AUF1 Gene Therapy for Limb-Girdle Muscular Dystrophy

Abstract

A method for treating or ameliorating the symptoms of limb-girdle muscular dystrophy is provided by administration of a therapeutically effective dose of an adeno-associated virus (AAV) or recombinant adeno-associated virus (rAAV) containing a transgene encoding AU-rich element-binding factor 1 (AUF1), which is effective for the treatment of limb-girdle muscular dystrophy. Also provided are AAV vectors and rAAV vectors encoding the AUF1 protein.

Description

Technical Field

[0001] This application claims the priority of U.S. Provisional Patent Application No. 63 / 391,252, filed on July 21, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] This invention was made with government support under Grant No. 5R01AR0744303 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Field The present disclosure relates to the treatment of myopathic diseases such as limb-girdle muscular dystrophy (LGMD) by administration of a dose of a gene therapy vector in which the transgene encodes AUF1, such as AAV and rAAV gene therapy vectors. Also provided are AAV gene therapy vectors encoding the AUF1 protein and methods of treatment using the same.

Background Art

[0004] Background Limb-girdle muscular dystrophy (LGMD) is a group of more than 30 different subtypes of muscular dystrophy that cause muscle weakness and wasting in the pelvic and upper limb girdles. LGMD has diverse clinical phenotypes, including (1) variation in age of onset, (2) rate of progression, (3) specific muscle wasting patterns, and (4) invasion of respiratory and cardiac muscles. The proteins encoded by LGMD disease genes have diverse cellular functions, including glycosylation and the integrity, maintenance, and repair of the sarcolemma, and disruption of all of these leads to muscle damage and degeneration. Currently, there is no effective treatment for any LGMD subtype.

[0005] LGMD type 1 is autosomal dominant and accounts for approximately 10% of all LGMDs. Examples of subtype 1 include LGMD1C caused by mutations in caveolin 3, and LGMD1G caused by mutations in HNRPDL, a protein involved in mRNA biogenesis and metabolism. LGMD type 2 is autosomal recessive. Several genes that cause LGMD2 encode proteins that directly associate with dystrophin. Sarcolemmal glycanopathies have loss-of-function mutations in genes encoding α-, β-, γ-, or δ-sarcoglycan, causing LGMD2C, LGMD2D, LGMD2E, and LGMD2F, respectively. LGMD2 dystrophinopathies include LGMD2I (mutation in FKRP), LGMD2K (mutation in POMT1), LGMD2M (mutation in FKTN), LGMD2N (mutation in POMT2), LGMD2O (mutation in POMGnT1), LGMD2P (mutation in DAG1), LGMD2T (mutation in GMPPB), and LGMD2U (mutation in ISP / CRPPA).

[0006] LGMD2 calpainopathies include LGMD2A caused by mutations in calpain 3 (CAPN3). LGMD2A is the most frequent LGMD worldwide. Calpain is an intracellular non-lysosomal cysteine protease regulated by calcium ions. LGMD2 dysferlinopathies include LGMD2B. Other LGMD2 subtypes include LGMD2L, LGMD2H, LGMD2W, and LGMD2X.

[0007] Sarcoglycans are a group of transmembrane proteins that associate with dystrophin. The sarcoglycan subcomplex tightly associates with β-dystroglycan and consists of four single-pass transmembrane proteins: α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, and δ-sarcoglycan. Mutations in the genes encoding α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, and δ-sarcoglycan cause LGMD2C–F, respectively.

[0008] Dystrophin is a cytoplasmic protein encoded by the DMD gene and has the function of linking the cytoskeletal actin filaments and membrane proteins. Normally, the dystrophin protein is mainly present in skeletal muscle and cardiac muscle, and is also expressed in small amounts in the brain. By linking the actin of the contractile apparatus to the connective tissue layer surrounding each muscle fiber, it functions as a buffer during muscle fiber contraction. In muscle, dystrophin is localized on the cytoplasmic side of the muscle cell membrane.

[0009] Muscle wasting disorders account for a large portion of human diseases. Among them, there are those that are genetic in origin (mainly muscular dystrophy), age-related (sarcopenia), or the result of traumatic muscle injury. There are few treatment options available for individuals with myopathy or those suffering from severe muscle trauma or age-related muscle mass loss (known as sarcopenia). The physiology of myopathy is well understood and is typically based on the common etiology in which the continuous cycle of muscle degeneration and regeneration functionally exhausts and renders unable to reactivate, and sometimes leads to the loss of, muscle stem cells (satellite cells) and their progenitor cells (Carlson & Conboy, “Loss of Stem Cell Regenerative Capacity Within Aged Niches,” Aging Cell 6(3):371-82(2007); Shefer et al., “Satellite-cell Pool Size Does Matter: Defining the Myogenic Potency of Aging Skeletal Muscle,” Dev. Biol. 294(1):50-66(2006); Bernet et al., “p38 MAPK Signaling Underlies a Cell-autonomous Loss of Stem Cell Self-renewal in Skeletal Muscle of Aged Mice,” Nat. Med. 20(3):265-71(2014); and Dumont et al., “Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function,” Development 142(9):1572-1581(2015)).

[0010] Skeletal muscle regeneration is initiated by skeletal muscle stem cells (satellite cells) that exist between the fibers of striated muscle (muscle fibers), which are bundles of contractile cells, and the basement membrane that covers those muscle fibers (Carlson & Conboy, “Loss of Stem Cell Regenerative Capacity within Aged Niches,” Aging Cell 6(3):371-382 (2007) and Schiaffino & Reggiani, “Fiber Types in Mammalian Skeletal Muscles,” Physiol. Rev. 91(4):1447-1531 (2011)). When muscle is physically damaged, the anatomical niche is disrupted, and normally quiescent satellite cells are activated and proliferate asymmetrically. Some satellite cells reconstitute the stem cell population, while the majority differentiate and fuse to form new muscle fibers (Hindi et al., “Signaling Mechanisms in Mammalian Myoblast Fusion,” Sci. Signal. 6(272):re2 (2013)).The specific importance of satellite cell / myoblast populations in muscle regeneration has been demonstrated by studies (Shefer et al., “Satellite-cell Pool Size Does Matter: Defining the Myogenic Potency of Aging Skeletal Muscle,” Dev. Biol. 294(1):50-66(2006); Dumont et al., “Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function,” Development 142(9):1572-1581(2015); Briggs & Morgan, “Recent Progress in Satellite Cell / Myoblast Engraftment -- Relevance for Therapy,” FEBS J. 280(17):4281-93(2013); Morgan & Zammit, “Direct Effects of the Pathogenic Mutation on Satellite Cell Function in Muscular Dystrophy,” Exp. Cell Res. 316(18):3100-8(2010); and Relaix & Zammit, “Satellite Cells are Essential for Skeletal Muscle Regeneration: The Cell on the Edge Returns Centre Stage,” Development 139(16):2845-56(2012)).

[0011] Skeletal muscle fibers are divided into two types that exhibit different contraction and metabolic properties, namely, slow-twitch (type I) and fast-twitch (type II). Slow-twitch and fast-twitch fibers are defined according to their contraction speed, metabolism, and the type of myosin gene expressed (Schiaffino & Reggiani, “Fiber Types in Mammalian Skeletal Muscles,” Physiol. Rev. 91(4):1447-1531(2011) and Bassel-Duby & Olson, “Signaling Pathways in Skeletal Muscle Remodeling,” Annu. Rev. Biochem. 75:19-37(2006)). Slow-twitch fibers are rich in mitochondria, preferentially utilize oxidative metabolism, and provide resistance to fatigue at the expense of contraction speed. Fast-twitch fibers atrophy easily in response to nutrient deprivation, traumatic injury, progressive weakness associated with aging (sarcopenia), and cachexia due to cancer, while slow-twitch fibers are highly resilient (Wang & Pessin, “Mechanisms for Fiber-Type Specificity of Skeletal Muscle Atrophy,” Curr. Opin. Clin. Nutr. Metab. Care 16(3):243-250(2013); Tonkin et al., “SIRT1 Signaling as Potential Modulator of Skeletal Muscle Diseases,” Curr. Opin. Pharmacol. 12(3):372-376(2012); and Arany, Z, “PGC-1 Coactivators and Skeletal Muscle Adaptations in Health and Disease,” Curr. Opin. Genet. Dev. 18(5):426-434(2008)).Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α or Ppargc1) is a major physiological regulator in mitochondrial biogenesis and specification of type I muscle fibers (Lin et al., “Transcriptional Co-Activator PGC-1 Alpha Drives the Formation of Slow-Twitch Muscle Fibres,” Nature 418(6899):797-801(2002)). PGC1α stimulates mitochondrial biogenesis and oxidative metabolism through increased expression of nuclear respiratory factors (NRF) such as NRF1 and 2 that stimulate mitochondrial biogenesis, mitochondrial transcription factor A (Tfam), and also promotes the formation of slow muscle fibers through increased expression of Mef2 protein in addition to mitochondrial biogenesis (Lin et al., “Transcriptional Co-Activator PGC-1 Alpha Drives the Formation of Slow-Twitch Muscle Fibres,” Nature 418(6899):797-801(2002); Lai et al., “Effect of Chronic Contractile Activity on mRNA Stability in Skeletal Muscle,” Am. J. Physiol. Cell. Physiol. 299(1):C155-163(2010); Ekstrand et al., “Mitochondrial Transcription Factor A Regulates mtDNA Copy Number in Mammals,” Hum. Mol. Genet. 13(9):935-944(2004); and Scarpulla, RC, “Transcriptional Paradigms in Mammalian Mitochondrial Biogenesis and Function,” Physiol. Rev. 88(2):611-638(2008)).Importantly, PGC1α protects muscle from atrophy due to disuse, certain myopathies, starvation, sarcopenia, cachexia, and other causes (Wiggs, M.P., “Can Endurance Exercise Preconditioning Prevention Disuse Muscle Atrophy?,” Front. Physiol. 6:63 (2015); Wing et al., “Proteolysis in Illness-Associated Skeletal Muscle Atrophy: From Pathways to Networks,” Crit. Rev. Clin. Lab. Sci. 48(2):49-70 (2011); Bost & Kaminski, “The Metabolic Modulator PGC-1alpha in Cancer,” Am. J. Cancer Res. 9(2):198-211 (2019); and Dos Santos et al., “The Effect of Exercise on Skeletal Muscle Glucose Uptake in type 2 Diabetes: An Epigenetic Perspective,” Metabolism 64(12):1619-1628 (2015)).

[0012] Skeletal muscles can remodel between slow and fast muscle fibers in response to physiological stimuli, muscle loading, atrophy, disease, and injury (Bassel-Duby & Olson, “Signaling Pathways in Skeletal Muscle Remodeling,” Annu. Rev. Biochem. 75:19-37 (2006)), which involves transcriptional, metabolic, and post-transcriptional regulatory mechanisms (Schiaffino & Reggiani, “Fiber Types in Mammalian Skeletal Muscles,” Physiol. Rev. 91(4):1447-1531 (2011) and Robinson & Dilworth, “Epigenetic Regulation of Adult Myogenesis,” Curr. Top Dev. Biol. 126:235-284 (2018)).Endurance slow-twitch type I muscle fibers are more resistant to muscle atrophy, so selectively promoting slow-twitch muscle has been a long-term goal (Talbot & Maves, “Skeletal Muscle Fiber Type: Using Insights from Muscle Developmental Biology to Dissect Targets for Susceptibility and Resistance to Muscle Disease,” Wiley Interdiscip. Rev. Dev. Biol. 5(4):518-534(2016)) and could be an effective therapy for sarcopenia, Duchenne muscular dystrophy, cachexia, and other muscle-wasting diseases (Selsby et al., “Rescue of Dystrophic Skeletal Muscle By PGC-1alpha Involves A Fast To Slow Fiber Type Shift In The Mdx Mouse,” PLoS One 7(1):e30063(2012); von Maltzahn et al., “Wnt7a Treatment Ameliorates Muscular Dystrophy,” Proc. Natl. Acad. Sci. USA 109(50):20614-20619(2012); and Ljubicic et al., “The Therapeutic Potential Of Skeletal Muscle Plasticity In Duchenne Muscular Dystrophy: Phenotypic Modifiers As Pharmacologic Targets,” FASEB J. 28(2):548-568(2014)).

[0013] The myogenic program is regulated by genes encoding myogenic regulatory factors (MRFs) (Mok & Sweetman, “Many Routes to the Same Destination: Lessons From Skeletal Muscle Development,” Reproduction 141(3):301-12(2011)), and this factor directs activated satellite cells to differentiate into myoblasts, arrest their proliferation, differentiate, and fuse into multinucleated muscle fibers (Mok & Sweetman, “Many Routes to the Same Destination: Lessons From Skeletal Muscle Development,” Reproduction 141(3):301-12(2011)). Skeletal muscle regeneration is identified and staged by specific expression markers. PAX7 is a transcription factor expressed by quiescent satellite cells and early-activated satellite cells (Brack, A.S., “Pax7 is Back,” Skelet. Muscle 4(1):24(2014) and Gunther, S., et al., “Myf5-positive Satellite Cells Contribute to Pax7-dependent Long-term Maintenance of Adult Muscle Stem Cells,” Cell Stem Cell 13(5):590-601(2013)).

[0014] Satellite cells lose their ability to maintain a quiescent population with aging (Dumont et al., “Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function,” Development 142(9):1572-1581(2015)), become depleted, or functionally exhausted, which is a major cause of sarcopenia (muscle loss) due to aging and myopathic diseases (Bernet et al., “p38 MAPK Signaling Underlies a Cell-autonomous Loss of Stem Cell Self-renewal in Skeletal Muscle of Aged Mice,” Nat. Med. 20(3):265-71(2014); Dumont et al., “Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function,” Development 142(9):1572-1581(2015); Kudryashova et al., “Satellite Cell Senescence Underlies Myopathy in a Mouse Model of Limb-girdle Muscular Dystrophy 2H,” J. Clin. Invest. 122(5):1764-76(2012); and Silva et al., “Inhibition of Stat3 Activation Suppresses Caspase-3 and the Ubiquitin-proteasome System, Leading to Preservation of Muscle Mass in Cancer Cachexia,” J. Biol. Chem. 290(17):11177-87(2015)).

[0015] Therefore, there is an urgent need for effective treatment options to address the consequences of LGMD, including, for example, alleviating the decline in muscle fiber strength, loss of basic muscle molecular functions, and the deleterious effects of pathological immune responses on muscle health and integrity.

[0016] The present disclosure aims to overcome these and other deficiencies in the art. SUMMARY OF THE INVENTION

[0017] Summary One aspect of the present disclosure relates to a method of treating limb-girdle muscular dystrophy (LGMD) in a subject in need thereof. The method involves administering to the subject an adeno-associated virus (AAV) particle or a recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid molecule encoding an AU-rich mRNA-binding factor 1 (AUF1) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by terminal inverted repeat (ITR) sequences. In some embodiments, the subject comprises a functional AUF1 protein isoform.

[0018] Another aspect of the present disclosure relates to a method of treating mitochondrial dysfunction associated with limb-girdle muscular dystrophy (LGMD) in a subject in need thereof. The method involves administering to the subject an adeno-associated virus (AAV) particle or a recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid molecule encoding an AU-rich mRNA-binding factor 1 (AUF1) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by terminal inverted repeat (ITR) sequences. In some embodiments, the subject comprises a functional AUF1 protein isoform.

[0019] As the use of adeno-associated virus (AAV)-mediated gene therapy, which has the potential to treat various rare diseases, progresses, there is expectation and interest in the use of AAV for the treatment of LGMD.

[0020] Accordingly, there is a need in the art for methods of administering AAV vectors and rAAV vectors encoding AUF1 for the treatment or amelioration of symptoms of LGMD, including sarcoglycanopathy, calpainopathy, dysferlinopathy, and dystrophinopathy, and minimizing the immune response to the therapeutic protein.

[0021] When the expression of AU-rich mRNA binding factor 1 (AUF1) increases in muscle cells, the amount, function, and performance of muscle are restored or increased, and muscle atrophy is decreased or reversed. AUF1 expression in muscle cells increases the expression of components of the dystrophin glycoprotein complex (DGC), also referred to herein as the dystrophin-binding protein complex or DAPC, increases the involvement of the components in the DGC, and can stabilize the muscle sheath. The DGC is composed of a sarcoglycan subcomplex of α, β, γ, and δ sarcoglycans and sarcospan (SSPN). AUF1 has also been shown to have activity in reducing muscle degeneration and improving muscle fiber size and endurance in δ-sarcoglycan null mice, supporting its activity in the treatment of LGMD, including sarcoglycanopathy, calpainopathy, dysferlinopathy, and dystrophinopathy.

[0022] AUF1 also promotes the expression of genes downstream of the CAPN3 gene in calpainopathy, correcting the physiological and phenotypic dysfunction of CAPN3-deficient mice, including mitochondrial biogenesis, normal muscle ultrastructure construction, and increased CAMKIIβ kinase activity, which is a central point of deficiency in this disease. Accordingly, provided are AUF1 monotherapy and combination therapies for the treatment and improvement of the symptoms of LGMD, including AUF1 therapies comprising an AUF1 gene therapy construct, optionally, a second therapy comprising an rAAV gene therapy vector expressing a therapeutic protein such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, calcium / calmodulin-dependent protein kinase II β isoform protein, other proteins (other than AUF1), or a portion thereof, and / or optionally, in combination with other therapies for LGMD. Also provided are AAV or rAAV gene therapy vectors for delivering AUF1, and methods of treatment for diseases associated with, for example, LGMD, muscle wasting, and muscle injury using those gene therapy vectors.

[0023] International Patent Application Publication No. WO2016 / 034794 by Schneider et al. (Schneider 2016) (incorporated herein by reference in its entirety) relates to compositions for the uptake of muscle cells (e.g., compositions encoding AUF1), compositions containing satellite cell populations and myosatellite cell populations, as well as pharmaceutical compositions, methods for producing myosatellite cell compositions, and methods for bringing about myogenesis via myosatellite cells. Schneider 2016 relates to treating muscle stem cells (satellite cells) with AUF1 gene therapy, and the AUF1 transgene is transcriptionally regulated by a transcriptional regulatory element specific to satellite cells. Schneider 2016 further relates to treating muscle cells with monoallelic gene therapy in which the wild-type AUF1 gene is replaced with a defective AUF1 gene. All of the examples provided were those that replaced the defective endogenous AUF1 gene with a normal AUF1 gene. In contrast, the present disclosure relates to a method for treating muscular dystrophies such as LGMD that express endogenous AUF1 by supplementing additional exogenous AUF1 to muscle cells. It was completely unexpected that muscle cells showing mitochondrial dysfunction including biogenesis, which have no defect in the expression of the endogenous AUF1 gene but have defects in the functions of other genes that promote the pathological conditions of muscular dystrophy diseases, could be effectively treated by supplementing additional AUF1. It is completely novel and unexpected that AUF1 supplementation gene therapy can effectively treat muscular dystrophies and mitochondrial dysfunctions in which defects in the expression or function of the AUF1 gene are not known to be involved, and it was not known or speculated in the prior art including Schneider 2016. The examples of the present disclosure surprisingly show that supplementation of AUF1 is effective in avoiding the constellation of defective genes in LGMD that does not contain AUF1.

[0024] International Patent Application Publication No. WO2021 / 146711 (Schneider 2021) (which is incorporated herein by reference in its entirety) relates to an adeno-associated virus (AAV) vector comprising a muscle cell-specific promoter and a nucleic acid molecule encoding an AU-rich mRNA-binding factor 1 (AUF1) protein or a functional fragment thereof, wherein the nucleic acid molecule is heterologous to the muscle cell-specific promoter and is operably linked to the muscle cell-specific promoter. Schneider 2021 also discloses a composition comprising the AAV vector, as well as a method for promoting muscle regeneration of damaged muscle, a method for treating degenerative skeletal muscle loss in a subject, a method for preventing traumatic muscle injury in a subject such as Duchenne muscular dystrophy, a method for treating traumatic muscle injury in a subject, and a method for treating muscle loss due to aging in a subject. In contrast, the present disclosure relates to the treatment of limb-girdle muscular dystrophy by bypassing the defective gene of LGMD and by increasing the function of a plurality of other genes that can complement the defective gene and do not include the AUF1 gene. The present disclosure is not related to a composition or method for muscle regeneration or prevention of injury after traumatic injury.

[0025] International Patent Application Publication No. WO2023 / 004331 (Schneider 2023) (incorporated herein by reference in its entirety) provides a method for treating or ameliorating the symptoms of dystrophinopathies such as Duchenne muscular dystrophy and Becker muscular dystrophy by administering a therapeutically effective dose of a recombinant adeno-associated virus (rAAV) containing a transgene encoding AUF1, and a second rAAV encoding another therapeutic method effective for treating dystrophinopathies by increasing muscle strength and endurance, which provides a dystrophin gene substitute that replaces microdystrophin or a defective dystrophin gene and AUF1. In contrast, the present disclosure relates to the treatment of limb-girdle muscular dystrophy (LGMD) by bypassing the defective gene of LGMD, and surprisingly shows that replenishment of AUF1 is effective for the treatment of LGMD without AUF1 mutations. The present disclosure does not relate to compositions or methods for replacing defective dystrophin genes not involved in LGMD.

[0026] A method for treating limb-girdle muscular dystrophy (LGMD), such as subtypes 1 and 2 of LGMD, is provided by administering to a subject in need thereof a gene therapy vector, particularly an AAV vector or an rAAV vector, the gene therapy vector comprising a genome having a transgene encoding AUF1 protein operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount. In other embodiments, the AUF1 protein or nucleic acid encoding AUF1 is administered in combination with another therapeutic method for use in the treatment of LGMD. The other therapeutic method can be a therapeutic method of gene therapy (including a therapeutic method co-expressed with AUF1 or a therapeutic method co-expressed as a separate gene therapy vector) or a therapeutic method of non-gene therapy.

[0027] The treatment methods provided herein include the treatment of human subjects having a type 1 LGMD, such as limb-girdle muscular dystrophy type 1C (LGMD1C) and limb-girdle muscular dystrophy type 1G (LGMD1G). The treatment methods also include the treatment of sarcoglycanopathies, such as limb-girdle muscular dystrophy type 2C (LGMD2C), limb-girdle muscular dystrophy type 2D (LGMD2D), limb-girdle muscular dystrophy type 2E (LGMD2E), and limb-girdle muscular dystrophy type 2F (LGMD2F); dystrophinopathies, such as limb-girdle muscular dystrophy type 2I (LGMD2I), limb-girdle muscular dystrophy type 2K (LGMD2K), limb-girdle muscular dystrophy type 2M (LGMD2M), limb-girdle muscular dystrophy type 2N (LGMD2N), limb-girdle muscular dystrophy type 2O (LGMD2O), limb-girdle muscular dystrophy type 2P (LGMD2P), limb-girdle muscular dystrophy type 2T (LGMD2T), and limb-girdle muscular dystrophy type 2U (LGMD2U); calpainopathies, such as limb-girdle muscular dystrophy type 2A (LGMD2A); dysferlinopathies, such as limb-girdle muscular dystrophy type 2B (LGMD2B); and other limb-girdle muscular dystrophy type 2 (LGMD2) subtypes, such as limb-girdle muscular dystrophy type 2L (LGMD2L), limb-girdle muscular dystrophy type 2H (LGMD2H), limb-girdle muscular dystrophy type 2W (LGMD2W), and limb-girdle muscular dystrophy type 2X (LGMD2X). In embodiments, methods for treating LGMD not associated with mutations in the gene encoding AUF1 are provided.

[0028] In embodiments, AUF1 is human AUF1 p37 AUF1 , p40 AUF1 , p42 AUF1 , or p45 AUF1 isoform (e.g., p40 AUF1(including isoforms) and can be encoded by a nucleotide sequence that has been codon-optimized and CpG-depleted, such as the nucleotide sequence of SEQ ID NO: 17. In additional embodiments, in an AUF1 gene therapy vector comprising an AAV and rAAV gene therapy vector, the muscle cell-specific promoter is the muscle creatine kinase (MCK) promoter, the syn100 promoter, the CK6 promoter, the CK7 promoter, the CK8 promoter, the CK9 promoter, the dMCK promoter, the tMCK promoter, the skeletal muscle 22 (SM22) promoter, the myo-3 promoter, the Spc5-12 promoter (including SpcV1 (SEQ ID NO: 127) or SpcV2 (SEQ ID NO: 128) which are modified Spc5-12 promoters), the creatine kinase (CK) 8e promoter, the U6 promoter, the H1 promoter, the desmin promoter, the Pitx3 promoter, the skeletal alpha-actin promoter, the MHCK7 promoter, or the Sp-301 promoter (see also Table 8).

[0029] In certain embodiments, provided is a method of administering an AAV particle or an rAAV particle comprising a recombinant genome having the nucleotide sequence of SEQ ID NO: 31 (spc-hu-opti-AUF1-CpG(-)), SEQ ID NO: 32 (tMCK-huAUF1), SEQ ID NO: 33 (spc5-12-hu-opti-AUF1-WPRE), SEQ ID NO: 34 (ss-CK7-hu-AUF1), SEQ ID NO: 35 (spc-hu-AUF1 without intron), or SEQ ID NO: 36 (D(+)-CK7AUF1). The rAAV particle is, in embodiments, of the AAV8 or AAV9 serotype and has a capsid that is at least 95% identical to SEQ ID NO: 114 (AAV8 capsid), SEQ ID NO: 115 (AAV9 capsid), or SEQ ID NO: 118 (AAVhu.32 capsid). In certain embodiments, the first therapy is administered systemically, such as intravenously, at a dose of 1E8 vector genomes (vg / kg) to 2E15 vg / kg per kg, 1E13 to 1E14 vg / kg, or 2E13 vg / kg (vector genomes / kg (vg / kg) and genome copies / kg (gc / kg) are EX and X10 XSimilarly, (used interchangeably herein). In other embodiments, provided is a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 17 encoding human AUF1 p40, which is a codon-optimized and CpG-reduced sequence. A vector comprising this sequence (SEQ ID NO: 17) operably linked to a muscle cell-specific promoter that can be the muscle creatine kinase (MCK) promoter, Syn promoter, syn100 promoter, CK6 promoter, CK7 promoter, CK8 promoter, CK9 promoter, dMCK promoter, tMCK promoter, skeletal muscle 22 (SM22) promoter, myo-3 promoter, Spc5-12 promoter (including Spc5v1 (SEQ ID NO: 127) and Spc5v2 (SEQ ID NO: 128) of the variant Spc5-12 promoter), creatine kinase (CK) 8e promoter, U6 promoter, H1 promoter, desmin promoter, Pitx3 promoter, skeletal alpha-actin promoter, MHCK7 promoter, or Sp-301 promoter (see, e.g., Table 8) is provided. In embodiments, in addition to being operably linked to a muscle-specific promoter sequence, the nucleotide sequence of SEQ ID NO: 17 is further operably linked to an intron sequence such as the VH4 intron sequence, a polyadenylation signal sequence such as the rabbit beta-globin polyadenylation signal sequence, and / or a WPRE sequence (disclosed herein). The vector can be a cis plasmid for packaging rAAV or an rAAV genome flanked by ITR sequences. The genome in the rAAV particles can be single-stranded or self-complementary. Additionally, considering the size of the human AUF1 p40 sequence, the rAAV vector sequence can include a 5' stuffer sequence and / or a 3' stuffer sequence (see Table 10) and / or an SV40 polyadenylation signal sequence.

[0030] In a specific embodiment, a method for treating LGMD and a pharmaceutical composition for use in the treatment of LGMD are, as disclosed herein, administered an AAV or rAAV generated from a vector comprising the nucleotide sequence of SEQ ID NO: 17 encoding human AUF1 p40 operably linked to a regulatory sequence that promotes expression in muscle, including a muscle-specific promoter (or a constitutive promoter) (see, for example, Table 8. In embodiments, it may have the nucleotide sequence of SEQ ID NO: 31 (spc-hu-opti-AUF1-CpG(-)), SEQ ID NO: 32 (tMCK-huAUF1), SEQ ID NO: 33 (spc5-12-hu-opti-AUF1-WPRE), SEQ ID NO: 34 (ss-CK7-hu-AUF1), SEQ ID NO: 35 (spc-hu-AUF1 without intron), or SEQ ID NO: 36 (D(+)-CK7AUF1). rAAV particles, pharmaceutical compositions, and methods of using the same are also further provided. The rAAV particles are, in embodiments, of AAV8, AAV9, or AAVhu.32 serotype, or a capsid of Table 11, and include having a capsid that is at least 95% identical to SEQ ID NO: 114 (AAV8 capsid), SEQ ID NO: 115 (AAV9 capsid), or SEQ ID NO: 118 (AAVhu.32 capsid).

[0031] Accordingly, one aspect of the disclosure relates to a method of treating limb-girdle muscular dystrophy (LGMD) in a subject in need thereof. The method involves administering to the subject an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AU-rich mRNA-binding factor 1 (AUF1) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by terminal inverted repeat (ITR) sequences. In some embodiments, the subject expresses functional AUF1. According to such embodiments, the subject has LGMD not associated with a mutation in the gene encoding AUF1. BRIEF DESCRIPTION OF THE DRAWINGS

[0032]

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Mode for Carrying Out the Invention

[0033] Detailed Description A method for treating limb-girdle muscular dystrophy (LGMD), such as subtypes 1 and 2 of LGMD, is provided by administering a gene therapy vector, particularly an AVV vector or an rAAV vector, to a subject in need thereof. The gene therapy vector comprises a genome having a transgene encoding an AUF1 protein operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount. In other embodiments, the AUF1 protein or nucleic acid encoding AUF1 is administered in combination with another therapy for the treatment of LGMD. The other therapy can be a gene therapy (including a therapy co-expressed with AUF1 or a therapy co-expressed as a separate gene therapy vector) or a non-gene therapy.

[0034] Accordingly, one aspect of the present disclosure relates to a method of treating limb-girdle muscular dystrophy (LGMD) in a subject in need thereof. The method involves administering to the subject an adeno-associated virus (AAV) particle or a recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by terminal inverted repeat (ITR) sequences. In some embodiments, the subject comprises a functional AUF1 protein isoform.

[0035] Another aspect of the present disclosure relates to a method of treating mitochondrial dysfunction associated with limb-girdle muscular dystrophy (LGMD) in a subject in need thereof. The method involves administering to the subject an adeno-associated virus (AAV) particle or a recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by terminal inverted repeat (ITR) sequences. In some embodiments, the subject comprises a functional AUF1 protein isoform.

[0036] The treatment methods provided herein include the treatment of human subjects having type 1 limb-girdle muscular dystrophy (LGMD), such as limb-girdle muscular dystrophy type 1C (LGMD1C) and limb-girdle muscular dystrophy type 1G (LGMD1G). The treatment methods also include sarcoglycan disorders, such as limb-girdle muscular dystrophy type 2C (LGMD2C), limb-girdle muscular dystrophy type 2D (LGMD2D), limb-girdle muscular dystrophy type 2E (LGMD2E), and limb-girdle muscular dystrophy type 2F (LGMD2F), dystrophin disorders, such as limb-girdle muscular dystrophy type 2I (LGMD2I), limb-girdle muscular dystrophy type 2K (LGMD2K), limb-girdle muscular dystrophy type 2M (LGMD2M), limb-girdle muscular dystrophy type 2N (LGMD2N), limb-girdle muscular dystrophy type 2O (LGMD2O), limb-girdle muscular dystrophy type 2P (LGMD2P), limb-girdle muscular dystrophy type 2T (LGMD2T), and limb-girdle muscular dystrophy type 2U (LGMD2U), calpain disorders, such as limb-girdle muscular dystrophy type 2A (LGMD2A), dysferlin disorders, such as limb-girdle muscular dystrophy type 2B (LGMD2B), and other LGMD2 subtypes, such as limb-girdle muscular dystrophy type 2L (LGMD2L), limb-girdle muscular dystrophy type 2H (LGMD2H), limb-girdle muscular dystrophy type 2W (LGMD2W), and limb-girdle muscular dystrophy type 2X (LGMD2X), to provide treatment of type 2 limb-girdle muscular dystrophy (LGMD). In embodiments, methods of treating LGMD not associated with mutations in the gene encoding AUF1 are provided.

[0037] In some embodiments, the AUF1 gene therapy is administered alone with a gene therapy vector comprising a transgene encoding an AUF1 gene effective to treat LGMD operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount.

[0038] In some embodiments, the AUF1 gene therapy is administered in combination with a gene therapy vector comprising a genome having a transgene encoding a therapeutic protein (such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, calcium / calmodulin-dependent protein kinase II β isoform protein, other proteins (other than AUF1), or a portion thereof) operably linked to a regulatory element that promotes expression in muscle cells, in a therapeutically effective amount.

[0039] In some embodiments, the AUF1 gene therapy is administered in combination with a small molecule drug. According to such embodiments, the small molecule drug is AMPBP (CID 11210285 hydrochloride; 2-amino-4-(3,4-methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride, N4-(1,3-benzodioxol-5-ylmethyl)-6-(3-methoxyphenyl)-2,4-pyrimidinediamine hydrochloride, a Wnt agonist); lansoprazole (2-[[[3-methyl-4-(2,2,2-trifluoroethoxy)-2-pyridinyl]methyl]sulfinyl]-1H-benzimidazole); parbendazole (methyl(5-butyl-1H-benzimidazol-2-yl)carbamate); and rabeprazole sodium salt (1H-benzimidazole, 2-[[[4-(3-methoxypropoxy)-3-methyl-2-pyridinyl]methyl]sulfinyl). As described herein, AMBP activates Wnt signaling without inhibiting GSK-3β, lansoprazole is a gastric proton pump inhibitor, parbendazole has broad anthelmintic activity, and rabeprazole sodium salt is a gastric pump H + / K + ATPase inhibitor.

[0040] In some embodiments, the AUF1 gene therapy is administered in a therapeutically effective amount in combination with a gene therapy vector comprising a genome having a transgene encoding α-sarcoglycan effective to treat LGMD2C operably linked to a regulatory element that promotes expression in muscle cells. In some embodiments, the AUF1 gene therapy is administered in a therapeutically effective amount in combination with a gene therapy vector comprising a genome having a transgene encoding β-sarcoglycan effective to treat LGMD2D operably linked to a regulatory element that promotes expression in muscle cells. In some embodiments, the AUF1 gene therapy is administered in a therapeutically effective amount in combination with a gene therapy vector comprising a genome having a transgene encoding γ-sarcoglycan effective to treat LGMD2E operably linked to a regulatory element that promotes expression in muscle cells. In some embodiments, the AUF1 gene therapy is administered in a therapeutically effective amount in combination with a gene therapy vector comprising a genome having a transgene encoding δ-sarcoglycan effective to treat LGMD2F operably linked to a regulatory element that promotes expression in muscle cells.

[0041] In other embodiments, the AUF1 protein or nucleic acid encoding AUF1 is administered in combination with another therapy for use in the treatment of LGMD.

[0042] Also provided is an AUF1 AAV gene therapy construct. The construct includes a regulatory element that promotes expression in muscle cells, and optionally, other regulatory elements such as a polyadenylation sequence, an intron sequence, WPRE or other elements, and / or a stuffer sequence including, for example, those disclosed herein, operably linked to a coding sequence (SEQ ID NO: 17) that has been codon-optimized and CpG-depleted for human p40 AUF1 (see, for example, Table 10) (see Table 2 for the nucleotide sequences of the construct components). Exemplary constructs are described, for example, in FIG. 1 (see also Table 3). Constructs that include adjacent ITR sequences can have the nucleotide sequences of SEQ ID NOs: 31-36. The gene therapy vector can be, for example, an AAV8 serotype vector, an AAV9 serotype vector, an AAVhu.32 serotype vector (see, for example, the capsids in Table 11) or other suitable AAV serotype capsids that promote delivery to or have tropism for skeletal muscle cells. Accordingly, provided are compositions comprising the AUF1 AAV gene therapy vectors described herein (e.g., those shown in FIG. 1), and methods of administering the same, for treating LGMD and subtypes of LGMD, such as LGMD calpainopathy, dystrophinopathy, dysferlinopathy, and sarcoglycanopathy, by restoring or increasing muscle mass, muscle function or performance, and / or by decreasing or reversing muscle atrophy. Such methods include, among other things, stabilizing the sarcolemma of muscle cells by providing the AUF1 protein, particularly by reducing efflux (e.g., as measured by creatine kinase levels), increasing the expression and / or presence of β-sarcoglycan or utrophin in the dystrophin-glycoprotein complex of muscle cells, increasing PGC1α and MEF gene expression levels, type I oxidative slow muscle fibers, and CAMKIIβ kinase activity.Such methods also include promoting an increase in muscle cell mass, the number of muscle fibers, the size of muscle fibers, a decrease or reversal of muscle cell atrophy, activation and differentiation of satellite cells, muscle cell function (e.g., by increasing mitochondrial oxidative capacity), improvement of mitochondrial biogenesis, and an increase in the proportion of slow muscle fibers in muscle (including that due to conversion of fast muscle fibers to slow muscle fibers).

[0043] Also provided are pharmaceutical compositions formulated for peripheral administration, such as intravenous administration, of rAAV encoding AUF1 as described herein.

[0044] 1. Definitions The term "vector" is used interchangeably with "expression vector". The term "vector" can refer to a viral or non-viral, prokaryotic or eukaryotic, DNA or RNA sequence that can be transfected into a cell called a "host cell" such that all or part of the sequence is transcribed. The transcript need not be expressed. Also, the vector need not contain a transgene with a coding sequence. Vectors are often assembled as a complex of elements derived from different viral, bacterial, or mammalian genes. Vectors contain various coding and non-coding sequences such as sequences encoding selectable markers, sequences that facilitate propagation in bacteria, or one or more transcription units that are expressed only in certain cell types. For example, mammalian expression vectors often contain both prokaryotic sequences that facilitate propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed only in eukaryotic cells. It will be understood by those skilled in the art that the design of an expression vector can depend on factors such as the choice of host cell to be transformed and the level of expression of the desired protein.

[0045] The term "promoter" is used interchangeably with "promoter element" and "promoter sequence". Similarly, the term "enhancer" is used interchangeably with "enhancer element" and "enhancer sequence". The term "promoter" refers to the minimal sequence of the transgene that is sufficient to initiate transcription of the coding sequence of the transgene. A promoter can be constitutive or inducible. A constitutive promoter is considered a strong promoter if it drives expression of the transgene at a level equivalent to that of the cytomegalovirus promoter (CMV) (Boshart et al., "A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus," Cell 41:521 (1985); which is incorporated herein by reference in its entirety). A promoter can be a synthetic promoter, a modified promoter, or a hybrid promoter. A promoter can bind to other regulatory sequences / elements that, when bound to appropriate intracellular regulatory factors, either promote ("enhancer") or repress ("repressor") promoter-dependent transcription. A promoter, enhancer, or repressor is said to be "operably linked" to a transgene if such element(s) control or affect the rate or efficiency of transcription of the transgene. For example, a promoter sequence located proximal to the 5' end of the transgene coding sequence is typically operably linked to the transgene. As used herein, the term "regulatory element" is used interchangeably with "regulatory sequence" and refers to a promoter, enhancer, and other expression control elements, or any combination of such elements.

[0046] A promoter is located on the 5’ (upstream) side of the gene it controls. Many eukaryotic promoters contain two types of recognition sequences, the TATA box and upstream promoter elements. The TATA box is located 25 - 30 bp upstream from the transcription start site and is thought to be involved in enabling RNA polymerase II to initiate RNA synthesis at the correct site. In contrast, upstream promoter elements determine the rate of transcription initiation. These elements can act regardless of their orientation but must be located within 100 - 200 bp upstream of the TATA box.

[0047] Enhancer elements can stimulate transcription from linked homologous or heterologous promoters by up to 1000 - fold. Enhancer elements often maintain their activity even when their orientation is reversed (Li et al., “High Level Desmin Expression Depends on a Muscle - Specific Enhancer,” J. Bio. Chem. 266(10):6562 - 6570(1991); the entire content is incorporated herein by reference). Furthermore, unlike promoter elements, enhancers can be active even when located downstream from the transcription start site, for example, within an intron, or when located quite far from the promoter (Yutzey et al., “An Internal Regulatory Element Controls Troponin I Gene Expression,” Mol. Cell. Bio. 9(4):1397 - 1405(1989); the entire content is incorporated herein by reference).

[0048] The term “muscle - specific” refers to the ability of regulatory elements such as promoters and enhancers to drive the expression of a nucleic acid molecule (e.g., a nucleic acid molecule encoding the AU - rich mRNA - binding factor 1 (AUF1) protein or a functional fragment thereof) operably linked thereto exclusively or preferentially in muscle cells or muscle tissue.

[0049] The term "AAV" or "adeno-associated virus" refers to a dependoparvovirus within the genus of viruses in the Parvoviridae family. AAV can be AAV derived from a naturally occurring "wild-type" virus, AAV derived from an rAAV genome packaged into a capsid containing a capsid protein encoded by a naturally occurring cap gene, and / or AAV derived from an rAAV genome packaged into a capsid containing a capsid protein encoded by a non-naturally occurring cap gene. Examples of the latter include rAAV having a capsid protein with an amino acid sequence modified from that of a naturally occurring capsid and / or a peptide insert.

[0050] The term "rAAV" refers to "recombinant AAV". In some embodiments, recombinant AAV has an AAV genome in which some or all of the rep gene and the cap gene are replaced with heterologous sequences.

[0051] The term "rep-cap helper plasmid" refers to a plasmid that provides the functions of the viral rep gene and cap gene and aids in the production of AAV from an rAAV genome lacking a functional rep gene and / or cap gene sequence.

[0052] The term "cap gene" refers to a nucleic acid sequence that encodes a capsid protein that forms or aids in the formation of the viral capsid coat. In the case of AAV, the capsid protein can be VP1, VP2, or VP3.

[0053] The term "rep gene" refers to a nucleic acid sequence that encodes non-structural proteins necessary for viral replication and production.

[0054] The terms "nucleic acid" and "nucleotide sequence" include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations or hybrids of DNA and RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be produced, for example, using nucleotide analogs, which include, but are not limited to, inosine or tritylated bases. Such analogs can also include DNA or RNA molecules having modified backbones that confer beneficial attributes on the molecule, such as, for example, nuclease resistance or increased ability to cross cell membranes. A nucleic acid or nucleotide sequence can be single-stranded, double-stranded, can contain both single-stranded and double-stranded portions, and can contain triple-stranded portions, but is preferably double-stranded DNA.

[0055] The amino acid residues disclosed herein can be modified by conservative substitutions that maintain or substantially maintain the overall polypeptide structure and / or function. As used herein, "conservative amino acid substitutions" indicate the following: hydrophobic amino acids (i.e., Ala, Cys, Gly, Pro, Met, Val, Ile, and Leu) can be substituted with other hydrophobic amino acids; hydrophobic amino acids with bulky side chains (i.e., Phe, Tyr, and Trp) can be substituted with other hydrophobic amino acids with bulky side chains; amino acids with positively charged side chains (i.e., Arg, His, and Lys) can be substituted with other amino acids with positively charged side chains; amino acids with negatively charged side chains (i.e., Asp and Glu) can be substituted with other amino acids with negatively charged side chains; amino acids with polar uncharged side chains (i.e., Ser, Thr, Asn, and Gln) can be substituted with other amino acids with polar uncharged side chains.

[0056] The terms "subject", "host", and "patient" are used interchangeably. A subject can be a mammal such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human), including humans.

[0057] The term "therapeutic agent" refers to any agent that can be used for the treatment, management, or amelioration of symptoms associated with a disease or disorder, where the disease or disorder is related to the function provided by the transgene. A "therapeutically effective amount" refers to the amount of an agent (e.g., the amount of a product expressed by the transgene) that, when administered to a subject suffering from the disease or disorder of interest, provides at least one therapeutic benefit in the treatment or management of the disease or disorder. Further, a therapeutically effective amount with respect to the agents of the present disclosure means the amount of the agent alone or in combination with other therapies that provides at least one therapeutic benefit in the treatment or management of the disease or disorder.

[0058] The term "preventive agent" refers to any agent that can be used in the prevention, reduction in the likelihood of occurrence, delay, or blunting of the progression of a disease or disorder, where the disease or disorder is related to the function provided by the transgene. A "preventively effective amount" refers to the amount of a preventive agent (e.g., the amount of a product expressed by the transgene) that, when administered to a subject at risk for the disease or disorder of interest, provides at least one preventive benefit in the prevention or delay of the disease or disorder. A preventively effective amount may also refer to an amount of the agent sufficient for the prevention, reduction in the likelihood of occurrence, or delay of the disease or disorder of interest, or for the delay of the progression of the disease or disorder of interest, or for the delay or minimization of the onset of the disease or disorder of interest, or for the prevention or delay of its recurrence or spread. A preventively effective amount may also refer to an amount of the agent sufficient for the prevention or delay of the worsening of the symptoms of the disease or disorder of interest. Further, a preventively effective amount with respect to the preventive agents of the present disclosure means the amount of the preventive agent alone or in combination with other agents that provides at least one preventive benefit in the prevention or delay of the disease or disorder.

[0059] The prophylactic agent of the present disclosure can be administered to a subject "predisposed" to the target disease or disorder. A subject "predisposed" to a disease or disorder is one who exhibits symptoms associated with the onset of the disease or disorder, or who has a genetic structure, environmental exposure, or other risk factor for such a disease or disorder, but does not yet have symptoms at a level that would be diagnosed as the disease or disorder. For example, a patient with a family history of a disease associated with a defective gene (such as that provided by a transgene) can be considered to be predisposed to that disease. Further, a patient with dormant tumors that persist after removal of a primary tumor can be considered to be predisposed to tumor recurrence.

[0060] The term "pharmaceutically acceptable carrier" refers to a carrier that does not cause an allergic reaction or other adverse effect in the patient to whom it is administered and is compatible with the other components of the formulation. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients, or carriers, which are preferably selected with respect to the intended mode of administration and are consistent with conventional pharmaceutical practice. For example, solid carriers / diluents include, but are not limited to, rubber, starch (such as corn starch, pregelatinized starch), sugars (such as lactose, mannitol, sucrose, dextrose), cellulosic materials (such as microcrystalline cellulose), acrylates (such as polymethyl acrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. Pharmaceutically acceptable carriers can further include small amounts of auxiliary substances such as wetting agents or emulsifiers, preservatives or buffers that enhance the shelf life or effectiveness of the nucleic acid molecules described herein.

[0061] The term "CpG island" refers to a characteristic region of the genome that contains the dinucleotide CpG (e.g., where a G (guanine) base follows a C (cytosine) base (CpG)) at a high frequency. Thus, the G+C content of CpG islands is significantly higher than that of non-island DNA. CpG islands can be identified by analysis of nucleotide length, nucleotide composition, and the frequency of CpG dinucleotides. The CpG island content in any given nucleotide sequence or genome can be measured using the following criteria: the island size is greater than 100, the GC percentage is greater than 50.0%, and the ratio of the observed number to the expected number of CG dinucleotides based on the number of Gs and Cs in the segment is greater than 0.6 (Obs / Exp > 0.6). Obs / Exp CpG = number of CpGs * N / (number of Cs * number of Gs) where N is the length of the sequence.

[0062] For such calculations, various software tools are available, such as world-wide-web.urogene.org / cgi-bin / methprimer / methprimer.cgi, world-wide-web.cpgislands.usc.edu / , world-wide-web.ebi.ac.uk / Tools / emboss / cpgplot / index.html, and world-wide-web.bioinformatics.org / sms2 / cpg_islands.html (see also Gardiner-Garden and Frommer, J. Mol. Biol. 196(2):261-82 (1987); Li LC and Dahiya R., “MethPrimer: Designing Primers for Methylation PCRs,” Bioinformatics 18(11):1427-31 (2002), the entireties of which are hereby incorporated by reference). In one embodiment, the algorithm for identifying CpG islands is found at www.urogene.org / cgi-bin / methprimer / methprimer.cgi.

[0063] 2. AU-rich mRNA Binding Factor 1 Vector 2.1. AU-rich mRNA Binding Factor 1 Transgene A nucleic acid comprising a transgene encoding AUF1 or a therapeutically functional fragment thereof, including the p37, p40, p42, and p45 isoforms of human and mouse AUF1, and a vector and a viral particle comprising rAAV containing the same, and methods of using the same in a method of treatment, prevention, or amelioration for a condition related to a decrease in muscle mass or performance, or when an increase in muscle mass or performance is desired or useful. The AUF1 gene therapy vector is used in a method of treating or ameliorating the symptoms of LGMD by administering the AUF1 gene therapy vector.

[0064] Genes involved in rapid responses to cell stimulation are highly regulated and typically encode mRNAs that are selectively and rapidly degraded, quickly terminating protein expression and reprogramming cells (Moore et al., “Physiological Networks and Disease Functions of RNA-binding Protein AUF1,” Wiley Interdiscip. Rev. RNA 5(4):549-64 (2014); which is hereby incorporated by reference in its entirety). These include growth factors, inflammatory cytokines (Moore et al., “Physiological Networks and Disease Functions of RNA-binding Protein AUF1,” Wiley Interdiscip Rev RNA 5(4):549-64 (2014) and Zhang et al., “Purification, Characterization, and cDNA Cloning of an AU-rich Element RNA-binding Protein, AUF1,” Mol. Cell. Biol. 13(12):7652-65 (1993); which are hereby incorporated by reference in their entirety), and tissue stem cell fate-determining mRNAs with extremely short half-lives of 5 to 30 minutes (Chenette et al., “Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,” Cell Rep. 16(5):1379-90 (2016); which is hereby incorporated by reference in its entirety).

[0065] Short-lived mRNAs typically contain AU-rich elements (AREs) in the 3’ untranslated region (3’UTR) of the mRNA and have the repeated sequence AUUUA, which results in rapid decay or, in some cases, stabilization (Moore et al., “Physiological Networks and Disease Functions of RNA-binding Protein AUF1,” Wiley Interdiscip Rev.RNA 5(4):549-64 (2014); which is hereby incorporated by reference in its entirety). AREs function as binding sites for regulatory proteins known as AU-rich binding proteins (AUBPs), which regulate mRNA stability and, in some cases, translation (Moore et al., “Physiological Networks and Disease Functions of RNA-binding Protein AUF1,” Wiley Interdiscip.Rev.RNA 5(4):549-64 (2014); Zhang et al., “Purification, Characterization, and cDNA Cloning of an AU-rich Element RNA-binding Protein, AUF1,” Mol.Cell.Biol. 13(12):7652-65 (1993); and Halees et al., “ARED Organism: Expansion of ARED Reveals AU-rich Element Cluster Variations Between Human And Mouse,” Nucleic Acids Res 36(Database issue):D137-40 (2008); which are hereby incorporated by reference in their entirety).

[0066] AU-rich mRNA-binding factor 1 (AUF1; heterogeneous nuclear ribonucleoprotein D0, hnRNP D0; also known as the HNRNPD gene) binds with high affinity to repeated AU-rich elements located in the 3’UTR present in approximately 5% of mRNAs. AUF1 typically targets ARE-mRNAs for rapid degradation, but conversely, although less well understood, it can stabilize some ARE-mRNAs and increase translation (Moore et al., “Physiological Networks and Disease Functions of RNA-Binding Protein AUF1,” Wiley Interdiscip. Rev. RNA 5(4):549-564 (2014); which is hereby incorporated by reference in its entirety). Mice lacking AUF1 have been previously reported to have accelerated reduction in muscle mass because they are unable to execute the myogenic program (Chenette et al., “Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,” Cell Rep. 16(5):1379-90 (2016); which is hereby incorporated by reference in its entirety).In skeletal muscle, AUF1 expression significantly decreases with aging, and this has been found to greatly contribute to muscle loss and atrophy, reduction in muscle mass, and decline in muscle strength (Abbadi et al., “Muscle Development and Regeneration Controlled by AUF1-mediated Stage-specific Degradation of Fate-determining Checkpoint mRNAs,” Proc. Natl. Acad. Sci. USA 116(23):11285-11290 (2019), and Abbadi et al. “AUF1 Gene Transfer Increases Exercise Performance and Improves Skeletal Muscle Deficit in Adult Mice” Molecular Therapy 22:222-236 (2021); the entireties of which are incorporated herein by reference). Furthermore, AUF1 has also been found to control all major stages of skeletal muscle development starting from satellite cell activation and differentiation lineage determination by selectively targeting major differentiation checkpoint mRNAs that interfere with entry into each subsequent step of muscle development for rapid degradation.

[0067] AUF1 is a group of four related protein isoforms identified by molecular weight (p37 AUF1 , p40 AUF1 , p42 AUF1 , p45 AUF1) exist, and these are obtained by alternative splicing of a single pre-mRNA (Moore et al., “Physiological Networks and Disease Functions of RNA-Binding Protein AUF1,” Wiley Interdiscip. Rev. RNA 5(4):549-564 (2014); Chen & Shyu, “AU-Rich Elements: Characterization and Importance in mRNA Degradation,” Trends Biochem. Sci. 20(11):465-470 (1995); and Kim et al., “Emerging Roles of RNA and RNA-Binding Protein Network in Cancer Cells,” BMB Rep. 42(3):125-130 (2009); the entirety of which are incorporated herein by reference). Each of these four isoforms contains two RNA recognition motifs (“RRMs”) of a centrally located tandem sequence and mediates RNA binding (DeMaria et al., “Structural Determinants in AUF 1 Required for High Affinity Binding to A+U-rich Elements,” J. Biol. Chem. 272:27635-27643 (1997); the entirety of which is incorporated herein by reference).

[0068] The general structure of an RRM is a β-α-β-β-α-β RNA-binding platform of anti-parallel β-sheets supported by α-helices (Zucconi & Wilson, “Modulation of Neoplastic Gene Regulatory Pathways by the RNA-binding Factor AUF1,” Front. Biosci. 16:2307-2325 (2013); Nagai et al., “The RNP Domain: A Sequence-specific RNA-binding Domain Involved in Processing and Transport of RNA,” Trends Biochem. Sci. 20:235-240 (1995); the entirety of which are incorporated herein by reference). The structure of the individual AUF1 RRM domains resolved by NMR is almost identical to this overall tertiary structure (Zucconi & Wilson, “Modulation of Neoplastic Gene Regulatory Pathways by the RNA-binding Factor AUF1,” Front. Biosci. 16:2307-2325 (2013); Nagata et al., “Structure and Interactions with RNA of the N-terminal UUAG-specific RNA-binding Domain of hnRNP D0,” J. Mol. Biol. 287:221-237 (1999); and Katahira et al., “Structure of the C-terminal RNA-binding Domain of hnRNP D0 (AUF1), its Interactions with RNA and DNA, and Change in Backbone Dynamics Upon Complex Formation with DNA,” J. Mol. Biol. 311:973-988 (2001); the entirety of which are incorporated herein by reference).

[0069] Mutations and / or polymorphisms in AUF1 are associated with human limb-girdle muscular dystrophy (LGMD) type 1G (Chenette et al., “Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,” Cell Rep. 16(5):1379-1390 (2016); which is hereby incorporated by reference in its entirety), suggesting that AUF1 is extremely important in the maintenance of postnatal skeletal muscle.

[0070] The term “fragment” or “portion” as used herein with respect to a given polypeptide sequence (e.g., AUF1) refers to a continuous stretch of amino acid sequence of a given polypeptide that is shorter than the full-length sequence of the given polypeptide. A fragment of a polypeptide can be defined by its first position and its last position, which correspond to positions in the sequence of the given full-length polypeptide, respectively. The sequence position corresponding to the first position is located N-terminal to the sequence position corresponding to the last position. The sequence of the fragment or portion is a continuous amino acid sequence or stretch of amino acids in the given polypeptide that begins at the sequence position corresponding to the first position and ends at the sequence position corresponding to the last position. A functional or active fragment is, for example, a fragment that retains a functional characteristic of a native sequence or other reference sequence. Typically, an active fragment is a fragment that retains substantially the same activity as the wild-type protein. A fragment can contain, for example, a functionally important domain such as a domain important for receptor or ligand binding. A functional fragment is at least 10, 15, 20, 50, 75, 100, 150, 200, 250 or 300 contiguous amino acids of full-length AUF1 (including its p37, p40, p42 or p45 isoforms) and retains one or more AUF1 functions.

[0071] Thus, in certain embodiments, the functional fragments of AUF1 described herein include at least one RNA recognition motif (RRM) domain. In certain embodiments, the functional fragments of AUF1 described herein include two RRM domains.

[0072] The AUF1 or functional fragments thereof described herein can be derived from mammalian AUF1. In one embodiment, AUF1 or a functional fragment thereof is human AUF1 or a functional fragment thereof. In another embodiment, AUF1 or a functional fragment thereof is mouse AUF1 or a functional fragment thereof. The AUF1 protein according to the embodiments described herein is the p37 isoform of AUF1 AUF1 , p40 AUF1 , p42 AUF1 , and p45 AUF1 and may include one or more of. The GenBank accession numbers corresponding to the nucleotide and amino acid sequences of each human and mouse isoform are found in Table 1 below, each of which is hereby incorporated by reference in its entirety.

[0073] (Table 1) Summary of GenBank accession numbers for AUF1 sequences TIFF2025523953000002.tif75163

[0074] The sequences referred to in Table 1 are reproduced below.

[0075] Human p37 of GenBank accession number NM_001003810.1 (SEQ ID NO: 1) AUF1 The nucleotide sequence is as follows: TIFF2025523953000003.tif207154

[0076] Human p37 of GenBank accession number NP_001003810.1 (SEQ ID NO: 2) AUF1 The amino acid sequence is as follows: TIFF2025523953000004.tif27152

[0077] Human p40 of GenBank accession number NM_002138.3 (SEQ ID NO: 5) AUF1 The nucleotide sequence is as follows: TIFF2025523953000005.tif212154

[0078] Human p40 of GenBank accession number NP_002129.2 (SEQ ID NO: 6) AUF1 The amino acid sequence is as follows: TIFF2025523953000006.tif33152

[0079] Human p42 of GenBank accession number NM_031369.2 (SEQ ID NO: 9) AUF1 The nucleotide sequence is as follows: TIFF2025523953000007.tif219154

[0080] Human p42 of GenBank accession number NP_112737.1 (SEQ ID NO: 10) AUF1 The amino acid sequence is as follows: TIFF2025523953000008.tif33152

[0081] Human p45 of GenBank accession number NM_031370.2 (SEQ ID NO: 13) AUF1 The nucleotide sequence is as follows: TIFF2025523953000009.tif224154

[0082] Human p45 of GenBank accession number NP_112738.1 (SEQ ID NO: 14) AUF1 The amino acid sequence is as follows: TIFF2025523953000010.tif33152

[0083] Mouse p37 with GenBank accession number NM_001077267.2 (SEQ ID NO: 3) AUF1 The nucleotide sequence is as follows: TIFF2025523953000011.tif51152TIFF2025523953000012.tif243154TIFF2025523953000013.tif243154TIFF2025523953000014.tif117154

[0084] Mouse p37 with GenBank accession number NP_001070735.1 (SEQ ID NO: 4) AUF1 The amino acid sequence is as follows: TIFF2025523953000015.tif27152

[0085] Mouse p40 with GenBank accession number NM_007516.3 (SEQ ID NO: 7) AUF1 The nucleotide sequence is as follows: TIFF2025523953000016.tif57152TIFF2025523953000017.tif243154TIFF2025523953000018.tif243154TIFF2025523953000019.tif117154

[0086] Mouse p40 with GenBank accession number NP_031542.2 (SEQ ID NO: 8) AUF1 The amino acid sequence is as follows: TIFF2025523953000020.tif33152

[0087] Mouse p42 with GenBank accession number NM_001077266.2 (SEQ ID NO: 11) AUF1 The nucleotide sequence is as follows: TIFF2025523953000021.tif51152TIFF2025523953000022.tif243154TIFF2025523953000023.tif243154TIFF2025523953000024.tif129154

[0088] Mouse p42 of GenBank accession number NP_001070734.1 (SEQ ID NO: 12) AUF1 The amino acid sequence is as follows: TIFF2025523953000025.tif33152

[0089] Mouse p45 of GenBank accession number NM_001077265.2 (SEQ ID NO: 15) AUF1 The nucleotide sequence is as follows: TIFF2025523953000026.tif39152TIFF2025523953000027.tif243154TIFF2025523953000028.tif243154TIFF2025523953000029.tif147154

[0090] Mouse p45 of GenBank accession number NP_001070733.1 (SEQ ID NO: 16) AUF1 The amino acid sequence is as follows: TIFF2025523953000030.tif33152

[0091] It should be noted that the sequences described in this specification can be described with reference to accession numbers, including, for example, coding or protein sequences that contain or do not contain additional sequence elements or portions (e.g., leader sequences, tags, immature portions, control regions, etc.), as provided in Table 1. Thus, a reference to such a sequence accession number or corresponding sequence identification number refers to either the sequence fully described therein or some portion thereof (e.g., the portion encoding a protein or polypeptide of interest for the technology described herein (e.g., AUF1 or a functional fragment thereof); the mature protein sequence described within a longer amino acid sequence; the control region of interest (e.g., promoter sequence or regulatory element) disclosed within a longer sequence described herein, etc.). Similarly, variants and isoforms of the accession numbers and corresponding sequence identification numbers described herein are also contemplated.

[0092] Accordingly, in certain embodiments, the AUF1 protein referred to herein has the amino acid sequences set forth in Table 1 and the sequences disclosed herein, or is a functional fragment thereof. In certain embodiments, AUF1 is the p37, p40, p42 or p45 form of human AUF1, having the amino acid sequences of SEQ ID NO: 2, 6, 10, or 14, respectively. In other embodiments, AUF1 is the p37, p40, p42 or p45 form of mouse AUF1, having the amino acid sequences of SEQ ID NO: 4, 8, 12, or 16, respectively. In certain embodiments, AUF1 has 90%, 95% or 99% sequence identity to the amino acid sequences of SEQ ID NO: 2, 6, 10, or 14 and has AUF1 functional activity. In certain embodiments, AUF1 has 90%, 95% or 99% sequence identity to the amino acid sequences of SEQ ID NO: 4, 8, 12, or 16 and has AUF1 functional activity. In one embodiment, the functional fragment referred to herein, in the case of human AUF1, has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequences of SEQ ID NO: 2, 6, 10, or 14, or, in other embodiments, in the case of mouse AUF1, has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to the amino acid sequences of SEQ ID NO: 4, 8, 12, or 16.

[0093] Also provided are nucleic acids comprising a nucleotide sequence encoding a human AUF1 protein or a functional fragment thereof, for example, the nucleotide sequences of SEQ ID NO: 1, 5, 9, or 13. Also provided are nucleic acids comprising a nucleotide sequence having 80%, 85%, 90%, 95%, or 99% sequence identity to one of the nucleotide sequences of SEQ ID NO: 1, 5, 9, or 13 and encoding a human AUF1 protein or a functional fragment thereof having the amino acid sequence of SEQ ID NO: 2, 6, 10, or 14. A codon-optimized sequence encoding the AUF1 protein is provided, including that the codon-optimized version of the human p40 AUF1 coding sequence is the nucleotide sequence of SEQ ID NO: 17. Also provided are nucleic acids comprising a nucleotide sequence having 80%, 85%, 90%, 95%, or 99% sequence identity to one of the nucleotide sequences of SEQ ID NO: 3, 7, 11, or 15 and encoding a mouse AUF1 protein or a functional fragment thereof having the amino acid sequence of SEQ ID NO: 4, 8, 12, or 16.

[0094] In some embodiments, the AAV vectors and virus particles described herein comprise a nucleic acid molecule comprising a nucleotide sequence described in Table 1 (or described herein) or a portion thereof encoding a functional fragment of the AUF1 protein described above, for expression in a cell of a subject, particularly a muscle cell of a subject, and particularly in the expression cassette described herein.

[0095] 2.2. AUF Gene Cassette Another aspect provided herein relates to a nucleic acid expression cassette comprising, for example, in muscle cells, a regulatory element, such as a promoter element, for enhancing or promoting the expression of a nucleic acid encoding AUF1 or a functional fragment thereof, and optionally an enhancer element and / or an intron, operably linked to a nucleic acid encoding AUF1 (including human p37, p40, p42 or p45 AUF1 and combinations thereof) or a functional fragment thereof. The expression cassette or transgene provided herein may comprise a nucleotide sequence encoding a human AUF1 protein having the amino acid sequence of SEQ ID NO: 2, 6, 10, or 14 or a functional fragment thereof (alternatively, for example, in a mouse model study, the expression cassette comprises a nucleotide sequence encoding a mouse AUF1 protein having the amino acid sequence of SEQ ID NO: 4, 8, 12, or 16 or a functional fragment thereof). In embodiments, the nucleotide sequence encoding human AUF1 is SEQ ID NO: 1, 5, 9, or 13 (or the nucleotide sequence encoding mouse AUF1 is SEQ ID NO: 3, 7, 11, or 15). In certain embodiments, the nucleotide sequence encodes human p40 AUF1 and is SEQ ID NO: 17, which is codon and CpG optimized. In certain embodiments, the AUF1 protein has only 1, 2, 3, 4, 5, 10, 15 amino acid substitutions, including conservative substitutions, relative to the amino acid sequence of SEQ ID NO: 2, 6, 10, or 14 or a functional fragment thereof (alternatively, for example, in a mouse model study, relative to the amino acid sequence of SEQ ID NO: 12, 16, 20 or 24), wherein the AUF1 protein has one or more AUF1 functions. In embodiments, the regulatory control element comprises a promoter and may be constitutive or tissue-specific, i.e., active only (or substantially more active or significantly more active) in the target cell / tissue. In particular, promoters and other regulatory elements that promote muscle-specific expression, such as those of Table 8 below, are provided.In embodiments including use as a transgene in a recombinant AAV particle, the expression cassette or transgene is flanked by terminal inverted repeats (ITRs) (e.g., including the form of the ITR for an AAV2 ITR, single-stranded AAV genome or self-complementary AAV genome). For example, the 5' ITR sequence and the 3' ITR sequence are SEQ ID NO: 28 and SEQ ID NO: 29, respectively. In one embodiment, the 5' ITR is mutated for use in a self-complementary vector and may have, for example, the nucleotide sequence of SEQ ID NO: 30.

[0096] 2.2.1. Codon optimization and CpG removal In one aspect, the nucleotide sequence encoding AUF1 is modified by codon optimization and removal of CpG dinucleotides and CpG islands. The immune response to transgenes is a concern in human clinical applications. The immune response to AAV can be suppressed by reducing the number of CpG dinucleotides in the AAV genome (Faust et al., “CpG-Depleted Adeno-Associated Virus Vectors Evade Immune Detection,” J. Clin. Invest. 123(7):2994-3001 (2013); which is hereby incorporated by reference in its entirety). By removing CpG motifs from the transgene sequence, the role of TLR9 in the activation of innate immunity upon recognition of the transgene as non-self is reduced, resulting in stable and long-term transgene expression (see also Wang et al., “Adeno-Associated Virus Vector as a Platform for Gene Therapy Delivery,” Nat. Rev. Drug Discov. 18(5):358-378 (2019); and Rabinowitz et al., “Adeno-Associated Virus (AAV) versus Immune Response,” Viruses 11(2) (2019), which are hereby incorporated by reference in their entirety). In embodiments, the AUF1 nucleotide sequence and expression cassette are human codon-optimized along with CpG removal. The nucleotide sequence that has been codon-optimized and CpG-removed can be designed by any method known in the art, including, for example, the Thermo Fisher Scientific GeneArt Gene Synthesis tool (Waltham, MA USA) that utilizes GeneOptimizer. The nucleotide sequence of SEQ ID NO: 17 described herein represents a sequence that has been codon-optimized and CpG-removed.

[0097] 2.2.2. AUF1 rAAV Genome Construct Provided is a construct useful as a cis plasmid for an rAAV construct comprising a nucleotide sequence encoding AUF1 (including its p37, p40, p42 or p45 isoforms, including those of mouse and human) operably linked to a regulatory sequence that promotes AUF1 expression in muscle cells.

[0098] Provided herein is an rAAV genomic construct comprising an AUF1 transgene comprising a codon-optimized, CpG-depleted human AUF1 p40 coding sequence of SEQ ID NO: 17 operably linked to a regulatory sequence that promotes expression in muscle cells. In certain embodiments, the construct has a muscle-specific promoter that can be Spc5-12 (Spc5v1 or Spc5v2, including SEQ ID NOs: 127 and 128, respectively, which are modified Spc5-12 promoters disclosed herein), tMCK or CK7 (see also Table 8 herein for promoters), and optionally, an intron sequence, such as the VH4 intron (see Table 9 for intron sequences), between the promoter and the AUF1 coding sequence, a polyA signal sequence, such as the rabbit beta-globin polyA signal sequence (SEQ ID NO: 23), and optionally, a WPRE sequence (SEQ ID NO: 24). The construct also includes a 5' stuffer sequence and / or a 3' stuffer sequence (SEQ ID NOs: 26 and 27 in Table 2, or any stuffer sequence known in the art, such as the stuffer sequences disclosed in Table 10 below), and may include an SV40 polyadenylation signal sequence that is in reverse orientation to the coding sequence and adjacent to the 3' ITR sequence. In certain embodiments, the construct has one or more components of Table 2.

[0099] (Table 2) Components of the AUF1 construct TIFF2025523953000031.tif219166TIFF2025523953000032.tif247166TIFF2025523953000033.tif247166TIFF2025523953000034.tif112166

[0100] In some embodiments, the rAAV genome comprises the following components: (1) AAV terminal inverted repeats flanking an expression cassette; (2) regulatory control elements, e.g., a) a promoter / enhancer, b) a polyA signal, and c) optionally an intron; and (3) a nucleic acid sequence encoding AUF1. In a specific embodiment, the construct described herein provides a transgene comprising the following components: (1) AAV2 or AAV8 terminal inverted repeats (ITRs) flanking an expression cassette; (2) a control element comprising a muscle-specific Spc5-12 promoter, tMCK promoter or CK7 promoter and a polyA signal comprising a rabbit beta globin polyA signal; and (3) a nucleic acid encoding AUF1 as described herein, comprising a codon-optimized, CpG-depleted AUF1 p40 coding sequence (e.g., encoding). In a specific embodiment, an rAAV AUF1 construct is provided that comprises the following components: (1) AAV2 or AAV8 ITRs flanking an expression cassette; (2) a control element comprising a) a muscle-specific Spc5-12 promoter, tMCK promoter or CK7 promoter; b) an intron (e.g., VH4) and c) a polyA signal sequence such as a rabbit beta globin polyA signal sequence; and (3) a nucleotide sequence encoding AUF1 as described herein, comprising a codon-optimized, CpG-depleted AUF1 p40 coding sequence (SEQ ID NO: 17). Optionally, the construct comprises a WPRE element 3' of the coding sequence and 5' of the polyA signal sequence. The construct may also include 5' and 3' "stuffer sequences" between the ITR sequence and an expression cassette comprising the coding sequence and regulatory sequences operably linked to the coding sequence, and an SV40 polyA signal sequence adjacent to the 5' side of the 3' ITR sequence. In certain embodiments, the vector is single-stranded and has 5' and 3' ITRs, provided in Table 2 as SEQ ID NO: 28 and SEQ ID NO: 29, respectively. In certain other embodiments, the vector is a self-complementary vector and has a modified 5' ITR, mITR, e.g., that of SEQ ID NO: 30 provided in Table 2, and a 3' ITR such as SEQ ID NO: 29.

[0101] The exemplary rAAV genomes and sequences contained within the cis plasmids are shown in FIG. 1 and Table 3 and include the following:

[0102] spc-hu-opti-AUF1-CpG(-): Codon-optimized, CpG-depleted human AUF1 sequence (including 5’ (141bp) stuffer and 3’ (893bp) stuffer) driven by the Spc5-12 promoter + VH4 intron - downstream SV40 polyA signal (inverted); has the nucleotide sequence of SEQ ID NO: 31 (including the ITR sequence).

[0103] tMCK-huAUF1: Codon-optimized, CpG-depleted human AUF1 sequence (including 5’ (141bp) stuffer and 3’ (893bp) stuffer) driven by the tMCK promoter (without intron) - downstream SV40 polyA signal (inverted); has the nucleotide sequence of SEQ ID NO: 32 (including the ITR sequence).

[0104] spc5-12-hu-opti-AUF1-WPRE: Codon-optimized, CpG-depleted human AUF1 sequence driven by the Spc5-12 promoter + VH4 intron, 3’ WPRE upstream of the polyA (including 5’ (141bp) stuffer and 3’ (893bp) stuffer) - downstream SV40 polyA signal (inverted); SEQ ID NO: 33 (including the ITR sequence).

[0105] ss-CK7-Hu-AUF1: Codon-optimized, CpG-depleted human AUF1 sequence (including 5’ (141bp) stuffer and 3’ (893bp) stuffer) driven by the CK7 promoter (without intron) - downstream SV40 polyA signal (inverted); SEQ ID NO: 34 (including the ITR sequence).

[0106] spc-hu-AUF1 without intron: Codon-optimized, CpG-depleted human AUF1 sequence (including 5’ (141bp) and 3’ (893bp) stuffers) driven by the Spc5-12 promoter (without intron) - downstream SV40 polyA signal (inverted); SEQ ID NO: 35 (including ITR sequence).

[0107] D(+)-CK7AUF1: Self-complementary vector; Codon-optimized, CpG-depleted human AUF1 sequence driven by the CK7 promoter (without stuffer); SEQ ID NO: 36 (including ITR sequence).

[0108] The nucleotide sequences of these AUF1 constructs are provided in Table 3.

[0109] (Table 3) TIFF2025523953000035.tif241161TIFF2025523953000036.tif246161TIFF2025523953000037.tif246161TIFF2025523953000038.tif246161TIFF2025523953000039.tif246161TIFF2025523953000040.tif246161TIFF2025523953000041.tif246161TIFF2025523953000042.tif184161

[0110] These recombinant genomes encoding AUF1, and cis plasmid vectors containing these sequences used to produce AAV particles or rAAV particles, comprising AAV8 serotype, AAV9 serotype or AAVhu.32 serotype particles described herein, are provided, which are useful in methods for treating, preventing, or ameliorating a disease or disorder in a subject, including a human subject in need thereof, by promoting or increasing muscle mass, muscle function or performance, and / or decreasing or reversing muscle atrophy, including types of LGMD, further described herein. In a further embodiment, these AAV genomes or rAAV genomes, and AAV particles or rAAV particles produced from these cis plasmids containing the sequences described herein including the sequences of Table 3, are administered in combination with AAV or rAAV containing a transgene encoding a DAPC protein component, such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan and δ-sarcoglycan, for the treatment of dystrophinopathies including limb-girdle muscular dystrophy (LGMD) in a subject, including a human subject in need thereof. In other embodiments, an AAV or rAAV gene therapy vector comprising a transgene encoding AUF1, comprising the AAV genome or rAAV genome of Table 3, is administered in combination with another therapy effective to treat LGMD, including sarcoglycanopathies, calpainopathies, dysferlinopathies, and dystrophinopathies, including those described herein, to provide a method for treating LGMD, including sarcoglycanopathies, calpainopathies, dysferlinopathies, and dystrophinopathies, in a subject, including a human subject in need thereof.

[0111] 3. Microdystrophin Vector 3.1. Microdystrophin Encoded by the Transgene In some embodiments, one of the transgenes provided herein for the methods of the invention encodes a micro-dystrophin consisting of dystrophin domains arranged from amino-terminus to carboxy-terminus as ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, where ABD is the actin-binding domain of dystrophin, H1 is the hinge 1 region of dystrophin, R1 is the spectrin 1 region of dystrophin, R2 is the spectrin 2 region of dystrophin, R3 is the spectrin 3 region of dystrophin, H3 is the hinge 3 region of dystrophin, R24 is the spectrin 24 region of dystrophin, H4 is the hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT is the C-terminal domain (including at least a portion of the CT domain containing the α1-syntrophin binding site (including SEQ ID NO: 50)).

[0112] The amino acid sequence of the minimal α-syntrophin binding site (SEQ ID NO: 50) is as follows: MENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQ

[0113] The present disclosure contemplates variants of micro-dystrophin, but only when the therapeutic efficacy of micro-dystrophin containing such variants is substantially maintained. Functional activity includes (1) binding to one, a combination, or all of actin, β-dystroglycan, α1-syntrophin, α-dystrobrevin, and nNOS; (2) improvement of muscle function in an animal model (e.g., mdx mouse model) or a human subject; and / or (3) cardiac protection or improvement of cardiac function in an animal model or a human patient.

[0114] Table 4 provides the amino acid sequences of embodiments of microdystrophin according to the present disclosure. In certain embodiments, microdystrophin has the amino acid sequence of SEQ ID NO: 176 (DYS1), 177 (DYS3), or 178 (DYS5). In other embodiments, microdystrophin has the amino acid sequence of SEQ ID NO: 179 (human MD1 (R4-R23 / ΔCT), SEQ ID NO: 180 (microdystrophin), SEQ ID NO: 181 (Dys3978), SEQ ID NO: 182 (MD3), or SEQ ID NO: 183 (MD4). Also, other embodiments are contemplated to be substitution variants of microdystrophin defined by SEQ ID NO: 176 (DYS1), 177 (DYS3), or 178 (DYS5). For example, conservative substitutions can be made to SEQ ID NO: 176, 177, or 178 (or SEQ ID NOs: 179-183) and its functional activity can be substantially maintained. In embodiments, microdystrophin can have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 176, 177, or 178 (or SEQ ID NO: 183) and can maintain functional microdystrophin activity, as determined by one or more of the in vitro assays or in vivo assays in the animal models disclosed below.

[0115] (Table 4) Amino Acid Sequences of RGX-DYS and Microdystrophin Proteins TIFF2025523953000043.tif150165TIFF2025523953000044.tif246165TIFF2025523953000045.tif246165TIFF2025523953000046.tif246165TIFF2025523953000047.tif188165

[0116] 3.2. Nucleic Acid Compositions Encoding Microdystrophin Another aspect of the disclosure is a nucleic acid comprising a nucleotide sequence encoding the microdystrophin described herein. Such a nucleic acid comprises a nucleotide sequence encoding a microdystrophin having domains arranged from the N-terminus to the C-terminus as follows: ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT, as detailed above. The nucleotide sequence can be any nucleotide sequence encoding the domains. The nucleotide sequence can be codon-optimized and / or have CpG islands removed for expression in appropriate contexts.

[0117] In various embodiments, the nucleic acid comprises a nucleotide sequence encoding a microdystrophin having the amino acid sequence of SEQ ID NO: 176, SEQ ID NO: 177, or SEQ ID NO: 178.

[0118] 3.2.1. Codon Optimization and CpG Removal In one aspect, the nucleotide sequence encoding the microdystrophin cassette is modified by codon optimization and removal of CpG dinucleotides and CpG islands. The immune response to the microdystrophin transgene is a concern in human clinical applications, as demonstrated in the first Duchenne muscular dystrophy (DMD) gene therapy clinical trial and several adeno-associated virus (AAV)-mini-dystrophin gene therapies in canine models [Mendell, J.R., et al., Dystrophin immunity in Duchenne’s muscular dystrophy. J. Med. 363(15):1429-37(2010); and Kornegay et al., “Widespread Muscle Expression of an AAV9 Human Mini-Dystrophin Vector after Intravenous Injection in Neonatal Dystrophin-Deficient Dogs.” Mol. Ther. 18(8):1501-1508(2010); which are incorporated herein by reference in their entirety].

[0119] In an embodiment, the microdystrophin cassette is human codon-optimized along with CpG depletion. The nucleotide sequence that has been codon-optimized and depleted of CpG can be designed by any method known in the art, including, for example, the Thermo Fisher Scientific GeneArt Gene Synthesis tool (Waltham, MA USA) that utilizes GeneOptimizer. The nucleotide sequences of SEQ ID NOs: 91, 92, and 93 described herein represent sequences that have been codon-optimized and depleted of CpG.

[0120] The amino acid sequence of DYS1 (SEQ ID NO: 91) is as follows:

[0121] The amino acid sequence of DYS3 (SEQ ID NO: 92) is as follows:

[0122] The amino acid sequence of DYS5 (SEQ ID NO: 93) is as follows:

[0123] There is provided a microdystrophin transgene in which the number of CpG dinucleotide sequences is reduced, and as a result, the number of CpG islands is decreased. In certain embodiments, the microdystrophin nucleotide sequence has less than two (2) CpG islands, or one (1) CpG island, or zero (0) CpG islands. In embodiments, there is provided a microdystrophin transgene having less than two or one CpG island, or zero CpG islands, with reduced immunogenicity as measured by anti-drug antibody titer, compared to a microdystrophin transgene having more than two CpG islands. In certain embodiments, the microdystrophin nucleotide sequence consisting essentially of SEQ ID NO: 91, 92, or 93 has zero (0) CpG islands. In other embodiments, the microdystrophin transgene nucleotide sequence consisting essentially of a microdystrophin gene operably linked to a promoter has less than two (2) CpG islands, and the microdystrophin consists of SEQ ID NO: 91, 92, or 93. In yet other embodiments, the microdystrophin transgene nucleotide sequence consisting essentially of a microdystrophin gene operably linked to a promoter has one (1) CpG island, and the microdystrophin consists of SEQ ID NO: 91, 92, or 93.

[0124] 3.2.2. Microdystrophin transgene construct There are provided microdystrophin transgene constructs and artificial rAAV genomes for use in the methods disclosed herein. The transgene comprises a nucleotide sequence encoding a microdystrophin disclosed herein, operably linked to a transcriptional regulatory sequence comprising a promoter that promotes expression in muscle cells and other regulatory sequences that promote expression of microdystrophin. The transgene is flanked by AAV ITR sequences.

[0125] In some embodiments, the rAAV genome comprises a vector comprising the following components: (1) AAV terminal inverted repeats flanking an expression cassette; (2) regulatory control elements, e.g., a) a promoter / enhancer, b) a polyA signal, and c) optionally an intron; and (3) a nucleic acid sequence encoding microdystrophin. In a specific embodiment, the construct described herein comprises the following components: (1) AAV2 or AAV8 terminal inverted repeat (ITR) flanking an expression cassette; (2) control elements comprising a muscle-specific Spc5-12 promoter and a small polyA signal; and (3) a microdystrophin-encoding nucleic acid as described herein, comprising a microdystrophin-encoding sequence of the RGX-DYS1 transgene (SEQ ID NO: 91) or the RGX-DYS5 transgene (SEQ ID NO: 93) (e.g., encoding). In a specific embodiment, the construct described herein comprises the following components: (1) AAV2 or AAV8 ITR flanking an expression cassette; (2) control elements comprising a) a muscle-specific Spc5-12 promoter, b) a small polyA signal; and (3) a microdystrophin cassette comprising ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT from N-terminus to C-terminus, wherein CT comprises a portion of CT that includes at least the α1-syntrophin binding site and comprises a CT having the amino acid sequence of SEQ ID NO: 48 or 49. In a specific embodiment, the construct described herein comprises the following components: (1) AAV2 or AAV8 ITR flanking an expression cassette; (2) control elements comprising a) a muscle-specific Spc5-12 promoter, b) an intron (e.g., VH4) and c) a small polyA signal; and (3) a microdystrophin cassette comprising ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT from N-terminus to C-terminus, wherein CT comprises a portion of CT that includes at least the α1-syntrophin binding site and comprises a CT having the amino acid sequence of SEQ ID NO: 48 or 49, and ABD1 is directly bound to VH4.

[0126] The amino acid sequence of the C-terminal domain (CT) (SEQ ID NO: 48) is as follows: TIFF2025523953000048.tif26163 (The coiled-coil motif H1 is shown in bold, motif H2 is shown in lowercase, and the dystrobrevin binding side is shown in italics).

[0127] The amino acid sequence of the minimal / truncated C-terminal domain (CT1.5) (SEQ ID NO: 49) is as follows: TIFF2025523953000049.tif18163 (The 1-syntrophin binding site is shown in italics).

[0128] In a specific embodiment, the construct described herein comprises the following components: (1) AAV2 ITRs flanking an expression cassette; (2) a muscle-specific Spc5-12 promoter (or a promoter such as the modified Spc5-12 promoter SPc5v1 or SPc5v2 (SEQ ID NO: 127 or 128), and b) control elements including a small polyA signal; and (3) a nucleic acid encoding AUF1. In some embodiments, the construct described herein comprises AAV ITRs flanking an AUF1 expression cassette comprising one or more of the AUF1 sequences disclosed herein.

[0129] In certain embodiments, the constructs described herein comprise the following components: (1) AAV2 ITRs flanking an expression cassette; (2) a muscle-specific Spc5-12 promoter (or modified Spc5-12 promoters SPc5v1 or SPc5v2 (SEQ ID NO: 127 or 128)), and b) control elements including a small polyA signal; and (3) a nucleic acid encoding an RGX-DYS1 microdystrophin having the amino acid sequence of SEQ ID NO: 176, which is encoded by the nucleotide sequence of SEQ ID NO: 91. In certain embodiments, the constructs described herein comprise the following components: (1) AAV2 ITRs flanking an expression cassette; (2) a muscle-specific Spc5-12 promoter, and b) control elements including a small polyA signal; and (3) a nucleic acid encoding an RXG-DYS5 microdystrophin having the amino acid sequence of SEQ ID NO: 178, which is encoded by the nucleotide sequence of SEQ ID NO: 93. In some embodiments, the constructs described herein comprising AAV ITRs flanking a microdystrophin expression cassette comprise ABD1-H1-R1-R2-R3-H2-R24-H4-CR-CT from the N-terminus to the C-terminus, where CT comprises a portion of CT that includes at least an α1-syntrophin binding site, comprises a CT having the amino acid sequence of SEQ ID NO: 48 or 49, and can be 4000 nt to 5000 nt in length. In some embodiments, such constructs are less than 4900 nt, 4800 nt, 4700 nt, 4600 nt, 4500 nt, 4400 nt, or 4300 nt in length.

[0130] Some nucleic acid embodiments of the present disclosure include an AAV vector or rAAV vector encoding dystrophin that comprises or consists of the nucleotide sequences of SEQ ID NO: 184, 185, or 186 provided in Table 5 below. In various embodiments, an AAV vector or rAAV vector that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequences of SEQ ID NO: 184, 185, or 186 or their reverse complementary strands, and that encodes a nucleotide sequence for an AAV vector or rAAV vector suitable for therapeutically effective expression of dystrophin in muscle cells. In embodiments, a construct having the nucleotide sequence of SEQ ID NO: 184, 185, or 186 is within a recombinant rAAV8 particle or a recombinant AAV9 particle.

[0131] (Table 5) RGX-DYS cassette nucleotide sequence TIFF2025523953000050.tif104163TIFF2025523953000051.tif248163TIFF2025523953000052.tif248163TIFF2025523953000053.tif248163TIFF2025523953000054.tif248163TIFF2025523953000055.tif248163TIFF2025523953000056.tif247163TIFF2025523953000057.tif248163TIFF2025523953000058.tif248163TIFF2025523953000059.tif248163TIFF2025523953000060.tif248163TIFF2025523953000061.tif248163TIFF2025523953000062.tif248163TIFF2025523953000063.tif148163

[0132] 4. α-, β-, γ- or δ-sarcoglycan vector 4.1. α-, β-, γ-, or δ-Sarcoglycan Encoded by the Introduced Gene Table 6 provides the amino acid sequences of embodiments of α-, β-, γ-, and δ-sarcoglycan according to the disclosure. Other embodiments are contemplated to be substitution variants of α-, β-, γ-, or δ-sarcoglycan defined by SEQ ID NO: 144 (α-sarcoglycan), 52 (α-sarcoglycan), 145 (β-sarcoglycan), 52 (β-sarcoglycan), 146 (γ-sarcoglycan), 54 (γ-sarcoglycan), 94 (γ-sarcoglycan), 95 (γ-sarcoglycan), 96 (γ-sarcoglycan), 147 (δ-sarcoglycan), or 129 (δ-sarcoglycan). For example, conservative substitutions can be made to SEQ ID NO: 144, 52, 145, 52, 146, 54, 94, 95, 96, 147, or 129, and their functional activity can be substantially maintained. In embodiments, the sarcoglycan can have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequences of SEQ ID NO: 144, 52, 145, 52, 146, 54, 94, 95, 96, 147, or 129 and can maintain functional sarcoglycan activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in the animal models disclosed below.

[0133] (Table 6) Amino Acid Sequences of Sarcoglycan Proteins TIFF2025523953000064.tif255160TIFF2025523953000065.tif216164TIFF2025523953000066.tif84164*The NCBI reference sequences are hereby incorporated by reference in their entirety.

[0134] 4.2. Nucleic Acid Compositions Encoding α-, β-, γ-, or δ-Sarcoglycan Another aspect of the disclosure is a nucleic acid comprising a nucleotide sequence encoding an α-, β-, γ-, or δ-sarcoglycan as described herein. The nucleotide sequence can be any nucleotide sequence encoding an α-, β-, γ-, or δ-sarcoglycan. The nucleotide sequence can be codon optimized and / or have CpG islands removed for expression in an appropriate context.

[0135] Table 7 provides nucleic acid sequences encoding embodiments of α-, β-, γ-, and δ-sarcoglycans according to the disclosure. Other embodiments are contemplated to be substitution variants of α-, β-, γ-, or δ-sarcoglycan defined by SEQ ID NO: 130 (α-sarcoglycan), 131 (α-sarcoglycan), 132 (β-sarcoglycan), 133 (β-sarcoglycan), 134 (γ-sarcoglycan), 135 (γ-sarcoglycan), 136 (γ-sarcoglycan), 137 (γ-sarcoglycan), 148 (γ-sarcoglycan), 149 (δ-sarcoglycan), or 150 (δ-sarcoglycan). For example, conservative substitutions can be made to SEQ ID NO: 130, 131, 132, 133, 134, 135, 136, 137, 148, 149, or 150 and its functional activity can be substantially maintained. In embodiments, the sarcoglycan can have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 130, 131, 132, 133, 134, 135, 136, 137, 148, 149, or 150 and can maintain functional sarcoglycan activity as determined by, for example, one or more of the in vitro or in vivo assays in the animal models disclosed below.

[0136] (Table 7) Nucleic Acid Sequences of Sarcoglycan Proteins TIFF2025523953000067.tif239161TIFF2025523953000068.tif246161TIFF2025523953000069.tif250161TIFF2025523953000070.tif246161TIFF2025523953000071.tif246161TIFF2025523953000072.tif246161TIFF2025523953000073.tif246161TIFF2025523953000074.tif246161TIFF2025523953000075.tif250161TIFF2025523953000076.tif250161TIFF2025523953000077.tif250161TIFF2025523953000078.tif250161TIFF2025523953000079.tif246161TIFF2025523953000080.tif246161TIFF2025523953000081.tif250161TIFF2025523953000082.tif250161TIFF2025523953000083.tif250161TIFF2025523953000084.tif246161TIFF2025523953000085.tif250161TIFF2025523953000086.tif250161TIFF2025523953000087.tif241161*The NCBI reference sequence is hereby incorporated by reference in its entirety.

[0137] 5. Regulatory element The expression cassettes, rAAV genomes or rAAV vectors disclosed herein include a regulatory element, such as a promoter element, for enhancing or promoting the expression of a transgene, and optionally, an enhancer element and / or an intron, operably linked to either AUF1 or a transgene encoding a therapeutic protein. In some embodiments, the AAV vector or rAAV vector also includes regulatory control elements known to those skilled in the art to affect the expression of the RNA and / or protein products encoded by the nucleic acid (transgene) within the target cells of a subject. The regulatory control elements can be tissue-specific, i.e., active only (or substantially more active or significantly more active) in the target cells / tissues.

[0138] 5.1. Promoter 5.1.1. Tissue-Specific Promoter In specific embodiments, the expression cassette of the AAV vector or rAAV vector includes regulatory sequences, such as a promoter operably linked to the transgene, that enable expression in the target tissue. The promoter can be a muscle promoter. In certain embodiments, the promoter is a muscle-specific promoter. The terms "muscle-specific," "muscle-selective," or "muscle-directed" refer to a nucleic acid element whose activity is adapted to muscle cells or muscle tissue by interaction of such element with the intracellular environment of muscle cells. Such muscle cells can include muscle cells, myotube cells, cardiomyocytes, etc. Also included are the specialized forms of muscle cells with distinct properties such as cardiomyocytes, skeletal cells, and smooth muscle cells. Various therapies are thought to benefit from the muscle-specific expression of transgenes. In particular, gene therapy for treating various forms of muscular dystrophy, which is delivered to muscle cells and enables high transduction efficiency, has the additional advantage of inducing the expression of the transgene in the cells where the transgene is most needed. Heart tissue can also benefit from the muscle-directed expression of transgenes. The muscle-specific promoter can be operably linked to the transgenes of the present disclosure.

[0139] The adeno-associated virus (AAV) vectors disclosed herein include a muscle cell-specific promoter operably linked to a nucleic acid encoding a therapeutic protein for the treatment of AUF1 and / or LGMD. In some embodiments, the muscle cell-specific promoter mediates cell-specific and / or tissue-specific expression of the AUF1 protein or a fragment thereof. The promoter can be a mammalian promoter. For example, the promoter can be selected from the group consisting of a human promoter, a mouse promoter, a porcine promoter, a feline promoter, a canine promoter, a sheep promoter, a non-human primate promoter, a equine promoter, a bovine promoter, and the like.

[0140] In some embodiments, the muscle cell-specific promoter is one of a muscle creatine kinase (MCK) promoter, a syn100 promoter, a creatine kinase (CK)6 promoter, a creatine kinase (CK)7 promoter, a dMCK promoter, a tMCK promoter, a smooth muscle 22 (SM22) promoter, a myo-3 promoter, a Spc5-12 promoter, a creatine kinase (CK)8 promoter, a creatine kinase (CK)8e promoter, a creatine kinase (CK)9 promoter, a U6 promoter, an H1 promoter, a desmin promoter, a Pitx3 promoter, a skeletal alpha-actin promoter, an MHCK7 promoter, and a Sp-301 promoter. Individual muscle cell-specific promoter sequences are well known in the art, and exemplary promoters are provided in Table 8 below (Malerba et al., “PABPN1 Gene Therapy for Oculopharyngeal Muscular Dystrophy,” Nat. Commun. 8:14848 (2017); Wang et al., “Construction and Analysis of Compact Muscle-Specific Promoters for AAV Vectors,” Gene. Ther. 15:1489-1499 (2008); Piekarowicz et al., “A Muscle Hybrid Promoter as a Novel Tool for Gene Therapy,” Mol. Ther. Methods Clin. Dev. 15:157-169 (2019); Salva et al., “Design of Tissue-Specific Regulatory Cassettes for High-Level rAAV-Mediated Expression in Skeletal and Cardiac Muscle,” Mol. Ther. 15(2):320-329 (2007); Lui et al.,“Synthetic Promoter for Efficient and Muscle-Specific Expression of Exogenous Genes,”Plasmid 106:102441(2019)、Li et al.,“Synthetic Muscle Promoters:Activities Exceeding Naturally Occurring Regulatory Sequences,”Nature Biotechnology 17:241-245(1999);Liu et al.,“Therapeutic Levels of Factor IX Expression using a Muscle-Specific Promoter and Adeno-Associated Virus Serotype 1 Vector,”Hum.Gene Ther.15:783-792(2004);Draghia-Akli et al.,“Myogenic Expression of an Injectable Protease-Resistant Growth Hormone-Releasing Hormone Augments Long-Term Growth in Pigs,”Nat.Biotechnol.17:1179-1183(1999);Hagstrom et al.,“Improved Muscle-Derived Expression of Human Coagulation Factor IX from a Skeletal Actin / CMV Hybrid Enhancer / Promoter,”Blood 95:2536-2542(2000);Li et al.,“rAAV Vector-Mediated Sarcoglycan Gene Transfer in a Hamster Model for Limb Girdle Muscular Dystrophy,”Gene Therapy 6:74-82(1999);Wang et al.,"Construction and Analysis of Compact Muscle-Specific Promoters for AAV Vectors," Gene Therapy 15:1489-1499(2008); and Qiao et al., "Muscle and Heart Function Restoration in a Limb Girdle Muscular Dystrophy 2I (LGMD2I) Mouse Model by Systemic FKRP Gene Delivery," Mol. Ther. 22(11):1890-1899(2014); the entireties of which are incorporated herein by reference).

[0141] (Table 8) Promoter sequences TIFF2025523953000088.tif232163TIFF2025523953000089.tif208163TIFF2025523953000090.tif232163TIFF2025523953000091.tif246163TIFF2025523953000092.tif151163

[0142] In some embodiments, the muscle cell-specific promoter is the muscle creatine kinase (MCK) promoter. The muscle creatine kinase (MCK) gene is highly active in all striated muscles. Creatine kinase plays an important role in ATP regeneration within the contraction and ion transport systems. Creatine kinase enables muscle contraction in the absence of glycolysis or respiration by transferring a phosphate group from phosphocreatine to ADP to form ATP. There are four known isoforms of creatine kinase: brain creatine kinase (CKB), muscle creatine kinase (MCK), and two mitochondrial types (CKMi). MCK is the most abundant non-mitochondrial mRNA, is expressed in all types of skeletal muscle fibers, and is also highly active in cardiac muscle. The MCK gene is not expressed in myoblasts but shows transcriptional activity as myoblasts undergo terminal differentiation into myocytes. The MCK gene regulatory region shows striated muscle-specific activity and has been extensively characterized in vivo and in vitro. The main regulatory regions known for the MCK gene include a muscle-specific enhancer located approximately 1.1 kb 5′ of the transcription start site in mice and a 358 bp proximal promoter. Additional sequences that regulate MCK expression are distributed over a 3.3 kb region 5′ of the transcription start site and the first intron of 3.3 kb. For mammalian MCK regulatory elements, including human and mouse promoter and enhancer elements, see Hauser et al., “Analysis of Muscle Creatine Kinase Regulatory Elements in Recombinant Adenoviral Vectors,” Mol. Therapy 2:16-25 (2000), which is incorporated herein by reference in its entirety.Suitable muscle creatine kinase (MCK) promoters include, but are not limited to, the wild-type MCK promoter, the dMCK promoter, and the tMCK promoter (Wang et al., “Construction and Analysis of Compact Muscle-Specific Promoters for AAV Vectors,” Gene Ther. 15(22):1489-1499(2008); which is incorporated herein by reference in its entirety).

[0143] In some embodiments, the muscle-specific promoter is selected from the Spc5-12 promoter (SEQ ID NO: 18 or 106) (including SPc5v1 or SPc5v2, which are modified Spc5-12 promoters (SEQ ID NO: 127 or 128, respectively)), the muscle creatine kinase myosin light chain (MLC) promoter, the myosin heavy chain (MHC) promoter, the desmin promoter (human - SEQ ID NO: 98), the MCK7 promoter (SEQ ID NO: 104), the CK6 promoter, the CK8 promoter (SEQ ID NO: 107), the MCK promoter (or a truncated form thereof) (SEQ ID NO: 105 or 21), the alpha-actin promoter, the beta-actin promoter, the gamma-actin promoter, the E-syn promoter, the cardiac troponin C promoter, the troponin I promoter, the myoD gene family promoter, or the muscle-selective promoter present within intron 1 of ocular Pitx3.

[0144] The synthetic promoter c5-12, known as the Spc5-12 promoter (Li et al., “Synthetic Muscle Promoters: Activities Exceeding Naturally Occurring Regulatory Sequences,” Nat. Biotechnol. 17(3):241-245(1999); which is hereby incorporated by reference in its entirety), has been found to have cell type-restricted expression, particularly expression specific to muscle cells. The Spc5-12 promoter is less than 350 bp in length, shorter than most endogenous promoters, and can be advantageous when the nucleic acid encoding the therapeutic protein is relatively long.

[0145] Alternatively, the promoter can be a constitutive promoter, such as the CB7 promoter. Additional promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the Rous sarcoma virus (RSV) promoter, the MMT promoter, the EF-1 alpha promoter (SEQ ID NO: 110), the UB6 promoter, the chicken beta-actin promoter, and the CAG promoter (SEQ ID NO: 108). In some embodiments, an inducible promoter, such as a hypoxia-inducible promoter or a rapamycin-inducible promoter, is used, particularly when it is desirable to turn off transgene expression.

[0146] 5.2. Intron A certain specific gene expression cassette further includes an intron that can promote proper splicing and thus transgene expression, for example, 5' to an AUF1 or a sequence encoding a therapeutic protein (such as dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or the calcium / calmodulin-dependent protein kinase II β isoform protein or a part thereof). Thus, in some embodiments, an intron is ligated to the 5' end of a sequence encoding an AUF1 or a therapeutic protein (such as dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or the calcium / calmodulin-dependent protein kinase II β isoform protein or a part thereof). In certain embodiments, the intron has a length of less than 100 nucleotides.

[0147] In an embodiment, the intron is a VH4 intron. The VH4 intron nucleic acid may include SEQ ID NO: 111 shown in Table 9 below.

[0148] (Table 9) Nucleotide sequences of various introns TIFF2025523953000093.tif67158

[0149] In other embodiments, the intron is a chimeric intron derived from human β-globin and Ig heavy chain (also known as β-globin splice donor / immunoglobulin heavy chain splice acceptor intron, or β-globin / IgG chimeric intron) (Table 9, SEQ ID NO: 112). Other introns well known to those skilled in the art, such as chicken β-actin intron, murine minute virus (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), β-globin splice donor / immunoglobulin heavy chain splice acceptor intron (Table 9, SEQ ID NO: 138), adenovirus splice donor / immunoglobulin splice acceptor intron, SV40 late splice donor / splice acceptor (19S / 16S) intron (Table 9, SEQ ID NO: 113), etc., may be employed.

[0150] 5.3. Other Regulatory Elements Another aspect of the disclosure relates to an expression cassette comprising a polyadenylation (polyA) site downstream of the coding region of a transgene for a therapeutic protein (such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or the calcium / calmodulin-dependent protein kinase II β isoform protein or a portion thereof). Any polyA site that signals the end of transcription and induces the synthesis of a polyA tail is suitable for use in the AAV vectors of the disclosure. Exemplary polyA signals are derived from, but not limited to: SV40 late gene, rabbit β-globin gene, bovine growth hormone (BPH) gene, human growth hormone (hGH) gene, and synthetic polyA (SPA) site. Exemplary polyA signal sequences useful in the constructs described herein are provided in Table 2 above.

[0151] Also provided are constructs comprising a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) that can enhance the expression of the transgene. The WPRE element can be inserted into the 3' untranslated region of the transgene on the 5' side of the polyadenylation signal sequence. See, for example, Zufferey et al, “Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors,” J. Virol. 73(4):2886-2892(1999), which is hereby incorporated by reference in its entirety. In certain embodiments, the WPRE element has the nucleotide sequence of SEQ ID NO: 24 (see Table 2 above).

[0152] Other elements that can be included in the construct are filler or stuffer sequences, particularly incorporated at the 5' and 3' ends between the ITR sequence and the expression cassette sequence to optimize the length of the nucleic acid between the ITR sequences and improve packaging efficiency. The SV40 polyadenylation sequence located adjacent to the ITR sequence can isolate the transcription of the transgene from the interference of the ITR. Exemplary stuffer sequences and SV40 polyA sequences are provided in Table 2 above. Alternative polyA sequences and stuffer sequences are known in the art; see, for example, Table 10.

[0153] Nucleic acids containing a stuffer (or filler) polynucleotide sequence expand the transgene size of any heterologous gene, such as the AUF1 gene in Table 2 or Table 3. In some embodiments, the stuffer (or filler) polynucleotide sequence comprises SEQ ID NO: 26 or 27. In some embodiments, the stuffer (or filler) polynucleotide sequence comprises SEQ ID NOs: 139-143 or a fragment of SEQ ID NOs: 139-143 (see Table 10) that are 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-600, 600-750, 750-1,000, 1,000-1,500, 1,500-1,601 nucleotides in length. In other embodiments, the stuffer polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 143 (see Table 10), or one or more fragments thereof.

[0154] In some embodiments, when combined with a heterologous gene sequence, the stuffer polynucleotide sequence has a combined total length of about 2.4 - 5.2 kb, or about 3.1 - 4.7 kb, when the heterologous gene sequence and the stuffer polynucleotide sequence are combined. The transgene can include, but is not limited to, any one of the genes or nucleic acids encoding the therapeutic AUF1 genes listed in Tables 2 and 3.

[0155] In the case of a stuffer sequence and an enhancer sequence such as an intron, the nucleic acid sequence is operably linked to the transgene either continuously or substantially continuously. Optionally, operably linked can mean that a coding region and a non-coding region, or two coding regions, are linked continuously, for example, within a reading frame. In some cases, for example, in the case of an enhancer that can function even several kilobases away from a promoter, such as an intron sequence and a stuffer sequence, these regulatory sequences can be considered to be operably linked even if they are not directly contiguous with a downstream or upstream promoter and / or a heterologous gene.

[0156] (Table 10) TIFF2025523953000094.tif119161TIFF2025523953000095.tif244161TIFF2025523953000096.tif248161TIFF2025523953000097.tif148161

[0157] 5.4. Reporter gene In some embodiments, the disclosed gene cassette and thus the adeno-associated virus vector contain a nucleic acid molecule encoding a reporter protein. The reporter protein can be selected, for example, from the group consisting of β-galactosidase, chloramphenicol acetyltransferase, luciferase, and fluorescent proteins.

[0158] In certain embodiments, the reporter protein is a fluorescent protein. Suitable fluorescent proteins include, but are not limited to, green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), or any other suitable fluorescent protein. In certain embodiments, the reporter protein is a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), and yellow fluorescent protein (YFP).

[0159] In some embodiments, the reporter protein is luciferase. As used herein, the term "luciferase" refers to a type of enzyme that catalyzes a reaction that generates light. Luciferases have been identified and cloned from a variety of organisms, including, among others, fireflies, click beetles, Renilla (sea pansy), marine shrimps, and bacteria. Examples of luciferases that can be used as reporter proteins include, for example, Renilla (e.g., Renilla reniformis) luciferase, Gaussia (e.g., Gaussia princeps) luciferase, Metridia luciferase, firefly (e.g., Photinus pyralis luciferase), click beetle (e.g., Pyrearinus termitilluminans) luciferase, deep-sea shrimp (e.g., Oplophorus gracilirostris) luciferase. Luciferase reporter proteins include both native proteins and engineered variants, which are designed to have one or more modified properties compared to the native protein, such as improved photostability, improved pH stability, increased fluorescence or luminescence, dimerization, oligomerization, reduced tendency to aggregate or be toxic to cells, modification of the emission spectrum, and / or modification of substrate utilization.

[0160] 5.5. Virus Vector The transgenes encoding AUF1 and other proteins disclosed herein can be included in AAV vectors for administration of gene therapy to human subjects. In some embodiments, an AAV or recombinant AAV (rAAV) vector can comprise an AAV viral capsid and a viral or artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs), the expression cassette being operably linked to one or more regulatory sequences that control the expression of the transgene in human muscle cells to express and deliver the AUF1 protein or optionally other therapeutic proteins, and comprising AUF1 or a coding transgene. The provided methods are useful in the production of any isolated recombinant AAV particles for delivery of the AUF1 protein or other therapeutic proteins described herein, or in the preparation of a composition comprising any isolated recombinant AAV particles encoding the AUF1 protein or other therapeutic proteins, or in a method for treating a disease or disorder comprising an LGMD type suitable for treatment by the AUF1 protein or a combination of the AUF1 protein and another therapeutic protein in a subject in need thereof, the method comprising administration of any isolated recombinant AAV particles encoding the AUF1 protein or administration of a combination (including separate administrations) of rAAV particles encoding the AUF1 protein and rAAV particles encoding another therapeutic protein described herein. Thus, the rAAV can be any serotype, variant, mutant, hybrid, or derivative known in the art, or any combination thereof (collectively referred to as "serotypes"). In certain embodiments, the AAV serotype has tropism for muscle tissue (including skeletal, cardiac, or smooth muscle).

[0161] In some embodiments, the AAV particle or rAAV particle has a capsid protein derived from the AAV8 serotype. In other embodiments, the AAV particle or rAAV particle has a capsid protein derived from the AAV9 serotype. In still other embodiments, the AAV particle or rAAV particle has a capsid protein derived from the hu.32 serotype. In particular, provided are vector spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1 without intron, or AUF1 constructs of D(+)-CK7AUF1 having the nucleotide sequences of SEQ ID NOs: 31-36 in AAV particles or rAAV particles having the AAV8 capsid. Also provided are RGX-DYS1 constructs in AAV particles or rAAV particles having the AAV8 capsid and RGX-DYS1 constructs in AAV particles or rAAV particles having the AAV9 capsid for use in the methods disclosed herein. Also provided are RGX-DYS5 constructs in AAV particles or rAAV particles having the AAV8 capsid and RGX-DYS5 constructs in rAAV particles having the AAV9 capsid.

[0162] In some embodiments, the AAV particle or rAAV particle comprises a capsid protein derived from an AAV capsid serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, or AAV2.5 serotypes, or can be of the AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, or AAVhu.32 serotypes.

[0163] In some embodiments, the AAV particles or rAAV particles comprise a capsid protein that is a derivative, variant, or pseudotype of the AAV8 capsid protein. In some embodiments, the AAV particles or rAAV particles have a capsid protein having a capsid protein that is at least 80% identical, such as 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences (SEQ ID NO: 114) (Table 11) of the AAV8 capsid protein. In some embodiments, the AAV particles or rAAV particles have a capsid protein having a capsid protein that is at least 80% identical, such as 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences (SEQ ID NO: 115) (Table 11) of the AAV9 capsid protein. In some embodiments, the AAV particles or rAAV particles have a capsid protein having a capsid protein that is at least 80% identical, such as 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical, to the VP1, VP2, and / or VP3 sequences (see Table 11) of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74 (SEQ ID NOs: 119 and 120), AAVhu.37 (SEQ ID NO: 116), AAVAAV.hu31 (SEQ ID NO: 117), or AAVhu.32 (SEQ ID NO: 118) serotype capsid proteins.

[0164] The nucleic acid sequences of AAV-based viral vectors and methods for producing recombinant AAVs and AAV capsids are taught, for example, in U.S. Pat. Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9,458,517; and 9,587,282; U.S. Patent Application Publication Nos. 2015 / 0374803; 2015 / 0126588; 2017 / 0067908; 2013 / 0224836; 2016 / 0215024; 2017 / 0051257; International Patent Application Nos. PCT / US2015 / 034799; PCT / EP2015 / 053335; WO2003 / 052051, WO2005 / 033321, WO03 / 042397, WO2006 / 068888, WO2006 / 110689, WO2009 / 104964, WO2010 / 127097, and WO2015 / 191508, as well as U.S. Application Publication No. 20150023924.

[0165] In certain embodiments, single-stranded AAV (ssAAV) can be used. In certain embodiments, self-complementary vectors, such as scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2):171-82, McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16, Pages 1248-1254; and U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety). Self-complementary vectors can include mutant ITR sequences, such as the mutant 5' ITR sequences of Table 2.

[0166] In additional embodiments, the rAAV particles include pseudotyped rAAV particles. In some embodiments, the pseudotyped rAAV particles comprise (a) a nucleic acid vector comprising AAV ITRs, and (b) a capsid composed of an AAVx (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, or AAVhu.32), particularly a capsid protein derived from AAV8. In additional embodiments, the rAAV particles include pseudotyped rAAV particles containing the AAV8 capsid protein. In some embodiments, the pseudotyped rAAV8 particles are rAAV2 / 8 pseudotyped particles.Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., “Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation,” J. Virol. 75(16):7662-7671 (2001); Halbert et al., “Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes,” J. Virol. 74(3):1524-1532 (2000); Zolotukhin et al., “Production and Purification of Serotype 1, 2, and 5 Recombinant Adeno-Associated Viral Vectors,” Methods 28(2):158-167 (2002); and Auricchio et al., “Exchange of Surface Proteins Impacts on Viral Vector Cellular Specificity and Transduction Characteristics: the Retina as a Model,” Hum. Molec. Genet. 10:3075-3081 (2001), each of which is hereby incorporated by reference in its entirety).

[0167] In some embodiments, the AAV particle or rAAV particle comprises an AAV capsid protein chimera of an AAV8 capsid protein and one or more AAV capsid proteins derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, or AAVhu.32.

[0168] In some embodiments, the AAV particle or rAAV particle comprises an AAV capsid protein of Clade A, B, E, or F. In some embodiments, the AAV particle or rAAV particle comprises an AAV capsid protein of Clade F. In some embodiments, the AAV particle or rAAV particle comprises an AAV capsid protein of Clade E.

[0169] Table 11 below provides examples of the amino acid sequences of the AAV8, AAV9, AAV.rh74, AAV.hu31, AAVhu.32, and AAV.hu37 capsid proteins. Exemplary ITR sequences are provided in Table 2.

[0170] (Table 11) TIFF2025523953000098.tif137163TIFF2025523953000099.tif248163TIFF2025523953000100.tif122163

[0171] 5.6. Method for producing rAAV particles Another aspect of the disclosure involves making the molecules disclosed herein. In some embodiments, a molecule according to the disclosure is made by providing nucleotides comprising a nucleic acid sequence encoding any of the capsid protein molecules herein, and preparing corresponding rAAV particles having a capsid coat composed of the capsid protein using a packaging cell line. Such capsid proteins are described in Section 5.6.5 above. In some embodiments, the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, such as 96%, 97%, 98%, 99% or 99.9% identity to the sequence of a capsid protein molecule described herein. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, such as 96%, 97%, 98%, 99% or 99.9% identity to the sequence of the AAV8 capsid protein and simultaneously retains (or substantially retains) the biological function of the AAV8 capsid protein. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, such as 96%, 97%, 98%, 99% or 99.9% identity to the sequence of the AAV9 capsid protein and simultaneously retains (or substantially retains) the biological function of the AAV9 capsid protein.

[0172] Capsid proteins, coats, and rAAV particles can be produced by techniques known in the art. In some embodiments, the viral genome includes at least one terminal inverted repeat sequence that enables packaging into a vector. In some embodiments, the viral genome further includes the cap gene and / or the rep gene for expression and splicing of the cap gene. In embodiments, the cap gene and the rep gene are provided by the packaging cell and are not present in the viral genome.

[0173] In some embodiments, instead of an existing capsid gene, a nucleic acid encoding an engineered capsid protein is cloned into an AAV Rep-Cap plasmid. When introduced together into a host cell, this plasmid helps package the rAAV genome into the engineered capsid protein as a capsid coat. A packaging cell can be any cell type that has the genes necessary to facilitate replication of the AAV genome, assembly of the capsid, and packaging.

[0174] Many cell culture systems for the production of rAAV particles are known in the art, and any of them can be used to carry out the methods disclosed herein. Cell culture systems include transfection, stable cell line production, and infectious hybrid virus production systems, including, but not limited to, adenovirus-AAV hybrids, herpesvirus-AAV hybrids, and baculovirus-AAV hybrids. An rAAV production culture for the production of rAAV virus particles requires: (1) a suitable host cell, including, for example, a human-derived cell line, a mammalian cell line, or an insect-derived cell line; (2) a suitable helper virus function provided by a wild-type or mutant adenovirus (such as a temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct that provides helper functions; (3) the AAV rep gene and cap gene and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences and optionally regulatory elements; and (5) a medium and medium components (nutrients) suitable for supporting cell growth / survival and rAAV production.

[0175] Non-limiting examples of host cells include A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, HEK293 and their derivatives (HEK293T cells, HEK293F cells), Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, myoblasts, CHO cells or CHO-derived cells, or insect-derived cell lines such as SF-9 (for example, in the case of a baculovirus production system). For an overview, reference is made to Aponte-Ubillus et al., “Molecular Design for Recombinant Adeno-Associated Virus (rAAV) Vector Production,” Appl. Microbiol. Biotechnol. 102:1045-1054 (2018), and for the production technology, the whole of it is incorporated herein by reference.

[0176] In one aspect, provided herein is a method for producing rAAV particles, comprising: (a) providing a cell culture comprising insect cells; (b) introducing into the cells one or more baculovirus vectors encoding at least one of (i) an rAAV genome to be packaged, (ii) an AAV rep protein sufficient for packaging, and (iii) an AAV cap protein sufficient for packaging; (c) adding sufficient nutrients to the cell culture and maintaining the cell culture under conditions that allow for the production of rAAV particles. In some embodiments, the method comprises using a first baculovirus vector encoding a rep gene and a cap gene and a second baculovirus vector encoding an rAAV genome. In some embodiments, the method comprises using a baculovirus encoding an rAAV genome and insect cells expressing a rep gene and a cap gene. In some embodiments, the method comprises using a baculovirus vector encoding a rep gene, a cap gene, and an rAAV genome. In some embodiments, the insect cells are Sf-9 cells. In some embodiments, the insect cells are Sf-9 cells comprising one or more stably integrated heterologous polynucleotides encoding a rep gene and a cap gene.

[0177] In some embodiments, the methods disclosed herein use a baculovirus production system. In some embodiments, the baculovirus production system uses a first baculovirus encoding a rep gene and a cap gene and a second baculovirus encoding an rAAV genome. In some embodiments, the baculovirus production system uses a baculovirus encoding an rAAV genome and a host cell expressing a rep gene and a cap gene. In some embodiments, the baculovirus production system uses a baculovirus encoding a rep gene, a cap gene, and an rAAV genome. In some embodiments, the baculovirus production system uses insect cells such as Sf-9 cells.

[0178] Those skilled in the art are aware of numerous methods by which the AAV rep gene and cap gene, AAV helper genes (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), and the rAAV genome (containing one or more genes of interest flanked by ITRs) can be introduced into cells to produce or package rAAV. The term "adenovirus helper function" refers to a number of viral helper genes that are expressed (either as RNA or protein) intracellularly such that AAV can grow efficiently within the cell. Those skilled in the art understand that helper viruses, including adenovirus and herpes simplex virus (HSV), facilitate AAV replication and that certain genes that provide essential functions have been identified. For example, the helper can induce changes to the cellular environment that facilitate the expression and replication of such AAV genes. In some embodiments of the methods disclosed herein, the AAV rep gene and cap gene, helper genes, and rAAV genome are introduced into cells by transfection using one or more plasmid vectors encoding the AAV rep gene and cap gene, helper genes, and rAAV genome. In some embodiments of the methods disclosed herein, the AAV rep gene and cap gene, helper genes, and rAAV genome can be introduced into cells by transduction using a viral vector, e.g., an rHSV vector encoding the AAV rep gene and cap gene, helper genes, and rAAV genome. In some embodiments of the methods disclosed herein, one or more of the AAV rep gene and cap gene, helper genes, and rAAV genome are introduced into cells by transduction using an rHSV vector. In some embodiments, the rHSV vector encodes the AAV rep gene and cap gene. In some embodiments, the rHSV vector encodes the helper genes. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep gene and cap gene.In some embodiments, the rHSV vector encodes a helper gene and an rAAV genome. In some embodiments, the rHSV vector encodes a helper gene as well as AAV rep and cap genes.

[0179] In one aspect, provided herein is a method for producing rAAV particles, comprising: (a) providing a cell culture comprising a host cell; (b) introducing into the cell one or more rHSV vectors encoding at least one of (i) an rAAV genome to be packaged, (ii) helper functions necessary for packaging of rAAV particles, (iii) AAV rep proteins sufficient for packaging, and (iv) AAV cap proteins sufficient for packaging; (c) adding nutrients sufficient for the cell culture and maintaining the cell culture under conditions that allow production of rAAV particles. In some embodiments, the rHSV vector encodes AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper functions. In some embodiments, the rHSV vector comprises one or more endogenous genes encoding helper functions. In some embodiments, the rHSV vector comprises one or more heterologous genes encoding helper functions. In some embodiments, the rHSV vector encodes an rAAV genome. In some embodiments, the rHSV vector encodes AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper functions and an rAAV genome. In some embodiments, the rHSV vector comprises helper functions as well as AAV rep and cap genes. In some embodiments, the cell comprises one or more stably integrated heterologous polynucleotides encoding rep and cap genes.

[0180] In one aspect, provided herein is a method for producing rAAV particles, comprising: (a) providing a cell culture comprising mammalian cells; (b) introducing into the cells one or more polynucleotides encoding at least one of: (i) an rAAV genome to be packaged, (ii) helper functions necessary for packaging of rAAV particles, (iii) AAV rep proteins sufficient for packaging, and (iv) AAV cap proteins sufficient for packaging; (c) adding sufficient nutrients to the cell culture and maintaining the cell culture under conditions that allow production of rAAV particles. In some embodiments, the helper functions are encoded by adenoviral genes. In some embodiments, the mammalian cells comprise one or more stably integrated heterologous polynucleotides encoding the rep gene and the cap gene.

[0181] Molecular biological techniques for developing plasmids or viral vectors encoding the AAV rep gene and cap gene, helper genes, and / or rAAV genome are generally known in the art. In some embodiments, the AAV rep gene and cap gene are encoded by one plasmid vector. In some embodiments, the AAV helper genes (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene) are encoded by one plasmid vector. In some embodiments, the E1a gene or E1b gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection with one viral vector. In some embodiments, the E1a gene and E1b gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection with one plasmid vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection with one plasmid vector. In some embodiments, the helper genes are stably expressed by the host cell. In some embodiments, the AAV rep gene and cap gene are encoded by one viral vector. In some embodiments, the AAV helper genes (e.g., adenovirus E1a gene, E1b gene, E4 gene, E2a gene, and VA gene) are encoded by one viral vector. In some embodiments, the E1a gene or E1b gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection with one viral vector. In some embodiments, the E1a gene and E1b gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection with one viral vector.In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection with one viral vector. In some embodiments, the AAV rep gene and cap gene, the adenoviral helper functions necessary for packaging, and the rAAV genome to be packaged are introduced into the cell by transfection with one or more polynucleotides, e.g., vectors. In some embodiments, the methods disclosed herein involve transfecting a cell with a mixture of three polynucleotides: one encoding the cap gene and the rep gene, one encoding the adenoviral helper functions necessary for packaging (e.g., the adenoviral E1a gene, E1b gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged. In some embodiments, the AAV cap gene is the AAV8 cap gene. In some embodiments, the AAV cap gene is the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, or AAVhu.32 cap gene. In some embodiments, the vector encoding the rAAV genome to be packaged comprises a gene of interest flanked by AAV ITRs. In certain embodiments, the ITR sequences are AAV2 ITR sequences and include the 5' and 3' sequences of SEQ ID NOs: 28 and 29, respectively, as set forth in Table 2.

[0182] Any combination of vectors can be used to introduce the AAV rep gene and cap gene, the AAV helper genes, and the rAAV genome into cells in which rAAV particles are produced or packaged. In some embodiments of the methods disclosed herein, a first plasmid vector encoding an rAAV genome comprising a gene of interest flanked by AAV inverted terminal repeats (ITRs), a second vector encoding the AAV rep gene and cap gene, and a third vector encoding a helper gene can be used. In some embodiments, a mixture of the three vectors is co-transfected into the cells. In some embodiments, a combination of transfection and infection is used by using both plasmid vectors and viral vectors.

[0183] In some embodiments, one or more of the rep gene and cap gene, and the AAV helper genes are constitutively expressed by the cells and need not be transfected or transduced into the cells. In some embodiments, the cells constitutively express the rep gene and / or the cap gene. In some embodiments, the cells constitutively express one or more AAV helper genes. In some embodiments, the cells constitutively express E1a. In some embodiments, the cells contain a stable transgene encoding the rAAV genome.

[0184] In some embodiments, the AAV rep gene, cap gene, and helper genes (e.g., the Ela gene, E1b gene, E4 gene, E2a gene, or VA gene) can be of any AAV serotype. In some embodiments, the AAV rep gene and cap gene for production of rAAV particles are from different serotypes. For example, the rep gene is from AAV2, while the cap gene is from AAV8.

[0185] In some embodiments, the rep gene is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, or AAVhu.32 or other AAV serotypes (e.g., hybrid serotypes having sequences derived from two or more serotypes). In other embodiments, the rep gene and the cap gene are derived from the same serotype. In still other embodiments, the rep gene and the cap gene are derived from the same serotype and the rep gene comprises at least one modified protein domain or modified promoter domain. In certain embodiments, the at least one modified domain comprises a nucleotide sequence of a serotype different from the capsid serotype. The modified domain within the rep gene can be a hybrid nucleotide sequence consisting of fragments of different serotypes.

[0186] The hybrid rep gene results in an improvement in the packaging efficiency of rAAV particles, including the packaging of a viral genome containing a therapeutic protein transgene (such as dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or the calcium / calmodulin-dependent protein kinase II β isoform protein or a part thereof) that is greater than 4 kb, greater than 4.1 kb, greater than 4.2 kB, greater than 4.3 kb, greater than 4.4 kB, greater than 4.5 kb, or greater than 4.6 kb. The AAV rep gene consists of a nucleic acid sequence encoding non-structural proteins required for viral replication and production. Transcription of the rep gene is initiated from the p5 or p19 promoter, generating two large non-structural Rep proteins (Rep78 and Rep68) and two small non-structural Rep proteins (Rep52 and Rep40), respectively. Furthermore, the Rep78 / 68 domain contains a DNA-binding domain that recognizes specific ITR sequences within the ITR. All four Rep proteins have a common helicase and ATPase domain and function in genome replication and / or capsid formation (Maurer and Weitzman, “Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction,” Hum. Gene Ther. 31(9-10):499-511(2020); which is incorporated herein by reference in its entirety). Transcription of the cap gene is initiated from the p40 promoter, but since this sequence is within the C-terminus of the rep gene, it has been suggested that other elements in the rep gene may induce p40 promoter activity.The p40 promoter domain contains the transcription factor binding elements EF1A, MLTF, and ATF, the Fos / Jun binding element (AP-1), the Sp1-like elements (Sp1 and GGT), and the TATA element (Pereira and Muzyczka, “The Adeno-Associated Virus Type 2 p40 Promoter Requires a Proximal Sp1 Interaction and a p19 CArG-like Element to Facilitate Rep Transactivation,” J. Virol. 71(6):4300-4309 (1997); which is hereby incorporated by reference in its entirety). In some embodiments, the rep gene comprises a modified p40 promoter. In some embodiments, the p40 promoter has one or more of the EF1A binding element, the MLTF binding element, the ATF binding element, the Fos / Jun binding element (AP-1), the Sp1-like element (Sp1 or GGT), or the TATA element modified. In other embodiments, the rep gene is serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, rh8, rh10, rh20, rh39, rh.74, RHM4-1, or hu37, and part or an element of the p40 promoter domain is modified to serotype 2. In yet other embodiments, the rep gene is serotype 8 or 9, and part or an element of the p40 promoter domain is modified to serotype 2.

[0187] The ITR contains the complementary sequences of A and A', B and B', and C and C', and the D sequence is contiguous with the ssDNA genome. The complementary sequences of the ITR form a hairpin structure by self-annealing (Berns, KI., "The Unusual Properties of the AAV Inverted Terminal Repeat," Hum. Gene Ther. 31(9-10):518-523(2020); which is hereby incorporated by reference in its entirety). The D sequence contains a Rep binding element (RBE) and a terminal resolution site (TRS), which together constitute the AAV origin of replication. The ITR is also required as a packaging signal for encapsidation of the replicated genome into the capsid. In some embodiments, the ITR sequence and the cap gene are derived from the same serotype, except that one or more of the complementary sequences of A and A', B and B', C and C', or the D sequence may be modified to contain sequences derived from a serotype different from the capsid. In some embodiments, the modified ITR sequence is derived from the same serotype as the rep gene. In other embodiments, the ITR sequence and the cap gene are derived from different serotypes, except that one or more of the ITR sequences selected from the complementary sequences of A and A', B and B', C and C', or the D sequence are derived from the same serotype as the capsid (cap gene), and one or more of the ITR sequences are derived from the same serotype as the rep gene.

[0188] In some embodiments, the rep gene and the cap gene are from the same serotype, and the rep gene comprises a modified Rep78 domain, DNA binding domain, endonuclease domain, ATPase domain, helicase domain, p5 promoter domain, Rep68 domain, p5 promoter domain, Rep52 domain, p19 promoter domain, Rep40 domain or p40 promoter domain. In other embodiments, the rep gene and the cap gene are from the same serotype, and the rep gene comprises at least one protein domain or promoter domain from a different serotype. In one embodiment, the rAAV comprises a transgene flanked by AAV2 ITR sequences, AAV8 cap, and hybrid AAV2 / 8 rep. In another embodiment, the AAV2 / 8 rep comprises serotype 8 rep except that the p40 promoter domain or a portion thereof is from serotype 2 rep. In other embodiments, the AAV2 / 8 rep comprises serotype 2 rep except that the p40 promoter domain or a portion thereof is from serotype 8 rep. In some embodiments, three or more serotypes may be utilized to construct the hybrid rep / cap plasmid.

[0189] For the transfection of cells, any suitable method known in the art can be used, and it can also be used for the production of rAAV particles by the methods disclosed herein. In some embodiments, the methods disclosed herein include transfecting cells using a chemical transfection method. In some embodiments, the chemical transfection method uses calcium phosphate, highly branched organic compounds (dendrimers), cationic polymers (e.g., DEAE dextran or polyethyleneimine (PEI)), lipofection. In some embodiments, the chemical transfection method uses a cationic polymer (e.g., DEAE dextran or polyethyleneimine (PEI)). In some embodiments, the chemical transfection method uses polyethyleneimine (PEI). In some embodiments, the chemical transfection method uses DEAE dextran. In some embodiments, the chemical transfection method uses calcium phosphate.

[0190] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques can be performed according to the specifications of the manufacturer or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, reference is made to Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is hereby incorporated by reference herein for all purposes. Unless otherwise defined, the nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as laboratory procedures and techniques, are those well known and commonly used in the art. Standard techniques can be used for chemical synthesis, chemical analysis, the preparation, formulation, and delivery of pharmaceuticals, and the treatment of patients.

[0191] Constructs encoding the rAUF1 protein disclosed herein, such as the constructs of SEQ ID NOs: 31-36 (spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1 without intron, and D(+)-CK7AUF1, respectively), or constructs encoding a therapeutic protein (such as dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or a calcium / calmodulin-dependent protein kinase II β isoform protein or a part thereof), a host cell line for producing rAAV particles containing SEQ ID NO: 184 or 186 (RGX-DYS1 or RGX-DYS5) is provided.

[0192] In a preferred embodiment, rAAV provides a transgene delivery vector that can be used in therapeutic and prophylactic applications, as discussed in more detail below.

[0193] The nucleic acid sequences of AAV-based viral vectors, as well as methods for making recombinant AAV and AAV capsids, are taught, for example, in US7,282,199; US7,790,449; US8,318,480; US8,962,332; and PCT / EP2014 / 076466, which are hereby incorporated by reference in their entirety.

[0194] 6. Therapeutic utility Methods are provided for testing the infectivity of recombinant vectors disclosed herein, such as AAV particles or rAAV particles. For example, the infectivity of recombinant gene therapy vectors in muscle cells can be tested in C2C12 myoblasts. Without limitation, several muscle cell lines or heart cell lines can be utilized, including cell lines of T0034 (human), L6 (rat), MM14 (mouse), P19 (mouse), G-7 (mouse), G-8 (mouse), QM7 (quail), H9c2(2-1) (rat), Hs 74.Ht (human), and Hs 171.Ht (human). Vector copy number can be evaluated using polymerase chain reaction technology, and the level of therapeutic protein expression can be tested by measuring the mRNA level of the therapeutic protein (such as dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or the calcium / calmodulin-dependent protein kinase II β isoform protein or a portion thereof) in the cells.

[0195] 6.1. Animal Model The efficacy of a viral vector containing a transgene encoding an AUF1 protein or a therapeutic protein described herein can be tested, for example, by administering to an animal model using a δ-sarcoglycan deletion mouse and / or a Golden Retriever muscular dystrophy (GRMD) model, replacing sarcoglycan, and evaluating the biodistribution, expression, and therapeutic effect of transgene expression. The therapeutic effect can be evaluated, for example, by evaluating the change in muscle strength in an animal administered the transgene. Also, the preclinical therapeutic efficacy of the vectors described herein can be evaluated using animal models that use large mammals as well as vertebrate and invertebrate non-mammalian animals. Accordingly, provided are compositions and methods for therapeutic administration comprising an amount of the vectors disclosed herein encoding AUF1 alone or in combination with a therapeutic protein such as dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, calcium / calmodulin-dependent protein kinase II β isoform protein, other proteins (other than AUF1), or a portion thereof, that has been demonstrated to be effective according to the methods for evaluating therapeutic efficacy disclosed herein.

[0196] 6.2. Mouse Model The efficacy of a gene therapy vector alone or in combination with a second therapy disclosed herein can be evaluated in a mouse model of LGMD. α-, β-, γ-, and δ-sarcoglycan null mice exhibit progressive muscle pathology and dysfunction from the first week. δ-sarcoglycan null mice have histological features of muscular dystrophy, including extensive necrosis, fibrosis, inflammation, calcification, and muscle dysfunction. δ-sarcoglycan deletion mice have fibrotic lesions and electrocardiogram abnormalities in the heart from 8 weeks and cardiomyopathy from 16 weeks (van Putten et al., “Mouse Models for Muscular Dystrophies: An Overview,” Dis. Models Mech. 13(2): dmm043562 (2020); which is incorporated herein by reference in its entirety).

[0197] 6.2.1. CAPN3-Deficient Calpainopathy Mouse Model The efficacy of the gene therapy vector alone or in combination with the second therapy disclosed herein can be evaluated in a mouse model of LGMD. CAPN3 - / - The deletion mouse model well recapitulates the LGMD2A disease, and the mice show myodegeneration, necrosis, mitochondrial and functional abnormalities, small muscle fibers, loss of slow muscle fibers, and reduced muscle strength. CAPN3 - / - As a limitation of the deletion mouse model, fibrosis may not develop, and patients may have an allele deletion and a second allele missense / nonsense mutation.

[0198] Small molecule activators of CAMKIIβ kinase activity have been reported to slightly restore muscle function in CAPN3-deficient mice (Liu et al., “A Small-Molecule Approach to Restore a Slow - Oxidative Phenotype and Defective CaMKIIβ Signaling in Limb Girdle Muscular Dystrophy,” Cell Reports 1:100122 (2020); which is hereby incorporated by reference in its entirety), providing a response for measuring AUF1 gene therapy in this disease model.

[0199] 6.3. Cardiac Function The evaluation of effectiveness against cardiac function can be measured in mice, including δ-sarcoglycan null mice. To measure blood pressure (BP), mice are sedated using 1.5% isoflurane, the anesthesia level is constantly monitored, and the body temperature is maintained at 36.5 - 37.58 °C. The heart rate is maintained at 450 - 550 beats per minute. A BP cuff is attached around the tail, and then the tail is placed in the sensor assembly to monitor non-invasive BP during anesthesia. BP measurements are performed 10 times continuously. Qualitative and quantitative measurements of tail BP are performed offline using analysis software, including systolic pressure, diastolic pressure, and mean pressure. For example, see Wehling-Henricks et al., “Cardiomyopathy in Dystrophin-Deficient Hearts is Prevented by Expression of a Neuronal Nitric Oxide Synthase Transgene in the Myocardium,” Hum. Mol, Genetics 14(14):1921 - 1933(2005) and Uaesoontrachoon et al., “Long-Term Treatment with Naproxcinod Significantly Improves Skeletal and Cardiac Disease Phenotype in the mdx Mouse Model of Dystrophy,” Hum. Mol. Genetics 23(12):3239 - 3249(2014), which are hereby incorporated by reference in their entirety).

[0200] To monitor the amplitude and interval time of the ECG of freely moving mice in the waking state, a wireless telemetry device is used. The transmitting unit is implanted into the abdominal cavity of anesthetized mice, and two electrical leads are fixed near the apex and the right acromion in the lead II direction. Each mouse is housed in a cage on top of an antenna receiver connected to a computer system for data recording. Unfiltered ECG data are collected for 10 seconds every hour for 35 days. Considering the recovery from surgery and to ensure that the effects of anesthesia have subsided, the data for the first 7 days are discarded. The data waveforms and parameters are analyzed with the DSI analysis package (ART 3.01 and Physiostat 4.01), and the measurements are tabulated and averaged to determine the heart rate, ECG amplitude, and interval time. For arrhythmias, two independent observers scrutinize the raw ECG waveforms.

[0201] To measure the degree of fibrosis in the hearts of the test mice, picrosirius red staining is performed. Briefly, at the end of the test, immediately after euthanasia, the myocardium is excised and fixed in 10% formalin for subsequent processing. The heart is sectioned, and the paraffin sections are deparaffinized with xylene, followed by nuclear staining with Weigert's hematoxylin for 8 minutes. Then, after washing, it is stained with picrosirius red (0.5 g of sirius red F3B, saturated aqueous picric acid solution) for an additional 30 minutes. The sections are cleared with xylene three times and mounted in Permount. Five digital images are randomly taken using an Eclipse E800 (Nikon, Japan) microscope, and blinded analysis is performed using Image J (NIH). Blood samples are collected by cardiac puncture when the animals are euthanized, and the collected serum is used to measure the CK level of the muscle.

[0202] 6.4. Dogs Most canine studies have been conducted in the Golden Retriever Muscular Dystrophy (GRMD) model (Korneygay et al., “The Golden Retriever Model of Duchenne Muscular Dystrophy,” Skelet. Muscle 7(1):9 (2017), which is hereby incorporated by reference in its entirety). Dogs with GRMD have phenotypes in skeletal and cardiac muscle, suffer from a progressive lethal disease with selective muscle lesions, which is a severe phenotype approximated by the DMD phenotype. Dogs with GRMD have a single nucleotide change that results in exon skipping and out-of-frame DMD transcripts. Phenotypic characteristics in dogs include elevated serum CK, CRD in EMG, and histopathological evidence of grouped muscle fiber necrosis and regeneration. Phenotypic variability is frequently observed in GRMD as well as in humans. Dogs with GRMD develop paradoxical muscle hypertrophy, which is thought to be involved in the phenotype of affected dogs, and general features include stiffness during walking, decreased joint range of motion, and difficulty in opening the mouth. Objective biomarkers for assessing disease progression include rigid flexion, tibiofibular joint angle, decreased rate of stretch contraction, maximum hip flexion angle, pelvic angle, peripheral anterior sartorius muscle, and quadriceps muscle mass.

[0203] 7. Treatment method with AUF1 gene therapy construct A method of treating a human subject with LGMD using the AUF1 gene therapy construct disclosed herein is provided. Thus, a method of treating or ameliorating the symptoms of LGMD in a subject in need thereof, the method comprising a nucleotide sequence encoding a human AUF1 protein (e.g., comprising the nucleotide sequence of SEQ ID NO: 17) operably linked to one or more regulatory sequences that promote the expression of the AUF1 protein in the muscle cells of the subject and flanked by ITR sequences (see Table 2 for the nucleotide sequences of the possible components of these recombinant genomes), and contacting the muscle cells with a therapeutically effective amount of an AAV vector or rAAV vector comprising an AAV8 vector, an AAV9 vector, or an AAVhu.32 vector, which can be one of SEQ ID NOs: 31-36 (vectors of spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1-intronless, or D(+)-CK7AUF1), under conditions effective to cause the exogenous expression of AUF1 in the muscle cells.

[0204] In some embodiments, the method of treating a human subject provides treatment for LGMD type 1. In some embodiments, the method of treating a human subject provides treatment for limb-girdle muscular dystrophy type 1C (LGMD1C) having a mutation in caveolin 3. In some embodiments, the method of treating a human subject provides treatment for limb-girdle muscular dystrophy type 1G (LGMD1G) having a mutation in HNRPDL, a protein involved in mRNA biogenesis and metabolism.

[0205] In some embodiments, a method of treating a human subject provides treatment for LGMD type 2. In some embodiments, the treatment method provides treatment for sarcoglycan abnormalities. In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2C (LGMD2C). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2D (LGMD2D). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2E (LGMD2E). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2F (LGMD2F).

[0206] In some embodiments, a method of treating a human subject provides treatment for LGMD dystrophin abnormalities. In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2I (LGMD2I) (mutation in FKRP). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2K (LGMD2K) (mutation in POMT1). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2M (LGMD2M) (mutation in FKTN). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2N (LGMD2N) (mutation in POMT2). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2O (LGMD2O) (mutation in POMGnT1). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2P (LGMD2P) (mutation in DAG1). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2T (LGMD2T) (mutation in GMPPB). In some embodiments, the treatment method provides treatment for limb-girdle muscular dystrophy type 2U (LGMD2U) (mutation in ISP / CRPPA).

[0207] In some embodiments, a method of treating a human subject provides treatment for dysferlinopathy. In some embodiments, a method of treating a human subject provides treatment for limb-girdle muscular dystrophy type 2B (LGMD2B) (mutation in DYSF).

[0208] In some embodiments, a method of treating a human subject provides treatment for limb-girdle muscular dystrophy type 2L (LGMD2L) (mutation in ANO5). In some embodiments, a method of treating a human subject provides treatment for limb-girdle muscular dystrophy type 2H (LGMD2H) (mutation in TRIM32). In some embodiments, a method of treating a human subject provides treatment for limb-girdle muscular dystrophy type 2W (LGMD2W) (mutation in LIMS2). In some embodiments, a method of treating a human subject provides treatment for limb-girdle muscular dystrophy type 2X (LGMD2X).

[0209] In some embodiments, a method of treating a human subject provides treatment for calpainopathy. In some embodiments, a method of treating a human subject provides treatment for limb-girdle muscular dystrophy type 2A (LGMD2A).

[0210] In an embodiment, a method of treating a human subject provides a gene therapy vector comprising a genome comprising a transgene encoding p37 AUF1 In an embodiment, a method of treating a human subject provides a gene therapy vector comprising a genome comprising a transgene encoding p40 AUF1 In an embodiment, a method of treating a human subject provides a gene therapy vector comprising a genome comprising a transgene encoding p42 AUF1 In an embodiment, a method of treating a human subject provides a gene therapy vector comprising a genome comprising a transgene encoding p45 AUF1 In some embodiments, the therapy is a monotherapy comprising an AAV vector or an rAAV vector.

[0211] In an embodiment, provided is a method of treating a human subject with a gene therapy vector having a combination of two or more AUF1 isoforms, namely p37AUF1, p40AUF1, p42AUF1, and / or p45AUF1.

[0212] In an embodiment, the method of treating a human subject comprises a treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a muscle creatine kinase (MCK) promoter, syn100 promoter, CK6 promoter, CK7 promoter, CK8 promoter, CK9 promoter, dMCK promoter, tMCK promoter, smooth muscle 22 (SM22) promoter, myo-3 promoter, SPc5-12 promoter, mutant SPc5-12 promoter, creatine kinase (CK) 8e promoter, U6 promoter, H1 promoter, desmin promoter, Pitx3 promoter, skeletal alpha-actin promoter, MHCK7 promoter or Sp-301 promoter.

[0213] In an embodiment, the method of treating a human subject utilizes a codon-optimized AUF1 gene therapy construct. In an embodiment, the method of treating a human subject utilizes a CpG-depleted AUF1 gene therapy construct. In an embodiment, the AUF1 gene therapy construct of the method has the nucleotide sequence of SEQ ID NO: 31. In an embodiment, the AUF1 gene therapy construct of the method has the nucleotide sequence of SEQ ID NO: 32. In an embodiment, the AUF1 gene therapy construct of the method has the nucleotide sequence of SEQ ID NO: 33. In an embodiment, the AUF1 gene therapy construct of the method has the nucleotide sequence of SEQ ID NO: 34. In an embodiment, the AUF1 gene therapy construct of the method has the nucleotide sequence of SEQ ID NO: 35. In an embodiment, the AUF1 gene therapy construct of the method has the nucleotide sequence of SEQ ID NO: 36.

[0214] In an embodiment, a method of treating a human subject comprises a treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 31 (spc-hu-opti-AUF1-CpG(-)). In an embodiment, a method of treating a human subject comprises a treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 32 (tMCK-huAUF1). In an embodiment, a method of treating a human subject comprises a treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 33 (spc5-12-hu-opti-AUF1-WPRE). In an embodiment, a method of treating a human subject comprises a treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 34 (ss-CK7-hu-AUF1). In an embodiment, a method of treating a human subject comprises a treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 35 (spc-hu-AUF1 without intron). In an embodiment, a method of treating a human subject comprises a treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 36 (D(+)-CK7AUF1).

[0215] In embodiments, the method of treating a human subject utilizes an AAV8 gene therapy vector. In embodiments, the method of treating a human subject utilizes an AAV9 gene therapy vector. In embodiments, the method of treating a human subject utilizes an AAVhu.37 gene therapy vector. In embodiments, the method of treating a human subject utilizes an AAVhu.31 gene therapy vector. In embodiments, the method of treating a human subject utilizes an AAV hu.32 gene therapy vector. In embodiments, the method of treating a human subject utilizes an AAV Rh.74 gene therapy vector. In embodiments, the method of treating a human subject utilizes an AAV having a capsid that is at least 95% identical to SEQ ID NO: 114 (AAV8 capsid). In embodiments, the method of treating a human subject utilizes an AAV having a capsid that is at least 95% identical to SEQ ID NO: 115 (AAV9 capsid). In embodiments, the method of treating a human subject utilizes an AAV having a capsid that is at least 95% identical to SEQ ID NO: 116 (hu.37 capsid). In embodiments, the method of treating a human subject utilizes an AAV having a capsid that is at least 95% identical to SEQ ID NO: 117 (hu.31 capsid). In embodiments, the method of treating a human subject utilizes an AAV having a capsid that is at least 95% identical to SEQ ID NO: 118 (hu.32 capsid). In embodiments, the method of treating a human subject utilizes an AAV having a capsid that is at least 95% identical to SEQ ID NO: 119 or SEQ ID NO: 120 (Rh.74 capsid).

[0216] In embodiments, the method results in, for example, a 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more (or 2-fold, 3-fold or more) increase in the amount of muscle cells, increase in endurance, and / or decrease in serum markers of muscle atrophy compared to the level or reference level in the subject prior to administration (e.g., 1 day, 1 week, or 2 weeks prior) 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration to the subject.

[0217] Treatment in the methods according to the present disclosure can have a duration of at least 1 week, at least 1 month, at least several months, at least 1 year, at least 2, 3, 4, 5, 6 years or more. In embodiments, a subject may experience a response to gene therapy treatment (e.g., an increase in muscle mass, muscle strength, or performance) 2 weeks, 4 weeks, 6 weeks, 2 months, 3 months, or 6 months after administration.

[0218] 8. Method of combination therapy A method of treating a human subject for any LGMD that can be treated by providing the functional AUF1 disclosed herein in combination with a second therapy, wherein the second therapy can treat LGMD or improve one or more of its symptoms. The gene therapy vector expressing AUF1 provided herein can be administered as a monotherapy or in combination with a second therapy described herein for treating LGMD. In some embodiments, the combination therapy is a combination of any one of the AUF1 gene therapy vectors disclosed herein and a gene therapy vector encoding another therapeutic protein or another therapy disclosed herein.

[0219] A method of treating a human subject with LGMD by combining the AUF1 gene therapy construct disclosed herein with other therapies is provided. In some embodiments, the method of combination therapy provides treatment for LGMD type 1. In some embodiments, the method of combination therapy provides treatment for limb-girdle muscular dystrophy type 1C (LGMD1C) having a mutation in caveolin 3. In some embodiments, the method of combination therapy provides treatment for limb-girdle muscular dystrophy type 1G (LGMD1G) having a mutation in HNRPDL, a protein involved in mRNA biogenesis and metabolism.

[0220] In some embodiments, the combination therapy method provides treatment for LGMD type 2. In some embodiments, the combination therapy method provides treatment for sarcoglycanopathy. In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2C (LGMD2C). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2D (LGMD2D). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2E (LGMD2E). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2F (LGMD2F).

[0221] In some embodiments, the combination therapy method provides treatment for dystrophinopathy of limb-girdle muscular dystrophy type 2 (LGMD2). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2I (LGMD2I) (mutation of FKRP). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2K (LGMD2K) (mutation of POMT1). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2M (LGMD2M) (mutation of FKTN). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2N (LGMD2N) (mutation of POMT2). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2O (LGMD2O) (mutation of POMGnT1). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2P (LGMD2P) (mutation of DAG1). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2T (LGMD2T) (mutation of GMPPB). In some embodiments, the combination therapy method provides treatment for limb-girdle muscular dystrophy type 2U (LGMD2U) (mutation of ISP / CRPPA).

[0222] In some embodiments, a method of combination therapy for a human subject provides treatment for dysferlinopathy. In some embodiments, the method of combination therapy provides treatment for limb-girdle muscular dystrophy type 2B (LGMD2B) (mutation in DYSF).

[0223] In some embodiments, the method of combination therapy provides treatment for limb-girdle muscular dystrophy type 2L (LGMD2L) (mutation in ANO5). In some embodiments, the method of combination therapy provides treatment for limb-girdle muscular dystrophy type 2H (LGMD2H) (mutation in TRIM32). In some embodiments, the method of combination therapy provides treatment for limb-girdle muscular dystrophy type 2W (LGMD2W) (mutation in LIMS2). In some embodiments, the method of combination therapy provides treatment for limb-girdle muscular dystrophy type 2X (LGMD2X).

[0224] In some embodiments, a method of monotherapy or combination therapy provides treatment for calpainopathy. In some embodiments, the method of combination therapy provides treatment for limb-girdle muscular dystrophy type 2A (LGMD2A).

[0225] In an embodiment, a method of treating a human subject provides a first gene therapy vector comprising a genome comprising a transgene encoding p37 AUF1 In an embodiment, a method of treating a human subject provides a first gene therapy vector comprising a genome comprising a transgene encoding p40 AUF1 In an embodiment, a method of treating a human subject provides a first gene therapy vector comprising a genome comprising a transgene encoding p42 AUF1 In an embodiment, a method of treating a human subject provides a first gene therapy vector comprising a genome comprising a transgene encoding p45 AUF1 In an embodiment, a method of treating a human subject with a gene therapy vector having a combination of two or more AUF1 isoforms, namely p37AUF1, p40AUF1, p42AUF1, and / or p45AUF1, is provided.

[0226] In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a muscle creatine kinase (MCK) promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a syn100 promoter.

[0227] In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a CK6 promoter.

[0228] In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a CK7 promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a CK8 promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a CK9 promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a dMCK promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a tMCK promoter.

[0229] In embodiments, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a smooth muscle 22 (SM22) promoter. In embodiments, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a myo-3 promoter. In embodiments, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a Spc5-12 promoter. In embodiments, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a creatine kinase (CK) 8e promoter. In embodiments, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a U6 promoter.

[0230] In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to an H1 promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a desmin promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a Pitx3 promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a skeletal alpha-actin promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an AAV particle or an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to an MHCK7 promoter. In an embodiment, a method of treating a human subject includes a first treatment method comprising an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a Sp-301 promoter.

[0231] In an embodiment, a method of treating a human subject comprises a first treatment comprising AAV particles or rAAV particles comprising a nucleic acid molecule comprising a codon-optimized AUF1 gene therapy construct. In an embodiment, a method of treating a human subject comprises a first treatment comprising AAV particles or rAAV particles comprising a nucleic acid molecule comprising a CpG-depleted AUF1 gene therapy construct. In an embodiment, the AUF1 gene therapy construct of the method of combination therapy has the nucleotide sequence of SEQ ID NO: 31. In an embodiment, the AUF1 gene therapy construct of the method of combination therapy has the nucleotide sequence of SEQ ID NO: 32. In an embodiment, the AUF1 gene therapy construct of the method of combination therapy has the nucleotide sequence of SEQ ID NO: 33. In an embodiment, the AUF1 gene therapy construct of the method of combination therapy has the nucleotide sequence of SEQ ID NO: 34. In an embodiment, the AUF1 gene therapy construct of the method of combination therapy has the nucleotide sequence of SEQ ID NO: 35. In an embodiment, the AUF1 gene therapy construct of the method of combination therapy has the nucleotide sequence of SEQ ID NO: 36.

[0232] In embodiments, a method of treating a human subject comprises a first treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 31 (spc-hu-opti-AUF1-CpG(-)). In embodiments, a method of treating a human subject comprises a first treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 32 (tMCK-huAUF1). In embodiments, a method of treating a human subject comprises a first treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 33 (spc5-12-hu-opti-AUF1-WPRE). In embodiments, a method of treating a human subject comprises a first treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 34 (ss-CK7-hu-AUF1). In embodiments, a method of treating a human subject comprises a first treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 35 (spc-hu-AUF1 without intron). In embodiments, a method of treating a human subject comprises a first treatment method comprising AAV particles or rAAV particles having the nucleotide sequence of SEQ ID NO: 36 (D(+)-CK7AUF1).

[0233] In embodiments, a method of treating a human subject comprises a first treatment method using an AAV8 gene therapy vector. In embodiments, a method of treating a human subject comprises a first treatment method using an AAV9 gene therapy vector. In embodiments, a method of treating a human subject comprises a first treatment method using an AAVhu.32 gene therapy vector. In embodiments, a method of treating a human subject comprises a first treatment method using an AAV having a capsid that is at least 95% identical to SEQ ID NO: 114 (AAV8 capsid). In embodiments, a method of treating a human subject comprises a first treatment method using an AAV having a capsid that is at least 95% identical to SEQ ID NO: 115 (AAV9 capsid). In embodiments, a method of treating a human subject comprises a first treatment method using an AAV having a capsid that is at least 95% identical to SEQ ID NO: 118 (AAVhu.32 capsid).

[0234] A method of treating LGMD in a subject in need thereof, comprising administering to the subject a first treatment in a therapeutically effective amount (either alone or in combination with a second treatment) and a second treatment different from the first treatment in a therapeutically effective amount (either alone or in combination with the first treatment), wherein the first treatment comprises a first AAV particle or rAAV particle encoding an AUF1 protein or a functional fragment thereof and comprising a nucleic acid molecule operably linked to a muscle cell-specific promoter. In embodiments, the AAV particle or rAAV particle comprises a construct having one of the nucleotide sequences of SEQ ID NOs: 31-36 (spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1 without intron, or D(+)-CK7AUF1), for example, the rAAV is of serotype AAV8 or AAV9 or AAVhu.32. In some embodiments, the first treatment is an AAV particle. In some embodiments, the second treatment is an AAV particle. In some embodiments, the first and second treatments are AAV particles.

[0235] In some embodiments, the first treatment is an rAAV particle. In some embodiments, the second treatment is an rAAV particle. In some embodiments, the first and second treatments are rAAV particles.

[0236] In an embodiment, the second treatment method is a second therapeutic protein and a pharmaceutical composition, and includes an AAV or rAAV vector particle containing a therapeutic protein construct containing dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or a calcium / calmodulin-dependent protein kinase II β isoform protein or a part thereof, and the AAV or rAAV is of AAV8 serotype or AAV9 serotype or AAVhu.32 serotype.

[0237] In certain embodiments, the product of AUF1 gene therapy and the product of gene therapy of a therapeutic protein (such as dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or a calcium / calmodulin-dependent protein kinase II β isoform protein or a part thereof) are delivered simultaneously or within 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 2 weeks, 3 weeks, or 4 weeks of each other, and the second product is administered before any immune response to the product of the first gene therapy. In other embodiments, the product of AUF1 gene therapy and the product of gene therapy of a therapeutic protein are delivered simultaneously or within 1 hour, 2 hours, or 3 hours, and the second product is administered before any immune response to the product of the first gene therapy. In still other embodiments, both the product of AUF1 gene therapy and the product of gene therapy of a therapeutic protein include an AAV vector or rAAV vector of the same serotype and are delivered simultaneously or without an interval of more than 1 hour.

[0238] In other embodiments, the second therapy is a mutation suppression therapy, a steroid therapy, an immunosuppressive / anti-inflammatory therapy, any therapy that treats one or more symptoms of LGMD, or any combination thereof, as disclosed in more detail herein. Alternatively, the therapy is administered as a third therapy in addition to the AUF1 gene therapy vector and the second therapeutic protein gene therapy vector, and the third therapy can be a mutation suppression therapy, a steroid therapy, an immunosuppressive / anti-inflammatory therapy, any therapy that treats one or more symptoms of LGMD, or any combination thereof, as disclosed in more detail herein. The administration of each second therapy may be at any of the dosages known for each administration of the second therapy.

[0239] In some embodiments, the second therapy (or optionally the third therapy) can be administered to alleviate or further alleviate one or more symptoms or features of LGMD, which can be evaluated by any of the following assays for the subject, without limitation: extension of time to inability to walk, improvement of muscle strength, improvement of the ability to lift heavy objects, improvement of the time required to rise from a supine position, improvement of the 9-meter walking time, improvement of the time required to climb four steps of stairs, improvement of the leg function grade, improvement of the lung function, improvement of the heart function, improvement of the quality of life. Each of these assays is known to those of ordinary skill in the art. As an example, the publication by Manzur et al. (Manzur et al., “Glucocorticoid Corticosteroids for Duchenne Muscular Dystrophy,” Cochrane Database Syst Rev 1:CD003725 (2008); which is hereby incorporated by reference in its entirety) provides an extensive description of these assays. For each of these assays, if a detectable improvement or extension of the parameter measured by the assay is observed, it may indicate that one or more symptoms of Duchenne muscular dystrophy in the individual have been alleviated using the methods of the present disclosure. The detectable improvement or extension can be a statistically significant improvement or extension as described by Hodgetts et al. (Hodgetts S., et al., “Reduced Necrosis of Dystrophic Muscle by Depletion of Host Neutrophils, or Blocking TNFalpha Function with Etanercept in mdx Mice,” Neuromuscul.Disord. 16(9-10):591-602 (2006); which is hereby incorporated by reference in its entirety). Alternatively, alleviation of one or more symptoms of Duchenne muscular dystrophy can be evaluated by measuring an improvement in the function, integrity and / or survival of muscle fibers, as defined herein.

[0240] Treatment in the methods according to the present disclosure can have a duration of at least 1 week, at least 1 month, at least several months, at least 1 year, at least 2, 3, 4, 5, 6 years or more. The frequency of administration of any of the second treatment methods, including those not delivered by gene therapy and those described herein, can depend on several parameters such as the age of the patient, the type of mutation, the number of molecules (dosage), the formulation of the molecule, etc. The frequency can range from once every at least 2 weeks, or between 3 weeks, 4 weeks, 5 weeks, or longer periods.

[0241] The first treatment method, the second treatment method, and optionally the third or further different treatment method can be administered to the individual in any order. When multiple second treatment methods (e.g., the third treatment method) are administered, they can also be administered to each other and to the first treatment method in any order. In one embodiment, the treatment methods are administered simultaneously (which means that the treatment methods are administered within 10 hours, for example, within 1 hour). In another embodiment, the treatment methods are administered sequentially. In some aspects, the administrations of the first and second treatment methods can be performed within 7 days, 10 days, or 14 days of each other. In some aspects, simultaneous administration means that the first and second treatment methods are formulated together in a single composition or can each be formulated separately. In some aspects, the third treatment method is administered simultaneously with the first and / or second treatment methods or at separate times, for example, according to a regular dosing schedule such as daily, weekly, or monthly.

[0242] In some embodiments, the first and second treatment methods provide a synergistic therapeutic effect with respect to one or more clinical endpoints in the treatment of the subject's LGMD. In particular, the therapeutic effect is greater than the additive therapeutic effect when the first and second treatment methods are administered alone. In some embodiments, the first and second treatment methods provide a synergistic effect in that the treatment methods result in improvements in different clinical endpoint sets, such that the combined therapeutic benefit is greater than the individual therapeutic benefits of each treatment method.

[0243] In some embodiments, when a third or additional therapy is administered, the first, second, and third therapies provide a synergistic therapeutic effect with respect to one or more clinical endpoints in the treatment of LGMD in a subject, and in particular, the therapeutic effect is greater than the additive therapeutic effect when the first, second, and third therapies are administered alone. In some embodiments, the first, second, and third therapies provide a synergistic effect in that the therapies result in improvement in different sets of clinical endpoints, such that the combined therapeutic benefit is greater than the individual therapeutic benefits of each therapy.

[0244] 8.1. Therapeutic protein therapy in combination therapy A method of treating LGMD in a subject in need thereof, comprising administering to the subject a first therapy and a second therapy, wherein the first therapy is an AAV vector or rAAV vector comprising a transgene encoding AUF1 disclosed herein, and the second therapy is a gene therapy vector comprising an rAAV gene therapy vector encoding a therapeutic protein disclosed herein, is disclosed.

[0245] In some embodiments, the transgene encoding the therapeutic protein encodes dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, calcium / calmodulin-dependent protein kinase II β isoform protein, other proteins (other than AUF1), or a portion thereof.

[0246] In some embodiments, AAV particles or rAAV particles encoding a therapeutic protein such as a therapeutically effective amount of dystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, calcium / calmodulin-dependent protein kinase II β isoform protein, other proteins (other than AUF1), or a portion thereof are in an amount of 1E8 vg / kg to 2E15 vg / kg, for example, 1×10 10 ~1×10 15 genome copies / kg; 5×10 10 ~1×10 15 genome copies / kg; 1×10 11 ~1×10 15 genome copies / kg; 5×10 11 ~1×10 15 genome copies / kg; 1×10 12 ~1×10 15 genome copies / kg; 2×10 12 ~1×10 15 genome copies / kg; 1×10 13 ~1×10 15 genome copies / kg; or 2×10 13 ~1×10 15 genome copies / kg and are administered intravenously or intramuscularly.

[0247] In certain embodiments, the first therapy is an AAV or rAAV particle, comprising a construct having one of the nucleotide sequences of SEQ ID NOs: 31-36 (spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1 without intron, or D(+)-CK7AUF1), and the AAV or rAAV is of AAV8 serotype or AAV9 serotype or AAVhu.32 serotype. The second therapy is an AAV particle or rAAV particle having a recombinant genome with the nucleotide sequence of a therapeutic protein disclosed herein, and the AAV or rAAV is of AAV8 serotype or AAV9 serotype or AAVhu.32 serotype. In embodiments, the ratio of the AAV particle or rAAV particle having a transgene encoding AUF1 to the AAV particle or rAAV particle having a transgene encoding a therapeutic protein (other than AUF1) is 1:1, 1:2, 1:4, 1:5, 1:10, 1:50, 1:100 or 1:1000. Alternatively, the ratio of the AUF1 gene therapy vector to the dystrophin gene therapy vector is 0.5:1, 0.25:1, 0.2:1, or 0.1:1.

[0248] 8.2. Dystrophin Gene Therapy Disclosed herein is a method of treating LGMD in a subject in need thereof, comprising administering to the subject an AAV vector or rAAV vector comprising a transgene encoding AUF1 disclosed herein. In some embodiments, an AAV vector is administered. In some embodiments, an rAAV vector is administered.

[0249] Also disclosed is a method of treating LGMD in a subject in need thereof, comprising administering to the subject a first treatment and a second treatment, wherein the first treatment is an AAV vector or rAAV vector comprising a transgene encoding AUF1 disclosed herein, and the second treatment is a gene therapy vector comprising an AAV or rAAV gene therapy vector encoding microdystrophin disclosed herein.

[0250] In some embodiments, the transgene encoding the microdystrophin protein consists of a dystrophin domain arranged from the amino terminus to the carboxy terminus as ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, where ABD is the actin-binding domain of dystrophin, H1 is the hinge 1 region of dystrophin, R1 is the spectrin 1 region of dystrophin, R2 is the spectrin 2 region of dystrophin, R3 is the spectrin 3 region of dystrophin, H3 is the hinge 3 region of dystrophin, R24 is the spectrin 24 region of dystrophin, H4 is the hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises a portion of CT containing at least the α1-syntrophin binding site.

[0251] In some embodiments, CT comprises, or consists of, the proximal 194 amino acids of the C-terminus of dystrophin, or the proximal portion of the C-terminus encoding at least human dystrophin amino acid residues 3361-3554 of SEQ ID NO: 51 (UniProtKB-P11532), or the proximal portion of the C-terminus encoded by at least exons 70-74 and the first 36 amino acids of the amino acid sequence encoded by the nucleotide sequence of exon 75.

[0252] The amino acid sequence of human dystrophin (UniProt KB-P11532) (SEQ ID NO: 51) is as follows:

[0253] In some embodiments, the microdystrophin protein has the amino acid sequence of microdystrophin (SEQ ID NO: 176, 177, or 178) encoded by DYS1, DYS3, or DYS5. Alternatively, the microdystrophin protein has one of the amino acid sequences of SEQ ID NOs: 179-183. In some embodiments, the microdystrophin protein is encoded by the nucleic acid sequence of SEQ ID NO: 91, 92, or 93. In an embodiment, the nucleic acid sequence encoding microdystrophin is operably linked to a regulatory sequence including the promoter listed in Table 7 and other regulatory elements such as those of Table 2 or Table 8. In certain embodiments, the rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 184, 185, or 186 (RGX-DYS-1, RGX-DYS-3, or RGX-DYS-5), or alternatively SpcV1-μDys1 (SEQ ID NO: 188) or SpcV2-μDys1 (SEQ ID NO: 190). In a specific embodiment, the rAAV is of the AAV8 serotype, the AAV9 serotype, or AAVhu.32 or any other serotype having tropism for muscle cells as disclosed above.

[0254] In other embodiments, the microdystrophin gene therapy is SGT-001, serotype AAV9, rAAVrh74.MHCK7.microdystrophin, SRP-9001 (Willcocks et al. “Assessment of rAAVrh.74.MHCK7.micro-dystrophin Gene Therapy Using Magnetic Resonance Imaging in Children with Duchenne Muscular Dystrophy,” JAMA Network Open 4:e2031851 (2021); which is incorporated herein by reference in its entirety); GNT-004 (Le Guiner et al. “Long-Term Microdystrophin Gene Therapy is Effective in a Canine Model of Duchenne Muscular Dystrophy,” Nat. Commun. 8:16105 (2017); which is incorporated herein by reference in its entirety); or Pfizer PF-06939926 (AAV9 mini-dystrophin) or any other mini-dystrophin or microdystrophin construct.

[0255] 8.2.1. α-, β-, γ- or δ-sarcoglycan gene therapy Disclosed herein is a method of treating sarcoglycanopathy in a subject in need thereof, the method comprising administering to the subject an AAV vector or an rAAV vector comprising a transgene encoding AUF1 disclosed herein. In some embodiments, an AAV vector is administered. In some embodiments, an rAAV vector is administered.

[0256] Also disclosed is a method of treating sarcoglycanopathy in a subject in need thereof, the method comprising administering to the subject a first treatment and a second treatment, wherein the first treatment is an AAV vector or an rAAV vector comprising a transgene encoding AUF1 disclosed herein, and the second treatment is a gene therapy vector comprising an AAV or rAAV gene therapy vector encoding an α-, β-, γ- or δ-sarcoglycan disclosed herein.

[0257] In some embodiments, the α-sarcoglycan protein has the amino acid sequence of SEQ ID NO: 144. In some embodiments, the β-sarcoglycan protein has the amino acid sequence of SEQ ID NO: 145. In some embodiments, the γ-sarcoglycan protein has the amino acid sequence of SEQ ID NO: 146. In some embodiments, the δ-sarcoglycan protein has the amino acid sequence of SEQ ID NO: 147. In some embodiments, the first treatment is an AAV vector. In some embodiments, the first treatment is an rAAV vector.

[0258] In some embodiments, the α-sarcoglycan protein is encoded by the nucleic acid sequence of SEQ ID NO: 144. In embodiments, the nucleic acid sequence encoding α-sarcoglycan is operably linked to a regulatory sequence comprising a promoter listed in Table 6 and other regulatory elements such as those of Table 2 or Table 9, for example. In certain embodiments, the AAV or rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 144. In specific embodiments, the AAV or rAAV is of the AAV8 serotype or the AAV9 serotype or AAVhu.32 or any other serotype having tropism for muscle cells as disclosed above.

[0259] In some embodiments, the β-sarcoglycan protein is encoded by the nucleic acid sequence of SEQ ID NO: 145. In an embodiment, the nucleic acid sequence encoding α-sarcoglycan is operably linked to a regulatory sequence comprising a promoter listed in Table 8 and other regulatory elements such as those in Table 2 or Table 9. In certain embodiments, AAV or rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 145. In a specific embodiment, AAV or rAAV is of serotype AAV8 or AAV9 or AAVhu.32 or any other serotype having tropism for muscle cells as disclosed above.

[0260] In some embodiments, the γ-sarcoglycan protein is encoded by the nucleic acid sequence of SEQ ID NO: 146. In an embodiment, the nucleic acid sequence encoding α-sarcoglycan is operably linked to a regulatory sequence comprising a promoter listed in Table 8 and other regulatory elements such as those in Table 2 or Table 9. In certain embodiments, AAV or rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 146. In a specific embodiment, AAV or rAAV is of serotype AAV8 or AAV9 or AAVhu.32 or any other serotype having tropism for muscle cells as disclosed above.

[0261] In some embodiments, the δ-sarcoglycan protein is encoded by the nucleic acid sequence of SEQ ID NO: 147. In an embodiment, the nucleic acid sequence encoding α-sarcoglycan is operably linked to a regulatory sequence comprising a promoter listed in Table 8 and other regulatory elements such as those in Table 2 or Table 9. In certain embodiments, AAV or rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 147. In a specific embodiment, AAV or rAAV is of serotype AAV8 or AAV9 or AAVhu.32 or any other serotype having tropism for muscle cells as disclosed above.

[0262] In other embodiments, the α-, β-, γ- or δ-sarcoglycan gene therapy is any other α-, β-, γ- or δ-sarcoglycan construct.

[0263] In some embodiments, AAV particles or rAAV particles encoding a therapeutically effective amount of α-sarcoglycan are administered intravenously or intramuscularly at a dose of 1×10 8 genome copies / kg to 2×10 15 genome copies / kg or 2×10 13 ~1×10 15 genome copies / kg. In some embodiments, AAV particles or rAAV particles encoding a therapeutically effective amount of β-sarcoglycan are administered intravenously or intramuscularly at a dose of 1×10 8 genome copies / kg to 2×10 15 genome copies / kg or 2×10 13 ~1×10 15 genome copies / kg. In some embodiments, AAV particles or rAAV particles encoding a therapeutically effective amount of γ-sarcoglycan are administered intravenously or intramuscularly at a dose of 1×10 8 genome copies / kg to 2×10 15 genome copies / kg or 2×10 13 ~1×10 15 genome copies / kg. In some embodiments, AAV particles or rAAV particles encoding a therapeutically effective amount of δ-sarcoglycan are administered intravenously or intramuscularly at a dose of 1×10 8 genome copies / kg to 2×10 15 genome copies / kg or 2×10 13 ~1×10 15 genome copies / kg.

[0264] In certain embodiments, the first therapy is an AAV particle or an rAAV particle comprising a construct having one of the nucleotide sequences of SEQ ID NOs: 31-36 (spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1 without intron, or D(+)-CK7AUF1), and the AAV or rAAV is of AAV serotype 8 or AAV serotype 9 or AAVhu.32 serotype. The second therapy is an rAAV particle having a recombinant genome having the nucleotide sequence of SEQ ID NO: 144, 145, 146, or 147, and the AAV or rAAV is of AAV serotype 8 or AAV serotype 9 or AAVhu.32 serotype. In embodiments, the ratio of the AAV particle or rAAV particle having a transgene encoding AUF1 to the AAV particle or rAAV particle having a transgene encoding α-, β-, γ-, or δ-sarcoglycan is 1:1, 1:2, 1:4, 1:5, 1:10, 1:50, 1:100, or 1:1000. Alternatively, the ratio of the AUF1 gene therapy vector to the α-, β-, γ-, or δ-sarcoglycan gene therapy vector is 0.5:1, 0.25:1, 0.2:1, or 0.1:1.

[0265] 8.3. Gene Therapy for Calpainopathy Disclosed herein is a method of treating calpainopathy in a subject in need thereof, the method comprising administering to the subject an AAV vector or an rAAV vector comprising a transgene encoding AUF1 as disclosed herein. In some embodiments, the AAV vector is administered. In some embodiments, the rAAV vector is administered.

[0266] Also disclosed is a method of treating calpainopathy in a subject in need thereof, comprising administering to the subject a first treatment and a second treatment, wherein the first treatment is an AAV vector or rAAV vector comprising a transgene encoding AUF1 disclosed herein, and the second treatment is a gene therapy vector comprising an AAV or rAAV gene therapy vector encoding calpain 3 (CAPN3) or calcium / calmodulin-dependent protein kinase II β isoform (CaMKIIβ) disclosed herein. In some embodiments, the first treatment is an AAV vector. In some embodiments, the first treatment is an rAAV vector.

[0267] In some embodiments, the calpain 3 protein has the amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, or 159. Table 12 provides the amino acid sequences of embodiments of calpain 3 according to the present disclosure. Further embodiments are substitution variants of calpain 3 defined by SEQ ID NO: 151 (human calpain 3 variant 1 / isoform a), 152 (human calpain 3 variant 2 / isoform b), 153 (human calpain 3 variant 3 / isoform c), 154 (human calpain 3 variant 4 / isoform d), 155 (human calpain 3 variant 5 / isoform e), 156 (human calpain 3 variant 6 / isoform f), 157 (mouse calpain 3 variant 1 / isoform a), 158 (mouse calpain 3 variant 2 / isoform a), or 159 (mouse calpain 3 variant 3 / isoform c). For example, conservative substitutions can be made to SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, or 159 while substantially maintaining its functional activity. In embodiments, calpain 3 can have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequences of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, or 159 and can maintain functional calpain 3 activity as determined, for example, by one or more of the in vitro or in vivo assays in the animal models disclosed below.

[0268] (Table 12) Amino acid sequence of calpain 3 protein TIFF2025523953000101.tif239160TIFF2025523953000102.tif248160TIFF2025523953000103.tif174160*The NCBI reference sequences are hereby incorporated by reference in their entirety.

[0269] In some embodiments, the calpain 3 protein is encoded by the nucleic acid sequence of SEQ ID NO: 160, 161, 162, 163, 164, 165, 166, 167, or 168. In an embodiment, the nucleic acid sequence encoding the calpain 3 protein is operably linked to a regulatory sequence comprising a promoter listed in Table 8 and other regulatory elements such as those of Table 2 or Table 9. In certain embodiments, the AAV vector or rAAV vector has a recombinant genome having the nucleotide sequence of SEQ ID NO: 160, 161, 162, 163, 164, 165, 166, 167, or 168. In a specific embodiment, the rAAV is of the AAV8 serotype or AAV9 serotype or AAVhu.32 or any other serotype having tropism for muscle cells as disclosed above.

[0270] Table 13 provides nucleic acid sequences encoding embodiments of calpain 3 according to the present disclosure. Other embodiments are substitution variants of calpain 3 defined by SEQ ID NO: 160 (human calpain 3 variant 1 / isoform a), 161 (human calpain 3 variant 2 / isoform b), 162 (human calpain 3 variant 3 / isoform c), 163 (human calpain 3 variant 4 / isoform d), 164 (human calpain 3 variant 5 / isoform e), 165 (human calpain 3 variant 6 / isoform f), 166 (mouse calpain 3 variant 1 / isoform a), 167 (mouse calpain 3 variant 2 / isoform a), or 168 (mouse calpain 3 variant 3 / isoform c). For example, conservative substitutions can be made to SEQ ID NO: 160, 161, 162, 163, 164, 165, 166, 167, or 168 and its functional activity can be substantially maintained. In embodiments, the calpain 3 sequence can have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequences of SEQ ID NO: 160, 161, 162, 163, 164, 165, 166, 167, or 168 and can maintain functional calpain 3 activity, as determined, for example, by one or more of the in vitro or in vivo assays in the animal models disclosed below.

[0271] (Table 13) Nucleic acid sequence of calpain 3 protein TIFF2025523953000104.tif133160TIFF2025523953000105.tif241160TIFF2025523953000106.tif250160TIFF2025523953000107.tif246160TIFF2025523953000108.tif246160TIFF2025523953000109.tif246160TIFF2025523953000110.tif246160TIFF2025523953000111.tif250160TIFF2025523953000112.tif246160TIFF2025523953000113.tif246160TIFF2025523953000114.tif150160*The NCBI reference sequence is hereby incorporated by reference in its entirety.

[0272] In some embodiments, the calcium / calmodulin-dependent protein kinase II β isoform protein has the amino acid sequence of SEQ ID NO: 169 or 170. Table 14 provides the amino acid sequences of embodiments of calcium / calmodulin-dependent protein kinase II β according to the present disclosure. Also contemplated are other embodiments that are substitution variants of calcium / calmodulin-dependent protein kinase II β defined by SEQ ID NO: 169 (human calcium / calmodulin-dependent protein kinase II β) or 170 (mouse calcium / calmodulin-dependent protein kinase II β). For example, conservative substitutions can be made to SEQ ID NO: 169 or 170 while substantially maintaining its functional activity. In embodiments, the calcium / calmodulin-dependent protein kinase II β can have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 169 or 170 and can maintain functional calcium / calmodulin-dependent protein kinase II β activity, as determined by, for example, one or more of the in vitro or in vivo assays in the animal models disclosed below.

[0273] (Table 14) Amino acid sequence of calcium / calmodulin-dependent protein kinase II β protein TIFF2025523953000115.tif148160*The NCBI reference sequence is hereby incorporated by reference in its entirety.

[0274] In some embodiments, the calcium / calmodulin-dependent protein kinase II β isoform protein is encoded by the nucleic acid sequence of SEQ ID NO: 171 or 172. In embodiments, the nucleic acid sequence encoding the calcium / calmodulin-dependent protein kinase II β isoform protein is operably linked to a regulatory sequence comprising a promoter listed in Table 8 and other regulatory elements such as those of Table 2 or Table 9. In certain embodiments, the AAV vector or rAAV vector has a recombinant genome having the nucleotide sequence of SEQ ID NO: 171 or 172. In specific embodiments, the rAAV is of serotype AAV8 or AAV9 or AAVhu.32 or any other serotype having tropism for muscle cells as disclosed above.

[0275] Table 15 provides nucleic acid sequences encoding embodiments of the calcium / calmodulin-dependent protein kinase II β protein according to the present disclosure. Other embodiments are contemplated to be substitution variants of the calcium / calmodulin-dependent protein kinase II β protein defined by SEQ ID NO: 171 (human calcium / calmodulin-dependent protein kinase II β protein) or 172 (mouse calcium / calmodulin-dependent protein kinase II β protein). For example, conservative substitutions can be made to SEQ ID NO: 171 or 172 while substantially maintaining its functional activity. In embodiments, the calcium / calmodulin-dependent protein kinase II β protein sequence can have at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 171 or 172 and can maintain functional calcium / calmodulin-dependent protein kinase II β protein activity as determined by, for example, one or more of the in vitro or in vivo assays in the animal models disclosed below.

[0276] (Table 15) Nucleic acid sequence of calcium / calmodulin-dependent protein kinase II β protein TIFF2025523953000116.tif119160TIFF2025523953000117.tif250160TIFF2025523953000118.tif246160TIFF2025523953000119.tif250160TIFF2025523953000120.tif73160*The NCBI reference sequence is hereby incorporated by reference in its entirety.

[0277] In other embodiments, calpain 3 (CAPN3) or calcium / calmodulin-dependent protein kinase II β isoform (CaMKIIβ) gene therapy is any other calpain 3 (CAPN3) or calcium / calmodulin-dependent protein kinase II β isoform (CaMKIIβ) construct.

[0278] In some embodiments, AAV particles or rAAV particles encoding a therapeutically effective amount of calpain 3 protein are 1×10 8 genome copies / kg ~ 2×10 15 genome copies / kg or 2×10 13 ~ 1×10 15 genome copies / kg and are administered intravenously or intramuscularly at a dose of.

[0279] In some embodiments, AAV particles or rAAV particles encoding a therapeutically effective amount of calcium / calmodulin-dependent protein kinase II β isoform protein are 1×10 8 genome copies / kg ~ 2×10 15 genome copies / kg or 2×10 13 ~ 1×10 15 genome copies / kg and are administered intravenously or intramuscularly at a dose of.

[0280] In certain embodiments, the first therapy is an AAV particle or an rAAV particle comprising a construct having one of the nucleotide sequences of SEQ ID NOs: 31-36 (spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1 without intron, or D(+)-CK7AUF1), wherein the AAV or rAAV is of AAV serotype 8 or AAV serotype 9 or AAVhu.32 serotype, and the second therapy is an AAV particle or an rAAV particle having a recombinant genome having the nucleotide sequence of SEQ ID NOs: 144, 145, 146 or 147, wherein the AAV or rAAV is of AAV serotype 8 or of AAV serotype 9 or AAVhu.32 serotype. In embodiments, the ratio of the AAV particle or rAAV particle having a transgene encoding AUF1 to the AAV particle or rAAV particle having a transgene encoding α-, β-, γ- or δ-sarcoglycan is 1:1, 1:2, 1:4, 1:5, 1:10, 1:50, 1:100 or 1:1000. Alternatively, the ratio of the AUF1 gene therapy vector to the α-, β-, γ- or δ-sarcoglycan gene therapy vector is 0.5:1, 0.25:1, 0.2:1, or 0.1:1.

[0281] 8.4. Mutation Suppression Therapy A method of treating LGMD in a subject in need thereof, comprising administering to the subject a first treatment method and a second treatment method, wherein the first treatment method is an AAV vector or an rAAV vector comprising a transgene encoding AUF1 disclosed herein, and the second treatment method is a mutation suppression therapy, is disclosed. In embodiments, a combination of an AAV or rAAV encoding AUF1, microdystrophin or an AAV or rAAV encoding α-, β-, γ-, or δ-sarcoglycan, and a mutation suppression therapy (third treatment method) is administered to treat or ameliorate the symptoms of LGMD in the subject. In some embodiments, the first treatment method is an AAV vector. In some embodiments, the first treatment method is an rAAV vector.

[0282] In some embodiments, the second treatment method (or the third treatment method) is ataluren. In some embodiments, ataluren is administered orally. In some embodiments, ataluren can be administered at a dose of 10 mg / kg / day to 200 mg / kg / day. In some embodiments, ataluren can be administered at a dose of 40 mg / kg. For example, the administration can be 10 mg / kg in the morning, 10 mg / kg at noon, and 20 mg / kg in the evening. The administration period of ataluren can be several weeks, several months, or several years. In some embodiments, the treatment increased the ability to walk / run a longer distance and / or the ability to climb stairs compared to pre-treatment levels.

[0283] In some embodiments, the second treatment method (or the third treatment method) is gentamicin. In some embodiments, gentamicin is administered intravenously. In some embodiments, gentamicin can be administered at a dose of 3 mg / kg / day to 25 mg / kg / day. In some embodiments, gentamicin can be administered at a dose of 7.5 mg / kg / day. The administration period of ataluren can be several weeks, several months, or several years. In some embodiments, the treatment increased hearing, renal function and / or muscle strength compared to pre-treatment levels.

[0284] In some embodiments, the mutation suppression therapy is a nonsense suppression mutation. For example, the subject may have a nonsense mutation, and the second therapy enables the ribosome to read through the premature nonsense mutation.

[0285] Nonsense suppression therapies can generally be of two classes. The first class includes compounds that interfere with codon-anticodon recognition during protein translation to promote readthrough of nonsense codons in eukaryotic cells. These agents can act, for example, by binding to ribosomes and affecting their activity in promoting translation initiation or polypeptide chain elongation, or both. For example, this class of nonsense suppressors can act by binding to rRNA (e.g., by reducing the binding affinity to 18S rRNA). The second class provides the eukaryotic translation machinery with tRNAs that incorporate amino acids into the polypeptide for which the mRNA normally encodes a stop codon, for example, suppressor tRNAs.

[0286] 8.5. Steroid Therapy A method of treating LGMD in a subject in need thereof, comprising administering to the subject a first therapy and a second therapy, wherein the first therapy is an rAAV comprising a transgene encoding AUF1 disclosed herein, and the second therapy is a steroid therapy, is disclosed. In some embodiments, the steroid therapy is a glucocorticoid steroid. In embodiments, a combination of rAAV encoding AUF1, microdystrophin, or rAAV encoding α-, β-, γ-, or δ-sarcoglycan, and steroid therapy (as a third therapy) is administered to treat or ameliorate the symptoms of LGMD in the subject.

[0287] In some embodiments, the steroid therapy is prednisone, deflazacort, bamololone, or spironolactone, or a combination thereof. Spironolactone is an aldosterone antagonist and may not be considered a steroid, but it is used in a similar manner to steroids and is often compared to corticosteroids.

[0288] In some embodiments, the daily dose of prednisone is 0.2 mg / kg / day to 10 mg / kg / day. In some embodiments, the daily dose of prednisone is 0.75 mg / kg / day. In some embodiments, the daily dose of deflazacort is 0.2 mg / kg / day to 40 mg / kg / day. In some embodiments, the daily dose of deflazacort is 0.9 mg / kg / day. In some embodiments, the daily dose of bamololone is 0.5 mg / kg to 40 mg / kg. In some embodiments, the daily dose of bamololone is 2 mg / kg, 6 mg / kg, or 20 mg / kg. In some embodiments, the daily dose of spironolactone is 5 mg to 40 mg. In some embodiments, the daily dose of spironolactone is 12.5 mg or 25 mg.

[0289] The steroid dose can be increased or decreased based on growth, weight, and other side effects experienced. In some embodiments, the administration can be either daily or at a high dose on weekends. For example, in some embodiments, a twice-weekly dose can be up to 250 mg / day of prednisone or 300 mg / day of deflazacort. In some embodiments, the administration can be, for example, administered for 10 days and then off for 10 days.

[0290] 8.6. Immunosuppressive / anti-inflammatory therapy A method of treating LGMD in a subject in need thereof, comprising administering to the subject a first treatment and a second treatment, wherein the first treatment is an rAAV comprising a transgene encoding AUF1 disclosed herein, and the second treatment is an immunosuppressive therapy or an anti-inflammatory therapy, is disclosed. In embodiments, a combination of an rAAV encoding AUF1, microdystrophin or an rAAV encoding α-, β-, γ-, or δ-sarcoglycan, and an immunosuppressive / anti-inflammatory therapy (as a third treatment) is administered to treat or ameliorate the symptoms of LGMD in the subject.

[0291] In some embodiments, the immunosuppressive therapy or anti-inflammatory therapy is edasalonexent.

[0292] In some embodiments, the immunosuppressive therapy or anti-inflammatory therapy is canakinumab. Canakinumab is a monoclonal antibody that targets IL1b, a cytokine involved in inflammation and the immune response. In some embodiments, canakinumab can be administered subcutaneously. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be weeks, months, or years. In some embodiments, canakinumab can be administered at a dose of 0.5 mg / kg to 20 mg / kg. In some embodiments, canakinumab can be administered at a dose of 2 mg / kg or 4 mg / kg. For example, the administration can be a single dose by subcutaneous injection at 2 mg / kg or 4 mg / kg.

[0293] In some embodiments, the immunosuppressive therapy or anti-inflammatory therapy is pembrolizumab. Pembrolizumab is an antibody therapy designed to block the activity of connective tissue growth factor (CTGF), a pro-inflammatory protein that promotes fibrosis (scarring) and is found at abnormally high levels in the muscles of people with DMD. Fibrosis is a characteristic of muscular dystrophy and is responsible for muscle weakness and muscle damage, including in the heart muscle. In some embodiments, inhibition of connective tissue growth factor (CTGF) by pembrolizumab can result in a reduction in muscle fibrosis that leads to an increase in muscle function. In some embodiments, pembrolizumab can be administered intravenously. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be weeks, months, or years. In some embodiments, pembrolizumab can be administered at a dose of 10 mg / kg to 200 mg / kg. In some embodiments, pembrolizumab can be administered at a dose of 35 mg / kg. For example, the administration can be via intravenous (IV) infusion of 35 mg / kg every two weeks.

[0294] In some embodiments, the immunosuppressive therapy or anti-inflammatory therapy is imlifidase. Imlifidase is an enzyme that rapidly cleaves IgG antibodies, thereby suppressing the immune response to AAV. Thus, if the immune response to AAV is suppressed, gene therapy treatments using AAV vectors can be used more efficiently. In some embodiments, imlifidase can be administered intravenously. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be weeks, months, or years. In some embodiments, imlifidase can be administered at a dose of 0.1 mg / kg to 10 mg / kg. In some embodiments, imlifidase can be administered at a dose of 0.25 mg / kg. For example, the administration can be a single dose via intravenous (IV) infusion of 0.25 mg / kg.

[0295] 8.7. Therapies for treating one or more symptoms of LGMD A method of treating LGMD in a subject in need thereof, comprising administering to the subject a first treatment and a second treatment, wherein the first treatment is an rAAV comprising a transgene encoding AUF1 disclosed herein, and the second treatment is a therapy that treats one or more symptoms of LGMD, is disclosed. In some embodiments, the therapy that treats one or more symptoms of LGMD can also include any of the mutation suppression therapies, steroid therapies, and immunosuppressive / anti-inflammatory therapies described herein. In embodiments, a combination of an rAAV encoding AUF1, an rAAV encoding microdystrophin or α-, β-, γ-, or δ-sarcoglycan, and a therapy (as a third treatment) that treats one or more symptoms of LGMD is administered to treat or ameliorate the symptoms of LGMD in the subject.

[0296] In some embodiments, one or more symptoms of LGMD are a decrease in muscle mass and / or muscle strength, wherein the second treatment improves muscle mass and / or muscle strength. For example, the second treatment can be spironolactone (the same as described for steroid therapy), follistatin, SERCA2a, EDG-5506, tamoxifen, gibinosat, ASP0367, or a combination thereof.

[0297] In some embodiments, follistatin or a follistatin variant can be used as the second treatment. In some embodiments, follistatin can be administered as a gene therapy using a viral vector such as AAV.

[0298] In some embodiments, SERCA2a can be used as a second therapy (or a third therapy). In some embodiments, SERCA2a can be administered as gene therapy with a viral vector such as AAV. In some embodiments, SERCA2a can be administered intravenously. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be several weeks, several months, or several years. In some embodiments, 1×10 11 ~1×10 14 vg is administered. In some embodiments, 6×10 12 vg is administered.

[0299] EDG-5506 is a small molecule therapy that can stabilize skeletal muscle fibers (muscles under voluntary control) and protect them from damage during contraction. In some embodiments, SERCA2a can be administered orally. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be several weeks, several months, or several years.

[0300] In some embodiments, the second therapy (or the third therapy) is tamoxifen. In some embodiments, tamoxifen can be administered orally. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be several weeks, several months, or several years. In some embodiments, tamoxifen can be administered at a dose of 0.1 mg / kg to 20 mg / kg. In some embodiments, tamoxifen can be administered at a dose of 0.6 mg / kg. In some embodiments, tamoxifen can be administered at a dose of 5 mg to 100 mg. For example, the administration can be a single oral dose of 0.6 mg / kg per day.

[0301] In some embodiments, Jibinostat is a molecule that inhibits an enzyme called histone deacetylase (HDAC), which can turn off gene expression and reduce the regenerative capacity of muscle. By inhibiting HDAC, Jibinostat can reduce fibrosis and muscle cell death and at the same time enable muscle regeneration. In some embodiments, Jibinostat is administered via an oral suspension. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be several weeks, several months, or several years. In some embodiments, Jibinostat can be administered at a dose of 1 mg / ml to 100 mg / ml. In some embodiments, Jibinostat can be administered at a dose of 10 mg / ml. For example, the administration can be via a 10 mg / ml oral suspension twice a day.

[0302] In some embodiments, ASP0367 is used to turn on the PPAR delta (δ) pathway. The PPAR-δ pathway controls mitochondria by turning on various genes within the cell. When the pathway is turned on, mitochondria use fatty acids more frequently and more mitochondria are created. More fatty acids are used as energy, increasing energy production. Thus, ASP0367 is a mitochondria-directed drug for the treatment of DMD and is designed to treat DMD by increasing fatty acid oxidation and mitochondrial biogenesis in muscle cells.

[0303] In some embodiments, the second treatment (or the third treatment) is a cell-based therapy. For example, the cell-based therapy is one or more myoblasts. In some embodiments, the myoblast-based therapy is described in NCT02196467. In some embodiments, myoblasts resuspended in saline can be transplanted into the extensor carpi radialis muscle of one forearm of the patient at a density of 1 million to 500 million per cubic centimeter. More specifically, 30 million myoblasts per cubic centimeter can be transplanted.

[0304] In some embodiments, the cell-based therapy is CAP-1002, which can improve respiratory function, cardiac function and upper limb function. Thus, in some embodiments, the cell-based therapy is cardiosphere-derived cells.

[0305] In some embodiments, one or more symptoms of LGMD are symptoms related to the cardiac condition. In some embodiments, the cardiac condition is cardiomyopathy, reduced cardiac function, cardiac fibrosis, or a combination thereof. Thus, in some embodiments, the second therapy (or the third therapy) is ifetroban, bisoprolol fumarate, eplerenone, or a combination thereof.

[0306] Iferoban is a potent and selective thromboxane receptor antagonist. In some embodiments, iferoban can stop important molecular signals that mediate the mechanisms of heart inflammation and fibrosis (scarring of tissues). In some embodiments, iferoban is administered orally. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be several weeks, several months, or several years. In some embodiments, iferoban can be administered at a dose of 50 mg to 400 mg. In some embodiments, iferoban can be administered at a dose of 200 mg. For example, the administration can be via once-daily capsules, such as four 50 mg capsules. In some embodiments, bisoprolol is administered at a dose of 0.05 mg / kg to 20 mg / kg. In some embodiments, bisoprolol is administered at a dose of 0.2 mg / kg. In some embodiments, bisoprolol is administered at a dose of 1.25 mg every 24 hours, and the subject is monitored for heart rate, blood pressure, and other heart-related symptoms. The dose of bisoprolol can be increased stepwise by 1.25 mg until a daily dose of 0.2 mg / kg or the maximum tolerated dose (resting heart rate <75 bpm and systolic blood pressure <90 mmHg) is reached. The administration can be increased based on the evaluation of the subject's heart rate, blood pressure, symptoms, and ECG.

[0307] In some embodiments, eplerenone is administered orally. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be several weeks, several months, or several years. In some embodiments, eplerenone can be administered at a dose of 10 mg to 200 mg. In some embodiments, eplerenone can be administered at a dose of 25 mg. For example, the administration can be via once-daily capsules, a single 25 mg capsule.

[0308] In some embodiments, one or more symptoms of LGMD are respiratory symptoms. Accordingly, the second treatment (or the third treatment) can be idebenone. In some embodiments, idebenone can be administered orally. In some embodiments, the administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be several weeks, several months, or several years. In some embodiments, idebenone can be administered at a dose of 250 mg / day to 2000 mg / day. In some embodiments, idebenone can be administered at a dose of 900 mg / day. For example, the administration can be oral three times a day, and each oral administration can be two tablets of 150 mg each. In some embodiments, the second treatment (or the third treatment) is orthopedic management, endocrine management, gastrointestinal management, urological management, or a combination thereof. In some embodiments, the second treatment (or the third treatment) is transcutaneous electrical stimulation (TENS). TENS can increase muscle strength, increase joint range of motion, and / or improve sleep. In some embodiments, TENS is applied using the VECTTOR system. The VT-200 or the VECTTOR system provides electrical stimulation via electrodes on the acupoints of the subject's feet / legs and hands / arms, and provides symptom relief of chronic refractory pain and / or postoperative pain management. In some embodiments, nerve stimulation therapy (e.g., TENS) can be performed once, twice, three times, four times, five times or more a day.

[0309] 8.8. Therapeutically Effective Dosage Disclosed is a method of treatment by peripheral administration, including intravenous administration, of a human patient (e.g., a subject) suitable for treatment with AAV or rAAV encoding functional AUF1, or a second therapeutic protein or a fragment thereof effective for the treatment or amelioration of one or more symptoms of functional AUF1 and LGMD. In some aspects, the patient / subject suitable for treatment with AAV or rAAV encoding AUF1 is a patient having LGMD, including LGMD type 1 or type 2.

[0310] In some embodiments, the first treatment is an AAV particle or an rAAV particle that includes a serotype AAV8 or AAV9 or AAVhu.32 and contains a construct encoding AUF1, the construct having a nucleotide sequence of SEQ ID NOs: 31-36 (spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1 without intron, and D(+)-CK7AUF1, respectively). Administration of the rAAV particle containing the construct encoding AUF1 described herein is at 1×10 8 vg / kg to 2×10 15 vg / kg or 2×10 13 to 1×10 15 of the dose, for example, can be performed at a dose of 2×10 14 vg / kg. The dose can range from 1×10 8 vector genomes (vg / kg) per kg to 2×10 15 vg / kg. In some embodiments, the dose is 2×10 13 , 3×10 13 , 1×10 14 , 3×10 14 , 5×10 14 vg / kg. In some embodiments, the dose, in combination with the second treatment, is 1×10 14 , 1.1×10 14 , 1.2×10 14 , 1.3×10 14 , 1.4×10 14 , 1.5×10 14 , 1.6×10 14 , 1.7×10 14 , 1.8×10 14 , 1.9×10 14 , 2×10 14 , 2.1×10 14 , 2.2×10 14 , 2.3×10 14 , 2.4×10 14 , 2.5×10 14 , 2.6×10 14 , 2.7×10 14, 2.8×10 14 , 2.9×10 14 , or 3×10 14 vg / kg may be.

[0311] In some embodiments, the second treatment method is AAV particles or rAAV particles containing a construct encoding microdystrophin, and the administration of AAV particles or rAAV particles containing a construct encoding microdystrophin as described herein, which contains a construct having the nucleotide sequence of SEQ ID NO: 94, 95 or 96 (serotype AAV8 or AAV9), is 1×10 8 genome copies / kg to 2×10 15 genome copies / kg or 2×10 13 to 1×10 15 of the dosage, for example, at a dosage of 2×10 14 vg / kg. The dosage can be in the range of 1×10 8 vector genomes (vg / kg) to 2×10 15 vg / kg. In some embodiments, the dosage is 2×10 13 , 3×10 13 , 1×10 14 , 3×10 14 , 5×10 14 vg / kg may be. In some embodiments, the dosage is 1×10 14 , 1.1×10 14 , 1.2×10 14 , 1.3×10 14 , 1.4×10 14 , 1.5×10 14 , 1.6×10 14 , 1.7×10 14 , 1.8×10 14 , 1.9×10 14 , 2×10 14 , 2.1×10 14 , 2.2×10 14 , 2.3×10 14 , 2.4×10 14 , 2.5×10 14 , 2.6×10 14 , 2.7×10 14 , 2.8×1014 , 2.9×10 14 , or 3×10 14 vg / kg. In some embodiments, the second therapy is AAV particles or rAAV particles containing a construct encoding an α-, β-, γ-, or δ-sarcoglycan as described herein.

[0312] In certain embodiments, the ratio of the AUF1 gene therapy vector to the second gene therapy vector is 1:1, 1:2, 1:4, 1:5; 1:10, 1:50, 1:100, or 1:1000. Alternatively, the ratio of the AUF1 gene therapy vector to the second gene therapy vector is 0.5:1, 0.25:1, 0.2:1, or 0.1:1.

[0313] A therapeutically effective dosage is administered as a single dose (e.g., simultaneously in a single composition or separate compositions), or within 1 hour, 2 hours, 3 hours, 4 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 2 weeks. In some embodiments, the therapy is monotherapy. According to such embodiments, only the AUF1 gene therapy vector is administered.

[0314] In some embodiments, the therapy is combination therapy. In embodiments, the AUF1 gene therapy vector of the first therapy is administered before the second therapy. In some embodiments, the AUF1 gene therapy vector of the first therapy is administered after the second therapy. If the second therapy is not gene therapy, or if a third therapy (or yet another therapy) that is not a gene therapy vector is administered, it may be administered in multiple doses during the course of the treatment regimen (i.e., for several days, weeks, months, etc.), before or after the first (and / or second) therapy or both before and after the first (and / or second) gene therapy vector.

[0315] The dosage is therapeutically effective and can be evaluated at an appropriate time point after administration, including 12 weeks, 26 weeks, 52 weeks or more, and includes an evaluation for improvement or amelioration of the symptoms and / or biomarkers of LGMD known in the art and detailed herein. The recombinant vectors used for delivery of the transgene encoding AUF1 are described herein. Such vectors need to be directed against human muscle cells (including skeletal muscle, skeletal and / or cardiac muscle), and may include non-replicating rAAV, particularly those having an AAV8 capsid, an AAV9 capsid or an AAVhu.32 capsid. Recombinant vectors for AUF1 expression, including vectors having constructs of spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1 without intron, and D(+)-CK7AUF1 (see Figure 1), can be administered in any manner such that the recombinant vector enters muscle tissue, including introducing the recombinant vector into the bloodstream, including intravenous administration.

[0316] Any such recombinant vector at a therapeutically effective dose should be administered in any manner such that the recombinant vector enters muscle (e.g., skeletal muscle or cardiac muscle), including introducing the recombinant vector into the bloodstream. In a specific embodiment, the vector is administered subcutaneously, intramuscularly or intravenously. Expression of the transgene product results in delivery and maintenance of the transgene product in muscle.

[0317] Pharmaceutical compositions suitable for intravenous, intramuscular, or subcutaneous administration include a suspension of recombinant AAV containing any of the transgenes disclosed herein in a formulation buffer containing a physiologically compatible aqueous buffer. The formulation buffer may include one or more of a polysaccharide, a surfactant, a polymer, or an oil. The disclosed pharmaceutical compositions may include any of the vectors described herein, particularly rAAV vectors containing a transgene encoding AUF1 or microdystrophin or sarcoglycan disclosed herein, and can be used in the disclosed methods.

[0318] The disclosed treatment method can result in one of many endpoints indicating therapeutic efficacy described herein. In some embodiments, the endpoint can be monitored 6 weeks, 12 weeks, 24 weeks, 30 weeks, 36 weeks, 42 weeks, 48 weeks, 1 year, 2 years, 3 years, 4 years, or 5 years after administration of rAAV particles containing a transgene encoding AUF1.

[0319] In some embodiments, creatine kinase activity can be used as an endpoint of the therapeutic efficacy of the treatment methods and administration methods disclosed herein. Creatine kinase activity in a subject can be decreased compared to the level prior to such administration (creatine kinase activity level). In some embodiments, creatine kinase activity in a subject can be decreased compared to the level in the subject prior to treatment (creatine kinase activity level) or compared to the level in an untreated subject having LGMD (creatine kinase activity level) (e.g., a reference level identified in a natural history study). Creatine kinase activity measured in a human subject after administration of AAV or rAAV containing a transgene encoding AUF1, including in combination with a second treatment, can be relative to the creatine kinase activity in the subject prior to administration, the creatine kinase activity in an untreated LGMD subject, the creatine kinase activity in a subject without LGMD, or a control value that can be a standard creatine kinase activity. In some embodiments, the administration results in a decrease in creatine kinase activity, which can be a decrease of 1000 to 10,000 units / liter compared to the value measured in the subject prior to administration of the control or treatment. In some embodiments, amounts of 1000, 2000, 3000, 4000, or 5000 units / liter at the endpoint after administration indicate a decrease.

[0320] In some embodiments, a decrease in the lesion of the gastrocnemius muscle (or other muscle) can be used as an endpoint indicator of the therapeutic efficacy of the treatment methods and administration methods disclosed herein. The lesion of the gastrocnemius muscle in a subject can be decreased compared to the level before administration of the treatment method (the lesion level of the gastrocnemius muscle). In some embodiments, the lesion of the gastrocnemius muscle in a subject can be decreased compared to the level (the lesion level of the gastrocnemius muscle) in an untreated subject having LGMD. The comparison of the lesions of the gastrocnemius muscle can be against a standard, which is a number or set of numbers representing the lesions of subjects without LGMD or untreated subjects with LGMD. Thus, in some embodiments, the comparison of the lesions of the gastrocnemius muscle after administration of the treatment method can be against a control subject. The control can be the lesion of the gastrocnemius muscle in the subject before administration, the lesion of the gastrocnemius muscle in an untreated subject with LGMD, the lesion of the gastrocnemius muscle in a subject without LGMD, or the lesion of the gastrocnemius muscle in the standard.

[0321] In some embodiments, the lesion in the gastrocnemius muscle of a subject is evaluated using magnetic resonance imaging (MRI). MRI can be a good tool for imaging muscles, ligaments, and tendons, and thus, by using MRI, muscle diseases can be detected and / or characterized. In some embodiments, administration of the treatment methods disclosed herein results in a decrease in the lesion of the gastrocnemius muscle of about 1-100%, 2-50%, or 3-10% after administration, compared to a control, for example, compared to the lesion of the gastrocnemius muscle of the subject before such administration. For example, a subject treated with AAV or rAAV containing a transgene encoding AUF1, including in combination with another treatment method, can have a lesion reduction of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or more compared to a control.

[0322] In some embodiments, the gastrocnemius muscle mass (or the muscle mass of any other muscle) can be used as an endpoint of therapeutic efficacy. The gastrocnemius muscle mass in a subject can be decreased compared to the level (level of gastrocnemius muscle mass) prior to administration of the AAV or rAAV containing the transgene encoding AUF1. In some embodiments, the gastrocnemius muscle mass in a subject can be decreased compared to the level (level of gastrocnemius muscle mass) in a subject without LGMD. In some embodiments, the gastrocnemius muscle mass in a subject can be decreased compared to the level (level of gastrocnemius muscle mass) in an untreated subject with LGMD. The comparison of gastrocnemius muscle mass can be to a standard, which is a number or set of numbers representing the amount in a subject without LGMD or the amount in an untreated subject with LGMD. Thus, in some embodiments, the comparison of gastrocnemius muscle mass after administration of the therapies disclosed herein can be to a control. The control can be the gastrocnemius muscle mass in the subject prior to administration, the gastrocnemius muscle mass in an untreated subject with LGMD, the gastrocnemius muscle mass in a subject without LGMD, or the gastrocnemius muscle mass in a standard.

[0323] In some embodiments, the gastrocnemius muscle mass of a subject can be evaluated using MRI. In some embodiments, the administration results in a decrease in gastrocnemius muscle mass of about 1-100%, 2-50%, or 3-20% compared to a control, e.g., compared to the gastrocnemius muscle mass prior to such administration. In some embodiments, the decrease in gastrocnemius muscle mass after administration of an AAV or rAAV containing a transgene encoding AUF1, including in combination with a second therapy, is about 2-400 mm 3 , 5-200 mm 3 , or 20-100 mm 3 compared to a control. For example, a subject treated with an rAAV containing a transgene encoding AUF1, including in combination with a second therapy, can have a decrease in gastrocnemius muscle mass of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mm 3 or more compared to a control.

[0324] In some embodiments, the muscle fat percentage can be used as an endpoint for the therapeutic efficacy of the method of administering the AAV or rAAV therapy disclosed herein. The muscle can be the muscles of the lower limb girdle and thighs (gluteus maximus, adductor magnus, rectus femoris, vastus lateralis, vastus medialis, biceps femoris, semitendinosus, and gracilis). The muscle fat percentage in a subject can be decreased compared to the pre-administration level (level of muscle fat percentage) of AAV or rAAV containing a transgene encoding AUF1, including in combination with a second therapy, as disclosed herein. In some embodiments, the muscle fat percentage can be decreased compared to the level (level of muscle fat percentage) in an untreated subject having LGMD. The comparison of the muscle fat percentage can be to a standard, which is a number or set of numbers representing the amount or percentage of muscle fat percentage in a subject without LGMD or the amount or percentage in an untreated subject having LGMD. Thus, in some embodiments, the comparison of the muscle fat percentage after administration of AAV or rAAV containing a transgene encoding AUF1, including in combination with a second therapy, can be to a control. The control can be the muscle fat percentage in the subject before administration, the muscle fat percentage in an untreated LGMD subject, the muscle fat percentage in a subject without LGMD, or the standard muscle fat percentage.

[0325] In some embodiments, the muscle fat percentage of a subject is evaluated using magnetic resonance imaging (MRI). In some embodiments, a method of treating LGMD is provided by peripheral administration, including intravenous administration of an AAV vector or rAAV vector containing an AUF1 construct, including a second therapy, resulting in a decrease in the muscle fat percentage after administration that can be, for example, about 1 to 100%, 2 to 50%, or 3 to 10% compared to a control, such as compared to the muscle fat percentage before the administration. For example, a subject administered as such can have a decrease in muscle fat percentage of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or more compared to a control.

[0326] In some embodiments, the walking score can be used as an endpoint of treatment. The walking score can be about -1 to 2 after administration. In some embodiments, the North Star Ambulatory Assessment (NSAA) can be used as an endpoint of treatment. The NSAA of the treated subject can be compared to the NSAA before administration. The NSAA of the treated subject can be compared to the NSAA of a subject without LGMD. The NSAA of the treated subject can be compared to an untreated subject with LGMD. The NSAA of the treated subject can be relative to a standard, where the standard is a score or set of scores representing the NSAA of a subject without LGMD or an untreated subject with LGMD. In some embodiments, the NSAA of the treated subject is compared to the pre-administration NSAA score or to any of the above NSAA comparisons. In some embodiments, the increase can be 0 to 1, 0 to 2, or 1 to 2.

[0327] 8.9. Cardiac Output Although skeletal muscle symptoms are considered a defining feature of LGMD, patients most commonly die of respiratory or heart failure. LGMD patients develop dilated cardiomyopathy (DCM) necessary for systolic function. This causes an influx of extracellular calcium, triggering protease activation, cardiomyocyte death, tissue necrosis, and inflammation, ultimately leading to fat accumulation and fibrosis. This process first affects the left ventricle (LV). The left ventricle is responsible for pumping blood to most of the body, is thicker, and thus experiences a greater workload. Atrophied cardiomyocytes show loss of striations, vacuolization, fragmentation, and nuclear degeneration. Functionally, atrophy and scarring cause structural destabilization and hypokinesis of the LV, ultimately progressing to general DCM. DMD can be associated with various ECG changes such as sinus tachycardia, decreased circadian index, decreased heart rate variability, shortened PR interval, right ventricular hypertrophy, S-T segment depression, and QTc prolongation.

[0328] The gene therapy treatment provided herein can delay or prevent the progression of LGMD, and in particular, can reduce or alleviate the progression of cardiac insufficiency and / or maintain or improve cardiac function. Efficacy can be monitored by periodically evaluating signs and symptoms of heart lesions or heart failure according to the age and disease stage of the test population, using continuous electrocardiogram, and non-invasive continuous imaging examinations (e.g., echocardiogram or cardiac magnetic resonance imaging (CMR)). CMR can be used to monitor changes from baseline in forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak expiratory flow during coughing, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), inflammation, and fibrosis. ECG can be used to monitor conduction abnormalities and arrhythmias. In particular, ECG can be used to evaluate normalization of PR interval, R wave in V1, Q wave in V6, ventricular repolarization, QS wave in the inferior and / or lateral wall, conduction disorders in right bundle branch block, QT C, and QRS.

[0329] The treatment methods disclosed herein can improve or maintain cardiac function, or delay the loss of cardiac function, by preventing, for example, a decrease to less than 45% in LVEF and / or a decrease in normalization of function (LVFS ≥ 28%), when measured by continuous electrocardiogram and / or non-invasive continuous imaging examinations (e.g., echocardiogram or cardiac magnetic resonance imaging (CMR)). The measurements can be compared to untreated controls or the subject prior to treatment. Alternatively, the treatment disclosed herein results in an improvement in cardiac function or a decrease in the loss of cardiac function when evaluated by monitoring changes from baseline in forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak expiratory flow during cough, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), inflammation, and fibrosis. An ECG can be used to monitor conduction abnormalities and arrhythmias. In particular, an ECG can be used to evaluate normalization of the PR interval, R wave in V1, Q wave in V6, ventricular repolarization, QS wave in the inferior and / or lateral wall, conduction disturbances in right bundle branch block, QTC, and QRS.

[0330] In some embodiments, cardiac function and / or pulmonary function can be used as endpoints for evaluating the therapeutic efficacy of administration. The cardiac function and / or pulmonary function in a subject can be improved or increased compared to the level before such administration (the level of cardiac function and / or pulmonary function). In some embodiments, the cardiac function and / or pulmonary function in a subject can be improved or increased compared to the level (the level of cardiac function and / or pulmonary function) in a subject without LGMD. In some embodiments, the cardiac function and / or pulmonary function in a subject can be decreased compared to the level (the level of cardiac function and / or pulmonary function) in an untreated subject with LGMD. The comparison of cardiac function and / or pulmonary function can be to a standard, which is a number or set of numbers representing cardiac function and / or pulmonary function in a subject without LGMD or cardiac function and / or pulmonary function in an untreated subject with LGMD. Thus, in some embodiments, the comparison of cardiac function and / or pulmonary function after administration can be to a control. The control can be cardiac function and / or pulmonary function in the subject before administration, cardiac function and / or pulmonary function in an untreated subject with LGMD, cardiac function and / or pulmonary function in a subject without LGMD, or cardiac function and / or pulmonary function in the standard.

[0331] In some embodiments, the improvement or increase in cardiac function and / or pulmonary function is, for example, 1 to 100% compared to a control, such as compared to the subject before administration. In some embodiments, cardiac function can be measured using impedance, electrical activity, and calcium handling.

[0332] 8.10. Primary evaluation items of the patient The effectiveness of the compositions (including the dosage of the compositions) and methods described herein can be evaluated in the clinical evaluation of the subject being treated. The primary assessment items for patients include changes from baseline in forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak expiratory flow during coughing, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), changes from baseline in NSAA, changes from baseline in Performance of Upper Limp (PUL) score, and changes from baseline in Brooke Upper Extremity Scale score (Brooke score), changes from baseline in grip strength, pinch strength, changes in myocardial fibrosis score by MRI, upper arm (biceps) muscle fat and fibrosis evaluated by MRI, leg strength measurement using a dynamometer, 6-minute walk test, 10-minute walk test, changes in gait analysis by 3D gait recording, changes in utrophin membrane staining via quantitative imaging of immunostained biopsy sections, and monitoring changes in regenerated fibers (via muscle biopsy) by measuring the combination of fiber size and fetal-type myosin positivity may be included.For example, see Mazzone E et al, “North Star Ambulatory Assessment, 6-Minute Walk Test and Timed Items in Ambulant Boys with Duchenne Muscular Dystrophy,” Neuromuscul. Disord. 20(11):712-716(2010); Abdelrahim et al., “Evaluation of Cardiac Functions in Children with Duchenne Muscular Dystrophy: A Prospective Case-Control Study,” Electron Physician 9(11):5732-5739(2017); Magrath et al., “Cardiac MRI Biomarkers for Duchenne Muscular Dystrophy,” Biomark. Med. 12(11):1271-1289(2018); and Pane et al., “Upper Limb Function in Duchenne Muscular Dystrophy: 24 Month Longitudinal Data,” PLoS One 13(6):e0199223(2018), the entireties of which are hereby incorporated by reference in their entireties).

Example

[0333] The following examples are intended to illustrate the implementation of the embodiments of the present application, but are by no means intended to limit the scope thereof.

[0334] Example 1 - AUF1 Gene Expression Cassette for Insertion into Cisplasmid A construct for preparing an rAAV8 vector encoding p40 AUF1 was synthesized. A nucleotide sequence encoding human p40 AUF1 (SEQ ID NO: 17) that was codon-optimized and CpG-depleted was identified, synthesized, and cloned into a cis plasmid. Using regulatory elements, an expression cassette incorporating the opti-CpG(−) AUF1 coding sequence (SEQ ID NO: 17) (the amino acid sequence is provided in Table 2) was generated. Constructs of spc-hu-opti-AUF1-CpG(−) (SEQ ID NO: 31), tMCK-huAUF1 (SEQ ID NO: 32), spc5-12-hu-opti-AUF1-WPRE (SEQ ID NO: 33), ss-CK7-hu-AUF1 (SEQ ID NO: 34), spc-hu-AUF1 without intron (SEQ ID NO: 35), or D(+)-CK7AUF1 (SEQ ID NO: 36) are shown in FIG. 1 (the nucleotide sequences are provided in Table 3). The constructs were introduced into a cis plasmid used for the production of rAAV, for example, rAAV8 particles containing a recombinant genome encoding AUF1. Methods for producing rAAV particles are known in the art, and in the aforementioned experiments using rAAV particles, triple transfection of HEK293 cells was performed with (1) a cis plasmid (a transgene flanked by AAV ITR sequences (such as a therapeutic transgene described herein)); (2) a rep / cap plasmid (the AAV rep gene and cap gene and gene products, for example, rep2 / cap8 in AAV8); and (3) a helper plasmid (suitable helper virus functions, usually a mutant adenovirus), and then the cells were cultured in a suitable medium and medium components to support rAAV production until the particles (rAAV vectors) were recovered and purified.

[0335] Example 2 - Calpain 3-deficient limb-girdle muscular dystrophy (LGMD) type 2A CAPN3 disease accounts for approximately 30% of all LGMD cases worldwide and is estimated to be 1 in 40,000 to 1 in 100,000 individuals, but in certain regions, it can be up to 100 times more frequent due to the founder effect. LGMD R1 (CAPN3) is on average 10 to 12 times less frequent than DMD.

[0336] Limb-girdle muscular dystrophy (LGMD) type 2A is caused by mutations in calpain 3, a sarcomere-specific titin-binding Ca2 + cysteine protease (CAPN3). CAPN3 is a protease specific to skeletal muscle and is required to activate muscle contraction by selective cleavage and activation of contractile proteins, thereby activating the muscle calcium pump. The main features include a marked loss of type I oxidative slow muscle fibers and changes in PGC1α activity due to impairment of calmodulin kinase IIβ (CaMKIIβ) activity. +

[0337] The diagnosis of calpainopathy is confirmed by molecular identification of biallelic disease variants of the CAPN3 gene or dominant heterozygous CAPN3 mutations, and most cases of calpainopathy are autosomal recessive. More than 400 pathogenic mutations (null deletions, missense, nonsense) are known for CAPN3.

[0338] The clinical features of CAPN3 mainly include proximal muscle wasting; no involvement of the myocardium or facial muscles; slow but progressive muscle necrosis; and loss of muscle regeneration. The age of onset of muscle weakness ranges from 2 to 40 years, with an average of 15 years. Further symptoms include difficulty walking, a tendency to walk on tiptoe, winged scapulae due to weakness of the scapulohumeral muscles, and shortness of breath at rest. When the upper airway muscle system is affected, speech and swallowing disorders begin to develop. Some fatigue, drowsiness, loss of appetite, weight loss, and decreased concentration may also occur. As the disease progresses, there may be a waddling gait, difficulty climbing stairs, difficulty lifting heavy objects, and difficulty rising from the floor or a chair.

[0339] Calpainopathy is less severe than Duchenne muscular dystrophy (DMD), generally has a later age of onset, no involvement of the myocardium or diaphragm muscle, and is mainly limited to the limb skeletal muscles. These features provide more quantitative means for measuring the outcomes of interventional clinical trials.

[0340] The average lifespan of patients with CAPN3 deficiency is near normal, but there are unmet needs for treatment methods.

[0341] CAPN3-deficient disease mice Findings in mouse LGMD-2A models and humans have shown mitochondrial dysfunction and muscle regeneration disorders, similar to Duchenne muscular dystrophy. In CAPN3-deficient mice mimicking LGMD-2A, drugs that improve mitochondrial function also improve muscle function and partially correct muscle regeneration.

[0342] CAPN3 - / - The deletion mouse model (JACS) well reproduces the LGMD2A disease, showing muscle degeneration, regeneration arrest, necrosis, mitochondrial and functional abnormalities, small muscle fibers, loss of slow muscle fibers, and reduced muscle strength. CAPN3 - / - As a limitation of the CAPN3 deletion mouse model (JACS), fibrosis may not develop, and patients may have allele deletions and a second allele missense / nonsense mutation.

[0343] Small molecule activators of CAMKIIβ kinase activity have been reported to slightly restore muscle function in CAPN3-deficient mice (Liu et al., “A Small-Molecule Approach to Restore a Slow - Oxidative Phenotype and Defective CaMKIIβ Signaling in Limb Girdle Muscular Dystrophy,” Cell Reports 1:100122 (2020); which is hereby incorporated by reference in its entirety), providing a response for measuring AUF1 gene therapy in this disease model.

[0344] AAV8 hAUF1 gene therapy restores muscle fiber integrity, size, and maturity in CAPN3-deficient disease mice To evaluate the effect of AUF1 therapy, codon-optimized humanized AUF1 (AAV8-hAUF1) was administered to CAPN3-deficient mice at 6×10 13Viral genome / kg mouse body weight (low dose), codon-optimized humanized AUF1 (AAV8-hAUF1) at 1×10 14 Viral genome / kg mouse body weight (high dose), or vector control (control) was administered.

[0345] When CAPN3-deficient mice were treated with AAV8-hAUF1 for 2 months, as shown in Figure 2D, muscle endurance and muscle strength increased beyond CAPN3 KO and wild-type levels. In particular, low-dose hAUF1 gene therapy was sufficient to significantly increase muscle endurance and muscle strength, as measured by time to fatigue (Figure 3A), distance to fatigue (Figure 3B), maximum speed (Figure 3C), and muscle grip strength (Figure 3D).

[0346] Three months of AAV8-hAUF1 gene therapy nearly fully restored muscle fiber morphology, with larger, more mature muscle fibers being prominent (Figure 2A), and the cross-sectional area (csa) containing two or more nuclei, a marker of mature muscle fibers, increased (Figure 2B). In CAPN3 deficiency, the diaphragm pathology was less severe than that of limb skeletal muscle, but AAV8-hAUF1 gene therapy corrected the pathology in both muscle types (Figure 2A–2B).

[0347] One of the main goals of CAPN3 deficiency therapy is to restore CAMKIIβ kinase activity, which can be determined by specific activation phosphorylation and the subsequent increase in downstream defective mitochondrial biogenesis. Two months of AAV8-hAUF1 gene therapy in CAPN3-deficient mice restored high levels of CAMKIIβ kinase expression and phosphorylation, and also increased mitochondrial content, as indicated by an increase in mitochondrial DNA levels (Figure 4).

[0348] Another major goal of the therapy for calpain-3 deficiency is to restore the levels and activities of the SERCA2 pump and PGC1α, which promote mitochondrial biogenesis and type I slow-twitch muscle fibers. Two months of AUF1 gene therapy increased the expression, activated phosphorylation, SERCA2A, and both of PGC1α in the gastrocnemius muscle (Figure 5).

[0349] AUF1 gene therapy also potently restored mitochondria, as shown in the TA muscle at two months of therapy, and it was also found that this occurred in the appropriate location under the muscle sheath (Figure 6).

[0350] The characteristics of calpain-3 deficiency are the destruction of the tissue and structure between the myofibrils within the muscle fibers, which prevents normal muscle contraction and generates mitochondria that are exhausted and on the verge of death. As shown in the gastrocnemius muscle, two months of AAV8-hAUF1 gene therapy almost normally restored the tissue and mitochondria between the myofibrils of calpain-3-deficient muscle (Figure 7).

[0351] In calpain-3 LGMD, there is a decrease in the activity of succinate dehydrogenase (SDH), which is characteristic of mitochondrial activity deficiency. AAV8-hAUF1 gene therapy for calpain-3-deficient TA muscle restores high levels of SDH activity in the deep part of the muscle (zone 1) (Figure 8).

[0352] The results presented in this specification show that AAV8-hAUF1 increases the muscle endurance and strength of CAPN3 KO mice to levels close to those of wild-type mice, that low-dose hAUF1 gene therapy is sufficient to increase the muscle endurance and strength of CAPN3 KO mice, that hAUF1 increases the CAMKIIβ mRNA level, protein level, and activated CAMKIIβ Thr286 phosphorylation in the tested muscles (gastrocnemius and TA muscles), that in CAPN3 KO mice treated with hAUF1 gene therapy, succinate dehydrogenase (SDH) and NADH staining, which indicate an increase in mitochondrial oxidative function, were strongly increased, that AUF1 slightly increased the muscle fiber area of TA and diaphragm in CAPN3 KO mice, that SERCA2 mitochondrial pump protein expression was increased by hAUF1 treatment in CAPN3 KO mice, and that phenotypic recovery is more prominent in female mice than in male mice.

[0353] Example 3 - Evaluation of AUF1 Gene Therapy Constructs in δ-Sarcoglycan Deficient Mice Sarcoglycanopathy diseases are caused by a dominant mutation in one of the four sarcoglycan protein genes (α, β, γ, δ). Six sarcoglycan proteins interact with dystrophin to form a complex, which stabilizes the muscle sheath, promotes contraction, maintains muscle integrity, and enables proper calcium function in contraction. The onset of the disease is severe, occurring from childhood to adulthood, and affects the extremities and girdle skeletal muscles, including the diaphragm and myocardium. The worldwide prevalence is low, at 2 to 3 per 100,000 people. It is considered a rare disease. The delta type of the disease is the rarest, and mutations in SCGD cause LGMD2F.

[0354] There is a well-established mouse model for sarcoglycan-deficient delta protein (δ-sarcoglycanopathy, SCGD). SCGD deficiency mimics the human disease. The SCGD protein is part of a sarcoglycan complex consisting of four proteins, which stabilizes the interaction between dystrophin and the muscle sheath (sarcolemma), resulting in stability and contractile ability.

[0355] Scgd with a C57BL / 6J genetic background tm1Mcn The mouse is a model of human sarcoglycan delta (dystrophin-binding glycoprotein) deficiency disorder. Homozygous mice lacking the Sgcd allele are viable and fertile, develop symptoms by 8 weeks of age, have muscle degeneration including myocardium by 12 weeks of age, and then die rapidly.

[0356] AAV8-hAUF1 (codon-optimized humanized AUF1) was administered to δ-sarcoglycan-deficient mice by intravenous (i.v.) injection into the orbital venous plexus at 6e13 viral genomes / kg mouse body weight. In the mouse model, 2 months of AAV8-hAUF1 gene therapy significantly restored the integrity of the diaphragm muscle, and the marked degeneration of SCGD muscle, evident as dark blue staining regions by H&E staining, was repaired (Figure 9).

[0357] In SCGD deficiency, the gastrocnemius muscle is affected. AAV8-hAUF1 (codon-optimized humanized AUF1 described in the provisional application) was administered to animals by intravenous (i.v.) injection into the orbital venous plexus at 6e13 viral genomes / kg mouse body weight. In the mouse model, 2 months of AAV8-hAUF1 gene therapy significantly restored the integrity of the muscle, and the marked degeneration of SCGD muscle, evident as dark blue staining regions by H&E staining, was repaired (Figure 10).

[0358] The expression of embryonic myosin heavy chain (eMHC) is a characteristic of muscle regeneration. However, SCGD muscle fails to regenerate well chronically and eMHC is continuously expressed. 2 months of AAV8 hAUF1 gene therapy restored more normal muscle regeneration, decreased the expression of eMHC, increased the size of more mature muscle fibers indicated by fibers with two or more nuclei, and also increased the cross-sectional area (csa) (Figure 11).

[0359] 2 months of AAV8-hAUF1 gene therapy in SCGD-deficient mice increased all forms of muscle fibers of type I slow muscle and multiple forms of type II fast muscle (Figure 12).

[0360] The muscle function of animals was tested for muscle grip strength, where a mouse was able to grasp a grid with its forelimbs while pulling on the mouse's tail. Figure 13 shows that in SCGD mice, a statistical increase was obtained by 2 months of AAV8 hAUF1 gene therapy, and in female mice it is close to wild type.

[0361] The results presented herein show that hAUF1 protects muscle from dystrophy and increases muscle fiber area (shown for the diaphragm and gastrocnemius muscle). In δ-sarcoglycan SCGD-deficient mice, hAUF1 gene therapy increased the area of all muscle fiber types. hAUF1 increases muscle strength in d-sarcoglycan SCGD-deficient mice.

[0362] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

[0363] The discussion herein is provided to better understand the nature of the problems faced by the art and should not be construed in any way as an admission as to prior art. Nor should any citation of documents herein be construed as an admission that such document constitutes "prior art" against the present application.

[0364] The present invention will be described in detail with reference to its specific embodiments, but it will be understood that equivalent variations in functionality are within the scope of the present invention. Indeed, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and the accompanying drawings in addition to those shown and described herein. Such modifications are intended to be within the scope of the appended claims. Those skilled in the art will be able to recognize or ascertain many equivalents of the specific embodiments of the present invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the following claims.

Claims

**Claim 1** A method for treating limb-girdle muscular dystrophy (LGMD) in a subject in need thereof, the method comprising administering to the subject, a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by terminal inverted repeat (ITR) sequences in an adeno-associated virus (AAV) particle or a recombinant adeno-associated virus (rAAV) particle containing the same. **Claim 2** A method for treating mitochondrial dysfunction associated with limb-girdle muscular dystrophy (LGMD) in a subject in need thereof, the method comprising administering to the subject, a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by terminal inverted repeat (ITR) sequences in an adeno-associated virus (AAV) particle or a recombinant adeno-associated virus (rAAV) particle containing the same. **Claim 3** The method according to claim 1 or claim 2, wherein the subject comprises a functional AUF1 protein isoform. **Claim 4** The method according to any one of claims 1 to 3, wherein the limb-girdle muscular dystrophy (LGMD) is subtype 1. **Claim 5** The method according to claim 4, wherein the LGMD is limb-girdle muscular dystrophy type 1C (LGMD1C). **Claim 6** The method according to claim 4, wherein the LGMD is limb-girdle muscular dystrophy type 1G (LGMD1G). **Claim 7** The method according to any one of claims 1 to 3, wherein the LGMD is limb-girdle muscular dystrophy type 2 (LGMD2). **Claim 8** The method according to claim 7, wherein the LGMD is sarcoglycanopathy. **Claim 9** The method according to claim 8, wherein the sarcoglycanopathy is limb-girdle muscular dystrophy type 2C (LGMD2C). **Claim 10** The method according to claim 8, wherein the sarcoglycanopathy is limb-girdle muscular dystrophy type 2D (LGMD2D). **Claim 11** The method according to claim 8, wherein the sarcoglycanopathy is limb-girdle muscular dystrophy type 2E (LGMD2E). **Claim 12** The method according to claim 8, wherein the sarcoglycanopathy is limb-girdle muscular dystrophy type 2F (LGMD2F). **Claim 13** The method according to claim 7, wherein the LGMD is distroglycanopathy.

14. The method according to claim 13, wherein the distroglycanopathy is limb-girdle muscular dystrophy type 2I (LGMD2I).

15. The method according to claim 13, wherein the distroglycanopathy is limb-girdle muscular dystrophy type 2K (LGMD2K).

16. The method according to claim 13, wherein the distroglycanopathy is limb-girdle muscular dystrophy type 2M (LGMD2M).

17. The method according to claim 13, wherein the distroglycanopathy is limb-girdle muscular dystrophy type 2N (LGMD2N).

18. The method according to claim 13, wherein the distroglycanopathy is limb-girdle muscular dystrophy type 2O (LGMD2O).

19. The method according to claim 13, wherein the distroglycanopathy is limb-girdle muscular dystrophy type 2P (LGMD2P).

20. The method according to claim 13, wherein the distroglycanopathy is limb-girdle muscular dystrophy type 2T (LGMD2T).

21. The method according to claim 13, wherein the distroglycanopathy is limb-girdle muscular dystrophy type 2U (LGMD2U).

22. The method according to claim 7, wherein the LGMD is dysferlinopathy.

23. The method according to claim 22, wherein the dysferlinopathy is limb-girdle muscular dystrophy type 2B (LGMD2B).

24. The method according to claim 7, wherein the LGMD is limb-girdle muscular dystrophy type 2L (LGMD2L).

25. The method according to claim 7, wherein the LGMD is limb-girdle muscular dystrophy type 2H (LGMD2H).

26. The method according to claim 7, wherein the LGMD is limb-girdle muscular dystrophy type 2W (LGMD2W).

27. The method according to claim 7, wherein the LGMD is limb-girdle muscular dystrophy type 2X (LGMD2X).

28. The method according to claim 7, wherein the LGMD is calpainopathy, and optionally, the subject comprises a calpain 3 (CAPN3) mutation.

29. The method according to claim 28, wherein the calpainopathy is limb-girdle muscular dystrophy recessive type 1 / limb-girdle muscular dystrophy type 2A (LGMD R1 / LGMD2A).

30. The method according to any one of the preceding claims, wherein the muscle cell-specific promoter is a muscle creatine kinase (MCK) promoter, syn100 promoter, CK6 promoter, CK7 promoter, CK8 promoter, or CK9 promoter, dMCK promoter, tMCK promoter, smooth muscle 22 (SM22) promoter, myo-3 promoter, Spc5-12 promoter, creatine kinase (CK) 8e promoter, U6 promoter, H1 promoter, desmin promoter, Pitx3 promoter, skeletal alpha-actin promoter, MHC-K7 promoter, or Sp-301 promoter.

31. The method according to claim 30, wherein the muscle cell-specific promoter is a tMCK promoter, Spc5-12 promoter, or CK7 promoter.

32. The nucleic acid molecule is p37 ＡＵＦ１ , p40 ＡＵＦ１ , p42 ＡＵＦ１ , or p45 ＡＵＦ１ The method according to any one of the preceding claims, encoding one or more of.

33. The method according to any one of the preceding claims, wherein the nucleotide sequence encoding the AUF1 protein is the nucleotide sequence of SEQ ID NO:

17.

34. The method according to any one of the preceding claims, wherein the AAV particle or the rAAV particle comprises a recombinant genome having the nucleotide sequence of SEQ ID NO: 31 (spc-hu-opti-AUF1-CpG(-)), SEQ ID NO: 32 (tMCK-huAUF1), SEQ ID NO: 33 (spc5-12-hu-opti-AUF1-WPRE), SEQ ID NO: 34 (ss-CK7-hu-AUF1), SEQ ID NO: 35 (spc-hu-AUF1-intronless), or SEQ ID NO: 36 (D(+)-CK7AUF1).

35. The method according to any one of the preceding claims, wherein the AAV or the rAAV has a capsid that is at least 95% identical to SEQ ID NO: 114 (AAV8 capsid) or SEQ ID NO: 115 (AAV9 capsid) or SEQ ID NO: 118 (hu.32 capsid).

36. The method according to any one of the preceding claims, wherein the AAV or the rAAV is administered at a dose of 1E8 to 1E15 vector genomes / kg or a dose of 2E15 vector genomes / kg.

37. The method according to any one of the preceding claims, wherein the AAV or the rAAV is administered intravenously.

38. The method according to any one of the preceding claims, wherein a second treatment method is administered.

39. The method according to claim 38, wherein the AAV or the rAAV and the second therapy are administered simultaneously, or within 1 week or within 2 weeks of each other.

40. The method according to claim 38 or claim 39, wherein the second therapy is a mutation suppression therapy, a steroid therapy, an immunosuppressive / anti-inflammatory therapy, or a therapy for treating one or more symptoms of LGMD.

41. The method according to any one of claims 38 to 40, wherein the second therapy is administered intravenously.

42. A nucleic acid molecule encoding a γ-sarcoglycan (SGCG) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter, and flanked by terminal inverted repeat (ITR) sequences The method according to claim 9, wherein a second therapy comprising an AAV particle or an rAAV particle containing the same is administered.

43. A nucleic acid molecule encoding an α-sarcoglycan (SGCA) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter, and flanked by terminal inverted repeat (ITR) sequences The method according to claim 10, wherein a second therapy comprising an AAV particle or an rAAV particle containing the same is administered.

44. A nucleic acid molecule encoding a β-sarcoglycan (SGCB) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter, and flanked by terminal inverted repeat (ITR) sequences The method according to claim 11, wherein a second therapy comprising an AAV particle or an rAAV particle containing the same is administered.

45. A nucleic acid molecule encoding a δ-sarcoglycan (SGCD) protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter, and flanked by terminal inverted repeat (ITR) sequences The method according to claim 12, wherein a second therapy comprising an AAV particle or an rAAV particle containing the same is administered.

46. A nucleic acid molecule encoding a calpain 3 protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter, and flanked by terminal inverted repeat sequences The method according to claim 28 or claim 29, wherein a second therapy comprising an AAV particle or an rAAV particle containing the same is administered.

47. A nucleic acid molecule encoding a calcium / calmodulin-dependent protein kinase II β isoform protein or a functional fragment thereof, operably linked to a muscle cell-specific promoter, and flanked by terminal inverted repeat sequences The method according to claim 28 or claim 29, wherein a second treatment method comprising AAV particles or rAAV particles containing is administered.

48. The method according to any one of the preceding claims, wherein the administration promotes phosphorylation of CAMKIIβ in the subject.

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