOGICAL / SAFETY STUDY OF RAAVRH.74.MHCK7.MICRODYSTROPHIN IN MICE. Cohort Number Study Agent Dose (vg / kg) Treatment / Day 0 Follow-up Day 1 Sacrificial endpoint Week 6 Extra (1) Low dose AAVrh.74.MHCK7.Micro-dys 8.0 x1013 Single iv injection in Weight at 24 h, 5M +2 (2) High dose AAVrh.74.MHCK7.Micro-dys 2.0 x1014 tail vein Observations 5M +2 (3) Control Vehicle (LRS) 0 mdx mice Clinical 5M +2 TOTAL MICE N=21 EXAMPLE 4 GENERATION OF THE PAAV.MCK.MICRODYSTROPHIN CONSTRUCT

[00125] The pAAV.MCK.microdystrophin plasmid was constructed by inserting the MCK expression cassette carrying a codon-optimized human microdystrophin cDNA sequence into the psub201 AAV cloning vector (Samulski et al., J. Virol. 61 (10): 30963101). A muscle-specific regulatory element was included in the construct to drive muscle-specific gene expression. This regulatory element comprised the mouse MCK core enhancer (206 bp) fused with the 351 bp (proximal) MCK core promoter. Following the main promoter, the construct comprises the 53 bp (untranslated) endogenous mouse MCK exon 1 for efficient transcription initiation, followed by the 97 bp SVS 16S / 19S late splice signals and a small 5' UTR (61 bp). The intron and 5' UTR were derived from the pCMVβ plasmid (Clontech).The microdystrophin cassette has a Kozak consensus immediately ahead of the ATG start and a small 53 bp synthetic polyA signal for mRNA termination. The human microdystrophin cassette contains the domains (R4 to R23 / Δ71 to 78), as previously described by Harper et al. Nat. Med. 8(3):253 to 261, 2002.

[00126] The pAAV.MCK.microdistrofina plasmid contained the human microdystrophin cDNA expression cassette flanked by AAV2 inverted-terminal repeat (ITR) sequences (see Figure 7). This sequence was encapsulated in AAVrh.74 virions. The Petition 870210023177, dated 11 / 03 / 2021, page 46 / 74 The 42 / 62 molecular clone of the AAVrh.74 serotype was cloned from a rhesus monkey lymph node and is described in Rodino-Klapac et al. Journal of Tran. Med. 45:(2007). EXAMPLE 5 Potency and Dose Analysis Using Raav.MCK.Microdystrophina

[00127] Expression studies were conducted with the human microdystrophin construct (rAAV.MCK.microdystrophin; described in Example 1) by intramuscular injection. 3 x 10⁹, 3 x 10¹⁰, or 1 x 10¹¹vg (n=3 per group) were injected into the tibialis anterior (TA) muscle of mdx mice (spontaneous mutant Dmdmdx mice that do not express dystrophin). Four weeks later, the muscles were harvested and stained for dystrophin expression using an antibody specific for N-terminal Dys3 and hematoxylin and eosin (HE) staining. Figure 8 shows a linear correlation between expression and dose, where there is very little expression (no effect level) at 3 x 10⁹vg and 89% expression at 1 x 10¹¹vg. EXAMPLE 6 Vascular delivery of RAAV.MCK.MICRODYSTROPHIN to MDX mice

[00128] Using an isolated limb perfusion model (Rodino-Klapac et al., J. Trans. Med. 5 (45): 1 to 11, 2007), mdx mice (n=10) received an injection of 1 x 1011vg of rAAVrh.74.MCK.microdystrophin via the femoral artery, and the results were analyzed. Three months after gene transfer, lower limb muscles were harvested, and efficacy studies demonstrated significant improvement in both strength and resistance to injury induced by eccentric contraction (Figure 9).

[00129] Immunostaining of the dystrophin protein in the extensor digitorum longus (EDL) muscle and TA muscle shows expression in mi Petition 870210023177, dated 11 / 03 / 2021, page 47 / 74 43 / 62 mdx fibers after treatment with rAAVrh.74-MCK-microdystrophin (Figure 9A). Cell-transfected infected muscle was stained identically, and exposures were compared over time. Figure 9B demonstrates that rAAVrh.74-MCK-microdystrophin significantly increased normalized specific strength compared to cell-transfected mdx muscles (P<0.05 vs. mdx). Furthermore, mdx muscles infected with rAAVrh.74-MCK-microdystrophin (human) were compared with cell-transfected contralateral mdx EDL muscles (blue) and wild-type EDL muscles (WT C57Bl / 10) for strength drop during repetitive eccentric contractions 12 weeks after gene transfer (Figure 9C). Treatment with rAAVrh.74-MCK-microdystrophin (Micro-dys) was found to significantly protect against strength loss compared to mdx muscles treated with cell transfection (P <0.001 vs. mdx). EXAMPLE 7 primate studies

[00130] In order to apply preclinical findings in mice to a clinical paradigm, a non-human primate (NHP) was systemically dosed to assess the safety and efficacy of future clinical trials. The effect of a total dose of 2x1014vg of AAVrh74.MHCK7.microdystrophin.FLAG administered intravenously via the cephalic vein in a non-human primate was studied. This dose was proportional (based on animal weight) to the systemic dose administered to mice and corresponded to the intermediate dose (Total Dose of 6.0 x 1012vg) administered to mice.

[00131] Baseline chemical analyses and immunological studies, including enzyme-linked immunosorbent assay (ELISpot), were performed to measure T cells against the AAVrh.74 capsid and microdystrophin, as well as anti-AAV antibody titers. They were Petition 870210023177, dated 11 / 03 / 2021, page 48 / 74 Three peptide sets for the AAVrh.74 capsid protein (Genemed Synthesis, San Antonio, TX) were used, each containing 34 to 36 peptides, 18 amino acids in length and overlapping by 11 residues. Four peptide sets comprising the microdystrophin.FLAG protein (Genemed Synthesis) were also used, with 18 amino acids in length and overlapping by 11 residues. Concanavalin A (ConA) (Sigma, 1 pg / ml) served as a positive control and 0.25% dimethyl sulfoxide (DMSO) as a negative control. These sets were repeated every two weeks throughout the study. Three months after treatment, animals were sacrificed for full tissue necropsy. Immunological assays showed no unexpected responses to the capsid or transgene by ELISpot (Figure 12A) and no unexpected antibody response to the AAVrh74 capsid by ELISA (Figure 12B).

[00132] In addition, complete blood count and chemistry panels showed slight elevation of liver enzymes that were normalized back to baseline without intervention or treatment required, as shown in Table 3 below. TABLE 3 BLOOD CHEMISTRY 13 to 176 24-hour Reference Baseline 2 weeks 4 weeks 6 weeks 8 weeks 12 weeks Total Protein (6.4 to 7 mg / dl) 7 7 6.7 6.6 6.6 6.6 6.7 Total Bilirubin (0.15 to 0.23 mg / dl) 0.2 0.4 0.3 0.2 0.3 0.4 0.4 ALT (31 to 50 U / l) 39 38 75 104 182 172 65 AST (19 to 38 U / l) 35 63 50 92 98 121 64 Alkaline Phosphatase (504 to 821 U / l) 417 396 332 383 598 608 578 GGT 77 77 120 106 131 156 134 CK 109 504 164 183 137 126 123

[00133] There were no other unexpected chemical values ​​throughout the duration of the study. Finally, a complete analysis of all skeletal muscles demonstrated broad expression in muscle fibers through immunofluorescence staining with a specific antibody for FLAG and western blot detection using a mouse monoclonal antibody for dystrophin (Figures 14A, B). Petition 870210023177, dated 11 / 03 / 2021, page 49 / 74 45 / 62

[00134] The data together demonstrate that systemic administration of AAVrh74.MHCK7.microdystrophin.FLAG established safety and efficacy with widespread expression in all skeletal muscles in a non-human primate. EXAMPLE 8 Pre-clinical study to demonstrate efficacy.

[00135] A preclinical study was conducted to demonstrate the efficacy of systemic administration of rAAVrh74.MHCK7.microdystrophin in the treatment of skeletal and cardiac muscle deficits in mdx mice. The AAVrh74 vector containing a human dystrophin microtransgene optimized for a muscle- and cardiac-specific promoter-driven codon, MHCK7, as described in Example 1, was used for this study.

[00136] Systemic injections of rAAVrh74.MHCK7.microdystrophin via the tail vein in mdx mice (dystrophin null) were used for a dose-response study. The results of this study demonstrated that systemic injections in mdx mice were effective in normalizing the histological and functional findings measured in the limb and diaphragm in a dose-dependent manner. Furthermore, no significant vector-associated toxicity was reported after formal histopathological review by a certified veterinary pathologist.

[00137] The vector for this study was produced by the Viral Vector Nucleus of the National Children's Hospital using a triple transfection method of HEK293 cells under research-grade conditions. Vector characterization after production included titer determination by qPCR with a supercoiled pattern, endotoxin level determination (EU / ml), and a sterility assessment. The produced vector was analyzed by SDS-PAGE to verify the consistency of the banding pattern with the expected rAAV. The vector was produced Petition 870210023177, dated 11 / 03 / 2021, pp. 50 / 74 46 / 62 using the plasmid containing the microdystrophin construct, a muscle-specific MHCK7 promoter to drive expression, a Kozak consensus sequence (CCACC), an SV40 chemical intron, the synthetic polyadenylation site (of 53 bp) (Figure 1 Error! Reference source not found.). The microdystrophin expression cassette was cloned between AAV2 ITRs packaged in an AAVrh74 vector for improved transduction into skeletal and cardiac tissue.

[00138] The potency of the test article rAAVrh74.MHCK7.microdistrofina was determined by performing intramuscular injections of the vector into mdx mice. Wild-type mice serve as a positive control and injection of sterile lactated mice into mdx mice serves as a negative control. Petition 870210023177, dated 11 / 03 / 2021, pp. 51 / 74 TABLE 4: OVERVIEW OF THE STUDY DESIGN OF RAAVRH74.MHCK7.MICRODYSTROPHIN Route of Delivery Strain Total Dose Number of Mice Evaluation Criteria Analysis IV (Efficacy) mdx 2E+12 5 3 months IF, H&E, Diaph Phys IV (Efficacy) mdx 6E+12 8 3 months IF, H&E, Diaph Phys, TA Phys, Pathway, Western Blot Biodistribution, IV (Efficacy) mdx 1,2E+13 8 3 months IF, H&E, Diaph Phys, TA Phys, Pathway, Western Blot Biodistribution, IV (Efficacy) C57BL / 6 6E+12 5 3 months IF, H&E, Diaph Phys, TA Phys, Pathway IV (Efficacy) mdx 6E+12 5 6 months IF, H&E, Diaph Phys, TA Phys, Pathway IV (Efficacy) mdx — 8 3 months IF, H&E, Diaph Phys, Pathway, IV (Efficacy) C57BL / 6 — 6 3 months IF, H&E, Diaph Phys, Pathway, IF: immunofluorescence; H&E: hematoxylin and eosin staining; Diaph / TA Phys: specific force measurements in the diaphragm and TA muscle; Pathway: formal histopathology; not injected All injected animals were treated at 4 to 5 weeks of age and necropsied 3 or 6 months after injection. Control mice were necropsied. 47 / 62 at 4 months of age and at 7 months of age. Petition 870210023177, dated 11 / 03 / 2021, pp. 52 / 74 48 / 62

[00139] The animals listed in Table 4 were dosed at the indicated age (4 to 5 weeks old) with an injection into the tail vein for systemic administration. To perform accurate dosing with an intramuscular injection, the animals were briefly anesthetized by inhalation of isoflurane. Doses were administered by direct injection into the anterior tibial muscle of the hind limb. Anesthesia was not required for accurate dosing with systemic administration. Doses were administered via the vasculature through the tail vein. Care was taken to accurately deposit the entire dose of vector into the vessel. After dosing, the animals were placed on a heating pad until spontaneous movement was recovered and then returned to the cage. Observations of each animal were conducted weekly throughout the duration of the study.

[00140] At the appropriate age, as listed in Table 4, mice were overdosed with a ketamine / xylazine mixture (200 mg / kg / 20 mg / kg). Blood was collected by cardiac puncture, and whole blood was sent for complete blood count (CBC) analysis. Serum was stored at -80 °C until serum chemistry was analyzed by Charles Rivers Laboratory. Tissues were then collected and sent for analysis by an independent, in-house veterinary histopathologist.

[00141] Intramuscular delivery of rAAVrh74.MHCK7.microdystrophin to dystrophin-null mice at a total dose of 1 x 1011vg resulted in ~70% dystrophin expression in the injected TA muscles. Immunofluorescence imaging of the vector-dosed mouse confirmed microdystrophin gene expression. Restoration of dystrophin expression after systemic treatment with RAAVRH74.MHCK7.MICRODYSTROPHIN Petition 870210023177, dated 11 / 03 / 2021, pp. 53 / 74 49 / 62

[00142] The efficacy of the test article rAAVrh74.MHCK7.microdystrophin was determined by performing systemic injections in mdx mice (genotype: C57BL / 10ScSn-Dmdmdx / J) using a dose scale of low, medium, and high doses (2.0 x 1012vg Total Dose; 6.0 x 1012vg Total Dose; 1.2 x 1013vg Total Dose) to evaluate transgenic expression and vector efficacy when administered systemically at 3 and 6 months post-injection. Mice received injections at 4–5 weeks of age, and a full necropsy was performed 3 and 6 months post-injection. Based on the average weights of the animals per group, these doses are equal: 8 x 10¹³vg / kg, 2 x 10¹⁴vg / kg, and 6 x 10¹⁴vg / kg. Injection of an equal volume of Ringer's lactate served as a negative control. Injection of an equal volume of Ringer's lactate into C57BL / 6 mice served as a positive control.Safety was determined by administering systemic injections to WT mice at a dose of 6.0 x 1012vg total doses (indicated as the WT TX mid-dose group). Immunofluorescence staining of the anterior tibial (TA), gastrocnemius (GAS), quadriceps (QUAD), gluteus (GLUT), psoas, triceps (TRI), diaphragm (DIA), and heart skeletal muscles was performed to determine dystrophin restoration and ensure the efficacy of the rAAVrh74.MHCK7.microdystrophin viral vector.

[00143] Skeletal muscles (TA, QUAD, GLUT, TRI) were extracted, along with the heart and diaphragm, for analysis. Organs were also removed for toxicology and biodistribution studies. Microdystrophin transgene expression remained high after 3–6 months of treatment. This was accompanied by improved muscle histopathology and function, with no adverse effects on non-target organs. Reversal of the dystrophic phenotype in MDX mice treated systemically with RAAVRH74.MHCK7.MI. Publication 870210023177, dated 11 / 03 / 2021, page 54 / 74 50 / 62 CRODYSTROPHEN

[00144] Hematoxylin and eosin (H&E) staining of skeletal muscles, diaphragm, and heart was performed to determine the reversal and improvement of dystrophic pathology after systemic injection of rAAVrh74.MHCK7.microdystrophin at a total dose of 2 x 10¹²vg (low dose; n=1), a total dose of 6 x 10¹²vg (medium dose; n=8), and a total dose of 1.2 x 10¹³vg (high dose; n=8) for each dose, with euthanasia 12 weeks after injection. Twenty-four weeks after injection, a second cohort of animals treated with the medium dose (total dose of 6 x 10¹²vg) was evaluated for reversal and improvement of dystrophin pathology (n=5).

[00145] Immunofluorescence staining for human microdystrophin protein was used to determine microdystrophin transgene expression on the left and right sides of six skeletal muscles (TA, GAS, QUAD, GLUT, psoas, TRI), as well as in the diaphragm and heart in all dystrophin-null mice that received microdystrophin vector injection. This was performed to determine dystrophin restoration and to ensure the efficacy of the rAAVrh74.MHCK7.microdystrophin viral vector at a total dose of 2 x 1012vg (low dose; n=2), a total dose of 6 x 1012vg (medium dose; n=8), and a total dose of 1.2 x 1013vg (high dose; n=8) for each dose with euthanasia 12 weeks after injection.

[00146] To evaluate expression and transduction efficiency, images from the three dosing cohorts and the left and right sides of each muscle were used for quantification. Four 20X images of each muscle were taken, and the percentage of microdystrophin-positive fibers was determined for each image, resulting in the average transduction percentage for each muscle. Figures 14 and 15 show representative images of mice treated with the average dose (6 x 1012vg; 2 x 1014vg / kg) and the high dose. Petition 870210023177, dated 11 / 03 / 2021, pp. 55 / 74 51 / 62 (1.2 x 1013vg; 6 x 1014vg / kg). Dystrophin-null mice that received Ringer's lactate injection and were age-matched were included as negative controls, and wild-type mice that received Ringer's lactate injection were included as positive controls. The heart showed >75% in all animals analyzed.

[00147] Muscles from untreated animals exhibited generalized myopathy, including fatty infiltration, central nucleation, fibrosis, and focal areas of necrosis. H&E staining in Figure 16 and Figure 17 illustrates this dystrophic phenotype in dystrophin-null mice when compared to normal WT mice and the improvement in muscle pathology after treatment at the medium dose (6 x 1012vg; 2 x 1014vg / kg) or the high dose (1.2 x 1013vg; 6 x 1014vg / kg). Quantification of histological parameters showed a reduction in central nucleation (Figure 18) and a normalization of mean fiber diameters (Figures 16 and 17) in treated mice in all muscles in a dose-dependent manner. Sirius Red staining demonstrated a reduction in collagen deposition in the diaphragm in the medium and high dose cohorts compared to the untreated cohorts (mdx LR) (Figure 19). Functional evaluation of systemic treatment with RAAVRH74.MHCK7.MICRODYSTROPHIN

[00148] To determine whether microdystrophin gene transfer provided a functional strength benefit to the diseased muscle, the functional properties of the diaphragm and tibialis anterior of mdx, WT, and vector-dosed mice were evaluated at three dose levels. The dose scale included low dose (8 x 1013vg / kg), medium dose (2 x 1014vg / kg), and high dose (6 x 1014vg / kg). Functional evaluation of systemic treatment with rAAVrh74.MHCK7.microdystrophin using ex vivo assessment of specific strength and di Petition 870210023177, dated 11 / 03 / 2021, pages 56 / 74 52 / 62 minution of force production after eccentric contractions in the TA was used 24 weeks after injection in animals that received a systemic injection of rAAVrh74.MHCK7.microdystrophin at a total dose of 6 x 1012vg (average dose). In addition, specific force production in the diaphragm was evaluated in the same animals.

[00149] As described in the previous figures, histopathology exhibited a more normalized environment with improvements in central nucleation, collagen deposition, and fiber size at the medium and high doses. Administration of rAAVrh74.MHCK7.microdystrophin into the tail veins led to a gradual improvement in specific force production in the diaphragm (176.9 mN / mm2 in the medium dose group versus 227.78 mN / mm2 in the high dose group). Furthermore, the long-term treated cohort represents mice 6 months after injection (medium dose 2 x 1014vg / kg) and there was no deviation in long-term diaphragm force production (176.9 mN / mm2 vs. 194.9 mN / mm2) (Figure 20).

[00150] Furthermore, functional deficits in the tibialis anterior muscle were observed in mdx mice compared to WT mice. Mdx mice demonstrated a 50% reduction in force production compared to WT mice (171.3 mN / mm2 vs. 291.65 mN / mm2) and greater force loss after eccentric contractions (32% loss in mdx; 5% loss in TP). Systemic administration of the mid-dose level of rAAVrh74.MHCK7.microdystrophin resulted in 65.5% dystrophin in the tibialis anterior muscle and restoration of specific force production that improved to 235.4 mN / mm2 and protected the muscle against repeated eccentric contraction damage with only a 25% reduction in force (Figure 21). The medium-dose WT group represents a wild-type treated cohort in order to demonstrate the absence of toxicity and maintenance of functional outcome measurements after treatment. Petition 870210023177, dated 11 / 03 / 2021, page 57 / 74 53 / 62 to with vector. SUMMARY

[00151] Following the initial demonstration of biopotency by intramuscular injection, a comparable or increased restoration of microdystrophin with vascular delivery was achieved while transducing skeletal, diaphragm, and heart muscles. Efficacy demonstrated dose-dependent reversal of dystrophic features by reducing inflammation, fewer degenerated fibers, and improved functional recovery, protecting against eccentric contractions in the tibialis anterior and diaphragm. Functional benefits of the vector include a gradual improvement in wild-type levels in diaphragm and TA force generation. EXAMPLE 9 Toxicology and Biodistribution of Systemic Treatment ÇOMRAAVRH74.MHÇK7.M1ÇRODYSTROPHIN

[00152] Organs and tissues of mdx mice that received systemic injection of rAAVrh74.MHCK7.microdystrophin were collected for quantitative real-time PCR to detect specific vector DNA sequences. Proteins extracted from all collected organs and tissues were processed by Western blot to detect microdystrophin in non-target organs.

[00153] The test article was administered at three dose levels: low (2 x 1012vg; 8 x 1013vg / kg), medium (6 x 1012vg; 8 x 1014vg / kg), and high (1.2 x 1013vg; 6 x 1014vg / kg) intravenously at 4 to 5 weeks of age. To assess vector safety, H&E staining was performed on cryosections of muscle tissue and all major organs harvested from the same mouse cohorts described previously. Organs and muscles from C57BL6 WT mice systemically treated with the vector at the medium dose were also included. mdx and WT mice treated with Ringer's lactate were also included. Petition 870210023177, dated 11 / 03 / 2021, pp. 58 / 74 54 / 62 were also included for histopathological analysis. These sections were formally reviewed for toxicity by a board-certified third-party veterinary pathologist, and no adverse effects were detected in any sample from any of the mice; the results are summarized below.

[00154] Group details and study plan are shown in 4 below. TABLE 5: SAFETY STUDY PROJECT FOR RAAVRH74.MHCK7.MICRODYSTROPHIN Delivery Method Animal Strain Total Dose (vg) Number of Mice Treatment Evaluation Criteria Pathology Report Number IV mdx 2x10¹² 5 3 months AAVrh74-mdx-MOUSE-001.1 IV mdx 6x10¹² 7 3 months AAVrh74-mdx-MOUSE-001.1 / 001.2 IV mdx 1.2 x 10¹³ 8 3 months AAVrh74-mdx-MOUSE-001.2 IV C57BL / 6 6x10¹² 5 3 months AAVrh74-mdx-MOUSE-001.2 IV mdx 6x10¹² 5 6 months AAVrh74-mdx-MOUSE-001.3 IV mdx — 8 3 months AAVrh74-mdx-MOUSE-001.2 IV C57BL / 6 — 6 3 months AAVrh74-mdx-MOUSE-001.2 HISTOPATHOLOGICAL REVIEW OF TISSUE TRANSDUCED BY VECTOR

[00155] Intravenous injection of rAAVrh74.MHCK7.microdystrophin did not cause microscopic changes in the myofibers of the examined skeletal muscles. Furthermore, no treatment-related lesions were observed in any of the tissues evaluated histologically. Any changes observed were seen in both treated and control mice and were considered incidental findings. Taken together, these data indicate that this test article was well-tolerated by the test subjects. Additionally, compared to age-matched, untreated mdx mouse reference samples, administration of rAAVrh74.MHCK7.microdystrophin decreased myofiber atrophy in treated mdx mice, thus showing that the test article may improve the degree of myopathy associated with mdx deficiencies.

[00156] In addition to the review of diseased mdx mice treated systemically with the vector, rAAVrh74.MHCK7.microdystrophin was delivered systemically to five C57BL / 6 WT mice in a Petition 870210023177, dated 11 / 03 / 2021, pp. 59 / 74 55 / 62 dose identical to the minimally effective dose (MED) established in the above studies in mdx mice, total dose of 6 x 1012vg (2 x 1014vg / kg). This allowed the intravenous delivery study of the test article in healthy WT mice to determine more definitively whether any adverse effects resulted solely due to the treatment. Here again, a variety of skeletal muscles, including the diaphragm, along with the heart, and five other organs were harvested and H&A sections of each tissue were formally reviewed by an independent veterinary pathologist. VECTOR GENOME BIODISTRIBUTION

[00157] The presence of specific DNA sequences for the test article was examined using a quantitative real-time PCR (qPCR) assay. Biodistribution analysis was performed on tissue samples collected from three animals with mdx vector doses at dose levels. A positive signal was anything greater than or equal to 100 single-stranded DNA copies / pg of genomic DNA detected. Tissues were harvested at necropsy and vector-specific primer probe sets specific for MHCK7 promoter sequences were used. Figure 22 and Table 6 below show the vector genome copies detected in each tissue sample from mice that received rAAVrh74.MHCK7.microdystrofin injection. TABLE 6: NUMBER OF VECTOR GENOME COPIES IN ORGANS AND MUSCLES OF THREE MDX MICE DOSED WITH VECTOR BY DOSE LEVEL. VALUES ARE SHOWN IN VG / pG OF GENOMIC DNA. Tissue 2.00E+12 6.00E+12 1.20E+13 (average number of copies in vg / ug) (average number of copies in vg / ug) (average number of copies in vg / ug) Color 2.84E+04 7.65E+05 5.35E+06 Lung 3.14E+04 2.52E+05 1.49E+06 Liver 4.36E+04 1.11E+07 1.80E+07 Kidney 1.96E+04 3.27E+05 1.06E+06 Spleen 5.69E+04 5.27E+05 5.78E+05 Gonad 5.74E+04 3.68E+04 3.50E+05 Petition 870210023177, dated 11 / 03 / 2021, pp. 60 / 74 56 / 62 Tissue 2.00E+12 6.00E+12 1.20E+13 (average copies in vg / ug) (average copies in vg / ug) (average copies in vg / ug) Day 2.22E+04 3.55E+05 2.32E+06 Pso 1.28E+05 1.60E+05 1.57E+06 Tri 1.60E+05 5.45E+05 2.50E+06 Qd 2.66E+06 6.57E+05 2.29E+06 Gas 1.69E+05 5.80E+05 2.93E+06 TA 5.86E+05 1.25E+05 1.32E+06

[00158] Transcription of rAAVrh74.MHCK7.microdystrophin was detected at varying levels in all tissues collected. As expected, the highest levels were observed in skeletal muscle and heart. The lowest levels were detected in the gonad, lung, kidney, and spleen. These data indicate that the test article was efficiently delivered to all investigated tissues of mice dosed with vectors.

[00159] As the qPCR results above indicate, intravenous administration of rAAVrh74.MHCK7.microdystrophin resulted in vector transcript distribution to varying levels in most tissues, with the highest levels occurring in the liver, heart, and quadriceps muscle (medium dose) and in the liver, heart, and gastrocnemius muscle (high dose). Therefore, the aim of this part of the study was to determine the protein expression of the human microdystrophin transgene in these tissues to ensure the functionality of the muscle-specific MHCK7 promoter. Western blotting was used to detect microdystrophin expression in tissue samples.

[00160] Protein expression and vector biodistribution were also evaluated using qPCR and western blotting (Figure 23), and these data indicate normal vector levels in external organs and minimal detection of microdystrophin protein in livers treated with high doses. These results were not correlated with any toxicity, as determined by the liver pathologist. Furthermore, serum chemistries were analyzed by an independent CRO (Charles River Laboratories), which indicated normal values ​​in all chemistries analyzed. There were three abnormal values ​​in the enzyme. Petition 870210023177, dated 11 / 03 / 2021, pages 61 / 74 57 / 62 hepatic AST, 2 of which were demonstrated in the mdx-LR group and 1 in the medium dose group (Figure 23). A subset of animals underwent creatine kinase (CK) analysis; however, samples were analyzed before and after physiological assessment. Serum analysis corroborates the lack of toxicity after delivery of the test article.

[00161] Microdystrophin protein expression was observed in varying amounts in all skeletal muscle samples, as well as in heart samples (Figure 24). However, a minimal amount of protein was detected in the high-dose liver cohorts. This is believed to be a benign finding and may be due to expression in liver smooth muscle. Importantly, no adverse histopathological effects were noted by the independent pathologist's report in the liver. SUMMARY

[00162] Histopathological review concluded that the mdx-LR cohort exhibited generalized myopathy affecting all seven skeletal muscles evaluated, as well as the right ventricular wall of the heart. Key findings of the histopathological review included marked and generalized myofiber atrophy (30–75% of normal myofiber size), minimal to mild mononuclear cell inflammation, increased interstitial space, and increased cytoplasmic mineral deposits. The diaphragm exhibited the most marked changes in mononuclear cell infiltration and myofiber atrophy. The heart exhibited some small foci of minimal mononuclear cell accumulation in the ventricular myocardium. Vector-dosed cohorts substantially reduced myopathy in all skeletal tissues and the heart. Reductions in histopathological findings were dose-dependent, with the high-dose group exhibiting substantially less degeneration and inflammation.There were no adverse effects due to vector treatment with rAAVrh74.MHCK7.micro. Petition 870210023177, dated 11 / 03 / 2021, pages 62 / 74 58 / 62 dystrophin, as documented in the cohort treated with WT and with the vector dosed with mdx. There were incidental findings in the liver and lung of mdx and WT mice, regardless of treatment, in which the mice exhibited mild vacuolization of hepatocyte cytoplasm. Therefore, the test article was safe, effective, and the protective effect was dose-dependent. REFERENCES 1. Hoffman, EP, Brown, RH, Jr. & Kunkel, LM Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 51, 919 to 928 (1987). 2. Straub, V. & Campbell, KP Muscular dystrophies and the dystrophin-glycoprotein complex. Curr Opin Neurol 10, 168 to 175 (1997). 3. Sacco, A., et al. Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx / mTR mice. Cell 143, 1059 a 1071 (2010). 4. Wallace, G.Q. & McNally, E.M. Mechanisms of muscle degeneration, regeneration, and repair in the muscular dystrophies. 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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 a 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 a 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 a 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 a 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, 1.105 a 1.110 (1992). 15. Stedman, H.H., et al. The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy. Nature 352, 536 a 539 (1991). 16. Deconinck, A.E., et al. Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell 90, 717 a 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 a 738 (1997). Petição 870210023177, de 11 / 03 / 2021, pág. 64 / 74 60 / 62 18. Love, D.R., et al. An autosomal transcript in skeletal muscle with homology to dystrophin. Nature 339, 55 a 58 (1989). 19. Tinsley, J.M., et al. Primary structure of dystrophinrelated protein. Nature 360, 591 a 593 (1992). 20. Tinsley, J., et al. Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat Med 4, 1.441 a 1.444 (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, 3.333 a 3.344 (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 a 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 a 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, 4.929 a 4.937 (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. 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Claims

CLAIMS 1. Recombinant AAVrh74 vector, characterized in that it comprises in the 5' to 3' direction an inverted terminal repeat (ITR), a muscle-specific control element MHCK7, a chimeric intron sequence, the nucleotide sequence of SEQ ID NO: 1, a poly A tail and an ITR.

2. Recombinant AAVrh74 vector, according to claim 1, characterized in that it further comprises an SV40 intron sequence.

3. Recombinant AAVrh74 vector, according to claim 1, characterized in that it further comprises a nucleotide sequence defined as nucleotides 7-5042 of SEQ ID NO:

3.

4. Recombinant AAVrh74 vector, according to claim 1, characterized in that it further comprises a chimeric intron sequence between said nucleotide sequence and said muscle-specific control element MHCK7, wherein the chimeric intron sequence is presented as nucleotides 1046-1195 of SEQ ID NO:

3.

5. Recombinant AAVrh74 vector, according to claim 1, characterized in that it further comprises a 3' poly A tail of said nucleotide sequence, wherein the sequence of said poly A tail is presented as nucleotides 4787 to 4842 of SEQ ID NO:

3.

6. Composition, characterized in that it comprises the recombinant AAVrh74 vector, as defined in any one of claims 1 to 5, and a pharmaceutically acceptable carrier.

7. Use of a recombinant AAVrh74 vector as defined in any of claims 1 to 5 or of a composition as defined in claim 6, characterized in that it is for Petition 870250052516, dated 06 / 23 / 2025, page 8 / 16 2 / 2 preparation of a medicament for the treatment of muscular dystrophy in an individual in need thereof.

8. Use of a recombinant AAVrh74 vector as defined in any one of claims 1 to 5 or of a composition as defined in claim 6, characterized in that it is for the preparation of a medicament to reduce or prevent fibrosis in an individual suffering from muscular dystrophy.

9. Use, according to claim 7 or 8, characterized in that the individual suffers from Duchenne muscular dystrophy.

10. Use, according to any one of claims 7 to 9, characterized in that the medicament is formulated for intramuscular injection or intravenous injection.

11. Use, according to any one of claims 7 to 9, characterized in that the medicament is formulated for systemic administration.

12. Use according to claim 11, characterized in that the medicament is formulated for parenteral administration by injection, infusion or implantation.

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