JP2025523953A - AUF1 gene therapy for limb-girdle muscular dystrophy - Google Patents

Abstract

Methods are provided for treating or ameliorating symptoms of limb-girdle muscular dystrophy 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) effective in treating limb-girdle muscular dystrophy. Also provided are AAV and rAAV vectors that encode the AUF1 protein.
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Description

This application claims priority to U.S. Provisional Patent Application No. 63/391,252, filed July 21, 2022, the entire contents of which are incorporated herein by reference.

This invention was made with government support under grant 5R01AR0744303 awarded by the National Institutes of Health. The Government has certain rights in this invention.

FIELD This disclosure relates to the treatment of myodegenerative diseases, such as limb-girdle muscular dystrophy (LGMD), by administering a dose of a gene therapy vector, such as AAV and rAAV gene therapy vectors, in which the transgene encodes AUF1. Also provided are AAV gene therapy vectors encoding the AUF1 protein and methods of treatment using the same.

2. Background Limb-girdle muscular dystrophies (LGMD) are a group of more than 30 different subtypes of muscular dystrophies that cause muscle weakness and wasting in the pelvis and upper limb girdles. LGMD has diverse clinical phenotypes, including (1) varying ages of onset, (2) rates of progression, (3) specific patterns of muscle wasting, and (4) involvement of respiratory and cardiac muscles. Proteins encoded by LGMD disease genes have diverse cellular functions, including glycosylation and muscle membrane integrity, maintenance, and repair, all of which, when disrupted, result in muscle damage and degeneration. Currently, there is no effective LGMD treatment for any LGMD subtype.

Type 1 LGMD is inherited dominantly and accounts for approximately 10% of all LGMD cases. 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. Type 2 LGMD is inherited recessively. Several genes responsible for LGMD2 code for proteins that directly associate with dystrophin. Sarcoglycanopathies have loss-of-function mutations in genes encoding α-, β-, γ-, or δ-sarcoglycan, causing LGMD2C, LGMD2D, LGMD2E, and LGMD2F, respectively. LGMD2 dystrophinopathy includes LGMD2I (FKRP mutation), LGMD2K (POMT1 mutation), LGMD2M (FKTN mutation), LGMD2N (POMT2 mutation), LGMD2O (POMGnT1 mutation), LGMD2P (DAG1 mutation), LGMD2T (GMPPB mutation), and LGMD2U (ISP/CRPPA mutation).

LGMD2 calpainopathy includes LGMD2A, which is caused by mutations in calpain 3 (CAPN3). LGMD2A is the most common LGMD worldwide. Calpains are intracellular non-lysosomal cysteine proteases that are regulated by calcium ions. LGMD2 dysferlinopathy includes LGMD2B. Other LGMD2 subtypes include LGMD2L, LGMD2H, LGMD2W, and LGMD2X.

Sarcoglycans are a group of transmembrane proteins that associate with dystrophin. The sarcoglycan subcomplex is tightly associated with β-dystroglycan and is composed 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.

Dystrophin is a cytoplasmic protein encoded by the DMD gene that functions to link cytoskeletal actin filaments and membrane proteins. Normally, dystrophin protein is found primarily in skeletal and cardiac muscles, with smaller amounts expressed in the brain, where it functions as a shock absorber during muscle fiber contraction by linking the actin of the contractile apparatus to the connective tissue layer surrounding each muscle fiber. In muscle, dystrophin is localized to the cytoplasmic face of the sarcolemma.

Muscle-wasting diseases represent a large proportion of human disease, some of which are genetic in origin (mainly muscular dystrophies), age-related (sarcopenia), or the result of traumatic muscle injury. Few treatment options are available for individuals with myopathies or those who suffer from severe muscle trauma or age-related loss of muscle mass (known as sarcopenia). The physiology of myopathies is well understood and is based on a common etiology in which muscle stem cells (satellite cells) and their progenitor cells typically become functionally exhausted, fail to reactivate, and are sometimes lost due to continuous cycles of muscle degeneration and regeneration (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 Muscles," Journal of Clinical Neuroscience 6(3):371-82 (2007)). 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)).

Muscle regeneration is initiated by skeletal muscle stem cells (satellite cells) that reside between the striated muscle fibers (myofibers), which are bundles of contractile cells, and the basement membrane that covers those 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 Mammary Skeletal Muscles," Physiol. Rev. 91(4):1447-1531(2011)). When muscles are physically injured, the anatomical niche is disrupted and normally quiescent satellite cells are activated and proliferate asymmetrically. Some satellite cells reconstitute the stem cell population, whereas most others differentiate and fuse to form new muscle fibers (Hindi et al., "Signaling Mechanisms in Mammalian Myoblast Fusion," Sci. Signal. 6(272):re2 (2013)). Studies have demonstrated the specific importance of satellite cell/myoblast populations in muscle regeneration (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 Center Stage,” Development 139(16):2845-56 (2012)).

Muscle fibers are divided into two types, slow (type I) and fast (type II), that exhibit different contractile and metabolic properties. Slow and fast fibers are defined according to their contractile 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 muscle fibers are rich in mitochondria and preferentially utilize oxidative metabolism, providing resistance to fatigue at the expense of contraction velocity. Fast-twitch muscle fibers atrophy easily in response to nutrient deficiency, traumatic injury, age-related progressive wasting (sarcopenia), and cancer-related cachexia, whereas slow-twitch muscle fibers are more 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 Atrophy," Journal of Neuropathology 16(3):243-250 (2013)). 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 of mitochondrial biogenesis and muscle fiber type I specification (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 (NRFs), such as NRF1 and 2, which stimulate mitochondrial biogenesis, and mitochondrial transcription factor A (Tfam). In addition to mitochondrial biogenesis, PGC1α also promotes the formation of slow-twitch muscle fibers through increased expression of Mef2 protein (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 Expression in Slow-Twitch Muscle Fibres," Nature 418(6899):797-801(2002); Stability in Skeletal Muscle,” Am. J. Physiol. Cell. Physiol. 299(1): C155-163 (2010); Transcription Factor A Regulations mtDNA Copy Number in Mammals, “Hum. Mol. Genet. 13(9):935-944 (2004); and Scarpulla, RC, “Transcrip- tional Paradigms in Mammalian Mitochondrial Biogenesis and Function, “Physiol. Rev. 88(2):611-638 (2008)). Importantly, PGC1α protects muscles 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-1 alpha 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)).

Skeletal muscles can remodel between slow and fast 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)). Selectively enhancing slow-twitch muscle type I muscle fibers has been a long-standing goal because endurance slow-twitch muscle fibers are more resistant to muscle atrophy (Talbot & Maves, “Skeletal Muscle Fiber Type: Using Insights from Muscle Developmental Biology to Dissect Targets for Susceptibility and Resistance to Muscle Disease,” Wiley Periodontics, 2011). Interdiscip. Rev. Dev. Biol. 5(4):518-534(2016)), and may 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 Lujubicic 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)).

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)), which direct the differentiation of activated satellite cells into myoblasts, their proliferation arrest, differentiation, and fusion into multinucleated muscle fibers (Mok & Sweetman, "Many Routes to the Same Destination: Lessons From Skeletal Muscle Development," Reproduction 141(3):301-12 (2011)). 141(3):301-12 (2011)). Skeletal muscle regeneration is identified and staged by unique expression markers. PAX7 is a transcription factor expressed by quiescent and early activated satellite cells (Brack, A.S., "Pax7 is Back," Skeleton. 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)).

With aging, satellite cells lose the ability to maintain a quiescent population (Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function," Development 142(9):1572-1581 (2015)), become exhausted, or become 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-girl 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)).

Therefore, there is an urgent need for effective treatment options that address the consequences of LGMD, including, for example, loss of muscle fiber strength, loss of fundamental muscle molecular function, and mitigating the destructive effects of a pathological immune response on muscle health and integrity.

The present disclosure is intended to overcome these and other deficiencies in the art.

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 inverted terminal repeat (ITR) sequences. In some embodiments, the subject comprises a functional AUF1 protein isoform.

Another aspect of the 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 inverted terminal repeat (ITR) sequences. In some embodiments, the subject comprises a functional AUF1 protein isoform.

As advances are made in the use of adeno-associated virus (AAV)-mediated gene therapy to potentially treat a variety of rare diseases, there is excitement and interest in the possibility of using AAV to treat LGMD.

Thus, there is a need in the art for methods of administering AAV and rAAV vectors encoding AUF1 to treat or ameliorate symptoms of LGMD, including sarcoglycanopathies, calpainopathies, dysferlinopathies, and dystrophinopathies, while minimizing immune responses to the therapeutic protein.

Increasing AU-rich mRNA binding factor 1 (AUF1) expression in muscle cells can restore or increase muscle mass, function and performance, and reduce or reverse muscle atrophy. AUF1 expression in muscle cells can increase the expression of components of the dystrophin glycoprotein complex (DGC), also referred to herein as the dystrophin-associated protein complex or DAPC, and increase their participation in the DGC and stabilize the sarcolemma. The DGC is composed of α, β, γ and δ sarcoglycans and the sarcospan (SSPN) sarcoglycan subcomplex. AUF1 has further demonstrated activity in reducing muscle degeneration and improving muscle fiber size and endurance in δ-sarcoglycan null mice, supporting activity in the treatment of LGMD, including sarcoglycanopathies, calpainopathies, dysferlinopathies and dystrophinopathies.

AUF1 also promotes expression of genes downstream of the CAPN3 gene in calpain disorders, correcting physiological and phenotypic dysfunction in CAPN3-deficient mice, including mitochondrial biogenesis, normal muscle ultrastructural architecture, and increased CAMKIIβ kinase activity, which is central to the deficiencies of this disease. Thus, AUF1 monotherapy and AUF1 combination therapy for the treatment and amelioration of symptoms of LGMD are provided, comprising an AUF1 therapy comprising an AUF1 gene therapy construct, optionally in combination with 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 portions 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 using those gene therapy vectors, for example, for diseases associated with LGMD, muscle wasting, and muscle damage.

Schneider et al., International Patent Publication No. WO 2016/034794 (Schneider 2016), which is incorporated herein by reference in its entirety, relates to compositions for incorporation of muscle cells (e.g., compositions encoding AUF1), satellite cell populations, and compositions containing muscle satellite cell populations, as well as pharmaceutical compositions, methods of making muscle satellite cell compositions, and methods of effecting muscle satellite cell-mediated myogenesis. Schneider 2016 relates to treating muscle stem cells (satellite cells) with AUF1 gene therapy, in which the AUF1 transgene is transcriptionally controlled by a satellite cell-specific transcriptional regulatory element. Schneider 2016 further relates to treating muscle cells with monoallelic gene therapy, in which a wild-type AUF1 gene is replaced with a defective AUF1 gene. All examples provided involved replacing a defective endogenous AUF1 gene with a normal AUF1 gene. In contrast, the present disclosure relates to a method of treating muscular dystrophies, such as LGMD, that express endogenous AUF1 by supplementing muscle cells with additional exogenous AUF1. It was completely unexpected that muscle cells that are not defective in the expression of the endogenous AUF1 gene, but are defective in the function of other genes that promote muscular dystrophic disease pathology and exhibit mitochondrial dysfunction, including biogenesis, could be effectively treated by supplementing with additional AUF1. It is completely novel and unexpected, and was not known or suspected in the prior art, including Schneider 2016, that AUF1 replacement gene therapy can effectively treat muscular dystrophies and mitochondrial dysfunction that are not known to involve defects in the expression or function of the AUF1 gene. The examples of the present disclosure surprisingly show that supplementing AUF1 is effective in circumventing the constellation of defective genes in LGMD that does not include AUF1.

International Patent Application Publication No. WO 2021/146711 (Schneider 2021), 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, the nucleic acid molecule being heterologous to the muscle cell-specific promoter and operably linked to the muscle cell-specific promoter. Schneider 2021 also discloses compositions comprising the AAV vector, as well as methods for promoting muscle regeneration of damaged muscle, treating degenerative skeletal muscle loss in a subject, preventing traumatic muscle injury in a subject, such as Duchenne muscular dystrophy, treating traumatic muscle injury in a subject, and 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 for LGMD and by increasing the function of multiple other genes that can compensate for the defective gene and do not include the AUF1 gene. The present disclosure does not relate to compositions or methods for preventing muscle regeneration or damage after traumatic injury.

International Patent Application Publication No. WO 2023/004331 (Schneider 2023), incorporated herein by reference in its entirety, provides a method for treating or ameliorating symptoms of dystrophinopathy, 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 microdystrophin or another therapy effective for treating dystrophinopathy by providing a defective dystophin gene and a dystophin gene substitute that replaces AUF1 to increase muscle strength and endurance. In contrast, the present disclosure relates to the treatment of limb-girdle muscular dystrophy by not passing through the defective gene for LGMD, and surprisingly shows that supplementation of AUF1 is effective in treating LGMD that does not involve AUF1 mutations. This disclosure does not relate to compositions or methods for replacing defective dystropin genes that are not involved in LGMD.

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

The therapeutic methods provided herein include treatment of human subjects with type 1 LGMD, e.g., limb-girdle muscular dystrophy type 1C (LGMD1C) and limb-girdle muscular dystrophy type 1G (LGMD1G). The methods of treatment also include those for 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); calpainopathy, e.g., limb-girdle muscular dystrophy type 2A (LGMD2A); dysferlinopathy, e.g., limb-girdle muscular dystrophy type 2B (LGMD2B); and other limb-girdle muscular dystrophy type 2 (LGMD2) subtypes, e.g., 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 an embodiment, a method of treating LGMD that is not associated with a mutation in the gene encoding AUF1 is provided.

In embodiments, AUF1 is a human AUF1 p37 ^AUF1 , p40 ^AUF1 , p42 ^AUF1 , or p45 ^AUF1 isoform (e.g., including the p40 ^AUF1 isoform) and can be encoded by a codon-optimized, CpG-removed nucleotide sequence, e.g., the nucleotide sequence of SEQ ID NO:17. In additional embodiments, in the AUF1 gene therapy vectors, including AAV and rAAV gene therapy vectors, the muscle cell specific promoter is a muscle creatine kinase (MCK) promoter, synlOO 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 modified Spc5-12 promoters SpcV1 (SEQ ID NO: 127) or SpcV2 (SEQ ID NO: 128)), creatine kinase (CK)8e promoter, U6 promoter, H1 promoter, desmin promoter, Pitx3 promoter, skeletal alpha-actin promoter, MHCK7 promoter, or Sp-301 promoter (see also Table 8).

In certain embodiments, methods are provided for administering AAV particles or rAAV particles that comprise 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). The rAAV particles, in embodiments, are of the AAV8 or AAV9 serotype and have 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 (AAVhu.32 capsid). In certain embodiments, the first treatment is administered systemically, e.g., intravenously, at a dose of 1E8 vector genomes per kg (vg/kg) to 2E15 vg/kg, 1E13-1E14 vg/kg, or at a dose of 2E13 vg/kg (vector genomes/kg (vg/kg) and genome copies/kg (gc/kg) are used interchangeably herein, as are EX and X10 ^X. ) In other embodiments, a nucleic acid is provided that comprises the nucleotide sequence of SEQ ID NO: 17 encoding human AUF1 p40, which is a codon-optimized, CpG-reduced sequence. Vectors are provided that contain this sequence (SEQ ID NO:17) operably linked to a muscle cell-specific promoter, which can be a 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 variant Spc5-12 promoters Spc5v1 (SEQ ID NO:127) and Spc5v2 (SEQ ID NO:128)), 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). In an embodiment, the nucleotide sequence of SEQ ID NO: 17, in addition to being operably linked to a muscle-specific promoter sequence, is further operably linked to an intron sequence, such as a VH4 intron sequence, a polyadenylation signal sequence, such as a rabbit beta globin polyadenylation signal sequence, and/or a WPRE sequence (disclosed herein). The vector may be a rAAV or a cis-plasmid for packaging the rAAV genome, flanked by ITR sequences. The genome in the rAAV particle may be single-stranded or self-complementary. In addition, taking into account the size of the human AUF1p40 sequence, the rAAV vector sequence may include a 5' stuffer sequence and/or a 3' stuffer sequence (see Table 10) and/or an SV40 polyadenylation signal sequence.

In specific embodiments, methods of treating LGMD and pharmaceutical compositions for use in treating LGMD include administering an AAV or rAAV produced 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), as disclosed herein (see, e.g., Table 8, in embodiments, 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), and rAAV particles, pharmaceutical compositions and methods of using the same comprising these nucleotide sequences are further provided. The rAAV particles, in embodiments, are of the AAV8, AAV9 or AAVhu.32 serotype, or a capsid of Table 11, including 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).

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 AAV particle or 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 inverted terminal repeat (ITR) sequences. In some embodiments, the subject expresses functional AUF1. According to such embodiments, the subject has LGMD that is not associated with a mutation in the gene encoding AUF1.

1 shows the vector gene expression cassettes and AUF1 constructs used in cis plasmids for the production of AAV gene therapy vectors. The DNA lengths of each construct are provided. Hu-AUF1-CpG(-): CpG-depleted human AUF1 p40 coding sequence; stuffer: non-coding stuffer or filler sequence; Spc5-12: synthetic muscle-specific promoter; vh-4 in: VH4: human immunoglobulin heavy chain variable region intron; tMCK: truncated muscle creatine kinase promoter; CK7: creatine kinase 7 promoter; RBG-PA: rabbit beta-globin polyA signal sequence; SV40 pA: SV40 polyA signal sequence; and WPRE: woodchuck hepatitis virus post-transcriptional regulatory element. 2A-2B are diagrams (FIG. 2A) and graphs (FIG. 2B) showing that AAV8 hAUF1 gene therapy restores muscle fiber integrity, size, and maturity in CAPN3-deficient disease mice. Animals received AAV8-hAUF1 (codon-optimized humanized AUF1) at either 6e13 or 1e14 vg/kg (6×10 ¹³ or 1×10 ¹⁴ viral genomes/kg mouse body weight), or vector control where indicated, by intravenous (i.v.) injection into the orbital venous plexus. FIG. 2A shows that in a mouse model, 3 months of AAV8-hAUF1 gene therapy restored muscle fiber morphology to near normal, with a notable increase in larger mature muscle fibers and larger cross-sectional area (csa) containing two or more nuclei, a marker of mature muscle fibers (FIG. 2B). The psoas muscle, one of the limb muscles most affected in CAPN3 deficiency, is shown. In CAPN3 deficiency, diaphragm pathology is less severe than limb skeletal muscles, but it is also a pathology that is corrected by AAV8-hAUF1 gene therapy. See legend to FIG. 2A. AAV8-hAUF1 gene therapy in CAPN3-deficient mice shows robust and significant gains in muscle function, endurance, and strength testing after 2 months of gene therapy at both low and high doses. Graph shows time to fatigue in CAPN3-deficient diseased (KO) mice treated with AAV8-hAUF1 at 6e13 ( ^6x1013 viral genomes/kg mouse body weight) and mice treated with AAV8-hAUF1 at 1e14 vg/kg ( ^1x1014 viral genomes/kg mouse body weight). Graph shows data for male and female mice combined). AAV8-hAUF1 gene therapy in CAPN3-deficient mice shows robust and significant gains in muscle function, endurance, and strength testing after 2 months of gene therapy at both low and high doses. Graph shows distance to exhaustion for CAPN3-deficient diseased (KO) mice treated with AAV8-hAUF1 at 6e13 ( ^6x1013 viral genomes/kg mouse body weight) and mice treated with AAV8-hAUF1 at 1e14 vg/kg ( ^1x1014 viral genomes/kg mouse body weight). Graph shows data for male and female mice combined). AAV8-hAUF1 gene therapy in CAPN3-deficient mice shows robust and significant gains in muscle function, endurance, and strength tests after 2 months of both low and high dose gene therapy. Graph shows maximum speed for CAPN3-deficient diseased (KO) mice treated with AAV8-hAUF1 at 6e13 ( ^6x1013 viral genomes/kg mouse body weight) and mice treated with AAV8-hAUF1 at 1e14 vg/kg ( ^1x1014 viral genomes/kg mouse body weight). Graph shows data for male and female mice combined). AAV8-hAUF1 gene therapy in CAPN3-deficient mice shows robust and significant gains in muscle function, endurance, and strength tests after 2 months of gene therapy at both low and high doses. Graph shows muscle grip strength in CAPN3-deficient diseased (KO) mice treated with AAV8-hAUF1 at 6e13 ( ^6x1013 viral genomes/kg mouse body weight) and mice treated with AAV8-hAUF1 at 1e14 vg/kg ( ^1x1014 viral genomes/kg mouse body weight). Graph shows data for male and female mice combined). We show that AAV8-hAUF1 gene therapy restores high levels of CAMKIIβ kinase expression and phosphorylation, and also increases mitochondrial content as indicated by increased mitochondrial DNA levels. One of the main goals of therapy for CAPN3 deficiency is to restore CAMKIIβ kinase activity, which can be determined by specific activation phosphorylation and an increase in downstream defective mitochondrial biogenesis. Data are shown for gastrocnemius muscles from two mice 2 months after gene therapy. This shows that hAUF1 supplementation increases CAMKIIb protein expression, activating phosphorylation, SERCA2A and PGC1a protein in CAPN3 KO mice. Another major goal of therapy for CAPN3 deficiency is to restore the levels and activity of SERCA2 pumps, as well as PGC1α levels, which promote mitochondrial biogenesis and type I endurance slow muscle fibers. AUF1 gene therapy increased both males and females. Data shows gastrocnemius muscle of two mice 2 months after gene therapy. See the description of Figure 5-1. We show that hAUF1 increases subsarcolemmal mitochondrial activity in TA muscle. AUF1 gene therapy strongly restores mitochondria, and found them to be in the proper location under the sarcolemma, as shown in TA muscle 2 months after therapy. These transmission electron micrographs show abnormal mitochondria in untreated CAPN3 KO tibialis anterior (TA) muscle with dark staining, whereas restored mitochondria within the sarcolemma (muscle cell membrane) are darker with well-defined cristae. We show that AUF1 gene therapy increases intermyofibrillar mitochondria and improves myofibrillar alignment in TA muscle. CAPN3 deficiency is characterized by disruption of intermyofibrillar organization and structure within muscle fibers, which impedes normal muscle contraction and results in exhausted and dying mitochondria. As shown in gastrocnemius muscle, 2 months of AAV8-hAUF1 gene therapy restored near-normal intermyofibrillar organization and mitochondria in CAPN3-deficient muscle. Similar data were seen in TA and other muscles. Figure 1 shows that succinate dehydrogenase (SDH) is increased in TA muscle of CAPN3 KO mice after 2 months of AAV8-hAUF1 treatment. CAPN3 LGMD results in reduced succinate dehydrogenase (SDH) activity, a hallmark of defective mitochondrial activity. AAV8-hAUF1 gene therapy to CAPN3-deficient TA muscle restores high levels of SDH activity in the deep muscle (zone 1). Quantification of deep muscle (zone 1) and superficial muscle (zone 3) shows increased SDH activity with hAUF1 gene therapy. See the description of Figure 8-1. We show that AAV8 hAUF1 gene transfer reduces degeneration of gastrocnemius and diaphragm muscles in LGMD δ-sarcoglycan KO mice. In SCGD deficiency, the diaphragm muscle is one of the most affected muscles. Animals were administered AAV8-hAUF1 (codon-optimized humanized AUF1) at 6e13 viral genomes/kg mouse body weight by intravenous (i.v.) injection into the orbital venous plexus. In the mouse model, 2 months of AAV8-hAUF1 gene therapy significantly restored the integrity of the diaphragm muscle and repaired the marked degeneration of SCGD muscle, evident as dark blue staining areas on H&E staining. See the description of Figure 9-1. We show that AAV8 hAUF1 gene transfer reduces degeneration of the gastrocnemius muscle in LGMD δ-sarcoglycan KO mice. In SCGD deficiency, the gastrocnemius muscle is the affected muscle. Animals were administered AAV8-hAUF1 (codon-optimized humanized AUF1 described in the provisional application) at 6e13 viral genomes/kg mouse body weight by intravenous (i.v.) injection into the orbital venous plexus. Two months of AAV8-hAUF1 gene therapy significantly restored muscle integrity in the mouse model, repairing the marked degeneration of the SCGD muscle, evident as dark blue stained areas on H&E staining. We show that hAUF gene transfer reduces eMHC gene expression in the diaphragm and improves muscle fiber integrity and size. Embryonic myosin heavy chain (eMHC) expression is a hallmark of muscle regeneration. However, SCGD muscles chronically fail to regenerate and continue to express eMHC. Two months of AAV8 hAUF1 gene therapy restored more normal muscle, reduced eMHC expression, and larger, more mature muscle fibers as indicated by fibers with more than two nuclei and larger cross-sectional area (csa). See the description of Figure 11-1. Figure 1 shows the effect of hAUF1 gene transfer on diaphragm muscle degeneration and muscle fiber type expression in δ-sarcoglycan KO mice 2 months after gene therapy. Two months of AAV8-hAUF1 gene therapy in SCGD-deficient mice increased all types of muscle fibers, including type I slow twitch and multiple forms of type II fast twitch. See the description of Figure 12-1. Figure 1 shows that AAV8 hAUF1 gene transfer increases muscle strength in delta-sarcoglycan KO mice. Animal muscle function was tested for muscle grip strength, where the mouse can grasp a grid with its forelimbs while pulling on its tail. Data show that 2 months of AAV8 hAUF1 gene therapy resulted in a statistical increase in SCGD mice, approaching wild type in female mice.

DETAILED DESCRIPTION A method is provided for treating limb-girdle muscular dystrophy (LGMD), e.g., LGMD type 1 and type 2 subtypes, by administering to a subject in need thereof a gene therapy vector, particularly an AAV vector or rAAV vector, the gene therapy vector comprising 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 another embodiment, the AUF1 protein or nucleic acid encoding AUF1 is administered in combination with another therapy used to treat LGMD. The other therapy can be a gene therapy therapy (including a therapy co-expressed with AUF1 or co-expressed as a separate gene therapy vector) or a non-gene therapy therapy.

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 inverted terminal repeat (ITR) sequences. In some embodiments, the subject comprises a functional AUF1 protein isoform.

Another aspect of the 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 inverted terminal repeat (ITR) sequences. In some embodiments, the subject comprises a functional AUF1 protein isoform.

The therapeutic methods provided herein include treatment of human subjects having type 1 limb-girdle muscular dystrophy (LGMD), e.g., limb-girdle muscular dystrophy type 1C (LGMD1C) and limb-girdle muscular dystrophy type 1G (LGMD1G). The methods of treatment also include those for 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, e.g., limb-girdle muscular dystrophy type 2A (LGMD2A), dysferlinopathies, e.g., limb-girdle muscular dystrophy type 2B (LGMD2B), as well as other LGMD2 subtypes, e.g., 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 an embodiment, a method of treating LGMD that is not associated with a mutation in the gene encoding AUF1 is provided.

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

In some embodiments, AUF1 gene therapy is administered in combination with a gene therapy vector that includes a genome having a transgene encoding a therapeutic protein effective to treat LGMD (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 portions thereof) operably linked to regulatory elements that promote expression in muscle cells in a therapeutically effective amount.

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 selected from the group consisting of AMPBP (CID 11210285 hydrochloride; 2-amino-4-(3,4-Wnt signaling activator (methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine hydrochloride, N4-(1,3-benzodioxol-5-ylmethyl)-6-(3-methoxyphenyl)-2,4-pyrimidinediamine hydrochloride, a Wnt agonist); Lanzoprazole (2-[[[3-methyl-4-(2,2,2-trimethylphenyl)pyrimidine hydrochloride, a Wnt agonist); fluoroethoxy)-2-pyridinyl]methyl]sulfinyl]-1H-benzimidazole); parbendazole (methyl(5-butyl-1H-benzimidazole-2-nonsteroidal antiyl)carbamate); and rabeprazole sodium salt (1H-benzimidazole, 2-[[[4-(3-gastric pump H+/K=ATPase methoxypropoxy)-3-methyl-2-pyridinyl]methyl]sulfinyl). As described herein, AMBP activates Wnt signaling without inhibiting GSK-3β, lanzaoprazole is a gastric proton pump inhibitor, parbendazole has broad spectrum anthelmintic activity, and rabeprazole sodium salt is a gastric pump H ⁺ /K ⁺ ATPase inhibitor.

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

In other embodiments, the AUF1 protein or nucleic acid encoding AUF1 is administered in combination with another therapy used to treat LGMD.

Also provided are AUF1 AAV gene therapy constructs. The constructs have a codon-optimized, CpG-depleted coding sequence for human p40 AUF1 (SEQ ID NO: 17) operably linked to regulatory elements that facilitate expression in muscle cells, and optionally other regulatory elements such as polyadenylation sequences, intron sequences, WPRE or other elements, and/or stuffer sequences, including, for example, those disclosed herein (see, e.g., Table 10) (see Table 2 for nucleotide sequences of construct components). Exemplary constructs are described, for example, in FIG. 1 (see also Table 3). Constructs including flanking ITR sequences can have the nucleotide sequences of SEQ ID NOs: 31-36. Gene therapy vectors can be, for example, AAV8 serotype vectors, AAV9 serotype vectors, AAVhu. The AAV gene therapy vector may be a 32 serotype vector (see, e.g., capsids in Table 11) or other suitable AAV serotype capsid that, for example, facilitates delivery to or has tropism for skeletal muscle cells. Thus, provided are compositions comprising the AUF1 AAV gene therapy vectors described herein (e.g., those shown in FIG. 1), as well as methods of administering the same, for treating LGMD and subtypes of LGMD, e.g., LGMD calpainopathies, dystrophinopathies, dysferlinopathies, and sarcoglycanopathies, by restoring or increasing muscle mass, muscle function or performance, and/or reducing or reversing muscle atrophy. Such methods include, among other things, stabilizing the sarcolemma of muscle cells by providing AUF1 protein, 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 oxidized slow muscle fibers, and CAMKIIβ kinase activity. Such methods also include promoting increased muscle cell mass, muscle fiber number, muscle fiber size, reducing or reversing muscle cell atrophy, satellite cell activation and differentiation, muscle cell function (e.g., by increasing mitochondrial oxidative capacity), improving mitochondrial biogenesis, and increasing the proportion of slow muscle fibers in muscle (including by converting fast muscle fibers to slow muscle fibers).

Also provided are pharmaceutical compositions formulated for peripheral administration, e.g., intravenous administration, of rAAV encoding AUF1 as described herein.

1. Definitions The term "vector" is used interchangeably with "expression vector." The term "vector" may 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," so that all or part of the sequence is transcribed. It is not necessary that the transcript be expressed. It is also not necessary that the vector 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 that code for 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. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the expression level of the desired protein, and the like.

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 a minimal sequence of a transgene sufficient to initiate transcription of the coding sequence of the transgene. Promoters can be constitutive or inducible. A constitutive promoter is considered a strong promoter if it drives expression of a transgene at a level equivalent to 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); incorporated herein by reference in its entirety). The promoter may be a synthetic promoter, a modified promoter, or a hybrid promoter. The promoter may be linked to other regulatory sequences/elements that, when combined with the appropriate intracellular regulatory factors, promote ("enhancer") or suppress ("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 transcription rate or efficiency of the transgene. For example, a promoter sequence that is located proximal to the 5' end of the transgene coding sequence is usually operably linked to the transgene. As used herein, the term "regulatory element" is used interchangeably with "regulatory sequence" and refers to promoters, enhancers, and other expression control elements, or any combination of such elements.

Promoters are located 5' (upstream) of the gene they control. Many eukaryotic promoters contain two types of recognition sequences: the TATA box and the upstream promoter element. The TATA box is located 25-30 bp upstream from the transcription start site and is thought to be involved in ensuring that RNA polymerase II begins RNA synthesis at the correct site. In contrast, the upstream promoter element determines the rate at which transcription is initiated. These elements can act regardless of their orientation, but must be located within 100-200 bp upstream of the TATA box.

Enhancer elements can stimulate transcription from linked homologous or heterologous promoters up to 1000-fold. Enhancer elements often remain active 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); incorporated herein by reference in its entirety). Furthermore, unlike promoter elements, enhancers can be active even when located downstream from the transcription start site, e.g., within an intron, or even when located far away from the promoter (Yutzey et al., "An Internal Regulatory Element Controls Troponin I Gene Expression," Mol. Cell. Bio. 9(4):1397-1405 (1989); incorporated herein by reference in its entirety).

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

The term "AAV" or "adeno-associated virus" refers to a dependoparvovirus within the virus genus of the Parvoviridae family. The AAV may be derived from a naturally occurring "wild-type" virus, from a rAAV genome packaged in a capsid that contains a capsid protein encoded by a naturally occurring cap gene, and/or from a rAAV genome packaged in a capsid that contains a capsid protein encoded by a non-naturally occurring capsid gene. Examples of the latter include rAAVs having capsid proteins with altered sequences and/or peptide insertions in the amino acid sequence of the naturally occurring capsid.

The term "rAAV" refers to "recombinant AAV." In some embodiments, a recombinant AAV has an AAV genome in which some or all of the rep and cap genes have been replaced with heterologous sequences.

The term "rep-cap helper plasmid" refers to a plasmid that provides viral rep and cap gene functions and aids in the production of AAV from rAAV genomes that lack functional rep and/or cap gene sequences.

The term "cap gene" refers to a nucleic acid sequence that encodes a capsid protein that forms or helps form the capsid coat of a virus. In the case of AAV, the capsid protein can be VP1, VP2, or VP3.

The term "rep genes" refers to nucleic acid sequences that encode nonstructural proteins necessary for viral replication and production.

The terms "nucleic acid" and "nucleotide sequence" include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules or hybrid DNA/RNA molecules, and analogs of DNA or RNA molecules. Such analogs can be generated, for example, using nucleotide analogs, including, but not limited to, inosine or tritylated bases. Such analogs can also include DNA or RNA molecules that contain modified backbones that confer beneficial attributes to the molecule, such as, for example, increased nuclease resistance or 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, can contain triple-stranded portions, but is preferably double-stranded DNA.

The amino acid residues disclosed herein may be modified by conservative substitutions that maintain or substantially maintain the overall polypeptide structure and/or function. As used herein, "conservative amino acid substitutions" refers to the following: hydrophobic amino acids (i.e., Ala, Cys, Gly, Pro, Met, Val, lie, 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, and 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.

The terms "subject," "host," and "patient" are used interchangeably. A subject can be a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans), including humans.

The term "therapeutic agent" refers to any agent that can be used to treat, manage, or ameliorate symptoms associated with a disease or disorder related to the function provided by the transgene. A "therapeutically effective amount" refers to an amount of an agent (e.g., the amount of a product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of a disease or disorder of interest when administered to an afflicted subject. Additionally, a therapeutically effective amount with respect to an agent of the present disclosure refers to an amount of an agent alone, or in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of a disease or disorder.

The term "prophylactic agent" refers to any agent that can be used in preventing, reducing the likelihood of, delaying, or slowing the progression of a disease or disorder that is related to the function provided by the transgene. A "prophylactically effective amount" refers to an amount of the prophylactic agent (e.g., the amount of a product expressed by the transgene) that provides at least one prophylactic benefit when administered to a predisposed subject in preventing or delaying a disease or disorder of interest. A prophylactically effective amount may also refer to an amount of agent sufficient to prevent, reduce the likelihood, or delay the occurrence of, or delay the progression of, a disease or disorder of interest, an amount sufficient to delay or minimize the onset of, or prevent or delay the recurrence or spread of, a disease or disorder of interest. A prophylactically effective amount may also refer to an amount of agent sufficient to prevent or delay the worsening of symptoms of a disease or disorder of interest. Additionally, a prophylactically effective amount for a prophylactic agent of the present disclosure refers to an amount of the prophylactic agent alone, or in combination with other agents, that provides at least one prophylactic benefit in preventing or delaying a disease or disorder.

The prophylactic agents of the present disclosure can be administered to a subject who is "predisposed" to a disease or disorder of interest. A subject who is "predisposed" to a disease or disorder is one who exhibits symptoms associated with the development of the disease or disorder, or who has the genetic makeup, environmental exposure, or other risk factors for such a disease or disorder, but is not yet symptomatic at a level that would result in a diagnosis of the disease or disorder. For example, a patient who has a family history of a disease associated with a defective gene (provided by the transgene) may be considered to be predisposed to the disease. Additionally, a patient with a dormant tumor that persists after removal of the primary tumor may be considered to be predisposed to tumor recurrence.

The term "pharmaceutical acceptable carrier" refers to a carrier that does not cause an allergic reaction or other untoward effects in a patient to whom it is administered and is compatible with other ingredients of the formulation. Pharmaceutically acceptable carriers include, for example, pharmaceutical diluents, excipients, or carriers, suitably selected with respect to the intended form of administration and consistent with conventional pharmaceutical practice. For example, solid carriers/diluents include, but are not limited to, gums, starches (e.g., corn starch, pregelatinized starch), sugars (e.g., lactose, mannitol, sucrose, dextrose), cellulosic materials (e.g., microcrystalline cellulose), acrylates (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof. Pharmaceutically acceptable carriers may further include minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the nucleic acid molecules described herein.

The term "CpG island" refers to a characteristic region of a genome that contains a high frequency of the dinucleotide CpG (e.g., a C (cytosine) base immediately followed by a G (guanine) base (CpG)), and thus the G+C content of CpG islands is significantly higher than non-island DNA. CpG islands can be identified by analysis of nucleotide length, nucleotide composition, and frequency of CpG dinucleotides. The CpG island content in any particular nucleotide sequence or genome can be measured using the following criteria: island size >100, GC percent >50.0%, and a ratio of observed to expected number of CG dinucleotides based on the number of Gs and Cs in the segment >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.

For such calculations, see 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. Various software tools are available, such as the MethPrimer.html (Gardiner-Garden and Frommer, J. Mol. Biol. 196(2):261-82 (1987); see also Li LC and Dahiya R., "MethPrimer: Designing Primers for Methylation PCRs," Bioinformatics 18(11):1427-31 (2002), which are incorporated by reference in their entireties). In one embodiment, an algorithm for identifying CpG islands is found at www.urogen.org/cgi-bin/methprimer/methprimer.cgi.

2. AU-rich mRNA-binding factor 1 vectors 2.1. AU-rich mRNA-binding factor 1 transgenes Nucleic acids comprising a transgene encoding AUF1 or a therapeutically functional fragment thereof, including the p37, p40, p42, and p45 isoforms of human and mouse AUF1, as well as vectors and viral particles, including rAAV, containing the same, and methods of using the same in methods of treatment, prevention, or amelioration of symptoms of conditions associated with reduced muscle mass or performance or where increased muscle mass or performance is desired or useful, are provided. The AUF1 gene therapy vectors are used in methods of treating or ameliorating symptoms of LGMD by administering the AUF1 gene therapy vector.

Genes involved in rapid responses to cellular stimuli are highly regulated and typically encode mRNAs that are selectively and rapidly degraded, rapidly terminating protein expression and reprogramming the cell (Moore et al., "Physiological Networks and Disease Functions of RNA-binding Protein AUF1," Wiley Interdiscip. Rev. RNA 5(4):549-64 (2014); incorporated herein 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,” Wiley Interdiscip Rev RNA 5(4):549-64 (2014)). Protein, AUF1," Mol. Cell. Biol. 13(12):7652-65 (1993); the entirety of which is incorporated herein by reference), 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); the entirety of which is incorporated herein by reference).

Short-lived mRNAs typically contain an AU-rich element (ARE) in the 3' untranslated region (3'UTR) of the mRNA, with the repeat sequence AUUUA that leads to rapid decay or possibly stabilization (Moore et al., "Physiological Networks and Disease Functions of RNA-binding Protein AUF1," Wiley Interdiscip Rev. RNA 5(4):549-64 (2014); incorporated herein by reference in its entirety). AREs serve 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," Wiley Interdiscip. Rev. RNA 5(4):549-64 (2014); 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); all of which are incorporated herein by reference in their entireties).

AU-rich mRNA-binding factor 1 (AUF1; also known as heterogeneous nuclear ribonucleoprotein D0, hnRNP D0; HNRNPD gene) binds with high affinity to repetitive AU-rich elements located in the 3'UTR, which are present in approximately 5% of mRNAs. AUF1 typically targets ARE-mRNAs for rapid degradation, but conversely, in a less understood way, it can stabilize some ARE-mRNAs, increasing their translation (Moore et al., "Physiological Networks and Disease Functions of RNA-Binding Protein AUF1," Wiley Interdiscip. Rev. RNA 5(4):549-564 (2014); incorporated herein by reference in its entirety). It has previously been reported that mice lacking AUF1 are unable to execute the myogenic program, leading to accelerated loss of muscle mass (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); incorporated herein by reference in its entirety). In addition, it has been shown that AUF1 expression in skeletal muscles is significantly decreased with age, which contributes greatly to muscle loss and atrophy, loss of muscle mass, and loss of 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 entire contents of which are incorporated herein by reference). Additionally, AUF1 has been shown to control all major stages of skeletal muscle development, beginning with satellite cell activation and lineage commitment, by selectively targeting for rapid degradation key differentiation checkpoint mRNAs that prevent entry into the next steps of muscle development.

AUF1 has four related protein isoforms (p37 ^AUF1 , p40 ^AUF1 , p42 ^AUF1 , and p45 ^AUF1 ) that are distinguished by molecular weight and are generated 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 & Syu, "AU-Rich Elements: Characterization and Importance in mRNA Expression and Functions," Wiley Interdiscip. Rev. RNA 5(4):549-564 (2014)). and Kim et al., "Emerging Roles of RNA and RNA-Binding Protein Network in Cancer Cells," BMB Rep. 42(3):125-130 (2009); which are incorporated herein by reference in their entireties. Each of these four isoforms contains two centrally located, tandemly arranged RNA recognition motifs ("RRMs") that mediate 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); incorporated herein by reference in its entirety).

The general structure of the RRM is a β-α-β-β-α-β RNA-binding platform of antiparallel 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); incorporated herein by reference in their entireties. The structures of individual AUF1 RRM domains resolved by NMR are largely consistent with 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 hnRNPs," Front. Biosci. 16:2307-2325 (2013)). 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); all of which are incorporated herein by reference in their entireties).

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); incorporated herein by reference in its entirety), suggesting that AUF1 is crucial in maintaining skeletal muscle after birth.

The term "fragment" or "portion" as used herein with respect to a given polypeptide sequence (e.g., AUF1) refers to a contiguous stretch of amino acids in the sequence of the given polypeptide that is shorter than the full-length sequence of the given polypeptide. A fragment of a polypeptide may be defined by its first position and its last position, which respectively correspond to positions in the sequence of the given full-length polypeptide. The sequence position corresponding to the first position is located N-terminally to the sequence position corresponding to the last position. A fragment or portion sequence is a contiguous amino acid sequence or stretch of amino acids in a 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 the functional characteristics 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 may contain a functionally important domain, such as, for example, 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.

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

The AUF1 or functional fragment thereof described herein may be derived from a mammalian AUF1. In one embodiment, the AUF1 or functional fragment thereof is human AUF1 or a functional fragment thereof. In another embodiment, the AUF1 or functional fragment thereof is mouse AUF1 or a functional fragment thereof. The AUF1 protein according to the embodiments described herein may include one or more of the AUF1 isoforms p37 ^AUF1 , p40 ^AUF1 , p42 ^AUF1 , and p45 ^AUF1 . 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 incorporated herein by reference in its entirety.

Table 1. Summary of GenBank accession numbers for AUF1 sequences.

The sequences referenced in Table 1 are reproduced below.

The human p37 ^AUF1 nucleotide sequence, GenBank Accession No. NM_001003810.1 (SEQ ID NO:1), is as follows:

The human p37 ^AUF1 amino acid sequence of GenBank Accession No. NP_001003810.1 (SEQ ID NO:2) is as follows:

The human p40 ^AUF1 nucleotide sequence of GenBank Accession No. NM_002138.3 (SEQ ID NO:5) is as follows:

The human p40 ^AUF1 amino acid sequence of GenBank Accession No. NP_002129.2 (SEQ ID NO:6) is as follows:

The human p42 ^AUF1 nucleotide sequence of GenBank Accession No. NM_031369.2 (SEQ ID NO:9) is as follows:

The human p42 ^AUF1 amino acid sequence of GenBank Accession No. NP_112737.1 (SEQ ID NO: 10) is as follows:

The human p45 ^AUF1 nucleotide sequence of GenBank Accession No. NM_031370.2 (SEQ ID NO:13) is as follows:

The human p45 ^AUF1 amino acid sequence of GenBank Accession No. NP_112738.1 (SEQ ID NO: 14) is as follows:

The mouse p37 ^AUF1 nucleotide sequence of GenBank Accession No. NM_001077267.2 (SEQ ID NO:3) is as follows:

The mouse p37 ^AUF1 amino acid sequence of GenBank Accession No. NP_001070735.1 (SEQ ID NO:4) is as follows:

The mouse p40 ^AUF1 nucleotide sequence of GenBank Accession No. NM_007516.3 (SEQ ID NO:7) is as follows:

The mouse p40 ^AUF1 amino acid sequence of GenBank Accession No. NP_031542.2 (SEQ ID NO:8) is as follows:

The nucleotide sequence of mouse p42 ^AUF1 , GenBank Accession No. NM_001077266.2 (SEQ ID NO:11), is as follows:

The mouse p42 ^AUF1 amino acid sequence of GenBank Accession No. NP_001070734.1 (SEQ ID NO: 12) is as follows:

The mouse p45 ^AUF1 nucleotide sequence of GenBank Accession No. NM_001077265.2 (SEQ ID NO: 15) is as follows:

The mouse p45 ^AUF1 amino acid sequence of GenBank Accession No. NP_001070733.1 (SEQ ID NO: 16) is as follows:

It should be noted that the sequences described herein may be described with reference to accession numbers, including, for example, coding sequences or protein sequences with or without additional sequence elements or portions (e.g., leader sequences, tags, immature portions, control regions, etc.), for example, as provided in Table 1. Thus, reference to such sequence accession numbers or corresponding sequence identification numbers refers to either the sequence fully described therein or some portion thereof (e.g., the portion encoding the protein or polypeptide of interest to the technology described herein (e.g., AUF1 or a functional fragment thereof); the mature protein sequence described within the longer amino acid sequence; the control region of interest (e.g., promoter sequence or regulatory element) disclosed within the longer sequence described herein, etc.). Similarly, variants and isoforms of the accession numbers and corresponding sequence identification numbers described herein are also contemplated.

Thus, in certain embodiments, the AUF1 protein referred to herein has an amino acid sequence as set forth in Table 1 and as disclosed herein, or is a functional fragment thereof. In certain embodiments, the AUF1 is a p37, p40, p42, or p45 form of human AUF1, having the amino acid sequence of SEQ ID NO: 2, 6, 10, or 14, respectively. In other embodiments, the AUF1 is a p37, p40, p42, or p45 form of mouse AUF1, having the amino acid sequence of SEQ ID NO: 4, 8, 12, or 16, respectively. In certain embodiments, the AUF1 has 90%, 95%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, 6, 10, or 14, and has AUF1 functional activity. In certain embodiments, the AUF1 has 90%, 95%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4, 8, 12, or 16, and has AUF1 functional activity. In one embodiment, the functional fragments referred to herein include an amino acid sequence having 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 sequence of SEQ ID NO: 2, 6, 10, or 14 for human AUF1, or to the amino acid sequence of SEQ ID NO: 4, 8, 12, or 16 for mouse AUF1.

Also provided is a nucleic acid comprising a nucleotide sequence encoding a human AUF1 protein or a functional fragment thereof, for example, a nucleotide sequence of SEQ ID NO: 1, 5, 9, or 13. Also provided is a nucleic acid 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 an amino acid sequence of SEQ ID NO: 2, 6, 10, or 14. Codon-optimized sequences encoding AUF1 proteins are provided, including the codon-optimized version of the human p40 AUF1 coding sequence being the nucleotide sequence of SEQ ID NO: 17. Also provided is a nucleic acid 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 an amino acid sequence of SEQ ID NO: 4, 8, 12, or 16.

In some embodiments, the AAV vectors and viral 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, particularly in an expression cassette described herein, for expression in a cell of a subject, particularly a muscle cell of a subject.

2.2. AUF Gene Cassettes Another aspect provided herein relates to a nucleic acid expression cassette comprising a nucleic acid encoding AUF1 (including human p37, p40, p42 or p45 AUF1, including combinations thereof) or a functional fragment thereof, operably linked to regulatory elements, e.g., promoter elements, and optionally enhancer elements and/or introns, for enhancing or promoting expression of the nucleic acid encoding AUF1 or a functional fragment thereof, e.g., in muscle cells. 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 (or, e.g., in the case of mouse model testing, 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 is SEQ ID NO: 17, encoding human p40 AUF1, and 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 (or, for example, in the case of mouse model studies, 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 elements include promoters and can be constitutive or tissue-specific, i.e., active (or substantially more active or significantly more active) only in target cells/tissues. In particular, promoters and other regulatory elements that promote muscle-specific expression are provided, such as those in Table 8 below. In embodiments involving use as a transgene in a recombinant AAV particle, the expression cassette or transgene is flanked by inverted terminal repeats (ITRs), including, for example, AAV2 ITRs, forms of ITRs for single-stranded AAV genomes, or self-complementary AAV genomes. For example, the 5' and 3' ITR sequences are SEQ ID NOs: 28 and 29, respectively. In one embodiment, the 5' ITR is mutated for a self-complementary vector, and may have, for example, the nucleotide sequence of SEQ ID NO: 30.

2.2.1. Codon Optimization and CpG Removal In one embodiment, the nucleotide sequence encoding AUF1 is modified by codon optimization and removal of CpG dinucleotides and CpG islands. Immune responses to transgenes are a concern in human clinical applications. AAV-induced immune responses 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); incorporated herein by reference in its entirety). Removal of CpG motifs from the transgene sequence may result in stable and long-term transgene expression by reducing the role of TLR9 in activating innate immunity upon recognition of the transgene as non-self (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 incorporated herein by reference in their entirety). In an embodiment, the AUF1 nucleotide sequence and expression cassette are human codon-optimized with CpG removal. The codon-optimized and CpG-removed nucleotide sequence can be designed by any method known in the art, including, for example, Thermo Fisher Scientific GeneArt Gene Synthesis tool utilizing GeneOptimizer (Waltham, MA USA). The nucleotide sequence of SEQ ID NO: 17 described herein represents a codon-optimized and CpG-removed sequence.

2.2.2. AUF1 rAAV Genomic Constructs Constructs useful as cis-plasmids for rAAV construction are provided that contain nucleotide sequences encoding AUF1 (including its p37, p40, p42 or p45 (including mouse and human) isoforms) operably linked to regulatory sequences that promote AUF1 expression in muscle cells.

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 regulatory sequences that promote expression in muscle cells. In certain embodiments, the construct has a muscle-specific promoter that can be Spc5-12 (the modified Spc5-12 promoters disclosed herein, Spc5v1 or Spc5v2 (including SEQ ID NOs: 127 and 128, respectively), tMCK or CK7 (see also Table 8 herein for promoters), and optionally an intron sequence between the promoter and the AUF1 coding sequence, e.g., a VH4 intron (see Table 9 for intron sequences), a polyA signal sequence, e.g., a rabbit beta globin polyA signal sequence, or ... The construct may also include a 5' stuffer sequence and/or a 3' stuffer sequence (including 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), as well as an SV40 polyadenylation signal sequence inverted relative to the coding sequence and adjacent to the 3' ITR sequence. In certain embodiments, the construct has one or more components of Table 2.

Table 2. Components of the AUF1 construct

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

Exemplary rAAV genomes and sequences contained within the cis plasmids are shown in Figure 1 and Table 3 and include:

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

tMCK-huAUF1: codon-optimized, CpG-depleted human AUF1 sequence (including 5' (141 bp) stuffer and 3' (893 bp) stuffer) driven by the tMCK promoter (no introns) - downstream SV40 polyA signal (reverse); has the nucleotide sequence of SEQ ID NO:32 (including ITR sequences).

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

ss-CK7-Hu-AUF1: codon-optimized, CpG-depleted human AUF1 sequence (including 5' (141 bp) stuffer and 3' (893 bp) stuffer) driven by the CK7 promoter (intron-free) - downstream SV40 polyA signal (reverse); SEQ ID NO: 34 (including ITR sequences).

spc-hu-AUF1-intronless: codon-optimized, CpG-depleted human AUF1 sequence (including 5' (141 bp) stuffer and 3' (893 bp) stuffer) driven by Spc5-12 promoter (intronless) - downstream SV40 polyA signal (reverse); SEQ ID NO:35 (including ITR sequence).

D(+)-CK7AUF1: Self-complementary vector; codon-optimized, CpG-deleted human AUF1 sequence driven by the CK7 promoter (no stuffer); SEQ ID NO:36 (including ITR sequence).

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

(Table 3)

Provided are AAV or rAAV particles comprising these recombinant genomes encoding AUF1 and cis-plasmid vectors comprising these sequences used to produce AAV or rAAV particles, including AAV8 serotype, AAV9 serotype or AAVhu.32 serotype particles described herein, which may be 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 reducing or reversing muscle atrophy, as further described herein, including types of LGMD. In further embodiments, these AAV genomes or rAAV genomes, and AAV particles or rAAV particles produced from cis-plasmids containing these sequences described herein, including those in Table 3, are administered in combination with AAV or rAAV containing transgenes encoding DAPC protein components, e.g., microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan and δ-sarcoglycan, for the treatment of dystrophinopathy, including limb-girdle muscular dystrophy (LGMD), in a subject, including a human subject in need thereof. In other embodiments, methods are provided for treating LGMD, including sarcoglycanopathies, calpainopathies, dysferlinopathies, and dystrophinopathies, in a subject, including a human subject in need thereof, by administering an AAV or rAAV gene therapy vector comprising a transgene encoding AUF1, including an AAV genome or rAAV genome of Table 3, in combination with another therapy effective to treat LGMD, including sarcoglycanopathies, calpainopathies, dysferlinopathies, and dystrophinopathies, including those described herein.

3. Micro-dystrophin vector 3.1. Transgene-encoded micro-dystrophin In some embodiments, encoded by one of the transgenes provided herein for the methods of the invention is a micro-dystrophin consisting of dystrophin domains arranged from amino terminus to carboxy terminus as follows: 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 the portion of the CT domain that contains the α1-syntrophin binding site (SEQ ID NO:50)).

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

The present disclosure contemplates variants of microdystrophin, provided that the therapeutic efficacy of microdystrophin including 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) improving muscle function in an animal model (e.g., mdx mouse model) or human subject; and/or (3) cardioprotection or improvement of myocardial function in an animal model or human patient.

Table 4 provides 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 as substitution variants of microdystrophin defined by SEQ ID NO: 176 (DYS1), 177 (DYS3), or 178 (DYS5). For example, SEQ ID NO: 176, 177, or 178 (or SEQ ID NOs: 179-183) and substantially maintain its functional activity. In embodiments, the microdystrophin may 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 NOs: 176, 177, or 178 (or SEQ ID NO: 183) and may maintain functional microdystrophin activity, e.g., as determined by one or more of the in vitro or in vivo assays in animal models disclosed below.

Table 4. Amino acid sequences of RGX-DYS and microdystrophin proteins

3.2. Micro-dystrophin-Encoding Nucleic Acid Compositions Another aspect of the present disclosure is a nucleic acid comprising a nucleotide sequence encoding micro-dystrophin as described herein. Such a nucleic acid comprises a nucleotide sequence encoding micro-dystrophin having domains arranged from N-terminus to C-terminus as follows: ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT, as detailed above. The nucleotide sequence may be any nucleotide sequence that encodes the domains. The nucleotide sequence may be codon optimized and/or CpG islands removed for expression in appropriate circumstances.

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.

3.2.1 Codon Optimization and CpG Removal In one embodiment, the nucleotide sequence encoding the microdystrophin cassette is modified by codon optimization and removal of CpG dinucleotides and CpG islands. Immune responses to the microdystrophin transgene are a concern in human clinical applications, as demonstrated in the first Duchenne muscular dystrophy (DMD) gene therapy clinical trial and in several adeno-associated virus (AAV)-minidystrophin 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. (2010); “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); incorporated herein by reference in their entireties.

In embodiments, the micro-dystrophin cassette is human codon optimized with CpG deletion. The codon optimized and CpG deleted nucleotide sequence can be designed by any method known in the art, including, for example, Thermo Fisher Scientific GeneArt Gene Synthesis tool utilizing GeneOptimizer (Waltham, MA USA). The nucleotide sequences of SEQ ID NOs: 91, 92, and 93 described herein represent codon optimized and CpG deleted sequences.

The amino acid sequence of DYS1 (SEQ ID NO:91) is as follows:
ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAAGATGTGCAGAAGAAAAACCTT CACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCAAGCAGCACATTGAGAGACCTGTTCAG TGACCTGCAGGATGGCAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAGAAGCTGCC TAAAGAGAAGGGCAGCACAAGAGTGCATGCCCTGAACAATGTGAACAAGGCCCTGAGAGTGCT GCAGAACAACAATGTGGACCTGGTCAATATTGGCAGCACAGACATTGTGGATGGCAACCACA AGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAAGTGAAGAATGTGATGAAGA ACATCATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTGCTGAGCTGGGTCAGACAGA GCACCAGAAAACTACCCTCAAGTGAATGTGATCAACTTCACCACCTCTTGGAGTGATGGACTGG CCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTTTGACTGGAACTCTGTTGTGTGC CAGCAGTCTGCCACAGAGAGACTGGAACATGCCTTCCAACATTGCCAGATACCAGCTGGGAATT GAGAAAACTGCTGGACCCTGAGGATGTGGACACCACCTATCCTGACAAGAAATCCATCCTCCATG TACATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGCCATTCAAGAGGTT GAGATGCTGCCCAGACCTCCTAAAGTGACCAAAGAGGAACACTTCCAGCTGCACCACCAGAT GCACTACTCTCAGCAGATCACAGTGTCTCTGGCCCAGGGATATGAGAGAACAAGCAGCCCCAA GCCTAGGTTCAAAGAGCTATGCCTACACACAGGCTGCCTATGTGACCACATCTGACCCCACAAG AAGCCCATTTCCAAGCCAGCATCTGGAAGCCCCTGAGGACAAGAGCTTTGGCAGCAGCCTGAT GGAATCTGAAGTGAACCTGGATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCTGGCTGC TGTCTGCTGAGGATACACTGCAGGCTCAGGGTGAAATCAGCAATGATGTGGAAGTGGTCAAGG ACCAGTTTCACACCCATGAGGGCTACATGATGGACCTGACAGCCCACCAGGGGCAGAGTGGGAA ATATCCTGCAGCTGGGCTCCAAGCTGATTGGCACAGGCAAGCTGTCTGAGGATGAAGAGACAG AGGTGCAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTCTGAGAGTGGCCAGCATG GAAAAGCAGAGCAACCTGCAGAGTGCTCATGGACCTGCAGAATCAGAAAACTGAAAGAACTG AATGACTGGCTGACCAAAGACAGAAGAAAGGACTAGGAAGATGGAAGAGGAACCTCTGGGACCA GACCTGGAAGATCTGAAAAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTTGAGCAA GAGCAAGTCAGAGTGAACAGCCTGACACACATGGTGGTGGTTGTGGATGAGTCCTCTGGGGA TCATGCCACAGCTGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGGGCCCAACATCTG TAGGTGGACAGAGGATAGATGGGTGCTGCTCCAGGACATTCTGCTGAAGTGGCAGAGACTGAC AGAGGAACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGATGCTGTCAACAAGATCCA TACCACAGGCTTCAAGGATCAGAATGAGATGCTCAGCTCCCTGCAGAAAACTGGCTGTGCTGA AGGCTGACCTGGAAAAGAAAAAAGCAGTCCATGGGCAAGCTCTAACAGCCTGAAGCAGGACCTGC TGTCTACCCTGAAGAACAAGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGAACAACTTTGCTA GATGCTGGGACAACCTGGTGCAGAAGCTGGAAAAGTCTACAGCCCAGATCAGCCAGCAACCTG ATCTTGCCCCTGGCCTGACCACAATTGGAGCCTCTCCAACACAGACTGTGACCCTGGTTACC CAGCCAGTGGTCACCAAAGAGACAGCCATCAGCAAAACTGGAAATGCCCAGCTCTCTGATGCTG GAAGTCCCACACTGGAAAGGCTGCAAGAACTTCAAAGAGGCCACAGATGAGCTGGACCTGAAG CTGAGACAGGCTGAAGTGATCAAAGGCAGCTGGCAGCCAGTTGGGGACCTGCTCATTGATAGC CTGCAGGACCATCTGGAAAAAAGTGAAAGCCCTGAGGGGAGAGATTGCCCCTCTGAAAGAAAA TGTGTCCCATGTGAATGACCTGGCCAGACAGCTGACCACACTGGGAATCCAGCTGAGCCCTA CAACCTGAGCACCCTTGAGGACCTGAACACCAGGTGGAAGCTCCTCCAGGTGGCAGTGGAAGA TAGAGTCAGGCAGCTGCATGAGGCCCCACAGAGATTTTGGACCAGCCAGCCAGCACTTTCTGTC TACCTCTGTGCAAGGCCCCTGGGAGAGAGCTATCTCTCCTAACAAGGTGCCCTACTACATCA ACCATGAGACACAGACCACCTGTTGGGATCACCCCAAGATGACAGAGCTGTACCAGAGTCTG CAGACCTCCAACAATGTCAGATTCAGTGCCTACAGGACTGCCATGAAGCTCAGAAGGCTCCAGA AAGCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCAGCTTGTGATGCCCTGGACCAGCACAATC TGAAGCAGAATGACCAGCCTATGGACATCCTCCAGATCATCAACTGCCTCACCACCATCTAT GATAGGCTGGAACAAGAGCACAACAATCTGGTCAATGTGCCCCTGTGTGTGGACATGTGCCTG AATTGGCTGCTGAATGTGTATGACACAGGCAGAACAGGCAGGATCAGAGTCCTGTCCTTCAAG ACAGGCATCATCTCCCTGTGCAAAGCCCACTTGGAGGACAAGTACAGATACCTGTTCAAGCAA GTGGCCTCCAGCACAGGCTTTGTGACCAGAGAAGGCTGGGCCTGCTCCTGCATGACAGCAT TCAGATCCCTAGACAGCTGGGAGAAGTGGCTTCCTTTGGAGGCAGCAATATTGAGCCATCAGT CAGGTCCTGTTTTCAGTTTGCCAAACAACAAGCCTGAGATTGAGGCTGCCCTGTTCCTGGACTG GATGAGACTTGAGCCTCAGAGCATGGTCTGGCTGCCTGTGCTTCATAGAGTGGCTGCTGCTGA GACTGCCAAGCACCAGGCCAAGTGCAAACATCTGCAAAGAGTGCCCCATCATTGGCTTCAGAT ACAGATCCCTGAAGCACTTCAACTATGATATCTGCCAGAGCTGCTTCTTTAGTGGCAGGGGTTG CCAAGGGCCACAAATGCACTACCCCATGGTGGAATACTGCACCCCCAACAAACCTCTGGGGAAG ATGTTAGAGACTTTGCCAAGGTGCTGAAAAAACAAGTTCAGGACCAAGAGATACTTTGCTAAGC ACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTGCTTGAGGGTGACAACATGGAAACCCCCT GTGACACTGATCAATTTCTGGCCAGTGGACTCTGCCCCTGCCTCAAGTCCACAGCTGTCCCAT GATGACACCCACAGCAGAATTGAGCACTATGCCTCCAGACTGGCAGAGATGGAAAACAGCAAT GGCAGCTACCTGAATGATAGCATCAGCCCCAATGAGAGCATTGATGATGAGCATCTGCTGATC CAGCACTACTGTCAGTCCCTGAACCAGGACTCTCCACTGAGCCAGCCTAGAAGCCCTGCTCAG ATCCTGATCAGCCTTGAGTCTGAGGAAAGGGGAGAGCTGGAAAGAATCCTGGCAGATCTTGAG GAAGAGAACAGAAACCTGCAGGCAGAGTATGACAGGCTCAAACAGCAGCATGAGCACAAGGGGA CTGAGCCCTCTGCCTTCTCCTCCTGAAATGATGCCCACCTCTCCACAGTCTCCAAGGTGATGA

The amino acid sequence of DYS3 (SEQ ID NO:92) is as follows:
ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAAGATGTGCAGAAAGAAAACCTTCACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCAAGCAGCACATT GAGAACCTGTTCAGTGACCTGCAGGATGGCAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAGAAGCTGCCTAAAAGAGAAGGGCAGCACAAGAGTGCATGCCCTGA ACAATGTGAACAAGGCCCTGAGAGTGCTGCAGAACAACAATGTGGACCTGGTCAATATTGGCAGCACAGACATTGTGGATGGCAACCACAAGCTGACCCTGGGCCTGATCT GGAACATCATCCTGCACTGGCAAGTGAAGAATGTGATGAAGAACATCATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTGCTGAGCTGGGTCAGACAGAGCACCAG AAAACTACCCTCAAGTGAATGTGATCAACTTCACCACCTCTTGGAGTGATGGACTGGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTGTTTGACTGGAACTCTGT TGTGTGCCAGCAGTCTGCCACAGAGACTGGAACATGCCTTCAACATTGCCAGATACCAGCTGGGAATTGAGAAAACTGCTGGACCCTGAGGATGTGGACACCACCTATCCT GACAAGAAATCCATCCTCATGTACATCACCAGCCTGTTCCAGGTGCTGCCCCAGCAAGTGTCCATTGAGGCCATTCAAGAGGTTGAGATGCTGCCCAGACCTCCTAAAGTGA CCAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACTACTCTCAGCAGATCACAGTGTCTCTGGCCCAGGGATATGAGAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAG CTATGCCTACACAGGCTGCCTATGTGACCACATCTGACCCCACAAGAAGCCCATTTCCAAGCCAGCATCTGGAAGCCCCTGAGGACAAGAGCTTTGGCAGCAGCCTGAT GGAATCTGAAGTGAACCTGGATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCTGGCTGCTGTCTGCTGAGGGATACACTGCAGGCTCAGGGTGAAATCAGCAATGATGTG GAAGTGGTCAAGGACCAGTTTCACACCCATGAGGGCTACATGATGGACCTGACAGCCCACCAGGGCAGAGTGGGAAATATCCTGCAGCTGGGCTCCAAGCTGATTGGCACA GGCAAGCTGTCTGAGGATGAAGAGACAGAGGTGCAAGAGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTCTGAGAGTGGCCAGCATGGAAAAGCAGAGCAACCTGCACA GAGTGCTCATGGACCTGCAGAATCAGAAAACTGAAAGAACTGAATGACTGGCTGACCAAGACAGAAGAAAGGACTAGGAAGATGGAAGAGGAACCTCTGGGACCAGACCTGG AAGATCTGAAAAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTTGAGCAAGAGCAAGTCAGAGTGAACAGCCTGACACACATGGTGGTGGTTGTGGATGAGTCCTC TGGGGATCATGCCACAGCTGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGGGCCCAACATCTGTAGGTGGACAGAGGATAGATGGGTGCTGCTCCAGGACATTCTG CTGAAGTGGCAGAGACTGACAGAGGAACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGATGCTGTCAAAGAGATCCATACCACAGGCTTCAAGGATCAGAATGAGA TGCTCAGCTCCCTGCAGAAACTGGCTGTGCTGAAGGCTGACCTGGAAAAGAAAAAAGCAGTCCATGGGCAAGCTCTACAGCCTGAAGCAGGACCTGCTGTCTACCCTGAAGA ACAAGTCTGTGACCCAGAAAACTGAGGCCTGGCTGGACAACTTTGCTAGATGCTGGGAACAACCTGGTGCAGAAGCTGGAAAAGTCTACAGCCCAGATCAGCCAGCAACCTGA TCTTGCCCCTGGCCTGACCACAATTGGAGCCTCTCCAACACAGACTGTGACCCTGGTTACCCAGCCAGTGGTCACCAAAGAGACAGCCATCAGCAAAACTGGAAATGCCCAG CTCTCTGATGCTGGAAGTCCCACACTGGAAAGGCTGCAAGAACTTCAAGAGGCCACAGATGAGCTGGACCTGAAGCTGAGACAGGCTGAAGTGATCAAAGGCAGCTGGCAG CCAGTTGGGGACCTGCTCATTGATAGCCTGCAGGACCATCTGGAAAAAAGTGAAAGCCCTGAGGGGAGAGATTGCCCCTGAAAGAAAATGTGTCCCATGTGAATGACCTG GCCAGACAGCTGACCACACTGGGAATCCAGCTGAGCCCCTACAACCTGAGCACCCTTGAGGACCTGAAACACCAGGTGGAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGTCA GGCAGCTGCATGAGGCCACAGAGATTTTGGACCAGCCAGCCAGCACTTTCTGTCTACCTCTGTGCAAGGCCCCTGGGAGAGAGCTATCTTCCTAACAAGGTGCCCTACTA CATCAACCATGAGACACAGACCACCTGTTGGGATCACCCCAAGATGACAGAGCTGTACCAGAGTCTGGCAGACCTCAACAATGTCAGATTCAGTGCCTACAGGACTGCCATG AAGCTCAGAAGGCTCCAGAAAGCTCTGTGCCTGGACCTGCTTTCCCTGAGTGCAGCTTGTGATGCCCTGGACCAGCACAATCTGAAGCAGAATGACCAGCCTATGGACATC CTCCAGATCATCAACTGCCTCACCACCATCTATGATAGGCTGGAACAAGAGCACAACAATCTGGTCCAATGTGCCCCTGTGTGTGGACATGTGCCTGAATTGGCTGCTGAATG TGTATGACACAGGCAGAACAGGCAGGATCAGAGTCCTGTCCTTCAAGACAGGCATCATCTCCCTGTGCAAAGCCCACTTGGAGGACAGTACAGATAACCTGTTCAAGCAAGT GGCCTCCAGCACAGGCTTTTGTGACCAGAGAAGGCTGGGCCTGCTCCTGCATGACAGCATTCAGATCCCTAGACAGCTGGGAGAAGTGGCTTCCTTTGGAGGCAGCAATATT GAGCCATCAGTCAGGTCCTGTTTTCAGTTTGCCAACAACAAGCCTGAGATTGAGGCTGCCCTGTTCCTGGACTGGATGAGACTTGAGCCTCAGAGCATGGTCTGGCTGCCT GTGCTTCATAGAGTGGCTGCTGCTGAGACTGCCAAGCACCAGGCCAAGTGCAACATCTGCAAAGAGTGCCCCATCATTGGCTTCAGATACAGATCCCTGAAGCACTTCAACT ATGATATCTGCCAGAGCTGCTTCTTTAGTGGCAGGGTTGCCAAGGGCACAAAATGCACTACCCCATGGTGGAATACTGCACCCCAACAACCTCTGGGGAAGATGTTAGAGA CTTTGCCAAGGTGCTGAAAAACAAGTTCAGGACCAAGAGATAACTTTGCTAAGCACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTGCTTGAGGGTGACAACATGGAAAACC

The amino acid sequence of DYS5 (SEQ ID NO:93) is as follows:
ATGCTTTGGTGGGAAGAGGTGGAAGATTGCTATGAGAGGGAAGATGTGCAGAAGAAAAACC TTCACCAAATGGGTCAATGCCCAGTTCAGCAAGTTTGGCAAGCAGCACATTGAGAACCTG TTCAGTGACCTGCAGGATGGCAGAAGGCTGCTGGATCTGCTGGAAGGCCTGACAGGCCAG AAGCTGCCTAAAGAGAAGGGGCAGCACAAGAGTGCATGCCCTGAACAATGTGAACAAGGCC CTGAGAGTGCTGCAGAACAACAATGTGGACCTGGTCAATATTGGCAGCACAGACATTGTG GATGGCAACCACAAGCTGACCCTGGGCCTGATCTGGAACATCATCCTGCACTGGCAAGTG AAGAATGTGATGAAGAACATCATGGCTGGCCTGCAGCAGACCAACTCTGAGAAGATCCTG CTGAGCTGGGTCAGACAGAGCACCAGAAAACTACCCTCAAGTGAATGTGATCAACTTCACCA CCTCTTGGAGTGATGGACTGGCCCTGAATGCCCTGATCCACAGCCACAGACCTGACCTGGT TTGACTGGAACTCTGTTGTGTGCCAGCAGTCTGCCACAGAGACTGGAACATGCCTTCA ACATTGCCAGATACCAGCTGGGAATTGAGAAAACTGCTGGACCCTGAGGATGTGGACACCA CCTATCCTGACAAGAAATCCATCCTCATGTACATCACCAGCCTGTTCCAGGTGCTGCCCCA GCAAGTGTCCATTGAGGCCATTCAAGAGGTTGAGATGCTGCCCAGACCTCCTAAAGTGAC CAAAGAGGAACACTTCCAGCTGCACCACCAGATGCACTACTCTCAGCAGATCACAGTGTC TCTGGCCCAGGGATATGAGAGAACAAGCAGCCCCAAGCCTAGGTTCAAGAGCTATGCCTA CACACAGGCTGCCTATGTGACCACATCTGACCCCACAAGAAGCCCATTTCCAAGCCAGCAT CTGGAAGCCCCTGAGGACAAGAGCTTTGGCAGCAGCCTGATGGAATCTGAAGTGAACCTG GATAGATACCAGACAGCCCTGGAAGAAGTGCTGTCCTGGCTGCTGTCTGCTGAGGATACA CTGCAGGCTCAGGGTGAAATCAGCAATGATGTGGAAGTGGTCAAGGACCAGTTTCACACC CATGAGGGCTACATGATGGACCTGACAGCCCACCAGGGCAGAGTGGGAAATATCCTGCAGC TGGGCTCCAAGCTGATTGGCACAGGCAAGCTGTCTGAGGATGAAGAGACAGAGGTGCAAG AGCAGATGAACCTGCTGAACAGCAGATGGGAGTGTCTGAGAGTGGCCAGCATGGAAAAGC AGAGCAAACCTGCACAGAGTGCTCATGGACCTGCAGAATCAGAAAACTGAAAGAACTGAATG ACTGGCTGACCAAAGACAGAAGAAAGGACTAGGAAGATGGAAGAGGAACCTCTGGGACCAGA CCTGGAAGATCTGAAAAGACAGGTGCAGCAGCATAAGGTGCTGCAAGAGGACCTTGAGCA AGAGCAAGTCAGAGTGAACAGCCTGACACACATGGTGGTGGTTGTGGATGAGTCCTCTG GGATCATGCCACAGCTGCTCTGGAAGAACAGCTGAAGGTGCTGGGAGACAGATGGGCCCAA CATCTGTAGGTGGACAGAGGATAGATGGGTGCTGCTCCAGGACATTCTGCTGAAGTGGCAG AGACTGACAGAGGAACAGTGCCTGTTTTCTGCCTGGCTCTCTGAGAAAGAGGATGCTGTC AACAAGATCCATACCACAGGCTTCAAGGATCAGAATGAGATGCTCAGCTCCCTGCAGAAA CTGGCTGTGCTGAAGGCTGACCTGGAAAAGAAAAAAGCAGTCCATGGGCAAGCTCTAACAGC CTGAAGCAGGACCTGCTGTCTACCCTGAAGAACAAGTCTGTGACCCAGAAAACTGAGGCCT GGCTGGACAACTTTGCTAGATGCTGGGACAACCTGGTGCAGAAGCTGGAAAAGTCTACAG CCCAGATCAGCCAGCAACCTGATCTTGCCCCTGGCCTGACCACAATTGGAGCCTCTCCAA CACAGACTGTGACCCTGGTTACCCAGCCAGTGGTCACCAAAGAGACAGCCATCAGCAAAC TGGAAATGCCCAGCTCTCTGATGCTGGAAGTCCCACACTGGAAAGGCTGCAAGAACTTCA AGAGGCACAGATGAGCTGGACCTGAAGCTGAGACAGGCTGAAGTGATCAAAGGCAGCTG GCAGCCAGTTGGGGACCTGCTCATTGATAGCCTGCAGGACCATCTGGAAAAAAGTGAAAGC CCTGAGGGAGAGATTGCCCCTCTGAAAGAAAATGTGTCCCATGTGAATGACCTGGCCAG ACAGCTGACCACACTGGGAATCCAGCTGAGCCCCTACAACCTGAGCCACCCTTGAGGACCTG AACACCAGGTGGAAGCTCCTCCAGGTGGCAGTGGAAGATAGAGTCAGGCAGCTGCATGAG GCCCACAGAGATTTTGGACCAGCCAGCCAGCACTTTCTGTCTACCTCTGTGCAAGGCCCC TGGGAGAGAGCTATCTCTCCTAACAAGGTGCCCTAACTACATCAACCATGAGACACAGACC ACCTGTTGGGGATCACCCCAAGATGACAGAGCTGTACCAGAGTCTGGCAGACCTCCAACAATG TCAGATTCAGTGCCTACAGGACTGCCATGAAGCTCAGAAGGCTCCAGAAAGCTCTGTGCC TGGACCTGCTTTCCCTGAGTGCAGCTTGTGATGCCCTGGACCAGCACAATCTGAAGCAGA ATGACCAGCCTATGGACATCCTCCAGATCATCAACTGCCTCACCACCATCTATGATAGGC TGGAACAAGAGCACAACAATCTGGTCAATGTGCCCCTGTGTGTGGACATGTGCCTGAATTG GCTGCTGAATGTGTATGACACAGGCAGAACAGGCAGGATCAGAGTCCTGTCCTTCAAGAC AGGCATCATCTCCCTGTGCAAAGCCCACTTGGAGGACAAGTACAGATAACCTGTTCAAGCA AGTGGCCTCCAGCACAGGCTTTTTGTGACCAGAGAAGGCTGGGCCTGCTCCTGCATGACAG CATTCAGATCCCTAGACAGCTGGGAGAAGTGGCTTCCTTTGGAGGCAGCAATATTGAGCCA TCAGTCAGGTCCTGTTTTCAGTTTGCCAACAACAAGCCTGAGATTGAGGCTGCCCTGTTC CTGGACTGGATGAGACTTGAGCCTCAGAGCATGGTCTGGCTGCCTGTGCTTCATAGAGTG GCTGCTGCTGAGACTGCCAAGCACCAGGCCAAGTGCAAACATCTGCAAAGAGTGCCCCATC ATTGGCTTCAGATACAGATCCCTGAAGCACTTCAACTATGATATCTGCCAGAGCTGCTTCT TTAGTGGCAGGGTTGCCAAGGCCACAAAAATGCACTACCCCATGGTGGAATACTGCACCC CAACAACCTCTGGGGAAGATGTTAGAGACTTTGCCAAGGTGCTGAAAAAACAAGTTCAGGA CCAAGAGATACTTTGCTAAGCACCCCAGAATGGGCTACCTGCCTGTCCAGACAGTGCTTG AGGGTGACAACATGGAAACCCCTGTGACACTGATCAATTTCTGGCCAGTGGACTCTGCCCC TGCCTCAAGTCCACAGCTGTCCCATGATGACACCCACAGCAGAATTGAGCACTATGCCTC CAGACTGGCAGAGATGGAAAACAGCAATGGCAGCTACCTGAATGATAGCATCAGCCCCAA TGAGAGCATTGATGATGAGCATCTGCTGATCCAGCACTACTGTCAGTCCCTGAACCAGGA CTCTCCACTGAGCCAGCCTAGAAGCCCTGCTCAGATCCTGATCAGCCTTGAGTCTTGATGA

Provided is a micro-dystrophin transgene having a reduced number of CpG dinucleotide sequences and, as a result, a reduced number of CpG islands. In certain embodiments, the micro-dystrophin nucleotide sequence has less than two (2) CpG islands, or one (1) CpG island, or zero (0) CpG islands. In embodiments, provided is a micro-dystrophin transgene having less than two or one CpG island, or zero CpG islands, with reduced immunogenicity as measured by anti-drug antibody titers compared to a micro-dystrophin transgene having more than two CpG islands. In certain embodiments, the micro-dystrophin nucleotide sequence consisting essentially of SEQ ID NO: 91, 92, or 93 has zero (0) CpG islands. In other embodiments, the micro-dystrophin transgene nucleotide sequence consisting essentially of a micro-dystrophin gene operably linked to a promoter has less than two (2) CpG islands, and the micro-dystrophin consists of SEQ ID NO: 91, 92, or 93. In yet another embodiment, the micro-dystrophin transgene nucleotide sequence essentially consisting of a micro-dystrophin gene operably linked to a promoter has one (1) CpG island, and the micro-dystrophin consists of SEQ ID NO: 91, 92, or 93.

3.2.2. Micro-dystrophin transgene constructs and artificial rAAV genomes for use in the methods disclosed herein are provided. The transgene comprises a nucleotide sequence encoding the micro-dystrophin disclosed herein operably linked to transcriptional regulatory sequences including a promoter that promotes expression in muscle cells and other regulatory sequences that promote expression of micro-dystrophin. The transgene is flanked by AAV ITR sequences.

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

The amino acid sequence of the C-terminal domain (CT) (SEQ ID NO:48) is as follows:
(The coiled-coil motif H1 is in bold, motif H2 in lower case, and the dystrobrevin binding side is in italics).

The minimal/truncated C-terminal domain (CT1.5) amino acid sequence (SEQ ID NO:49) is as follows:
(1-syntrophin binding sites are shown in italics).

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

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

Some nucleic acid embodiments of the present disclosure include an AAV vector or rAAV vector encoding a micro-dystrophin comprising or consisting of the nucleotide sequence of SEQ ID NO: 184, 185, or 186, as provided in Table 5 below. In various embodiments, the AAV vector or rAAV vector comprises a nucleotide sequence having 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 sequence of SEQ ID NO: 184, 185, or 186, or their reverse complement, and encoding an AAV vector or rAAV vector suitable for expressing therapeutically effective micro-dystrophin in muscle cells. In embodiments, the construct having the nucleotide sequence of SEQ ID NO: 184, 185, or 186 is within a recombinant rAAV8 particle or a recombinant AAV9 particle.

Table 5: RGX-DYS cassette nucleotide sequence

Table 6 provides amino acid sequences of embodiments of α-, β-, γ-, and δ-sarcoglycans according to the disclosure. Also contemplated are substitution variants of α-, β-, γ-, or δ-sarcoglycans 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 substantially maintain its functional activity. 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 sequence of SEQ ID NO: 144, 52, 145, 52, 146, 54, 94, 95, 96, 147, or 129 and can maintain functional sarcoglycan activity, e.g., as determined by one or more of the in vitro or in vivo assays in animal models disclosed below.

Table 6. Amino acid sequences of sarcoglycan proteins
*NCBI reference sequences are incorporated herein by reference in their entirety.

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

Table 7 provides nucleic acid sequences encoding embodiments of α-, β-, γ-, and δ-sarcoglycans according to the present disclosure. Other embodiments are also contemplated to be substitution variants of α-, β-, γ-, or δ-sarcoglycans 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 substantially maintain its functional activity. 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, e.g., as determined by one or more of the in vitro or in vivo assays in animal models disclosed below.

Table 7. Nucleic acid sequences of sarcoglycan proteins
*NCBI reference sequences are incorporated herein by reference in their entirety.

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

5.1. Promoters 5.1.1. Tissue-specific promoters In specific embodiments, the expression cassette of the AAV or rAAV vector includes a regulatory sequence, such as a promoter, operably linked to the transgene, that allows 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-targeted" refer to a nucleic acid element whose activity is adapted to muscle cells or muscle tissue by the interaction of such element with the intracellular environment of the muscle cell. Such muscle cells can include myocytes, myotubes, cardiomyocytes, and the like. Specialized forms of muscle cells with distinct properties, such as cardiomyocytes, skeletal cells, and smooth muscle cells, are also included. Various therapies would benefit from muscle-specific expression of transgenes. In particular, gene therapies to treat various forms of muscular dystrophy that are delivered to muscle cells and allow high transduction efficiency have the added advantage of directing expression of the transgene in cells where the transgene is most needed. Cardiac tissue may also benefit from muscle-targeted expression of the transgene. The muscle-specific promoter can be operably linked to a transgene of the present disclosure.

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 may be a mammalian promoter. For example, the promoter may be selected from the group consisting of a human promoter, a mouse promoter, a porcine promoter, a feline promoter, a canine promoter, an ovine promoter, a non-human primate promoter, an equine promoter, a bovine promoter, and the like.

In some embodiments, the muscle cell-specific promoter is one of muscle creatine kinase (MCK) promoter, syn100 promoter, creatine kinase (CK) 6 promoter, creatine kinase (CK) 7 promoter, dMCK promoter, tMCK promoter, smooth muscle 22 (SM22) promoter, myo-3 promoter, Spc5-12 promoter, creatine kinase (CK) 8 promoter, creatine kinase (CK) 8e promoter, creatine kinase (CK) 9 promoter, U6 promoter, H1 promoter, desmin promoter, Pitx3 promoter, skeletal alpha-actin promoter, MHCK7 promoter, and Sp-301 promoter. Specific muscle cell-specific promoter sequences are well known in the art, and exemplary promoters are provided in Table 8 below (see, for example, 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 Promoter for AAV Vectors," Gene. Ther. 15:1489-1499 (2008); Piekarowicz et al., "A Muscle Cell-Specific Promoter Sequence," Gene. Ther. 15:1489-1499 (2008)). 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 Regulation 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. 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 Girl 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 Girl Muscular Dystrophy 2I (LGMD2I) Mouse Model by Systemic FKRP Gene Delivery," Mol. Ther. 22(11):1890-1899 (2014); the entire contents of which are incorporated herein by reference).

Table 8. Promoter sequences

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 a key role in contraction and ATP regeneration within the ion transport system. Creatine kinase transfers a phosphate group from phosphocreatine to ADP to form ATP, allowing muscle contraction in the absence of glycolysis and respiration. There are four known isoforms of creatine kinase: brain creatine kinase (CKB), muscle creatine kinase (MCK), and two mitochondrial forms (CKMi). MCK is the most abundant non-mitochondrial mRNA, 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 is transcriptionally active as myoblasts undergo terminal differentiation into myocytes. The MCK gene regulatory region exhibits striated muscle specific activity and has been extensively characterized in vivo and in vitro. The major regulatory regions known for the MCK gene include a muscle-specific enhancer located approximately 1.1 kb 5' of the transcription start site in mouse and 358 bp of the proximal promoter. Additional sequences regulating MCK expression are distributed over a 3.3 kb region 5' of the transcription start site and over the first 3.3 kb intron. Mammalian MCK regulatory elements, including human and mouse promoter and enhancer elements, are described in 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 Promoter for AAV Vectors," Gene Ther. 15(22):1489-1499 (2008); the entire contents of which are incorporated herein by reference).

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

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

Alternatively, the promoter can be a constitutive promoter, e.g., 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, particularly when it is desirable to turn off transgene expression, an inducible promoter is used, e.g., a hypoxia-inducible promoter or a rapamycin-inducible promoter.

5.2. Introns Certain gene expression cassettes further comprise an intron, e.g., 5' of the sequence encoding AUF1 or a therapeutic protein (such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II β isoform protein or a portion thereof), which may facilitate proper splicing and thus transgene expression. Thus, in some embodiments, an intron is attached to the 5' end of the sequence encoding AUF1 or a therapeutic protein (such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II β isoform protein or a portion thereof). In certain embodiments, the intron is less than 100 nucleotides in length.

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

Table 9. Nucleotide sequences of various introns

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 may be employed, such as chicken β-actin intron, minute virus of mice (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).

5.3. Other Regulatory Elements Another aspect of the present disclosure relates to expression cassettes that include 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 calcium/calmodulin-dependent protein kinase II β isoform protein or a portion thereof. Any polyA site that signals the end of transcription and directs the synthesis of a polyA tail is suitable for use in the AAV vectors of the present disclosure. Exemplary polyA signals are derived from, but are not limited to, the SV40 late gene, rabbit β-globin gene, bovine growth hormone (BPH) gene, human growth hormone (hGH) gene, and synthetic polyA (SPA) sites. Exemplary polyA signal sequences useful in the constructs described herein are provided in Table 2 above.

Also provided are constructs that include a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) that can enhance expression of a transgene. The WPRE element can be inserted into the 3' untranslated region of a transgene 5' to the polyadenylation signal sequence. See, e.g., 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 incorporated by reference in its entirety. In a particular embodiment, the WPRE element has the nucleotide sequence of SEQ ID NO:24 (see Table 2 above).

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

A nucleic acid comprising a stuffer (or filler) polynucleotide sequence expands the transgene size of any heterologous gene, for example, the AUF1 gene of 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 NO:139-143 or a fragment of SEQ ID NO:139-143 (see Table 10) that is 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.

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

In the case of enhancer sequences, such as stuffer sequences and introns, the nucleic acid sequence is operably linked to the transgene contiguously or substantially contiguously. Where appropriate, operably linked can refer to coding and non-coding regions, or two coding regions, being contiguously linked, e.g., in reading frame. In some cases, such as enhancers that can function even several kilobases away from the promoter, such as intron sequences and stuffer sequences, these regulatory sequences can be operably linked even if they are not directly contiguous with the downstream or upstream promoter and/or heterologous gene.

(Table 10)

5.4 Reporter Genes In some embodiments, the disclosed gene cassettes, and thus the adeno-associated viral vectors, comprise a nucleic acid molecule encoding a reporter protein. The reporter protein can be selected from the group consisting of, for example, β-galactosidase, chloramphenicol acetyltransferase, luciferase, and fluorescent proteins.

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, ZsGreenl), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellowl), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyanl, Mid, etc.) orishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, 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).

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 produces light. Luciferases have been identified and cloned from a variety of organisms, including fireflies, click beetles, Renilla, marine copepods, and bacteria, among others. 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, and deep-sea shrimp (e.g., Oplophorus gracilirostris) luciferase. Luciferase reporter proteins include both native proteins and engineered variants that are designed to have one or more altered properties compared to the native protein, such as improved photostability, improved pH stability, increased fluorescence or luminescence, reduced tendency to dimerize, oligomerize, aggregate or be toxic to cells, altered emission spectra, and/or altered substrate utilization.

5.5. Viral Vectors The transgenes encoding AUF1 and other proteins disclosed herein can be included in AAV vectors for gene therapy administration to human subjects. In some embodiments, an AAV or recombinant AAV (rAAV) vector can include an AAV viral capsid and a viral genome or artificial genome that includes an expression cassette flanked by AAV inverted terminal repeats (ITRs), the expression cassette including AUF1 or a coding transgene operably linked to one or more regulatory sequences that control expression of the transgene in human muscle cells to express and deliver the AUF1 protein or, optionally, other therapeutic proteins. The provided method is suitable for use in producing any isolated recombinant AAV particle for delivery of AUF1 protein or other therapeutic proteins described herein, or in making compositions comprising any isolated recombinant AAV particle encoding AUF1 protein or other therapeutic proteins, or in a method for treating a disease or disorder, including LGMD type, suitable for treatment with AUF1 protein or a combination of AUF1 protein and another therapeutic protein in a subject in need thereof, comprising administering any isolated recombinant AAV particle encoding AUF1 protein or administering (including separately) a combination of rAAV particles encoding AUF1 protein and rAAV particles encoding another therapeutic protein described herein. Thus, the rAAV can be any serotype, variant, modification, hybrid, or derivative thereof, or any combination thereof (collectively referred to as "serotype") known in the art. In certain embodiments, the AAV serotype has tropism for muscle tissue, including skeletal muscle, cardiac muscle, or smooth muscle.

In some embodiments, the AAV particles or rAAV particles have capsid proteins derived from the AAV8 serotype. In other embodiments, the AAV particles or rAAV particles have capsid proteins derived from the AAV9 serotype. In yet other embodiments, the AAV particles or rAAV particles have capsid proteins derived from the hu. 32 serotype. In particular, AUF1 constructs of vectors spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1-intronless, or D(+)-CK7AUF1 having the nucleotide sequence of SEQ ID NOs: 31-36 in AAV particles or rAAV particles having an AAV8 capsid are provided. Further provided are RGX-DYS1 constructs in AAV particles or rAAV particles having AAV8 capsids and RGX-DYS1 constructs in AAV particles or rAAV particles having AAV9 capsids for use in the methods disclosed herein. Also provided are RGX-DYS5 constructs in AAV particles or rAAV particles having AAV8 capsids and RGX-DYS5 constructs in rAAV particles having AAV9 capsids.

In some embodiments, the AAV or rAAV particles comprise capsid proteins 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 may be an AAVrh. 8, AAVrh. 10, AAVrh. 43, AAVrh. 74, AAVhu. 37, AAVAAV. hu31, or AAVhu. 32 serotype.

In some embodiments, the AAV particles or rAAV particles comprise capsid proteins that are derivatives, variants, or pseudotypes of the AAV8 capsid protein. In some embodiments, the AAV particles or rAAV particles comprise capsid proteins having capsid proteins that are at least 80% identical, e.g., 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 of the AAV8 capsid protein (SEQ ID NO:114) (Table 11). In some embodiments, the AAV or rAAV particle comprises a capsid protein having a capsid protein that is at least 80% identical or greater, e.g., 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 sequence of the AAV9 capsid protein (SEQ ID NO: 115) (Table 11). In some embodiments, the AAV or rAAV particle comprises a capsid protein having a capsid protein that is at least 80% identical or greater, e.g., 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 sequence of the AAV9 capsid protein (SEQ ID NO: 115) (Table 11). 8, AAVrh. 10, AAVrh. 43, AAVrh. 74 (SEQ ID NO: 119 and 120), AAVhu. 37 (SEQ ID NO: 116), AAVAAV. hu31 (SEQ ID NO: 117), or AAVhu. 32 (SEQ ID NO: 118) serotype capsid protein VP1, VP2 and/or VP3 sequences (see Table 11), including capsid proteins having capsid proteins that are at least 80% identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e., up to 100% identical.

Nucleic acid sequences of AAV-based viral vectors and methods for producing recombinant AAV and AAV capsids are described, for example, in U.S. Patent 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; 9458517; and 9,587,282; U.S. Patent Application Publication Nos. 2015/0374803; 2015/01265; and U.S. Patent Application Publication Nos. 2015/01265 ... 88; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; International Patent Application No. PCT/US2015/034799; PCT/EP2015/053335; WO2003/052051, WO2005/033321, WO03/042397, WO2006/068888, WO2006/110689, WO2009/104964, WO2010/127097, and WO2015/191508, and U.S. Publication No. 20150023924.

In certain embodiments, single stranded AAV (ssAAV) may be used. In certain embodiments, self-complementary vectors, e.g., scAAV, may 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. Patent 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 may include mutant ITR sequences, e.g., mutant 5'ITR sequences of Table 2.

In additional embodiments, the rAAV particle comprises a pseudotyped rAAV particle. In some embodiments, the pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs, and (b) a capsid comprised of capsid proteins derived from 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 AAV8. In additional embodiments, the rAAV particles comprise pseudotyped rAAV particles containing AAV8 capsid proteins, hi some embodiments, the pseudotyped rAAV8 particles are rAAV2/8 pseudotyped particles. Methods for producing and using pseudotyped rAAV particles are known in the art (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 Type 5," J. Virol. 75(16):7662-7671 (2001); 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. al., “Exchange of Surface Proteins Impacts on Viral Vector Cellular See, "Specificity and Transduction Characteristics: the Retina as a Model," Hum. Molec. Genet. 10:3075-3081 (2001), which are incorporated herein by reference in their entireties.

In some embodiments, the AAV or rAAV particles comprise an AAV capsid protein chimera of an AAV8 capsid protein and one or more AAV capsid proteins 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.

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

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

(Table 11)

5.6. Methods of Making rAAV Particles Another aspect of the disclosure includes making the molecules disclosed herein. In some embodiments, the molecules according to the disclosure are made by providing a nucleotide sequence comprising a nucleic acid sequence encoding any of the capsid protein molecules herein and using a packaging cell line to prepare a corresponding rAAV particle having a capsid coat composed of capsid proteins. 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%, e.g., 96%, 97%, 98%, 99%, or 99.9% identity to the sequence of the capsid protein molecule described herein. In some embodiments, the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, e.g., 96%, 97%, 98%, 99% or 99.9% identity to the sequence of the AAV8 capsid protein, while retaining (or substantially retaining) 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%, e.g., 96%, 97%, 98%, 99% or 99.9% identity to the sequence of the AAV9 capsid protein, while retaining (or substantially retaining) the biological function of the AAV9 capsid protein.

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 inverted terminal repeat sequence that allows packaging into a vector. In some embodiments, the viral genome further includes a cap gene and/or a rep gene for expression and splicing of the cap gene. In embodiments, the cap and rep genes are provided by the packaging cell and are not present in the viral genome.

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

Many cell culture systems are known in the art for the production of rAAV particles, any of which can be used to practice 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. rAAV production cultures for the production of rAAV viral particles require: (1) a suitable host cell, including, for example, a human-derived cell line, a mammalian cell line, or an insect-derived cell line; (2) suitable helper virus functions provided by wild-type or mutant adenovirus (such as a temperature-sensitive adenovirus), herpesvirus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences and optionally regulatory elements; and (5) suitable media and media components (nutrients) to support cell growth/survival and rAAV production.

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 (e.g., in the case of baculovirus production systems). For a review, see Aponte-Ubillus et al. , "Molecular Design for Recombinant Adeno-Associated Virus (rAAV) Vector Production," Appl. Microbiol. Biotechnol. 102:1045-1054 (2018), the entire contents of which are incorporated herein by reference for production techniques.

In one aspect, provided herein is a method of 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. a rAAV genome to be packaged; ii. AAV rep proteins sufficient for packaging; and iii. AAV cap proteins sufficient for packaging; and (c) adding sufficient nutrients to the cell culture and maintaining the cell culture under conditions that permit 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 a rAAV genome. In some embodiments, the method comprises using a baculovirus encoding a rAAV genome and an insect cell expressing the rep gene and the cap gene. In some embodiments, the method comprises using a baculovirus vector encoding the rep gene and the cap gene and the rAAV genome. In some embodiments, the insect cell is an Sf-9 cell. In some embodiments, the insect cell is an Sf-9 cell that contains one or more stably integrated heterologous polynucleotides encoding rep and cap genes.

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

Those skilled in the art are aware of the numerous ways in which AAV rep and cap genes, AAV helper genes (e.g., adenovirus E1a, E1b, E4, E2a, and VA genes), and rAAV genomes (including one or more genes of interest flanked by ITRs) can be introduced into cells to produce or package rAAV. The phrase "adenovirus helper functions" refers to the numerous viral helper genes that are expressed (as RNA or protein) in cells so that AAV can grow efficiently in the cells. Those skilled in the art are aware that helper viruses, including adenovirus and herpes simplex virus (HSV), facilitate AAV replication, and that certain genes that provide essential functions have been identified, e.g., helpers 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 and cap genes, helper genes, and rAAV genome are introduced into the cell by transfection with one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of the methods disclosed herein, the AAV rep and cap genes, helper genes, and rAAV genome can be introduced into the cell by transduction with a viral vector, for example, an rHSV vector encoding the AAV rep and cap genes, helper genes, and rAAV genome. In some embodiments of the methods disclosed herein, one or more of the AAV rep and cap genes, helper genes, and rAAV genome are introduced into the cell by transduction with an rHSV vector. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper genes. 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 genes and an rAAV genome. In some embodiments, the rHSV vector encodes helper genes and AAV rep and cap genes.

In one aspect, provided herein is a method of 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. a rAAV genome to be packaged; ii. helper functions necessary for packaging of the rAAV particles; iii. AAV rep proteins sufficient for packaging; and iv. AAV cap proteins sufficient for packaging; and (c) adding sufficient nutrients to the cell culture and maintaining the cell culture under conditions that permit the 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 the 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 and AAV rep and cap genes. In some embodiments, the cell comprises one or more stably integrated heterologous polynucleotides encoding rep and cap genes.

In one aspect, provided herein is a method of 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. a rAAV genome to be packaged; ii. helper functions necessary for packaging of the rAAV particles; iii. AAV rep proteins sufficient for packaging; and iv. AAV cap proteins sufficient for packaging; and (c) adding sufficient nutrients to the cell culture and maintaining the cell culture under conditions that permit the production of rAAV particles. In some embodiments, the helper functions are encoded by adenovirus genes. In some embodiments, the mammalian cells comprise one or more stably integrated heterologous polynucleotides encoding rep and cap genes.

Molecular biology techniques for developing plasmids or viral vectors encoding AAV rep and cap genes, helper genes, and/or rAAV genomes are generally known in the art. In some embodiments, the AAV rep and cap genes are encoded by a single plasmid vector. In some embodiments, the AAV helper genes (e.g., the adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single plasmid vector. In some embodiments, the E1a or E1b genes are stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection with a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by the host cell, and the E4, E2a, and VA genes are introduced into the cell by transfection with a single 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 a single plasmid vector. In some embodiments, the helper genes are stably expressed by the host cell. In some embodiments, the AAV rep and cap genes are encoded by a single viral vector. In some embodiments, the AAV helper genes (e.g., the adenovirus E1a, E1b, E4, E2a, and VA genes) are encoded by a single viral vector. In some embodiments, the E1a or E1b gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection with a single viral vector. In some embodiments, the E1a and E1b genes are stably expressed by the host cell, and the E4, E2a, and VA genes are introduced into the cell by transfection with a single 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 a viral vector. In some embodiments, the AAV rep and cap genes, the adenoviral helper functions required 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 include transfecting a cell with a mixture of three polynucleotides: one encoding the cap and rep genes, one encoding the adenoviral helper functions required for packaging (e.g., the adenoviral E1a, E1b, E4, E2a, and VA genes), 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 an 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 contains a gene of interest flanked by AAV ITRs. In certain embodiments, the ITR sequence is an AAV2 ITR sequence and includes the 5' and 3' sequences of SEQ ID NOs:28 and 29, respectively, as set forth in Table 2.

Any combination of vectors can be used to introduce the AAV rep and cap genes, AAV helper genes, and the rAAV genome into the cells where rAAV particles are produced or packaged. In some embodiments of the methods disclosed herein, a first plasmid vector encoding a rAAV genome containing a gene of interest flanked by AAV inverted terminal repeats (ITRs), a second vector encoding AAV rep and cap genes, and a third vector encoding a helper gene may 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 a plasmid vector and a viral vector.

In some embodiments, the rep and cap genes, and one or more of the AAV helper genes, are constitutively expressed by the cell and do not need to be transfected or transduced into the cell. In some embodiments, the cell constitutively expresses the rep and/or cap genes. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses E1a. In some embodiments, the cell contains a stable transgene encoding a rAAV genome.

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

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 from two or more serotypes). In other embodiments, the rep gene and the cap gene are derived from the same serotype. In yet 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, at least one modified domain comprises a nucleotide sequence of a serotype different from the capsid serotype. The modified domain in the rep gene can be a hybrid nucleotide sequence consisting of fragments of different serotypes.

The hybrid rep gene provides improved packaging efficiency of rAAV particles, including packaging of viral genomes containing therapeutic protein transgenes of 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, such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II β isoform proteins or portions thereof. The AAV rep gene consists of nucleic acid sequences encoding nonstructural proteins required for viral replication and production. Transcription of the rep gene is initiated from the p5 or p19 promoter, which produces two large (Rep78 and Rep68) and two small (Rep52 and Rep40) nonstructural Rep proteins, respectively. Additionally, the Rep78/68 domain contains a DNA-binding domain that recognizes specific ITR sequences within the ITRs. All four Rep proteins share common helicase and ATPase domains and function in genome replication and/or encapsidation (Maurer and Weitzman, "Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction," Hum. Gene Ther. 31(9-10):499-511 (2020); incorporated herein by reference in its entirety). Although transcription of the cap gene is initiated from the p40 promoter, this sequence is within the C-terminus of the rep gene, suggesting the possibility that other elements in the rep gene may induce p40 promoter activity. The p40 promoter domain contains transcription factor binding elements EF1A, MLTF, and ATF, Fos/Jun binding element (AP-1), Sp1-like elements (Sp1 and GGT), and a TATA element (Pereira and Muzyczka, "The Adeno-Associated Virus Type 2 p40 Promoter Required a Proximal Sp1 Interaction and a p19 CArG-like Element to Facilitate Rep19 ). Transactivation," J. Virol. 71(6):4300-4309 (1997); incorporated herein by reference in its entirety. In some embodiments, the rep gene comprises a modified p40 promoter. In some embodiments, the p40 promoter is modified in any one or more of an EF1A binding element, an MLTF binding element, an ATF binding element, a Fos/Jun binding element (AP-1), an Sp1-like element (Sp1 or GGT), or a TATA element. In other embodiments, the rep gene is serotype 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, rh8, rhlO, rh20, rh39, rh.74, RHM4-1, or hu37, and a portion or element of the p40 promoter domain is modified to serotype 2. In yet another embodiment, the rep gene is serotype 8 or 9, and a portion or element of the p40 promoter domain is modified to serotype 2.

The ITRs contain the complementary sequences of A and A', B and B', and C and C', and the D sequence is continuous with the ssDNA genome. The complementary sequences of the ITRs form a hairpin structure by self-annealing (Berns, K. I., "The Unusual Properties of the AAV Inverted Terminal Repeat," Hum. Gene Ther. 31(9-10):518-523 (2020); incorporated herein 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 replication origin. The ITRs are also required as packaging signals for encapsidation of the genome after replication. In some embodiments, the ITR sequences and the cap gene are from the same serotype, except that one or more of the A and A' complementary sequences, the B and B' complementary sequences, the C and C' complementary sequences, or the D sequences may be modified to contain sequences from a different serotype than the capsid. In some embodiments, the modified ITR sequences are from the same serotype as the rep gene. In other embodiments, the ITR sequences and the cap gene are from different serotypes, except that one or more of the ITR sequences selected from the A and A' complementary sequences, the B and B' complementary sequences, the C and C' complementary sequences, or the D sequences are from the same serotype as the capsid (cap gene), and one or more of the ITR sequences are from the same serotype as the rep gene.

In some embodiments, the rep and cap genes are from the same serotype, and the rep gene comprises a modified Rep78 domain, a DNA binding domain, an endonuclease domain, an ATPase domain, a helicase domain, a p5 promoter domain, a Rep68 domain, a p5 promoter domain, a Rep52 domain, a p19 promoter domain, a Rep40 domain, or a p40 promoter domain. In other embodiments, the rep and cap genes 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, an AAV8 cap, and a 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 derived 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 derived from serotype 8 rep. In some embodiments, three or more serotypes may be utilized to construct a hybrid rep/cap plasmid.

Any suitable method known in the art can be used to transfect cells and can be used to produce 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)), or lipofection. In some embodiments, the chemical transfection method uses cationic polymers (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.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The techniques and procedures described above 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. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for all purposes. Unless specific definitions are provided, the nomenclature utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

Host cell lines are provided for producing rAAV particles containing constructs encoding the rAUF1 protein disclosed herein, e.g., constructs of SEQ ID NOs: 31 to 36 (spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1-intronless, and D(+)-CK7AUF1, respectively), or constructs encoding therapeutic proteins (such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II β isoform protein or a portion thereof), SEQ ID NO: 184 or 186 (RGX-DYS1 or RGX-DYS5).

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.

Nucleic acid sequences for AAV-based viral vectors, as well as methods for producing recombinant AAV and AAV capsids, are taught, for example, in US 7,282,199; US 7,790,449; US 8,318,480; US 8,962,332; and PCT/EP2014/076466, which are incorporated herein by reference in their entireties.

6. Therapeutic Utility Methods are provided for testing the infectivity of recombinant vectors, e.g., AAV or rAAV particles, disclosed herein. For example, the infectivity of recombinant gene therapy vectors in muscle cells can be tested in C2C12 myoblast cells. Several muscle or cardiac cell lines can be utilized, including, but not limited to, 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) cell lines. Vector copy number can be assessed using polymerase chain reaction technology, and the level of therapeutic protein expression can be tested by measuring the mRNA levels of the therapeutic protein (such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II β isoform protein or a portion thereof) in the cells.

6.1 Animal Models The efficacy of viral vectors containing transgenes encoding the AUF1 protein or therapeutic proteins described herein can be tested by administering to an animal model, replacing sarcoglycan, and evaluating the biodistribution, expression, and therapeutic effect of transgene expression, for example, using the δ-sarcoglycan deficient mouse and/or golden retriever muscular dystrophy (GRMD) models. Therapeutic effect can be evaluated, for example, by evaluating changes in muscle strength in animals administered the transgene. Animal models using large mammals and non-mammalian vertebrates and invertebrates can also be used to evaluate the preclinical therapeutic efficacy of the vectors described herein. Thus, compositions and methods are provided for therapeutic administration comprising a dose of a vector disclosed herein encoding AUF1 alone or in combination with 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 portions thereof, in an amount demonstrated to be effective according to the methods for assessing therapeutic efficacy disclosed herein.

6.2 Mouse Models The efficacy of gene therapy vectors alone or in combination with a second therapeutic modality disclosed herein can be assessed in mouse models of LGMD. α-, β-, γ-, and δ-sarcoglycan null mice show progressive muscle pathology and dysfunction from the first week. δ-sarcoglycan null mice have histological hallmarks of muscular dystrophy, including extensive necrosis, fibrosis, inflammation, calcification, and muscle dysfunction. δ-sarcoglycan-deficient mice have fibrotic lesions and electrocardiographic 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); incorporated herein by reference in its entirety).

6.2.1 CAPN3-Deficient Calpainopathy Mouse Model The efficacy of gene therapy vectors alone or in combination with a second therapeutic modality disclosed herein can be assessed in a mouse model of LGMD. The ^CAPN3-/- deletion mouse model closely recapitulates LGMD2A disease, with mice exhibiting muscle degeneration, necrosis, mitochondrial and functional abnormalities, small muscle fibers, loss of slow muscle fibers, and muscle weakness. Limitations of the ^CAPN3-/- deletion mouse model include the failure to develop fibrosis, and patients may harbor both an allelic deletion and a second allelic missense/nonsense mutation.

Small molecule activators of CAMKIIβ kinase activity have been reported to modestly 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 Guard Muscular Dystrophy,” Cell Reports 1:100122 (2020); incorporated herein by reference in its entirety), providing a response to measure AUF1 gene therapy in this disease model.

6.3. Cardiac Function Assessment of efficacy on cardiac function can be measured in mice, including δ-sarcoglycan null mice. To measure blood pressure (BP), mice are sedated using 1.5% isofluorane, anesthesia levels are constantly monitored, and body temperature is maintained at 36.5-37.58°C. Heart rate is maintained at 450-550 beats/min. A BP cuff is placed around the tail, which is then placed on a sensor assembly to monitor non-invasive BP during anesthesia. Ten consecutive BP measurements are taken. Qualitative and quantitative measurements of tail BP, including systolic, diastolic, and mean pressures, are made offline using analysis software. See, for example, 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 UAEontrachoon et al. (See, e.g., Wang, 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 is incorporated by reference in its entirety.

A wireless telemetry device is used to monitor ECG peaks and interval times in awake, freely moving mice. A transmitter unit is implanted intraperitoneally in anesthetized mice, and two electrical leads are fixed in lead II orientation near the apex and right acromion. Mice are individually housed in cages above an antenna receiver connected to a computer system for data recording. Unfiltered ECG data are collected for 10 seconds every hour for 35 days. The first 7 days of data are discarded to allow for recovery from surgery and to ensure that the effects of anesthesia have subsided. Data waveforms and parameters are analyzed with DSI analysis packages (ART 3.01 and Physiostat 4.01), and measurements are tabulated and averaged to determine heart rate, ECG peaks, and interval times. Raw ECG waveforms are reviewed by two independent observers for arrhythmias.

Picrosirius red staining is performed to measure the degree of fibrosis in the hearts of the test mice. Briefly, at the end of the study, immediately after euthanasia, the myocardium is removed and fixed in 10% formalin for further processing. The hearts are sectioned and the paraffin sections are deparaffinized with xylene followed by nuclear staining with Weigert's hematoxylin for 8 minutes. They are then washed and stained with picrosirius red (0.5 g Sirius Red F3B, saturated aqueous picric acid) for an additional 30 minutes. The sections are cleared three times with xylene and mounted in Permount. Five random digital images are taken using an Eclipse E800 (Nikon, Japan) microscope and blinded analysis is performed using Image J (NIH). Blood samples are taken by cardiac puncture when the animals are euthanized and the collected serum is used to measure muscle CK levels.

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," Skeleton. Muscle 7(1):9 (2017), incorporated by reference in its entirety). Dogs with GRMD have a skeletal and cardiac phenotype and suffer from a progressively fatal disease with selective muscle pathology, a severe phenotype that more closely resembles that of DMD. Dogs with GRMD have a single nucleotide change that results in exon skipping and out-of-frame DMD transcripts. Phenotypic features in dogs include elevated serum CK, CRD in EMG, and histopathological evidence of clustered muscle fiber necrosis and regeneration. Phenotypic variability is frequently observed in GRMD as in humans. Dogs with GRMD develop paradoxical muscle hypertrophy that may contribute to the phenotype of affected dogs, and are commonly characterized by stiffness when gaited, reduced range of motion, and trismus. Objective biomarkers to assess disease progression include tonic flexion, tibiofibular joint angle, reduced eccentric contraction rate, maximum hip flexion angle, pelvic angle, anterior sartorius circumference, and quadriceps mass.

7. Methods of Treatment with AUF1 Gene Therapy Constructs Methods of treating a human subject with LGMD using the AUF1 gene therapy constructs disclosed herein are provided. Accordingly, there is provided a method of treating or ameliorating a symptom of LGMD in a subject in need thereof, comprising a recombinant genome 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 facilitate expression of the AUF1 protein in muscle cells of the subject and flanked by ITR sequences (see Table 2 for nucleotide sequences of possible components of these recombinant genomes), and comprising an AAV8 vector, an AAV9 vector or an AAVhu. Methods are provided by contacting a muscle cell with a therapeutically effective amount of an AAV vector or rAAV vector, including a 32 vector, under conditions effective to express exogenous AUF1 in the muscle cell.

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

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

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

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

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

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

In embodiments, the method of treating a human subject provides a gene therapy vector comprising a genome comprising a transgene encoding p37 ^AUF1 . In embodiments, the method of treating a human subject provides a gene therapy vector comprising a genome comprising a transgene encoding p40 ^AUF1 . In embodiments, the method of treating a human subject provides a gene therapy vector comprising a genome comprising a transgene encoding p42 ^AUF1 . In embodiments, the 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 a rAAV vector.

In embodiments, a method is provided for treating a human subject with a gene therapy vector having a combination of two or more AUF1 isoforms, i.e., p37AUF1, p40AUF1, p42AUF1, and/or p45AUF1.

In embodiments, a method of treating a human subject includes a therapeutic method comprising an AAV particle or 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, a syn100 promoter, a CK6 promoter, a CK7 promoter, a CK8 promoter, a CK9 promoter, a dMCK promoter, a tMCK promoter, a smooth muscle 22 (SM22) promoter, a myo-3 promoter, a SPc5-12 promoter, a mutant SPc5-12 promoter, a creatine kinase (CK)8e promoter, a U6 promoter, a H1 promoter, a desmin promoter, a Pitx3 promoter, a skeletal alpha-actin promoter, a MHCK7 promoter, or a Sp-301 promoter.

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-deleted AUF1 gene therapy construct. In an embodiment, the AUF1 gene therapy construct of the method has a nucleotide sequence of SEQ ID NO:31. In an embodiment, the AUF1 gene therapy construct of the method has a nucleotide sequence of SEQ ID NO:32. In an embodiment, the AUF1 gene therapy construct of the method has a nucleotide sequence of SEQ ID NO:33. In an embodiment, the AUF1 gene therapy construct of the method has a nucleotide sequence of SEQ ID NO:34. In an embodiment, the AUF1 gene therapy construct of the method has a nucleotide sequence of SEQ ID NO:35. In an embodiment, the AUF1 gene therapy construct of the method has a nucleotide sequence of SEQ ID NO:36.

In an embodiment, a method of treating a human subject includes a method of therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:31 (spc-hu-opti-AUF1-CpG(-)). In an embodiment, a method of treating a human subject includes a method of therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:32 (tMCK-huAUF1). In an embodiment, a method of treating a human subject includes a method of therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:33 (spc5-12-hu-opti-AUF1-WPRE). In an embodiment, a method of treating a human subject includes a method of therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:34 (ss-CK7-hu-AUF1). In an embodiment, the method of treating a human subject includes a method of treatment comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:35 (spc-hu-AUF1-intronless). In an embodiment, the method of treating a human subject includes a method of treatment comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:36 (D(+)-CK7AUF1).

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 an embodiment, the method of treating a human subject utilizes an AAV having a capsid at least 95% identical to SEQ ID NO: 116 (hu.37 capsid). In an embodiment, the method of treating a human subject utilizes an AAV having a capsid at least 95% identical to SEQ ID NO: 117 (hu.31 capsid). In an embodiment, the method of treating a human subject utilizes an AAV having a capsid at least 95% identical to SEQ ID NO: 118 (hu.32 capsid). In an embodiment, the method of treating a human subject utilizes an AAV having a capsid at least 95% identical to SEQ ID NO: 119 or SEQ ID NO: 120 (Rh.74 capsid).

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

Treatment in the methods disclosed herein may have a duration of at least one week, at least one month, at least several months, at least one year, at least two, three, four, five, six years or more. In embodiments, the subject may experience a response to the gene therapy treatment (e.g., increased muscle mass, strength, or performance) two weeks, four weeks, six weeks, two months, three months, or six months after administration.

8. Methods of Combination Therapy Methods of treating a human subject for any LGMD that can be treated by providing a functional AUF1 as disclosed herein in combination with a second therapy are provided, where the second therapy can treat LGMD or improve one or more symptoms thereof. The gene therapy vector expressing AUF1 provided herein can be administered as a monotherapy or in combination with a second therapy described herein to treat LGMD. In some aspects, the combination therapy is a combination of any one of the AUF1 gene therapy vectors disclosed herein with a gene therapy vector encoding another therapeutic protein or another therapy disclosed herein.

Methods are provided for treating a human subject with LGMD using the AUF1 gene therapy constructs disclosed herein in combination with other therapies. In some embodiments, the combination treatment methods provide for treatment of LGMD type 1. In some embodiments, the combination treatment methods provide for treatment of limb-girdle muscular dystrophy type 1C (LGMD1C) with a mutation in caveolin-3. In some embodiments, the combination treatment methods provide for treatment of limb-girdle muscular dystrophy type 1G (LGMD1G) with a mutation in HNRPDL, a protein involved in mRNA biogenesis and metabolism.

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

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

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

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

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

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 provides a gene therapy vector having a combination of two or more AUF1 isoforms, i.e., p37AUF1, p40AUF1, p42AUF1, and/or p45AUF1.

In an embodiment, the method of treating a human subject includes a first treatment comprising an AAV particle or 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. In an embodiment, the method of treating a human subject includes a first treatment comprising an AAV particle or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a syn100 promoter.

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

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

In an embodiment, the method of treating a human subject includes a first treatment comprising an AAV particle or 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 an embodiment, the method of treating a human subject includes a first treatment comprising an AAV particle or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a myo-3 promoter. In an embodiment, the method of treating a human subject includes a first treatment comprising an AAV particle or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a Spc5-12 promoter. In an embodiment, the method of treating a human subject includes a first treatment comprising an AAV particle or 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 an embodiment, the method of treating a human subject includes a first treatment comprising an AAV particle or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof, operably linked to a U6 promoter.

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

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

In an embodiment, a method of treating a human subject includes a first therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:31 (spc-hu-opti-AUF1-CpG(-)). In an embodiment, a method of treating a human subject includes a first therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:32 (tMCK-huAUF1). In an embodiment, a method of treating a human subject includes a first therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:33 (spc5-12-hu-opti-AUF1-WPRE). In an embodiment, a method of treating a human subject includes a first therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:34 (ss-CK7-hu-AUF1). In an embodiment, the method of treating a human subject includes a first therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:35 (spc-hu-AUF1-intronless). In an embodiment, the method of treating a human subject includes a first therapy comprising an AAV particle or rAAV particle having a nucleotide sequence of SEQ ID NO:36 (D(+)-CK7AUF1).

In an embodiment, the method of treating a human subject includes a first therapy utilizing an AAV8 gene therapy vector. In an embodiment, the method of treating a human subject includes a first therapy utilizing an AAV9 gene therapy vector. In an embodiment, the method of treating a human subject includes a first therapy utilizing an AAVhu.32 gene therapy vector. In an embodiment, the method of treating a human subject includes a first therapy utilizing an AAV having a capsid that is at least 95% identical to SEQ ID NO:114 (AAV8 capsid). In an embodiment, the method of treating a human subject includes a first therapy utilizing an AAV having a capsid that is at least 95% identical to SEQ ID NO:115 (AAV9 capsid). In an embodiment, the method of treating a human subject includes a first therapy utilizing an AAV having a capsid that is at least 95% identical to SEQ ID NO:118 (AAVhu.32 capsid).

Disclosed is a method of treating LGMD in a subject in need thereof, comprising administering to the subject a therapeutically effective amount (either when administered alone or in combination with a second therapy) of a first therapy and a therapeutically effective amount (either when administered alone or in combination with the first therapy) of a second therapy different from the first therapy, wherein the first therapy is a first AAV particle or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein or a functional fragment thereof and operably linked to a muscle cell-specific promoter. In embodiments, the AAV or rAAV particles include a construct having a nucleotide sequence of one 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-intronless, or D(+)-CK7AUF1, respectively), e.g., the rAAV is an AAV8 serotype or an AAV9 serotype or an AAVhu.32 serotype. In some embodiments, the first therapy is an AAV particle. In some embodiments, the second therapy is an AAV particle. In some embodiments, the first and second therapies are AAV particles.

In some embodiments, the first therapy is an rAAV particle. In some embodiments, the second therapy is an rAAV particle. In some embodiments, the first and second therapies are rAAV particles.

In embodiments, the second therapy is a second therapeutic protein, pharmaceutical composition, comprising an AAV or rAAV vector particle comprising a therapeutic protein construct comprising microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II β isoform protein or a portion thereof, wherein the AAV or rAAV is an AAV8 serotype or an AAV9 serotype or an AAVhu.32 serotype.

In certain embodiments, the AUF1 gene therapy product and the therapeutic protein (such as microdystrophin, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II β isoform protein or portion thereof) gene therapy product 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 prior to any immune response to the first gene therapy product. In other embodiments, the AUF1 gene therapy product and the therapeutic protein gene therapy product are delivered simultaneously or within 1 hour, 2 hours, or 3 hours, including the second product being administered prior to any immune response to the first gene therapy product. In yet other embodiments, both the AUF1 gene therapy product and the therapeutic protein gene therapy product comprise an AAV vector or rAAV vector of the same serotype and are delivered simultaneously or within an hour or less of each other.

In other embodiments, the second therapy is a mutation suppression therapy, a steroid therapy, an immunosuppressant/anti-inflammatory therapy, any therapy treating 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 immunosuppressant/anti-inflammatory therapy, any therapy treating one or more symptoms of LGMD, or any combination thereof, as disclosed in more detail herein. The administration of each second therapy can be at any of the doses known for the respective administration of the second therapy.

In some embodiments, the second therapy (or optionally a third therapy) can be administered to alleviate or further alleviate one or more symptoms or characteristics of LGMD, which can be assessed in a subject by any of the following assays, including, but not limited to, delay in time to ambulatory failure, improvement in muscle strength, improvement in ability to lift weights, improvement in time to stand from supine position, improvement in 30-foot walk time, improvement in time to climb 4 flights of stairs, improvement in leg function grade, improvement in pulmonary function, improvement in cardiac function, and improvement in quality of life. Each of these assays is known to those of skill in the art. As an example, see Manzur et al. The publication by Manzur et al., "Glucocorticoid Corticosteroids for Duchenne Muscular Dystrophy," Cochrane Database Syst Rev 1:CD003725 (2008); incorporated herein by reference in its entirety, provides an extensive description of these assays. For each of these assays, a detectable improvement or prolongation of the parameter measured in the assay may indicate that one or more symptoms of Duchenne muscular dystrophy have been alleviated in an individual using the methods of the present disclosure. A detectable improvement or prolongation may be achieved by administering to the individual a medicament for which the method of the present disclosure is disclosed. (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); incorporated herein by reference in its entirety). Alternatively, alleviation of one or more symptoms of Duchenne muscular dystrophy may be assessed by measuring improvements in muscle fiber function, integrity and/or survival, as defined herein.

Treatment in the methods disclosed herein may have a duration of at least one week, at least one month, at least several months, at least one year, at least two, three, four, five, six years or more. The frequency of administration of any of the second therapies, including those not delivered by gene therapy and those described herein, may depend on several parameters such as the age of the patient, the type of mutation, the number of molecules (dosage), the formulation of the molecules, etc. The frequency may range from at least once every two weeks, or for periods of three, four, five weeks, or longer.

The first and second therapies, and optionally a third or further therapy, can be administered to an individual in any order. When multiple second therapies (e.g., a third therapy) are administered, they can also be administered in any order with respect to each other and to the first therapy. In one embodiment, the therapies are administered simultaneously (meaning that the therapies are administered within 10 hours, e.g., within 1 hour). In another embodiment, the therapies are administered sequentially. In some aspects, administration of the first and second therapies can occur within 7 days, 10 days, or 14 days of each other. In some aspects, administration simultaneously means that the first and second therapies can be formulated together in a single composition or each can be formulated alone. In some aspects, the third therapy is administered at the same time as the first and/or second therapy, or at a separate time, e.g., on a regular dosing schedule, such as daily, weekly, or monthly.

In some embodiments, the first and second therapies provide a synergistic therapeutic effect with respect to one or more clinical endpoints in treating LGMD in a subject, and in particular, the therapeutic effect is greater than the additive therapeutic effect of the first and second therapies when administered alone. In some embodiments, the first and second therapies provide a synergistic effect in that the therapies provide improvement in a different set of clinical endpoints, such that the therapeutic benefit of the combination is greater than the therapeutic benefit of each therapy individually.

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 treating LGMD in a subject, and in particular, the therapeutic effect is greater than the additive therapeutic effect of the first, second, and third therapies when administered alone. In some embodiments, the first, second, and third therapies provide a synergistic effect in that the therapies provide improvements in a different set of clinical endpoints, such that the therapeutic benefit of the combination is greater than the therapeutic benefit of each therapy individually.

8.1. Therapeutic Protein Therapy in Combination Therapy Disclosed are methods of treating LGMD in a subject in need thereof comprising administering to the subject a first therapy and a second therapy, where 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 a rAAV gene therapy vector encoding a therapeutic protein disclosed herein.

In some embodiments, the transgene encoding a therapeutic protein encodes 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 portions thereof.

In some embodiments, AAV particles or rAAV particles 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 portions thereof, are administered at a therapeutic concentration of between 1E8vg/kg and 2E15vg/kg, e.g., between 1×10 ¹⁰ and 1×10 ¹⁵ genome copies/kg; between 5×10 ¹⁰ and 1×10 ¹⁵ genome copies/kg; between 1×10 ¹¹ and 1×10 ¹⁵ genome copies/kg; between 5×10 ¹¹ and 1×10 ¹⁵ genome copies/kg; between 1×10 ¹² and 1×10 ¹⁵ genome copies/kg; between 2×10 ¹² and 1×10 ¹⁵ genome copies/kg; between 1×10 ¹³ and 1×10 or 2×10 ¹³ to 1× ^{10 15 genome} copies/kg, administered intravenously or intramuscularly.

In certain embodiments, the first treatment is an AAV or rAAV particle and comprises a construct having a nucleotide sequence of one 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-intronless, or D(+)-CK7AUF1, respectively), wherein the AAV or rAAV is an AAV8 serotype or an AAV9 serotype or an AAVhu. 32 serotype, and the second therapy is an AAV particle or rAAV particle having a recombinant genome having a nucleotide sequence of a therapeutic protein disclosed herein, the AAV or rAAV being an AAV8 serotype, or an AAV9 serotype, or an AAVhu.32 serotype. In embodiments, the ratio of AAV particles or rAAV particles having a transgene encoding AUF1 to AAV particles or rAAV particles 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 AUF1 gene therapy vector to micro-dystrophin gene therapy vector is 0.5:1, 0.25:1, 0.2:1, or 0.1:1.

8.2. Micro-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 as disclosed herein. In some embodiments, an AAV vector is administered. In some embodiments, a rAAV vector is administered.

Also disclosed is 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 as disclosed herein, and the second therapy is a gene therapy vector comprising an AAV or rAAV gene therapy vector encoding micro-dystrophin as disclosed herein.

In some embodiments, the transgene encoding the micro-dystrophin protein consists of dystrophin domains arranged from amino terminus to carboxy terminus as follows: 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 a cysteine-rich region of dystrophin, and CT includes at least a portion of CT that includes the α1-syntrophin binding site.

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

The amino acid sequence of human dystrophin (UniProt KB-P11532) (SEQ ID NO:51) is as follows:
MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNII LHWQVKNVMKNIMAGLQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILM YITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQ TALEEVLSWLLSAEDTLQAQGEISNDVEVVKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLMDLQNQKLKEL NDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATAALEEQLKVLGDRWANICRWTEDRWVLLQDILLKWQRLTEEQCLFSAWL SEKEDAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTTQPSLTQTTVMETVTTVTT REQILVKHAQEELPPPPQKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQSPEFAIFRKEGNFSDLKEKVNAIEREKAEKFRKLQDASRSAQALVEQMVNEGVNADSIKQAS EQLNSRWIEFCQLLSERLNWLEYQNNIIAFYNQLQQLEQMTTTAENWLKIQPTTPSEP TAIKSQLKICKDEVNRLSDLQPQIERLKIQSIALKEKGQGPMFLDADFVAFTNHFKQV FSDVQAREKELQTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSLQEQQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEIEIEGRWKKLSSQL VEHCQKLEEQMNKLRKIQNHIQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTIQPSLNSVNEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQQVYARKEA LKGGLEKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTESVNSVIAQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEVWA CWHELLSYLEKANKWLNEVEFKLKTTENIPGGAEISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMDELINEELETFNSRWRELHEEAVRRQKLLEQSIQSAQETEKSLHL IQESLTFIDKQLAAYIADKVDAAQMPQEAQKIQSDLTSHEISLEEMKKHNQGKEAAQRVLSQIDVAQKKLQDVSMKFRLFQKPANFEQRLQESKMILDEVKMHLPALETKSVEQE VVQSQLNHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEV AWGKATQKEIEKQKVHLKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLLLEYQKHMETFDQNVDHITKWIIQADTLLDSEKKKPQQKEDVLKRLKAELND IRPKVDSTRDQAANLMANRGDHCRKLVEPQISELNHRFAAISHRIKTGKASIPLKELE QFNSDIQKLLEPLEAEIQQGVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQRITDER KREEIKIKQQLLQTKHNALKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIKEIDRELQKKKEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWR EIESKFAQFRRLNFAQIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERV KLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGS LNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQW IKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLL QELRAKQPDLAPGLTTIGASPTQTVTLVTQPVVTKETAISKLEMPSSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVMVGDLEDINEMIIKQKATMQDLEQRRPQLEELIT AAQNLKNKTSNQEARTIITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEAEQVLGQARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKL LRDYSADDTRKVHMITENINASWRSIHKRVSEREAALEETHRLQQFPLDLEKFLAWL TEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGEIEAHTDVYHNLDENSQKIL RSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPL EGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVS HVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRT AMKLRRLQKALCLDLLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHN NLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVAS STGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQS CFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSY LNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHK GRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPM LLRVVGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM

In some embodiments, the microdystrophin protein has the amino acid sequence of microdystrophin encoded by DYS1, DYS3 or DYS5 (SEQ ID NO: 176, 177, or 178). Alternatively, the microdystrophin protein has the amino acid sequence of one 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 embodiments, the nucleic acid sequence encoding microdystrophin is operably linked to a regulatory sequence including a promoter listed in Table 7 and other regulatory elements, for example, in Table 2 or Table 8. In certain embodiments, the rAAV has a recombinant genome having a 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 specific embodiments, the rAAV is an AAV8 serotype, an AAV9 serotype, or an AAVhu.32 or any other serotype with tropism for muscle cells as disclosed above.

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

8.2.1 α-, β-, γ-, or δ-Sarcoglycan Gene Therapy Disclosed herein are methods of treating a sarcoglycanopathy in a subject in need thereof, comprising administering to the subject an AAV vector or rAAV vector comprising a transgene encoding AUF1 as disclosed herein. In some embodiments, an AAV vector is administered. In some embodiments, a rAAV vector is administered.

Also disclosed is a method of treating a sarcoglycanopathy 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 as disclosed herein, and the second therapy is a gene therapy vector comprising an AAV or rAAV gene therapy vector encoding α-, β-, γ-, or δ-sarcoglycan as disclosed herein.

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 therapy is an AAV vector. In some embodiments, the first therapy is a rAAV vector.

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 including a promoter listed in Table 6 and other regulatory elements, e.g., of Table 2 or Table 9. In certain embodiments, the AAV or rAAV has a recombinant genome having a nucleotide sequence of SEQ ID NO: 144. In specific embodiments, the AAV or rAAV is an AAV8 serotype or an AAV9 serotype or an AAVhu.32 or any other serotype with tropism for muscle cells as disclosed above.

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

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

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

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

In some embodiments, a therapeutically effective amount of AAV particles or rAAV particles encoding α-sarcoglycan is 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 to 1×10 ¹⁵ genome copies/kg. In some embodiments, a therapeutically effective amount of AAV particles or rAAV particles encoding β-sarcoglycan is 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 to 1×10 ¹⁵ genome copies/kg. In some embodiments, a therapeutically effective amount of AAV particles or rAAV particles encoding γ-sarcoglycan is 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 to 1×10 ¹⁵ genome copies/kg. In some embodiments, a therapeutically effective amount of AAV particles or rAAV particles encoding δ-sarcoglycan is 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 to 1×10 ¹⁵ genome copies/kg.

In certain embodiments, the first treatment is an AAV particle or rAAV particle comprising a construct having a nucleotide sequence of one 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-intronless, or D(+)-CK7AUF1, respectively), wherein the AAV or rAAV is an AAV8 serotype or an AAV9 serotype or an AAVhu. and the second therapy is a rAAV particle having a recombinant genome having a nucleotide sequence of SEQ ID NO: 144, 145, 146 or 147, and the AAV or rAAV is an AAV8 serotype, or an AAV9 serotype, or an AAVhu.32 serotype. In embodiments, the ratio of AAV particles or rAAV particles having a transgene encoding AUF1 to AAV particles or rAAV particles 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 AUF1 gene therapy vector to α-, β-, γ- or δ-sarcoglycan gene therapy vector is 0.5:1, 0.25:1, 0.2:1, or 0.1:1.

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

Also disclosed is a method of treating a calpainopathy 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 as disclosed herein, and the second therapy 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) as disclosed herein. In some embodiments, the first therapy is an AAV vector. In some embodiments, the first therapy is a rAAV vector.

In some embodiments, the calpain 3 protein has an amino acid sequence of SEQ ID NO: 151, 152, 153, 154, 155, 156, 157, 158, or 159. Table 12 provides amino acid sequences of embodiments of calpain 3 according to the present disclosure. Also contemplated are other embodiments substituted 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 and substantially maintain its functional activity. In embodiments, calpain 3 may 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: 151, 152, 153, 154, 155, 156, 157, 158, or 159, and may maintain functional calpain 3 activity, e.g., as determined by one or more of the in vitro or in vivo assays in animal models disclosed below.

Table 12. Amino acid sequence of calpain 3 protein
*NCBI reference sequences are incorporated herein by reference in their entirety.

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

Table 13 provides nucleic acid sequences encoding embodiments of calpain 3 according to the present disclosure. Also contemplated 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 substantially maintain its functional activity. 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 sequence of SEQ ID NO: 160, 161, 162, 163, 164, 165, 166, 167, or 168 and can maintain functional calpain 3 activity, e.g., as determined by one or more of the in vitro or in vivo assays in animal models disclosed below.

Table 13. Nucleic acid sequence of calpain 3 protein
*NCBI reference sequences are incorporated herein by reference in their entirety.

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 amino acid sequences of embodiments of calcium/calmodulin-dependent protein kinase II β according to the present disclosure. Other embodiments are also contemplated to be 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 into 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, e.g., as determined by one or more of the in vitro or in vivo assays in animal models disclosed below.

Table 14. Amino acid sequence of calcium/calmodulin-dependent protein kinase II β protein
*NCBI reference sequences are incorporated herein by reference in their entirety.

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 including a promoter listed in Table 8 and other regulatory elements, e.g., in Table 2 or Table 9. In certain embodiments, the AAV vector or rAAV vector has a recombinant genome having a nucleotide sequence of SEQ ID NO: 171 or 172. In specific embodiments, the rAAV is an AAV8 serotype or an AAV9 serotype or an AAVhu.32 or any other serotype with tropism for muscle cells as disclosed above.

Table 15 provides nucleic acid sequences encoding embodiments of calcium/calmodulin-dependent protein kinase II β proteins according to the present disclosure. Other embodiments are also contemplated as substitution variants of calcium/calmodulin-dependent protein kinase II β proteins 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 into SEQ ID NO: 171 or 172 while substantially maintaining its functional activity. In embodiments, the calcium/calmodulin-dependent protein kinase II β protein sequence may 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 may maintain functional calcium/calmodulin-dependent protein kinase II β protein activity, e.g., as determined by one or more of the in vitro or in vivo assays in animal models disclosed below.

Table 15. Nucleic acid sequence of calcium/calmodulin-dependent protein kinase II β protein
*NCBI reference sequences are incorporated herein by reference in their entirety.

In other embodiments, the 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.

In some embodiments, a therapeutically effective amount of AAV or rAAV particles encoding calpain 3 protein is 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 to 1×10 ¹⁵ genome copies/kg.

In some embodiments, a therapeutically effective amount of AAV particles or rAAV particles encoding a calcium/calmodulin-dependent protein kinase II β isoform protein is 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 to 1×10 ¹⁵ genome copies/kg.

In certain embodiments, the first treatment is an AAV particle or rAAV particle comprising a construct having a nucleotide sequence of one 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-intronless, or D(+)-CK7AUF1, respectively), wherein the AAV or rAAV is an AAV8 serotype or an AAV9 serotype or an AAVhu. and the first treatment is an AAV particle or rAAV particle having a recombinant genome having a nucleotide sequence of SEQ ID NO: 144, 145, 146 or 147, and the AAV or rAAV is an AAV8 serotype, or an AAV9 serotype or an 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 AUF1 gene therapy vector to α-, β-, γ- or δ-sarcoglycan gene therapy vector is 0.5:1, 0.25:1, 0.2:1, or 0.1:1.

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

In some embodiments, the second therapy (or third therapy) is ataluren. In some embodiments, ataluren is administered orally. In some embodiments, ataluren may be administered at a dose of 10 mg/kg/day to 200 mg/kg/day. In some embodiments, ataluren may be administered at a dose of 40 mg/kg. For example, administration may be 10 mg/kg in the morning, 10 mg/kg in the midday, and 20 mg/kg in the evening. The duration of administration of ataluren may be weeks, months, or years. In some embodiments, treatment increases the ability to walk/run greater distances and/or increase the ability to climb stairs compared to pre-treatment levels.

In some embodiments, the second therapy (or third therapy) is gentamicin. In some embodiments, gentamicin is administered intravenously. In some embodiments, gentamicin may be administered at a dose of 3 mg/kg/day to 25 mg/kg/day. In some embodiments, gentamicin may be administered at a dose of 7.5 mg/kg/day. The duration of administration of ataluren may be weeks, months, or years. In some embodiments, treatment results in increased hearing, renal function, and/or muscle strength compared to pre-treatment levels.

In some embodiments, the mutation suppression therapy is a nonsense suppressor mutation. For example, the subject may have a nonsense mutation and the second therapy allows the ribosome to read the premature nonsense mutation.

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

8.5. STEROID THERAPY Disclosed are methods of treating LGMD in a subject in need thereof comprising administering to the subject a first therapy and a second therapy, where the first therapy is a rAAV comprising a transgene encoding AUF1 disclosed herein and the second therapy is steroid therapy. In some embodiments, the steroid therapy is a glucocorticoid steroid. In embodiments, a combination of a rAAV encoding AUF1, a rAAV encoding microdystrophin or α-, β-, γ-, or δ-sarcoglycan, and steroid therapy (as a third therapy) is administered to treat or ameliorate symptoms of LGMD in a subject.

In some embodiments, the steroid therapy is prednisone, deflazacort, vamorolone, or spironolactone, or a combination thereof. Spironolactone is an aldosterone antagonist and may not be considered a steroid, but is used similarly to steroids and is often compared to corticosteroids.

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 vamorolone is 0.5 mg/kg to 40 mg/kg. In some embodiments, the daily dose of vamorolone 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.

Steroid doses can be increased or decreased based on growth, weight, and other side effects experienced. In some embodiments, dosing can be either daily or with higher doses on weekends. For example, in some embodiments, twice weekly doses can be up to 250 mg/day of prednisone or 300 mg/day of deflazacort. In some embodiments, dosing can be 10 days on, 10 days off, etc.

8.6 Immunosuppressive/Anti-inflammatory Therapy Disclosed are methods of treating LGMD in a subject in need thereof comprising administering to the subject a first therapy and a second therapy, where the first therapy is a rAAV comprising a transgene encoding AUF1 as disclosed herein, and the second therapy is an immunosuppressive therapy or an anti-inflammatory therapy. In embodiments, a combination of a rAAV encoding AUF1, a rAAV encoding microdystrophin or α-, β-, γ-, or δ-sarcoglycan, and an immunosuppressive/anti-inflammatory therapy (as a third therapy) is administered to treat or ameliorate symptoms of LGMD in a subject.

In some embodiments, the immunosuppressive or anti-inflammatory therapy is edasalonexant.

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

In some embodiments, the immunosuppressive or anti-inflammatory therapy is pamrevlumab. Pamrevlumab 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 hallmark of muscular dystrophies and contributes to muscle weakness and damage, including in the heart muscle. In some embodiments, inhibition of connective tissue growth factor (CTGF) with pamrevlumab can result in a decrease in muscle fibrosis leading to increased muscle function. In some embodiments, pamrevlumab can be administered intravenously. In some embodiments, administration can be daily, weekly, or monthly. In some embodiments, the duration of treatment can be weeks, months, or years. In some embodiments, pamrevlumab can be administered at a dose of 10 mg/kg to 200 mg/kg. In some embodiments, pamrevlumab can be administered at a dose of 35 mg/kg. For example, administration can be via intravenous (IV) infusion of 35 mg/kg every two weeks.

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

8.7. Therapies Treating One or More Symptoms of LGMD Disclosed are methods of treating LGMD in a subject in need thereof, comprising administering to the subject a first therapy and a second therapy, where the first therapy is a rAAV comprising a transgene encoding AUF1 as disclosed herein, and the second therapy is a therapy treating one or more symptoms of LGMD. In some embodiments, the therapy treating one or more symptoms of LGMD may also include any of the mutation suppression therapy, steroid therapy, and immunosuppressant/anti-inflammatory therapy described herein. In embodiments, a combination of a rAAV encoding AUF1, a rAAV encoding microdystrophin or α-, β-, γ-, or δ-sarcoglycan, and a therapy treating one or more symptoms of LGMD (as a third therapy) is administered to treat or ameliorate a symptom of LGMD in a subject.

In some embodiments, one or more symptoms of LGMD is loss of muscle mass and/or strength, and wherein the second therapy improves muscle mass and/or strength. For example, the second therapy can be spironolactone (as described for steroid therapy), follistatin, SERCA2a, EDG-5506, tamoxifen, divinostat, ASP0367, or combinations thereof.

In some embodiments, follistatin or a follistatin variant may be used as a second treatment. In some embodiments, follistatin may be administered as a gene therapy with a viral vector, such as AAV.

In some embodiments, SERCA2a may be used as a second therapy (or third therapy). In some embodiments, SERCA2a may be administered as gene therapy with a viral vector, such as AAV. In some embodiments, SERCA2a may be administered intravenously. In some embodiments, administration may be daily, weekly, or monthly. In some embodiments, the duration of treatment may be weeks, months, or years. In some embodiments, 1×10 ¹¹ -1×10 ¹⁴ vg are administered. In some embodiments, 6×10 ¹² vg are administered.

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, administration can be daily, weekly, or monthly. In some embodiments, the duration of treatment can be weeks, months, or years.

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

In some embodiments, givinostat is a molecule that inhibits an enzyme called histone deacetylase (HDAC), which can turn off gene expression and reduce muscle's ability to regenerate. By inhibiting HDAC, givinostat can reduce fibrosis and muscle cell death while allowing muscle to regenerate. In some embodiments, givinostat is administered via oral suspension. In some embodiments, administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be weeks, months, or years. In some embodiments, givinostat can be administered at a dose of 1 mg/ml to 100 mg/ml. In some embodiments, givinostat can be administered at a dose of 10 mg/ml. For example, administration can be via a 10 mg/ml oral suspension twice daily.

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 in the cell. When the pathway is turned on, mitochondria use fatty acids more frequently and more mitochondria are made. With more fatty acids used for energy, energy production increases. 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.

In some embodiments, the second therapy (or third therapy) is a cell-based therapy. For example, the cell-based therapy is one or more myoblasts. In some embodiments, a myoblast-based therapy is described in NCT02196467. In some embodiments, myoblasts resuspended in saline can be implanted into the extensor carpi radialis muscle of one of the patient's forearms at between 1 million and 500 million per cubic centimeter. More specifically, 30 million myoblasts can be implanted per cubic centimeter.

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

In some embodiments, the one or more symptoms of LGMD are symptoms related to a 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 third therapy) is ifetroban, bisoprolol fumarate, eplerenone, or a combination thereof.

Ifetroban is a potent selective thromboxane receptor antagonist. In some embodiments, ifetroban can halt important molecular signals that mediate cardiac inflammation and fibrosis (tissue scarring) mechanisms. In some embodiments, ifetroban is administered orally. In some embodiments, administration can be daily, weekly, or monthly. In some embodiments, the duration of treatment can be weeks, months, or years. In some embodiments, ifetroban can be administered at a dose of 50 mg to 400 mg. In some embodiments, ifetroban can be administered at a dose of 200 mg. For example, administration can be via capsules once a day, e.g., 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 cardiac-related symptoms. The dose of bisoprolol can be increased in increments of 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. Dosing may be increased depending on evaluation of the subject's heart rate, blood pressure, symptoms, and ECG.

In some embodiments, eplerenone is administered orally. In some embodiments, administration may be daily, weekly, or monthly. In some embodiments, the duration of treatment may be weeks, months, or years. In some embodiments, eplerenone may be administered in doses of 10 mg to 200 mg. In some embodiments, eplerenone may be administered in doses of 25 mg. For example, administration may be via a once-daily capsule, a single 25 mg capsule.

In some embodiments, the one or more symptoms of LGMD are respiratory symptoms. Thus, the second therapy (or third therapy) can be idebenone. In some embodiments, idebenone can be administered orally. In some embodiments, administration can be daily, weekly, or monthly. In some embodiments, the treatment period can be weeks, months, or 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, administration can be oral three times a day, with each oral administration being two tablets of 150 mg each. In some embodiments, the second therapy (or third therapy) is orthopedic management, endocrine management, gastrointestinal management, urological management, or a combination thereof. In some embodiments, the second therapy (or third therapy) is transcutaneous electrical stimulation (TENS). TENS can increase muscle strength, increase range of motion, and/or improve sleep. In some embodiments, TENS is administered using a VECTTOR system. The VT-200 or VECTTOR system delivers electrical stimulation via electrodes on acupuncture points on the subject's feet/legs and hands/arms to provide symptomatic relief of chronic intractable pain and/or post-surgical pain management. In some embodiments, neurostimulation therapy (e.g., TENS) can be administered once, twice, three times, four times, five times, or more times per day.

8.8. THERAPEUTIC EFFECTIVE DOSE Methods of treating a human patient (e.g., subject) suitable for treatment with an AAV or rAAV encoding a functional AUF1, or a functional AUF1 and a second therapeutic protein or fragment thereof effective to treat or ameliorate one or more symptoms of LGMD, by peripheral administration, including intravenous administration, are disclosed. In some embodiments, the patient/subject suitable for treatment with an AAV or rAAV encoding AUF1 is a patient with LGMD, including type 1 or type 2 LGMD.

In some embodiments, the first therapy is an AAV8 serotype or an AAV9 serotype or an AAVhu. Administration of rAAV particles containing an AUF1-encoding construct as described herein, including AAV particles or rAAV particles, including serotype 32, including constructs having 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-intronless, and D(+)-CK7AUF1, respectively), can be at a dose of 1 x ¹⁰⁸ vg/kg to 2 x ¹⁰¹⁵ vg/kg or 2 x ¹⁰¹³ to 1 x ¹⁰¹⁵ , e.g., 2 x ¹⁰¹⁴ vg/kg. Doses may range from ^1x108 vector genomes per kg (vg/kg) to ^2x1015 vg/kg. In some embodiments, doses may be ^2x1013 , ^3x1013 , ^1x1014 , ^3x1014 , ^5x1014 vg/kg. In some embodiments, the dose may be 1x10, ^{1.1x10, 1.2x10 ,} 1.3x10, ^1.4x10 , 1.5x10, ^1.6x10 , ^{1.7x10 ,} 1.8x10, 1.9x10, ^2x10 , 2.1x10, ^{2.2x10 , 2.3x10} , ^2.4x10 , ^2.5x10 , ^2.6x10 , ^2.7x10 , ^2.8x10 , ^2.9x10 , ^{or 3x10} vg/ ^{kg in} combination with a second ^therapy .

In some aspects, the second therapy is an AAV particle or rAAV particle containing a micro-dystrophin-encoding construct, and administration of an AAV particle or rAAV particle containing a micro-dystrophin-encoding construct described herein, including a construct having a nucleotide sequence of SEQ ID NO: 94, 95, or 96 (serotype AAV8 or AAV9), can be at a dose of 1× ¹⁰ genome copies/kg to 2× ¹⁰ genome copies/kg or a dose of 2× ¹⁰ to 1× ¹⁰ , e.g., 2× ¹⁰ vg/kg. Doses can range from 1× ¹⁰ vector genomes per kg (vg/kg) to 2× ¹⁰ vg/kg. In some embodiments, doses can be 2× ¹⁰ , 3× ¹⁰ , 1× ¹⁰ , 3× ¹⁰ , 5× ¹⁰ vg/kg. In some embodiments, the dose can be 1x10, ^{1.1x10, 1.2x10 ,} 1.3x10, ^1.4x10 , ^1.5x10 , 1.6x10, ^1.7x10 , ^1.8x10 , ^{1.9x10, 2x10 , 2.1x10 , 2.2x10 , 2.3x10} , ^2.4x10 , ^2.5x10 , ^2.6x10 , ^2.7x10 , ^{2.8x10 , 2.9x10 ,} or ^{3x10 vg} /kg. In some embodiments, the second therapeutic is an AAV particle or rAAV particle containing a construct encoding an α-, β-, γ-, or δ-sarcoglycan described herein.

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.

The therapeutically effective dose 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 a monotherapy. According to such embodiments, only the AUF1 gene therapy vector is administered.

In some embodiments, the therapy is a 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 a gene therapy, or if a third therapy (or additional therapy) that is not a gene therapy vector is administered, it may be administered in multiple doses over the course of the treatment regimen (i.e., days, weeks, months, etc.), and may be administered before or after the first (and/or second) therapy, or both before and after the first (and/or second) gene therapy vector.

The dosage is therapeutically effective and can be evaluated at appropriate time points after administration, including 12 weeks, 26 weeks, 52 weeks or more, including evaluation for improvement or amelioration of symptoms and/or biomarkers of LGMD as known in the art and detailed herein. Recombinant vectors used to deliver the transgene encoding AUF1 are described herein. Such vectors should have tropism for human muscle cells (including skeletal, skeletal and/or cardiac cells) and may include non-replicating rAAV, particularly those having an AAV8 capsid, an AAV9 capsid or an AAVhu.32 capsid. Recombinant vectors for expressing AUF1, including vectors having the constructs spc-hu-opti-AUF1-CpG(-), tMCK-huAUF1, spc5-12-hu-opti-AUF1-WPRE, ss-CK7-hu-AUF1, spc-hu-AUF1-intronless, and D(+)-CK7AUF1 (see FIG. 1), can be administered in any manner that allows the recombinant vector to enter muscle tissue, including introducing the recombinant vector into the bloodstream, including intravenous administration.

A therapeutically effective dose of any such recombinant vector should be administered by any method that results in the recombinant vector entering muscle (e.g., skeletal or cardiac muscle), including introducing the recombinant vector into the bloodstream. In specific embodiments, the vector is administered subcutaneously, intramuscularly, or intravenously. Expression of the transgene product results in delivery and maintenance of the transgene product in muscle.

Pharmaceutical compositions suitable for intravenous, intramuscular, or subcutaneous administration include suspensions of recombinant AAV containing any of the transgenes disclosed herein in a formulation buffer, including 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 transgenes encoding AUF1 or microdystrophin or sarcoglycan disclosed herein, and may be used in the disclosed methods.

The disclosed therapeutic methods can result in one of a number of endpoints indicative of therapeutic efficacy as described herein. In some embodiments, the endpoints 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.

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

In some embodiments, the reduction in gastrocnemius (or other muscle) pathology can be used as an endpoint indicator of the therapeutic efficacy of the treatment and administration methods disclosed herein. The gastrocnemius pathology in the subject can be reduced compared to levels (gastrocnemius pathology levels) prior to administration of the therapy. In some embodiments, the gastrocnemius pathology in the subject can be reduced compared to levels (gastrocnemius pathology levels) in untreated subjects with LGMD. The comparison of gastrocnemius pathology can be to a standard, which is a number or set of numbers representing the pathology of subjects without LGMD or the pathology of untreated subjects with LGMD. Thus, in some embodiments, the comparison of gastrocnemius pathology after administration of the therapy can be to a control subject. The control can be the gastrocnemius pathology in the subject prior to administration, the gastrocnemius pathology in untreated LGMD subjects, the gastrocnemius pathology in subjects without LGMD, or the gastrocnemius pathology in a standard.

In some embodiments, the lesions in the subject's gastrocnemius muscle are assessed using magnetic resonance imaging (MRI). MRI can be a good tool for imaging muscles, ligaments, and tendons, and thus can be used to detect and/or characterize muscle disease. In some embodiments, administration of a therapy disclosed herein results in a reduction in the lesions in the gastrocnemius muscle after administration, compared to a control, e.g., compared to the lesions in the gastrocnemius muscle of the subject before said administration, that is about 1-100%, 2-50%, or 3-10%. For example, a subject treated with an AAV or rAAV comprising a transgene encoding AUF1, including in combination with another therapy, can have a lesion reduction of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or more compared to a control.

In some embodiments, gastrocnemius muscle mass (or any other muscle mass) 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) before administration of an AAV or rAAV containing a 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 of a subject without LGMD or the amount of an untreated subject with LGMD. Thus, in some embodiments, the comparison of gastrocnemius muscle mass after administration of a therapeutic method disclosed herein can be to a control. The control can be the gastrocnemius muscle mass in a subject before administration, the gastrocnemius muscle mass in a subject with untreated LGMD, the gastrocnemius muscle mass in a subject without LGMD, or the gastrocnemius muscle mass in a standard.

In some embodiments, the subject's gastrocnemius muscle mass may be assessed using MRI. In some embodiments, 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 said administration. In some embodiments, the decrease in gastrocnemius muscle mass following administration of an AAV or rAAV comprising a transgene encoding AUF1, including in combination with a second therapy, may be about 2-400 mm 3 , 5-200 mm ³ , or 20-100 mm ³ compared to a control. For example, a subject treated with a rAAV comprising a transgene encoding AUF1, including in combination with a second therapy, may 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.

In some embodiments, muscle fat percentage can be used as an endpoint of therapeutic efficacy of the method of administering an AAV or rAAV therapy disclosed herein. The muscles can be the muscles of the lower leg girdle and thigh (gluteus maximus, adductor magnus, rectus femoris, vastus lateralis, vastus medialis, biceps femoris, semitendinosus, and gracilis). The muscle fat percentage in the subject can be reduced compared to the level (level of muscle fat percentage) prior to administration of an AAV or rAAV comprising a transgene encoding AUF1, including in combination with a second therapy, as disclosed herein. In some embodiments, the muscle fat percentage can be reduced compared to the level (level of muscle fat percentage) in an untreated subject with LGMD. The comparison of 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 subjects without LGMD or the amount or percentage in untreated subjects with LGMD. Thus, in some embodiments, the comparison of muscle fat percentage after administration of an AAV or rAAV containing a transgene encoding AUF1, including in combination with a second therapy, can be to a control. The control can be muscle fat percentage in a subject prior to administration, muscle fat percentage in a subject with untreated LGMD, muscle fat percentage in a subject without LGMD, or normal muscle fat percentage.

In some embodiments, the subject's muscle fat percentage is assessed using magnetic resonance imaging (MRI). In some embodiments, methods are provided for treating LGMD by peripheral administration, including intravenous administration, of an AAV vector or rAAV vector containing an AUF1 construct, including a second therapy, resulting in a reduction in muscle fat percentage after administration, which may be about 1-100%, 2-50%, or 3-10%, compared to a control, e.g., compared to the muscle fat percentage before said administration. For example, subjects so administered may have a reduction in muscle fat percentage of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or more, compared to a control.

In some embodiments, the ambulation score can be used as an endpoint of treatment. The ambulation 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 subjects without LGMD. The NSAA of the treated subject can be compared to untreated subjects with LGMD. The NSAA of the treated subject can be relative to a standard, which is a score or set of scores representing the NSAA of subjects without LGMD or the NSAA of untreated subjects with LGMD. In some embodiments, the NSAA of the treated subject is compared to the NSAA score before administration or to any of the NSAA comparisons above. In some embodiments, the increase can be 0 to 1, 0 to 2, or 1 to 2.

8.9. Cardiac Output Although skeletal muscle involvement is considered the defining feature of LGMD, patients most commonly die of respiratory or cardiac failure. LGMD patients develop dilated cardiomyopathy (DCM) that is necessary for contractile function. This results in an influx of extracellular calcium, triggering protease activation, myocardial cell death, tissue necrosis, and inflammation, ultimately resulting in fat accumulation and fibrosis. This process first affects the left ventricle (LV), which is responsible for pumping blood to the majority of the body and is thicker and therefore experiences a greater workload. Atrophied cardiomyocytes show loss of striated muscle, vacuolization, fragmentation, and nuclear degeneration. Functionally, atrophy and scarring cause structural instability and reduced motion of the LV, ultimately progressing to generalized DCM. DMD can be associated with a variety of ECG changes, including sinus tachycardia, decreased circadian index, reduced heart rate variability, shortened PR interval, right ventricular hypertrophy, ST segment depression, and QTc prolongation.

The gene therapy treatments provided herein can slow or prevent the progression of LGMD, and in particular can reduce or ameliorate the progression of cardiac dysfunction and/or maintain or improve cardiac function. Efficacy can be monitored by periodically assessing cardiac pathology or signs and symptoms of heart failure according to the age and stage of the study population using serial electrocardiograms and non-invasive serial imaging tests (e.g., echocardiography 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), maximum expiratory flow (PEF), maximum 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, the ECG can be used to assess the PR interval, the R wave in V1, the Q wave in V6, ventricular repolarization, QS waves in the inferior and/or superior walls, conduction defects in right bundle branch block, QTC, and normalization of the QRS.

The therapeutic methods disclosed herein can improve or maintain cardiac function or slow the loss of cardiac function, for example, by preventing a decline in LVEF below 45% and/or normalization of function (LVFS ≥ 28%), as measured by serial electrocardiograms and/or non-invasive serial imaging (e.g., echocardiography or cardiac magnetic resonance imaging (CMR)). Measurements can be compared to untreated controls or pre-treatment subjects. Alternatively, the therapeutic methods disclosed herein result in improved cardiac function or reduced loss of cardiac function, as assessed 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), maximum expiratory flow (PEF), maximum 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, the ECG can be used to assess the PR interval, the R wave in V1, the Q wave in V6, ventricular repolarization, QS waves in the inferior and/or superior walls, conduction defects in right bundle branch block, QTC, and normalization of the QRS.

In some embodiments, cardiac and/or pulmonary function can be used as an endpoint to assess the therapeutic efficacy of administration. The cardiac and/or pulmonary function in a subject can be improved or increased compared to levels (levels of cardiac and/or pulmonary function) prior to said administration. In some embodiments, the cardiac and/or pulmonary function in a subject can be improved or increased compared to levels (levels of cardiac and/or pulmonary function) in subjects without LGMD. In some embodiments, the cardiac and/or pulmonary function in a subject can be decreased compared to levels (levels of cardiac and/or pulmonary function) in untreated subjects with LGMD. The comparison of cardiac and/or pulmonary function can be relative to a standard, which is a number or set of numbers representing cardiac and/or pulmonary function in subjects without LGMD or cardiac and/or pulmonary function in untreated subjects with LGMD. Thus, in some embodiments, the comparison of cardiac and/or pulmonary function after administration can be relative to a control. The control can be cardiac and/or pulmonary function in a subject prior to administration, cardiac and/or pulmonary function in a subject with untreated LGMD, cardiac and/or pulmonary function in a subject without LGMD, or cardiac and/or pulmonary function in a standard.

In some embodiments, the improvement or increase in cardiac and/or pulmonary function is 1-100% compared to a control, e.g., compared to the subject prior to administration. In some embodiments, cardiac function can be measured using impedance, electrical activity, and calcium handling.

8.10. Primary Patient Outcomes The efficacy of the compositions (including dosages of the compositions) and methods described herein may be assessed in clinical evaluations of treated subjects. Primary patient outcomes include change from baseline in forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), maximum expiratory flow (PEF), peak expiratory flow during cough, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), change from baseline in NSAA, change from baseline in Performance of Upper Limp (PUL) score, and Brooke Upper Extremity Score (BSE). These may include monitoring changes from baseline in Scale score (Brook score), changes from baseline in grip strength, pinch strength, changes in myocardial fibrosis score by MRI, biceps muscle fat and fibrosis assessed by MRI, leg strength measurements using a dynamometer, changes in 6-minute walk test, 10-minute walk test, gait analysis by 3D gait recording, changes in utrophin membrane staining via quantitative imaging of immunostained biopsy sections, and changes in regenerating fibers (via muscle biopsy) by measuring a combination of fiber size and fetal myosin positivity. For example, see Mazzone E et al, “North Star Ambulant 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. (See, e.g., Schneider, R., “Upper Limb Function in Duchenne Muscular Dystrophy: 24 Month Longitudinal Data,” PLoS One 13(6):e0199223 (2018), which are incorporated by reference in their entireties.

The following examples are intended to illustrate the practice of embodiments of the present application, but are in no way intended to limit its scope.

Example 1 - AUF1 Gene Expression Cassette for Insertion into Cis Plasmid A construct was synthesized to prepare a rAAV8 vector encoding p40 AUF1. A codon-optimized, CpG-depleted nucleotide sequence encoding human p40 AUF1 (SEQ ID NO:17) was identified, synthesized, and cloned into a cis plasmid. Regulatory elements were used to generate an expression cassette incorporating the opti-CpG(-) AUF1 coding sequence (SEQ ID NO:17) (amino acid sequence provided in Table 2). The 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-intronless (SEQ ID NO:35), or D(+)-CK7AUF1 (SEQ ID NO:36) are shown in Figure 1 (nucleotide sequences are provided in Table 3). The constructs were introduced into a cis plasmid used to produce rAAV, e.g., rAAV8 particles containing a recombinant genome encoding AUF1. Methods for producing rAAV particles are known in the art, and in the experiments described above using rAAV particles, triple transfection of HEK293 cells was performed with: (1) a cis plasmid (a transgene (such as a therapeutic transgene described herein) flanked by AAV ITR sequences); (2) a rep/cap plasmid (AAV rep and cap genes and gene products, e.g., rep2/cap8 for AAV8); and (3) a helper plasmid (suitable helper virus functions, usually a mutated adenovirus), and the cells were then cultured in suitable media and media components to support rAAV production until harvest and purification of the particles (rAAV vectors).

Example 2 - Calpain 3-Deficient Limb-Girdle Muscular Dystrophy (LGMD) Type 2A CAPN3 disease accounts for approximately 30% of all LGMD cases worldwide, estimated at 1:40,000-1:100,000, but may be 100-fold more prevalent in certain geographic regions due to founder effects. LGMD R1 (CAPN3) is on average 10-12 times less common than DMD.

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 skeletal muscle-specific protease that activates muscle contraction by selective cleavage and activation of contractile proteins, thereby activating the muscle calcium pump. Key features include a marked loss of type I oxidative slow-twitch fibers and altered PGC1α activity due to impaired Ca2 ⁺ calmodulin kinase IIβ (CaMKIIβ) activity.

The diagnosis of calpainopathy is confirmed by molecular identification of biallelic pathogenic variants in the CAPN3 gene or dominant heterozygous CAPN3 mutations; most cases of calpainopathy are autosomal recessive. There are over 400 known pathogenic variants in CAPN3 (null deletions, missense, nonsense).

Clinical features of CAPN3 include primarily proximal muscle wasting; no involvement of myocardium or facial muscles; slow but progressive muscle necrosis; and loss of muscle regeneration. The age at onset of muscle weakness ranges from 2 to 40 years, with an average of 15 years. Further symptoms include difficulty running, tendency to walk on tiptoes, and winging of the scapula due to weakness of the shoulder girdle muscles, as well as shortness of breath at rest. As the upper airway musculature is affected, speech and swallowing disorders begin to develop. Some fatigue, lethargy, loss of appetite, weight loss, and difficulty concentrating may also occur. As the disease progresses, there may be a swaying gait, difficulty climbing stairs, difficulty lifting heavy objects, and difficulty rising from the floor or a chair.

Calpainopathy is milder than Duchenne muscular dystrophy (DMD), generally occurs at a later age of onset, and is primarily restricted to the skeletal muscles of the appendages, without involvement of the myocardium or diaphragm. These characteristics provide a more quantitative means of measuring outcomes in interventional clinical trials.

Although life expectancy in CAPN3 deficiency is close to normal, there is an unmet need for treatments.

CAPN3-deficient disease mice Mouse LGMD-2A models and findings in humans show mitochondrial dysfunction and impaired muscle regeneration, similar to Duchenne muscular dystrophy. Drugs that improve mitochondrial function also improve muscle function and partially correct muscle regeneration in CAPN3-deficient mice that mimic LGMD-2A.

The CAPN3 ^-/- deletion mouse model (JACS) closely recapitulates LGMD2A disease, showing muscle degeneration, arrest of regeneration, necrosis, mitochondrial and functional abnormalities, small muscle fibers, loss of slow muscle fibers, and muscle weakness. Limitations of the CAPN3 ^-/- deletion mouse model (JACS) include the lack of fibrosis, and patients may harbor both an allelic deletion and a second allelic missense/nonsense mutation.

Small molecule activators of CAMKIIβ kinase activity have been reported to modestly 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 Guard Muscular Dystrophy,” Cell Reports 1:100122 (2020); incorporated herein by reference in its entirety), providing a response to measure AUF1 gene therapy in this disease model.

AAV8 hAUF1 Gene Therapy Restores Muscle Fiber Integrity, Size, and Maturity in CAPN3-Deficient Disease Mice To evaluate the efficacy of AUF1 therapy, CAPN3-deficient mice were administered codon-optimized humanized AUF1 (AAV8-hAUF1) at 6 x 10 ¹³ viral genomes/kg mouse body weight (low dose), codon-optimized humanized AUF1 (AAV8-hAUF1) at 1 x 10 ¹⁴ viral genomes/kg mouse body weight (high dose), or vector control (control).

Treatment of CAPN3-deficient mice with AAV8-hAUF1 for 2 months increased muscle endurance and strength above CAPN3 KO and wild-type levels, as shown in Figure 2D. Notably, low doses of hAUF1 gene therapy were sufficient to significantly increase muscle endurance and strength as measured by time to exhaustion (Figure 3A), distance to exhaustion (Figure 3B), maximum velocity (Figure 3C), and muscle grip strength (Figure 3D).

Three months of AAV8-hAUF1 gene therapy restored muscle fiber morphology to near normal, with a notable increase in larger mature muscle fibers (Figure 2A) and increased cross-sectional area (csa) containing two or more nuclei, a marker of mature muscle fibers (Figure 2B). In CAPN3 deficiency, diaphragm pathology was less severe than limb skeletal muscles, but AAV8-hAUF1 gene therapy corrected pathology in both muscle types (Figures 2A-2B).

One of the main goals of therapy for CAPN3 deficiency is to restore CAMKIIβ kinase activity, which can be determined by specific activation phosphorylation and an 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, as well as increased mitochondrial content, as indicated by increased mitochondrial DNA levels (Figure 4).

Another major goal of therapy for CAPN3 deficiency is to restore the levels and activity of the SERCA2 pump, as well as PGC1α levels, which promote mitochondrial biogenesis and type I endurance slow muscle fibers. Two months of AUF1 gene therapy increased both CAMKIIβ protein expression, activated phosphorylation, SERCA2A, and PGC1α in gastrocnemius muscle (Figure 5).

AUF1 gene therapy also potently restored mitochondria in the appropriate subsarcolemmal location, as shown in TA muscle 2 months after therapy (Figure 6).

CAPN3 deficiency is characterized by disruption of intermyofibrillar organization and structure within muscle fibers, which impedes normal muscle contraction and leads to exhausted and dying mitochondria. As shown in gastrocnemius muscle, 2 months of AAV8-hAUF1 gene therapy restored near-normal intermyofibrillar organization and mitochondria in CAPN3-deficient muscle (Figure 7).

In CAPN3 LGMD, there is reduced activity of succinate dehydrogenase (SDH), a hallmark of defective mitochondrial activity. AAV8-hAUF1 gene therapy of CAPN3-deficient TA muscle restores high levels of SDH activity in the deep muscle (zone 1) (Figure 8).

The results presented herein show that AAV8-hAUF1 increases muscle endurance and strength in CAPN3 KO mice to near wild-type levels; low-dose hAUF1 gene therapy is sufficient to increase muscle endurance and strength in CAPN3 KO mice; hAUF1 increases CAMKIIβ mRNA levels, protein levels, and activated CAMKIIβ Thr286 phosphorylation in the muscles tested (gastrocnemius and TA); CAPN3 KO mice treated with hAUF1 gene therapy showed robust increases in succinate dehydrogenase (SDH) and NADH staining, indicative of increased mitochondrial oxidative function; AUF1 modestly increases TA and diaphragm muscle fiber area in CAPN3 KO mice; SERCA2 mitochondrial pump protein expression is increased in CAPN3 KO mice; These results show that hAUF1 treatment increased the phenotype in KO mice, and that the phenotype was more significantly restored in female mice than in male mice.

Example 3 - Evaluation of AUF1 Gene Therapy Constructs in δ-Sarcoglycan Deficient Mice Sarcoglycanopathies are caused by dominant mutations in one of the four sarcoglycan protein genes (α, β, γ, δ). Six sarcoglycan proteins interact with dystrophin to form a complex that stabilizes the sarcolemma, promotes contraction, maintains muscle integrity and proper calcium function in contraction. Disease onset is severe, occurring from childhood to adulthood, affecting limb and girdle skeletal muscles, including the diaphragm and cardiac muscle. Global prevalence is low, at 2-3 per 100,000. It is considered a rare disease. The delta form of the disease is the rarest, with mutations in SCGD causing LGMD2F.

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 four-protein sarcoglycan complex that stabilizes the interaction of dystrophin with the sarcolemma (muscle fiber cell membrane), providing stability and contractile capacity.

The Scgd ^tm1Mcn mouse on a C57BL/6J genetic background is a model of human sarcoglycan delta (dystrophin-associated glycoprotein) deficiency disease. Mice homozygous for deletion of the Sgcd allele are viable and fertile, develop symptoms by 8 weeks of age, and develop degeneration of muscle, including the cardiac muscle, by 12 weeks of age, followed by rapid death.

δ-sarcoglycan-deficient mice were administered AAV8-hAUF1 (codon-optimized humanized AUF1) 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 repaired the marked degeneration of the SCGD muscle, evident as dark blue stained areas on H&E staining (Figure 9).

In SCGD deficiency, the gastrocnemius muscle is affected. Animals were administered AAV8-hAUF1 (codon-optimized humanized AUF1 described in the provisional application) 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 muscle integrity and repaired the marked degeneration of the SCGD muscle, evident as dark blue stained areas on H&E staining (Figure 10).

Embryonic myosin heavy chain (eMHC) expression is a hallmark of muscle regeneration. However, SCGD muscles chronically fail to regenerate and continue to express eMHC. Two months of AAV8 hAUF1 gene therapy restored more normal muscle regeneration, with reduced eMHC expression and larger, more mature muscle fibers as indicated by fibers with more than two nuclei and larger cross-sectional area (csa) (Figure 11).

Two months of AAV8-hAUF1 gene therapy in SCGD-deficient mice increased all types of muscle fibers, including type I slow-twitch and multiple forms of type II fast-twitch muscle fibers (Figure 12).

The muscle function of the animals was tested for muscle grip strength, which allows the mouse to grasp the grid with its forelimbs while pulling on its tail. Figure 13 shows that 2 months of AAV8 hAUF1 gene therapy resulted in a statistical increase in SCGD mice, approaching wild type in female mice.

The results presented here show that hAUF1 protects muscles from dystrophy and increases muscle fiber area (demonstrated for diaphragm and gastrocnemius muscles). 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.

All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

The discussion herein is provided to better understand the nature of the problems facing the art and should not be construed as an admission of prior art in any manner, nor should the citation of any document herein be construed as an admission that such document constitutes "prior art" to the present application.

Although the present invention has been described in detail with reference to specific embodiments thereof, it will be understood that functionally equivalent variations are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed within the scope of the following claims.

Claims (48)

1. A method of treating limb-girdle muscular dystrophy (LGMD) in a subject in need thereof, comprising administering to the subject:
The method comprises administering an adeno-associated virus (AAV) particle or a recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid molecule encoding AU-rich mRNA-binding factor 1 (AUF1) protein, or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by inverted terminal repeat (ITR) sequences. 1. A method of treating mitochondrial dysfunction associated with limb-girdle muscular dystrophy (LGMD) in a subject in need thereof, comprising administering to the subject:
The method comprises administering an adeno-associated virus (AAV) particle or a recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid molecule encoding AU-rich mRNA-binding factor 1 (AUF1) protein, or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by inverted terminal repeat (ITR) sequences. The method of claim 1 or claim 2, wherein the subject comprises a functional AUF1 protein isoform. The method according to any one of claims 1 to 3, wherein the limb-girdle muscular dystrophy (LGMD) is subtype 1. The method of claim 4, wherein the LGMD is limb-girdle muscular dystrophy type 1C (LGMD1C). The method of claim 4, wherein the LGMD is limb-girdle muscular dystrophy type 1G (LGMD1G). The method according to any one of claims 1 to 3, wherein the LGMD is limb-girdle muscular dystrophy type 2 (LGMD2). The method of claim 7, wherein the LGMD is a sarcoglycanopathy. The method according to claim 8, wherein the sarcoglycanopathy is limb-girdle muscular dystrophy type 2C (LGMD2C). The method according to claim 8, wherein the sarcoglycanopathy is limb-girdle muscular dystrophy type 2D (LGMD2D). The method according to claim 8, wherein the sarcoglycanopathy is limb-girdle muscular dystrophy type 2E (LGMD2E). The method according to claim 8, wherein the sarcoglycanopathy is limb-girdle muscular dystrophy type 2F (LGMD2F). The method of claim 7, wherein the LGMD is dystroglycanopathy. The method of claim 13, wherein the dystroglycanopathy is limb-girdle muscular dystrophy type 2I (LGMD2I). The method of claim 13, wherein the dystroglycanopathy is limb-girdle muscular dystrophy type 2K (LGMD2K). The method of claim 13, wherein the dystroglycanopathy is limb-girdle muscular dystrophy type 2M (LGMD2M). The method of claim 13, wherein the dystroglycanopathy is limb-girdle muscular dystrophy type 2N (LGMD2N). The method of claim 13, wherein the dystroglycanopathy is limb-girdle muscular dystrophy type 2O (LGMD2O). The method of claim 13, wherein the dystroglycanopathy is limb-girdle muscular dystrophy type 2P (LGMD2P). The method of claim 13, wherein the dystroglycanopathy is limb-girdle muscular dystrophy type 2T (LGMD2T). The method of claim 13, wherein the dystroglycanopathy is limb-girdle muscular dystrophy type 2U (LGMD2U). The method of claim 7, wherein the LGMD is dysferlinopathy. 23. The method of claim 22, wherein the dysferlinopathy is limb-girdle muscular dystrophy type 2B (LGMD2B). The method of claim 7, wherein the LGMD is limb-girdle muscular dystrophy type 2L (LGMD2L). The method of claim 7, wherein the LGMD is limb-girdle muscular dystrophy type 2H (LGMD2H). The method of claim 7, wherein the LGMD is limb-girdle muscular dystrophy type 2W (LGMD2W). The method of claim 7, wherein the LGMD is limb-girdle muscular dystrophy type 2X (LGMD2X). The method of claim 7, wherein the LGMD is a calpainopathy and, optionally, the subject comprises a calpain 3 (CAPN3) mutation. 29. The method of claim 28, wherein the calpain deficiency is recessive limb-girdle muscular dystrophy type 1/limb-girdle muscular dystrophy type 2A (LGMDR1/LGMD2A). The method of 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, a dMCK promoter, a tMCK promoter, a smooth muscle 22 (SM22) promoter, a myo-3 promoter, a SPc5-12 promoter, a creatine kinase (CK) 8e promoter, a U6 promoter, a H1 promoter, a desmin promoter, a Pitx3 promoter, a skeletal alpha-actin promoter, a MHCK7 promoter, or a Sp-301 promoter. The method of claim 30, wherein the muscle cell-specific promoter is a tMCK promoter, an SPc5-12 promoter, or a CK7 promoter. 2. A method according to any one of the preceding claims, wherein the nucleic acid molecule encodes one or more of ^p37AUF1 , ^p40AUF1 , ^p42AUF1 , or ^p45AUF1 . The method of any one of the preceding claims, wherein the nucleotide sequence encoding the AUF1 protein is the nucleotide sequence of SEQ ID NO: 17. The method of 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). The method of any one of the preceding claims, wherein the AAV or 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). The method of any one of the preceding claims, wherein the AAV or rAAV is administered at a dose of 1E8-1E15 vector genomes/kg or at a dose of 2E15 vector genomes/kg. The method of any one of the preceding claims, wherein the AAV or rAAV is administered intravenously. The method of any one of the preceding claims, wherein a second therapeutic agent is administered. 39. The method of claim 38, wherein the AAV or rAAV and the second therapy are administered simultaneously or within 1 week or 2 weeks of each other. The method of claim 38 or 39, wherein the second therapy is a mutation suppression therapy, a steroid therapy, an immunosuppressant/anti-inflammatory therapy, or a therapy that treats one or more symptoms of LGMD. The method of any one of claims 38 to 40, wherein the second treatment is administered intravenously. 10. The method of claim 9, wherein a second therapy is administered comprising an AAV particle or rAAV particle comprising 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 inverted terminal repeat (ITR) sequences. 11. The method of claim 10, wherein a second therapy is administered comprising an AAV particle or rAAV particle comprising a nucleic acid molecule encoding alpha-sarcoglycan (SGCA) protein, or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by inverted terminal repeat (ITR) sequences. 12. The method of claim 11, wherein a second therapy is administered comprising an AAV particle or rAAV particle comprising 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 inverted terminal repeat (ITR) sequences. 13. The method of claim 12, wherein a second therapy is administered comprising an AAV particle or rAAV particle comprising a nucleic acid molecule encoding a delta-sarcoglycan (SGCD) protein, or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by inverted terminal repeat (ITR) sequences. 30. The method of claim 28 or claim 29, wherein a second therapy is administered comprising an AAV particle or rAAV particle comprising a nucleic acid molecule encoding calpain 3 protein, or a functional fragment thereof, operably linked to a muscle cell-specific promoter and flanked by inverted terminal repeats. 30. The method of claim 28 or claim 29, wherein a second therapy is administered comprising an AAV particle or rAAV particle comprising 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 inverted terminal repeats. The method of any one of the preceding claims, wherein the administration promotes phosphorylation of CAMKIIβ in the subject.