WO2024020574A2 - Thérapie génique auf1 pour myopathie des ceintures - Google Patents

Thérapie génique auf1 pour myopathie des ceintures Download PDF

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WO2024020574A2
WO2024020574A2 PCT/US2023/070765 US2023070765W WO2024020574A2 WO 2024020574 A2 WO2024020574 A2 WO 2024020574A2 US 2023070765 W US2023070765 W US 2023070765W WO 2024020574 A2 WO2024020574 A2 WO 2024020574A2
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promoter
auf1
aav
muscular dystrophy
muscle
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WO2024020574A3 (fr
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Dounia ABBADI
Robert J. Schneider
Subha KARUMUTHIL-MELETHIL
Chunping Qiao
Kirk Elliott
Ye Liu
Olivier Danos
Steven FOLTZ
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New York University
Regenxbio, Inc.
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Definitions

  • the present disclosure relates to treatment of muscle degenerative disease, such as limb girdle muscular dystrophy (LGMD), by administration of doses of gene therapy vectors, such as AAV and rAAV gene therapy vectors in which the transgene encodes AUF1. Also provided are AAV gene therapy vectors encoding an AUF1 protein and methods of treatment using same.
  • gene therapy vectors such as AAV and rAAV gene therapy vectors in which the transgene encodes AUF1.
  • AAV gene therapy vectors encoding an AUF1 protein and methods of treatment using same.
  • LGMD Limb girdle muscular dystrophy
  • the proteins encoded by LGMD disease genes have a diverse array of cellular functions including glycosylation and muscle membrane integrity, maintenance, and repair which when disrupted all result in muscle damage and degeneration.
  • an effective LGMD treatment does not exist for any LGMD subtype.
  • Type 1 LGMDs are dominantly inherited and account for about 10% of all LGMDs.
  • subtype 1 include LGMD1C, which is caused by a mutation in caveolin 3, and LGMD1G, which is caused by a mutation in HNRPDL, a protein involved in mRNA biogenesis and metabolism.
  • Type 2 LGMDs are recessively inherited.
  • Several genes that cause LGMD2s encode for proteins that directly associate with dystrophin.
  • Sarcoglycanopathies have loss of function mutations in the genes encoding a-, P-, y- or 5-sarcoglycan and cause LGMD2C, LGMD2D, LGMD2E, and LGMD2F, respectively.
  • the LGMD2 dystrophinopathies include LGMD2I (mutation in FKRP), LGMD2K (mutation in POMT ), LGMD2M (mutation in FKTN), LGMD2N (mutation in POMT2 LGMD2O (mutation in POMGnTl LGMD2P (mutation in DAG1), LGMD2T (mutation in GMPPB) and LGMD2U (mutation m ISP/CRPPA).
  • LGMD2 calpainopathies include LGMD2A, which is caused by mutations in Calpain 3 (CAPN3). LGMD2A is the most frequent LGMD worldwide. Calpains are intracellular nonlysosomal cysteine proteases modulated by calcium ions.
  • the LGMD2 dysferlinopathies include LGMD2B.
  • Other LGMD2 subtypes include LGMD2L, LGMD2H, LGMD2W, and LGMD2X.
  • the sarcoglycans are a group of transmembrane proteins that associate with dystrophin.
  • the sarcoglycan subcomplex is tightly associated with 0-dystroglycan and is made of 4 single-pass transmembrane proteins, a-sarcoglycan, P-sarcoglycan, y-sarcoglycan and 5- sarcoglycan. Mutations in the genes encoding a-sarcoglycan, P-sarcoglycan, y-sarcoglycan and 5-sarcoglycan cause LGMD2C-F, respectively.
  • Dystrophin is a cytoplasmic protein encoded by the DMD gene, and functions to link cytoskeletal actin filaments to membrane proteins. Normally, the dystrophin protein, located primarily in skeletal and cardiac muscles, with smaller amounts expressed in the brain, acts as a shock absorber during muscle fiber contraction by linking the actin of the contractile apparatus to the layer of connective tissue that surrounds each muscle fiber. In muscle, dystrophin is localized at the cytoplasmic face of the sarcolemma membrane.
  • Muscle wasting diseases represent a major segment of human disease. They can be genetic in origin (primarily muscular dystrophies), related to aging (sarcopenia), or the result of traumatic muscle injury, among others. There are few treatment options available for individuals with myopathies, or those who have suffered severe muscle trauma, or the loss of muscle mass with aging (known as sarcopenia).
  • myopathies The physiology of myopathies is well understood and founded on a common pathogenesis of relentless cycles of muscle degeneration and regeneration, typically leading to functional exhaustion of muscle stem (satellite) cells and their progenitor cells that fail to reactivate, and at times their loss as well (Carlson & Conboy, “Loss of Stem Cell Regenerative Capacity Within Aged Niches,” Aging Cell 6(3):371-82 (2007); Shefer et al., “Satellite-cell Pool Size Does Matter: Defining the Myogenic Potency of Aging Skeletal Muscle,” Dev. Biol.
  • Muscle regeneration is initiated by skeletal muscle stem (satellite) cells that reside between striated muscle fibers (myofibers), which are the contractile cellular bundles, and the basal lamina that surrounds them (Carlson & Conboy, “Loss of Stem Cell Regenerative Capacity within Aged Niches,” Aging Cell 6(3): 371 -382 (2007) and Schiaffino & Reggiani, “Fiber Types in Mammalian Skeletal Muscles,” Physiol. Rev. 91(4): 1447-1531 (2011)).
  • striated muscle fibers myofibers
  • Satellite cells reconstitute the stem cell population while most others differentiate and fuse to form new myofibers (Hindi et al., “Signaling Mechanisms in Mammalian Myoblast Fusion,” Sci. Signal. 6(2Hy.XQ2 (2013)). Studies have demonstrated the singular importance of the satellite cell/myoblast population in muscle regeneration (Shefer et al., “Satellite-cell Pool Size Does Matter: Defining the Myogenic Potency of Aging Skeletal Muscle,” Dev. Biol.
  • Myofibers are divided into two types that display different contractile and metabolic properties: slow-twitch (Type I) and fast-twitch (Type II).
  • Slow- and fast-twitch myofibers are defined according to their contraction speed, metabolism, and 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 myofibers are rich in mitochondria, preferentially utilize oxidative metabolism, and provide resistance to fatigue at the expense of speed of contraction. Fast-twitch myofibers more readily atrophy in response to nutrient deprivation, traumatic damage, advanced age-related loss (sarcopenia), and cancer- mediated cachexia, whereas slow-twitch myofibers 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 Diseases,” Curr. Opin. Pharmacol.
  • Peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGCla or Ppargcl) is a major physiological regulator of mitochondrial biogenesis and Type I myofiber specification (Lin et al., “Transcriptional Co- Activator PGC-1 Alpha Drives the Formation of Slow-Twitch Muscle Fibres,” Nature 418 (6899):797-801 (2002)).
  • NRFs nuclear respiratory factors
  • Tfam mitochondria transcription factor A
  • Mef2 proteins Lai et al., “Effect of Chronic Contractile Activity on mRNA Stability in Skeletal Muscle,” Am. J. Physiol. Cell. Physiol.
  • Skeletal muscle can remodel between slow- and fast-twitch myofibers in response to physiological stimuli, load bearing, atrophy, disease, and injury (Bassel-Duby & Olson, “Signaling Pathways in Skeletal Muscle Remodeling,” Annu. Rev. Biochem. 75: 19-37 (2006)), involving transcriptional, metabolic, and post-transcriptional control 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 (2016)).
  • the myogenesis program is controlled by genes that encode myogenic regulatory factors (MRFs) (Mok & Sweetman, “Many Routes to the Same Destination: Lessons From Skeletal Muscle Development,” Reproduction 141 (3) :301 - 12 (2011)), which orchestrate differentiation of the activated satellite cell to become myoblasts, arrest their proliferation, cause them to differentiate, and fuse with multi-nucleated myofibers (Mok & Sweetman, “Many Routes to the Same Destination: Lessons From Skeletal Muscle Development,” Reproduction 141 (3): 301 -12 (2011)).
  • MRFs myogenic regulatory factors
  • PAX7 is a transcription factor expressed by quiescent and early activated satellite cells (Brack, A.S., “Pax7 is Back,” Skelet. Muscle 4(1):24 (2014) and Gunther, S., et al., “Myf5- positive Satellite Cells Contribute to Pax7-dependent Long-term Maintenance of Adult Muscle Stem Cells,” Cell Stem Cell 13(5):590-601 (2013)).
  • LGMD which include, e.g., loss of muscle fiber strength, loss of fundamental muscle molecular functions, and attenuation of the exacerbating destructive effects of the pathological immune response on muscle health and integrity.
  • One aspect of the present disclosure is directed to a method of treating limb girdle muscular dystrophy (LGMD) in a subject in need thereof.
  • This method involves administering to the subject an adeno-associated virus (AAV) or recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein, or functional fragment thereof, operatively coupled to a muscle cell-specific promoter and flanked by inverted terminal repeat (ITR) sequences.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • ITR inverted terminal repeat
  • the subject comprises a functional AUF1 protein isoform.
  • Another aspect of the present disclosure is directed to a method of treating limb girdle muscular dystrophy (LGMD) associated mitochondrial dysfunction in a subject in need thereof.
  • This method involves administering to the subject an adeno-associated virus (AAV) or recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein, or functional fragment thereof, operatively coupled to a muscle cell-specific promoter and flanked by inverted terminal repeat (ITR) sequences.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • ITR inverted terminal repeat
  • the subject comprises a functional AUF1 protein isoform.
  • AAV adeno-associated virus
  • AUF1 expression in muscle cells restores or increases muscle mass, function and performance, and reduces or reverses muscle atrophy.
  • AUF1 expression in muscle cells increases expression of components of the dystrophin glycoprotein complex (DGC), also referred to herein as the dystrophin associated protein complex or DAPC, and increases participation of components in the DGC, which can stabilize the sarcolemma.
  • DGC dystrophin glycoprotein complex
  • the DGC consists of the sarcoglycan subcomplex of a, P, y and 5 sarcoglycan and sarcospan (SSPN).
  • AUF1 has further shown activity in reducing muscle degeneration and improving muscle fiber size and endurance in 3-sarcoglycan null mice, supporting activity in treatment of LGMD, including sarcoglycanopathies, calpainopathies, dysferlinopathies and dystrophinopathies. [0022] AUF1 also promotes the expression of genes that lie downstream of the CAPN3 gene in calpainopathies, correcting the physiological and phenotypic dysfunction of CAPN3 deficient mice, including increasing mitochondrial biogenesis, normal muscle ultrastructural architecture, and CAMKIip kinase activity, a central point of deficiency in this disease.
  • AUF1 monotherapies and AUF1 combination therapies for treatment and amelioration of symptoms of LGMD comprising AUF1 therapeutics, including AUF1 gene therapy constructs, optionally in combination with a second therapeutic, including rAAV gene therapy vectors expressing a therapeutic protein such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y-sarcoglycan, 5-sarcoglycan, calpain 3, calcium/calmodulin-dependent protein kinase II P isoform proteins, other protein (other than AUF1), or portions thereof, and/or optionally other therapies for LGMD.
  • AAV or rAAV gene therapy vectors for delivery of AUF1 and methods of treatment including for LGMD, diseases associated with muscle wasting and muscle injury, using those gene therapy vectors.
  • Schneider 2016 further pertains to treating muscle cells with mono-allelic gene therapy, where a wild type AUF1 gene replaces a defective AUF1 gene. All examples provided were the replacement of a defective endogenous AUF1 gene with a normal AUF1 gene.
  • the present disclosure relates to methods of treating muscular dystrophies such as LGMDs, which express endogenous AUF1 by supplementing with additional exogenous AUF1 in muscle cells. It is totally unexpected that muscle cells which are not defective in expression of the endogenous AUF1 gene but are defective in the function of other genes that promote muscular dystrophy disease pathology, and display dysfunction in mitochondria, including in their biogenesis, would be effectively treated by supplementing with additional AUF1.
  • AUF1 supplemental gene therapy to effectively treat muscular dystrophies and mitochondrial dysfunction that are not known to involve defective AUF1 gene expression or function, is entirely novel and unexpected and were not known or suspected in the prior art, including Schneider 2016.
  • the examples of the present disclosure surprisingly demonstrate that supplementation of AUF1 is effective to bypass the constellation of defective genes in LGMDs, which do not include AUF1.
  • WO 2021/146711 relates to an adeno-associated viral (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, where the nucleic acid molecule is heterologous to and operatively coupled to the muscle cellspecific promoter.
  • AAV adeno-associated viral
  • Schneider 2021 also discloses compositions comprising the AAV vector, as well as methods of promoting muscle regeneration in injured muscle, a method of treating degenerative skeletal muscle loss in a subject, methods of preventing traumatic muscle injury in a subject such as Duchenne Muscular Dystrophy, methods of treating traumatic muscle injury in a subject, and methods of treating muscle loss due to aging in a subject.
  • the present disclosure relates to the treatment of limb girdle muscular dystrophies by bypassing the defective genes in the LGMDs and providing increased 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 regeneration of muscle following traumatic injury nor prevention of injury.
  • WO 2023/004331 provides methods of treating or ameliorating the symptoms of dystrophinopathies, such as Duchenne muscular dystrophy and Becker muscular dystrophy by administration of therapeutically effective doses of recombinant adeno-associated viruses (rAAV) containing a transgene encoding AUF1 and a second rAAV encoding a microdystrophin or other therapeutic effective to treat the dystrophinopathy by providing a dystophin gene surrogate to replace the defective dystophin gene and AUF1 to increase muscle strength and stamina.
  • rAAV recombinant adeno-associated viruses
  • the present disclosure relates to the treatment of limb girdle muscular dystrophies by bypassing the defective genes in the LGMDs and surprisingly demonstrate that supplementation of AUF1 is effective in treating LGMDs, which do not include AUF1 mutations.
  • the present disclosure does not relate to compositions or methods for replacing a defective dystropin gene, which is not involved in LGMDs.
  • LGMD limb girdle muscular dystrophy
  • a gene therapy vector particularly, AAV or rAAV vectors
  • the gene therapy vector comprises a genome with a transgene encoding an AUF1 protein operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount.
  • AUF1 protein or nucleic acid encoding AUF1 is administered in combination with another therapeutic for use in treating an LGMD.
  • the other therapeutic may be a gene therapy therapeutic (including a therapeutic co-expressed with AUF1 or as a separate gene therapy vector) or non-gene therapy therapeutic.
  • the methods of treatment provided herein include treatment of human subjects having Type 1 LGMDs, including limb-girdle muscular dystrophy type 1C (LGMD1C) and limb-girdle muscular dystrophy type 1G (LGMD1G).
  • the methods of treatment also provide for the treatment of Type 2 LGMDs, including sarcoglycanopathies, such as limb-girdle muscular dystrophy type 2C (LGMD2C), limb-girdle muscular dystrophy type 2D (LGMD2D), limbgirdle muscular dystrophy type 2E (LGMD2E), and limb-girdle muscular dystrophy type 2F (LGMD2F); dystrophinopathies such as limb-girdle muscular dystrophy type 21 (LGMD2I), limb-girdle muscular dystrophy type 2K (LGMD2K), limb-girdle muscular dystrophy type 2M (LGMD2M), limb-girdle muscular dystrophy type 2N (LGMD2N), limb-girdle
  • the AUF1 is a human AUF1 p37 AUF1 , p40 AUF1 , p42 AUF1 , or p45 AUF1 isoform, including, for example, the p40 AUF1 isoform, and may be encoded by a codon optimized, CpG deleted nucleotide sequence, for example, the nucleotide sequence of SEQ ID NO: 17.
  • the muscle cell-specific promoter is a muscle creatine kinase (MCK) promoter, a synlOO 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 (including modified Spc5-12 promoters SpcVl (SEQ ID NO: 127) or SpcV2 (SEQ ID NO: 128), a creatine kinase (CK) 8e promoter, a U6 promoter, a Hl promoter, a desmin promoter, a Pitx3 promoter, a skeletal alpha-actin promoter, a MHCK7 promoter, or a Sp-301 promoter (see also Table 8).
  • MCK muscle creatine kinase
  • the provided methods administer an AAV or rAAV particle, which comprises a recombinant genome having the nucleotide sequence of SEQ ID NO: 31 (spc-hu-opti-AUFl-CpG(-)), SEQ ID NO: 32 (tMCK-huAUFl), SEQ ID NO: 33 (spc5-12- hu-opti-AUFl-WPRE), SEQ ID NO: 34 (ss-CK7-hu-AUFl), SEQ ID NO: 35 (spc-hu-AUFl -nointron), or SEQ ID NO: 36 (D(+)-CK7AUFl).
  • the rAAV particle is, in embodiments, an AAV8 or AAV9 serotype and 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 (AAVhu.32 capsid).
  • the first therapeutic is administered systemically, including intravenously at a dose of 1E8 vector genomes per kg (vg/kg) to 2E15 vg/kg, lE13 to 1E14 vg/kg, or 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 XI 0 x ).
  • a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 17 encoding human AUF1 p40, which is a codon optimized, reduced CpG sequence.
  • vectors comprising this sequence (SEQ ID NO: 17) operably linked to a muscle cell-specific promoter, which may a muscle creatine kinase (MCK) promoter, a Syn promoter, a synlOO 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 (including variant Spc5-12 promoters Spc5vl (SEQ ID NO: 127) and Spc5v2 (SEQ ID NO: 128), a creatine kinase (CK) 8e promoter, a U6 promoter, a Hl promoter, a desmin promoter, a Pitx3 promoter, a skeletal alpha-actin promoter, a MHCK7 promoter, or a Sp-301 promoter (
  • the nucleotide sequence of SEQ ID NO: 17, in addition to being operably linked to the 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 (as disclosed herein).
  • the vector may be a cis plasmid for packaging rAAV or an rAAV genome, which is flanked by ITR sequences.
  • the genome in the rAAV particle may be single stranded or may be self complementary.
  • the rAAV vector sequence may also comprise 5’ and/or 3’ stuffer sequences (see Table 10) and/or a SV40 polyadenylation signal sequence.
  • methods of treating LGMD and pharmaceutical compositions for use in treating LGMD administer AAVs or rAAVs generated from a vector, which comprise a nucleotide sequence of SEQ ID NO: 17, encoding human AUF1 p40, operably linked to regulatory sequence that promotes expression in muscle, including muscle specific promoters (or constitutive promoters) as disclosed herein (see, for example, Table 8, and may have, in embodiments, a nucleotide sequence of SEQ ID NO: 31 (spc-hu-opti-AUFl-CpG(-)), SEQ ID NO: 32 (tMCK-huAUFl), SEQ ID NO: 33 (spc5-12-hu-opti-AUFl-WPRE), SEQ ID NO: 34 (ss-CK7-hu-AUFl), SEQ ID NO: 35 (spc-hu-AUFl -no-intron), or SEQ ID NO: 36 (D(+)-CK7AU
  • the rAAV particle is, in embodiments an AAV8, AAV9 or AAVhu.32 serotype, or capsid in 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).
  • one aspect of the present disclosure relates to a method of treating limb girdle muscular dystrophy (LGMD) in a subject in need thereof.
  • This method involves administering to the subject an AAV or rAAV particle comprising a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein, or functional fragment thereof, operatively coupled to a muscle cell-specific promoter and flanked by inverted terminal repeat (ITR) sequences.
  • the subject expresses a functional AUF1.
  • the subject has a LGMD which is not associated with a mutation in the gene encoding AUF1.
  • FIG. 1 illustrates vector gene expression cassettes and AUF1 constructs for use in a cis plasmid for production of AAV gene therapy vectors. DNA length for each construct is provided.
  • Hu-AUFl-CpG(-) CpG depleted human AUF1 p40 coding sequence; Stuffer: noncoding 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.
  • FIGS. 2A-2B are images (FIG. 2A) and graphs (FIG. 2B) demonstrating that AAV8 hAUFl gene therapy restores muscle fiber integrity, size, and maturity in CAPN3 deficient disease mice.
  • Animals were administered AAV8-hAUFl (codon optimized humanized AUF1) by intravenous (i.v.) injection in the retro-orbital sinus at either 6el3 or lel4 vg/kg (6xl0 13 or IxlO 14 viral genomes/kg mouse weight) or, when indicated, vector control.
  • FIG. 2A demonstrates that, in the mouse model, AAV8-hAUFl gene therapy at 3 months restores near normal muscle fiber morphology, with larger mature myofibers evident with greater cross sectional area (csa) containing >2 nuclei (FIG. 2B), a marker of mature muscle fibers. Shown for the psoas muscle, one of the most affected limb muscles in CAPN3 deficiency. While pathology is not as severe in diaphragm as in limb skeletal muscles in CAPN3 deficiency, there is pathology which is also corrected by AAV8-hAUFl gene therapy.
  • FIGS. 3A-3D demonstrate that AAV8-hAUFl gene therapy in CAPN3 deficient mice recovers a strong and significant increase in muscle function, stamina, and strength tests at both the low and high dose at 2 months of gene therapy.
  • Graphs show time to exhaustion (FIG. 3A), distance to exhaustion (FIG. 3B), maximum speed (FIG. 3C), and muscle grip strength (FIG. 3D) of CAPN3 deficient disease mice (KO) mice administered AAV8-hAUFl at 6el3 (6xl0 13 viral genomes/kg mouse weight) and mice administered AAV8-hAUFl at lel4 vg/kg (IxlO 14 viral genomes/kg mouse weight). Graphs show combined data for male and female mice).
  • FIG. 4 demonstrates that AAV8-hAUFl gene therapy restores a high level of CAMKII0 kinase expression and phosphorylation, as well increased mitochondria content, shown by increased levels of mitochondrial DNA.
  • One of the main goals of therapy for CAPN3 deficiency is to restore CAMKII0 kinase activity, which can be determined by specific activating phosphorylation and the downstream increase in deficient mitochondrial biogenesis. Data is shown for two mice in the gastrocnemius muscle at 2 months of gene therapy.
  • FIG. 5 demonstrates that hAUFl supplementation increases CAMKIIb protein expression, activating phosphorylation, SERCA2A and PGCla proteins in CAPN3 KO mice.
  • Another one of the main goals of therapy for CAPN3 deficiency is to restore SERCA2 pump levels and activity, and PGCla levels that promote mitochondrial biogenesis and slow twitch Type I stamina muscle fibers.
  • AUF1 gene therapy increases both in males and females. Data shown for two mice in the gastrocnemius muscle at 2 months of gene therapy.
  • FIG. 6 demonstrates that hAUFl increases subsarcolemmal mitochondria activity in the TA muscle.
  • AUF1 gene therapy was found to strongly restore mitochondria and to do so at the proper location in the sub-sarcolemma, as shown in the TA muscle at 2 months of therapy. Shown in these transmission electron micrographs in dark stain are abnormal mitochondria in the untreated CAPN3 KO tibialis anterior (TA) muscle, and restored mitochondria within the sarcolemma (muscle cell membrane) that are darker, with ample well-defined cisternae.
  • FIG. 7 demonstrates that AUF1 gene therapy increases intermyofibrillar mitochondira in the TA muscle and improves myofibrillar alignment.
  • a hallmark of CAPN3 deficiency is the disruption of intermyofibrillar organization and structure within muscle fibers, which prevents normal muscle contraction, as well as generation of exhausted and dying mitochondria.
  • AAV8-hAUFl gene therapy at two months restores near-normal intermyofibrillar organization and mitochondria to CAPN3 deficient muscle. Identical data were found in the TA and other muscles.
  • FIG. 8 demonstrates that succinate dehydrogenase (SDH) is increased in TA muscle of CAPN3 KO mice after 2 months of AAV8-hAUFl treatment.
  • Loss of succinate dehydrogenase (SDH) activity a hallmark of mitochondrial activity deficiency, occurs in CAPN3 LGMD.
  • AAV8-hAUFl gene therapy to CAPN3 deficient TA muscle restores a high level of SDH activity deep into muscle (Zone 1). Quantification of deep muscle (Zone 1) and superficial muscle (Zone 3) shows increased SDH activity with hAUFl gene therapy.
  • FIG. 9 demonstrates that AAV8 hAUFl gene transfer reduces gastrocnemius and diaphragm muscle degeneration in LGMD 5-sarcoglycan KO mice.
  • the diaphragm muscle is one of the most affected muscles in SCGD deficiency.
  • Animals were administered AAV8- hAUFl (codon optimized humanized AUF1) by intravenous (i.v.) injection in the retro-orbital sinus at 6el3 viral genomes/kg mouse weight.
  • AAV8-hAUFl gene therapy at 2 months restores significant diaphragm muscle integrity, repairs the profound degeneration of SCGD muscle which is apparent in the H&E stains as a dark blue stained area.
  • FIG. 10 demonstrates that AAV8 hAUFl gene transfer reduces gastrocnemius muscle degeneration in LGMD 5-sarcoglycan KO mice.
  • the gastrocnemius muscle is an affected muscle in SCGD deficiency.
  • Animals were administered by intravenous (i.v.) injection in the retro-orbital sinus AAV8-hAUFl (codon optimized humanized AUF1, described in the provisional application) at 6el3 viral genomes/kg mouse weight.
  • AAV8- hAUFl gene therapy at 2 months restores significant muscle integrity, repairs the profound degeneration of SCGD muscle which is apparent in the H&E stains as a dark blue stained area.
  • hAUF gene transfer reduces diaphragm eMHC gene expression and improves muscle fiber integrity and size.
  • Expression of embryonic myosin heavy chain (eMHC) is a hallmark of muscle renewal.
  • SCGD muscle undergoes chronic and unsuccessful renewal with continuous expression of eMHC.
  • AAV8 hAUFl gene therapy at 2 months restores more normal muscle, reduced expression of eMHC, larger more mature muscle fibers shown by fibers with >2 nuclei, greater cross sectional area (csa).
  • FIG. 12 demonstrates the effect of hAUFl gene transfer on diaphragm muscle degeneration and myofiber type expression 2 months post-gene therapy in 5-sarcoglycan KO mice.
  • AAV8-hAUFl gene therapy in SCGD deficient mice at two months increases all forms of muscle fibers, Type I slow twitch, and multiple forms of Type II fast twitch.
  • FIG. 13 demonstrates that AAV8 hAUFl gene transfer increases muscle strength in 5-sarcoglycan KO mice.
  • Animal muscle function was tested for muscle grip strength, the ability of the mouse to grasp a grid with its front limbs while tugging on the tail.
  • Data show statistically increased gains by AAV8 hAUFl gene therapy at 2 months in SCGD mice which approaches wild type for female mice.
  • LGMD limb girdle muscular dystrophy
  • AVV limb girdle muscular dystrophy
  • rAAV vectors in which the gene therapy vector comprises a genome with a transgene encoding an AUF1 protein operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount.
  • AUF1 protein or nucleic acid encoding AUF1 is administered in combination with another therapeutic for use in treating an LGMD.
  • the other therapeutic may be a gene therapy therapeutic (including a therapeutic co-expressed with AUF1 or as a separate gene therapy vector) or not a gene therapy therapeutic.
  • one aspect of the present disclosure is directed to a method of treating limb girdle muscular dystrophy (LGMD) in a subject in need thereof.
  • This method involves administering to the subject an adeno-associated virus (AAV) or recombinant adeno- associated virus (rAAV) particle comprising a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein, or functional fragment thereof, operatively coupled to a muscle cell-specific promoter and flanked by inverted terminal repeat (ITR) sequences.
  • AAV adeno-associated virus
  • rAAV recombinant adeno- associated virus
  • the subject comprises a functional AUF1 protein isoform.
  • Another aspect of the present disclosure is directed to a method of treating limb girdle muscular dystrophy (LGMD) associated mitochondrial dysfunction in a subject in need thereof.
  • This method involves administering to the subject an adeno-associated virus (AAV) or recombinant adeno-associated virus (rAAV) particle comprising a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein, or functional fragment thereof, operatively coupled to a muscle cell-specific promoter and flanked by inverted terminal repeat (ITR) sequences.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • ITR inverted terminal repeat
  • the subject comprises a functional AUF1 protein isoform.
  • the methods of treatment provided herein include treatment of human subjects having Type 1 limb-girdle muscular dystrophies (LGMDs), including limb-girdle muscular dystrophy type 1C (LGMD1C) and limb-girdle muscular dystrophy type 1G (LGMD1G).
  • LGMDs Type 1 limb-girdle muscular dystrophies
  • LGMD1C limb-girdle muscular dystrophy type 1C
  • LGMD1G limb-girdle muscular dystrophy type 1G
  • the methods of treatment also provide for the treatment of Type 2 limb-girdle muscular dystrophies (LGMDs), including 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 21 (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 20 (LGMD20), limb-girdle muscular dystrophy type 2P (LGMD2P), limb-girdle muscular dystrophy type 2T (LGMD2T),
  • AUF1 gene therapy is administered alone in a gene therapy vector comprising a genome with a transgene encoding the AUF1 gene effective to treat the LGMD operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount.
  • AUF1 gene therapy is administered in combination with a gene therapy vector comprising a genome with a transgene encoding a therapeutic protein (such as microdystrophin, a-sarcoglycan, P-sarcoglycan, y-sarcoglycan, 5-sarcoglycan, calpain 3, calcium/calmodulin-dependent protein kinase II P isoform proteins, other protein (other than AUF1), or portions thereof) effective to treat the LGMD operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount.
  • a therapeutic protein such as microdystrophin, a-sarcoglycan, P-sarcoglycan, y-sarcoglycan, 5-sarcoglycan, calpain 3, calcium/calmodulin-dependent protein kinase II P isoform proteins, other protein (other than AUF1), or portions thereof
  • the AUF1 gene therapy is administered in combination with a small molecule drug.
  • the small molecule drug is AMPBP (CID 11210285 hydrochloride; 2-Amino-4-(3,4- Activator of Wnt signaling
  • AMBP activates Wnt signaling without inhibiting GSK-3P
  • Lanzaoprazole is a gastric proton pump inhibitor
  • Parbendazole has a broad-spectrum anthelmintic activity
  • Rabeprazole sodium salt is a gastric H + /K + ATPase pump inhibitor.
  • AUF1 gene therapy is administered in combination with a gene therapy vector comprising a genome with a transgene encoding an a-sarcoglycan effective to treat LGMD2C operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount.
  • AUF1 gene therapy is administered in combination with a gene therapy vector comprising a genome with a transgene encoding a P-sarcoglycan effective to treat LGMD2D operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount.
  • AUF1 gene therapy is administered in combination with a gene therapy vector comprising a genome with a transgene encoding a y-sarcoglycan effective to treat LGMD2E operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount.
  • AUF1 gene therapy is administered in combination with a gene therapy vector comprising a genome with a transgene encoding a 5- sarcoglycan effective to treat LGMD2F operably linked to a regulatory element that promotes expression in muscle cells in a therapeutically effective amount.
  • AUF1 protein or nucleic acid encoding AUF1 is administered in combination with another therapeutic for use in treating a LGMD.
  • AUF1 AAV gene therapy constructs have a codon optimized, CpG depleted coding sequence for human p40 AUF1 (SEQ ID NO: 17) operably linked to a regulatory element that promotes expression in muscle cells (see, e.g., Table 10) and optionally other regulatory elements such as polyadenylation sequences, intron sequences, WPRE or other element, and/or stuffer sequences, including, for example, as disclosed herein (see Table 2 for nucleotide sequences of construct components). Exemplary constructs are depicted, for example, in FIG. 1 (see also Table 3).
  • the constructs, including flanking ITR sequences, may have nucleotide sequences of SEQ ID NOs: 31 to 36.
  • the gene therapy vectors may be, e.g., AAV8 serotype vectors, AAV9 serotype vectors, AAVhu.32 serotype vectors (see, for example, capsids in Table 11) or other appropriate AAV serotype capsids, for example, that promote delivery to or have a tropism for skeletal muscle cells. Accordingly, provided are compositions comprising, and methods of administering, the AUF1 AAV gene therapy vectors described herein (for example, as depicted in FIG.
  • LGMD for treating LGMD, and the subtypes of LGMD, including LGMD calpainopathies, dystrophinopathies, dysferlinopathies and sarcoglycanopathies, including by restoring or increasing muscle mass, muscle function or performance, and/or reducing or reversing muscle atrophy.
  • Such methods include stabilizing the sarcolemma of the muscle cell by reducing leakiness (for example, as measured by creatine kinase levels), increasing expression of [3-sarcoglycan or utrophin and/or its presence in the dystrophin-gly coprotein complex of muscle cells, increasing levels of PGCla and MEF gene expression, type I slow oxidative muscle fibers, and CAMKIip kinase activity, among others, by providing AUF1 protein.
  • Such methods also include promoting an increase in muscle cell mass, number of muscle fibers, size of muscle fibers, reduction in or reverse of muscle cell atrophy, satellite cell activation and differentiation, improvement in muscle cell function (for example, by increasing mitochondrial oxidative capacity), mitochondrial biogenesis, and increasing proportion of slow twitch fiber in muscle (including by conversion of fast to slow twitch muscle fibers).
  • compositions formulated for peripheral including, intravenous, administration of the AUF1 -encoding rAAV described herein.
  • vector is used interchangeably with “expression vector.”
  • the term “vector” may refer to viral or non-viral, prokaryotic or eukaryotic, DNA or RNA sequences that are capable of being transfected into a cell, referred to as “host cell,” so that all or a part of the sequences are transcribed. It is not necessary for the transcript to be expressed. It is also not necessary for a vector to comprise a transgene having a coding sequence. Vectors are frequently assembled as composites of elements derived from different viral, bacterial, or mammalian genes.
  • Vectors contain various coding and non-coding sequences, such as sequences coding for selectable markers, sequences that facilitate their propagation in bacteria, or one or more transcription units that are expressed only in certain cell types.
  • mammalian expression vectors often contain both prokaryotic sequences that facilitate the propagation of the vector in bacteria and one or more eukaryotic transcription units that are expressed only in eukaryotic cells. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • promoter is used interchangeably with “promoter element” and “promoter sequence.”
  • enhancer is used interchangeably with “enhancer element” and “enhancer sequence.”
  • promoter refers to a minimal sequence of a transgene that is sufficient to initiate transcription of a coding sequence of the transgene. Promoters may be constitutive or inducible.
  • a constitutive promoter is considered to be a strong promoter if it drives expression of a transgene at a level comparable to that of the cytomegalovirus promoter (CMV) (Boshart et al., “A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus,” Cell 41 :521 (1985), which is hereby incorporated by reference in its entirety).
  • Promoters may be synthetic, modified, or hybrid promoters. Promoters may be coupled with other regulatory sequences/elements which, when bound to appropriate intracellular regulatory factors, enhance (“enhancers”) or repress (“repressors”) promoter-dependent transcription.
  • a promoter, enhancer, or repressor is said to be “operably linked” to a transgene when such element(s) control(s) or affect(s) transgene transcription rate or efficiency.
  • a promoter sequence located proximally to the 5' end of a transgene coding sequence is usually operably linked with the transgene.
  • regulatory elements is used interchangeably with “regulatory sequences” and refers to promoters, enhancers, and other expression control elements, or any combination of such elements.
  • Promoters are positioned 5' (upstream) to the genes that they control.
  • Many eukaryotic promoters contain two types of recognition sequences: TATA box and the upstream promoter elements.
  • TATA box located 25-30 bp upstream of the transcription initiation site, is thought to be involved in directing RNA polymerase II to begin RNA synthesis at the correct site.
  • the upstream promoter elements determine the rate at which transcription is initiated. These elements can act regardless of their orientation, but they must be located within 100 to 200 bp upstream of the TATA box.
  • Enhancer elements can stimulate transcription up to 1000-fold from linked homologous or heterologous promoters. Enhancer elements often remain active even if 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), which is hereby incorporated by reference in its entirety). Furthermore, unlike promoter elements, enhancers can be active when placed downstream from the transcription initiation site, e.g., within an intron, or even at a considerable distance from the promoter (Yutzey et al., “An Internal Regulatory Element Controls Troponin I Gene Expression,” Mol. Cell. Bio. 9(4): 1397-1405 (1989), which is hereby incorporated by reference in its entirety).
  • muscle cell-specific refers to the capability of regulatory elements, such as promoters and enhancers, to drive expression of an operatively 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.
  • AAV or “adeno-associated virus” refers to a Dependoparvovirus within the Parvoviridae genus of viruses.
  • the AAV can be an AAV derived from a naturally occurring “wild-type” virus, an AAV derived from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a naturally occurring cap gene and/or from a rAAV genome packaged into a capsid comprising capsid proteins encoded by a non-naturally occurring capsid cap gene.
  • An example of the latter includes a rAAV having a capsid protein having a modified sequence and/or a peptide insertion into the amino acid sequence of the naturally- occurring capsid.
  • rAAV refers to a “recombinant AAV.”
  • a recombinant AAV has an AAV genome in which part or all of the rep and cap genes have been replaced with heterologous sequences.
  • rep-cap helper plasmid refers to a plasmid that provides the viral rep and cap gene function and aids the production of AAVs from rAAV genomes lacking functional rep and/or the cap gene sequences.
  • capsid protein refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid coat of the virus.
  • the capsid protein may be VP1, VP2, or VP3.
  • replica gene refers to the nucleic acid sequences that encode the non- structural protein needed for replication and production of virus.
  • nucleic acids and “nucleotide sequences” 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 using, for example, nucleotide analogs, which include, but are not limited to, inosine or tritylated bases. Such analogs can also comprise DNA or RNA molecules comprising modified backbones that lend beneficial attributes to the molecules such as, for example, nuclease resistance or an increased ability to cross cellular membranes.
  • the nucleic acids or nucleotide sequences can be single-stranded, double-stranded, may contain both singlestranded and double-stranded portions, and may contain triple-stranded portions, but preferably is double-stranded DNA.
  • Amino acid residues as disclosed herein can be modified by conservative substitutions to maintain, or substantially maintain, overall polypeptide structure and/or function.
  • “conservative amino acid substitution” indicates that: hydrophobic amino acids (i.e., Ala, Cys, Gly, Pro, Met, Vai, 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 (z.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 Gin) can be substituted with other amino acids with polar uncharged side chains.
  • subject may be a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) or a primate (e.g., monkey and human), and includes a human.
  • a non-primate e.g., cows, pigs, horses, cats, dogs, rats etc.
  • a primate e.g., monkey and human
  • therapeutic agent refers to any agent which can be used in treating, managing, or ameliorating symptoms associated with a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene.
  • a “therapeutically effective amount” refers to the amount of agent, (e.g., an amount of product expressed by the transgene) that provides at least one therapeutic benefit in the treatment or management of the target disease or disorder, when administered to a subject suffering therefrom.
  • a therapeutically effective amount with respect to an agent of the disclosure means that amount of agent alone, or when in combination with other therapies, that provides at least one therapeutic benefit in the treatment or management of the disease or disorder.
  • prophylactic agent refers to any agent which can be used in the prevention, reducing the likelihood of, delay, or slowing down of the progression of a disease or disorder, where the disease or disorder is associated with a function to be provided by a transgene.
  • a “prophylactically effective amount” refers to the amount of the prophylactic agent (e.g., an amount of product expressed by the transgene) that provides at least one prophylactic benefit in the prevention or delay of the target disease or disorder, when administered to a subject predisposed thereto.
  • a prophylactically effective amount also may refer to the amount of agent sufficient to prevent, reduce the likelihood of, or delay the occurrence of the target disease or disorder; or slow the progression of the target disease or disorder; the amount sufficient to delay or minimize the onset of the target disease or disorder; or the amount sufficient to prevent or delay the recurrence or spread thereof.
  • a prophylactically effective amount also may refer to the amount of agent sufficient to prevent or delay the exacerbation of symptoms of a target disease or disorder.
  • a prophylactically effective amount with respect to a prophylactic agent of the disclosure means that amount of prophylactic agent alone, or when in combination with other agents, that provides at least one prophylactic benefit in the prevention or delay of the disease or disorder.
  • a prophylactic agent of the disclosure can be administered to a subject “predisposed” to a target disease or disorder.
  • a subject that is “pre-disposed” to a disease or disorder is one that shows symptoms associated with the development of the disease or disorder, or that has a genetic makeup, environmental exposure, or other risk factor for such a disease or disorder, but where the symptoms are not yet at the level to be diagnosed as the disease or disorder.
  • a patient with a family history of a disease associated with a missing gene may qualify as one predisposed thereto.
  • a patient with a dormant tumor that persists after removal of a primary tumor may qualify as one predisposed to recurrence of a tumor.
  • pharmaceutically acceptable carrier refers to a carrier that does not cause an allergic reaction or other untoward effect in patients to whom it is administered and are compatible with the other ingredients in 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 practices.
  • solid carriers/diluents include, but are not limited to, a gum, a starch (e.g., corn starch, pregelatinized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g., microcrystalline cellulose), an acrylate (e.g., polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • Pharmaceutically acceptable carriers may further comprise 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 molecule described herein.
  • CpG islands means those distinctive regions of the genome that contain the dinucleotide CpG (e.g., C (cytosine) base followed immediately by a G (guanine) base (a CpG)) at high frequency, thus the G+C content of CpG islands is significantly higher than that of non-island DNA.
  • CpG islands can be identified by analysis of nucleotide length, nucleotide composition, and frequency of CpG dinucleotides.
  • CpG island content in any particular nucleotide sequence or genome may be measured using the following criteria: island size greater than 100, GC Percent greater than 50.0 %, and ratio greater than 0.6 of observed number of CG dinucleotides to the expected number on the basis of the number of Gs and Cs in the segment (Obs/Exp greater than 0.6).
  • nucleic acids including transgenes, encoding AUFls, including the p37, p40, p42, and p45 isoforms of human and mouse AUF1, or therapeutically functional fragments thereof, and vectors and viral particles, including rAAVs, containing same and methods of using same in methods of treatment, prevention, or amelioration of symptoms of conditions associated with loss of muscle mass or performance or where an increase in muscle mass or performance is desired or useful.
  • the AUF1 gene therapy vectors are used in methods of treating or ameliorating the symptoms of LGMD by administering the AUF1 gene therapy vectors.
  • Genes involved in rapid response to cell stimuli are highly regulated and typically encode mRNAs that are selectively and rapidly degraded to quickly terminate protein expression and reprogram the cell (Moore et al., “Physiological Networks and Disease Functions of RNA- binding Protein AUF1,” Wiley Interdiscip. Rev. RNA 5(4):549-64 (2014), which is hereby incorporated by reference in its entirety).
  • RNA-binding Protein AUF1 growth factors
  • inflammatory cytokines Physiological Networks and Disease Functions of RNA-binding Protein AUF1
  • Zhang et al. “Purification, Characterization, and cDNA Cloning of an AU-rich Element RNA-binding Protein, AUF1,” Mol. Cell. Biol.
  • tissue stem cell fate-determining mRNAs (Chenette et al., “Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,” Cell Rep. 16(5): 1379-90 (2016), which is hereby incorporated by reference in its entirety) that have very short half-lives of 5-30 minutes.
  • Short-lived mRNAs typically contain an AU-rich element (ARE) in the 3' untranslated region (3' UTR) of the mRNA, having the repeated sequence AUUUA (Moore et al., “Physiological Networks and Disease Functions of RNA-binding Protein AUF1,” Wiley Interdiscip Rev. RNA 5(4):549-64 (2014), which is hereby incorporated by reference in its entirety), which confers rapid decay or in some cases stabilization.
  • ARE AU-rich element
  • the ARE serves as a binding site for regulatory proteins known as AU-rich binding proteins (AUBPs) that control the stability and in some cases the translation of the mRNA (Moore et al., “Physiological Networks and Disease Functions of RNA-binding Protein AUF1,” Wiley Interdiscip. Rev. RNA 5(4):549-64 (2014); Zhang et al., “Purification, Characterization, and cDNA Cloning of an AU-rich Element RNA-binding Protein, AUF1,” Mol. Cell. Biol.
  • AUBPs regulatory proteins known as AU-rich binding proteins
  • AU-rich mRNA binding factor 1 (AUF1; also known as Heterogeneous Nuclear Ribonucleoprotein DO, hnRNP DO; HNRNPD gene) binds with high affinity to repeated AU-rich elements located in the 3' UTR found in approximately 5% of mRNAs.
  • AUF1 typically targets ARE-mRNAs for rapid degradation, while not as well understood, it can oppositely stabilize and increase the translation of some ARE-mRNAs (Moore et al., “Physiological Networks and Disease Functions of RNA-Binding Protein AUF1,” Wiley Interdiscip. Rev. RNA 5(4):549-564 (2014), which is hereby incorporated by reference in its entirety).
  • mice with AUF1 deficiency undergo an accelerated loss of muscle mass due to an inability to carry out the myogenesis program (Chenette et al., “Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,” Cell Rep. 16(5): 1379-90 (2016), which is hereby incorporated by reference in its entirety).
  • AUF1 expression is severely reduced with age in skeletal muscle, and this significantly contributes to loss and atrophy of muscle, loss of muscle mass, and reduced 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), which are hereby incorporated by reference in their entireties). It was also found that AUF1 controls all major stages of skeletal muscle development, starting with satellite cell activation and lineage commitment, by selectively targeting for rapid degradation the major differentiation checkpoint mRNAs that block entry into each next step of muscle development.
  • AUF1 has four related protein isoforms identified by their molecular weight (p37 AUF1 , p40 AUF1 , p42 AUF1 , p45 AUF1 ) derived by differential splicing of a single pre-mRNA (Moore et al., “Physiological Networks and Disease Functions of RNA-Binding Protein AUF1,” Wiley Interdiscip. Rev. RNA 5(4):549-564 (2014); Chen & Shyu, “AU-Rich Elements: Characterization and Importance in mRNA Degradation,” Trends Biochem. Sci.
  • RRM The general organization of an RRM is a P-a-P-P-a-P RNA binding platform of anti-parallel P-sheets backed by the a-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), which are hereby incorporated by reference in their entirety).
  • AUF1 Mutations and/or polymorphisms in AUF1 are linked to human limb girdle muscular dystrophy (LGMD) type 1G (Chenette et al., “Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,” Cell Rep. 16(5): 1379-1390 (2016), which is hereby incroproated by reference in its entirety), suggesting a critical requirement for AUF1 in post-natal skeletal muscle maintenance.
  • LGMD human limb girdle muscular dystrophy
  • fragment refers to a contiguous stretch of amino acids of the given polypeptide’s sequence that is shorter than the given polypeptide’s full-length sequence.
  • a fragment of a polypeptide may be defined by its first position and its final position, in which the first and final positions each correspond to a position in the sequence of the given full-length polypeptide. The sequence position corresponding to the first position is situated N-terminal to the sequence position corresponding to the final position.
  • the sequence of the fragment or portion is the contiguous amino acid sequence or stretch of amino acids in the given polypeptide that begins at the sequence position corresponding to the first position and ends at the sequence position corresponding to the final position.
  • Functional or active fragments are fragments that retain functional characteristics, e.g., of the native sequence or other reference sequence. Typically, active fragments are fragments that retain substantially the same activity as the wildtype protein.
  • a fragment may, for example, contain a functionally important domain, such as a domain that is important for receptor or ligand binding.
  • Functional fragments are at least 10, 15, 20, 50, 75, 100, 150, 200, 250 or 300 contiguous amino acids of a full length AUF1 (including the p37, p40, p42 or p45 isoforms thereof) and retain one or more AUF1 functions.
  • functional fragments of AUF1 as described herein include at least one RNA recognition domain (RRM) domain.
  • functional fragments of AUF1 as described herein include two RRM domains.
  • AUF1 or functional fragments thereof as described herein may be derived from a mammalian AUF 1.
  • the AUF 1 or functional fragment thereof is a human AUF1 or functional fragment thereof.
  • the AUF1 or functional fragment thereof is a murine AUF1 or a functional fragment thereof.
  • the AUF1 protein according to 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 is found in Table 1 below, each of which is hereby incorporated by reference in its entirety.
  • Table 1 Summary of GenBank Accession Numbers of AUF1 Sequences [0085] The sequences referred to in Table 1 are reproduced below.
  • NM_001003810.1 (SEQ ID NO: 1) is as follows:
  • NP_001003810.1 (SEQ ID NO: 2) is as follows: MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAATQGAAAA AGSGAGTGGG TASGGTEGGS 60
  • NM_002138.3 (SEQ ID NO: 5) is as follows:
  • AAAGTACCCC TTTGCACAGA TAGTTAATGT TTTATGCTTC CATTAAATAA AAAGGACTTA 1980 AAATCTGTTA ATTATAATAG AAATGCGGCT AGTTCAGAGA GATTTTTAGA GCTGTGGTGG 2040 ACTTCATAGA TGAATTCAAG TGTTGAGGGA GGATTAAAGA AATATATACC GTGTTTATGT 2100 GTGTGTGCTT
  • NP-002129.2 (SEQ ID NO: 6) is as follows:
  • NM_03 1369.2 (SEQ ID NO: 9) is as follows:
  • NP-l 12737.1 (SEQ ID NO: 10) is as follows:
  • NM_03 1370.2 (SEQ ID NO: 13) is as follows:
  • NP-l 12738.1 (SEQ ID NO: 14) is as follows:
  • NM_00 1077267.2 (SEQ ID NO: 3) is as follows:
  • TTGTTTGGCA CAAGTCTTCC CAGGCCAGTG AGACCTCCGT GTCCTCTCAG CACCATAGGG 3240 GTAGGTAACC CTGGTTAGGC TGGACAGGGG TTTGCTGAGG GAGTTTGTTC ATTTGAATCT 3300
  • CTGGTTTCTA CAATTTAAAT TAAAAACAGA ATTCATCTTA GAATTTTTCA
  • CCCTCTTCCC 3540 CAACTATTCT AATCAATCTT AAGTATGCCC TTCATCTTTT TTCCTTCCTA AGGCTTTTAC 3600
  • NP_001070735.1 (SEQ ID NO: 4) is as follows:
  • NM_007516.3 (SEQ ID NO: 7) is as follows:
  • NP-031542.2 (SEQ ID NO: 8) is as follows:
  • NM_00 1077266.2 (SEQ ID NO: 11) is as follows:
  • TTCTTCCTGC AGTGGGTTTT TATGGTTTTG GTTTGTTTTT TGTTGTTTTG GTTCTTTGTT 3600
  • CTCTCAGTTA TCAGTTGGAA GGAGGTTAAT TCTAATGTAC TTGGAAGAGG CCTGTAGACC 5400
  • mouse p42 AUF1 amino acid sequence of GenBank Accession No. NP_001070734.1 (SEQ ID NO: 12) is as follows:
  • mouse p45 AUF1 nucleotide sequence of GenBank Accession No NM_00 1077265.2 (SEQ ID NO: 15) is as follows:
  • ATGAAGGCCA TTCAAACTCC TCCCCACGAC ACACTGAAGC AGCGGCGGCA CAGCGGGAAG 600
  • CTTATCCTCA CGCCTACCTG AGTTAATTAT CTGTTGACCT AGATATCTTT CTGGCCACTC 3240
  • NP_001070733.1 (SEQ ID NO: 16) is as follows:
  • accession numbers for example, as provided in Table 1, that include, e.g., a coding sequence or protein sequence with or without additional sequence elements or portions (e.g., leader sequences, tags, immature portions, regulatory regions, etc.).
  • sequence accession numbers or corresponding sequence identification numbers refers to either the sequence fully described therein or some portion thereof (e.g., that portion encoding a protein or polypeptide of interest to the technology described herein (e.g., AUF1 or a functional fragment thereof); the mature protein sequence that is described within a longer amino acid sequence; a regulatory region of interest e.g., promoter sequence or regulatory element) disclosed within a longer sequence described herein; etc.).
  • variants and isoforms of accession numbers and corresponding sequence identification numbers described herein are also contemplated.
  • the AUF1 protein referred to herein has an amino acid sequence as set forth in Table 1 and the sequences disclosed herein, or is a functional fragment thereof.
  • the AUF1 is a p37, p40, p42 or p45 form of human AUF1 and has an amino acid sequence of SEQ ID NO: 2, 6, 10, or 14, respectively.
  • the AUF1 is a p37, p40, p42 or p45 form of mouse AUF1 and has an amino acid sequence of SEQ ID NO: 4, 8, 12, or 16, respectively.
  • 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.
  • the functional fragment as referred to herein includes an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity to amino acid sequence of SEQ ID NO: 2, 6, 10, or 14 for human AUF1 or in other embodiments to the amino acid sequence of SEQ ID NO: 4, 8, 12, or 16 for mouse AUF1.
  • nucleic acids comprising nucleotide sequences encoding a human AUF1 protein, or functional fragment thereof, for example, the nucleotide sequences of SEQ ID NO: 1, 5, 9, or 13. Also provided are nucleic acids comprising nucleotide sequences 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 having an amino acid sequence of SEQ ID NO: 2, 6, 10, or 14, or a functional fragment thereof.
  • codon optimized sequences encoding an AUF1 protein including, a codon optimized version of the human p40 AUF1 coding sequence is the nucleotide sequence of SEQ ID NO: 17. Also provided are nucleic acids comprising nucleotide sequences 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 having an amino acid sequence of SEQ ID NO: 4, 8, 12, or 16, or a functional fragment thereof.
  • the AAV vectors and viral particles described herein comprise a nucleic acid molecule comprising a nucleotide sequence set forth in Table 1 (or described herein), or portions thereof that encode a functional fragment of an AUF1 protein as described supra, particularly in an expression cassette as described herein for expression in the cells of a subject, particularly, muscle cells of a subject.
  • nucleic acid expression cassettes comprising a nucleic acid encoding an AUF 1 (including human p37, p40, p42 or p45 AUF1, including a combination thereof) or a functional fragment thereof operably linked to regulatory elements, including promoter elements, and optionally enhancer elements and/or introns, to enhance or facilitate expression of the nucleic acid encoding the AUF1 or functional fragment thereof, including, for example, in muscle cells.
  • AUF 1 including human p37, p40, p42 or p45 AUF1, including a combination thereof
  • regulatory elements including promoter elements, and optionally enhancer elements and/or introns
  • the expression cassettes or transgenes provided herein may comprise nucleotide sequences encoding a human AUF1 protein having an amino acid sequence of SEQ ID NO: 2, 6, 10, or 14, or a functional fragment thereof (or, alternatively, for example, for mouse model studies, the expression cassette comprises a nucleotide sequence encoding a mouse AUF1 protein having an amino acid sequence of SEQ ID NO: 4, 8, 12, or 16, or a functional fragment thereof).
  • the nucleotide sequence encoding the 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).
  • the nucleotide sequence is SEQ ID NO: 17, which encodes human p40 AUF1 and codon and CpG optimized.
  • the AUF1 protein has no more than 1, 2, 3, 4, 5, 10, 15 amino acid substitutions, including conservative substitutions, with respect to the amino acid sequence of SEQ ID NO: 2, 6, 10, or 14, or a functional fragment thereof (or, alternatively, for example, for mouse model studies, with respect to the amino acid sequence of SEQ ID NO: 12, 16, 20 or 24), where the AUF1 protein has one or more AUF1 functions.
  • the regulatory control elements include promoters and may be either constitutive or may be tissue-specific, that is, active (or substantially more active or significantly more active) only in the target cell/tissue.
  • promoter and other regulatory elements that promote muscle specific expression, such as those in Table 8 infra.
  • the expression cassette or transgene is flanked by inverted terminal repeats (ITRs) (for example AAV2 ITR, including forms of ITRs for single-stranded AAV genomes or self-complementary AAV genomes.
  • ITRs inverted terminal repeats
  • the 5’ and 3’ ITR sequences are SEQ ID NO: 28 and 29, respectively.
  • 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 Depletion
  • the nucleotide sequence encoding the AUF1 is modified by codon optimization and CpG dinucleotide and CpG island depletion. Immune response against a transgene is a concern for human clinical application. AAV-directed immune responses can be inhibited by reducing the number of CpG di-nucleotides in the AAV genome (Faust et al., “CpG- Depleted Adeno-Associated Virus Vectors Evade Immune Detection,” J. Clin. Invest. 123(7): 2994-3001 (2013), which is hereby incorporated by reference in its entirety).
  • the transgene sequence of CpG motifs may diminish the role of TLR9 in activation of innate immunity upon recognition of the transgene as non-self, and thus provide stable and prolonged transgene expression (See also Wang et al., “Adeno-Associated Virus Vector as a Platform for Gene Therapy Delivery,” Nat. Rev. Drug Discov. 18(5): 358-378 (2019); and Rabinowitz et al., “Adeno-Associated Virus (AAV) versus Immune Response,” Viruses 11(2) (2019), which are hereby incorporated by reference in their entirety).
  • the AUF1 nucleotide sequence and the expression cassette is human codon-optimized with CpG depletion.
  • Codon- optimized and CpG depleted nucleotide sequences may be designed by any method known in the art, including for example, by Thermo Fisher Scientific GeneArt Gene Synthesis tools utilizing GeneOptimizer (Waltham, MA USA)). Nucleotide sequence SEQ ID NO: 17 described herein represents codon-optimized and CpG depleted sequence.
  • constructs that are useful as cis plasmids for rAAV construction that comprise a nucleotide sequence that encodes AUF1, including the p37, p40, p42 or p45 (including mouse and human) isoform thereof, operably linked to regulatory sequences that promote AUF1 expression in muscle cells.
  • rAAV genome constructs comprising an AUF1 transgene, including the codon optimized, CpG deleted human AUF1 p40 coding sequence of SEQ ID NO: 17, operably linked to regulatory sequences that promote expression in muscle cells, are provided herein.
  • the constructs have a muscle specific promoter, which may be Spc5-12 (including modified Spc5-12 promoters Spc5vl or Spc5v2 (SEQ ID Nos: 127 and 128, respectively, disclosed herein), tMCK or CK7 (see also Table 8 herein for promoters), optionally with an intron sequence between the promoter and the AUF1 coding sequence, such as a VH4 intron (see Table 9 for intron sequences), poly A signal sequences, such as rabbit beta globin poly A signal sequence (SEQ ID NO: 23), and optionally an WPRE sequence (SEQ ID NO: 24).
  • a muscle specific promoter which may be Spc5-12 (including modified Spc5-12 promoters Spc5vl or Spc5v2 (SEQ ID Nos: 127 and 128, respectively, disclosed herein), tMCK or CK7 (see also Table 8 herein for promoters), optionally with an intron sequence between the promoter and the AUF1 coding sequence, such as
  • the constructs may also include 5’ and/or 3’ stuffer sequences (SEQ ID Nos: 26 and 27 in Table 2, or any stuffer sequence known in the art, including, for example, stuffer sequences disclosed in Table 10, infra), and a SV40 polyadenylation signal sequence reversed with respect to the coding sequence and adjacent to the 3’ ITR sequence.
  • the constructs have one or more components from Table 2.
  • the rAAV genome comprises the following components: (1) AAV inverted terminal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter/enhancers, b) a poly A signal, and c) optionally an intron; and (3) nucleic acid sequences coding for AUF1.
  • the constructs described herein comprise the following components: (1) AAV2 or AAV8 inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a muscle-specific Spc5-12 promoter, tMCK promoter or CK7 promoter and a poly A signal, including a rabbit beta globin poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid encoding AUF1 as described herein, including the codon optimized, CpG depleted AUF1 p40 coding sequence.
  • ITRs inverted terminal repeats
  • rAAV AUF1 constructs comprising the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) the muscle-specific Spc5-12 promoter, the tMCK promoter or the CK7 promoter; b) an intron (e.g., a VH4) and c) a poly A signal sequence, such as a rabbit beta globin poly A signal sequence; and (3) a nucleotide sequence encoding AUF1 as described herein, including the codon optimized, CpG depleted AUF1 p40 coding sequence (SEQ ID NO: 17).
  • the construct includes a WPRE element 3’ of the coding sequence and 5’ of the polyA signal sequence.
  • the construct may also include 5’ and 3’ “stuffer sequences” between the ITR sequences and the expression cassette comprising the coding sequence and the regulatory operably linked thereto and an SV40 poly A signal sequence adjacent to and 5’ of the 3’ ITR sequence.
  • the vectors are single stranded and have a 5’ITR and a 3’ ITR, for example, as provided in Table 2 as SEQ ID NO: 28 and SEQ ID NO: 29, respectively.
  • the vectors are self-complementary vectors and have an altered 5’ ITR, an mITR, for example, that of SEQ ID NO: 30 and a 3’ ITR, as provided in Table 2, such as SEQ ID NO: 29.
  • rAAV genomes and sequences contained within cis plasmids are depicted in FIG. 1 and Table 3, and include:
  • spc-hu-opti-AUF l-CpG( ⁇ ) Codon optimized, CpG depleted Human AUF1 sequence driven by Spc5-12 promoter+VH4 intron, including 5’ (141 bp) stuffer and 3’ (893 bp) stuffer with a downstream SV40 polyA signal (reverse); having a nucleotide sequence of SEQ ID NO: 31 (including the ITR sequences).
  • tMCK-huAUFF Codon optimized, CpG depleted Human AUF1 sequence driven by tMCK promoter (no intron), including 5’ (141 bp) stuffer and 3’ (893 bp) stuffer-downstream SV40 polyA signal (reverse); having a nucleotide sequence of SEQ ID NO: 32 (including the ITR sequences)
  • spc5-12-hu-opti-AUFl-WPRE' Codon optimized, CpG depleted Human AUF1 sequence driven by Spc5-12 promoter+ VH4 intron, including 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 the ITR sequences).
  • ss-CK7-Hu-AUFF Codon optimized, CpG depleted Human AUF1 sequence driven by CK7 promoter (no intron), including 5’ (141 bp) stuffer and 3’ (893 bp) stuffer) - downstream SV40 polyA signal (reverse); SEQ ID NO: 34 (including the ITR sequences).
  • spc-hu-AUF 1 -No-Intron' Codon optimized, CpG depleted Human AUF1 sequence driven by Spc5-12 promoter (no intron) (including 5’ (141 bp) stuffer and 3’ (893 bp) stuffer)- downstream SV40 polyA signal (reverse); SEQ ID NO: 35 (including ITR sequences).
  • D(+)-CK7AUFF Self-complementary vector, Codon optimized, CpG depleted Human AUF1 sequence driven by CK7 promoter (no stuffers); SEQ ID NO:36 (including ITR sequences).
  • 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 as described herein, which may be useful in the methods for treating, preventing or ameliorating diseases or disorders in subjects, including human subjects, in need thereof by promoting or increasing muscle mass, muscle function or performance, and/or reducing or reversing muscle atrophy as described further herein, including types of LGMD.
  • these AAV or rAAV genomes and AAV or rAAV particles produced from cis plasmids comprising these sequences described herein, including those in Table 3, are administered in combination with an AAV or rAAV comprising a transgene encoding a DAPC protein component, e.g., microdystrophin, a-sarcoglycan, P-sarcoglycan, y-sarcoglycan and 5-sarcoglycan, for treatment of dystrophinopathies in subjects, including human subjects, in need thereof, including limbgirdle muscular dystrophy (LGMD).
  • a DAPC protein component e.g., microdystrophin, a-sarcoglycan, P-sarcoglycan, y-sarcoglycan and 5-sarcoglycan
  • LGMD including sarcoglycanopathies, calpainopathies, dysferlinopathies, and dystrophinopathies
  • methods of treating LGMD including sarcoglycanopathies, calpainopathies, dysferlinopathies, and dystrophinopathies in subjects, including human subjects, in need thereof by administering an AAV or rAAV gene therapy vector comprising a transgene encoding AUF1, including the AAV or rAAV genomes in Table 3, in combination with another therapy effective to treat LGMD, including sarcoglycanopathies, calpainopathies, dysferlinopathies, and dystrophinopathies, including those described herein.
  • microdystrophins that consist of dystrophin domains arranged amino-terminus to the carboxy terminus: ABD-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, Hl is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is a hinge 4 region of dystrophin, CR is a cysteine-rich region of dystrophin and CT is the C terminal domain (and comprises at least the portion of the CT domain containing the al- syntrophin binding site, including SEQ ID NO: 50
  • amino acid sequence of minimal alpha-syntrophin binding site (SEQ ID NO: 50) is as follows:
  • the present disclosure contemplates variants of microdystrophin so long as the therapeutic efficacy of microdystrophin comprising such variants is substantially maintained.
  • Functional activity includes (1) binding to one of, a combination of, or all of actin, P- dystroglycan, al-syntrophin, a-dystrobrevin, and nNOS; (2) improved muscle function in an animal model (for example, in the mdx mouse model) or in human subjects; and/or (3) cardioprotective or improvement in cardiac muscle function in animal models or human patients.
  • Table 4 provides the amino acid sequences of the microdystrophin embodiments in accordance with the present disclosure.
  • the microdystrophin has an amino acid sequence of SEQ ID NOs: 176 (DYS1), 177 (DYS3), or 178 (DYS5). In other embodiments, the microdystrophin has an amino acid sequence of SEQ ID NO: 179 (human MD1 (R4-R23/ACT), SEQ ID NO: 180 (microdystrophin), SEQ ID NO: 181 (Dys3978), SEQ ID NO: 182 (MD3), or SEQ ID NO: 183 (MD4). It is also contemplated that other embodiments are substituted variants of microdystrophins as defined by SEQ ID NOs: 176 (DYS1), 177 (DYS3), or 178 (DYS5).
  • 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 alternatively SEQ ID NO: 183) and maintain functional microdystrophin activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in animal models disclosed infra.
  • nucleic acids comprising a nucleotide sequence encoding a microdystrophin as described herein.
  • Such nucleic acids comprise nucleotide sequences that encode the microdystrophin that has the domains arranged N-terminal to C-terminal as follows: ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT as detailed, supra.
  • the nucleotide sequence can be any nucleotide sequence that encodes the domains.
  • the nucleotide sequence may be codon optimized and/or depleted of CpG islands for expression in the appropriate context.
  • the nucleic acid comprises a nucleotide sequence encoding the microdystrophin having the amino acid sequence of SEQ ID NO: 176, SEQ ID NO: 177, or SEQ ID NO: 178.
  • the nucleotide sequence encoding the microdystrophin cassette is modified by codon optimization and CpG dinucleotide and CpG island depletion.
  • Immune response against microdystrophin transgene is a concern for human clinical application, as evidenced in the first Duchenne Muscular Dystrophy (DMD) gene therapy clinical trials and in several adeno-associated vial (AAV)-minidystrophin gene therapy in canine models (Mendell et al., “Dystrophin Immunity in Duchenne's Muscular Dystrophy,” N. Engl. J. Med.
  • the microdystrophin cassette is human codon-optimized with CpG depletion. Codon-optimized and CpG depleted nucleotide sequences may be designed by any method known in the art, including for example, by Thermo Fisher Scientific GeneArt Gene Synthesis tools utilizing GeneOptimizer (Waltham, MA USA)). Nucleotide sequences SEQ ID NOs: 91, 92, 93 described herein represent codon-optimized and CpG depleted sequences.
  • microdystrophin transgenes that have reduced numbers of CpG dinucleotide sequences and, as a result, have reduced number of CpG islands.
  • the microdystrophin nucleotide sequence has fewer than two (2) CpG islands, or one (1) CpG island or zero (0) CpG islands.
  • the microdystrophin nucleotide sequence consisting essentially of SEQ ID NO: 91, 92, or 93 has zero (0) CpG islands.
  • the microdystrophin transgene nucleotide sequence consisting essentially of a microdystrophin gene operably linked to a promoter, wherein the microdystrophin consists of SEQ ID NO: 91, 92, or 93, has one (1) CpG island.
  • microdystrophin transgene constructs and artificial rAAV genomes.
  • the transgenes comprise nucleotide sequences encoding microdystrophins disclosed herein operably linked to transcriptional regulatory sequences, including promoters, that promote expression in muscle cells and other regulatory sequences that promote expression of the microdystrophin.
  • the transgenes are flanked by AAV ITR sequences.
  • the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter/enhancers, b) a poly A signal, and c) optionally an intron; and (3) nucleic acid sequences coding for the microdystrophin.
  • the constructs described herein comprise the following components: (1) AAV2 or AAV8 inverted terminal repeats (ITRs) that flank the expression cassette; (2) control elements, which include a muscle-specific Spc5-12 promoter and a small poly A signal; and (3) transgene providing (e.g., coding for) a nucleic acid encoding microdystrophin as described herein, including the microdystrophin coding sequence of the RGX-DYS1 transgene (SEQ ID NO:91) or the RGX-DYS5 transgene (SEQ ID NO:93).
  • ITRs inverted terminal repeats
  • the constructs described herein comprise the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) the muscle-specific Spc5-12 promoter, b) a small poly A signal; and (3) microdystrophin cassette, which includes from the N- terminus to the C-terminus, ABD1-H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein CT comprises at least the portion of the CT comprising an al-syntrophin binding site, including the CT having an amino acid sequence of SEQ ID NOs: 48 or 49.
  • the constructs described herein comprise the following components: (1) AAV2 or AAV8 ITRs that flank the expression cassette; (2) control elements, which include a) the muscle-specific Spc5-12 promoter, b) an intron (e.g., VH4) and c) a small poly A signal; and (3) microdystrophin cassette, which includes from the N-terminus to the C-terminus ABD1-H1-R1-R2-R3-H3-R24-H4-CR- CT, wherein the CT comprises at least the portion of the CT comprising an al-syntrophin binding site, including the CT having an amino acid sequence of SEQ ID NOs: 48 or 49, ABDI being directly coupled to VH4.
  • C-terminal Domain SEQ ID NO: 48.
  • the constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) control elements, which include a promoter, such as the muscle-specific Spc5-12 promoter (or modified Spc5-12 promoter SPc5vl or SPc5v2 (SEQ ID NOs: 127 or 128), and b) a small poly A signal; and (3) the nucleic acid encoding an AUF1.
  • constructs described herein comprising AAV ITRs flanking an AUF1 expression cassette, which includes one or more of the AUF1 sequences disclosed herein.
  • the constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) control elements, which include the muscle-specific Spc5-12 promoter (or modified Spc5-12 promoter SPc5vl or SPc5v2 (SEQ ID Nos: 127 or 128)), and b) a small poly A signal; and (3) the nucleic acid encoding the RGX-DYS1 microdystrophin having an amino acid sequence of SEQ ID NO: 176, including encoded by a nucleotide sequence of SEQ ID NO: 91.
  • the constructs described herein comprise the following components: (1) AAV2 ITRs that flank the expression cassette; (2) control elements, which include the muscle-specific Spc5-12 promoter, and b) a small poly A signal; and (3) the nucleic acid encoding the RXG-DYS5 microdystrophin having an amino acid sequence of SEQ ID NO: 178, including encoded by a nucleotide sequence of SEQ ID NO: 93.
  • constructs described herein comprising AAV ITRs flanking a microdystrophin expression cassette, which includes from the N-terminus to the C- terminus ABD1-H1-R1-R2-R3-H2-R24-H4-CR-CT, wherein the CT comprises at least the portion of the CT comprising an al-syntrophin binding site, including the CT having an amino acid sequence of SEQ ID NO: 48 or 49, can be between 4000 nt and 5000 nt in length. In some embodiments, such constructs are less than 4900 nt, 4800 nt, 4700 nt, 4600 nt, 4500 nt, 4400 nt, or 4300 nt in length.
  • nucleic acid embodiments of the present disclosure comprise AAV or rAAV vectors encoding microdystrophin comprising or consisting of a nucleotide sequence of SEQ ID NO: 184, 185, or 186 provided in Table 5 below.
  • an AAV or rAAV vector comprising a nucleotide sequence that has at least 50%, at least 60%, at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 184, 185, or 186 or the reverse complement thereof and encodes an AAV or rAAV vector suitable for expression of a therapeutically effective microdystrophin in muscle cells.
  • the constructs having the nucleotide sequence of SEQ ID NO: 184, 185, or 186 are in a recombinant rAAV8 or recombinant AAV9 particle.
  • Table 6 provides the amino acid sequences of the a-, P-, y-, and 6-Sarcoglycan embodiments in accordance with the present disclosure. It is also contemplated that other embodiments are substituted variant of a-, P-, y-, or 6-Sarcoglycan as defined by SEQ ID NOs: 144 (a-sarcoglycan), 52 (a-sarcoglycan), 145 (P-sarcoglycan), 52 (P-sarcoglycan), 146 (y- sarcoglycan), 54 (y-sarcoglycan), 94 (y-sarcoglycan), 95 (y-sarcoglycan), 96 (y-sarcoglycan), 147 (8-sarcoglycan), or 129 (5-sarcoglycan).
  • sarcoglycan 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: 144, 52, 145, 52, 146, 54, 94, 95, 96, 147, or 129 and maintain functional sarcoglycan activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in animal models disclosed infra.
  • nucleic acids comprising a nucleotide sequence encoding a-, P-, y- or 5-Sarcoglycan as described herein.
  • the nucleotide sequence can be any nucleotide sequence that encodes a-, P-, y- or 5-Sarcoglycan.
  • the nucleotide sequence may be codon optimized and/or depleted of CpG islands for expression in the appropriate context.
  • Table 7 provides nucleic acid sequences encoding a-, P-, y-, and 5-Sarcoglycan embodiments in accordance with the present disclosure. It is also contemplated that other embodiments are substituted variant of a-, P-, y-, or 5-Sarcoglycan as defined by SEQ ID NOs: 130 (a-sarcoglycan), 131 (a-sarcoglycan), 132 (P-sarcoglycan), 133 (P-sarcoglycan), 134 (y- sarcoglycan), 135 (y-sarcoglycan), 136 (y-sarcoglycan), 137 (y-sarcoglycan), 148 (y- sarcoglycan), 149 (5-sarcoglycan), or 150 (5-sarcoglycan).
  • SEQ ID NOs 130 (a-sarcoglycan), 131 (a-sarcoglycan), 132 (P-sarcog
  • sarcoglycan 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 NOs: 130, 131, 132, 133, 134, 135, 136, 137, 148, 149, or 150 and maintain functional sarcoglycan activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in animal models disclosed infra.
  • Table 7 Nucleic Acid Sequences of Sarcoglycan Proteins
  • the expression cassettes, rAAV or rAAV genomes, and AAV or rAAV vectors disclosed herein comprise transgenes encoding either AUF1 or a therapeutic protein operably linked to regulatory elements, including promoter elements, and, optionally, enhancer elements and/or introns, to enhance or facilitate expression of the transgene.
  • the AAV or rAAV vector also includes such regulatory control elements known to one skilled in the art to influence the expression of the RNA and/or protein products encoded by nucleic acids (transgenes) within target cells of the subject.
  • Regulatory control elements and may be tissuespecific, that is, active (or substantially more active or significantly more active) only in the target cell/tissue.
  • the expression cassette of an AAV or rAAV vector comprises a regulatory sequence, such as a promoter, operably linked to the transgene that allows for expression in target tissues.
  • the promoter may be a muscle promoter.
  • the promoter is a muscle-specific promoter.
  • the phrase “muscle-specific”, “muscle-selective” or “muscle-directed” refers to nucleic acid elements that have adapted their activity in muscle cells or tissue due to the interaction of such elements with the intracellular environment of the muscle cells.
  • muscle cells may include myocytes, myotubes, cardiomyocytes, and the like. Specialized forms of myocytes with distinct properties such as cardiac, skeletal, and smooth muscle cells are included.
  • Various therapeutics may benefit from muscle-specific expression of a transgene.
  • gene therapies that treat various forms of muscular dystrophy delivered to and enabling high transduction efficiency in muscle cells have the added benefit of directing expression of the transgene in the cells where the transgene is most needed.
  • Cardiac tissue may also benefit from muscle-directed expression of the transgene.
  • Muscle-specific promoters may be operably linked to the transgenes of the disclosure.
  • Adeno-associated viral (AAV) vectors disclosed herein comprise a muscle cellspecific promoter operatively linked to the nucleic acid encoding the AUF1 and/or the therapeutic protein for treatment of a LGMD.
  • the muscle cell-specific promoter mediates cell-specific and/or tissue-specific expression of an AUF1 protein or fragment thereof.
  • the promoter may be a mammalian promoter.
  • the promoter may be selected from the group consisting of a human promoter, a murine 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.
  • the muscle cell-specific promoter is one of a muscle creatine kinase (MCK) promoter, a synlOO promoter, a creatine kinase (CK) 6 promoter, a creatine kinase (CK) 7 promoter, a dMCK promoter, a tMCK promoter, a smooth muscle 22 (SM22) promoter, a myo-3 promoter, a Spc5-12 promoter, a creatine kinase (CK) 8 promoter, a creatine kinase (CK) 8e promoter, a creatine kinase (CK) 9 promoter, a U6 promoter, a Hl promoter, a desmin promoter, a Pitx3 promoter, a skeletal alpha-actin promoter, a MHCK7 promoter, and a Sp-301 promoter.
  • MCK muscle creatine kinase
  • CK creatine kinase
  • synlOO promoter a
  • Suitable muscle cell-specific promoter sequences are well known in the art and exemplary promoters are provided in Table 8 below (Malerba et al., “PABPN1 Gene Therapy for Oculopharyngeal Muscular Dystrophy,” Nat. Commun. 8: 14848 (2017); Wang et al., “Construction and Analysis of Compact Muscle-Specific Promoters for AAV Vectors,” Gene. Ther. 15: 1489-1499 (2008); Piekarowicz et al., “A Muscle Hybrid Promoter as a Novel Tool for Gene Therapy,” Mol. Ther. Methods Clin. Dev.
  • the muscle cell-specific promoter is a muscle creatinekinase (MCK) promoter.
  • the muscle creatine kinase (MCK) gene is highly active in all striated muscles. Creatine kinase plays an important role in the regeneration of ATP within contractile and ion transport systems. It allows for muscle contraction when neither glycolysis nor respiration is present by transferring a phosphate group from phosphocreatine to ADP to form ATP.
  • CKB brain creatine kinase
  • MCK muscle creatine kinase
  • CKMi two mitochondrial forms
  • MCK is the most abundant non- mitochondrial mRNA that is expressed in all skeletal muscle fiber types and is also highly active in cardiac muscle.
  • the MCK gene is not expressed in myoblasts, but becomes transcriptionally active when myoblasts commit to terminal differentiation into myocytes.
  • MCK gene regulatory regions display striated muscle-specific activity and have been extensively characterized in vivo and in vitro.
  • the major known regulatory regions in the MCK gene include a muscle-specific enhancer located approximately 1.1 kb 5' of the transcriptional start site in mouse and a 358-bp proximal promoter. Additional sequences that modulate MCK expression are distributed over 3.3 kb region 5' of the transcriptional start site and in the 3.3-kb first intron.
  • 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 hereby incorporated by reference in its entirety.
  • Suitable muscle creatine kinase (MCK) promoters include, without limitation, a wild type MCK promoter, a dMCK promoter, and a tMCK promoter (Wang et al., “Construction and Analysis of Compact Muscle-Specific Promoters for AAV Vectors,” Gene Ther. 15(22): 1489- 1499 (2008), which is hereby incorporated by reference in its entirety).
  • the muscle-specific promoter is selected from an Spc5-12 promoter (SEQ ID NO: 18 or 106)(including a modified Spc5-12 promoter SPc5vl or SPc5v2 (SEQ ID NO: 127 or 128, respectively), a muscle creatine kinase myosin light chain (MLC) promoter, a myosin heavy chain (MHC) promoter, a desmin promoter (human— SEQ ID NO: 98), a MCK7 promoter (SEQ ID NO: 104), a CK6 promoter, a CK8 promoter (SEQ ID NO: 107), a MCK promoter (or a truncated form thereof) (SEQ ID NO: 105 or 21), an alpha actin promoter, a beta actin promoter, an gamma actin promoter, an E-syn promoter, a cardiac troponin C promoter, a troponin I promoter, a myoD gene family
  • Synthetic promoter c5-12 (Li et al., “Synthetic Muscle Promoters: Activities Exceeding Naturally Occurring Regulatory Sequences,” Nat. Biotechnol. 17(3): 241-245 (1999), which is hereby incorporated by reference in its entirety), known as the Spc5-12 promoter, has been shown to have cell type restricted expression, specifically muscle-cell specific expression. At less than 350 bp in length, the Spc5-12 promoter is smaller in length than most endogenous promoters, which can be advantageous when the length of the nucleic acid encoding the therapeutic protein is relatively long.
  • the promoter may be a constitutive promoter, for example, the CB7 promoter.
  • Additional promoters include, without limitation, cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1 alpha promoter (SEQ ID NO: 110), UB6 promoter, chicken beta-actin promoter, and CAG promoter (SEQ ID NO: 108).
  • CMV cytomegalovirus
  • RSV Rous sarcoma virus
  • MMT Rous sarcoma virus
  • EF-1 alpha promoter SEQ ID NO: 110
  • UB6 promoter EF-1 alpha promoter
  • UB6 promoter e.g., UB6 promoter
  • chicken beta-actin promoter e.g., chicken beta-actin promoter
  • CAG promoter e.g., CAG promoter
  • Certain gene expression cassettes further include an intron, for example, 5’ of the AUF1 or therapeutic protein coding sequence (such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof) which may enhance proper splicing and, thus, transgene expression.
  • an intron for example, 5’ of the AUF1 or therapeutic protein coding sequence (such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof) which may enhance proper splicing and, thus, transgene expression.
  • an intron is coupled to the 5’ end of a sequence encoding an AUF1 or therapeutif protein (such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof).
  • an AUF1 or therapeutif protein such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof.
  • the intron is less than 100 nucleotides in length.
  • the intron is a VH4 intron.
  • the VH4 intron nucleic acid can comprise SEQ ID NO: 111 as shown in Table 9 below.
  • the intron is a chimeric intron derived from human P- globin and Ig heavy chain (also known as P-globin splice donor/immunoglobulin heavy chain splice acceptor intron, or P-globin/IgG chimeric intron) (Table 9, SEQ ID NO: 112).
  • introns well known to the skilled person may be employed, such as the chicken P-actin intron, minute virus of mice (MVM) intron, human factor IX intron (e.g., FIX truncated intron 1), P- 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
  • poly A polyadenylation
  • the therapeutic protein such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof
  • Any polyA site that signals termination of transcription and directs the synthesis of a polyA tail is suitable for use in AAV vectors of the present disclosure.
  • Exemplary polyA signals are derived from, but not limited to, the following: the SV40 late gene, the rabbit P-globin gene, the bovine growth hormone (BPH) gene, the human growth hormone (hGH) gene, and the synthetic polyA (SPA) site.
  • Exemplary polyA signal sequences useful in the constructs described herein are provided in Table 2 supra.
  • WPRE Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element
  • the WPRE element may be inserted into 3’ untranslated regions of the transgene 5’ of 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 hereby incorporated by reference in its entirety.
  • the WPRE element has a nucleotide sequence of SEQ ID NO: 24 (see Table 2 supra).
  • Other elements that may be included in the construct are filler or stuffer sequences that may be incorporated particularly at the 5’ and 3’ ends between the ITR sequences and the expression cassette sequences to optimized the length of nucleic acid between the ITR sequences to improve packaging efficiency.
  • An SV40 polyadenylation sequence positioned adjacent to an ITR sequence can insulate transgene transcription from interference from the ITRs.
  • Exemplary stuffer sequences and the SV40 polyA sequence are provided in Table 2, supra.
  • Alternative polyA sequences and stuffer sequences are known in the art, see e.g. Table 10.
  • Nucleic acids comprising a stuffer (or filler) polynucleotide sequence extend the transgene size of any heterologous gene, for example an AUF1 gene of Table 2 or 3.
  • a stuffer (or filler) polynucleotide sequence comprises SEQ ID NO: 26 or 27.
  • a stuffer (or filler) polynucleotide sequence comprises SEQ ID NO: 139-143, or a fragment of SEQ ID NO: 139-143 (see Table 10) between 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.
  • the stuffer polynucleotide comprises a nucleic acid sequence SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO141, SEQ ID NO: 142, or SEQ ID NO: 143 (see Table 10), or a fragment or fragments thereof.
  • the stuffer polynucleotide sequence has a length that when combined with the heterologous gene sequence, the total combined length of the heterologous gene sequence and stuffer polynucleotide sequence is between about 2.4-5.2 kb, or between about 3.1-4.7 kb.
  • the transgene may comprise any one of the genes or nucleic acids encoding a therapeutic AUF1 gene listed in, but not limited to, Tables 2 and 3.
  • the nucleic acid sequences are operably linked to the transgene in a contiguous, or substantially contiguous manner.
  • operably linked may refer to joining a coding region and a non-coding region, or two coding regions in a contiguous manner, e.g. in reading frame.
  • enhancers which may function when separated from the promoter by several kilobases, such as intronic sequences and stuffer sequences, these regulatory sequences may be operably linked while not directly contiguous with a downstream or upstream promoter and/or heterologous gene.
  • the disclosed gene cassettes, and thus the adeno-associated viral vectors comprise a nucleic acid molecule encoding a reporter protein.
  • the reporter protein may be selected from the group consisting of, e.g., P-galactosidase, chloramphenicol acetyl transferase, luciferase, and fluorescent proteins.
  • the reporter protein is a fluorescent protein.
  • Suitable fluorescent proteins include, without limitation, 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, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFPl, DsRed-Express, DsRed2, DsRed-
  • the reporter protein is luciferase.
  • luciferase refers to members of a class of enzymes that catalyze reactions that result in production of light. Luciferases have been identified in and cloned from a variety of organisms including fireflies, click beetles, sea pansy (Renilla), marine copepods, and bacteria among others.
  • luciferases that may be used as reporter proteins include, e.g., Renilla (e.g., Renilla reniformis) luciferase, Gaussia (e.g., Gaussia princeps) luciferase), Metridia luciferase, firefly (e.g, Photinus pyralis luciferase), click beetle (e.g., Pyrearinus termitilluminans) luciferase, deep sea shrimp (e.g., Oplophorus gracilirostris) luciferase).
  • Renilla e.g., Renilla reniformis
  • Gaussia e.g., Gaussia princeps
  • Metridia luciferase e.g., firefly (e.g, Photinus pyralis luciferase), click beetle (e.g., Pyrearinus termitillumina
  • Luciferase reporter proteins include both naturally occurring proteins and engineered variants designed to have one or more altered properties relative to the naturally occurring protein, such as increased photostability, increased pH stability, increased fluorescence or light output, reduced tendency to dimerize, oligomerize, aggregate or be toxic to cells, an altered emission spectrum, and/or altered substrate utilization.
  • AUF1 and other protein encoding transgenes disclosed herein can be included in an AAV vector for gene therapy administration to a human subject.
  • AAV or recombinant AAV (rAAV) vectors can comprise an AAV viral capsid and a viral or artificial genome comprising an expression cassette flanked by AAV inverted terminal repeats (ITRs) wherein the expression cassette comprises an AUF1 or encoding 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 other therapeutic protein as the case may be.
  • ITRs AAV inverted terminal repeats
  • the provided methods are suitable for use in the production of any isolated recombinant AAV particles for delivery of an AUF1 protein or other therapeutic protein described herein, in the production of a composition comprising any isolated recombinant AAV particles encoding an AUF1 protein or other therapeutic protein, or in the method for treating a disease or disorder amenable for treatment, including LGMD types with an AUF1 protein or a combination of an AUF1 protein and another therapeutic protein in a subject in need thereof comprising the administration of any isolated recombinant AAV particles encoding an AUF1 protein or a combination (including administered separately) of an rAAV particle encoding an AUF1 protein and an rAAV particle encoding another therapeutic protein described herein.
  • the rAAV can be of any serotype, variant, modification, hybrid, or derivative thereof, known in the art, or any combination thereof (collectively referred to as “serotype”).
  • serotype has a tropism for muscle tissue (including skeletal muscle, cardiac muscle or smooth muscle).
  • AAV or rAAV particles have a capsid protein from an AAV8 serotype. In other embodiments, AAV or rAAV particles have a capsid protein from an AAV9 serotype. In still other embodiments, AAV or rAAV particles have a capsid protein from an hu.32 serotype.
  • RGX-DYS1 construct in an AAV or rAAV particle having an AAV8 capsid and the RGX-DYS1 construct in an AAV or rAAV particle having an AAV9 capsid.
  • RGX-DYS5 construct in an AAV or rAAV particle having an AAV8 capsid and the RGX- DYS5 construct in an rAAV particle having an AAV9 capsid.
  • the AAV or rAAV particles comprise a capsid protein 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 serotype or alternatively may be an AAVrh.8, AAVrh.10, AAVrh.43, AAVrh.74, AAVhu.37, AAVAAV.hu31, or AAVhu.32 serotype.
  • AAV or rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV8 capsid protein.
  • AAV or rAAV particles comprise a capsid protein that has a capsid protein at least 80% or more 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 sequence of AAV8 capsid protein (SEQ ID NO: 114) (Table 11).
  • AAV or rAAV particles comprise a capsid protein that has a capsid protein at least 80% or more 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 sequence of AAV9 capsid protein (SEQ ID NO: 115) (Table 11).
  • AAV or rAAV particles comprise a capsid protein that has capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e.
  • AAV1, VP2 and/or VP3 sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.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
  • AAVhu.32 SEQ ID NO: 118 serotype capsid protein (see Table 11).
  • Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; International Patent Application Nos. PCT/US2015/034799;
  • a single-stranded AAV can be used.
  • a self-complementary vector e.g., scAAV
  • Self-complementary vectors may include a mutant ITR sequence, for example, the mutant 5’ ITR sequence in Table 2.
  • rAAV particles comprise a pseudotyped rAAV particle.
  • the pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs and (b) a capsid comprised of capsid proteins derived from 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), in particular AAV8.
  • AAVx e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV2i8, AAV2.5, AAVrh.8, AAVrh.10, AAVrh.43,
  • rAAV particles comprise a pseudotyped rAAV particle containing AAV8 capsid protein.
  • the pseudotyped rAAV8 particle is an rAAV2/8 pseudotyped particle.
  • Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., “Enhancement of Muscle Gene Delivery with Pseudotyped Adeno-Associated Virus Type 5 Correlates with Myoblast Differentiation,” J. Virol. 75(16):7662-7671 (2001); Halbert et al., “Repeat Transduction in the Mouse Lung by Using Adeno-Associated Virus Vectors with Different Serotypes,” J.
  • the AAV or rAAV particles comprise an AAV capsid protein chimeric of 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.
  • the AAV or rAAV particles comprises a Clade A, B, E, or F AAV capsid protein. In some embodiments, the AAV or rAAV particles comprises a Clade F AAV capsid protein. In some embodiments the AAV or rAAV particles comprises a Clade E
  • AAV capsid protein AAV capsid protein
  • Table 11 below provides examples of amino acid sequences for an AAV8, AAV9, AAV.rh74, AAV.hu31, AAVhu.32, and AAV.hu37 capsid proteins. Exemplary ITR sequences are provided in Table 2. Table 11
  • a molecule according to the disclosure is made by providing a nucleotide comprising the nucleic acid sequence encoding any of the capsid protein molecules herein; and using a packaging cell system to prepare corresponding rAAV particles with capsid coats made up of the capsid protein.
  • capsid proteins are described in Section 5.6.5, supra.
  • the nucleic acid sequence encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, including 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of a capsid protein molecule described herein.
  • the nucleic acid encodes a sequence having at least 60%, 70%, 80%, 85%, 90%, or 95%, including 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of the AAV8 capsid protein, while retaining (or substantially retaining) 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%, including 96%, 97%, 98%, 99% or 99.9%, identity to the sequence of the AAV9 capsid protein, while retaining (or substantially retaining) biological function of the AAV9 capsid protein.
  • the capsid protein, coat, and rAAV particles may be produced by techniques known in the art.
  • the viral genome comprises at least one inverted terminal repeat to allow packaging into a vector.
  • the viral genome further comprises a cap gene and/or a rep gene for expression and splicing of the cap gene.
  • the cap and rep genes are provided by a packaging cell and not present in the viral genome.
  • the nucleic acid encoding the engineered capsid protein is cloned into an AAV Rep-Cap plasmid in place of the existing capsid gene.
  • this plasmid helps package an rAAV genome into the engineered capsid protein as the capsid coat.
  • Packaging cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging.
  • cell culture-based systems are known in the art for production of rAAV particles, any of which can be used to practice a method disclosed herein.
  • the cell culture-based systems include transfection, stable cell line production, and infectious hybrid virus production systems which include, but are not limited to, adenovirus-AAV hybrids, herpesvirus- AAV hybrids and baculovirus-AAV hybrids.
  • rAAV production cultures for the production of rAAV virus particles require: (1) suitable host cells, including, for example, human-derived cell lines, mammalian cell lines, or insect-derived cell lines; (2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperature-sensitive adenovirus), herpes virus, 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.
  • suitable host cells including, for example, human-derived cell lines, mammalian cell lines, or insect-derived cell lines
  • suitable helper virus function provided by wild type or mutant adenovirus (such as temperature-sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing help
  • Nonlimiting examples of host cells include: A549, WEHI, 10T1/2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, HEK293 and their derivatives (HEK293T cells, HEK293F cells), Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, myoblast cells, CHO cells or CHO-derived cells, or insect-derived cell lines such as SF-9 (e.g. in the case of baculovirus production systems).
  • SF-9 insect-derived cell lines
  • a method of producing rAAV particles comprising (a) providing a cell culture comprising an insect cell; (b) introducing into the cell one or more baculovirus vectors encoding at least one of: i. an rAAV genome to be packaged, ii. an AAV rep protein sufficient for packaging, and iii. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles.
  • the method comprises using a first baculovirus vector encoding the rep and cap genes and a second baculovirus vector encoding the rAAV genome.
  • the method comprises using a baculovirus encoding the rAAV genome and an insect cell expressing the rep and cap genes. In some embodiments, the method comprises using a baculovirus vector encoding the rep and cap genes and the rAAV genome.
  • the insect cell is an Sf-9 cell. In some embodiments, the insect cell is an Sf-9 cell comprising one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.
  • a method disclosed herein uses a baculovirus production system.
  • the baculovirus production system uses a first baculovirus encoding the rep and cap genes and a second baculovirus encoding the rAAV genome.
  • the baculovirus production system uses a baculovirus encoding the rAAV genome and a host cell expressing the rep and cap genes.
  • the baculovirus production system uses a baculovirus encoding the rep and cap genes and the rAAV genome.
  • the baculovirus production system uses insect cells, such as Sf-9 cells.
  • a skilled artisan is aware of the numerous methods by which AAV rep and cap genes, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and rAAV genomes (comprising one or more genes of interest flanked by ITRs) can be introduced into cells to produce or package rAAV.
  • AAV helper genes e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene
  • rAAV genomes comprising one or more genes of interest flanked by ITRs
  • helper viruses including adenovirus and herpes simplex virus (HSV), promote AAV replication and certain genes have been identified that provide the essential functions, e.g. the helper may induce changes to the cellular environment that facilitate such AAV gene expression and replication.
  • AAV rep and cap genes, helper genes, and rAAV genomes are introduced into cells by transfection of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.
  • AAV rep and cap genes, helper genes, and rAAV genomes can be introduced into cells by transduction with viral vectors, for example, rHSV vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.
  • viral vectors for example, rHSV vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.
  • one or more of AAV rep and cap genes, helper genes, and rAAV genomes are introduced into the cells by transduction with an rHSV vector.
  • the rHSV vector encodes the AAV rep and cap genes.
  • the rHSV vector encodes the helper genes.
  • the rHSV vector encodes the rAAV genome.
  • the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes the helper genes and the rAAV genome. In some embodiments, the rHSV vector encodes the helper genes and the AAV rep and cap genes.
  • 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. an rAAV genome to be packaged, ii. helper functions necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles.
  • the rHSV vector encodes the AAV rep and cap genes.
  • the rHSV vector encodes helper functions.
  • the rHSV vector comprises one or more endogenous genes that encode helper functions. In some embodiments, the rHSV vector comprises one or more heterogeneous genes that encode helper functions. In some embodiments, the rHSV vector encodes the rAAV genome. In some embodiments, the rHSV vector encodes the AAV rep and cap genes. In some embodiments, the rHSV vector encodes helper functions and the rAAV genome. In some embodiments, the rHSV vector encodes helper functions and the AAV rep and cap genes. In some embodiments, the cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.
  • a method of producing rAAV particles comprising (a) providing a cell culture comprising a mammalian cell; (b) introducing into the cell one or more polynucleotides encoding at least one of: i. an rAAV genome to be packaged, ii. helper functions necessary for packaging the rAAV particles, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; (c) adding to the cell culture sufficient nutrients and maintaining the cell culture under conditions that allow production of the rAAV particles.
  • the helper functions are encoded by adenovirus genes.
  • the mammalian cell comprises one or more stably integrated heterologous polynucleotide encoding the rep and cap genes.
  • AAV rep and cap genes are encoded by one plasmid vector.
  • AAV helper genes e.g., adenovirus El a gene, Elb gene, E4 gene, E2a gene, and VA gene
  • the El a gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector.
  • the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one plasmid vector.
  • 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 by one plasmid vector.
  • the helper genes are stably expressed by the host cell.
  • AAV rep and cap genes are encoded by one viral vector.
  • AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one viral vector.
  • the Ela gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector.
  • the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one viral vector.
  • 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 by one viral vector.
  • the AAV rep and cap genes, the adenovirus helper functions necessary for packaging, and the rAAV genome to be packaged are introduced to the cells by transfection with one or more polynucleotides, e.g., vectors.
  • a method disclosed herein comprises transfecting the cells with a mixture of three polynucleotides: one encoding the cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged.
  • the AAV cap gene is an AAV8 cap gene.
  • 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.
  • the vector encoding the rAAV genome to be packaged comprises a gene of interest flanked by AAV ITRs.
  • the ITR sequences are AAV2 ITR sequences and include 5’ and 3’ sequences of SEQ ID NO: 28 and 29, respectively, as set forth in Table 2.
  • Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and rAAV genome to a cell in which rAAV particles are to be produced or packaged.
  • a first plasmid vector encoding an rAAV genome comprising 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 helper genes can be used.
  • ITRs AAV inverted terminal repeats
  • a second vector encoding AAV rep and cap genes a third vector encoding helper genes
  • a mixture of the three vectors is co-transfected into a cell.
  • a combination of transfection and infection is used by using both plasmid vectors as well as viral vectors.
  • one or more of rep and cap genes, and AAV helper genes are constitutively expressed by the cells and does not need to be transfected or transduced into the cells.
  • the cell constitutively expresses rep and/or cap genes.
  • the cell constitutively expresses one or more AAV helper genes.
  • the cell constitutively expresses El a.
  • the cell comprises a stable transgene encoding the rAAV genome.
  • AAV rep, cap, and helper genes can be of any AAV serotype.
  • AAV rep and cap genes for the production of a rAAV particle are from different serotypes.
  • the rep gene is from AAV2 whereas the cap gene is from AAV8.
  • the rep gene is 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.32or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype).
  • the rep and the cap genes are from the same serotype.
  • the rep and the cap genes are from the same serotype, and the rep gene comprises at least one modified protein domain or modified promoter domain.
  • the at least one modified domain comprises a nucleotide sequence of a serotype that is different from the capsid serotype.
  • the modified domain within the rep gene may be a hybrid nucleotide sequence consisting fragments different serotypes.
  • Hybrid rep genes provide improved packaging efficiency of rAAV particles, including packaging of a viral genome comprising a therapeutic protein transgene (such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof) 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.
  • a therapeutic protein transgene such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof
  • AAV rep genes consist of nucleic acid sequences that encode the non- structural proteins needed for replication and production of virus. Transcription of the rep gene initiates from the p5 or pl9 promoters to produce two large (Rep78 and Rep68) and two small (Rep52 and Rep40) nonstructural Rep proteins, respectively. Additionally, Rep78/68 domain contains a DNA-binding domain that recognizes specific ITR sequences within the ITR. All four Rep proteins have common helicase and ATPase domains that function in genome replication and/or encapsidation (Maurer and Weitzman, “Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction,” Hum. Gene Ther.
  • cap gene initiates from a p40 promoter, which sequence is within the C- terminus of the rep gene, and it has been suggested that other elements in the rep gene may induce p40 promoter activity.
  • the p40 promoter domain includes transcription factor binding elements EFl A, MLTF, and ATF, Fos/Jun binding elements (AP-1), Sp 1 -like elements (Spl and GGT), and the TATA element (Pereira and Muzyczka, “The Adeno-Associated Virus Type 2 p40 Promoter Requires a Proximal Spl Interaction and a pl9 CArG-like Element to Facilitate Rep Transactivation,” J. Virol. 71(6): 4300-4309 (1997), which is hereby incorporated by reference in its entirety).
  • the rep gene comprises a modified p40 promoter.
  • the p40 promoter is modified at any one or more of the EFl A binding element, MLTF binding element, ATF binding element, Fos/Jun binding elements (AP- 1), Sp 1 -like elements (Spl or GGT), or the TATA element.
  • the rep gene is of 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 the portion or element of the p40 promoter domain is modified to serotype 2.
  • the rep gene is of serotype 8 or 9, and the portion or element of the p40 promoter domain is modified to serotype 2.
  • ITRs contain A and A’ complimentary sequences, B and B’ complimentary sequences, and C and C’ complimentary sequences; and the D sequence is contiguous with the ssDNA genome.
  • the complimentary sequences of the ITRs form hairpin structures by selfannealing (Berns, KI., “The Unusual Properties of the AAV Inverted Terminal Repeat,” Hum. Gene Ther. 31 (9-10): 518-523 (2020), which is hereby incorporated by reference in its entirety).
  • the D sequence contains a Rep Binding Element (RBE) and a terminal resolution site (TRS), which together constitute the AAV origin of replication.
  • RBE Rep Binding Element
  • TRS terminal resolution site
  • the ITRs are also required as packaging signals for genome encapsidation following replication.
  • the ITR sequences and the cap genes are from the same serotype, except that one or more of the A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence 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.
  • the ITR sequences and the cap genes are from different serotypes, except that one or more of the ITR sequences selected from A and A’ complimentary sequences, B and B’ complimentary sequences, C and C’ complimentary sequences, or the D sequence 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.
  • the rep and the cap genes are from the same serotype, and the rep gene comprises a modified Rep78 domain, DNA binding domain, endonuclease domain, ATPase domain, helicase domain, p5 promoter domain, Rep68 domain, p5 promoter domain, Rep52 domain, pl9 promoter domain, Rep40 domain or p40 promoter domain.
  • the rep and the cap genes are from the same serotype, and the rep gene comprises at least one protein domain or promoter domain from a different serotype.
  • an rAAV comprises a transgene flanked by AAV2 ITR sequences, an AAV8 cap, and a hybrid AAV2/8 rep.
  • the AAV2/8 rep comprises serotype 8 rep except for the p40 promoter domain or a portion thereof is from serotype 2 rep. In other embodiments, the AAV2/8 rep comprises serotype 2 rep except for the p40 promoter domain or a portion thereof is from serotype 8 rep. In some embodiments, more than two serotypes may be utilized to construct a hybrid replcap plasmid.
  • a method disclosed herein comprises transfecting a cell using a chemical based transfection method.
  • the chemical-based transfection method uses calcium phosphate, highly branched organic compounds (dendrimers), cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)), lipofection.
  • the chemicalbased transfection method uses cationic polymers (e.g., DEAE dextran or polyethylenimine (PEI)).
  • the chemical-based transfection method uses polyethylenimine (PEI).
  • the chemical-based transfection method uses DEAE dextran.
  • the chemical-based 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 foregoing techniques and procedures can be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., 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 any purpose.
  • the rAAVs provide transgene delivery vectors that can be used in therapeutic and prophylactic applications, as discussed in more detail below.
  • Nucleic acid sequences of AAV-based viral vectors, and methods of making recombinant AAV and AAV capsids, are taught, e.g., in US 7,282,199; US 7,790,449; US 8,318,480; US 8,962,332; and PCT/EP2014/076466, which are hereby incorporated by reference in their entirety.
  • a recombinant vector disclosed herein for example AAV or rAAV particles.
  • the infectivity of recombinant gene therapy vectors in muscle cells can be tested in C2C12 myoblasts.
  • muscle or heart cell lines may be utilized, including but not limited to T0034 (human), L6 (rat), MM 14 (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 numbers may be assessed using polymerase chain reaction techniques and level of therapeutic protein expression may be tested by measuring levels of therapeutic protein (such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof) mRNA in the cells.
  • therapeutic protein such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof
  • the efficacy of a viral vector containing a transgene encoding an AUF1 protein or a therapeutic protein as described herein may be tested by administering to an animal model to replace sarcoglycan, for example, by using the 5-sarcoglycan deleted mouse and/or the golden retriever muscular dystrophy (GRMD) model and to assess the biodistribution, expression and therapeutic effect of the transgene expression.
  • the therapeutic effect may be assessed, for example, by assessing change in muscle strength in the animal receiving the transgene.
  • Animal models using larger mammals as well as nonmammalian vertebrates and invertebrates can also be used to assess pre-clinical therapeutic efficacy of a vector described herein.
  • compositions and methods for therapeutic administration comprising a dose of an AUF1 alone or in combination with a therapeutic protein such as microdystrophin, a- sarcoglycan, P-sarcoglycan, y-sarcoglycan, 5-sarcoglycan, calpain 3, calcium/calmodulin- dependent protein kinase II P isoform proteins, other protein (other than AUF1), or portions thereof encoding vector disclosed herein in an amount demonstrated to be effective according to the methods for assessing therapeutic efficacy disclosed here.
  • a therapeutic protein such as microdystrophin, a- sarcoglycan, P-sarcoglycan, y-sarcoglycan, 5-sarcoglycan, calpain 3, calcium/calmodulin- dependent protein kinase II P isoform proteins, other protein (other than AUF1), or portions thereof encoding vector disclosed herein in an amount demonstrated to be effective according to the methods for assessing therapeutic efficacy disclosed
  • the efficacy of gene therapy vectors alone or in combination with the second therapeutics disclosed herein may be assessed in murine models of LGMD.
  • the a-, P-, y-, and 5- sarcoglycan null mice display progressive muscle pathology and functional impairments starting at 1 week.
  • the 5-sarcoglycan null mouse has the histological features muscular dystrophy, including areas of abundant necrosis, fibrosis, inflammation, calcification, and impaired muscle function.
  • the 5-sarcoglycan deleted mouse has fibrotic lesions and electrocardiogram abnormalities beginning at 8 weeks and cardiomyopathy from 16 weeks (van Putten et al., “Mouse Models for Muscular Dystrophies: An Overview,” Dis. Models Meeh. 13(2):dmm043562 (2020), which is hereby incorporated by reference in its entirety).
  • the efficacy of gene therapy vectors alone or in combination with the second therapeutics disclosed herein may be assessed in murine models of LGMD.
  • the CAPN3' 1 ' deletion mouse model replicates LGMD2A disease well, with mice displaying muscle degeneration, necrosis, abnormal mitochondria and function, small myofibers, loss of slow fibers, weakness.
  • Limitations of the CAPN3' 1 ' deletion mouse model include the following: does not develop fibrosis, patients may have allele deletion and 2 nd allele missense/nonsense mutations.
  • mice including 5- sarcoglycan null mice.
  • BP blood pressure
  • mice are sedated using 1.5% isofluorane with constant monitoring of the plane of anesthesia and maintenance of the body temperature at 36.5-37.58°C. The heart rate is maintained at 450-550 beats/minute.
  • a BP cuff is placed around the tail, and the tail is then placed in a sensor assembly for noninvasive BP monitoring during anesthesia.
  • Ten consecutive BP measurements are taken.
  • Qualitative and quantitative measurements of tail BP including systolic pressure, diastolic pressure and mean pressure, are made offline using analytic software.
  • Radio telemetry devices are used to monitor ECG wave heights and interval durations in awake, freely moving mice.
  • Transmitter units are implanted in the peritoneal cavity of anesthetized mice and the two electrical leads are secured near the apex of the heart and the right acromion in a lead II orientation.
  • Mice are housed singly in cages over antenna receivers connected to a computer system for data recording. Unfiltered ECG data is collected for 10 seconds each hour for 35 days. The first 7 days of data are discarded to allow for recovery from the surgical procedure and ensure any effects of anesthesia has subsided.
  • Data waveforms and parameters are analyzed with the DSI analysis packages (ART 3.01 and Physiostat 4.01) and measurements are compiled and averaged to determine heart rates, ECG wave heights and interval durations.
  • Raw ECG waveforms are scanned for arrhythmias by two independent observers.
  • GRMD golden retriever muscular dystrophy
  • Phenotypic features in dogs include elevation of serum CK, CRDs on EMG, and histopathologic evidence of grouped muscle fiber necrosis and regeneration. Phenotypic variability is frequently observed in GRMD, as in humans. GRMD dogs develop paradoxical muscle hypertrophy which seems to play a role in the phenotype of affected dogs, with stiffness at gait, decreased joint range of motion, and trismus being common features. Objective biomarkers to evaluate disease progression include tetanic flexion, tibiotarsal joint angle, % eccentric contraction decrement, maximum hip flexion angle, pelvis angle, cranial sartorius circumference, and quadriceps femoris weight.
  • an AAV or rAAV vector including an AAV8 vector, an AAV9 vector or AAVhu.32 vector, that comprises a recombinant genome comprising a nucleotide sequence encoding a human AUF1 protein, including, for example, the nucleotide sequence of SEQ ID NO; 17, operably linked to one or more regulatory sequences that promote expression of the AUF1 protein in muscle cells of the subject, flanked by ITR sequences (see Table 2 for nucleotide sequences of potential components of these recombinant genomes), and, may be one of SEQ ID NO:31 to 36 (vectors spc-hu-opti-AUFl-CpG(-), tMCK-huAUFl, sp
  • the methods of treating human subjects provide for the treatment of LGMD type 1.
  • the methods of treating human subjects provide for the treatment of limb-girdle muscular dystrophy type 1C (LGMD1C), which has a mutation in caveolin 3.
  • the methods of treating human subjects provide for the treatment of limb-girdle muscular dystrophy type 1G (LGMD1G), which has a mutation in HNRPDL, a protein involved in mRNA biogenesis and metabolism.
  • the methods of treating human subjects provide for the treatment of LGMD type 2.
  • the methods of treatment provide for the treatment of a sarcoglycanopathy.
  • the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 2C (LGMD2C).
  • the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 2D (LGMD2D).
  • the methods of treatment provide for the treatment of limbgirdle muscular dystrophy type 2E (LGMD2E).
  • the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 2F (LGMD2F).
  • the methods of treatment of human subjects provide for the treatment of a LGMD2 dystrophinopathy.
  • the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 21 (LGMD2I) (mutation in FKRP).
  • the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 2K (LGMD2K) (mutation in POMTl).
  • the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 2M (LGMD2M) (mutation in FKTN).
  • the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 2N (LGMD2N) (mutation in POMT ). In some embodiments, the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 20 (LGMD20) (mutation in POMGnTl). In some embodiments, the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 2P (LGMD2P) (mutation in DAG1). In some embodiments, the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 2T (LGMD2T) (mutation in GMPPB). In some embodiments, the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 2U (LGMD2U) (mutation in ISP/CRPPA).
  • LGMD2N limb-girdle muscular dystrophy type 2N
  • the methods of treatment provide for the treatment of limb-girdle muscular dystrophy type 20 (LGMD20) (mutation in PO
  • the methods of treating human subjects provide for the treatment of a dysferlinopathy. In some embodiments, the methods of treating human subjects provide for the treatment of limb-girdle muscular dystrophy type 2B (LGMD2B) (mutation in DYSF).
  • LGMD2B limb-girdle muscular dystrophy type 2B
  • the methods of treating human subjects provide for the treatment of limb-girdle muscular dystrophy type 2L (LGMD2L) (mutation in ANO5). In some embodiments, the methods of treating human subjects provide for the treatment of limb-girdle muscular dystrophy type 2H (LGMD2H) (mutation in TRIM32). In some embodiments, the methods of treating human subjects provide for the treatment of limb-girdle muscular dystrophy type 2W (LGMD2W) (mutation in LIMS2). In some embodiments, the methods of treating human subjects provide for the treatment of limb-girdle muscular dystrophy type 2X (LGMD2X).
  • the methods of treating human subjects provide for the treatment of a calpainopathy. In some embodiments, the methods of treating human subjects provide for the treatment of limb-girdle muscular dystrophy type 2A (LGMD2A).
  • LGMD2A limb-girdle muscular dystrophy type 2A
  • the methods of treating human subjects provide a gene therapy vector comprising a genome comprising a transgene encoding p37 AUF1 . In embodiments, the methods of treating human subjects provide a gene therapy vector comprising a genome comprising a transgene encoding p40 AUF1 . In embodiments, the methods of treating human subjects provide a gene therapy vector comprising a genome comprising a transgene encoding p42 AUF1 . In embodiments, the methods of treating human subjects provide a gene therapy vector comprising a genome comprising a transgene encoding p45 AUF1 . In some embodiments, the therapy is a monotherapy comprising the AAV or rAAV vector.
  • kits for treating human subjects with gene therapy vectors with two or more AUF1 isoforms i.e., a combination of p37AUFl, p40AUFl, p42AUFl, and/or p45AUFl.
  • the methods of treating human subjects comprise a therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a muscle creatine kinase (MCK) promoter, synlOO promoter, CK6 promoter, CK7 promoter, CK8 promoter, CK9 promoter, dMCK promoter, tMCK promoter, smooth muscle 22 (SM22) promoter, myo-3 promoter, SPc5- 12 promoter, a mutant SPc5-12 promoter, creatine kinase (CK) 8e promoter, U6 promoter, Hl promoter, desmin promoter, Pitx3 promoter, skeletal alpha-actin promoter, MHCK7 promoter or a Sp-301 promoter.
  • MCK muscle creatine kinase
  • the methods of treating human subjects utilize AUF1 gene therapy constructs that have been codon-optimized. In embodiments, the methods of treating human subjects utilize AUF1 gene therapy constructs that have been CpG depleted. In embodiments, the AUF1 gene therapy constructs of the methods have the nucleotide sequences of SEQ ID NO: 31. In embodiments, the AUF 1 gene therapy constructs of the methods have the nucleotide sequences of SEQ ID NO: 32. In embodiments, the AUF1 gene therapy constructs of the methods have the nucleotide sequences of SEQ ID NO: 33. In embodiments, the AUF1 gene therapy constructs of the methods have the nucleotide sequences of SEQ ID NO: 34.
  • the AUF1 gene therapy constructs of the methods have the nucleotide sequences of SEQ ID NO: 35. In embodiments, the AUF1 gene therapy constructs of the methods have the nucleotide sequences of SEQ ID NO: 36.
  • the methods of treating human subjects comprise a therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 31 (spc- hu-opti-AUFl-CpG(-)). In embodiments, the methods of treating human subjects comprise a therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 32 (tMCK-huAUFl). In embodiments, the methods of treating human subjects comprise a therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 33 (spc5-12-hu-opti-AUFl-WPRE).
  • the methods of treating human subjects comprise a therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 34 (ss-CK7-hu-AUFl). In embodiments, the methods of treating human subjects comprise a therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 35 (spc-hu-AUFl -no-intron). In embodiments, the methods of treating human subjects comprise a therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 36 (D(+)-CK7AUFl).
  • the methods of treating human subjects utilize AAV8 gene therapy vectors. In embodiments, the methods of treating human subjects utilize AAV9 gene therapy vectors. In embodiments, the methods of treating human subjects utilize AAVhu.37 gene therapy vectors. In embodiments, the methods of treating human subjects utilize AAVhu.31 gene therapy vectors. In embodiments, the methods of treating human subjects utilize AAV hu.32 gene therapy vectors. In embodiments, the methods of treating human subjects utilize AAV Rh.74 gene therapy vectors. In embodiments, the methods of treating human subjects utilize AAV having a capsid that is at least 95% identical to SEQ ID NO: 114 (AAV8 capsid).
  • the methods of treating human subjects utilize AAV having a capsid that is at least 95% identical to SEQ ID NO: 115 (AAV9 capsid). In embodiments, the methods of treating human subjects utilize AAV having a capsid that is at least 95% identical to SEQ ID NO: 116 (hu.37 capsid). In embodiments, the methods of treating human subjects utilize AAV having a capsid that is at least 95% identical to SEQ ID NO: 117 (hu.31 capsid). In embodiments, the methods of treating human subjects utilize AAV having a capsid that is at least 95% identical to SEQ ID NO: 118 (hu.32 capsid).
  • the methods of treating human subjects utilize AAV having a capsid that is at least 95% identical to SEQ ID NO: 119 or SEQ ID NO: 120 (Rh.74 capsid).
  • the method results, for example, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration to the subject, in an increase in muscle cell mass, endurance and/or reduction in serum markers of muscle atrophy by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or greater (or 2 fold, 3 fold, or greater) relative to levels in the subject prior (for example 1 day, 1 week, or 2 weeks prior) to the administration or to reference levels.
  • a treatment in a method according to the disclosure may have a duration of at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6, years or more.
  • the subject may experience response to the gene therapy treatment (for example, increase in muscle mass, strength, or performance) 2 weeks, 4 weeks, 6 weeks, 2 months, 3 months, or 6 months after administration.
  • kits for treating human subjects for any LGMD that can be treated by providing a functional AUF1, as disclosed herein, in combination with a second therapeutic, wherein the second therapeutic can treat a LGMD or ameliorate one or more symptoms thereof.
  • Gene therapy vectors that express AUF1 provided herein can be administered as a monotherapy or in combination with a second therapeutic described herein to treat a LGMD.
  • 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 with another therapeutic as disclosed herein.
  • the methods of combination treatment provide for the treatment of LGMD type 1.
  • the methods of combination treatment provide for the treatment of limbgirdle muscular dystrophy type 1C (LGMD1C), which has a mutation in caveolin 3.
  • the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 1G (LGMD1G), which has a mutation in HNRPDL, a protein involved in mRNA biogenesis and metabolism.
  • the methods of combination treatment provide for the treatment of LGMD type 2. In some embodiments the methods of combination treatment provide for the treatment of a sarcoglycanopathy. In some embodiments the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2C (LGMD2C). In some embodiments the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2D (LGMD2D). In some embodiments the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2E (LGMD2E). In some embodiments the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2F (LGMD2F).
  • LGMD2C limb-girdle muscular dystrophy type 2C
  • LGMD2D limb-girdle muscular dystrophy type 2D
  • LGMD2E limb-girdle muscular dystrophy type 2E
  • the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2F (
  • the methods of combination treatment provide for the treatment of a limb-girdle muscular dystrophy type 2 (LGMD2) dystrophinopathy.
  • the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 21 (LGMD2I) (mutation in FKRP ⁇
  • the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2K (LGMD2K) (mutation in POMTl).
  • the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2M (LGMD2M) (mutation in FKTN).
  • the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2N (LGMD2N) (mutation in P0MT2'). In some embodiments, the methods of combination treatment provide for the treatment of limbgirdle muscular dystrophy type 20 (LGMD20) (mutation in POMGriTl). In some embodiments, the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2P (LGMD2P) (mutation in DAG1). In some embodiments, the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2T (LGMD2T) (mutation in GMPPB). In some embodiments, the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2U (LGMD2U) (mutation in ISP/CRPPA).
  • the methods of combination treatment of human subjects provide for the treatment of a dysferlinopathy.
  • the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2B (LGMD2B) (mutation in DESF).
  • LGMD2B limb-girdle muscular dystrophy type 2B
  • the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2L (LGMD2L) (mutation in ANO5). In some embodiments, the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2H (LGMD2H) (mutation in TRIM32). In some embodiments, the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2W (LGMD2W) (mutation in LIMS2'). In some embodiments, the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2X (LGMD2X).
  • the methods of monotherapy or combination treatment provide for the treatment of a calpainopathy. In some embodiments, the methods of combination treatment provide for the treatment of limb-girdle muscular dystrophy type 2A (LGMD2A).
  • the methods of treating human subjects provide a first gene therapy vector comprising a genome comprising a transgene encoding p37 AUF1 . In embodiments, the methods of treating human subjects provide a first gene therapy vector comprising a genome comprising a transgene encoding p40 AUF1 . In embodiments, the methods of treating human subjects provide a first gene therapy vector comprising a genome comprising a transgene encoding p42 AUF1 .
  • the methods of treating human subjects provide a first gene therapy vector comprising a genome comprising a transgene encoding p45 AUF1 .
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a muscle creatine kinase (MCK) promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a synlOO promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a CK6 promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a CK7 promoter. In embodiments, the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a CK8 promoter. In embodiments, the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a CK9 promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a dMCK promoter. In embodiments, the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a tMCK promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a smooth muscle 22 (SM22) promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a myo-3 promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a Spc5-12 promoter. In embodiments, the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a creatine kinase (CK) 8e promoter. In embodiments, the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a U6 promoter.
  • CK creatine kinase
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a Hl promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a desmin promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a Pitx3 promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a skeletal alpha-actin promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a MHCK7 promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an rAAV particle comprising a nucleic acid molecule encoding an AUF1 protein, or functional fragment thereof, operably coupled to a Sp-301 promoter.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule comprising an AUF1 gene therapy construct that has been codon-optimized.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle comprising a nucleic acid molecule comprising an AUF1 gene therapy construct that has been CpG depleted.
  • the AUF1 gene therapy constructs of the methods of combination treatment have the nucleotide sequences of SEQ ID NO: 31.
  • the AUF1 gene therapy constructs of the methods of combination treatment have the nucleotide sequences of SEQ ID NO: 32.
  • the AUF1 gene therapy constructs of the methods of combination treatment have the nucleotide sequences of SEQ ID NO: 33. In embodiments, the AUF1 gene therapy constructs of the methods of combination treatment have the nucleotide sequences of SEQ ID NO: 34. In embodiments, the AUF1 gene therapy constructs of the methods of combination treatment have the nucleotide sequences of SEQ ID NO: 35. In embodiments, the AUF1 gene therapy constructs of the methods of combination treatment have the nucleotide sequences of SEQ ID NO: 36.
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 31 (spc-hu-opti-AUFl-CpG(-)). In embodiments, the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 32 (tMCK-huAUFl). In embodiments, the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 33 (spc5-12-hu-opti-AUFl-WPRE).
  • the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 34 (ss-CK7-hu-AUFl). In embodiments, the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 35 (spc-hu-AUFl -no-intron). In embodiments, the methods of treating human subjects comprise a first therapeutic comprising an AAV or rAAV particle having the nucleotide sequence of SEQ ID NO: 36 (D(+)-CK7AUFl).
  • the methods of treating human subjects comprise a first therapeutic utilizing AAV8 gene therapy vectors. In embodiments, the methods of treating human subjects comprise a first therapeutic utilizing AAV9 gene therapy vectors. In embodiments, the methods of treating human subjects comprise a first therapeutic utilizing AAVhu.32 gene therapy vectors. In embodiments, the methods of treating human subjects comprise a first therapeutic utilizing AAV having a capsid that is at least 95% identical to SEQ ID NO: 114 (AAV8 capsid). In embodiments, the methods of treating human subjects comprise a first therapeutic utilizing AAV having a capsid that is at least 95% identical to SEQ ID NO: 115 (AAV9 capsid). In embodiments, the methods of treating human subjects comprise a first therapeutic utilizing AAV having a capsid that is at least 95% identical to SEQ ID NO: 118 (AAVhu.32 capsid).
  • the AAV or rAAVparticle comprises a construct having the nucleotide sequence of one of SEQ ID Nos: 31 to 36 (spc-hu-opti-AUFl-CpG(-), tMCK-huAUFl, spc5-12-hu-opti-AUFl-WPRE, ss-CK7-hu- AUF1, spc-hu-AUFl -no-intron, or D(+)-CK7AUFl, respectively), including where the rAAV is an AAV8 serotype or an AAV9 serotype or AAVhu.32 serotype.
  • the first therapeutic is an AAV particle.
  • the second therapeutic is an AAV particle.
  • the first and second therapeutics are AAV particles.
  • the first therapeutic is a rAAV particle.
  • the second therapeutic is a rAAV particle.
  • the first and second therapeutics are rAAV particles.
  • the second therapeutic is a second therapeutic protein , pharmaceutical composition, including an AAV or rAAV vector particle comprising a therapeutic protein construct, including microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof, including where the AAV or rAAV is an AAV8 serotype or an AAV9 serotype or AAVhu.32 serotype.
  • a therapeutic protein construct including microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof, including where the AAV or rAAV is an AAV8 serotype or an AAV9 sero
  • the AUF1 gene therapy product and the therapeutic protein are delivered at the same time or are delivered 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, including that the second product is administered prior to any immune response against the first gene therapy product.
  • the therapeutic protein such as microdystrophin, a-sarcoglycan, P- sarcoglycan, y- sarcoglycan, 5-sarcoglycan, calpain 3, or calcium/calmodulin-dependent protein kinase II P isoform proteins or portions thereof
  • the AUF1 gene therapy product and the therapeutic protein gene therapy product are delivered simultaneously or are delivered within 1 hour, 2 hours, or 3 hours, including that the second product is administered prior to any immune response against the first gene therapy product.
  • the AUF1 gene therapy product and the therapeutic protein gene therapy product both comprise an AAV or rAAV vector of the same serotype and are delivered simultaneously or are delivered no more than 1 hour apart.
  • the second therapeutic is a mutation suppression therapy, a steroid therapy, an immunosuppressive/anti-inflammatory therapy, any therapy that treats one or more symptoms of the LGMD, as disclosed herein in more detail or any combination thereof.
  • a therapeutic is administered in addition to the AUF1 gene therapy vector and the second therapeutic protein gene therapy vector, as a third therapeutic, which may be a mutation suppression therapy, a steroid therapy, an immunosuppressive/anti-inflammatory therapy, any therapy that treats one or more symptoms of the LGMD, as disclosed herein in more detail or any combination thereof.
  • Dosing for each second therapeutic can be any of the known doses for administering each of the second therapeutics.
  • the second therapeutic can be administered to alleviate or further alleviate one or more symptoms or characteristics of LGMD which may be assessed by any of, but not limited to, the following assays on the subject: prolongation of time to loss of walking, improvement of muscle strength, improvement of the ability to lift weight, improvement of the time taken to rise from the floor, improvement in the nine-meter walking time, improvement in the time taken for four-stairs climbing, improvement of the leg function grade, improvement of the pulmonary function, improvement of cardiac function, improvement of the quality of life.
  • Assays is known to the skilled person.
  • the alleviation of one or more symptom(s) of Duchenne Muscular Dystrophy may be assessed by measuring an improvement of a muscle fiber function, integrity and/or survival as later defined herein.
  • a treatment in a method according to the disclosure may have a duration of at least one week, at least one month, at least several months, at least one year, at least 2, 3, 4, 5, 6 years, or more.
  • the frequency of administration of any of the second therapeutics, including those not delivered by gene therapy and described herein may depend on several parameters such as the age of the patient, the type of mutation, the number of molecules (dose), the formulation of said molecule.
  • the frequency may be ranged between at least once in two weeks, or three weeks or four weeks or five weeks or a longer time period.
  • the first therapeutic and second therapeutic, and optionally a third or even further therapeutics can be administered to an individual in any order.
  • a third therapeutic e.g., a third therapeutic
  • said therapeutics are administered simultaneously (meaning that said therapeutics are administered within 10 hours, including within one hour).
  • said therapeutics are administered sequentially.
  • administration of the first and second therapeutic can occur within 7, 10, or 14 days of each other.
  • simultaneous administration can mean the first and second therapeutic are formulated together in a single composition or each can be formulated by itself.
  • a third therapeutic is administered concurrently with the first and/or second therapeutic, or is administered at a separate time, including on a regular dosing schedule, such as daily, weekly, or monthly.
  • the first and second therapeutics provide a synergistic therapeutic effect with respect to one or more clinical end points in the treatment of a LGMD in a subject, in particular, where the therapeutic effect is greater than the additive therapeutic effects of the first and second therapeutics when administered alone.
  • the first and second therapeutics provide a synergistic effect in that the therapeutics result in improvements in different sets of clinical endpoints such that the therapeutic benefit of the combination is greater than the therapeutic benefit of each therapeutic individually.
  • the first, second and third therapeutics when a third or further therapeutics are administered, provide a synergistic therapeutic effect with respect to one or more clinical end points in the treatment of a LGMD in a subject, in particular, where the therapeutic effect is greater than the additive therapeutic effects of the first, second and third therapeutics when administered alone.
  • the first, second and third therapeutics provide a synergistic effect in that the therapeutics result in improvements in different sets of clinical endpoints such that the therapeutic benefit of the combination is greater than the therapeutic benefit of each therapeutic individually.
  • first therapeutic is an AAV or rAAV vector comprising a transgene encoding a AUF1 disclosed herein
  • the second therapeutic is a gene therapy vector, including an rAAV gene therapy vector encoding a thereapeutic protein as disclosed herein.
  • the transgene that encodes a therapeutic protein encodes microdystrophin, a-sarcoglycan, P-sarcoglycan, y-sarcoglycan, 5-sarcoglycan, calpain 3, calcium/calmodulin-dependent protein kinase II P isoform proteins, other protein (other than AUF1), or portions thereof.
  • the therapeutically effective amount of the AAV or rAAV particle encoding the therapeutic protein such as microdystrophin, a-sarcoglycan, P-sarcoglycan, y-sarcoglycan, 5-sarcoglycan, calpain 3, calcium/calmodulin-dependent protein kinase II P isoform proteins, other protein (other than AUF1), or portions thereof is administered intravenously or intramuscularly at dose of 1E8 vg/kg to 2E15 vg/kg, such as IxlO 10 to IxlO 15 genome copies/kg; 5xl0 10 to IxlO 15 genome copies/kg; IxlO 11 to IxlO 15 genome copies/kg; 5xl0 u to IxlO 15 genome copies/kg; IxlO 12 to IxlO 15 genome copies/kg; 2xl0 12 to IxlO 15 genome copies/kg; IxlO 13 to IxlO 15 genome
  • the first therapeutic is an AAV or rAAV particle comprises a construct having the nucleotide sequence of one of SEQ ID Nos: 31 to 36 (spc-hu- opti-AUFl-CpG(-), tMCK-huAUFl, spc5-12-hu-opti-AUFl-WPRE, ss-CK7-hu-AUFl, spc-hu- AUF1 -no-intron, or D(+)-CK7AUFl, respectively), including where the AAV or rAAV is an AAV8 serotype or an AAV9 serotype or an AAVhu.32 serotype, and the second therapeutic is an AAV or rAAV particle which has a recombinant genome having the nucleotide sequence of a therapeutic protein as disclosed herein, including where the AAV or rAAV is an AAV8 serotype or is an AAV9 serotype or is an
  • the ratio of the AAV or rAAV particle having a transgene encoding AUF1 and the AAV or rAAV particle having a transgene encoding the therapeutic protein (other than AUF1) is 1 : 1, 1 :2, 1 :4, 1 :5; 1 : 10, 1 :50, 1 : 100 or 1 : 1000.
  • the ratio of the AUF1 gene therapy vector and the microdystrophin gene therapy vector is 0.5: 1, 0.25: 1, 0.2: 1, or 0.1 : 1. 8.2.
  • the AAV vector is administered.
  • the rAAV vector is administered.
  • first therapeutic is an AAV or rAAV vector comprising a transgene encoding a AUF1 disclosed herein
  • the second therapeutic is a gene therapy vector, including an AAV or rAAV gene therapy vector encoding microdystrophin as disclosed herein.
  • the transgene that encodes a microdystrophin protein consists of dystrophin domains arranged from amino-terminus to the carboxy terminus: ABD- H1-R1-R2-R3-H3-R24-H4-CR-CT, wherein ABD is an actin-binding domain of dystrophin, Hl is a hinge 1 region of dystrophin, R1 is a spectrin 1 region of dystrophin, R2 is a spectrin 2 region of dystrophin, R3 is a spectrin 3 region of dystrophin, H3 is a hinge 3 region of dystrophin, R24 is a spectrin 24 region of dystrophin, H4 is hinge 4 region of dystrophin, CR is the cysteine-rich region of dystrophin, and CT comprises at least the portion of the CT comprising an al-syntrophin binding site.
  • ABD is an actin-binding domain of dystrophin
  • Hl is a hinge 1 region of dystrophin
  • the CT comprises or consists of the proximal 194 amino acids of the C-terminus of dystrophin or at least the proximal portion of the C-terminus encoding human dystrophin amino acid residues 3361-3554 of SEQ ID NO: 51 (UniProtKB-Pl 1532) or at least the proximal portion of the C-terminus encoded by exons 70 to 74 and the first 36 amino acids of the amino acid sequence encoded by the nucleotide sequence of exon 75.
  • the microdystrophin protein has the amino acid sequence of the microdystrophin encoded by DYS1, DYS3 or DYS5 (SEQ ID NO: 176, 177, or 178). Alternatively, the microdystrophin protein has an amino acid sequence of one of SEQ ID NO: 179 to 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 coding for the microdystrophin is operably linked to regulatory sequences, including promoters as listed in Table 7 and other regulatory elements, for example, as in Table 2 or 8.
  • the rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 184, 185, or 186 (RGX-DYS-1, RGX-DYS-3, or RGX-DYS-5) or alternatively SpcVl-pDysl (SEQ ID NO: 188) or SpcV2-pDysl (SEQ ID NO: 190).
  • the rAAV is an AAV8 serotype, AAV9 serotype, or AAVhu.32 or any other serotype, including with a tropism for muscle cells, as disclosed supra.
  • the microdystrophin gene therapy is SGT-001, serotype AAV9, rAAVrh74.MHCK7. micro-dystrophin, SRP-9001 (see, Willcocks et al. “Assessment of rAAVrh.74. MHCK7. micro-dystrophin Gene Therapy Using Magnetic Resonance Imaging in Children with Duchenne Muscular Dystrophy,” JAMA Network Open 4:e2031851 (2021), which is hereby incorporated by reference in its entirety); GNT-004 (Le Guiner et al. “Long-Term Microdystrophin Gene Therapy is Effective in a Canine Model of Duchenne Muscular Dystrophy,” Nat.
  • Pfizer PF-06939926 AAV9 mini-dystrophin or any other mini-dystrophin or micro-dystrophin construct.
  • the AAV vector is administered.
  • the rAAV vector is administered.
  • the a-sarcoglycan protein has the amino acid sequence of SEQ ID NO: 144.
  • the P-sarcoglycan protein has the amino acid sequence of SEQ ID NO: 145.
  • the y-sarcoglycan protein has the amino acid sequence of SEQ ID NO: 146.
  • the 5-sarcoglycan protein has the amino acid sequence of SEQ ID NO: 147.
  • the first therapeutic is a AAV vector. In some embodiments, the first therapeutic is a rAAV vector.
  • the a-sarcoglycan protein is encoded by the nucleic acid sequence of SEQ ID NO: 144.
  • the nucleic acid sequence coding for the a- sarcoglycan is operably linked to regulatory sequences, including promoters as listed in Table 6 and other regulatory elements, for example, as in Table 2 or 9.
  • the AAV or rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 144.
  • the AAV or rAAV is an AAV8 serotype or AAV9 serotype or AAV hu.32 or any other serotype, including with a tropism for muscle cells, as disclosed supra.
  • the P-sarcoglycan protein is encoded by the nucleic acid sequence of SEQ ID NO: 145.
  • the nucleic acid sequence coding for the a- sarcoglycan is operably linked to regulatory sequences, including promoters as listed in Table 8 and other regulatory elements, for example, as in Table 2 or 9.
  • the AAV or rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 145.
  • the AAV or rAAV is an AAV8 serotype or AAV9 serotype or AAV hu.32 or any other serotype, including with a tropism for muscle cells, as disclosed supra.
  • the y-sarcoglycan protein is encoded by the nucleic acid sequence of SEQ ID NO: 146.
  • the nucleic acid sequence coding for the a- sarcoglycan is operably linked to regulatory sequences, including promoters as listed in Table 8 and other regulatory elements, for example, as in Table 2 or 9.
  • the AAV or rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 146.
  • the AAV or rAAV is an AAV8 serotype or AAV9 serotype or AAV hu.32 or any other serotype, including with a tropism for muscle cells, as disclosed supra.
  • the 5-sarcoglycan protein is encoded by the nucleic acid sequence of SEQ ID NO: 147.
  • the nucleic acid sequence coding for the a- sarcoglycan is operably linked to regulatory sequences, including promoters as listed in Table 8 and other regulatory elements, for example, as in Table 2 or 9.
  • the AAV or rAAV has a recombinant genome having the nucleotide sequence of SEQ ID NO: 147.
  • the AAV or rAAV is an AAV8 serotype or AAV9 serotype or AAV hu.32 or any other serotype, including with a tropism for muscle cells, as disclosed supra.
  • the a-, P-, y- or 5-sarcoglycan gene therapy is any other a-, P-, y- or 5-sarcoglycan construct.
  • the therapeutically effective amount of the AAV or rAAV particle encoding the a-sarcoglycan is administered intravenously or intramuscularly at dose of 1 * 10 8 genome copies/kg to 2* 10 15 genome copies/kg or 2* 10 13 to IxlO 15 genome copies/kg.
  • the therapeutically effective amount of the AAV or rAAV particle encoding the P-sarcoglycan is administered intravenously or intramuscularly at dose of 1 x 10 8 genome copies/kg to 2* 10 15 genome copies/kg or 2* 10 13 to IxlO 15 genome copies/kg.
  • the therapeutically effective amount of the AAV or rAAV particle encoding the y- sarcoglycan is administered intravenously or intramuscularly at dose of 1 x 10 8 genome copies/kg to 2x 10 15 genome copies/kg or 2x 10 13 to IxlO 15 genome copies/kg.
  • the therapeutically effective amount of the AAV or rAAV particle encoding the 5-sarcoglycan is administered intravenously or intramuscularly at dose of 1 x 10 8 genome copies/kg to 2x 10 15 genome copies/kg or 2x l0 13 to IxlO 15 genome copies/kg.
  • the first therapeutic is an AAV or rAAV particle comprising a construct having the nucleotide sequence of one of SEQ ID Nos: 31 to 36 (spc-hu- opti-AUFl-CpG(-), tMCK-huAUFl, spc5-12-hu-opti-AUFl-WPRE, ss-CK7-hu-AUFl, spc-hu- AUF1 -no-intron, or D(+)-CK7AUFl, respectively), including where the AAV or rAAV is an AAV8 serotype or an AAV9 serotype or an AAV hu.32 serotype, and the second therapeutic is an rAAV particle which has a recombinant genome having the nucleotide sequence of SEQ ID NO: 144, 145, 146 or 147, including where the AAV or rAAV is an AAV8 serotype or is an AAV
  • the ratio of the AAV or rAAV particle having a transgene encoding AUF1 and the AAV or rAAV particle having a transgene encoding the a-, P-, y- or 5-sarcoglycan is 1 : 1, 1 :2, 1 :4, 1 :5; 1 : 10, 1 :50, 1 : 100, or 1 : 1000.
  • the ratio of the AUF1 gene therapy vector and the a-, P-, y- or 5-sarcoglycan gene therapy vector is 0.5: 1, 0.25: 1, 0.2: 1, or 0.1 : 1.
  • a calpainopathy in a subject in need thereof, comprising administering to the subject an AAV or rAAV vector comprising a transgene encoding an AUF1 disclosed herein.
  • the AAV vector is administered.
  • the rAAV vector is administered.
  • the first therapeutic is an AAV vector.
  • the first therapeutic is a rAAV vector.
  • the calpain 3 protein has the amino acid sequence of SEQ ID NOs: 151, 152, 153, 154, 155, 156, 157, 158, or 159.
  • Table 12 provides amino acid sequences of the calpain 3 embodiments in accordance with the present disclosure.
  • calpain 3 as defined by SEQ ID NOs: 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 (murine calpain 3 variant 1/ isoform a), 158 (murine calpain 3 variant 2/ isoform a), or 159 (murine calpain 3 variant 3/ isoform c).
  • 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 NOs: 151, 152, 153, 154, 155, 156, 157, 158, or 159 and maintain functional calpain 3 activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in animal models disclosed infra. Table 12: Amino Acid Sequences of Calpain 3 Proteins
  • the calpain 3 protein is encoded by the nucleic acid sequence of SEQ ID NOs: 160, 161, 162, 163, 164, 165, 166, 167, or 168.
  • the nucleic acid sequence coding for the calpain 3 protein is operably linked to regulatory sequences, including promoters as listed in Table 8 and other regulatory elements, for example, as in Table 2 or 9.
  • the AAV or rAAV vector has a recombinant genome having the nucleotide sequence of SEQ ID NO: 160, 161, 162, 163, 164, 165, 166, 167, or 168.
  • the rAAV is an AAV8 serotype or AAV9 serotype or AAV hu.32 or any other serotype, including with a tropism for muscle cells, as disclosed supra.
  • Table 13 provides nucleic acid sequences encoding calpain 3 embodiments in accordance with the present disclosure.
  • calpain 3 as defined by SEQ ID NOs: 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
  • calpain 3 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 NOs: 160, 161, 162, 163, 164, 165, 166, 167, or 168 and maintain functional calpain 3 activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in animal models disclosed infra.
  • Table 13 Nucleic Acid Sequences of Calpain 3 Proteins
  • the calcium/calmodulin-dependent protein kinase II P isoform protein has the amino acid sequence of SEQ ID NO: 169 or 170.
  • Table 14 provides amino acid sequences of the calcium/calmodulin-dependent protein kinase II P embodiments in accordance with the present disclosure. It is also contemplated that other embodiments are substituted variant of calcium/calmodulin-dependent protein kinase II P as defined by SEQ ID NOs: 169 (human calcium/calmodulin-dependent protein kinase II P) or 170 (murine calcium/calmodulin-dependent protein kinase II P). For example, conservative substitutions can be made to SEQ ID NOs: 169 or 170 and substantially maintain its functional activity.
  • calcium/calmodulin-dependent protein kinase II P 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 NOs: 169 or 170 and maintain functional calcium/calmodulin-dependent protein kinase II P activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in animal models disclosed infra.
  • the calcium/calmodulin-dependent protein kinase II P isoform protein is encoded by the nucleic acid sequence of SEQ ID NO: 171 or 172.
  • the nucleic acid sequence coding for the calcium/calmodulin-dependent protein kinase II P isoform protein is operably linked to regulatory sequences, including promoters as listed in Table 8 and other regulatory elements, for example, as in Table 2 or 9.
  • the AAV or rAAV vector has a recombinant genome having the nucleotide sequence of SEQ ID NO: 171 or 172.
  • the rAAV is an AAV8 serotype or AAV9 serotype or AAV hu.32 or any other serotype, including with a tropism for muscle cells, as disclosed supra.
  • Table 15 provides nucleic acid sequences encoding calcium/calmodulin- dependent protein kinase II P protein embodiments in accordance with the present disclosure. It is also contemplated that other embodiments are substituted variant of calcium/calmodulin- dependent protein kinase II P protein as defined by SEQ ID NOs: 171 (human calcium/calmodulin-dependent protein kinase II P protein) or 172 (murine calcium/calmodulin- dependent protein kinase II P protein). For example, conservative substitutions can be made to SEQ ID NOs: 171 or 172 and substantially maintain its functional activity.
  • the calcium/calmodulin-dependent protein kinase II P 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 NOs: 171 or 172 and maintain functional calcium/calmodulin-dependent protein kinase II P protein activity, as determined, for example, by one or more of the in vitro assays or in vivo assays in animal models disclosed infra.
  • the calpain 3 (G47W3) or calcium/calmodulin-dependent protein kinase II P isoform (CaMKIIfi) gene therapy is any other calpain 3 (G47W3) or calcium/calmodulin-dependent protein kinase II P isoform (CaMKIIfi) construct.
  • the therapeutically effective amount of the AAV or rAAV particle encoding the calpain 3 protein is administered intravenously or intramuscularly at dose of P I O 8 genome copies/kg to 2> ⁇ 10 15 genome copies/kg or 2> ⁇ 10 13 to IxlO 15 genome copies/kg.
  • the therapeutically effective amount of the AAV or rAAV particle encoding the calcium/calmodulin-dependent protein kinase II P isoform protein is administered intravenously or intramuscularly at dose of 1 x 10 8 genome copies/kg to 2* 10 15 genome copies/kg or 2> ⁇ 10 13 to IxlO 15 genome copies/kg.
  • the first therapeutic is an AAV or a rAAV particle comprising a construct having the nucleotide sequence of one of SEQ ID Nos: 31 to 36 (spc-hu- opti-AUFl-CpG(-), tMCK-huAUFl, spc5-12-hu-opti-AUFl-WPRE, ss-CK7-hu-AUFl, spc-hu- AUF1 -no-intron, or D(+)-CK7AUFl, respectively), including where the AAV or rAAV is an AAV8 serotype or an AAV9 serotype or an AAV hu.32 serotype, and the second therapeutic is an AAV or rAAV particle which has a recombinant genome having the nucleotide sequence of SEQ ID NO: 144, 145, 146 or 147, including where the AAV or rAAV is an AAV8 serotype
  • the ratio of the AAV or rAAV particle having a transgene encoding AUF1 and the AAV or rAAV particle having a transgene encoding the a-, P-, y- or 5-sarcoglycan is 1 : 1, 1 :2, 1 :4, 1 :5; 1: 10, 1 :50, 1 : 100 or 1 : 1000.
  • the ratio of the AUF1 gene therapy vector and the a-, P-, y- or 5- sarcoglycan gene therapy vector is 0.5: 1, 0.25: 1, 0.2: 1, or 0.1 : 1. 8.4. Mutation Suppression Therapy
  • a combination of the AAV or rAAV encoding AUF1, the AAV or rAAV encoding microdystrophin or a-, P-, y-, or 5-sarcoglycan, and the mutation suppression therapeutic (as a third therapeutic) is administered to treat or ameliorate the symptoms of the LGMD of the subject.
  • the first therapeutic is an AAV vector.
  • the first therapeutic is a rAAV vector.
  • the second therapeutic is ataluren.
  • ataluren is administered orally.
  • ataluren can be administered in a dose of 10 mg/kg/day to 200 mg/kg/day.
  • ataluren can be administered in a dose of 40 mg/kg.
  • the dosing can be 10 mg/kg in the morning, 10 mg/kg at midday, and 20 mg/kg in the evening.
  • the length of time for ataluren administration can be weeks, months, or years.
  • treatment resulted in increased ability to walk/run longer distances and/or increased ability to climb stairs compared to pre-treatment levels.
  • the second therapeutic (or third therapeutic is gentamicin.
  • gentamicin is administered intravenously.
  • gentamicin can be administered in a dose of 3 mg/kg/day to 25 mg/kg/day.
  • gentamicin can be administered in a dose of 7.5 mg/kg/day.
  • the length of time for ataluren administration can be weeks, months, or years.
  • treatment resulted in increased hearing, kidney function and/or muscle strength compared to pre-treatment levels.
  • the mutation suppressor therapy is a nonsense suppressor mutation.
  • the subject can have a nonsense mutation and the second therapeutic enables a ribosome to read through a premature nonsense mutation.
  • Nonsense suppressor therapies can be of two general classes.
  • a first class includes compounds that disrupt codon-anticodon recognition during protein translation in a eukaryotic cell, so as to promote readthrough of a nonsense codon. These agents can act by, for example, binding to a ribosome so as to affect its activity in initiating translation or promoting polypeptide chain elongation, or both.
  • nonsense suppressor agents of this class can act by binding to rRNA (e.g., by reducing binding affinity to 18S rRNA).
  • a second class are those that provide the eukaryotic translational machinery with a tRNA that provides for incorporation of an amino acid in a polypeptide where the mRNA normally encodes a stop codon, e.g., suppressor tRNAs.
  • the steroid therapy is a glucocorticoid steroid.
  • a combination of the rAAV encoding AUF1, the rAAV encoding microdystrophin or a-, P-, y-, or 5-sarcoglycan, and the steroid therapy (as a third therapeutic) is administered to treat or ameliorate the symptoms of the LGMD of the subject.
  • the steroid therapy is prednisone, deflazacort, Vamorolone, or Spironolactone, or a combination thereof.
  • Spironolactone is an aldosterone antagonist and although may not be considered a steroid, it is used in a similar manner to steroids and is often compared to corticosteroids.
  • 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.
  • the steroid dose can be increased or decreased based on growth, weight, and other side effects experienced.
  • dosing can be either daily or high dose weekends.
  • doses of twice weekly can go up to 250 mg/day of prednisone or 300 mg/day of deflazacort.
  • dosing can be 10 days on, 10 days off, etc.
  • the first therapeutic is an rAAV comprising a transgene encoding an AUF1 disclosed herein and the second therapeutic is an immunosuppressive or anti-inflammatory therapy.
  • a combination of the rAAV encoding AUF1, the rAAV encoding microdystrophin or a-, P-, y-, or 5-sarcoglycan, and the immunosuppressive/anti-inflammatory therapeutic is administered to treat or ameliorate the symptoms of the LGMD of the subject.
  • the immunosuppressive or anti-inflammatory therapy is edasalonexent.
  • the immunosuppressive or anti-inflammatory therapy is canakinumab.
  • Canakinumab is a monoclonal antibody, targeting ILlb, which is a cytokine that plays a role in inflammation and immune responses.
  • canakinumab can be administered subcutaneously.
  • administration can be daily, weekly, or monthly.
  • the length of treatment can be weeks, months or years.
  • canakinumab can be administered in a dose of 0.5 mg/kg to 20 mg/kg.
  • canakinumab can be administered in a dose of 2 mg/kg or 4 mg/kg.
  • administration can be a single dose via subcutaneous injection of 2 or 4 mg/kg.
  • 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 unusually high levels in the muscles of people with DMD. Fibrosis is a hallmark of muscular dystrophies, contributing to muscle weakness and injury, including to cardiac muscle.
  • CTGF connective tissue growth factor
  • pamrevlumab can be administered intravenously. In some embodiments, administration can be daily, weekly, or monthly.
  • the length of treatment can be weeks, months or years.
  • Pamrevlumab can be administered in a dose of 10 mg/kg to 200 mg/kg.
  • Pamrevlumab can be administered in a dose of 35 mg/kg.
  • administration can be every two weeks via intravenous (IV) infusions of 35 mg/kg.
  • the immunosuppressive or anti-inflammatory therapy is imlifidase.
  • Imlifidase is an enzyme that rapidly cleaves IgG antibodies, thereby suppressing the immune response against AAVs. Thus, once the immune response against AAVs has been suppressed, gene therapy treatments using an AAV vector can be used more efficiently.
  • imlifidase can be administered intravenously. In some embodiments, administration can be daily, weekly, or monthly. In some embodiments, the length of treatment can be weeks, months or years.
  • imlifidase can be administered in a dose of 0.1 mg/kg to 10 mg/kg. In some embodiments, imlifidase can be administered in a dose of 0.25 mg/kg. For example, administration can a single dose via intravenous (IV) infusions of 0.25 mg/kg.
  • a therapy that treats one or more symptoms of the LGMD can also include any of the mutation suppression therapies, steroid therapies, and immunosuppressive/anti-inflammatory therapies described herein.
  • a combination of the rAAV encoding AUF1, the rAAV encoding microdystrophin or a-, P-, y-, or 5-sarcoglycan, and therapy that treats one or more symptoms of the LGMD (as a third therapeutic) is administered to treat or ameliorate the symptoms of the LGMD of the subject.
  • the one or more symptoms of the LGMD is decreased muscle mass and/or strength, wherein the second therapeutic improves muscle mass and/or strength.
  • the second therapeutic can be spironolactone (same as described for steroid therapy), Follistatin, SERCA2a, EDG-5506, Tamoxifen, Givinostat, ASP0367, or a combination thereof.
  • follistatin or follistatin variants can be used as the second therapeutic.
  • follistatin can be administered as a gene therapy in a viral vector such as AAV.
  • SERCA2a can be used as the second therapeutic (or a third therapeutic).
  • SERCA2a can be administered as a gene therapy in a viral vector such as AAV.
  • SERCA2a can be administered intravenously.
  • administration can be daily, weekly, or monthly.
  • the length of treatment can be weeks, months or years.
  • IxlO 11 to IxlO 14 vg is administered.
  • 6xl0 12 vg is administered.
  • EDG-5506 is a small molecule therapy that can stabilize skeletal muscle fibers
  • SERCA2a can be administered orally. In some embodiments, administration can be daily, weekly, or monthly. In some embodiments, the length of treatment can be weeks, months or years.
  • the second therapeutic (or third therapeutic) is tamoxifen. In some embodiments, tamoxifen can be administered orally. In some embodiments, administration can be daily, weekly, or monthly. In some embodiments, the length of treatment can be weeks, months or years. In some embodiments, tamoxifen can be administered in a dose of 0.1 mg/kg to 20 mg/kg.
  • tamoxifen can be administered in a dose of 0.6 mg/kg. In some embodiments, tamoxifen can be administered in a dose of 5 mg to 100 mg. For example, administration can be a single oral dose of 0.6 mg/kg daily.
  • Givinostat is a molecule that inhibits enzymes called histone deacetylases (HDACs) that turn off gene expression and can reduce a muscle’s ability to regenerate. By inhibiting HDACs, givinostat may reduce fibrosis and the death of muscle cells while also enabling muscles to regenerate.
  • Givinostat is administered via oral suspension. In some embodiments, administration can be daily, weekly, or monthly. In some embodiments, the length of treatment can be weeks, months or years. In some embodiments, Givinostat can be administered in a dose of 1 mg/ml to 100 mg/ml. In some embodiments, Givinostat can be administered in a dose of 10 mg/ml. For example, administration can be twice daily via oral suspension of 10 mg/ml.
  • ASP0367 is used turn on the PPAR delta (5) pathway.
  • the PPAR-5 pathway regulates mitochondria by turning on different genes in the cell. When the pathway is on, the mitochondria use fatty acids more often and more mitochondria are made. Using more fatty acids for energy results in increased energy production.
  • ASP0367 is a mitochondrial-directed medicine for the treatment of DMD, which is designed to treat DMD by increasing fatty acid oxidation and mitochondrial biogenesis in muscle cells.
  • the second therapeutic is a cell based therapy.
  • the cell based therapy is one or more myoblasts.
  • the myoblast cell based therapy is as described in NCT02196467.
  • 1-500 million myoblasts can be transplanted per centimeter cube in the Extensor carpi radialis of one of the patient's forearms, resuspended in saline. More specifically, 30 million myoblasts can be transplanted per centimeter cube can be transplanted.
  • the cell based therapy is CAP- 1002 and can improve respiratory, cardiac and upper limb function.
  • the cell based therapy is a cardiosphere derived cell.
  • the one or more symptoms of the LGMD is a symptom related to a cardiac condition.
  • the cardiac condition is cardiomyopathy, decreased cardiac function, fibrosis in the heart, or a combination thereof.
  • the second therapeutic (or third therapeutic) is Ifetroban, Bisoprolol fumarate, Eplerenone, or a combination thereof.
  • Ifetroban is a potent and selective thromboxane receptor antagonist. In some embodiments ifetroban can stop important molecular signals that mediate inflammation and fibrosis (tissue scaring) mechanisms in the heart. In some embodiments, ifetroban is administered orally. In some embodiments, administration can be daily, weekly, or monthly. In some embodiments, the length of treatment can be weeks, months or years. In some embodiments, ifetroban can be administered in a dose of 50 mg to 400 mg. In some embodiments, ifetroban can be administered in a dose of 200 mg. For example, administration can be once daily via capsule - four 50 mg capsules.
  • 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 24hr and the subject is monitored for heart rate, blood pressure, and other heart related symptoms. The bisoprolol dose can be increased 1.25mg progressively until a daily dose of 0.2mg/kg or the maximum tolerated dose (resting heart rate ⁇ 75bpm and systolic blood pressure ⁇ 90mmHg) is achieved. Dosing can be increased with an assessment of the subject’s heart rate, blood pressure, symptoms and ECG.
  • eplerenone is administered orally.
  • administration can be daily, weekly, or monthly.
  • the length of treatment can be weeks, months or years.
  • eplerenone can be administered in a dose of 10 mg to 200 mg.
  • eplerenone can be administered in a dose of 25 mg.
  • administration can be once daily via capsule in a single 25 mg capsule.
  • the one or more symptoms of the LGMD is a respiratory symptom.
  • the second therapeutic can be Idebenone.
  • Idebenone can be administered orally.
  • administration can be daily, weekly, or monthly.
  • the length of treatment can be weeks, months or years.
  • Idebenone can be administered in a dose of 250 mg/day to 2000 mg/day.
  • Idebenone can be administered in a dose of 900 mg/day.
  • administration can be three times a day, orally, wherein each oral administration is two tablets each of 150 mg.
  • the second therapeutic is orthopedic management, endocrinologic management, gastrointestinal management, urologic management, or a combination thereof.
  • the second therapeutic (or third therapeutic) is transcutaneous electrical nerve stimulation (TENS).
  • TENS can increase muscle strength, increase range of joint motions and/or improve sleep.
  • the TENS is applied using VECTTOR system.
  • the VT-200, or VECTTOR system delivers electrical stimulation via electrodes on the acupuncture points of a subject's feet/legs and hands/arms to provide symptomatic relief of chronic intractable pain and/or management of post-surgical pain.
  • nerve stimulator treatment e.g. TENS
  • TENS can be administered one time, two times, three times, four times, five times or more daily.
  • a patient/ subject amenable to treatment with the AAV or rAAV encoding an AUF1 is a patient having a LGMD, including a type 1 or type 2 LGMD.
  • the first therapeutic is an AAV or rAAV particle, including an AAV8 serotype or an AAV9 serotype or an AAVhu.32 serotype, containing a construct encoding AUF1 and administration of an rAAV particle containing a construct encoding AUF1 as described herein, including the constructs having nucleotide sequences of SEQ ID NO:31 to 36 (spc-hu-opti-AUFl-CpG(-), tMCK-huAUFl, spc5-12-hu-opti-AUFl-WPRE, ss-CK7-hu-AUFl, spc-hu-AUFl -no-intron, and D(+)-CK7AUFl, respectively), can occur at a dosage of I x lO 8 vg/kg to 2* 10 15 vg/kg or 2* 10 13 to IxlO 15 , including a dose of 2* 10
  • Doses can range from 1 * 10 8 vector genomes per kg 10 15 vg/kg.
  • the dose can be 2* 10 13 , 3* 10 13 , I x lO 14 , 3x l0 14 , .
  • the dose can be I x lO 14 , Ll x lO 14 , 1.2x l0 14 , 1.3x l0 14 , 1.4x l , 1.6x l0 14 , 1.7x l0 14 , 1.8xl0 14 , 1.9x l0 14 , 2x l0 14 , 2.
  • the second therapeutic is an AAV or rAAV particle containing a construct encoding a microdystrophin and administration of an AAV or rAAV particle containing a construct encoding a microdystrophin described herein, including constructs having a nucleotide sequence of SEQ ID NO: 94, 95 or 96 (serotype AAV8 or AAV9) can occur at a dosage of 1 x 10 8 genome copies/kg to 2x 10 15 genome copies/kg or 2x 10 13 to IxlO 15 , including a dose of 2x 10 14 vg/kg.
  • Doses can range from I x lO 8 vector genomes per kg (vg/kg) to 2x 10 15 vg/kg.
  • the dose can be 2x l0 13 , 3 x lO 13 , I x lO 14 , 3x l0 14 , 5x l0 14 vg/kg.
  • the dose can be l x 10 14 , l.l x 10 14 , 1.2x l0 14 , 1.3xl0 14 , 1.4x l0 14 , 1.5xl0 14 , 1.6x l0 14 , 1.7x l0 14 , 1.8x l0 14 , 1.9x l0 14 , 2x l0 14 , 2.1 xl0 14 , 2.2x l0 14 , 2.3x l0 14 , 2.4xl0 14 , 2.5x l0 14 , 2.6* 10 14 , 2.7* 10 14 , 2.8* 10 14 , 2.9* 10 14 , or 3* 10 14 vg/kg.
  • the second therapeutic is an AAV or rAAV particle containing a construct encoding an a-, P-, y- or 5-sarcoglycan described herein.
  • the ratio of the AUF1 gene therapy vector and the second gene therapy vector is 1 : 1, 1 :2, 1 :4, 1 :5; 1 : 10, 1 :50, 1 : 100 or 1 : 1000.
  • the ratio of the AUF1 gene therapy vector and the second gene therapy vector is 0.5: 1, 0.25: 1, 0.2: 1, or 0.1 : 1.
  • Therapeutically effective dosages are administered as a single dosage (for example, simultaneously in a single composition or separate compositions) or within 1 hour, 2 hours, 3 hours, 4 hours, 12 hours, 1 day, 2 day, 3, days, 4 days, 5 days, 6 days, 7 days, or 2 weeks.
  • the therapy is a monotherapy. In accordance with such embodiments, only the AUF1 gene therapy vector is administered.
  • the therapy is a combination therapy.
  • the first therapeutic, the AUF1 gene therapy vector is administered prior to the second therapeutic.
  • the first therapeutic, the AUF1 gene therapy vector is administered subsequent to the second therapeutic. If the second therapeutic is not a gene therapy or if a third therapeutic (or even further therapeutics) are administered which are not gene therapy vectors, it may be administered in multiple doses during the course of a treatment regimen (z.e., days, weeks, months, etc.) and may be administered before or after the first (and/or the second) therapeutic or both before and after the first (and or second) gene therapy vector.
  • a treatment regimen z.e., days, weeks, months, etc.
  • the dosages are therapeutically effective, which can be assessed at appropriate times after the administration, including 12 weeks, 26 weeks, 52 weeks or more, and include assessments for improvement or amelioration of symptoms and/or biomarkers of the LGMD as known in the art and detailed herein.
  • Recombinant vectors used for delivering the transgene encoding AUF1 are described herein. Such vectors should have a tropism for human muscle cells (including skeletal muscle, smooth muscle and/or cardiac muscle) and can include nonreplicating rAAV, particularly those bearing an AAV8 capsid, an AAV9 capsid or an AAVhu.32 capsid.
  • the recombinant vectors including vectors having the construct spc-hu-opti-AUFl- CpG(-), tMCK-huAUFl, spc5-12-hu-opti-AUFl-WPRE, ss-CK7-hu-AUFl, spc-hu-AUFl -nointron, and D(+)-CK7AUFl (see FIG. 1), for AUF1 expression can be administered in any manner such that the recombinant vector enters the muscle tissue, including by introducing the recombinant vector into the bloodstream, including intravenous administration.
  • Therapeutically effective doses of any such recombinant vector should be administered in any manner such that the recombinant vector enters the muscle (e.g., skeletal muscle or cardiac muscle), including by introducing the recombinant vector into the bloodstream.
  • the vector is administered subcutaneously, intramuscularly or intravenously.
  • the expression of the transgene product results in delivery and maintenance of the transgene product in the muscle.
  • compositions suitable for intravenous, intramuscular, or subcutaneous administration comprise a suspension of the recombinant AAV comprising any of the transgenes disclosed herein in a formulation buffer comprising a physiologically compatible aqueous buffer.
  • the formulation buffer can comprise one or more of a polysaccharide, a surfactant, polymer, or oil.
  • the disclosed pharmaceutical compositions can comprise any of the vectors described herein, particularly the rAAV vectors comprising a transgene encoding AUF1 or the microdystrophins or the sarcoglycans, disclosed herein and can be used in the disclosed methods.
  • the disclosed methods of treatment can result in one of many endpoints indicative of therapeutic efficacy described herein.
  • 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 the administration of a rAAV particle comprising a transgene that encodes AUF1.
  • creatine kinase activity can be used as an endpoint for therapeutic efficacy of the methods of treatment and administration disclosed herein.
  • the creatine kinase activity can decrease in the subject relative to the level (of creatine kinase activity) prior to said administration.
  • the creatine kinase activity can decrease in the subject relative to the level (of creatine kinase activity) in the subject prior to treatment or relative to the level (of creatine kinase activity) in a non-treated subject having a LGMD (for example, a reference level identified in a natural history study).
  • LGMD for example, a reference level identified in a natural history study.
  • the creatine kinase activity measured in the human subject after administration of an AAV or rAAV with a transgene encoding AUF1, including in combination with a second therapeutic can be to a control value which can be the creatine kinase activity in the subject prior to administration, creatine kinase activity in a subject with a LGMD that has not be treated, creatine kinase activity in a subject that does not have a LGMD, creatine kinase activity in a standard.
  • administration results in a decrease in creatine kinase activity, which can be a decrease of 1000 to 10,000 units/liter compared to a control or the value measured in the subject amount prior to administration of the therapeutic.
  • an amount of 1000, 2000, 3000, 4000, or 5000 units/liter in the after-administration endpoint is indicative of a decrease.
  • reduction in lesions in a gastrocnemius muscle can be used as an endpoint measure for therapeutic efficacy for the methods of treatment and administration disclosed herein.
  • the lesions in a gastrocnemius muscle can decrease in the subject relative to the level (of lesions in the gastrocnemius muscle) prior to administration of the therapeutics.
  • the lesions in the gastrocnemius muscle can decrease in the subject relative to the level (of lesions in the gastrocnemius muscle) in a non-treated subject having a LGMD.
  • the comparison of lesions in the gastrocnemius muscle can be to a standard, wherein the standard is a number or set of numbers that represent the lesions in a subject that does not have a LGMD or the lesions in a non-treated subject having a LGMD.
  • the comparison of lesions in the gastrocnemius muscle after administration of a therapeutic can be to a control subject.
  • the control can be the lesions in the gastrocnemius muscle in the subject prior to administration lesions in the gastrocnemius muscle in a subject with a LGMD that has not be treated, lesions in the gastrocnemius muscle in a subject that does not have a LGMD, or lesions in the gastrocnemius muscle in a standard.
  • the lesions in the gastrocnemius muscle of the subject are assessed using magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • administration of therapeutics disclosed herein results in a decrease of lesions in gastrocnemius muscle after administration is about 1-100%, 2- 50%, or 3-10% compared a control, for example, compared to the lesions in the gastrocnemius muscle of the subject prior to said administration.
  • a subject treated with an AAV or rAAV with a transgene encoding AUF1, including in combination with another therapeutic can have 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or greater decrease in lesions compared to a control.
  • gastrocnemius muscle volume (or muscle volume of any other muscle) can be used as an endpoint for treatment efficacy.
  • the gastrocnemius muscle volume can decrease in the subject relative to the level (of gastrocnemius muscle volume) prior to said administration of an AAV or rAAV with a transgene encoding AUF1.
  • the gastrocnemius muscle volume can decrease in the subject relative to the level (of gastrocnemius muscle volume) in a subject that does not have a LGMD.
  • the gastrocnemius muscle volume can decrease in the subject relative to the level (of gastrocnemius muscle volume) in a non-treated subject having a LGMD.
  • the comparison of gastrocnemius muscle volume can be to a standard, wherein the standard is a number or set of numbers that represent the volume in a subject that does not have a LGMD or the volume in a non-treated subject having a LGMD.
  • the comparison of gastrocnemius muscle volume after administration of the therapeutics disclosed herein can be to a control.
  • the control can be the gastrocnemius muscle volume in the subject prior to administration, gastrocnemius muscle volume in a subject with a LGMD that has not be treated, gastrocnemius muscle volume in a subject that does not have a LGMD, or gastrocnemius muscle volume in a standard.
  • the gastrocnemius muscle volume of the subject can be assessed using MRI.
  • the administration results in a decrease in gastrocnemius muscle volume of about 1-100%, 2-50%, or 3-20% compared a control, for example, compared to the gastrocnemius muscle volume prior to said administration.
  • a decrease of gastrocnemius muscle volume after administration of an AAV or rAAV comprising a transgene that encodes AUF1, including in combination with a second therapeutic can be about 2-400 mm 3 , 5-200 mm 3 , or 20-100 mm 3 compared a control.
  • a subject treated with a rAAV with a transgene encoding AUF1, including in combination with a second therapeutic can have 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 mm 3 or greater decrease in gastrocnemius muscle volume compared to a control.
  • a fat fraction of muscle can be used as an endpoint for therapeutic efficacy of the methods of administering AAV or rAAV therapeutics disclosed herein.
  • the muscle can be muscles in the pelvic girdle and thigh (gluteus maximus, adductor magnus, rectus femoris, vastus lateralis, vastus medialis, biceps femoris, semitendinosus, and gracilis).
  • the fat fraction of muscle can decrease in the subject relative to the level (of fat fraction of muscle) prior to said administration of AAV or rAAV with a transgene encoding AUF1, including in combination with a second therapeutic, as disclosed herein.
  • the fat fraction of muscle can decrease in the subject relative to the level (of fat fraction of muscle) in a non-treated subject having a LGMD.
  • the comparison of fat fraction of muscle can be to a standard, wherein the standard is a number or set of numbers that represent the amount or percent of fat fraction of muscle in a subject that does not have a LGMD or the amount or percent in a non-treated subject having a LGMD.
  • the comparison of fat fraction of muscle after administration of an AAV or rAAV with a transgene encoding an AUF1, including in combination with a second therapeutic can be to a control.
  • the control can be the fat fraction of muscle in the subject prior to administration, fat fraction of muscle in a subject with a LGMD that has not be treated, fat fraction of muscle in a subject that does not have a LGMD, or fat fraction of muscle of a standard.
  • the fat fraction of muscle of the subject are assessed using magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • a subject so administered can have 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or greater decrease in fat fraction of muscle compared to a control.
  • gait score can be used as an endpoint for treatment.
  • the gait score can be about -1 to 2 after administration.
  • the North Star Ambulatory Assessment (NSAA) can be used as an endpoint for treatment.
  • the NSAA of the treated subject can be compared to NSAA prior to administration.
  • the NSAA of the treated subject can be compared to NSAA in a subject that does not have a LGMD.
  • the NSAA of the treated subject can be compared to a non-treated subject having a LGMD.
  • the NSAA of the treated subject can be compared to a standard, wherein the standard is a score or set of scores that represent the NSAA in a subject that does not have a LGMD or the NSAA in a non-treated subject having a LGMD.
  • the increase can be from 0 to 1, 0 to 2, or from 1 to 2.
  • LGMD skeletal muscle symptoms
  • DCM dilated cardiomyopathy
  • LV left ventricle
  • Atrophic cardiomyocytes exhibit a loss of striations, vacuolization, fragmentation, and nuclear degeneration.
  • DMD may be associated with various ECG changes like sinus tachycardia, reduction of circadian index, decreased heart rate variability, short PR interval, right ventricular hypertrophy, S-T segment depression and prolonged QTc.
  • Gene therapy treatment provided herein can slow or arrest the progression of LGMD, particularly to reduce the progression of or attenuate cardiac dysfunction and/or maintain or improve cardiac function. Efficacy may be monitored by periodic evaluation of signs and symptoms of cardiac involvement or heart failure that are appropriate for the age and disease stage of the trial population, using serial electrocardiograms, and serial noninvasive imaging studies (e.g., echocardiography or cardiac magnetic resonance imaging (CMR)).
  • CMR cardiac magnetic resonance imaging
  • ECG may be used to monitor changes from baseline in forced vital capacity (FVC), forced expiratory volume (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak cough flow, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), inflammation, and fibrosis.
  • FVC forced vital capacity
  • FEV1 forced expiratory volume
  • MIP maximum inspiratory pressure
  • MEP maximum expiratory pressure
  • PEF peak expiratory flow
  • LVEF left ventricular ejection fraction
  • LVFS left ventricular fractional shortening
  • inflammation and fibrosis.
  • ECG may be used to monitor conduction abnormalities and arrythmias.
  • ECG may be used to assess normalization of the PR interval, R waves in VI, Q waves in V6, ventricular repolarization, QS waves in inferior and/or upper lateral wall, conduction disturbances in right bundle branch, QT C, and QRS.
  • Therapeutic methods disclosed herein can improve or maintain cardiac function or slow the loss of cardiac function, for example, by preventing reductions in decreasing LVEF below 45% and/or normalization of function (LVFS > 28%) as measured by serial electrocardiograms, and/or serial noninvasive imaging studies (e.g., echocardiography or cardiac magnetic resonance imaging (CMR)). Measurements may be compared to an untreated control or to the subject prior to treatment.
  • serial electrocardiograms e.g., echocardiography or cardiac magnetic resonance imaging (CMR)
  • CMR cardiac magnetic resonance imaging
  • ECG may be used to monitor conduction abnormalities and arrythmias.
  • ECG may be used to assess normalization of the PR interval, R waves in VI, Q waves in V6, ventricular repolarization, QS waves in inferior and/or upper lateral wall, conduction disturbances in right bundle branch, QT C, and QRS.
  • cardiac function and/or pulmonary function can be used as an endpoint for assessment of therapeutic efficacy of the administration.
  • the cardiac function and/or pulmonary function can improve or increase in the subject relative to the level (of cardiac function and/or pulmonary function) prior to said administration.
  • the cardiac function and/or pulmonary function can improve or increase in the subject relative to the level (of cardiac function and/or pulmonary function) in a subject that does not have a LGMD.
  • the cardiac function and/or pulmonary function can decrease in the subject relative to the level (of cardiac function and/or pulmonary function) in a non-treated subject having a LGMD.
  • the comparison of cardiac function and/or pulmonary function can be to a standard, wherein the standard is a number or set of numbers that represent the cardiac function and/or pulmonary function in a subject that does not have a LGMD or the cardiac function and/or pulmonary function in a non-treated subject having a LGMD.
  • the comparison of cardiac function and/or pulmonary function after administration can be to a control.
  • the control can be the cardiac function and/or pulmonary function in the subject prior to administration, cardiac function and/or pulmonary function in a subject with a LGMD that has not be treated, cardiac function and/or pulmonary function in a subject that does not have a LGMD, cardiac function and/or pulmonary function in a standard.
  • an improvement or increase in cardiac function and/or pulmonary function is 1 to 100% compared to a control, for example, compared to the subject prior to administration.
  • cardiac function can be measured using impedance, electric activities, and calcium handling.
  • Patient primary endpoints may include monitoring the change from baseline in forced vital capacity (FVC), forced expiratory volume (FEV1), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), peak expiratory flow (PEF), peak cough flow, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), change from baseline in the NSAA, change from baseline in the Performance of Upper Limp (PUL) score, and change from baseline in the Brooke Upper Extremity Scale score (Brooke score), change from baseline in grip strength, pinch strength, change in cardiac fibrosis score by MRI, change in upper arm (bicep) muscle fat and fibrosis assessed by MRI, measurement of leg strength using a dynamometer, walk test 6-minutes, walk test 10-minutes, walk analysis - 3D recording of walking, change in utrophin membrane staining via quantifiable imaging
  • Example 1 AUF1 Gene Expression Cassettes for Insertion Into Cis Plasmids
  • Constructs for preparing rAAV8 vectors encoding p40 AUF1 were synthesized.
  • a codon optimized, CpG depleted nucleotide sequence encoding human p40 AUF1 (SEQ ID NO: 17) was identified, synthesized and cloned into a cis plasmid.
  • Expression cassettes were generated incorporating the opti-CpG(-) AUF1 coding sequence (SEQ ID NO: 17) using regulatory elements, the amino acid sequence of which are provided in Table 2.
  • FIG. 1 The constructs, spc-hu-opti-AUFl-CpG(-)(SEQ ID NO: 31), tMCK-huAUFl (SEQ ID NO: 32), spc5-12-hu- opti-AUFl-WPRE (SEQ ID NO: 33), ss-CK7-hu-AUFl (SEQ ID NO: 34), spc-hu-AUFl -nointron (SEQ ID NO: 35), or D(+)-CK7AUFl (SEQ ID NO: 36) are depicted in FIG. 1 (nucleotide sequences provided in Table 3).
  • rAAV cis plasmids to be used in producing rAAV, e.g. rAAV8 particles containing the recombinant genome encoding AUF1.
  • Production methods for rAAV particles are known in the art, and for the foregoing experiments using rAAV particles, triple transfection of HEK293 cells was performed with (1) the cis plasmid (transgene (such as the therapeutic transgenes described herein) flanked by AAV ITR sequences); (2) rep/cap plasmid (AAV rep and cap genes and gene products, e.g.
  • helper plasmid suitable helper virus function, usually mutant adenovirus
  • Example 2 - Calpain 3 Deficient Limb Girdle Muscular Dystrophy (LGMD) Type 2A CAPN3 disease comprises approximately 30% of all LGMD cases worldwide, estimated at 1 :40,000 to 1 : 100,000 individuals, but can be 100-fold higher in certain regions due to founder effects.
  • LGMD R1 (CAPN3) averages 10-12-fold less frequent than DMD.
  • Limb girdle muscular dystrophy (LGMD) type 2A is caused by mutations in calpain 3, sarcomere-specific, titin-binding Ca2 + cysteine protease (CAPN3).
  • CAPN3 is a skeletal muscle specific protease that is required to activate muscle contraction by selective cleavage and activation of contraction proteins, thereby activating muscle calcium pumps.
  • Key features include pronounced loss of type I slow oxidative myofibers and changes in PGCla activity, due to impaired Ca2 + calmodulin kinase IIJ3 (CaMKIip) activity.
  • Clinical features of CAPN3 deficiency include primarily proximal muscle wasting; no involvement of cardiac or facial muscles; slow, but progressive, muscle necrosis; and loss of muscle regeneration.
  • the age at onset of muscle weakness ranges between 2 and 40 years, but averages 15 years. Additional symptoms include difficulty in running, the tendency to walk on tiptoes, and scapular winging caused by weakness of scapular girdle muscles, as well as breathlessness at rest.
  • speech and swallowing difficulties start to develop. Some fatigue, lethargy, poor appetite, weight loss, and impaired concentration can occur.
  • waddling gait difficultly climbing stairs, difficulty in lifting weights, and difficulty getting up from the floor or a chair may occur.
  • Calpainopathy presents as a less severe disease than Duchenne Muscular Dystrophy (DMD), has a generally later age of onset, does not involve cardiac or diaphragm muscles, and is largely limited to limb skeletal muscles. These features provide more quantitative means for measuring outcomes in interventive clinical trials.
  • DMD Duchenne Muscular Dystrophy
  • the CAPNS' ⁇ deletion mouse model (JACS), replicates LGMD2A disease well: muscle degeneration, halted regeneration, necrosis, abnormal mitochondria and function, small myofibers, loss of slow fibers, weakness.
  • the limitations of the CAPN3 ⁇ ⁇ deletion mouse model include the following: does not develop fibrosis, patients may have allele deletion and 2nd allele missense/nonsense mutations.
  • a small molecule activator of CAMKIIP kinase activity has been reported to modestly recover muscle function in the CAPN3 deficient mouse (Liu et al., “A Small-Molecule Approach to Restore a Slow - Oxidative Phenotype and Defective CaMKIip Signaling in Limb Girdle Muscular Dystrophy,” Cell Reports 1 : 100122 (2020), which is hereby incorporated by reference in its entirety), which provides a response against which to measure AUF1 gene therapy in this disease model.
  • CAPN3 deficient mice were administered: codon optimized humanized AUF1 (AAV8-hAUFl) at 6xl0 13 viral genomes/kg mouse weight (low dose), codon optimized humanized AUF1 (AAV8-hAUFl) at IxlO 14 viral genomes/kg mouse weight (high dose), or vector control (control).
  • FIG. 2A Three months of AAV8-hAUFl gene therapy restored near normal muscle fiber morphology, with larger mature myofibers evident (FIG. 2A) with greater cross-sectional area (csa) containing > 2 nuclei (FIG. 2B), which is a marker of mature muscle fibers. While pathology was not as severe in diaphragm as in limb skeletal muscles in CAPN3 deficiency, pathology was corrected in both muscle types by AAV8-hAUFl gene therapy (FIGS. 2A-2B).
  • One of the main goals of therapy for CAPN3 deficiency is to restore CAMKIIP kinase activity, which can be determined by specific activating phosphorylation and the downstream increase in deficient mitochondrial biogenesis.
  • Another one of the main goals of therapy for CAPN3 deficiency is to restore SERCA2 pump levels and activity, and PGCla levels that promote mitochondrial biogenesis and slow twitch Type I stamina muscle fibers.
  • Two months of AUF1 gene therapy increases CAMKIip protein expression, activating phosphorylation, SERCA2A, and PGCla both in the gastrocnemius muscle (FIG. 5).
  • AUF1 gene therapy was also found to strongly restore mitochondria and to do so at the proper location in the sub-sarcolemma, as shown in the TA muscle at 2 months of therapy (FIG. 6)
  • CAPN3 deficiency A hallmark of CAPN3 deficiency is the disruption of intermyofibrillar organization and structure within muscle fibers, which prevents normal muscle contraction, as well as generation of exhausted and dying mitochondria. As shown in the gastrocnemius muscle, AAV8-hAUFl gene therapy at two months restores near-normal intermyofibrillar organization and mitochondria to CAPN3 deficient muscle (FIG. 7).
  • SDH succinate dehydrogenase
  • Sarcoglycanopathy disease results from a dominant mutation in one of four sarcoglycan protein genes (a, P, y, 5). Six sarcoglycan proteins form a complex that interacts with dystrophin to stabilize the sarcolemma, promote contraction, maintain muscle integrity and proper calcium function in contraction. Disease onset is severe, anywhere from childhood to adulthood, affects limb and girdle skeletal muscles including diaphragm and cardiac muscle. Worldwide prevalence is low, 2-3/100,000 individuals. Considered a rare disease. The delta form of disease is the rarest, with mutations in SCGD causing LGMD2F.
  • SCGD sarcoglycan deficient delta protein
  • SCGD protein is part of the sarcoglycan complex of four proteins that stabilize dystrophin interaction with the sarcolemma (muscle fiber cell membrane), providing stability and contraction ability.
  • Scgd m 1 Mcn mice in a C57BL/6J genetic background are a model for human sarcoglycan, delta (dystrophin-associated glycoprotein) deficient disease.
  • Homozygous mice deleted for the Sgcd allele are viable, fertile, and show disease onset by 8 weeks of age with muscle degeneration including cardiac muscle by 12 weeks, followed by rapid death.
  • 5-sarcoglycan deleted mice were administered AAV8-hAUFl (codon optimized humanized AUF1) by intravenous (i.v.) injection in the retro-orbital sinus at 6el3 viral genomes/kg mouse weight.
  • AAV8-hAUFl gene therapy at 2 months restores significant diaphragm muscle integrity, repairs the profound degeneration of SCGD muscle which is apparent in the H&E stains as a dark blue stained area (FIG. 9).
  • the gastrocnemius muscle is affected in SCGD deficiency.
  • Animals were administered by intravenous (i.v.) injection in the retro-orbital sinus AAV8-hAUFl (codon optimized humanized AUF1, described in the provisional application) at 6el3 viral genomes/kg mouse weight.
  • AAV8-hAUFl gene therapy at 2 months restores significant muscle integrity, repairs the profound degeneration of SCGD muscle which is apparent in the H&E stains as a dark blue stained area (FIG. 10).
  • eMHC embryonic myosin heavy chain
  • SCGD muscle undergoes chronic and unsuccessful regeneration with continuous expression of eMHC.
  • AAV8 hAUFl gene therapy at 2 months restores more normal muscle regeneration, reduced expression of eMHC, larger more mature muscle fibers shown by fibers with >2 nuclei, and greater cross-sectional area (csa) (FIG. 11).
  • AAV8-hAUFl gene therapy in SCGD deficient mice at two months increases all forms of muscle fibers, Type I slow twitch, and multiple forms of Type II fast twitch (FIG. 12).
  • Animal muscle function was tested for muscle grip strength, the ability of the mouse to grasp a grid with its front limbs while tugging on the tail.
  • FIG. 13 demonstrates statistically increased gains by AAV8 hAUFl gene therapy at 2 months in SCGD mice which approaches wild type for female mice.

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Abstract

L'invention concerne des méthodes de traitement ou d'amélioration des symptômes de la dystrophie musculaire des ceintures par administration de doses thérapeutiquement efficaces de virus adéno-associés (AAV) ou de virus adéno-associés recombinants (rAAV) contenant un transgène codant pour le facteur 1 de liaison à un élément riche en AU (AUF1) efficace pour traiter la dystrophie musculaire des ceintures. L'invention concerne également des vecteurs AAV et rAAV codant pour des protéines AUF1.
PCT/US2023/070765 2022-07-21 2023-07-21 Thérapie génique auf1 pour myopathie des ceintures WO2024020574A2 (fr)

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