CA3164622A1 - Adeno-associated viral vector, compositions, methods of promoting muscle regeneration, and treatment methods - Google Patents

Abstract

The present application 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 cell-specific promoter. Also disclosed are 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.

Description

ADENO-ASSOCIATED VIRAL VECTOR, COMPOSITIONS, METHODS OF
PROMOTING MUSCLE REGENERATION, AND TREATMENT METHODS
[00011 This application claims the priority benefit of U.S.
Provisional Patent Application Serial No. 62/962,712, filed January 17, 2020, and U.S. Provisional Patent Application Serial No. 63/128,047, filed December 19, 2020, which are hereby incorporated by reference in their entirety.
[00021 This invention was made with government support under RO1 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD
[00031 The present application relates to adeno-associated viral (AAV) vectors and lentiviral vectors comprising a nucleic acid molecule encoding an AU-rich mRNA
binding factor 1 (AUF1) protein or a functional fragment thereof, as well as compositions and methods of use thereof.
BACKGROUND
[00041 Muscle wasting diseases represent a major source 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). 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. 294(1):50-66 (2006); Bernet et al., "p38 MAPK
Signaling Underlies a Cell-autonomous Loss of Stem Cell Self-renewal in Skeletal Muscle of Aged Mice,"
Nat. Med. 20(3):265-71 (2014); and Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function," Development 142(9):1572-1581 (2015)).
[00051 Age-related skeletal muscle loss and atrophy is characterized by the progressive loss of muscle mass, strength, and endurance with age. It can be a significant source of frailty, increased fractures, and mortality in the elderly population (Vermeiren et al., "Frailty and the

- 2 -Prediction of Negative Health Outcomes: A Meta-Analysis," J. Am. Med. Dir.
Assoc.
17(12):1163.e1-1163.e17 (2016) and Buford, T. W., "Sarcopenia: Relocating the Forest among the Trees," Toxicol. Pathot 45(7):957-960 (2017)). Although different strategies have been investigated to counter muscle loss and atrophy, regular resistance exercise is the most effective in slowing muscle loss and atrophy, but compliance and physical limitations are significant barriers (Wilkinson et al., "The Age-Related loss of Skeletal Muscle Mass and Function:
Measurement and Physiology of Muscle Fibre Atrophy and Muscle Fibre Loss in Humans,-Ageing Res. Rev. 47:123-132 (2018)). Consequently, with an aging global population, therapeutic strategies need to be developed to reverse age-related muscle decline.
[0006] 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," Physiot Rev. 91(4):1447-1531 (2011)). Upon physical injury to muscle, the anatomical niche is disrupted, normally quiescent satellite cells become activated and proliferate asymmetrically. Some 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(272):re2 (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. 294(1):50-66 (2006); Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function," Development 142(9):1572-1581 (2015);
Briggs & Morgan, "Recent Progress in Satellite Cell/Myoblast Engraftment -- Relevance for Therapy, FEBS J.
280(17):4281-93 (2013); Morgan & Zammit, "Direct Effects of the Pathogenic Mutation on Satellite Cell Function in Muscular Dystrophy," Exp. Cell Res. 316(18):3100-8 (2010); and Relaix & Zammit, "Satellite Cells are Essential for Skeletal Muscle Regeneration: The Cell on the Edge Returns Centre Stage," Development 139(16):2845-56 (2012)).
[0007] 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,-Physiot 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

- 3 -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.
Op/n. Cl/n. Nutr.
Metab. Care 16(3):243-250 (2013); Tonkin et al., "SIRT1 Signaling as Potential Modulator of Skeletal Muscle Diseases," Curr. Op/n. PharmacoL 12(3):372-376 (2012); and Arany, Z, "PGC-1 Coactivators and Skeletal Muscle Adaptations in Health and Disease,- Cum Op/n. Genet. Dev.
18(5):426-434 (2008)). 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)) PGCla stimulates mitochondrial biogenesis and oxidative metabolism through increased expression of nuclear respiratory factors (NRFs) such as NRF1 and 2 that stimulate mitochondrial biosynthesis, mitochondria transcription factor A (Tfam), and in addition to mitochondrial biosynthesis, also promote slow myofiber formation through increased expression of Mef2 proteins (Lin et al., "Transcriptional Co-Activator PGC-1 Alpha Drives the Formation of Slow-Twitch Muscle Fibres," Nature 418 (6899):797-801 (2002); Lai et al., "Effect of Chronic Contractile Activity on mRNA Stability in Skeletal Muscle," Am. J. Physiol.
Cell. Physiol.
299(1):C155-163 (2010); Ekstrand et al., "Mitochondrial Transcription Factor A
Regulates mtDNA Copy Number in Mammals," Hum. Mol. Genet. 13(9):935-944 (2004); and Scarpulla, RC, "Transcriptional Paradigms in Mammalian Mitochondrial Biogenesis and Function,"
Physiol. Rev. 88(2): 611-638 (2008)). Importantly, PGC la protects muscle from atrophy due to disuse, certain myopathies, starvation, sarcopenia, cachexia, and other causes (Wiggs, M. P., "Can Endurance Exercise Preconditioning Prevention Disuse Muscle Atrophy?,"
Front. Physiol.
6:63 (2015); Wing et al., "Proteolysis in Illness-Associated Skeletal Muscle Atrophy: From Pathways to Networks," Crit. Rev. Clin. Lab. Sci. 48(2).49-70 (2011); Bost &
Kaminski, "The Metabolic Modulator PGC-lalpha in Cancer," Am. J. Cancer Res. 9(2):198-211 (2019); and Dos Santos et al., "The Effect of Exercise on Skeletal Muscle Glucose Uptake in type 2 Diabetes: An Epigenetic Perspective," Metabolism 64(12):1619-1628 (2015)).
[0008] 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," Anntt. 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)

- 4 -and Robinson & Dilworth, "Epigenetic Regulation of Adult Myogenesis," Curr.
Top Dev. Biol.
126:235-284 (2018)). The ability to selectively promote slow-twitch muscle has been along-standing goal, because endurance slow-twitch Type I myofibers provide greater resistance to muscle atrophy (Talbot & Mayes, "Skeletal Muscle Fiber Type: Using Insights from Muscle Developmental Biology to Dissect Targets for Susceptibility and Resistance to Muscle Disease,"
Wiley Interdiscip. Rev. Dev. Biol. 5(4):518-534 (2016)), and could be an effective therapy for sarcopenia, Duchenne Muscular Dystrophy, cachexia, and other muscle wasting diseases (Selsby et al., "Rescue of Dystrophic Skeletal Muscle By PGC-lalpha Involves A Fast To Slow Fiber Type Shift In The Mdx Mouse," PLoS One 7(1):e30063 (2012); von Maltzahn et al., "Wnt7a Treatment Ameliorates Muscular Dystrophy," Proc. Natl. Acad. Sci. USA
109(50):20614-20619 (2012); and Ljubicic et al., "The Therapeutic Potential Of Skeletal Muscle Plasticity In Duchenne Muscular Dystrophy: Phenotypic Modifiers As Pharmacologic Targets,"
FASEB
28(2):548-568 (2014)).
[0009] Duchenne Muscular Dystrophy ("DMD") is one of the most severe disorders of muscle degeneration known as myopathies. Inherited in an X-linked recessive manner, the disorder is caused by mutations in the dystrophin gene, resulting in a near-absence of expression of the protein, which plays a key role in stabilization of muscle cell membranes (Bonilla et al., "Duchenne Muscular Dystrophy: Deficiency of Dystrophin at the Muscle Cell Surface," Cell 54(4):447-452 (1988) and Hoffman et al., "Dystrophin: The Protein Product of the Duchenne Muscular Dystrophy Locus," Cell 51(6):919-928 (1987)). Consequently, only males with the mutation are afflicted with DMD, which affects 1 in 3500 live births. There are no cures for DMD, and currently approved approaches involve limited use of corticosteroids to dampen inflammatory immune responses, a secondary exacerbating effect of muscle atrophy. While the inflammatory response is generally beneficial in normal muscle wound repair and regeneration, in DMD the response is no longer self-limiting due to the chronic nature of muscle damage. This results in exacerbation of necrosis of existing muscle and depletion of muscle fibers (myofibers) with replacement by connective and adipose tissue (Camwath & Shotton, "Muscular Dystrophy in the mdx Mouse: Histopathology of the Soleus and Extensor Digitorum Longus Muscles,"
Neurol. Sci. 80(1):39-54 (1987); Tanabe et al., "Skeletal Muscle Pathology in X Chromosome-Linked Muscular Dystrophy (mdx) Mouse," Acta Neuropathol. 69(1-2):91-95 (1986); and Fairclough et al., "Pharmacologically Targeting the Primary Defect and Downstream Pathology in Duchenne Muscular Dystrophy," CUM Gene Ther. 12(3):206-244 (2012)). While steroids can provide short-term increased muscle strength, long-term treatment is ultimately ineffective and can exacerbate disease. Steroids do not target the underlying cause of disease. There is therefore

- 5 -an urgent need for pharmacologic approaches that address the primary underlying cause of D1VID: loss of muscle fiber strength, loss of muscle stem cells, loss of muscle regenerative capacity, and attenuation of the exacerbating destructive effects of the pathological immune response on muscle health and integrity (Fairclough et al., "Pharmacologically Targeting the Primary Defect and Downstream Pathology in Duchenne Muscular Dystrophy," Curr.
Gene Ther. 12(3):206-244 (2012)).
[0010] Dystrophin functions to assemble the dystroglycan complex at the sarcolemma, which connects the extracellular matrix to the cytoplasmic intermediate filaments of the muscle cell, providing physical strength and structural integrity to muscle fibers which are readily damaged in the absence of dystrophin (Yiu & Kornberg, "Duchenne Muscular Dystrophy,"
1Veurol. India 56(3).236-247 (2008)). Dystrophin-defective myofibers are very easily damaged by minor stresses and micro-tears in DMD. This triggers continuous cycles of muscle repair and regeneration, depletes the muscle stem cell population, and provokes a destructive immune response that increases with age (Yiu & Kornberg, "Duchenne Muscular Dystrophy," Neurol India 56(3):236-247 (2008); Smythe et al., "Age Influences The Early Events of Skeletal Muscle Regeneration: Studies of Whole Muscle Grafts Transplanted Between Young (8 Weeks) and Old (13-21 Months) Mice," Exp. Gerontol. 43(6):550-562 (2008); Heslop et al., "Evidence for a Myogenic Stem Cell that is Exhausted in Dystrophic Muscle," I Cell Sci .
113(Pt 12):2299-32208 (2000); Cros et al., "Muscle Hypertrophy in Duchenne Muscular Dystrophy.
A
Pathological and Morphometric Study," I. Neurol 236(1):43-47 (1989); and Abdel-Salam et al., "Markers of Degeneration and Regeneration in Duchenne Muscular Dystrophy,"
Acta Myol.
28(3):94-100 (2009)). In this regard, it has been shown that the progressive loss of muscle and its regenerative capacity in MID results from exhaustion (inability to activate) and depletion of the muscle stem cell population (i.e., satellite cells) (Carlson 8z Conboy, "Loss of Stem Cell Regenerative Capacity Within Aged Niches," Aging Cell 6(3):371-82 (2007);
Kudryashova et al., "Satellite Cell Senescence Underlies Myopathy in a Mouse Model of Limb-girdle Muscular Dystrophy 2H," 1 Clin. Invest. 122(5):1764-76 (2012); Gopinath & Rando, "Stem Cell Review Series: Aging of the Skeletal Muscle Stem Cell Niche," Aging Cell 7(4):590-8 (2008); Morgan &
Zammit, "Direct Effects of the Pathogenic Mutation on Satellite Cell Function in Muscular Dystrophy," Exp. Cell Res. 316(18):3100-8 (2010); and Collins et al., "Stem Cell Function, Self-renewal, and Behavioral Heterogeneity of Cells From the Adult Muscle Satellite Cell Niche,"
Cell 122(2):289-301 (2005)). Typically, in normal muscle, the small pool of satellite cells that do not differentiate following injury repopulate muscle and re-enter the quiescent state in their niche, only to be activated again upon muscle damage to differentiate and fuse into myofibers.

- 6 -The niche is defined both structurally and morphologically as sites where satellite cells reside adjacent to muscle fibers, in which quiescence is maintained by the structural integrity of the micro-environment, identified by laminin and other structural proteins (Carlson & Conboy, "Loss of Stem Cell Regenerative Capacity Within Aged Niches," Aging Cell 6(3):371-82 (2007);
Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function,"
Development 142(9):1572-1581 (2015); Gopinath & Rando, "Stem Cell Review Series: Aging of the Skeletal Muscle Stem Cell Niche,- Aging Cell 7(4):590-8 (2008); Seale &
Rudnicki, "A New Look at the Origin, Function, and "Stem-cell" Status of Muscle Satellite Cells," Dev. Biol.
218(2):115-24 (2000); Briggs & Morgan, "Recent Progress in Satellite Cell/N1yoblast Engraftment -- Relevance for Therapy, FEBS J280(17).4281-93 (2013); Collins et al., "Stem Cell Function, Self-renewal, and Behavioral Heterogeneity of Cells From the Adult Muscle Satellite Cell Niche," Cell 122(2):289-301 (2005); and Murphy et al., "Satellite Cells, Connective Tissue Fibroblasts and Their Interactions are Crucial for Muscle Regeneration,"
Development 138(17):3625-37 (2011)). Studies suggest that it is the continuous damage to muscle in DMD that destroys this satellite cell niche, preventing these stem cells from renewing and ultimately leading to their functional exhaustion and cessation of muscle repair.
[0011] 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)). Unique expression markers identify and stage skeletal muscle regeneration. PAX7 is a transcription factor expressed by quiescent and early activated satellite cells (Brack, A.S., "Pax7 is Back," Sk-elet. 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)).
[0012] As satellite cells age, they lose their ability to maintain a quiescent population (Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function,"
Development 142(9):1572-1581 (2015)), and become depleted or functionally exhausted, a primary cause of sarcopenia (muscle loss) with aging and in myopathic diseases (Bernet et al., "p38 MAPK Signaling Underlies a Cell-autonomous Loss of Stem Cell Self-renewal in Skeletal Muscle of Aged Mice," Nat. Med. 20(3):265-71 (2014); Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function," Development 142(9): 1572-1581(2015);

- 7 -Kudryashova et al., "Satellite Cell Senescence Underlies Myopathy in a Mouse Model of Limb-girdle Muscular Dystrophy 2H," I Cl/n. Invest. 122(5):1764-76 (2012); and Silva et al., "Inhibition of Stat3 Activation Suppresses Caspase-3 and the Ubiquitin-proteasome System, Leading to Preservation of Muscle Mass in Cancer Cachexia,"1 Biol. Chem.
290(17):11177-87 (2015)).
[0013] Thus, there remains an urgent need for effective therapeutic options that address the primary underlying cause myopathic diseases (e.g., sarcopenia, Duchenne muscular dystrophy, traumatic muscle injury), which include, e.g., loss of muscle fiber strength, loss of muscle stem cells, loss of muscle regenerative capacity, and attenuation of the exacerbating destructive effects of the pathological immune response on muscle health and integrity.
[0014] The present application is directed to overcoming these and other deficiencies in the art.
SUMMARY
[0015] One aspect of the present application 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 (AIJF1) protein or a functional fragment thereof, where the nucleic acid molecule is heterologous to and operatively coupled to the muscle cell-specific promoter.
[0016] Another aspect of the present application relates to a composition comprising an adeno-associated viral (AAV) vector as described herein.
[0017] A further aspect of the present application relates to a pharmaceutical composition comprising an adeno-associated viral (AAV) vector described herein and a pharmaceutically-acceptable carrier.
[0018] Another aspect of the present application relates to a method of promoting muscle regeneration. This method involves contacting muscle cells with an adeno-associated viral (AAV) vector described herein or a composition described herein under conditions effective to express exogenous AUF1 in the muscle cells to increase muscle cell mass, increase muscle cell endurance, and/or reduce serum markers of muscle atrophy.
[0019] A further aspect of the present application relates to a method of treating degenerative skeletal muscle loss in a subject. This method involves selecting a subject in need of treatment for skeletal muscle loss and administering to the selected subject an adeno-associated viral (AAV) vector described herein or a composition described herein under conditions effective to cause skeletal muscle regeneration in the selected subject

-8-100201 Yet a further aspect of the present application relates to a method of preventing traumatic muscle injury in a subject. This method involves selecting a subject at risk of traumatic muscle injury and administering to the selected subject an adeno-associated viral (AAV) vector described herein, a composition described herein, or a lentiviral 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 cell-specific promoter.
[0021] Still another aspect of the present application relates to a method of treating traumatic muscle injury in a subject. This method involves selecting a subject having traumatic muscle injury and administering to the selected subject an adeno-associated viral (AAV) vector described herein, a composition described herein, or alentiviral 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 cell-specific promoter.
[0022] Prior studies have demonstrated that supplying ALTF1 to an animal model in which AUF1 had been experimentally deleted could result in new muscle regeneration (see PCT
Publication No. WO 2016/196350, which is hereby incorporated by reference in its entirety).
The present application is based, in part, on the surprising discovery that AUF1 supplementation by gene delivery restores muscle regeneration and function in degenerative muscle diseases such as Duchenne Muscular Dystrophy when there is no mutation or limitation of AUF1 expression.
This is particularly surprising in view of the fact that providing supplementary AUF1 has no impact on normal muscle and does not induce regeneration of normal muscle.
[0023] While not wishing to be bound by any theory as to how the mechanism works, the data presented herein demonstrate, inter alia, that in animal models of degenerative muscle diseases: (i) AUF1 gene transfer in Duchenne Muscular Dystrophy compensates for loss of mutated dystrophin by upregulating the dystrophin homolog utrophin, restoring muscle function;
(ii) AUF1 gene delivery does not activate regeneration of normal muscle; (iii) supplementation by gene transfer accelerates regeneration of wounded muscle and promotes muscle function despite normal levels of AUF1 expression in wounded muscle;
and (iv) AUF1 supplementation restores muscle regeneration, muscle mass, and function in aging muscle.
[0024] As described in the Examples, infra, AUF1 supplementation by gene transfer restores muscle regeneration, muscle mass, and muscle function in degenerative muscle diseases such as Duchenne Muscular Dystrophy in age-related loss of muscle mass and function and in traumatic muscle injury.

-9-100251 The Examples disclosed herein demonstrate that loss of expression of AUF1 occurs naturally during aging in skeletal muscle, and underlies age-related muscle loss and atrophy in sedentary animals, but can be reversed by AAV8-AUF1 skeletal muscle gene transfer.
Mice receiving AUF1 gene therapy regain significant and durable skeletal muscle mass and exercise endurance; an increase in Pax7+ activated satellite cells and myoblasts, a key indicator of sustainable muscle regeneration; increased expression of PGCla through stabilization of its mRNA; increased mitochondrial biogenesis; and decreased markers of muscle degeneration.
The Examples disclosed herein further demonstrate that muscle cell-specific AUF1 gene therapy restores skeletal muscle mass and function in a mouse model of Duchenne muscular dystrophy.
AUF1 gene therapy (e.g., by lentivirus vector delivery directly to muscle or systemic delivery of AUF1 by AAV8 vector) is also shown to be effective to: (1) activate muscle stem (satellite) cells; (2) reduce expression of established biomarkers of muscle atrophy; (3) accelerated the regeneration of mature muscle fibers (myofibers); (4) enhanced expression of muscle regeneration factors; (5) strongly accelerate the regeneration of injured muscle; (6) increase regeneration of both major types of muscle (i.e., slow-twitch (Type I) or fast-twitch (Type II) fibers); and restore muscle mass, muscle strength, and create normal muscle.
[0026] AAV8-AUF1 gene therapy may provide a potential long-term therapeutic intervention for debilitating human muscle loss and atrophy.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGs. IA-IL show AUF1 supplementation in skeletal muscle improves exercise endurance in 12 and 28 month old mice. FIG. 1A is a pair of photographic images showing representative staining of AAV GFP control and AAV AUF1/GFP positive myofibers in TA
muscle 40 d post-administration. FIG. 1B is a graph showing quantification of GFP positive myofibers in TA muscle 40 d post-AAV administration. n=5 mice. FIG. 1C is a pair of graphs showing relative fold increased expression of aufl mRNA in gastrocnemius, TA, EDL, and soleus muscles 40 d post-AAV administration. n=8-9 mice. FIGs. 1D-H are graphs showing strength and exercise endurance in 3 and 12 month old mice and 40 d post-AAV
administration:
grip strength time (FIG. ID), maximum speed (FIG. 1E), work performance (FIG.
IF), time to exhaustion (FIG. 1G), and distance to exhaustion (FIG. IH). n=5-9 mice. FIGs.
1I-L are graphs showing strength and exercise endurance 6 months post-AAV administration in 18 month old mice: maximum speed (FIG 1I), work performance (FIG 1J), time to exhaustion (FIG. 1K), and distance to exhaustion (FIG. IL). n=4 mice. Mean SEM from 5 or more independent studies *P<0.05, **P<0.01 by unpaired Mann-Whitney U test.

-10-100281 FIGs. 2A-2J show AUF1 gene therapy induces muscle mass along with an increase in myofiber capacity. FIGs. 2A-B are graphs showing muscle weight relative to total body weight 40 d post-AAV administration for gastrocnemius and TA muscles, respectively.
n=8-9 mice. FIGs. 2C-D are graphs showing frequency distribution of gastrocnemius myofiber CSA and mean area at 40 d post-AAV administration. n=6 mice/group. FIGs. 2E-F
are graphs showing frequency distribution of TA muscle CSA and mean area at 40 d post-AAV

administration. n=5 mice. FIG. 2G is a pair of photographic images showing representative immunostain of slow myofiber (red) and nuclei (DAPI blue) in gastrocnemius muscle at 40 d post-therapy. Scale bar: 200 p.m. FIG. 2H is a pair of graphs showing slow myofibers per field and mean CSA of slow and fast myofibers in gastrocnemius muscle at 40 d post-AAV
administration. FIG. 21 is a pair of photographic images showing representative immunostain of slow myofiber (red) and nuclei (blue) in soleus muscle 40 d after AAV AUF1-GFP
or AAV GFP
administration. Scale bar: 200 ium. FIG. 2J is a graph showing slow-twitch soleus muscle myofiber 40 d after AAV AUF1 or AAV GFP administration. Mean cross surface area (CSA).
n=3 mice per group.
[0029] FIGs. 3A-3J show molecular markers of skeletal muscle myogenesis in AAV8 AUF1-GFP gene transferred mice. FIGs. 3A-B are graphs showing relative myh7 mRNA levels in gastrocnemius (FIG. 3A) and soleus (FIG. 3B) muscles normalized to invariant nuclear TATA-box binding protein (tbp) mRNA at 40 d post-gene transfer. FIGs. 3C-D are graphs showing relative fast myosin mRNA levels in gastrocnemius (FIG. 3C) and soleus (FIG. 3D) muscles normalized to tbp mRNA at 40 d gene transfer. FIG. 3E is a graph showing expression levels of mRNAs as indicated in gastrocnemius muscle at 40 d post-gene transfer. FIG. 3F is a graph showing DNA mitochondrial content in gastrocnemius muscle 40 d or 6 months post gene transfer. FIG. 3G is a graph showing mil and nrf2 mRNA levels in gastrocnemius muscle 40 d after gene transfer. FIG. 3H is a graph showing nrfl and nrf2 mRNA levels in the soleus muscle 40 d after gene transfer. FIG. 31 is a pair of graphs showing mitochondrial DNA content in the gastrocnemius muscle 40 d and 6 months after gene transfer. FIG. 3J is a graph showing mitochondrial DNA content in the soleus muscle 40 d after gene transfer. Mean SEM from 3 or more independent studies. *P<0.05; **P<0.01 by unpaired Mann-Whitney U
test.
[0030] FIGs. 4A-4H show AUF1 is highly expressed in slow-twitch-enriched soleus muscle and stabilizes pgc la mRNA. FIG. 4A is a pair of graphs showing relative anfl mRNA
expression in 3 and 12 month old WT mice in TA, gastrocnemius, EDL, and soleus muscles.
n=5-7 mice. FIG. 4B is a representative immunoblot of AUF1 protein level and quantification in TA, gastrocnemius, EDL, and soleus muscle in 3 month old mice. FIG 4C is a graph showing

11 relative myh7 mRNA expression in 3 month old mouse TA, gastrocnemius, EDL, and soleus muscles. FIG. 4D shows relative pgcla mRNA expression and protein levels in WT

myoblasts and AUF1 KO myoblasts. FIG. 4E is a pair of graphs showing relative pgcla mRNA
expression in TA, gastrocnemius, and EDL muscles 40 d post-treatment, and in gastrocnemius at 6 months. FIG. 4F is a representative immunoblot of two AAV8-GFP control and GFP animals (left) and quantification of AUF1 and PGC1ct in three animals per group (right) at 6 months after treatment. FIG. 4G is a graph showing Pgcla mRNA
immunoprecipitation with endogenous AUF1 protein in myoblasts 48 h after myotube induction of differentiation in WT
C2C12 cells. n=3. FIG. 4H is a graph showing Pgcla mRNA decay rate in WT and C2C12 cells. Mean SEM from 3 or more independent studies. Panels A and B:
****P<0.001 by Kruskall -Wallis test. All other panels *P<0.05, **P<0.01, ***P<0.001 by unpaired Mann¨
Whitney U test.
100311 FIGs. 5A-5H show loss of AUF1 expression induces atrophy of slow-twitch myofibers. FIG. 5A is a graph showing body weight of WT and AUF1 KO mice at 3 months.
FIG. 5B shows TA, gastrocnemius, EDL, and soleus muscle mass in 3 month old WT
and AUF1 KO mice. Representative image of WT and AUF1 KO soleus muscles shown. FIG. 5C
shows photographic images of a representative immunostain of slow (top) or fast (bottom) myosin (red) and laminin (green) in the soleus muscle from 3 month old WT and AUF1 KO mice.
Scale bar:
200 lam. FIGs. 5D-E are graphs showing slow-twitch myofibers per field of percentage and number, respectively, in 3 month old WT and AUF1 KO mice. FIGs. 5F-G are graphs showing fast-twitch myofibers per field of percentage and number, respectively, in 3 month old WT and AUF1 KO mice. FIG. 5H is a graph showing mean soleus slow- and fast-twitch myofiber CSA
in 3 month old WT and AUF1 KO mice, n=6-7 mice.
100321 FIGs. 6A-6I show AUF1 deletion induces slow- and fast-twitch muscle atrophy at 6 months of age. FIG. 6A is a graph showing body weight of WT and AUF1 KO mice at 6 months, n=5-6 mice. FIG. 6B shows TA, EDL, gastrocnemius, and soleus muscle weight in 6 month old WT and AUF1 KO mice. FIG. 6C shows representative photographic images of excised muscles from 6 month old WT and AUF1 KO mice. FIG. 6D are photographic images showing representative immunostain of slow myosin (red) and laminin (green) in soleus muscle from 6 month old WT and AUF1 KO mice. Scale bar: 500 lam. FIG. 6E is a graph showing mean CSA of slow- and fast-twitch myofibers in soleus muscle of 6 month old WT
and AUF1 KO mice. FIG. 6F is a graph showing percentage of slow-twitch myofibers in 6 month old WT
and AUF1 KO mice in soleus muscle. FIG. 6G is a pair of photographic images showing representative staining of slow myosin (red) and laminin (green) in 6 month old WT and AUF1

- 12 -KO gastrocnemius muscle. Nuclei were stained by DAPI (blue), scale bar, 200 pm. FIG. 6H is a graph showing the number of slow-twitch myofibers per field in gastrocnemius muscle of 6 month old WT and AUF1 KO mice. n=4 mice per group. FIG. 61 is a graph showing mean gastrocnemius myofiber CSA of slow- and fast-twitch myofibers in 6 month old WT and AUF1 KO mice. n=4 mice per group. Mean SEM from 4 or more independent studies.
*P<0.05, **P<0.01 by unpaired Mann-Whitney U test.
[0033] FIGs. 7A-7G show AUF1 supplementation in skeletal muscle improves exercise endurance in 12-month old (middle-aged) and 18 month old mice. FIG. 7A is a graph showing relative expression of aufl mRNA in the TA, gastrocnemius, EDL, and soleus muscles normalized to invariant TBP mRNA at 3 and 12 months of age in WT mice. FIG. 7B
shows representative immunoblot and quantification of AUF1 protein levels in the TA
muscle of WT
mice with age at 3, 12, and 18 months. GAPDH is a loading control. n=3 mice per group per lane. FIG. 7C are graphs showing TA, gastrocnemius, EDL muscle mass, and soleus in 3, 12, and 18 month old WT mice normalized to total body weight. FIG. 7D is an immunoblot of AUF1 andp-tubulin in TA muscle as in FIG. 7A, 40 d after AAV8 administration.
FIG. 7E is a graph showing cuff] mRNA expression normalized to invariant gapdh mRNA in various organs of 12 month old mice, 40 d after AAV8 AUF1-GFP or AAV8 GFP control administration. FIG.
7F shows representative Pax7 staining in TA muscle in 12 month old mice 40 d after AAV8 AUF1-GFP or AAV8 GFP control vector administration. Scale bar, 100 pm.
Quantification of Pax7 mRNA expression normalized to invariant TBP mRNA, in TA muscle of 12 month old mice 40 d after AAV8 AUF1-GFP or AAV8 GFP control vector administration. n=8-9 per mice group. FIG. 7G is a graph showing relative expression of Trim63 and Fbxo32 mRNAs in TA
muscle normalized to TBP mRNA 40 d after AAV administration. Mean SEM from 3 or more independent studies. FIG. 7A-B: *P<0.05, **P<0.01 by Kruskall-Wallis test. All other panels *P<0.05, **P<0.01 by unpaired Mann-Whitney U test.
[0034] FIG. 8A-8B show AUF1 controls myosin and MEF2C
expression. The graphs of FIG. 8A show relative expression of fast and slow myosin mRNAs normalized to gapdh mRNA
in differentiating (48 h) WT myotubes and AUF1 KO C2C12 cells. n=5 mice per group. FIG.
8B is a graph showing mef2c mRNA expression normalized to TBP mRNA in gastrocnemius muscle 6 months after AAV AUF1-GFP or AAV GFP injection. n=5 mice per group.
Mean SEM from 5 or more independent studies. *P<0.05 by unpaired Mann-Whitney U
test.
[0035] FIGs. 9A-9G show AUF1 deletion induces slow-twitch muscle atrophy at a young age. FIG. 9A shows representative photographic images of TA, EDL, and gastrocnemius muscles in 3 month old WT and AUF1 KO mice. FIG. 9B shows representative immunostain

- 13 -images of slow and fast myosin (red) myofibers in the soleus of WT and AUF1 KO
mice. DAPI
stain (blue) of nuclei, laminin (green) stain of extracellular matrix. Scale bar: 500 lam. FIG. 9C
shows photographic images of representative stains of slow myosin (red) and laminin (green) in 3 month old WT and AUF1 KO gastrocnemius muscle (scale bar, 200 pm). FIGs. 9D-E are graphs showing percentage and number, respectively, of slow-twitch myofibers per field in gastrocnemius muscle of 3 month old WT and AUF1 KO mice. FIG. 9F is a graph showing mean gastrocnemius muscle area of slow- and fast-twitch myofibers in 3 month old WT and AUF1 KO mice. n=4 mice per group. FIG. 9G shows levels of PGC la, AUF1, and control GAPDH protein in gastrocnemius and soleus muscles of 3 month old WT and AUF1 KO mice.
Each lane corresponds to one mouse. Lower band in AUF1 gastrocnemius muscle lanes is a non-specific protein. Mean SEM from 3 or more independent studies. *13<0.05 by unpaired Mann-Whitney U test. ns, (not significant).
100361 FIGs. 10A-10C illustrate the development of AAV8 expression vectors. FIG.
10A is a schematic illustration of the development of AAV8 expression vectors.
The cDNA of the murine p40AuFI cDNA was cloned into an AAV8 vector under the tMCK promoter (AAV8-tMCK-AUF1-1RES-eGFP) (Vector Biolabs). The tMCK promoter was generated by the addition of a triple tandem of 2RS5 enhancer sequences (3-Ebox) ligated to the truncated regulation region of the MCK (muscle creatine kinase) promoter, which induces high muscle specificity (Blankinship et al., "Efficient Transduction of Skeletal Muscle Using Vectors Based on Adeno-associated Virus Serotype 6," Mol. Ther. 10(4):671-8 (2004), which is hereby incorporated by reference in its entirety). AAV8 vectors express AUF1 and GFP
(AUF1-GFP, with GFP translated from the same mRNA by the HCV IRES), or as a control only GFP.
Expression of both genes is controlled by the creatine kinase tMCK promoter that is selectively active in skeletal muscle cells. The AAV8-tMCK-1RES-eGFP construct was used as a control vector. FIG. 10B shows the amino acid sequence of the encoded p40AuF1 isoform (SEQ ID
NO:27) expressed in transduced cells by the AAV8 vector in FIG. 10A. FIG. 10C
shows the nucleotide sequence (SEQ ID NO:28) of the coding region of the p40Aun isoform.
100371 FIGs. 11A-11B show AAV8 transduction frequency in mdx mice. AAV8 AUF1-GFP and AAV8 GFP control vector-treated mdx mice displayed similar vector transduction and retention rates, shown by tibialis anterior (TA) muscle GFP staining. FIG. 11A
shows representative photographic images of GFP immunofluorescence staining of TA
muscle (green) to highlight AAV8 transduction efficiency and laminin-a2 staining (red) to highlight muscle fiber architecture and integrity. FIG. 11B is a graph showing quantification of 3 animals per condition

- 14 -for AAV8 GFP transduction in TA muscle. There is no statistical difference (ns) in transduction efficiency between control AAV8 GFP and treatment AAV8 AUF1 GFP groups.
[0038] FIGs. 12A-12F show AUF1 gene therapy enhances muscle mass and endurance in mcbc mice. One month old C57BL/10ScSn male DMD mice (herein mdx mice, JACS) were administered 2x1011 genome copies of AAV8 AUF1-GFP or control AAV8 GFP as a single retro-orbital injection of 50 ul containing 2.5x1011 AAV particles. Two months following AAV8 administration, mdx mice transduced with AAV8 AUF1-GFP or AAV8 GFP as a control were tested by standard procedures for exercise performance (see Examples, infra).
FIG. 12A is a graph showing Inc& control mice receiving only AAV8 GFP at three months old had an average body weight of 29 gm compared to 30 gm for wild type (WT) C57BL
mice.
In contrast, when compared to control AAV8 GFP treated mcbc mice, AAV8 AUF1-GFP
supplemented mcbc mice had an average body weight of 31 gm, a significant increase compared to control /H&c mice. FIG. 12B is a graph showing when normalized to body weight and at 2 months post-gene therapy transduction, AAV8 AUF1-GFP treated mdx mice demonstrated a 10% increase in tibialis anterior (TA) muscle mass, an 11%
increase in extensor digitorum longus (EDL) muscle mass, and an 8.5% increase in gastrocnemius muscle mass. There was no difference in soleus muscle mass. Compared to control AAV8 GFP
treated 111dX mice, AUF1 supplemented inclx mice showed a ¨40% improvement in grid hanging time (FIG. 12C), a measure of limb-girdle skeletal muscle strength and endurance. When tested by treadmill, AAV AUF1-GFP mcbc mice displayed 16% higher maximum speed (FIG.
12D), a 35% greater time to exhaustion (FIG 12E), and a 37% increased distance to exhaustion (FIG.
12F). These data demonstrate a substantial and statistically significant increase in exercise performance and endurance in nictx mice as a result of AUF1 gene transfer. All results are expressed as the mean+ SEM. Two group comparisons were analyzed by the unpaired Mann-Whitney test. *, P<0.05.
[0039] FIGs. 13A-13D show AUF1 gene therapy does not increase WT
muscle mass or endurance. Normal WT C57BL mice, the same background as mdx mice, were administered at 1 month of age AAV8 GFP control or AAV8 AUF1-GFP at 2x1011 genome copies by retro-orbital injection as described in FIGs. 12A-12F. Mice were analyzed at 3 months post-gene transfer. These data are in contrast to the significant increase in muscle mass and exercise endurance found in mcbc mice. Rather, WT mice administered with AAV8 compared to control AAV8 GFP mice of the same genetic background, show no statistically significant increase in body weight (FIG. 13A), treadmill time to exhaustion (FIG. 13B), maximum speed (FIG. 13C), and distance to exhaustion (FIG. 13D). All results are expressed SUBSTITUTE SHEET (RULE 26)

- 15 -as the mean SEM. Two group comparisons were analyzed by the unpaired Mann-Whitney test.
No results were found to be significantly different at P<0.05.
[0040] FIG. 14 shows AAV8 AUF1 gene therapy reduces serum creatine kinase levels in mdx mice. mdx mice at 1 month old were administered AAV8 AUF1-GFP or control AAV8 GFP as described in FIGs. 12A-12F. At 3 months, mice were tested for levels of serum creatine kinase (CK) activity, a measure of sarcolemma leakiness and muscle atrophy. Top:
Raw data showing serum CD activity results for WT control, mdx mice treated with AAV8 GFP vector alone, and mdx mice treated with AAV8 AUF1 GFP. Bolton':
Quantification of three replicate studies of 3 mice each. Control AAV8 GFP "mix mice displayed high levels of serum CK activity, mt.& mice that received AAV8 AUF1-GFP gene therapy were reduced in serum CK activity by more than 4-fold, a highly significant reduction. WT
C57BL mice had no detectable level of serum CK activity. ND, not detected. All results are expressed as the mean SEM. Two group comparisons were analyzed by the unpaired Mann-Whitney test. ", P<0.01, *** P<0.001.
[0041] FIGs. 15A-15B show AAV8 AUF1 gene therapy reduces muscle necrosis and fibrosis in mdx mouse diaphragm. mcbc mice at 1 month old were administered GFP or control AAV8 GFP as described in FIGs. 12A-12F. At 3 months, diaphragms were reduced from AAV8 GFP control and AAV8 AUF1-GFP mice, embedded FFPE and stained with H&E (FIG. 15A). The percent degenerative diaphragm muscle was scored and found to be reduced by 74% by AUF1 gene transfer. WT C57BL mouse diaphragm served as a control. Diaphragm muscle from mdx mice was stained with Masson Trichome to quantify muscle fibrosis (FIG. 15B). Shown are representative muscle sections. AUF1 gene transfer reduced fibrosis by 2-fold compared to control AAV8 GFP treated animals. All results are expressed as the mean SEM. Two group comparisons were analyzed by the unpaired Mann-Whitney test. **, P<0.01. Otherwise analyzed by Fisher Exact test as indicated.
[0042] FIGs. 16A-16B show AAV8 AUF1 gene therapy reduces muscle immune cell invasion. mdx mice at 1 month old were administered AAV8 AUF1-GFP or control GFP as described in FIGs. 12A-12F. At 3 months, diaphragms were resected from GFP control and AAV8 AUF1-GFP treated mice, embedded in FFPE, and stained with an antibody to the macrophage biomarker CD68 coupled with the red fluorescence marker Alexa Fluor 555. Representative images show strong reduction in macrophage CD68 staining in AAV8 AUF1-GFP treated animals compared to AAV8 GFP controls (FIG. 16A).
Quantification of 5 fields per specimen from 3 mice per group for CD68 staining (FIG. 16B).

SUBSTITUTE SHEET (RULE 26)

- 16 -All results are expressed as the mean SEM. Two group comparisons were analyzed by the unpaired Mann-Whitney test *, P<0.05.
[0043] FIGs. 17A-17E show AAV8 AUF1 gene therapy suppresses expression of embryonic myosin heavy chain (eMHC) in mdx mice. eMHC is a clinical marker of muscle degeneration in DMD. mdx mice at 1 month old were administered AAV8 AUF1-GFP
or control AAV8 GFP as described in FIGs. 12A-12F. At 3 months, diaphragm muscle was removed, fixed in FFPE, and stained with antibodies to eMHC (green), nuclei (DAPI, blue), and laminin (red). Immunotluorescence was carried out and representative images shown compared to WT C57BL6 mice (FIG. 17A). AAV8 AUF1-GFP gene transfer strongly reduced eHMC expression in diaphragm. High magnification of diaphragm stained as in FIG.
17A showing strong reduction in eMHC expression by AUF1 gene transfer (FIG.
17B).
Quantification of eMHC staining in myofibers, showing a 75% reduction in eMHC
expression by AUF1 gene transfer (FIG. 17C). The percent of centro-nuclei per myofiber/field was quantified, a measure of normal muscle fiber maturation (FIG. 17D). AUF1 gene transfer reduced the percentage of centro-nuclei by 52% compared to AAV8 GFP controls.
Myofiber cross sectional area (CSA) was quantified (FIG. 17E). AUF1 gene transfer strongly increased the CSA of the larger myofibers, indicative of mature regenerative muscle. All results are expressed as the mean SEM. Two group comparisons were analyzed by the unpaired Mann-Whitney test. Multiple group comparisons were performed using one-way analysis of variance (ANOVA). The non-parametric Kruskal¨Wallis test followed by the Dunn's comparison of pairs was used to analyze groups when suitable. *, P<0.05; *** P<0.001.
[0044] FIGs. 18A-18C show AAV8 AUF1 gene transfer increases expression of endogenous utrophin-A in mdx mice. mdx mice at 1 month old were administered AUF1-GFP or control AAV8 GFP as described in FIGs. 12A-12F. The gastrocnemius muscle was removed at 3 months, fixed in FFPE, and stained with DAPI (blue for nuclei, antibodies to utrophin (red) and laminin (green) (FIG. 18A). Representative images from 3 mice for each group are shown. AUF1 gene therapy strongly increased expression of utrophin and showed evidence for normalization of myofiber integrity (laminin staining).
Immunoblot analysis for utrophin, AUF1, and GAPDH (invariant control) proteins was conducted on the gastrocnemius muscle of 3 AAV8 GFP and 3 AAV8 AUF1-GFP mdx mice at 3 months (FIG.
18B). Gastrocnemius utrophin protein levels were increased by an average of 20-fold in animals receiving AUF1 gene therapy. AUF1 protein levels were increased an average of 3-4 fold. Utrophin mRNA levels were quantified by qRT-PCR and normalized to invariant TBP

SUBSTITUTE SHEET (RULE 26)

- 17 -mRNA (FIG. 18C). There was no statistically significant difference between samples n=3 animals for each condition_ [0045] FIGs. 19A-19C show AAV8 AUF1 gene transfer increases expression of satellite cell activation gene Pax7, key muscle regeneration genes pgc I a and mef2c, slow twitch determination genes and mitochondrial DNA content in mdx mice. mdx mice at 1 month old were administered AAV8 AUF1-GFP or control AAV8 GFP as described in FIGs.
12A-12F. The gastrocnemius muscle was removed at 3 months, mRNA extracted and quantified by qRT-PCR relative to invariant tbp mRNA. AUF1 gene therapy increased expression of pgc I a, mef2c, and Pax7 mRNAs in the gastrocnemius of mdx mice relative to controls receiving vector alone (FIG. 19A). Wild type non-mdx animals (WT) served as a control for normal muscle levels in age-matched animals. AAV8 AUF1 gene therapy restored near WT levels or exceeded WT levels of gene expression. AUF1 gene therapy increased expression of slow-twitch lineage determination myosin mRNAs in the gastrocnemius muscle in mdx animals relative to controls receiving vector alone (FIG. 19B) AAV8 AUF1 gene therapy restored near WT levels or exceeded WT levels of gene expression. AUF1 gene therapy increased expression of mitochondrial DNA in the gastrocnemius muscle of mdx mice, consistent with increased slow-twitch muscle mass (FIG 19C). All results are expressed as the mean+ SEM. Two group comparisons were analyzed by the unpaired Mann-Whitney test.
*, P<0.05; **, P<0.01; *** P<0.001.
[0046] FIG. 20 shows genome-wide transcriptomic and translatomic studies demonstrate AUF1 activation of C2C12 myoblast muscle fiber development. Proliferating C2C12 mouse cardiac myoblasts were transduced with lentivirus control vectors or lentivirus vectors expressing p45 AUF1, and induced to differentiate into myotubes by culturing in differentiation medium as described in the Examples infra. Proliferating myoblasts were used because they are activated in p38 MAPK and other signaling pathways that promote myogenesis, which is representative of the activated state and population of muscle cells following muscle damage from wounding, or the state of muscle in myogenic diseases, such as chronic regenerative attempts that occur in Duchene Muscular Dystrophy (DMD). Overview of the experimental approach. At 48 h, when myotubes begin to form, polyribosomes were separated by sucrose sedimentation corresponding to poorly translated (2 & 3 ribosome) fraction and well translated (>4 polysome) fractions, total mRNA and mRNA in polyribosome fractions were independently purified (polyA+ fraction devoid of rRNA), bacterial libraries were generated and subjected to deep sequencing using RNAseq, in two independent studies. Genome-wide mRNA
abundance SUBSTITUTE SHEET (RULE 26)

- 18 -used 10g2 ratios of translated/total mRNA. Procedures and bioinformatic pipeline used for analysis are described in the Examples infra [0047] FIGs. 21A-21B show AUF1 supplementation stimulates expression of major muscle development pathways and decreases expression of inflammatory cytokine, inflammation, cell proliferation, cell death, and anti-muscle regeneration pathways. Data from FIG. 20 genome-wide mRNA expression and translation analysis. Major upregulated pathways at the levels of transcription, translation, or both with AUF1 supplementation in C2C12 myoblasts (FIG. 21A). Analyzed by KEGG. Major downregulated pathways at the levels of transcription, translation, or both with AUF1 supplementation in C2C12 myoblasts (FIG. 21B).
Analyzed by KEGG.
[0048] FIGs. 22A-22B show AUF1 supplementation of C2C12 myoblasts upregulates pathways for major biological processes and molecular functions in muscle development and regeneration. Data from FIG. 20 genome-wide mRNA expression and translation analysis.
Major upregulated biological processes at the levels of transcription, translation, or both with AUF1 supplementation in C2C12 myoblasts (FIG. 22A). Analyzed by KEGG. Major upregulated molecular functions at the levels of transcription, translation, or both with AUF1 supplementation in C2C12 myoblasts (FIG. 22B). Analyzed by KEGG.
[0049] FIGs. 23A-23B show AUF1 supplementation of C2C12 myoblasts decreases muscle inflammation, inflammatory cytokine, and signaling pathways that oppose muscle regeneration. Data from FIG. 20 genome-wide mRNA expression and translation analysis.
Major downregulated biological processes at the levels of transcription, translation, or both with AUF1 supplementation in C2C12 myoblasts (FIG. 23A). Analyzed by KEGG. Major downregulated molecular functions at the levels of transcription, translation, or both with AUF1 supplementation in C2C12 myoblasts (FIG. 23B). Analyzed by KEGG.
[0050] FIG. 24 shows AUF1 supplementation of C2C12 myoblasts decreases expression of muscle genes associated with development of fibrosis. Data from FIG. 20 genome-wide mRNA expression and translation analysis. Major downregulated pathways and functions at the levels of transcription, translation, or both with AUF1 supplementation in C2C12 myoblasts.
Analyzed by KEGG.
[0051] FIGs. 25A-25D show lentivirus transduction of injured TA muscle with p45 AUF1 in mice activates satellite cells and reduces biomarkers of muscle atrophy. A lentivirus vector was developed expressing cDNA for p45 AUF1 under control of the CMV
promoter (Abbadi et al., "Muscle Development and Regeneration Controlled by AUF1-mediated Stage-specific Degradation of Fate-determining Checkpoint mRNAs," Proc. Nat'l. Acad.
Sci. USA

- 19 -116:11285-90 (2019), which is hereby incorporated by reference in its entirety). Three month old male mice were administered an intramuscular injection of 50 ill of filtered 1.2% BaC12 in sterile saline with control lentivirus vector or with lentivirus AUF1 vector (1x108 genome copies) (total volume 100 111) into the left Tibialis Anterior (TA) muscle (FIG. 25A). The right TA muscle remained uninjured as a control. Mice were sacrificed at 7 days post-injection. TA
muscles were excised, weighed, and normalized to mouse body weight in grams.
TA injury reduced TA weight by 27% which was restored to near-uninjured levels by concurrent AUF1 gene therapy. In FIG. 25B, immunoblot analysis of AUF1 normalized to invariant GAPDH
protein for TA muscle at 7 days post-lentivirus p45 AUF1 administration as in FIG. 25A.
Shown is a representative uninjured, two injured, and injured TA muscles with concurrent p45 AUF1 gene therapy from independent animals. Lentivirus p45 AUF1 gene transfer strongly increased levels of the p45 AUF1 isoform but not p42 AUF1 and p40 AUF1 that were not encoded (p37 AUF1 =
is undetectable). In FIG. 25C, TA muscles as in FIG. 25A were probed by qRT-PCR for Pax7 mRNA levels, a biomarker of muscle satellite (stem) cell activation, and normalized to invariant TATA-box binding protein (TBP) mRNA. AUF1 gene therapy increased Pax7 expression by >3-fold. In FIG. 25D, TA muscles as in FIG. 25A were probed by qRT-PCR for expression of muscle atrophy biomarker genes TRIM63 and Fbxo32, normalized to TBP mRNA.
TA muscle injury strongly induced expression of TRIM63 and Fbxo32 mRNA, which were downregulated to uninjured TA muscle levels by p45 Au" gene therapy, indicating strong cessation of muscle injury due to AUF1 intramuscular administration. No statistical difference (ns). All results are expressed as the mean + SEM with at least three independent trials of 3 or more animals per condition. Two group comparisons were analyzed by the unpaired Mann-Whitney test. *, P<0.05; **, P<0.01; ***, P<0.001.
[0052] FIGs. 26A-26D show p45 AUF1 lentivirus transduction enhances expression of muscle regeneration factors (MRFs) following TA muscle injury. Three month old male mice were injured in the TA muscle with BaC12 and administered with an intramuscular injection of control lentivirus vector or lentivirus AUF1 vector (see FIGs. 25A-D). Mice were sacrificed at 7 days post-injection. TA muscles were probed by qRT-PCR for identified mRNAs normalized to invariant TBP mRNA. In FIG. 26A, myogenin and MyoD mRNA levels, biomarkers of myoblast activation, differentiation, and muscle regeneration (Zammit, "Function of the Myogenic Regulatory Factors Myf5, MyoD, Myogenin and MRF4 in Skeletal Muscle, Satellite Cells and Regenerative Myogenesis," Semin. Cell. Dev. Biol. 72:19-32 (2017), which is hereby incorporated by reference in its entirety), were increased ¨2-fold by AUF1 gene therapy relative to injured control vector specimens. In FIG. 25B, myh8 mRNA, an embryonic myosin only SUBSTITUTE SHEET (RULE 26)

- 20 -expressed in adult muscle during muscle regeneration and a marker of co-expression of utrophin (Guiraud et al., "Embryonic Myosin is a Regeneration Marker to Monitor Utrophin-based Therapies for DMD," HUM. Mol. Genet. 28:307-19 (2019), which is hereby incorporated by reference in its entirety), was increased in expression by 5-fold in injured muscle with AUF1 gene therapy relative to injured control vector specimens. In FIG. 26C, myh7 mRNA, a myosin that specifies slow-twitch muscle (Zammit, "Function of the Myogenic Regulatory Factors Myf5, MyoD, Myogenin and MRF4 in Skeletal Muscle, Satellite Cells and Regenerative Myogenesis," Seinin. Cell. Dev. Biol. 72:19-32 (2017), which is hereby incorporated by reference in its entirety), was increased in expression by -2-fold in injured muscle with AUF1 gene therapy relative to injured control vector specimens. In FIG. 26D, myh4 mRNA, a myosin that specifies fast-twitch muscle (Zammit, -Function of the Myogenic Regulatory Factors Myf5, MyoD, Myogenin and MRF4 in Skeletal Muscle, Satellite Cells and Regenerative Myogenesis,"
Semin. Cell. Dev. Biol. 72:19-32 (2017), which is hereby incorporated by reference in its entirety), was increased in expression by -2-fold in injured muscle with AUF1 gene therapy relative to injured control vector specimens. All results are expressed as the mean SEM with at least three independent trials of 3 or more animals per condition. Two group comparisons were analyzed by the unpaired Mann-Whitney test. *, P<0.05; **, P<0.01.
[00531 FIGs. 27A-27D show p45 AUF1 lentivirus gene therapy promotes rapid regeneration of injured muscle. Three month old male mice were injured in the TA muscle with BaC12, and administered with an intramuscular injection of control lentivirus vector or lentivirus AUF1 vector, as in FIGs. 25A-25D. Mice were sacrificed at 3 days and 7 days post-injury. FIG.
27A shows photographic images of muscle fibers provide evidence for accelerated but normal muscle regeneration of myofibers in animals administered lentiviral AUF1 gene therapy. TA
muscle in OCT was sectioned and stained for immunofluorescence microscopy analysis for Laminin alpha 2 (red), Nuclei are stained with DAN (blue). Note the disrupted myofiber architecture and high level of central nuclei in the injured TA muscle treated with vector alone compared to the injured TA muscle administered lentiviral AUF1 gene therapy, consistent with accelerated muscle regeneration and mature myofibers. Scale bar, 2001.1m. FIG.
27B is a graph showing the percent muscle loss (atrophy) or gain (increase in mass) determined for the injured TA muscle compared to uninjured control or injured muscle receiving control lentivirus vector or lentivirus p45 AUF1, measured at sacrifice at 3 days and 7 days post-injury.
Injured TA muscle receiving sham gene therapy sustained a 20% loss in mass by day 3 following injury, which only very slightly improved by day 7. In contrast, injured TA muscle receiving AUF1 gene therapy showed a trend to less atrophy by day 3, which was almost fully recovered by day 7, SUBSTITUTE SHEET (RULE 26)

- 21 -demonstrating near normal mass. FIG 27C is a graph showing high levels of myotube central nuclei are a marker of immature myofiber development (Yin et al., "Satellite Cells and the Muscle Stem Cell Niche," Physiol. Rev. 93:23-67 (2013) and Schiaffino &
Reggiani, "Fiber Types in Mammalian Skeletal Muscles," Physiol. Rev. 91:1447-531 (2011), which are hereby incorporated by reference in their entirety). TA muscle analyzed at day 7 post-injury administered p45 AUF1 gene therapy were reduced by half in the percent of myofibers with central nuclei compared to vector only control injured muscle. This is consistent with accelerated muscle regeneration provided by AUF1 gene transfer. FIG. 27D is a graph showing a wider cross-sectional area of myofibers (cross-sectional area, CSA) with low numbers of central nuclei are indicative of mature myofiber development (Yin et al., "Satellite Cells and the Muscle Stem Cell Niche," Physiol. Rev. 93:23-67 (2013) and Schiaffino &
Reggiani, "Fiber Types in Mammalian Skeletal Muscles," Physiol. Rev. 91:1447-531 (2011), which are hereby incorporated by reference in their entirety). AUF1 gene transfer in injured TA
muscle produced a striking increase in CSA with reduced central nuclei per myofiber, consistent with generation of mature myofibers. All results are expressed as the mean+ SEM with at least three independent trials of 3 or more animals per condition. Two group comparisons were analyzed by the unpaired Mann-Whitney test. *, P<0.05; ", P<0.01, ***, P<0.001.
[00541 FIGs. 28A-28F show AUF1 is essential to promote repair of injured muscle, and can provide injury protection benefit when delivered by AAV8 gene transfer.
FIG. 28A is a schematic illustration of an AUF1 conditional knockout mouse developed as party of the technology described herein. Shown is a schematic of the exon 3 LoxP site insertions in the AUF1 gene. Lox sites were cloned to flank exon 3 of AUF1, which is maintained in all 4 AUF1 isoforms and contains the RNA binding domain. AUFIFloVFlox mice were derived, syblings mated to homogeneic purity generated, then mated with a Pax7cre ERT2 (B6;129-Pax7tm21(cre/ERT2)Fan/J mouse) (Jackson Labs). This provides cre recombinase induction by tamoxifen administration only in PAX7 expressing muscle satellite and myoblast cells. FIG.
28B is a graph showing results of three month old mice induced for cre expression with 5 daily i.p. injections of tamoxifen (3 mg/kg). There was no change in body weight of cre-induced mice.
FIG. 28C is a graph showing weight of non-injured skeletal muscles in mice were not significantly different in uninduced and tamoxifen induced cre mice. FIG. 28D
shows tamoxifen induction of cre for 3 months specifically deletes the aufl gene in skeletal muscle and abolishes skeletal muscle AUF1 protein expression. A representative immunoblot is shown for AUF1 levels in TA skeletal muscle and kidney, normalized to invariant GAPDH in control AUF1Hox/Flox and AUF1Hox/Hox x )7C"ERT2 mice after 5 days of cre induction and analyzed SUBSTITUTE SHEET (RULE 26)

- 22 -at day 7. There is no evidence for expression of AUF1 after Pax7-specific cre induction in muscle, whereas abundant AUF1 is present in kidney. FIG 28E is a graph showing one month old AUF1F1'/Fl" x PAX7c"ERT2 mice were either sham injected or injected with tamoxifen for days as above, then maintained on a diet that included oral tamoxifen for 5 months daily at 500 5 mg/kg (Envigo). Wild type (WT) BL6 mice and AUF1Flox/Flox x pAx-,cre ERT2 mice were either not induced for cre-expression (labeled AUF 1iFfl/Fax7) or induced for 5 months and deleted in the AUF1 gene (labeled AAUF lfuflIF'). One set of AAUF 1 filfl/Pax7 mice induced for cre expression for 5 months were also administered at 1 month of age with 2.0x10" AAV8 AUF1 particles (2x10" genome copies) by single retro-orbital injection of 50 pl. All mice were then injured by 1.2% BaC12 injection in the TA muscle, as described in FIGs. 25A-D. AUF1 is controlled by the creatine kinase tMCK promoter that is selectively active in skeletal muscle cells. TA muscle was excised at 7 days post-BaC12 injection and the percent of muscle atrophy determined by weight. TA muscle of AUF1Fl0x/Fl0x x p crc ERT2 mice expressing AUF1 and WT mice expressing AUF1 (not induced for cre) showed 16-18% atrophy that was not statistically different. In contrast, deletion of the AUF1 gene caused strongly increased atrophy of the TA
muscle, doubling atrophy levels to 35%. However, animals deleted for the AUF1 gene but prophylactically administered AAV8 AUF1 gene therapy demonstrated dramatically reduced levels of TA muscle atrophy, averaging ¨3%. FIG. 28F is a graph showing AUF1 control and cre-induced skeletal muscle AUF1 deleted mice were tested at 5 months for grip strength, a measure of limb-girdle skeletal muscle strength and endurance. AUF1 deleted mice showed a ¨50% reduction in grip strength. Collectively, these data demonstrate that AUF1 is essential for maintenance of muscle strength and muscle regeneration following injury, and that AUF1 gene therapy provides a remarkable ability to promote muscle regeneration and protect muscle from extensive damage despite traumatic injury. All results are expressed as the mean SEM with at least three independent trials of 3 or more animals per condition. Two group comparisons were analyzed by the unpaired Mann-Whitney test. *, P<0.05.
DETAILED DESCRIPTION
[0055] One aspect of the present application 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 cell-specific promoter.
[0056] The term "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 SUBSTITUTE SHEET (RULE 26)

- 23 -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. For example, 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.
[0057] The term "promoter" is used interchangeably with "promoter element" and "promoter sequence." Likewise, the term "enhancer" is used interchangeably with "enhancer element" and -enhancer sequence." The term "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," Ce// 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. For example, a promoter sequence located proximally to the 5' end of a transgene coding sequence is usually operably linked with the transgene. As used herein, the term "regulatory elements" is used interchangeably with "regulatory sequences" and refers to promoters, enhancers, and other expression control elements, or any combination of such elements.
[0058] 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. The 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

- 24 -correct site. In contrast, 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.
[0059] 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,- I Bto. 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).
[0060] The term "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.
[0061] Adeno-associated viral (AAV) vectors disclosed herein comprise a muscle cell-specific promoter. In some embodiments, 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. For example, 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.
[0062] In some embodiments, the muscle cell-specific promoter is selected from the group consisting of a muscle creatine kinase (MCK) promoter, a C5-12 promoter, a CK6-CK9 promoter, a dMCK promoter, a tMCK promoter, a smooth muscle 22 (SM22) promoter, a myo-3 promoter, a Spc512 promoter, a creatine kinase (CK) 8 promoter, a creatine kinase (CK) 8e promoter, a U6 promoter, a H1 promoter, a desmin promoter, a Pitx3 promoter, a skeletal alpha-actin promoter, a MHCK7 promoter, and a Sp-301 promoter. Suitable muscle cell-specific promoter sequences are well known in the art and are provided in Table 1 below (Malerba et al., "PABPN1 Gene Therapy for Oculopharyngeal Muscular Dystrophy," Nat.
C01111111112. 8:14848 (2017); Wang et al., "Construction and Analysis of Compact Muscle-Specific Promoters for AAV Vectors," Gene. Ther. 15:1489-1499 (2008); Piekarowicz etal., "A Muscle Hybrid SUBSTITUTE SHEET (RULE 26)

-25 -Promoter as a Novel Tool for Gene Therapy," Mol. Ther. Methods Cl/n. Dev.
15:157-169 (2019);
Salva et al., "Design of Tissue-Specific Regulatory Cassettes for High-Level rAAV-Mediated Expression in Skeletal and Cardiac Muscle," Mol. Ther. 15(2):320-329 (2007);
Lui et al., "Synthetic Promoter for Efficient and Muscle-Specific Expression of Exogenous Genes,"
Plasmid 106:102441 (2019), which are hereby incorporated by reference in their entirety.).
Table 1: Muscle Specific-Promoter Sequences Promoter Sequence*
SEQ
ID NO:
Human AGCCAGCCTCAGTTTCCCCTCCACTCAGTCCCTAGGAGGAAGGGGCGCCC 1 muscle AAGCGCGGGTTTCTGGGGTTAGACTGCCCTCCATTGCAATTGGTCCTTCT
creatine CCCGGCCTCTGCTTCCTCCAGCTCACAGGGTATCTGCTCCTCCTGGAGCC
kinase ACACCTTGGTTCCCCGAGGTGCCGCTGGGACTCGGGTAGGGGTGAGGGCC
CAGGGGGCACAGGGGGAGCCGAGGGCCACAGGAAGGGCTGGTGGCTGAAG
(MCK) GAGACTCAGGGGCCAGGGGACGGIGGCTICTACGTGCTTGGGACGTTCCC
AGCCACCGTCCCATGTTCCCGGCGGGGGGCCAGCTGTCCCCACCGCCAGC
CCAACTCAGCACTIGGTCAGGGTATCAGCTTGGIGGGGGGGCGTGAGCCC
AGCCCCTGGGGCGGCTCAGCCCATACAAGGCCATGGGGCTGGGCGCAAAG
CATGCCTGGGTTCAGGGTGGGTATGGTGCGGGAGCAGGGAGGTGAGAGGC
TCAGCTGCCCTCCAGAACTCCTCCCTGGGGACAACCCCTCCCAGCCAATA
GCACAGCCTAGGTCCCCCTATATAAGGCCACGGCTGCTGGCCCTTCCTTT
(NCBI sequence ID No. 1158) Human CTGAGGCTCAGGGCTAGCTCGCCCATAGACATACATGGCAGGCAGGCTTT 2 desmin GGCCAGGATCCCTCCGCCTGCCAGGCGTCTCCCTGCCCTCCCTTCCTGCC
TAGAGACCCCCACCCTCAAGCCTGGCTGGTCTTTGCCTGAGACCCAAACC
TCTTCGACTTCAAGAGAATATTTAGGAACAAGGTGGTTTAGGGCCTTTCC
TGGGAACAGGCCTTGACCCITTAAGAAATGACCCAAAGTCTCTCCTTGAC
CAAAAAGGGGACCCTCAAACTAAAGGGAAGCCTCTCTTCTGCTGICTCCC
CTGACCCCACTCCCCCCCACCCCAGGACGAGGAGATAACCAGGGCTGAAA
GAGGCCCGCCTGGGGGCTGCAGACATGCTTGCTGCCTGCCCTGGCGAAGG
ATTGGCAGGCTTGCCCGTCACAGGACCCCCGCTGGCTGACTCAGGGGCGC
AGGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCACGGCCACGGGCCGC
CCTTTCCTGGCAGGACAGCGGGATCTTGCAGCTGTCAGGGGAGGGGAGGC
GGGGGCTGATGTCAGGAGGGATACAAATAGTGCCGACGGCTGGGGGCCCT
(NCBI sequence ID No. 1674) Human GGAGTTCCAGGGGCGTAAAGGAGAGGGAGTTCGCCTICCITCCCITCCTG

skeletal ACACTCAGGAGTGACTGCTICTCCAATCCTCCCAACCCCACCACTCCACA
muscle CGACTCCCTCTTCCCGGTAGTCGCAAGTGGGAGITTGGGGATCTGAGCAA
AGAACCCGAAGAGGAGTTGAAATATTGGAAGTCAGCAGTCAGGCACCTIC
alpha CCGAGCGCCCAGGGCGCTCAGAGTGGACATGGTTGGGGAGGCCTITGGGA
actin actal CAGGTGCGGTTCCCGGAGCGCAGGCGCACACATGCACCCACCGGCGAACG
CGGTGACCCTCGCCCCACCCCATCCCCTCCGGCGGGCAACTGGGICGGGT
CAGGAGGGGCAAACCCGCTAGGGAGACACTCCATATACGGCCCGGCCCGC
GTTACCTGGGACCGGGCCAACCCGCTCCTICTTTGGTCAACGCAGGGGAC
CCGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGAGGGGGCTCTAGTGC
CCAACACCCAAATATGGCTCGAGAAGGGCAGCGACATTCCTGCGGGGTGG
CGCGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGAGGGACAAGC
(NCBI sequence ID No. 58)

-26 -Promoter Sequence*
SEQ
ID NO:
Mouse AGAAACCTGTGGTCTAGAGGCGGGGCGGGGCCGATGGAGGCAACGCACGC

muscle CCCCGCAGGCGCCCAGGCCACGCCCTCTGCCGCAGCATTCGGTGAAACCT
creatine GCGTTCCGAGAACTTCTGAAAACTTTATCTGGGGGCCTTCGAGAAGGCTC
kinase AGACAGTAAGGGTGCATGCTGCCAATCCTGAGGAGCTGAGTTCGATCCCT
(MCK) GAGACCTTCAGGGTGGACAGAGACGGACTCCCACATGTTGTTTTCTGACT
TCTACATGTGICCAGTCATACATACACAAATATGGAATAAACAGATGGCT
CATCAGGTAAGAGTGCTGGCTGCTTTTGCAGAGGACCCAGGTTCGATTTC
CAGAACCCACATGTCGGCTCAAAATCATCTGTAATTCCAGTTCCAGGGAG
ATCCAGCACTTTCTICCAGGGCCTCCACAGACACACATAAAATAAAGATA
AAAATCTCCAAAAAATATTGTTTTAATAATTACAACCTGAAGACCTTGCA
CAACTATTCCTGGCTGAGAAGATGGTAAGGGCGCTAGCTGCCAAGCTTGA
CAGCCTGAGITTCATCTCCAAGAACCATGAAAACTGACTCCTGGGAATTA
(NCBI sequence ID No. 12715) Molise GGAAGCAGAAGGCCAACATTCCTCCCAAGGGAAACTGAGGCTCAGAGTTA

desmin AAACCCAGGTATCAGTGATATGCATGTGCCCCGGCCAGGGTCACTCTCTG
ACTAACCGGTACCTACCCTACAGGCCTACCTAGAGACTCTTTTGAAAGGA
TGGTAGAGACCTGTCCGGGCTITGCCCACAGTCGTTGGAAACCTCAGCAT
TTTCTAGGCAACTTGTGCGAATAAAACACTTCGGGGGTCCTTCTTGTTCA
TTCCAATAACCTAAAACCTCTCCTCGGAGAAAATAGGGGGCCTCAAACAA
ACGAAATTCTCTAGCCCGCTTTCCCCAGGATAAGGCAGGCATCCAAATGG
AAAAAAAGGGGCCGGCCGGGGGTCTCCTGICAGCTCCTTGCCCTGTGAAA
CCCAGCAGGCCTGCCTGTCTICTGICCTCTTGGGGCTGTCCAGGGGCGCA
GGCCTCTTGCGGGGGAGCTGGCCTCCCCGCCCCCTCGCCTGTGGCCGCCC
TTTTCCTGGCAGGACAGAGGGATCCTGCAGCTGTCAGGGGAGGGGCGCCG
GGGGGTGATGTCAGGAGGGCTACAAATAGTGCAGACAGCTAAGGGGCTCC
(NCBI sequence ID No. 13346) Mouse GGGGTGATGTGTGICAGATCTCTGGATTGGGGGAGCTTCAAAGTGGGAAA

skeletal GAAAATGGAGTTCAAATGIGGGGCTTATTTTCCATCCCTACCTGGAGCCC
muscle ATGACTCCTCCCGGCTCACCTGACCACAGGGCTACCTCCCCTGAGCTTAA
GCATCAAGGCTTAGTAGTCTGAGTTAAGdAACCCATAAATGGGGTGCATT
alpha GTGGCAGGTCAGCAATCGTGIGTCCAGGIGGGCAGAACTGGGGAGACCTT
actin actal TCAAACAGGTAAATCTTGGGAAGTACAGACCAGCAGTCTGCAAAGCAGTG
ACCTTTGGCCCAGCACAGCCCTTCCGTGAGCCTTGGAGCCAGTTGGGAGG
GGCAGACAGCTGGGGATACTCTCCATATACGGCCTGGTCCGGTCCTAGCT
ACCTGGGCCAGGGCCAGTCCTCTCCTTCTTTGGTCAGTGCAGGAGACCCG
GGCGGGGACCCAGGCTGAGAACCAGCCGAAGGAAGGGACTCTAGTGCCCG
ACACCCAAATATGGCTTGGGAAGGGCAGCAACATTCCTTCGGGGCGGTGT
GGGGAGAGCTCCCGGGACTATATAAAAACCTGTGCAAGGGGACAGGCGGT
(NCBI sequence ID No. 11459)

-27 -Promoter Sequence*
SEQ
ID NO:

TAAGGGCCTGGGTAGGGGAGGTGGT GT GAGACGCT CC TGTCTCTCCT CTA
T CT GCCCATCGGCCCT T TGGGGAGGAGGAAT GT GCCCAAGGACTAAAAAA
AGGCCATGGAGCCAGAGGGGCGAGGGCAACAGACCTT TCATGGGCAAACC
T TGGGGCCCTGCT GT CTAGCATGCCCCACTACGGGTC TAGGCT GCCCAT G
TAAGGAGGCAAGGCCT GGGGACACCCGAGAT GCCT GGT TATAAT TAACCC
AGACATGTGGC TGCCCCCCCCCCCCCAACACCT GCTGCCTCTAAAAATAA
CCCTGTCCCTGGTGGATCCCCTGCATGCGAAGATCTT CGAACAAGGCT GT
GGGGGACTGAGGGCAGGCTGTAACAGGCTTGGGGGCCAGGGCT TATACGT
GCCTGGGACTCCCAAAGTATTACTGTT CCAT GT TCCCGGCGAAGGGCCAG
CTGTCCCCCGCCAGCTAGACT CAGCACTTAGT T TAGGAACCAGTGAGCAA
GTCAGCCCTIGGGGCAGCCCATACAAGGCCAT GGGGC TGGGCAAGCT GCA
CGCCT GGGTCC GGGGT GGGCACGGT GC CCGGGCAACGAGCT GAAAGCT CA
T CT GCTCTCAGGGGCCCCTCCCT GGGGACAGCCCCTC CTGGCTAGTCACA
CCCTGTAGGCT CCICTATATAACCCAGGGGCACAGGGGCTGCCCT CAT T C
TACCACCACCT CCACAGCAC
Spc5-12 CGAGCTCCACC GCGGT GGCGGCCGT CC GCCCT CGGCACCAT CCTCACGAC

ACCCAAATATGGCGACGGGTGAGGAAT GGTGGGGAGT TAT T TT TAGAGCG
GTGAGGAAGGT GGGCAGGCAGCAGGTGT TGGCGCT CT AAAAATAACT CCC
GGGAGT TAT T T TTAGAGCGGAGGAATGGTGGACACCCAAATATGGCGACG
GT T CCTCACCC GT CGCCATAT TTGGGT GTCCGCCCTCGGCCGGGGCCGCA
T TCCT GGGGGC CGGGCGGTGCTCCCGC CCGCCT CGAT AAAAGGCT CCGGG
GCCGGCGGCGGCCCACGAGCT ACCCGGAGGAGCGGGAGGCGCCAAGCT CT
AGAACTAGTGGATCCCCCGGGCTGCAGGAAT IC
*See Malerba et al., "PABPN1 Gene Therapy for Owl pilau fieal Muscular Dystrophy,- Nat.
Coimnun. 8:14848 (2017); Wang et al., "Construction and Analysis of Compact Muscle-Specific Promoters for AAV Vectors," Gene. /her. 15:1489-1499 (2008); Piekarowicz et al., "A Muscle Hybrid Promoter as a Novel Tool for Gene Therapy," Mol. 'her. Methods ('fin.
Dev. 15:157-169 (2019); and Salva et al., "Design of Tissue-Specific Regulatory Cassettes for High-Level rAAV-Mediated Expression in Skeletal and Cardiac Muscle," Mol. Ther. 15(2):320-329 (2007), which are hereby incorporated by reference in their entirety.
[0063] In some embodiments, the muscle cell-specific promoter is a muscle creatine-kinase ("MCK") promoter. The muscle creatine kinase (MCK) gene is highly active in all striated muscles. Creatine kinase plays an important role in 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. There are four known isoforms of creatine kinase: brain creatine kinase (CKB), muscle creatine kinase (MCK), and two mitochondrial forms (CKMi). MCK is the most abundant non-mitochondrial mRNA 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

-28 -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.
Mammalian MCK regulatory elements, including human and mouse promoter and enhancer elements, are described in Hauser et al., "Analysis of Muscle Creatine Kinase Regulatory Elements in Recombinant Adenoviral Vectors," Mol. Therapy 2:16-25 (2000), which is 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).
[0064] 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," Wiky Interdiscip. Rev. RNA 5(4):549-64 (2014), which is hereby incorporated by reference in its entirety). These include growth factors, inflammatory cytokines (Moore et al., "Physiological Networks and Disease Functions of RNA-binding Protein AUF1,"
Wiley Interdiscip Rev RNA 5(4):549-64 (2014) and Zhang et al., "Purification, Characterization, and cDNA Cloning of an AU-rich Element RNA-binding Protein, AUF1," Mol. Cell.
Biol.
13(12):7652-65 (1993), which are hereby incorporated by reference in their entirety), and tissue stem cell fate-determining mRNAs (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.
[0065] 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. 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.,

-29 -"Purification, Characterization, and cDNA Cloning of an AU-rich Element RNA-binding Protein, AUF'1,"Mol. Cell. Biol. 13(12)-7652-65 (1993); and Halees et al., "ARED Organism:
Expansion of ARED Reveals AU-rich Element Cluster Variations Between Human And Mouse,"
Nucleic Acids Res 36(Database issue):D137-40 (2008), which are hereby incorporated by reference in their entirety).
[0066] AU-rich mRNA binding factor 1 (AUF1; HNRNPD) binds with high affinity to repeated AU-rich elements ("AREs") located in the 3' untranslated region ("3' UTR") found in approximately 5% of mRNAs. Although 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). It was previously reported that 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). It was also found that 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), which is hereby incorporated by reference in its entirety). 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.
[0067]
AUF1 has four related protein isoforms identified by their molecular weight (p37AuFi, p40 AUFi p42'1, p45 AUF I) 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.
20(11):465-470 (1995); and Kim et al., "Emerging Roles of RNA and RNA-Binding Protein Network in Cancer Cells," Bil4B Rep. 42(3):125-130 (2009), which are hereby incorporated by reference in their entirety). Each of these four isoforms include two centrally-positioned, tandemly arranged RNA
recognition motifs ("RRIVIO which mediate RNA binding (DeMaria et al., "Structural

- 30 -Determinants in AUF 1 Required for High Affinity Binding to A+U-rich Elements," I Biol.
Chem. 272.27635-27643 (1997), which is hereby incorporated by reference in its entirety) [0068] The general organization of an RRM is a 13-a-3-p-a-13 RNA
binding platform of anti-parallel 3-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 RN? 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). Structures of individual AUF1 RRNI domains resolved by NMR are largely consistent with this overall tertiary fold (Zucconi & Wilson, "Modulation of Neoplastic Gene Regulatory Pathways by the RNA-binding Factor AUF1,"
Front. Biosci. 16:2307-2325 (2013); Nagata et al., "Structure and Interactions with RNA of the N-terminal UUAG-specific RNA-binding Domain of hnRNP DO," I. Mol. Biol.
287:221-237 (1999); and Katahira et al., "Structure of the C-terminal RNA-binding Domain of hnRNF' DO
(AUF1), its Interactions with RNA and DNA, and Change in Backbone Dynamics Upon Complex Formation with DNA," I. Mol. Biol. 311:973-988 (2001), which are hereby incorporated by reference in their entirety).
[0069] 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 regeneration and maintenance.
[0070] The term "fragment- or "portion- when used herein with respect to a given polypeptide sequence (e.g., AUF1), 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 wild-

- 31 -type protein. A fragment may, for example, contain a functionally important domain, such as a domain that is important for receptor or ligand binding_ [0071] Accordingly, in certain embodiments, functional fragments of AUF1 as described herein include at least one RNA recognition domain ("RRM") domain. In certain embodiments, functional fragments of AUF1 as described herein include two RRM domains.
[0072] AUF1 or functional fragments thereof as described herein may be derived from a mammalian AUF1. In one embodiment, the AUF1 or functional fragment thereof is a human AUF1 or functional fragment thereof. In another embodiment, 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 p37A1JF1 ,p40AUF%
p42Au", and p45AuFl. The GenBank accession numbers corresponding to the nucleotide and amino acid sequences of each human and mouse isoform is found in Table 2 below, each of which is hereby incorporated by reference in its entirety.
Table 2: Summary of GenBank Accession Numbers of AUF1 Sequences Isoform Human Mouse Nucleotide Amino Acid Nucleotide Amino Acid p37AuFt NM 001003810.2 NP 001003810.1 NM 001077267.2 NP 001070735.1 (SEQ ID NO:9) (SEQ ID NO:10) (SEQ ID NO: 11) (SEQ ID
NO:12) p 40AuF1 NM 002138.3 NP 002129.2 NM 007516.3 NP
031542.2 (SEQ ID NO: 13) (SEQ ID NO:14) (SEQ ID NO: 15) (SEQ ID
NO:16) p42AuFt NM 031369.2 NP 112737.1 NM 001077266.2 NP
001070734.1 (SEQ ID NO: 17) (SEQ ID NO:18) (SEQ ID NO: 19) (SEQ ID
NO:20) p45AUFl NM _031370.2 NP 112738.1 NM 001077265.2 NP
001070733.1 (SEQ ID NO:21) (SEQ ID NO:22) (SEQ ID NO:23) (SEQ ID
NO:24) [0073] The sequences referred to in Table 2 are reproduced below.
[0074] The human p37AuF1 nucleotide sequence of GenBank Accession No.
NM 001003810.1 (SEQ ID NO:9) is as follows:
CTTCCGTCGG CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA

GCGCGAGAGT GGGAGGCGAA GGGGGCAGGC CAGGGAGAGG CGCAGGAGCC TTTGCAGCCA

CGCGCGCGCC TTCCCTGTCT TGTGTGCTTC GCGAGGTAGA GCGGGCGCGC GGCAGCGGCG

GGGATTACTT TGCTGCTAGT TTCGGTTCGC GGCAGCGGCG GGTGTAGTCT CGGCGGCAGC

GGCGGAGACA CTAGCACTAT GTCGGAGGAG CAGTTCGGCG GGGACGGGGC GGCGGCAGCG

- 32 -CAGGGGGCAG CGGCGGCGGC GGGAAGCGGA GCCGGGACCG GGGGCGGAAC CGCGTCTGGA

GGCACCGAAG GGGGCAGCGC CGAGTCGGAG GGGGCGAAGA TTGACGCCAG TAAGAACGAG

GAGGATGAAG GGAAAATGTT TATAGGAGGC CTTAGCTGGG ACACTACAAA GAAAGATCTG

AAGGACTACT TTTCCAAATT TGGTGAAGTT GTAGACTGCA CTCTGAAGTT AGATCCTATC

ACAGGGCGAT CAAGGGGTTT TGGCTTTGTG CTATTTAAAG AATCGGAGAG TGTAGATAAG

GTCATGGATC AAAAAGAACA TAAATTGAAT GGGAAGGTGA TTGATCCTAA AAGGGCCAAA

GCCATGAAAA CAAAAGAGCC GGTTAAAAAA ATTTTTGTTG GTGGCCTTTC TCCAGATACA

CCTGAAGAGA AAATAAGGGA GTACTTTGGT GGTTTTGGTG AGGTGGAATC CATAGAGCTC

CCCATGGACA ACAAGACCAA TAAGAGGCGT GGGTTCTGCT TTATTACCTT TAAGGAAGAA

ATAAAAGTAG CCATGTCGAA GGAACAATAT CAGCAACAGC AACAGTGGGG ATCTAGAGGA

GGATTTGCAG GAAGAGCTCG TGGAAGAGGT GGTGACCAGC AGAGTGGTTA TGGGAAGGTA

TCCAGGCGAG GTGGTCATCA AAATAGCTAC AAACCATACT AAATTATTCC ATTTGCAACT

TATCCCCAAC AGGTGGTGAA GCAGIATTTT CCAATTTGAA GATTCATTTG AAGGTGGCTC

ATGACGTTGG GTCCCTCTGA AGTTTAATTC TGAGTTCTCA TTAAAAGAAA TTTGCTTTCA

TTGTTTTATT TCTTAATTGC TATGCTTCAG AATCAATTTG TGTTTTATGC CCTTTCCCCC

AGTATTGTAG AGCAAGTCTT GTGTTAAAAG CCCAGTGTGA CAGTGTCATG ATGTAGTAGT

GTCTTACTGG TTTTTTAATA AATCCTTTTG TATAAAAATG TATT GGCT CT TTTATCAT CA

GCCGAAATTG AGGACATGAT TAAAATTGCA GTGAAGTTTG AAATGTTTTT AGCAAAATCT

AATTTTTGCC ATAATGTGTC CTCCCTGTCC AAATTGGGAA TGACTTAATG TCAATTTGTT

TGTTGGTTGT TTTAATAATA CTTCCTTATG TAGCCATTAA GATTTATATG AATATTTTCC

CAAATGCCCA GTTTTTGCTT AATATGTATT GTGCTTTTTA GAACAAATCT GGATAAATGT

TTAAAATCTG TTAATTATAA TAGAAATGCG GCTAGTTCAG AGAGATTTTT AGAGCTGTGG

TGGACTTCAT AGATGAATTC AAGTGTTGAG GGAGGATTAA AGAAATATAT ACCGTGTTTA

TGTGTGTGTG CTT
[0075] The human p37AuF1 amino acid sequence of GenBank Accession No.
NP 001003810.1 (SEQ ID NO:10) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAATQGAAAA AGSGAGTGGG TASGGTEGGS

AESEGAKIDA SKNEEDEGKM FIGGLSWDTT KKDLKDYFSK FGEVVDCTLK LDPITGRSRG

FGFVLFKESE SVDKVMDQKE HKLNGKVIDP KRAKAMKTKE PVKKIFVGGL SPDTPEEKIR

EYFGGFGEVE SIELPMDNKT NKRRGFCFIT FKEEEPVKKI MEKKYHNVGL SKCEIKVAMS

KEQYQQQQQW GSRGGFAGRA RGRGGDQQSG YGKVSRRGGH QNSYKPY
[0076] The human p40AuF1 nucleotide sequence of GenBank Accession No.
NM 002138.3 (SEQ ID NO:13) is as follows:
CTTCCGTCGG CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA

GCGGCCGCCG CTGGTGCTTA TTCTTTTTTA GTGCAGCGGG AGAGAGCGGG AGTGTGCGCC

CGCGCGCGCC TTCCCTGTCT TGTGTGCTTC GCGAGGTAGA GCGGGCGCGC GGCAGCGGCG

- 33 -GGGATTACTT TGCTGCTAGT TTCGGTTCGC GGCAGCGGCG GGTGTAGTCT CGGCGGCAGC

GGCGGAGACA CTAGCACTAT GTCGGAGGAG GAGTTCGGCG GGGACGGGGC GGCGGCAGCG

GCAACGGCGG CGGTAGGCGG CTCGGCGGGC GAGCAGGAGG GAGCCATGGT GGCGGCGACA

CAGGGGGCAG CGGCGGCGGC GGGAAGCGGA GCCGGGACCG GGGGCGGAAC CGCGTCTGGA

GGCACCGAAG GGGGCAGCGC CGAGTCGGAG GGGGCGAAGA TTGACGCCAG TAAGAACGAG

GAGGATGAAG GCCATTCAAA CTCCTCCCCA CGACACTCTG AAGCAGCGAC GGCACAGCGG

GAAGAATGGA AAATGTTTAT AGGAGGCCTT AGCTGGGACA CTACAAAGAA AGATCTGAAG

GACTACTTTT CCAAATTTGG TGAAGTTGTA GACTGCACTC TGAAGTTAGA TCCTATCACA

GGGCGATCAA GGGGTTTTGG CTTTGTGCTA TTTAAAGAAT CGGAGAGTGT AGATAAGGTC

ATGAAAACAA AAGAGCCGGT TAAAAAAATT TTTGTTGGTG GCCTTTCTCC AGATACACCT

GAAGAGAAAA TAAGGGAGTA CTTTGGTGGT TTTGGTGAGG TGGAATCCAT AGAGCTCCCC

ATGGACAACA AGACCAATAA GAGGCGTGGG TTCTGCTTTA TTACCTTTAA GGAAGAAGAA

CCAGTGAAGA AGATAATGGA AAAGAAATAC CACAATGTTG GTCTTAGTAA ATGTGAAATA

TTTGCAGGAA GAGCTCGTGG AAGAGGTGGT GACCAGCAGA GTGGTTATGG GAAGGTATCC

AGGCGAGGTG GTCATCAAAA TAGCTACAAA CCATACTAAA TTATTCCATT TGCAACTTAT

CCCCAACAGG TGGTGAAGCA GTATTTTCCA ATTTGAAGAT TCATTTGAAG GTGGCTCCTG

CCACCTGCTA ATAGCAGTTC AAACTAAATT TTTTGTATCA AGTCCCTGAA TGGAAGTATG

TTTTATTTCT TAATTGCTAT GCTTCAGAAT CAATTTGTGT TTTATGCCCT TTCCCCCAGT

ATTGTAGAGC AAGTCTTGTG TTAAAAGCCC AGTGTGACAG TGTCATGATG TAGTAGTGTC

TTACTGGTTT TTTAATAAAT CCTTTTGTAT AAAAATGTAT TGGCTCTTTT ATCATCAGAA

TAGGAAAAAT TGTCATGGAT TCAAGTTATT AAAAGCATAA GTTTGGAAGA CAGGCTTGCC

TTTTGCCATA ATGTGTCCTC CCTGTCCAAA TTGGGAATGA CTTAATGTCA ATTTGTTTGT

TGGTTGTTTT AATAATACTT CCTTATGTAG CCATTAAGAT TTATATGAAT ATTTTCCCAA

ATGCCCAGTT TTTGCTTAAT ATGTATTGTG CTTTTTAGAA GAAATCTGGA TAAATGTGCA

AAAGTACCCC TTTGCACAGA TAGTTAATGT TTTATGCTTC CATTAAATAA AAAGGACTTA

AAATCTGTTA ATTATAATAG AAATGCGGCT AGTTCAGAGA aATTTTTAGA GCTGTGGTGG 2040 ACTTCATAGA TGAATTCAAG TGTTGAGGGA GGATTAAAGA AATATATACC GTGTTTATGT

GTGTGTGCTT
[0077] The human p40Aun amino acid sequence of GenBank Accession No.
NP 002129.2 (SEQ ID NO:14) is as follows:

AESEGAKIDA SKNEEDEGHS NSSPRHSEAA TAQREEWKMF IGGLSWDTTK KDLKDYFSKF

GEVVDCTLKL DPITGRSRGF GFVLFKESES VDKVMDQKEH KLNGKVIDPK RAKAMKTKEP

VKKIFVGGLS PDTPEEKIRE YFGGFGEVES IELPMDNKTN KRRGFCFITF KEEEPVKKIM

EKKYHNVGLS KCEIKVAMSK EQYQQQQQWG SRGGFAGRAR GRGGDQQSGY GKVSRRGGHQ

NSYKPY

- 34 -[0078] The human p42AuF1 nucleotide sequence of GenBank Accession No.
NM 031369.2 (SEQ ID NO:17) is as follows:

AACCAGaAGA GTGGTTATGG GAAGGTATCC AGGCGAGGTG GTCATCAAAA TAGCTACAAA 1320

35 GGATTAAAGA AATATATACC GTGTTTAT GT GTGTGTGCTT
40 [0079] The human p42AuF1 amino acid sequence of GenBank Accession No.
NP 112737.1 (SEQ ID NO:18) is as follows:

YTGYNNYYGY GDYSNQQSGY GKVSRRGGHQ NSYKPY
WOW The human p45AuFl nucleotide sequence of GenBank Accession No.
INM 031370.2 (SEQ ID NO:21) is as follows:

GCGCGAGAGT GGGAGGCGAA GGGGGCAGGC aAGGGAGAGG CGCAGGAGCC TTTGCAGCCA 180 GGCGGAGACA CTAGCACTAT GTCGGAGGAG aAGTTCGGCG GGGACGGGGC GGCGGCAGCG 360 CCAGTGAAGA AGATAATGGA AAAGAAATAC aACAATGTTG GTCTTAGTAA ATGTGAAATA 1080 TACTAAATTA TTCCATTTGC AACTTATCCC aAACAGGTGG TGAAGCAGTA TTTTCCAATT 1440 AATGTATTGG CTCTTTTATC ATCAGAATAG GAAAAATTGT aATGGATTCA AGTTATTAAA 1800

- 36 -TTAAAGAAAT ATATACCGTG TTTATGTGTG TGTGCTT
[0081] The human p45AuF1 amino acid sequence of GenBank Accession No.
NP 112738.1 (SEQ ID NO:22) is as follows:

GYGNYGYNSQ GYGGYGGYDY TGYNNYYGYG DYSNQQSGYG KVSRRGGHQN SYKPY
[0082] The mouse p37AuF1 nucleotide sequence of GenBank Accession No.
NM 001077267.2 (SEQ ID NO: 11) is as follows:

20 TACTTTGCTG CTAGTTTCGG TTCGCGGCGG CGGCGGCGTC GGCGGGTGTC GTCTTCGGCG .. 300 ATGAAGGGAA AATGTTTATA GGAGGCCTTA GCTGGGACAC CACAAAGAAA aATCTGAAGG 600 TGAAAACAAA AGAGCCTGTC AAAAAAATTT TTGTTGGTGG CCTTTCTCCA aACACACCTG 840

- 37 -

- 38 -GCCTTTGTAG GGGTGTTTTG AGACCCCCTC aATGCTAACA AATATACAGG TTTCTTAACA 4140 ACCAGGTGAG TCATTTCCTG GGGTTCTGTT TTCTTTTAAA ATCTTTCCCT AAACTTAATA __ 4560 TTATATGACA ATTGCTGTTC CCAAGTCAGA ATTCAGTGTG CTGATTTGAC ATCAGTTCGT __ 4680 TTGAGGTGTT AGGATTCTTG ACAGCCAGAA AGACTGAACC aACTATCTGG GCACAGTGTT 4860 25 GTACACACCT CTCATCAGTT ACCGGAGTCA TTGCATTGCG GACTAACTGG CTGACTCAAG __ 5520 TTAAAGTCGA AAGGATGTCT GGTTGTGGCT TTATTGTAAA aAGCCTTAGG TAAAGAGGGG 5640 GAGGGTGTTA GAACAGTCCA AATTTGTTAT TGTAGACTTG aAGTGGGAGG AATTTTTAAA 5820

- 39 -TTCAAATGAA GCTTACAGTG TGCTTTCTTG GGGTTTTGAT TTGCACTAAA TTTTATTTTC

TGAAAGATCA CTTATGTTTA TAATGTAGTG CTTTGTCTTA ACAATTAAAC TTTCCAGCAC

TCATGCA
[0083] The mouse p37AuF1 amino acid sequence of GenBank Accession No.
NP 001070735.1 (SEQ ID NO:12) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGEQEGAM VAAAAQGPAA AAGSGSGGGG SAAGGTEGGS

AEAEGAKIDA SKNEEDEGKM FIGGLSWDTT KKDLKDYFSK FGEVVDCTLK LDPITGRSRG

FGFVLFKESE SVDKVMDQKE HKLNGKVIDP KRAKAMKTKE PVKKIFVGGL SPDTPEEKIR

EYFGGFGEVE SIELPMDNKT NKRRGFCFIT FKEEEPVKKI MEKKYHNVGL SKCEIKVAMS

KEQYQQQQQW GSRGGFAGRA RGRGGDQQSG YGKVSRRGGH QNSYKPY
[0084] The mouse p40AuF1 nucleotide sequence of GenBank Accession No.
NM 007516.3 (SEQ ID NO:15) is as follows:
CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA GTGGCCGCCG

CTGCTACTTC ATTCTTTTTT TTTTCAGTGC AGCCGGGGAG AGCGAGAGAG CGCGCTGCGC

CGCGCCTTGT CTAGGGTGCC TCGCGAGGTA GAGCGGGCAT CGCGCGGCGG CGGCGGGGAT

TACTTTGCTG CTAGTTTCGG TTCGCGGCGG CGGCGGCGTC GGCGGGTGTC GTCTTCGGCG

GCGGCAGTAG CACTATGTCG GAGGAGCAGT TCGGAGGGGA CGGGGCGGCG GCGGCGGCAA

CGGCGGCGGT AGGCGGCTCG GCGGGCGAGC AGGAGGGAGC aATGGTGGCG GCGGCGGCGC

CCGAAGGAGG CAGCGCC GAG GCAGAGGGAG CCAAGATCGA CGCCAGTAAG AACGAGGAGG

ATGAAGGCCA TTCAAACTCC TCCCCACGAC ACACTGAAGC AGCGGCGGCA CAGCGGGAAG

AATGGAAAAT GTTTATAGGA GGCCTTAGCT GGGACACCAC AAAGAAAGAT CTGAAGGACT

ACTTTTCCAA ATTTGGTGAA GTTGTAGACT GCACTCTGAA GTTAGATCCT ATCACAGGGC

ATCAGAAAGA ACATAAATTG AATGGGAAAG TCATTGATCC TAAAAGGGCC AAAGCCATGA

AAACAAAAGA GCCTGTCAAA AAAATTTTTG TTGGTGGCCT TTCTCCAGAC ACACCTGAAG

AAAAAATAAG AGAGTACTTT GGTGGTTTTG GTGAGGTTGA ATCCATAGAG CTCCCTATGG

ACAACAAGAC CAATAAGAGG CGTGGGTTCT GTTTTATTAC CTTTAAGGAA GAGGAGCCAG

TAGCCATGTC AAAGGAACAG TATCAGCAGC AGCAGCAGTG GGGATCTAGA GGAGGGTTTG

CAGGCAGAGC TCGCGGAAGA GGTGGAGATC AGCAGAGTGG TTATGGGAAA GTATCCAGGC

GAGGTGGACA TCAAAATAGC TACAAACCAT ACTAAATTAT TCCATTTGCA ACTTATCCCC

AACAGGTGGT GAAGCAGTAT TTTCCAATTT GAAGATTCAT TTGAAGGTGG CTCCTGCCAC

TGGGTCCCTC TGAAGTTTAA TTCTGAGTTC TCATTAAAAG AATTTGCTTT CATTGTTTTA

TTTCTTAATT GCTATGCTTC AGTATCAATT TGTGTTTTAT GCCCCCCCTC CCCCCCAGTA

TTGTAGAGCA AGTCTTGTGT TAAAAAAAGC CCAGTGTGAC AGTGTCATGA TGTAGTAGTG

TCTTACTGGT TTTTTAATAA ATCCTTTTGT ATAAAAATGT ATTGGCTCTT TTATCATCAG

ATTTGAGGGG ATAAAAACAA CGGTAAACTT TGTCTGAAAG AGGGCATGGT TAAAAATGTA

-40 -AGTGCTGTAA AGTTGAAAGT TCATGAaAGA CATACAATGA GGGCTGCAGC CCATTTTTAA 2560 30 GTTTGTTTTT TGTTGTTTTG GTTCTTTGTT aAGCCCTGGA CAAAAACTTC CCTAGTTCTG 3540 AAGGCT GAG C T GG GT GT T GG CAAGAAC C CA GC T TAGAACA AACACAT G CA AGGC CAT

T CT T T GCT TC CACTGAGGAG TGGAACACTT TAGAATGAAC CT CTAGATAG ATATTTTTAT

-41 -GCTCATAAAC TTGTAAAGCT TACTTTCTCT TAATCCACCC aACATTTAAC AAGCCCTGGT 4260 AGGTGTTAGG ATTCTTGACA GCCAGAAAGA CTGAACCCAC TATCTGGGCA aAGTGTTCGT 4920 AGCCGTGGCT GGGCTCACTG ACCGTGGCTG TAAGTTACGG AGGaAGCACA aACTTCTGTA 5520 TCTTGCTACT GAAGTCTTGA GTTGGTCTCA TGCATTTACC CTGTTGACTT aAGCACCTTA 5640

-42 -TGCA
[0085] The mouse p40AuF1 amino acid sequence of GenBank Accession No.
NP 031542.2 (SEQ ID NO:16) is as follows:

NSYKPY
[0086] The mouse p42AuF1 nucleotide sequence of GenBank Accession No.
NM 001077266.2 (SEQ ID NO: 19) is as follows:

GAGAGTGGGA GGCGAGGGGG GCAGGCCGGG GAGAGGCGCA GGAGCCCTTG aAGCCACGCG 180 GCGGCAGTAG CACTATGTCG GAGGAGaAGT TCGGAGGGGA CGGGGCGGCG GCGGCGGCAA 360 AGAGCCTGTC AAAAAAATTT TTGTTGGTGG CCTTTCTCCA aACACACCTG 840

-43 -CATACAATGA GGGCTGaAGC CCATTTTTAA AAACATTATA ATACAAAAGT ATGCACATTT 2700 CTCACTGACT TATTTCTTGA ACTTTTGCCA TTTGaATAAA TCTTGTCAGC TTTGTTCTTG 3240 CATCTTAGAA TTTTTCACCC TCTTCCCCAA CTATTCTAAT aAATCTTAAG TATGCCCTTC 3720

-44 -TAATCCACCC CAGATTTAAC AAGCCCTGGT ACTTAGAATT TCAGAAGAGT AATGGCAGGT .. 4380 CCCAAGTCTT AGCCGTGGTG ATGGCTACAG TGTGGAAGAT GGTGAGCATT CTAGTGAGTA .. 5520 GTTTCAGAAT TAGATTCTGT GGAAGGTGAG GGTGTTAGAA aAGTCCAAAT TTGTTATTGT 5940 30 GATAAAGGTG GAAACGaATG TGGCAACACT TCTAAGTTCA TTTGTATATG TTTGTAATTT 6060 AGGAATGAAA TAATTGCCGT TTTTGTTGTT aAGTGAATGG GTGTGGTTTA ATCAGCGTAA 6300

-45 -TTTTGATTTG CACTAAATTT TATTITCTGA AAGATCACTT ATGTTTATAA TGTAGTGCTT

TGTCTTAACA ATTAAACTTT CCAGCACTCA TGCA
[0087] The mouse p42AuF1 amino acid sequence of GenBank Accession No.
NP 001070734.1 (SEQ ID NO:20) is as follows:

AEAEGAKIDA SKNEEDEGKM FIGGLSWDTT KKDLKDYFSK FGEVVDCTLK LDPITGRSRG

FGFVLFKESE SVDKVMDQKE HKLNGKVIDP KRAKAMKTKE PVKKIFVGGL SPDTPEEKIR

EYFGGFGEVE SIELPMDNKT NKRRGFCFIT FKEEEPVKKI MEKKYHNVGL SKCEIKVAMS

KEQYQQQQQW GSRGGFAGRA RGRGGGPSQN WNQGYSNYWN QGYGNYGYNS QGYGGYGGYD

YTGYNNYYGY GDYSNQQSGY GKVSRRGGHQ NSYKPY
[0088] The mouse p45AuF1 nucleotide sequence of GenBank Accession No.
NM 001077265.2 (SEQ ID NO:23) is as follows:
CCATTTTAGG TGGTCCGCGG CGGCGCCATT AAAGCGAGGA GGAGGCGAGA GTGGCCGCCG

CTGCTACTTC ATTCTTTTTT TTTTCAGTGC AGCCGGGGAG AGCGAGAGAG CGCGCTGCGC

CGCGCCTTGT CTAGGGTGCC TCGCGAGGTA GAGCGGGCAT CGCGCGGCGG CGGCGGGGAT

TACTTTGCTG CTAGTTTCGG TTCGCGGCGG CGGCGGCGTC GGCGGGTGTC GTCTTCGGCG

GCGGCAGTAG CACTATGTCG GAGGAGCAGT TCGGAGGGGA CGGGGCGGCG GCGGCGGCAA

CGGCGGCGGT AGGCGGCTCG GCGGGCGAGC AGGAGGGAGC aATGGTGGCG GCGGCGGCGC

CCGAAGGAGG CAGCGCC GAG GCAGAGGGAG CCAAGATCGA CGCCAGTAAG AACGAGGAGG

ATGAAGGCCA TTCAAACTCC TCCCCACGAC ACACTGAAGC AGCGGCGGCA CAGCGGGAAG

AATGGAAAAT GTTTATAGGA GGCCTTAGCT GGGACACCAC AAAGAAAGAT CTGAAGGACT

ACTTTTCCAA ATTTGGTGAA GTTGTAGACT GCACTCTGAA GTTAGATCCT ATCACAGGGC

ATCAGAAAGA ACATAAATTG AATGGGAAAG TCATTGATCC TAAAAGGGCC AAAGCCATGA

AAACAAAAGA GCCTGTCAAA AAAATTTTTG TTGGTGGCCT TTCTCCAGAC ACACCTGAAG

AAAAAATAAG AGAGTACTTT GGTGGTTTTG GTGAGGTTGA ATCCATAGAG CTCCCTATGG

ACAACAAGAC CAATAAGAGG CGTGGGTTCT GTTTTATTAC CTTTAAGGAA GAGGAGCCAG

TAGCCATGTC AAAGGAACAG TATCAGCAGC AGCAGCAGTG GGGATCTAGA GGAGGGTTTG

CAGGCAGAGC TCGCGGAAGA GGTGGAGGCC CCAGTCAAAA CTGGAACCAG GGATATAGTA

ACTATTGGAA TCAAGGCTAT GGCAACTATG GATATAACAG CCAAGGTTAC GGAGGTTATG

GAGGATATGA CTACACTGGT TACAACAACT ACTATGGATA TGGTGATTAT AGCAATCAGC

AAATTATTCC ATTTGCAACT TATCCCCAAC AGGTGGTGAA GCAGTATTTT CCAATTTGAA

GATTCATTTG AAGGTGGCTC CTGCCACCTG CTAATAGCAG TTCAAACTAA ATTTTTTCTA

TCAAGTTCCT GAATGGAAGT ATGACGTTGG GTCCCTCTGA AGTTTAATTC TGAGTTCTCA

TTAAAAGAAT TTGCTTTCAT TGTTTTATTT CTTAATTGCT ATGCTTCAGT ATCAATTTGT

GTGTGACAGT GTCATGATGT AGTAGTGTCT TACTGGTTTT TTAATAAATC CTTTTGTATA

-46 -ACAATGAGGG CTGCAGCCCA TTTTTAAAAA aATTATAATA CAAAAGTATG CACATTTGTT 2760 AACTTGCTTG GGGCAGTTTG AGCCTAGTTC AT GAGCT GCT AT CAGAT T GG T CT T GAT C CT

AGTAGATAAA TAACTTGGTT TTTAATGTTA ACTTTGTTTC aATTAAGTCA CATTTAAAAA 3180 CCCTGGACAA AAACTTCCCT AGTTCTGGTT TCTACAATTT AAATTAAAAA aAGAATTCAT 3720 T CAAAGT T TT TAGT GGAT GC TAAGT GAT CT TTGCTTCCAC TGAGGAGT GG AACACT T TAG

AATGAACCTC TAGATAGATA T TT TTAT T GT CT GGTGAGGG TTACTGGAGT T T CCCACC CT

GCCTGAAGGG T GAAT CT GGC TTACAGT GTT CT CATCT CAA AGGGAAGAAG GCAGATGGCT

-47 -15 AACCCACTAT CTGGGCACAG TGTTCGTGTT GCTCTATAAA TGTATGCTTT TTTTaATTTG 5100

-48 -TTGTAGAAGC TTCCTTATAG ATTCTTCAAA TGAAGCTTAC AGTGTGCTTT CTTGGGGTTT

TGATTTGCAC TAAATTTTAT TTTCTGAAAG ATCACTTATG TTTATAATGT AGTGCTTTGT

CTTAACAATT AAACTTTCCA GCACTCATGC A
[0089] The mouse p45AuF1 amino acid sequence of GenBank Accession No.
NP 001070733.1 (SEQ ID NO:24) is as follows:
MSEEQFGGDG AAAAATAAVG GSAGFQEGAM VAAAAQGPAA AAGSGSGGGG SAAGGTEGGS

AEAEGAKIDA SKNEEDEGHS NSSPRHTEAA AAQRFEWKMF IGGLSWDTTK KDLKDYFSKF

GEVVDCTLKL DPITGRSRGF GFVLFKESES VDKVMDQKEH KLNGKVIDPK RAKAMKTKEP

VKKIFVGGLS PDTPEEKIRE YFGGFGEVES IELPMDNKTN KRRGFCFITF KEEEPVKKIM

GYGNYGYNSQ GYGGYGGYDY TGYNNYYGYG DYSNQQSGYG KVSRRGGHQN SYKPY
[0090] It is noted that the sequences described herein may be described with reference to accession numbers 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.). Thus, reference to such sequence accession numbers or corresponding sequence identification numbers refers to either the sequence fully described therein or some portion thereof (e.g., that portion encoding a protein or polypepti de 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).
Likewise, variants and isoforms of accession numbers and corresponding sequence identification numbers described herein are also contemplated.
[0091] Accordingly, in certain embodiments, the AUF1 protein referred to herein has an amino acid sequence as set forth in Table 2 and the sequences disclosed herein, or is a functional fragment thereof. In one embodiment, 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 an amino acid sequence disclosed herein.
[0092] In some embodiments, the AAV vector described herein includes a nucleic acid molecule encoding a nucleotide sequence set forth in Table 2 (or described herein), or portions thereof that encode a functional fragment of an AUF1 protein as described supra.
[0093] As described in more detail below, compositions according to the present application may be useful in gene therapy, which includes both ex vivo and in vivo techniques.
Thus, host cells can be genetically engineered ex vivo with a nucleic acid molecule (or polynucleotide), with the engineered cells then being provided to a patient to be treated.
Delivery of the active agent of a composition described herein in vivo may involve a process that effectively introduces a molecule of interest (e.g., AUF1 protein or a functional fragment

-49 -thereof) into the cells or tissue being treated. In the case of polypeptide-based active agents, this can be carried out directly or, alternatively, by transfecting transcriptionally active DNA into living cells such that the active polypeptide coding sequence is expressed and the polypeptide is produced by cellular machinery. Transcriptionally active DNA may be delivered into the cells or tissue, e.g., muscle, being treated using transfection methods including, but not limited to, electroporation, microinjection, calcium phosphate coprecipitation, DEAE
dextran facilitated transfection, cationic liposomes, and retroviruses. In certain embodiments, the DNA to be transfected is cloned into a vector.
[0094] Alternatively, cells can be engineered in vivo by administration of the polynucleotide using techniques known in the art. For example, by direct injection of a "naked"
polynucleotide (Feigner et al., "Gene Therapeutics,- Nature 349:351-352 (1991); U.S. Patent No. 5,679,647; Wolff et al., "The Mechanism of Naked DNA Uptake and Expression,- Adv.
Genet. 54:3-20 (2005), which are hereby incorporated by reference in their entirety) or a polynucleotide formulated in a composition with one or more other targeting elements which facilitate uptake of the polynucleotide by a cell.
[0095] Host cells that can be used with the vectors described herein include, without limitation, myocytes. The term "myocyte," as used herein, refers a cell that has been differentiated from a progenitor myoblast such that it is capable of expressing muscle-specific phenotype under appropriate conditions. Terminally differentiated myocytes fuse with one another to form myotubes, a major constituent of muscle fibers. The term "myocyte" also refers to myocytes that are de-differentiated. The term includes cells in vivo and cells cultured ex vivo regardless of whether such cells are primary or passaged. Myocytes are found in all muscle types, e.g., skeletal muscle, cardiac muscle, smooth muscle, etc. Myocytes are found and can be isolated from any vertebrate species, including, without limitation, human, orangutan, monkey, chimpanzee, dog, cat, rat, rabbit, mouse, horse, cow, pig, elephant, etc.
Alternatively, the host cell can be a prokaryotic cell, e.g., a bacterial cell such as E. coil, that is used, for example, to propagate the vectors.
[0096] It may be desirable in certain circumstances to utilize myocyte progenitor cells such as mesenchymal precursor cells or myoblasts rather than fully differentiated myoblasts.
Examples of tissue from which such cells can be isolated include placenta, umbilical cord, bone marrow, skin, muscle, periosteum, or perichondrium. Myocytes can be derived from such cells, for example, by inducing their differentiation in tissue culture. The present application encompasses not only myocyte precursor/progenitor cells, but also cells that can be trans-differentiated into myocytes, e.g., adipocytes and fibroblasts.

- 50 -[0097] Also encompassed are expression systems comprising nucleic acid molecules described herein Generally, the use of recombinant expression systems involves inserting a nucleic acid molecule encoding the amino acid sequence of a desired peptide into an expression system to which the molecule is heterologous (i.e., not native or not normally present). One or more desired nucleic acid molecules encoding a peptide described herein (e.g., AUF1) may be inserted into the vector. When multiple nucleic acid molecules are inserted, the multiple nucleic acid molecules may encode the same or different peptides. The heterologous nucleic acid molecule is inserted into the expression system or vector in proper sense (5'3') orientation relative to the promoter and any other 5' regulatory molecules, and correct reading frame.
[0098] The preparation of the nucleic acid constructs can be carried out using standard cloning procedures well known in the art as described by Joseph Sambrook et al., MOLECULAR
CLONING: A LABORATORY MANUAL (Cold Springs Harbor 2012), which is hereby incorporated by reference in its entirety. U.S. Patent No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase.
These recombinant plasmids are then introduced by means of transformation and replicated in a suitable host cell.
[0099] A nucleic acid molecule encoding an AUF1 protein or functional fragment thereof and that is operatively coupled to a muscle-cell specific promoter (e.g., muscle creatine kinase (MCK) promoter) may include an additional elements including, without limitation, a leader sequence, a suitable 3' regulatory region to allow transcription in the host or a certain medium, and/or any additional desired component, such as reporter or marker genes.
Such additional elements may be cloned into the vector of choice using standard cloning procedures in the art, such as described in Joseph Sambrook et al., MOLECULAR CLONING: A LABORATORY
MANUAL
(Cold Springs Harbor 2012); Frederick M. Ausubel, SHORT PROTOCOLS IN MOLECULAR
BIOLOGY
(Wiley 2002); and U.S. Patent No. 4,237,224 to Cohen and Boyer, which are hereby incorporated by reference in their entirety.
[01001 In some embodiments, the adeno-associated viral vector comprises a nucleic acid molecule encoding a reporter protein. The reporter protein may be selected from the group consisting of, e.g., I3-galactosidase, chloramphenicol acetyl transferase, luciferase, and fluorescent proteins.
[01011 In certain embodiments, 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,

-51 -AceGFP, ZsGreen1), 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, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), or any other suitable fluorescent protein. In certain embodiments, the reporter protein is a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), and yellow fluorescent protein (YFP).
[0102] In some embodiments, the reporter protein is luciferase.
As used herein, the term "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 (Rendla), marine copepods, and bacteria among others. Examples of luciferases that may be used as reporter proteins include, e.g., Rendla (e.g., Renilla reniformis) luciferase, Gaussia (e.g., Gaussia princeps) luciferase), Metridia luciferase, firefly (e.g., Photinus pyrahs luciferase), click beetle (e.g., Pyrearinus termitillunnnans) luciferase, deep sea shrimp (e.g., Oplophorus gracihrostris) luciferase).
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.
[0103] Purine-rich element binding protein f3 (Purr.) is a transcriptional repressor of smooth muscle a-actin (SMA) gene expression in growth-activated vascular smooth muscle cells. In some embodiments, the adeno-associated viral vector comprises a nucleic acid molecule encoding a purine-rich element binding protein 13 (Purfl) inhibitor.
[0104] siRNAs are double stranded synthetic RNA molecules approximately 20-25 nucleotides in length with short 2-3 nucleotide 3' overhangs on both ends. The double stranded siRNA molecule represents the sense and anti-sense strand of a portion of the target mRNA
molecule, in this case a portion of a Purfl mRNA. The sequence of Pur13 mRNA
is readily known in the art and accessible to one of skill in the art for purposes of designing siRNA and shRNA oligonucleotides.

- 52 -[0105] siRNA molecules are typically designed to target a region of the mRNA target approximately 50-100 nucleotides downstream from the start codon Methods and online tools for designing suitable siRNA sequences based on the target mRNA sequences are readily available in the art (see, e.g., Reynolds et al., "Rational siRNA Design for RNA Interference,"
Nat. Biotech. 2:326-330 (2004); Chalk et al., "Improved and Automated Prediction of Effective siRNA," Bioehem. Biophys. Res. Comm. 319(1):264-274 (2004), Zhang et al., "Weak Base Pairing in Both Seed and 3' Regions Reduces RNAi Off-targets and Enhances si/shRNA
Designs," Nucleic Acids Res. 42(19):12169-76 (2014), which are hereby incorporated by reference in their entirety). Upon introduction into a cell, the siRNA complex triggers the endogenous RNA interference (RNAi) pathway, resulting in the cleavage and degradation of the target mRNA molecule.
[0106] Short or small hairpin RNA ("shRNA") molecules are similar to siRNA
molecules in function, but comprise longer RNA sequences that make a tight hairpin turn.
shRNA is cleaved by cellular machinery into siRNA and gene expression is silenced via the cellular RNA interference pathway. Methods and tools for designing suitable shRNA sequences based on the target mRNA sequences (e.g., Purf3 mRNA sequences) are readily available in the art (see e.g., Taxman et al., "Criteria for Effective Design, Constructions, and Gene Knockdown shRNA Vectors," BMC Biotech. 6:7 (2006) and Taxman et al., "Short Hairpin RNA
(shRNA):
Design, Delivery, and Assessment of Gene Knockdown," Meth. Mol. Biol. 629: 139-156 (2010), which are hereby incorporated by reference in their entirety).
[0107] Other suitable agents that can be encoded by the recombinant construct disclosed herein for purposes of inhibiting Pur13 include microRNAs ("miRNAs"). miRNAs are small, regulatory, noncoding RNA molecules that control the expression of their target mRNAs predominantly by binding to the 3' untranslated region (UTR). A single UTR may have binding sites for many miRNAs or multiple sites for a single miRNA, suggesting a complex post-transcriptional control of gene expression exerted by these regulatory RNAs (Shulka et al., "MicroRNAs: Processing, Maturation, Target Recognition and Regulatory Functions," Mol.
Cell. Pharmacol. 3(3):83-92 (2011), which is hereby incorporated by reference in its entirety).
Mature miRNA are initially expressed as primary transcripts known as a pri-miRNAs which are processed, in the cell nucleus, to 70-nucleotide stem-loop structures called pre-miRNAs by the microprocessor complex. The dsRNA portion of the pre-miRNA is bound and cleaved by Dicer to produce a mature 22 bp double-stranded miRNA molecule that can be integrated into the RISC complex; thus, miRNA and siRNA share the same cellular machinery downstream of their initial processing.

- 53 -[0108] microRNAs known to inhibit the expression of Pur13 molecules are known in the art and suitable for incorporation into the recombinant genetic const.ruct described herein For example, miR-22,miR-208b, and miR-499 are known to modulate expression of Pur13 (see, e.g., Gurha et al., "Targeted Deletion of MicroRNA-22 Promotes Stress-Induced Cardiac Dilation and Contractile Dysfunction," Circulation 125(22):2751-2761 (2012) and Simionescu-Bankston &
Kumar, "Noncoding RNAs in the Regulation of Skeletal Muscle Biology in Health and Disease,"
Mol Med. 94(8):853-866 (2017), which are hereby incorporated by reference in their entirety).
[0109] A variety of genetic signals and processing events that control many levels of gene expression (e.g., DNA transcription and messenger RNA ("mRNA") translation) can be incorporated into the nucleic acid construct to maximize protein production.
For the purposes of expressing a cloned nucleic acid sequence encoding a desired protein, it is advantageous to use strong promoters to obtain a high level of transcription.
[0110] There are other specific initiation signals required for efficient gene transcription and translation in eukaryotic cells that can be included in the nucleic acid construct to maximize protein production. Depending on the vector system and host utilized, any number of suitable transcription and/or translation elements, including constitutive, inducible, and repressible promoters, as well as minimal 5' promoter elements, enhancers or leader sequences may be used.
[0111] In some embodiments, the Puri3 inhibitor is a polypeptide. In a more specific embodiment, the Pur13 inhibitor is an antibody. As used herein, the term "antibody" is meant to include intact immunoglobulins derived from natural sources or from recombinant sources, as well as immunoreactive portions (i.e. antigen binding portions) of intact immunoglobulins.
Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies, antibody fragments (e.g. Fv, Fab, and F(ab)2), single chain antibodies (scFv), single-domain antibodies, chimeric antibodies, and humanized antibodies (Ed Harlow and David Lane, USING ANTIBODIES: A LABORATORY MANUAL
(Cold Spring Harbor Laboratory Press, 1999); Houston et al., "Protein Engineering of Antibody Binding Sites: Recovery of Specific Activity in an Anti-Digoxin Single-Chain Fv Analogue Produced in Escherichia coli," Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988);
Bird et al, "Single-Chain Antigen-Binding Proteins," Science 242:423-426 (1988), which are hereby incorporated by reference in their entirety).
[0112] Adeno-associated viral vectors and recombinant adeno-associated virus (AAV) vectors are well known delivery vehicles that can be constructed and used to deliver a nucleic acid molecule to cells, as described in Shi et al., "Therapeutic Expression of an Anti-Death Receptor-5 Single-Chain Fixed Variable Region Prevents Tumor Growth in Mice,"
Cancer Res.

- 54 -66:11946-53 (2006); Fukuchi etal., "Anti-A13 Single-Chain Antibody Delivery via Adeno-Associated Vim s for Treatment of Alzheimer's Disease," Neurohiol. Dis. 23:502-511 (2006);
Chatterjee et al., "Dual-Target Inhibition of HIV-1 In Vitro by Means of an Adeno-Associated Virus Antisense Vector," Science 258:1485-1488 (1992); Ponnazhagan et al., "Suppression of Human Alpha-globin Gene Expression Mediated by the Recombinant Adeno-associated Virus 2-based Antisense Vectors," J. Exp. Med. 179:733-738 (1994), which are hereby incorporated by reference in their entirety. In vivo use of these vehicles is described in Flotte etal., "Stable In Vivo Expression of the Cystic Fibrosis Transmembrane Conductance Regulator With an Adeno-Associated Virus Vector," Proc. Nat'l. Acad. Sci. 90:10613-10617 (1993), which is hereby incorporated by reference in its entirety.
[0113] Recombinant adeno-associated virus (AAV) vectors provide the ability to stably transduce and express genes with very long-term (many years) duration in skeletal muscle, and depending on the AAV vector serotype and its modification, to do so with high muscle-tropism and selectivity whether using local intramuscular injection or systemic routes of delivery (Phillips et al., "Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors,"
Methods MoL Biol. 709:141-51(2011) and Muraine et al., "Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle," Hum.
Gene Ther. 31(3-4):233-240 (2020), which are hereby incorporated by reference in their entirety). Moreover, for certain AAV serotypes and engineered variants, particularly AAV8 and its engineered variants, studies in mice have been shown to be predictive of human skeletal muscle transduction and gene expression, as found in clinical trials for skeletal muscle transmission and expression (Phillips et al., "Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors,- Methods Mot Biol. 709:141-51(2011) and Muraine et al., "Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle," Hum. Gene Ther. 31(3-4):233-240 (2020), which are hereby incorporated by reference in their entirety).
[0114] The AAV vector described herein may comprise a sequence isolated or derived from an AAV vector of serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), 10 (AAV10), 11 (AAV11) or any combination thereof.
[0115] In some embodiments, the adeno-associated viral (AAV) vector is a recombinant vector.
[0116] In one particular embodiment, the AAV vector is AAV8.
AAV8 derived from macaques is very poorly immunogenic, resulting in long-term expression of the encoded

- 55 -transgene (for many years), and efficiently transduce skeletal muscle with high tropism and selectivity in both human and mouse (Phillips et a!, "Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors," Methods Mol. Biol. 709:141-51 (2011); Muraine et al., "Transduction Efficiency of Adeno-Associated Virus Serotypes After Local Injection in Mouse and Human Skeletal Muscle," Hum. Gene Ther. 31(3-4):233-240 (2020);
Blankinship et al., "Efficient Transduction of Skeletal Muscle Using Vectors Based on Adeno-associated Virus Serotype 6," Mol. Ther. 10(4):671-8 (2004), and Gregorevic et al., "Viral Vectors for Gene Transfer to Striated Muscle," Curr. Opin. Mol. Ther. 6(5):491-8 (2004), which are hereby incorporated by reference in their entirety). AAV8 shows essentially no liver tropism, is largely specific for skeletal fibers and satellite cells, and has been shown to transduce skeletal muscles throughout the body (Wang et al., "Construction and Analysis of Compact Muscle-specific Promoters for AAV Vectors," Gene Ther. 15(22):1489-99 (2008), which is hereby incorporated by reference in its entirety).
[0117]
According to one embodiment, the adeno-associated viral (AAV) vector is an AAV8 vector with the nucleotide sequence of SEQ ID NO:25.
AAV8 AtTF1 Construct Sequence (SEQ ID NO:25) CCTGCAGGCA GCTGCGCGCT CGCTCGCTCA CTGAGGCCGC CCGGGCAAAG CCCGGGCGTC

GGGCGACCTT TGGTCGCCCG GCCTCAGTGA GCGAGCGAGC GCGaAGAGAG GGAGTGGCCA 120 ACTCCATCAC TAGGGGTTCC TGCGGCCTAA GGCAATTGGC CACTACGGGT CTAGGCTGCC

CAT GTAAGGA GGCAAGGCCT GGGGACACCC GAGATGCCTG GT TATAAT TA ACCCCAACAC

CTGCTGCCCC CCCCCCCCAA CACCTGCTGC CTGAGCCTGA GCGGTTACCC CACCCCGGTG

CCTGGGTCTT AGGCTCTGTA CACCATGGAG GAGAAGCTCG CTCTAAAAAT AACCCTGTCC

CCGAGATGCC TGGTTATAAT TAACCCCAAC ACCTGCTGCC CCCCCCCCCC AACACCTGCT

GCCTGAGCCT GAGCGGTTAC CCCACCCCGG TGCCTGGGTC TTAGGCTCTG TACACCATGG

AGGAGAAGCT CGCTCTAAAA ATAACCCTGT CCCTGGTGGA TCGCCACTAC GGGTCTAGGC

TGCCCATGTA AGGAGGCAAG GCCTGGGGAC ACCCGAGATG CCTGGTTATA ATTAACCCCA

GGTGCCTGGG TCTTAGGCTC TGTACACCAT GGAGGAGAAG CTCGCTCTAA AAATAACCCT

GTCCCTGGTG GATCCCTCCC TGGGGAaAGC CCCTCCTGGC TAGTCACACC CTGTAGGCTC

CTCTATATAA CCCAGGGGCA CAGGGGCTGC CCCCGGGTCA CCGCTAGCCA AAGCTTCTCG

AGGCTGGCTA GTTAAGCTAT CAACAAGTTT GTACAGAAAA GCAGGCTTTA AAGGAACCAA

TCGGAGGGGA CGGGGCGGCG GCGGCGGCAA CGGCGGCGGT AGGCGGCTCG GCGGGCGAGC

AGGAGGGAGC CATGGTGGCG GCGGCGGCGC AGGGGCCGGC GGCGGCGGCG GGAAGCGGGA

GCGGCGGCGG CGGCTCTGCG GCCGGAGGCA CCGAAGGAGG aAGCGCCaAG GCAGAGGGAG

CCAAGATCGA CGCCAGTAAG AACGAGGAGG ATGAAGGCCA TTCAAACTCC TCCCCACGAC

SUBSTITUTE SHEET (RULE 26)

- 56 -10 AGCAGCAGTG GGGATCTAGA GGAGGGTTTG aAGGCAGAGC TCGCGGAAGA GGTGGAGATC .. 1860 TTCTTGAAGA CAAACAACGT CTGTAGCGAC CCTTTGCAGG aAGCGGAACC CCCCACCTGG 2340 CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC aATTGTATGG GATCTGATCT 2520 CCACCCTCGT GACCACCCTG ACCTACGGCG TGCAGTGCTT aAGCCGCTAC CCCGACCACA 2880 ACCACTACCT GAGCACCCAG TCCGCCCTGA GCAAAGACCC aAACGAGAAG CGCGATCACA 3300 AACTAGTTGA GCGGCCGCAA CTCGAGACTC TAGAGGTTAA TCGATAATCA ACCTCTGGAT .. 3480 SUBSTITUTE SHEET (RULE 26)

- 57 -GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA aAGCAAGGGG GAGGATTGGG 4260 SUBSTITUTE SHEET (RULE 26)

- 58 -CGAAATAGAC AGATCGCTGA GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC

CAAGTTTACT CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC

TAGGTGAAGA TCCTTTTTGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC

CACTGAGCGT CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC TTTTTTTCTG

GATCAAGAGC TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA

AATACTGTCC TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG

CCTACATACC TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG

TGTCTTACCG GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA

CTACAGCGTG AGCTATGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT

CCGGTAAGCG GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC

TGGTATCTTT ATAGICCTGT CGGGTTTCGC aACCTCTGAC TTGAGCGTCG ATTTTTGTGA

TGCTCGTCAG GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC

[0118] Another aspect of the present application relates to a composition comprising an adeno-associated viral (AAV) vector as described herein.
[0119] In some embodiments, the composition of the present application further comprises a buffer solution.
[0120] The composition of the present application may further comprise one or more targeting elements. Suitable targeting elements include, without limitation, agents such as saponins or cationic polyamides (see, e.g.,U U.S. Patent Nos. 5,739,118 and 5,837,533, which are hereby incorporated by reference in their entirety); microparticles, microcapsules, liposomes, or other vesicles; lipids; cell-surface receptors; transfecting agents; peptides (e.g., one known to enter the nucleus); or ligands (such as one subject to receptor-mediated endocytosis). Suitable means for using such targeting elements include, without limitation:
microparticle bombardment;
coating the polynucleotide with lipids, cell-surface receptors, or transfecting agents;
encapsulation of the polynucleotide in liposomes, microparticles, or microcapsules;
administration of the polynucleotide linked to a peptide which is known to enter the nucleus; or administration of the polynucleotide linked to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu et al., "Receptor-Mediated in vitro Gene Transformation by a Soluble DNA
Carrier System," I. Biol. Chem. 262:4429-4432 (1987), which is hereby incorporated by reference in its entirety), which can be used to target cell types specifically expressing the receptors. Alternatively, a polynucleotide-ligand complex can be formed allowing the polynucleotide to be targeted for cell specific uptake and expression in vivo by targeting a specific receptor (see, e.g., PCT Application Publication Nos. WO 92/06180, WO
92/22635, WO

SUBSTITUTE SHEET (RULE 26)

- 59 -92/203167, WO 93/14188, and WO 93/20221, which are hereby incorporated by reference in their entirety) [0121] Thus, in some embodiments, the composition further includes a transfection reagent. The transfection reagent may be a positively charged transfection reagent. Suitable transfection reagents are well known in the art and include, e.g., Lipofectamine" RNAiMAX
(Invitrogen"), Lipofectamine 2000 (Invitrogen"), Lipofectamine" 3000 (Invitrogen"), Invivofectamine 3.0 (Invitrogen"), Lipofectamine" MessengerMAX" (Invitrogen"), Lipofectin' (Invitrogen'), siLentFet" (Bio-Rad), DharmaFECT" (Dharmacon), HiPerFect (Qiagen), TransIT-X2' (Mirus), jetMESSENGER" (Polyplus), Trans-Hi', JetPEI"
(Polyplus), and ViaFectTM (Promega).
[0122] In some embodiments, the composition is an aqueous composition. Aqueous compositions of the present application comprise an effective amount of the vector, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
[0123] A further aspect of the present application relates to a pharmaceutical composition comprising an adeno-associated viral (AAV) vector described herein and a pharmaceutically-acceptable carrier.
[0124] The term "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. For example, 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.
[0125] The vector(s) (i.e., adeno-associated viral (AAV) vector and/or lentiviral vectors disclosed herein) and/or pharmaceutical composition(s) disclosed herein can be formulated according to any available conventional method. Examples of preferred dosage forms include a tablet, a powder, a subtle granule, a granule, a coated tablet, a capsule, a syrup, a troche, an inhalant, a suppository, an injectable, an ointment, an ophthalmic ointment, an eye drop, a nasal drop, an ear drop, a cataplasm, a lotion and the like. In the formulation, generally used additives

- 60 -such as a diluent, a binder, a disintegrant, a lubricant, a colorant, a flavoring agent, and if necessary, a stabilizer, an emulsifier, an absorption enhancer, a surfactant, a pH adjuster, an antiseptic, an antioxidant, and the like can be used.
[0126] In addition, formulating a pharmaceutical composition can be carried out by combining compositions that are generally used as a raw material for pharmaceutical formulation, according to conventional methods. Examples of these compositions include, for example, (1) an oil such as a soybean oil, a beef tallow and synthetic glyceride; (2) hydrocarbon such as liquid paraffin, squalene, and solid paraffin; (3) ester oil such as octyldodecyl myristic acid and isopropyl myristic acid; (4) higher alcohol such as cetostearyl alcohol and behenyl alcohol, (5) a silicon resin, (6) a silicon oil, (7) a surfactant such as polyoxyethylene fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, a solid polyoxyethylene castor oil and polyoxyethylene polyoxypropylene block co-polymer; (8) water soluble macromolecule such as hydroxyethyl cellulose, polyacrylic acid, carboxyvinyl polymer, polyethyleneglycol, polyvinylpyrrolidone and methylcellulose;
(9)lower alcohol such as ethanol and isopropanol; (10) multivalent alcohol such as glycerin, propyleneglycol, dipropyleneglycol and sorbitol; (11) a sugar such as glucose and cane sugar;
(12) an inorganic powder such as anhydrous silicic acid, aluminum magnesium silicicate, and aluminum silicate;
(13) purified water, and the like.
[0127] Additives for use in the above formulations may include, for example, (1) lactose, corn starch, sucrose, glucose, mannitol, sorbitol, crystalline cellulose, and silicon dioxide as the diluent; (2) polyvinyl alcohol, polyvinyl ether, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatine, shellac, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, polypropylene glycol-poly oxyethylene-block co-polymer, meglumine, calcium citrate, dextrin, pectin, and the like as the binder; (3) starch, agar, gelatine powder, crystalline cellulose, calcium carbonate, sodium bicarbonate, calcium citrate, dextrin, pectic, carboxymethylcellulose/calcium, and the like as the disintegrant, (4) magnesium stearate, talc, polyethyleneglycol, silica, condensed plant oil, and the like as the lubricant; (5) any colorant whose addition is pharmaceutically acceptable is adequate as the colorant; (6) cocoa powder, menthol, aromatizer, peppermint oil, cinnamon powder as the flavoring agent;
(7) antioxidants whose addition is pharmaceutically accepted such as ascorbic acid or alpha-tophenol.
[0128] For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous

- 61 -media are employed as is known to those of skill in the art, particularly in light of the present application By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see, for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580, which is hereby incorporated by reference in its entirety). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologies standards.
Age-Related Muscle Loss and Sarcopenia Muscle Atrophy [0129] As described herein, advancing age and sedentary life-style promotes significant muscle loss that becomes largely irreversible with advancing age, including very severe muscle loss and atrophy with age (sarcopenia). Sarcopenia and age-related muscle loss is a significant source of morbidity and mortality in the aging and the elderly population.
Only physical exercise is considered an effective strategy to improve muscle maintenance and function, but it must begin well before the onset of disease. There are few effective therapeutic options.
[0130] The Examples of the present application demonstrate that skeletal muscle expression of the AUF1 gene is downregulated with age in mice. It is hypothesized that skeletal muscle expression of the AUF1 gene is also downregulated with age in humans, thereby possibly contributing to muscle loss with age. The results presented herein demonstrate that AUF1 skeletal muscle gene transfer: (1) strongly enhances exercise endurance in middle-aged (12 month; equivalent to 50-60 year old humans) and old mice (18 months;
equivalent to >70 years of age humans) to levels of performance displayed by young mice (3 months old;
equivalent to late teens, early 20's in humans); (2) stimulates both fast and slow muscle, but specifically specifies slow muscle lineage by increasing levels of expression of the gene pgcla (Peroxisome proliferator-activated receptor gamma co-activator 1-alpha), a major activator of mitochondrial biogenesis and slow-twitch myofiber specification; (3) significantly increases skeletal muscle mass and normal muscle fiber formation in middle age and old mice in age-related muscle loss;
and (4) reduces expression of established biomarkers of muscle atrophy and muscle inflammation in age-related muscle loss.
[0131] Thus, another aspect of the present application relates to a method of promoting muscle regeneration. This method involves contacting muscle cells with an adeno-associated viral (AAV) vector or a composition described herein under conditions effective to express

- 62 -exogenous AUF1 in the muscle cells to increase muscle cell mass, increase muscle cell endurance, and/or reduce seam markers of muscle atrophy [0132] As used herein, the terms "promote," "promotion," and "promoting" refer to an increase in an activity, response, condition, or other biological parameter, including the production, presence, expression, or function of cells, biomolecules or bioactive molecules. The terms "promote," "promotion," and "promoting include, but are not limited to, initiation of an activity, response, or condition, as well as initiation of the production, presence, or expression of cells, biomolecules, or bioactive molecules. The terms "promote," "promotion,"
and "promoting" may also include measurably increasing an activity, response, or condition, or measurably increasing the production, presence, expression, or function of cells, biomolecules, or bioactive molecules, as compared to a native or control level.
[0133] Suitable cells for use according to the methods of the present application include, without limitation, mammalian cells such as rodent (mouse or rat) cells, cat cells, dog cells, rabbit cells, horse cells, sheep cells, pig cells, cow cells, and non-human primate cells. In some embodiments the cells are human cells.
[0134] In some embodiments, the muscle cells are selected from the group consisting of a myocyte, a myoblast, a skeletal muscle cell, a cardiac muscle cell, a smooth muscle cell, and a muscle stem cells (e.g., a satellite cell).
[0135] The method may be carried out in vitro or ex vivo.
[0136] In some embodiments, the method further involves culturing the muscle cells ex vivo under conditions effective to express exogenous AUF1.
[0137] In some embodiments, the method is carried out in vivo.
[0138] In some embodiments, the method further involves contacting the muscle cells with a purine-rich element binding protein 13 (Pur13) inhibitor. The Pur13 inhibitor may be a nucleic acid molecule, a polypeptide, or a small molecule. In some embodiments, the nucleic acid molecule is selected from the group consisting of siRNA, shRNA, and miRNA. Suitable nucleic acid molecules are described in detail supra.
[0139] Contacting, according to the methods of the present application, may be carried out by oral administration, topical administration, transdermal administration, parenteral administration, subcutaneous administration, intravenous administration, intramuscular administration, intraperitoneal administration, by intranasal instillation administration, by intracavitary or intravesical instillation, intraocular administration, intraarterial administration, intralesional administration, or by application to mucous membranes. Thus, in some embodiments, the contacting is carried out by intramuscular administration, intravenous

- 63 -administration, subcutaneous administration, oral administration, or intraperitoneal administration to a subject In specific embodiments, the administering is carried out by intramuscular injection.
[0140] A further aspect of the present application relates to a method of treating degenerative skeletal muscle loss in a subject. This method involves selecting a subject in need of treatment for skeletal muscle loss and administering to the selected subject an adeno-associated viral (AAV) vector described herein or a composition described herein under conditions effective to cause skeletal muscle regeneration in the selected subject.
[0141] In carrying out the methods of the present application, "treating" or "treatment"
includes inhibiting, preventing, ameliorating or delaying onset of a particular condition. Treating and treatment also encompasses any improvement in one or more symptoms of the condition or disorder. 'Treating and treatment encompasses any modification to the condition or course of disease progression as compared to the condition or disease in the absence of therapeutic intervention.
[0142] Suitable subjects for treatment according to the methods of the present application include, without limitation, domesticated and undomesticated animals such as rodents (mouse or rat), cats, dogs, rabbits, horses, sheep, pigs, and non-human primates. In some embodiments the subject is a human subject. Exemplary human subjects include, without limitation, infants, children, adults, and elderly subjects.
[0143] In some embodiments, the subject has a degenerative muscle condition. As used herein, the term "degenerative muscle condition" refers to conditions, disorders, diseases and injuries characterized by one or more of muscle loss, muscle degeneration or wasting, muscle weakness, and defects or deficiencies in proteins associated with normal muscle function, growth or maintenance. In certain embodiments, a degenerative muscle condition is sarcopenia or cachexia. In other embodiments, a degenerative muscle condition is one or more of muscular dystrophy, muscle injury, including acute muscle injury, resulting in loss of muscle tissue, muscle atrophy, wasting or degeneration, muscle overuse, muscle disuse atrophy, muscle disuse atrophy, denervation muscle atrophy, dysferlinopathy, AIDS/HIV, diabetes, chronic obstructive pulmonary disease, kidney disease, cancer, aging, autoimmune disease, polymyositis, and dermatomyositis. Thus, in some embodiments, the subject has a degenerative muscle condition selected from the group consisting of sarcopenia or myopathy.
[0144] The compositions and methods described herein may be used in combination with other known treatments or standards of care for given diseases, injury, or conditions. For example, in the context of muscular dystrophy, a composition of the invention for promoting

- 64 -muscle satellite cell expansion can be administered in conjunction with such compounds as CT-1, pregnisone, or myostatin The treatments (and any combination treatments provided herein) may be administered together, separately or sequentially.
[0145] The subject may have a muscle disorder mediated by functional AUF1 deficiency or a muscle disorder not mediated by functional AUF deficiency.
[0146] In some embodiments, the subject has an adult-onset myopathy or muscle disorder.
[0147] As used herein, the term "muscular dystrophy" includes, for example, Duchenne, Becker, Limb-girdle, Congenital, Facioscapulohumeral, Myotonic, Oculopharyngeal, Distal, and Emery-Dreifuss muscular dystrophies. In particular embodiments, the muscular dystrophy is characterized, at least in part, by a deficiency or dysfunction of the protein dystrophin. Such muscular dystrophies may include Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (DMD). In other embodiments, the muscular dystrophy is associated with degenerative muscle conditions such as muscle disuse atrophy, denervation muscle atrophy, dysferlinopathy, AIDS/HIV, diabetes, chronic obstructive pulmonary disease, kidney disease, cancer, aging, autoimmune disease, polymyositis, and dermatomyositis.
[0148] In some embodiments of the methods disclosed herein, the subject has Duchenne Muscular Dystrophy (DMD). As described above, DMD is an X-linked muscle wasting disease that is quite common (1/3500 live births), generally but not exclusively found in males, caused by mutations in the dystrophin gene that impair its expression for which there are few therapeutic options that have been shown to be effective. Muscle satellite cells are unresponsive in DMD
and are said to be functionally exhausted, thereby limiting or preventing new muscle development and regeneration. DMD typically presents in the second year after birth and progresses over the next two to three decades to death in young men.
[0149] In some embodiments of the methods disclosed herein, the subject has Becker muscular dystrophy. As described above, Becker muscular dystrophy is a less severe form of the disease that also involves mutations that impair dystrophin function or expression but less severely. There are few therapeutic options that have been shown to be effective for Becker muscular dystrophy. There are no cures for DMD or Becker disease.
[0150] In some embodiments of the methods disclosed herein, the subject has traumatic muscle injury. As used herein, the term "traumatic muscle injury" refers to a condition resulting from a wide variety of incidents, ranging from, e.g., everyday accidents, falls, sporting accidents, automobile accidents, to surgical resections to injuries on the battlefield, and many more. Non-

- 65 -limiting examples of traumatic muscle injuries include battlefield muscle injuries, auto accident-related muscle injuries, and sports-related muscle injuries [0151] In some embodiments, the administering is effective to treat a subject having degenerative skeletal muscle loss For example, the administering may be effective to activate muscle stem cells, accelerate the regeneration of mature muscle fibers (myofibers), enhance expression of muscle regeneration factors, accelerate the regeneration of injured skeletal muscle, increase regeneration of slow-twitch (Type I) and/or fast-twitch (Type II) fibers), and/or restore muscle mass, muscle strength, and create normal muscle following in the selected subject.
[0152] In some embodiments, the administering is effective to transduce skeletal muscle cells (e.g-., cardiac diaphragm cells) and/or provide long-term (e.g., lasting at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or more) muscle cell-specific AUF1 expression in the selected subject.
[0153] In other embodiments, the administering is effective to (i) activate high levels of satellite cells and myoblasts; (ii) significantly increase skeletal muscle mass and normal muscle fiber formation; and/or (iii) significantly enhanced exercise endurance in the selected subject as compared to when the administering is not carried out.
[0154] In further embodiments, the administering is effective to reduce (i) biomarkers of muscle atrophy and muscle cell death; (ii) inflammatory immune cell invasion in skeletal muscle (including diaphragm); and/or (iii) muscle fibrosis and necrosis in skeletal muscle (including diaphragm) in the selected subject, as compared to when the administering is not carried out.
[0155] In certain embodiments, the administering is effective to (i) increase expression of endogenous utrophin in DMD muscle cells and/or (ii) suppress expression of embryonic dystrophin, a marker of muscle degeneration in DMD in the selected subject, as compared to when the administering is not carried out. In some embodiments of the methods disclosed herein, said administering is effective to upregulate endogenous utrophin protein expression in the selected subject, as compared to when the administering is not carried out. In some embodiments of the methods disclosed herein, said administering is effective to upregulate endogenous utrophin protein expression in said muscle cells, as compared to when the administering is not carried out.
[0156] In some embodiments, the administering is effective to (i) increase normal expression of genes involved in muscle development and regeneration and/or (ii) suppress genes involved in muscle cell fibrosis, death, and muscle-expressed inflammatory cytokines in the selected subject, as compared to when the administering is not carried out.

- 66 -[0157] In further embodiments, the administering does not increase muscle mass, endurance, or activate satellite cells in normal skeletal muscle [0158] In some embodiments, the administering is effective to accelerate muscle gain in the selected subject, as compared to when said administering is not carried out.
[0159] In certain embodiments, the administering is effective to reduce expression of established biomarkers of muscle atrophy in a subject having degenerative skeletal muscle loss.
Suitable biomarkers of muscle atrophy include, without limitation, TRIM63 and Fbxo32 mRNA.
In some embodiments, the administering is effective to enhance expression of established biomarkers of muscle myoblast activation, differentiation, and muscle regeneration in the selected subject. Suitable biomarkers of muscle atrophy include, without limitation, myogenin and MyoD mRNA levels, biomarkers of myoblast activation, differentiation and muscle regeneration (Zammit, "Function of the Myogenic Regulatory Factors Myf5, MyoD, Myogenin and MRF4 in Skeletal Muscle, Satellite Cells and Regenerative Myogenesis,-Semin. Cell. Dev.
Biol. 72:19-32 (2017), which is hereby incorporated by reference in its entirety).
[0160] In some embodiments, the method further involves administering a purine-rich element binding protein 3 (Pur13) inhibitor. The Pur13 inhibitor may be a nucleic acid molecule, a polypeptide, or a small molecule. In some embodiments, the nucleic acid molecule is selected from the group consisting of siRNA, shRNA, and miRNA. Suitable nucleic acid molecules are describe in detail supra.
[0161] Administering, according to the methods of the present application, may be carried out orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes.
Thus, in some embodiments, the administering is carried out intramuscularly, intravenously, subcutaneously, orally, or intraperitoneally. In specific embodiments, the administering is carried out by intramuscular injection. In some embodiments, an adeno-associated virus (AAV) vector is administered by intramuscular injection.
[0162] In some embodiments, the administering is carried out by intramuscular injection.
Traumatic Muscle Injury [0163] A further aspect of the present application relates to a method of preventing traumatic muscle injury in a subject. This method involves selecting a subject at risk of traumatic muscle injury and administering to the selected subject an adeno-associated viral (AAV) vector described herein, a composition described herein, or a lentiviral vector comprising

- 67 -a muscle cell specific promoter and a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF 1) protein or a functional fragment thereof, where the nucleic acid molecule is heterologous to and operatively coupled to the muscle cell-specific promoter.
[0164] Still another aspect of the present application relates to a method of treating traumatic muscle injury in a subject. This method involves selecting a subject having traumatic muscle injury and administering to the selected subject an adeno-associated viral (AAV) vector described herein, a composition described herein, or a lentiviral 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 cell-specific promoter.
[0165] Suitable subjects for treatment according to the methods of the present application include, without limitation, domesticated and undomesticated animals such as rodents (mouse or rat), cats, dogs, rabbits, horses, sheep, pigs, and non-human primates. In some embodiments the subject is a human subject. Exemplary human subjects include, without limitation, infants, children, adults, and elderly subjects.
[0166] As described supra, the term "traumatic muscle injury"
refers to a condition resulting from a wide variety of incidents, ranging from, e.g., everyday accidents, falls, sporting accidents, automobile accidents, to surgical resections to injuries on the battlefield, and many more. Non-limiting examples of traumatic muscle injuries include battlefield muscle injuries, auto accident-related muscle injuries, and sports-related muscle injuries.
[0167] In some embodiments, the subject is at risk of developing or is in need of treatment for a traumatic muscle injury selected from the group consisting of a laceration, a blunt force contusion, a shrapnel wound, a muscle pull, a muscle tear, a bum, an acute strain, a chronic strain, a weight or force stress injury, a repetitive stress injury, an avulsion muscle injury, and compartment syndrome.
[0168] In some embodiments, the subject is at risk of developing or is in need of treatment for a traumatic muscle injury that involves volumetric muscle loss ("VML"). The terms "volumetric muscle loss" or "VML" refer to skeletal muscle injuries in which endogenous mechanisms of repair and regeneration are unable to fully restore muscle function in a subject.
The consequences of VML are substantial functional deficits in joint range of motion and skeletal muscle strength, resulting in life-long dysfunction and disability.
[0169] In some embodiments, the administering is carried out in a subject at risk of developing a traumatic muscle injury and a prophylactically effective amount of the adeno-associated viral (AAV) vector, composition, or lentiviral vector of the present application is

- 68 -administered. The term "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result [0170] In some embodiments, the administering is carried to treat a subject having traumatic muscle injury and said administering is carried out immediately after the traumatic muscle injury (for example, within one minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 60 minutes, or any amount of time there between) of the traumatic muscle injury. In certain embodiments, said administering is carryout out within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours of the traumatic muscle injury. In other embodiments, said administering is carried out within 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 14 days of the traumatic muscle injury. In further embodiments, said administering may be carried out within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 52 weeks, or any amount of time there between of the traumatic muscle injury.
[0171] Adeno-associated virus (AAV) vectors and lentiviral vectors are currently the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred polynucleotides are stably integrated into the chromosomal DNA of the host.
[0172] The adeno-associated viral (AAV) vector and/or the lentiviral vector for use in the methods disclosed herein may encode AUF1 isoform p37AuF1, p40AUF1, p42AuF1 , or p45AUF1.
Suitable AUF isoform nucleic acid and amino acid sequences are identified supra. In certain embodiments, the adeno-associated viral (AAV) vector and/or the lentiviral vector for use in the methods disclosed herein encodes AUF isoform p45AuF1 .
[0173] In some embodiments, the adeno-associated virus (AAV) vector is AAV8-0/ICK-AUF1 or another human AAV including but not limited to AAV1, AAV2, AAV5, AAV6, or AAV9 vector encoding AUF1 (e.g., AUF 1 isoforms 37 AUF1 p40AUF1, p42Aun, and/or p45AuF1).
In other embodiments, the AAV is a human novel AAV capsid variant engineered for enhanced muscle-specific tropism including but not limited to AAV2i8 or AAV2.5. In yet other embodiments, the AAV vector is a non-human primate AAV vector including but not limited to AAVrh.8, AAVrh.10, AAVrh.43, or AAVrh.74.
[0174] In some embodiments, the lentiviral vector is a lentivirus p45 AUF1 vector, or a lentivirus expressing another AUF1 isoform (e.g., p 37 ALA', p40 AUF1 or p42 Aum) or

- 69 -combinations thereof (Abbadi et al., "Muscle Development and Regeneration Controlled by AUF1-mediated Stage-specific Degradation of Fate-determining Checkpoint mRNAs," Proc.
Nat'l. Acad. Sci. USA 116:11285-90 (2019), which is hereby incorporated by reference in its entirety). Other embodiments include expression of p37 AUF1, p40 AUF1, p42 AUF1, p45 MT", or combinations thereof from non-human lentivirus vectors including but not limited to simian, feline, and other mammalian lentivirus gene transfer vectors.
[0175] In one particular embodiment, the AUF1 p45 lentivirus vector has the following nucleotide sequence:
AUF1 p45 Lentivirus Vector Shuttle Plasmid (SEQ ID NO:26) GATTGACTGA GTCGCCCGGG TACCCGTGTA TCCAATAAAC CCTCTTGaAG TTGCATCCGA 420 CGGCTCGGCG GGCGAGCAGG AGGGAGCCAT GGTGGCGGCG ACAaAGGGGG CAGCGGCGGC 1500 CGCCGAGTCG GAGGGGGCGA AGATTGACGC aAGTAAGAAC GAGGAGGATG AAGGCCATTC 1620 SUBSTITUTE SHEET (RULE 26)

- 70 -CGTATTCAAC AAGGGGCTGA AGGATGCCCA GAAGGTACCC aATTGTATGG GATCTGATCT 2880 CACTACATGG CGTGATTTCA TATGCGCGAT TGCTGATCCC aATGTGTATC ACTGGCAAAC 3480 CCTGACGGAC AATGGCCGCA TAACAGCGGT aATTGACTGG AGCGAGGCGA TGTTCGGGGA 3660 ACTCGCCGAT AGTGGAAACC GACGCCCCAG aACTCGTCCG AGGGCAAAGG AATAGAGTAG 4020 SUBSTITUTE SHEET (RULE 26)

- 71 -CTTGAGTCCA ACCCGGTAAG ACACGACTTA TCGCCACTGG aAGCAGCCAC TGGTAACAGG 5340 GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCC

SUBSTITUTE SHEET (RULE 26)

- 72 -[0176] In some embodiments, the administering is effective to prevent muscle atrophy and/or muscle loss following traumatic muscle injury to the selected subject.
In other embodiments, the administering is effective to activate muscle stem cells following traumatic muscle injury to the selected subject. In further embodiments, the administering is effective to accelerate the regeneration of mature muscle fibers (myofibers), enhance expression of muscle regeneration factors, accelerate the regeneration of injured muscle, increased regeneration of slow-twitch (Type I) and/or fast-twitch (Type II) fibers), and/or restore muscle mass, muscle, strength and create normal muscle following traumatic muscle injury in the selected subject.
[0177] In some embodiments, the administering is effective to accelerate muscle gain following traumatic muscle injury in the selected subject, as compared to when said administering is not carried out.
[0178] In certain embodiments, the administering is effective to reduce expression of established biomarkers of muscle atrophy following traumatic muscle injury to the selected subject. Suitable biomarkers of muscle atrophy include, without limitation, TRIM63 and Fbxo32 mRNA. In some embodiments, the administering is effective to enhance expression of established biomarkers of muscle myoblast activation, differentiation and muscle regeneration following traumatic muscle injury to the selected subject. Suitable biomarkers of muscle atrophy include, without limitation, myogenin and MyoD mRNA levels, biomarkers of myoblast activation, differentiation and muscle regeneration (Zammit, "Function of the Myogenic Regulatory Factors Myf5, MyoD, Myogenin and MRF4 in Skeletal Muscle, Satellite Cells and Regenerative Myogenesis," Semin. Cell. Dev. Biol. 72:19-32 (2017), which is hereby incorporated by reference in its entirety).
[0179] In some embodiments, the administering is effective to deliver the vector or pharmaceutical composition described herein to a specific tissue in the subject. The tissue may be muscle tissue. For example, the muscle tissue may be all types of skeletal muscle, smooth muscle, or cardiac muscle.
[0180] Administering, according to the methods of the present application, may be carried out orally, topically, transdermally, parenterally, subcutaneously, intravenously, intramuscularly, intraperitoneally, by intranasal instillation, by intracavitary or intravesical instillation, intraocularly, intraarterially, intralesionally, or by application to mucous membranes.
Thus, in some embodiments, the administering is carried out intramuscularly, intravenously, subcutaneously, orally, or intraperitoneally. In specific embodiments, the administering is carried out by intramuscular injection. In some embodiments, an adeno-associated virus (AAV) vector is administered by intramuscular injection.

- 73 -[0181] In other embodiments, the administering is carried out by systemic administration. Thus, in some embodiments, a lentiviral 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 cell-specific promoter is administered systemically. In some embodiments, the lentiviral vectors administered systemically is a lentivirus expressing p37 AUF1, p40 AUF1, 02 AUF1, and/or p45 AUF1 AUF1 vector (Abbadi et al., "Muscle Development and Regeneration Controlled by AUF1-mediated Stage-specific Degradation of Fate-determining Checkpoint mRNAs," Proc. Nat 'I. Acad. Sci. USA 116:11285-90 (2019), which is hereby incorporated by reference in its entirety).
[0182] Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
[0183] Suitable regimens for initial contacting and further doses or for sequential contacting steps may all be the same or may be variable. Appropriate regimens can be ascertained by the skilled artisan, from the disclosure of the present application, the documents cited herein, and the knowledge in the art.
[0184] A dosage unit to be administered in methods of the present application will vary depending on the vector used, the route of administration, the type of tissue and cell being targeted, and the purpose of treatment, among other parameters. Dosage for treatment can be determined by a skilled person who would know how to determine dose using methods standard in the art. A dosage unit, corresponding to genome copy number, for example, could range from about, lx101 to lx1011, 1x102 to lx10", 1x103 to lx10", 1x104 to lx1011, 1x105 to lx10", 1x106 to lx1011, 1x107 to lx1011, 1x108 to 1x1011, 1x109 to lx1011, lx101 to lx1011, lx101 to lx101 , 1x102 to lx101 , 1x103 to lx101 , 1x104 to lx101 , 1x105 to lx101 , 1x106 to lx101 , 1x107 to 1x1010, 1x108 to 1x1010, 1x109 to lx10", lx101 to 1x109, 1x102 to 1x109, 1x103 to 1x109, 1x104 to lx109, 1X105 to lx109, lx106 to lx109, lx107 to lx109, lx1OS to lx109, lx101 to lx108, lx102 to 1x108, 1)(103 to 1x108, 1x104 to 1x108, 1x105 to 1x108, 1x106 to 1x108, or 1x107 to 1x108 genome copies of a vector disclosed herein. In some embodiments, a dosage unit, corresponding to genome copy number, for example, is administered in the range of lx101 to lx1012, 1x102 to lx1012, 1x103 to lx1012, 1X104 to 1X1012, 1X105 to lx1012, 1X106 to lx1012, 1x107 to lx1012, 1x108 to lx1012, 1X109 to 1X1012, 1X101 to lx1012, or lx1011 to lx1012genome copies; lx101 to lx1013, lx102 to lx1013, lx103 to 1x1013, lx104 to lx1013, lx105 to 1x1013, lx106 to lx1013,

- 74 -1x107 to lx1013, 1x108 to lx1013, 1x109 to lx1 013, lx101 to lx1013, lx10" to lx1013, or 1x1012 to lx1013 genome copies; lx101 to lx1014, 1X102 to 1x1014, 1x103 to 1x1014, 1x104 to 1x1014, 1)(105 to 1x1014, 1x106 to 1x1014, 1x107 to 1x1011, 1x108 to 1x1014, 1x109 to 1x1014, 1x101 to 1x1011, 1x1011 to 1x1011, 1x1012 to 1x1011, or lx1013 to 1x1011 genome copies;
lx101 to lx1015, 1x102 to 1x1015, 1x103 to lx1015, 1x104 to 1x1015, 1x105 to lx1015, 1x106 to lx1015, 1x107 to 1x1015, 1x108 to 1x1015, 1x109 to 1x105, 1x1019 to 1x1015, 1x1011 to 1x1015, 1x1012 to 1x1015, 1x1013 to lx1015, or lx1014 to 1x1015 genome copies; 1x101 to 1x1016, 1x102 to 1x1016, 1x103 to 1x1016, 1x104 to 1x1016, 1x105 to 1x1016, 1x106 to 1x1016, 1x107 to 1x1016, 1x108 to 1x1016, 1x109 to 1x1016, 1x1019 to 1x106, 1x1011 to 1x1016, 1x1012 to 1x1016, 1x1013 to 1x1016, 1x1014 to 1x1016, or 1x10" to 1x1016 genome copies; 1x101 to 3x1016, 1x102 to 3x1016, 1x103 to 3x1016, lx 104 to 3x 1016, lx1 05 to 3x1016, lx 1 06 to 3x1 016, 1 x 107 to 3x1016, lx 108 to 3x 1016, lx 1 09 to 3x1016, 1x1010 to 3x1016, 1x1011 to 3)(101-6, 1x1012 to 3x1016, 1x1013 to 3x1016, 1x1014 to 3x1016, or lx 1015 to 3x1016 genome copies; and any amount there between. Dosage will depend on route of administration, type of tissue and cells to receive the vector, timing of administration to human subjects, whether dosage is determined based on total genome copies to be delivered, and whether administration is determined by genome copies per kilogram body weight.
101851 In some embodiments, a subject is administered a vector or pharmaceutical composition described herein in one dose. In other embodiments, the subject is administered the vector or pharmaceutical composition described herein in a series of two or more doses in succession. In some other embodiments, where the subject is administered the vector or pharmaceutical composition described herein in a single dose, in two doses, and/or more than two doses, the doses may be the same or different, and they are administered with equal or with unequal intervals between them.
101861 A subject may be administered the vector or pharmaceutical composition described herein in many frequencies over a wide range of times. In some embodiments, the subject is administered the vector or pharmaceutical composition described herein over a period of less than one day. In other embodiments, the subject is contacted over two, three, four, five, or six days. In some embodiments, the contacting is carried out one or more times per week, over a period of weeks. In other embodiments, the contacting is carried out over a period of weeks for one to several months. In various embodiments, the contacting is carried out over a period of months. In others, the contacting may be carried out over a period of one or more years. Generally, lengths of treatment will be proportional to the length of the ischemic disease process, the effectiveness of the therapies being applied, and the condition and response of the subject being treated. According to some embodiments, the contacting is carried out daily.

- 75 -[0187] The choice of formulation for administered the vector or pharmaceutical composition described herein will depend on a variety of factors Prominent among these will be the species of subject, the nature of the disorder, dysfunction, or disease being treated and its state and distribution in the subject, the nature of other therapies and agents that are being administered, the optimum route for administration, survivability via the route, the dosing regimen, and other factors that will be apparent to those skilled in the art.
In particular, for instance, the choice of suitable carriers and other additives will depend on the exact route of contacting and the nature of the particular dosage form.
[0188] In the methods described herein, rather than administering a vector, other means of administering AUF can be carried out including by direct injection of: (i) encoding p37AuF1 , p40 AUF1, p42 AUF1, and/or p45 AUF1 DNA by plasmid; (ii) mRNA encoding p37 AUF1, p 40 AUF1, p42 AUF1, and/or p45 AUF1; and/or (iii) nanoparticle incorporation of AUF1 encoding DNA or mRNA.
EXAMPLES
[0189] The examples below are intended to exemplify the practice of embodiments of the present application but are by no means intended to limit the scope thereof.
Materials and Methods for Examples 1 ¨ 8 Mice [0190] All animal studies were approved by the NYU School of Medicine Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with IACUC
guidelines. All ant- KO mice and WT mice are of the 129/B6-background, bred at the F3 and F4 generations from aut- heterozygous mice (Pont et al., "mRNA Decay Factor Maintains Normal Aging, Telomere Maintenance, and Suppression of Senescence by Activation of Telomerase Transcription," Molecular Cell 47(1):5-15 (2012) and Lu et al., "Endotoxic Shock in AUF1 Knockout Mice Mediated by Failure to Degrade Proinflammatory Cytokine mRNAs,"
Genes Dev. 20(22):3174-3 184 (2006), which are hereby incorproated by reference in their entirety). 12 month old C57BL6 mice (Jackson) for AUF1 supplementation during AAV
experiments. One month old C57BL10 and C57BL/10ScSn-Dmc1nth/J mice (Jackson) were used for A AV experiments in Example S.
Cells [0191] C2C12 cells were obtained from the American Type Culture Collection (ATCC), authenticated by STR profiling and routinely checked for mycoplasma contamination. C2C12

- 76 -cells were maintained in DMEM (Corning), 10% FBS (Gibco), and 1% penicillin streptomycin (Life Technologies). To differentiate cells, media was switched to DMEM
(Corning), 2% Horse Serum (Gibco), and 1% penicillin streptomycin (Life Technologies) during 96 hours (Panda et al., "RNA-Binding Protein AUF1 Promotes Myogenesis by Regulating MEF2C
Expression Levels," Mol. Cell Biol. 34(16): 3106-3119 (2014), which is hereby incorporated by reference in its entirety). aufl KO C2C12 cells were created with Crispr-Cas9 methods (Abbadi et al., "Muscle Development and Regeneration Controlled by AUF I-Mediated Stage-Specific Degradation of Fate-Determining Checkpoint mRNAs," Proc. Natl. Acad. Sci. USA
116(23):11285-11290 (2019), which is hereby incorporated by refernce in its entirety). For assays performed in the presence of actinomycin D to determine mRNA stability, myoblasts cells were treated with 0.2 jig/ml of actinomycin D (Sigma). RNA
immune-precipitation experiments were done in WT C2C12 before and 48 hours of differentiation using a normal IgG rabbit control or a rabbit-anti AUF1 antibody (07-260, Millipore).
Immunofluorescence [0192] Mice had skeletal muscles removed as indicated in the text, put in OCT, frozen in dry ice-cooled isopentane (Tissue-Tek), fixed in 4% paraformaldehyde, and blocked in 3% BSA
in TBS. C2C12 cells were fixed in 4% paraformaldehyde and blocked in 3% BSA in PBS.
Samples were immunostained overnight with antibodies: AUF1 (07-260, Millipore), Slow myosin (N0Q7 5 4D, Sigma), Fast myosin (MY-32, Sigma), Laminin alpha 2 (4T-T8-2, Sigma), and GFP (2956, Cell signaling). Slow and fast myosin staining were done using MOM kit (Vector biolabs). Alexa Fluor donkey 488 and 555 secondary antibodies were used at 1:300 and incubated for 1 hour at room temperature. Slides were sealed with Vectashield with DAPI
(Vector). Images were processed using ImageJ.
Microscopy, Image Processing, and Analysis [0193] Images were acquired using a Zeiss LSM 700 confocal microscope, primarily with the 20X lens. Images were processed using ImageJ. If needed, color balance was adjusted linearly for the entire image and all images in experimental sets.
Immunoblot Studies [0194] C2C12 cells or muscle tissues were lysed using lysis buffer (50 mmol/L Tris-HC1, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% TritonX100) supplemented with complete protease inhibitor cocktail (Complete mini, ROCHE). Equal amounts of total protein were loaded on a polyacrylamide gel, resolved and transferred to PVDF
membrane.
Membrane was blocked with 5% nonfat milk in TBS-Tween 20 (0.1%) for 1 hour and probed

- 77 -with Antibody against AUF1 (07-260, Millipore) or against PGClalpha (Novus biologicals NBP1-04676). Bands were detected by peroxidase conjugated secondary antibodies (GE
healthcare) and visualized with the ECL chemiluminescence system. The immunoblots were also probed with a rabbit antibody to 13-tubulin (Cell Signaling 2146S) or GAPDH (Cell Signaling 2118S) as a control for loading. Quantification was performed by ImageJ.
Real-Time PCR Analysis [0195] RNA was extracted using Trizol (Invitrogen) according to the manufacturer's instructions. DNase treatment was systematically performed. Quantification of extracted RNA
was assessed using Nanodrop. The cDNA was synthesized using High Capacity cDNA
Reverse Transcription Kit (Applied Biosystems). mRNA was analyzed by real-time PCR
using the iTaq Universal SYBR Green Supermix (Bio Rad) probe. Relative quantification was determined using the comparative CT method with data normalized to housekeeping gene and calibrated to the average of control groups.
AA V-AUF1 Expression/AAV AUF1 Gene Transfer [0196] AUF1 was integrated into an AAV8 vector under the tMCK promoter (AAV8-tMCK-AUF1-1RES-eGFP) (Vector Biolabs) (FIG. 10). AAV8-tMCK-IRES-eGFP was used as a control vector. This promoter was generated by the addition of a triple tandem of 2RS5 enhancer sequences (3-Ebox) ligated to the truncated regulation region of the MCK
(muscle creatine kinase) promoter, which induced high muscle specificity (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).
C57B16 mice were injected with a single retro-orbital injection of 50 n1 (final concentration:
2.5x10" particles).
Muscle Function Tests [0197] Grid hanging time. Mice were placed in the center of a grid, 30 cm above soft bedding to prevent injury should they fall. The grid was then inverted. Grid hanging time was measured as the amount of time mice held on before dropping off the grid. Each mouse was analyzed twice with 5 repetitions per mouse.
[0198] Time, distance to exhaustion, and maximum speed. After 1 week of acclimation, mice were placed on a treadmill and the speed was increased by 1 m/min every 3 minutes and the slope was increased every 9 minutes by 5 cm to a maximum of 15 cm. Mice were considered to be exhausted when they stayed on the electric grid more than 10 seconds. Based on their weight and running performance, work performance was calculated in Joules (J).
Each mouse was analyzed twice with 5 repetitions per mouse.

- 78 -[0199] Strength by grip test (Examples 8 and 9): In this test, mice grasp a horizon tall grid connected to a dynamometer and are pulled backwards five times by tugging on the tail.
The force applied to the grid each time before the animal loses its grip is recorded in Newtons.
The average of the five tests is then normalized to the whole-body weight of each mouse. Mice are typically analyzed twice with 5 repetitions per mouse.
Dexa Muscle Mass Non-Invasive Quantitative Analysis (Example 7) [0200] Dual energy X-ray absorptiometry (DEXA) was used to record lean muscle mass and changes in muscle mass upon injury or age previously published (Chenette et al., "Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity,- Cell Rep. 16(5):1379-1390 (2016), which is hereby incorporated by reference in its entirety).
Quantification of satellite cells (Example 7) [0201] Muscles are excised and digested in collagenase type I.
Cell numbers are quantified by flow cytometry gating for Sdc4+ CD45- CD31- Scal- satellite cell populations (Shefer et al," Satellite-Cell Pool Size Does Matter. Defining the Myogenic Potency of Aging Skeletal Muscle," Dev. Biol. 294(1):50-66 (2006) and Brack et al., "Pax7 is Back," Skelet.
Muscle 4(1):24 (2014), which are hereby incorporated by reference in their entirety).
Muscle Fiber Type Analysis (Example 7) [0202] Skeletal muscles were removed, put in OCT compound, fixed in 4%
paraformaldehyde, and immunostained with antibodies to AUF1 (07-260, Millipore), slow myosin (N0Q7 5 4D, Sigma), fast myosin (MY-32, Sigma), and laminin alpha 2 membrane component (4H8-2, Sigma).
Histological Studies and Biochemical Analysis of Muscle Tissues (Examples 7 and 8) [0203] Muscles were removed and frozen in OCT compound, fixed in 4%
paraformaldehyde, and blocked in 3% BSA in TBS. Immunofluorescence or immunochemistry (Hematoxylin and Eosin, Masson Trichome) was performed. Fibrosis was assessed by staining of muscle sections with Masson trichrome to visualize areas of collagen deposition and quantified using ImageJ software. Immunofluorescence images were acquired using a Zeiss LSM 700 confocal microscope. Images and morphometric analysis (Feret diameter, Cross sectional area) were processed using ImageJ as recently described (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-(2019), which is hereby incorporated by reference in its entirety). Muscles were harvested for

- 79 -biochemical analysis including immunoblot, RNAseq, and RT-PCR analysis.
Evan Blue Dye Analysis (Example 7) [0204] Evan Blue dye was used as an in vivo marker of muscle damage. It identifies permeable skeletal myofibers that have become damaged (Wooddell et al., "Myofiber Damage Evaluation by Evans Blue Dye Injection," Curr. Protoc. Mouse Biol. 1(4):463-488 (2011), which is hereby incorporated by reference in its entirety).
Serum Creatine Kinase (CK) Activity (Example 7) [0205] Serum CK was evaluated at 37 C by standard spectrophotometric analysis using a creatine kinase activity assay kit (abcam). The results are expressed in mU/mL.
Blood Harvesting (Example 7) 102061 Peripheral blood was harvested to quantify creatine kinase levels, and levels of cytokines, cells and inflammatory markers.
Quantification and Statistical Analysis [0207] All results are expressed as the mean SEM. Two group comparisons were analyzed by the unpaired Mann-Whitney test. Multiple group comparisons were performed using one-way analysis of variance (ANOVA). The non-parametric Kruskal¨Wallis test followed by the Dunn's comparison of pairs was used to analyze groups when suitable. P-values of <0.05 were considered significant. All statistical analyses were performed using GraphPad Prism (version 7) software.
Genome- Wide Transcriptomic and Trans latomic Studies and Bioinformatic Data Analysis (Example 7) [0208] Polysome fractionation and inRNA isolation. Polysome isolation was performed by separation of ribosome-bound mRNAs by sucrose gradient centrifugation using cytoplasmic extracts as previously described (de la Parra et al., "A Widespread Alternate form of Cap-Dependent mRNA Translation Initiation," Nat. Commun. 9(1):3068 (2018) and Badura et al., "DNA Damage and eIF4G1 in Breast Cancer Cells Reprogram Translation for Survival and DNA Repair mRNAs," Proc. Natl. Acad.
USA 109(46):18767-72 (2012), which are hereby incorporated by reference in their entirety). Post-fractionation samples were pooled based on enriched for mRNAs bound to 2-3 ribosomes and >4 ribosomes corresponding to poorly translated and well translated fractions respectively, and used for RNA
sequencing (RNAseq).
RNA quality was measured by a Bioanalyzer (Agilent Technologies).
[0209] RNA sequencing and data analysis. Paired-end RNA-seq was carried out by the New York University School of Medicine Genome Technology Core using the Illumina HiSeq

- 80 -4000 single read. The low-quality reads (less than 20) were trimmed with Trimmomatic (Bolger et al., "Trimmomatic: A Flexible Trimmer for Illumina Sequence Data,"
Bioinformatics, 30(15):2114-20 (2014), which is hereby incorporated by reference in its entirety) (version 0.36) with the reads lower than 35 nt being excluded. The resulted sequences were aligned with STAR
(Dobin et al., "STAR: Ultrafast Universal RNA-Seq Aligner," Bioinformatics 29(1):15-21 (2013), which is hereby incorporated by reference in its entirety) (version 2.6.0a) to the hg38 reference genome in the single-end mode. The alignment results were sorted with SAMtools (Li et al., "The Sequence Alignment/Map format and SAMtools," Biontfarmatics 25(16):2078-2079 (2009), which is hereby incorporated by reference in its entirety) (version 1.9), after which supplied to HT Seq (Anders et al., -HTSeq--A Python Framework to Work with High-Throughput Sequencing Data," BioinfOrmatics 31(2):166-9 (2015), which is hereby incorporated by reference in its entirety) (version 0.10.0) to obtain the feature counts.
The feature counts tables from different samples were concatenated with a custom R script. To examine differences in transcription and translation, total mRNA and polysome mRNA were quantile-normalized separately. Regulation by transcription and translation and accompanying statistical analysis was performed using RIVET (Ernlund et al., "RIVET: Comprehensive Graphic User Interface for Analysis and Exploration of Genome-Wide Translatomics Data," BMC Genomics 19(1):809 (2018), which is hereby incorporated by reference in its entirety), where significant genes were identified as P<0.05 and >1 log fold change. Reactome pathway analysis was performed on genes that were up- and down-regulated by transcription and translation using Metascape (Zhou et al., "Metascape Provides a Biologist-Oriented Resource for the Analysis of Systems-Level Datasets," Nat. Commun. 10(1):1523 (2019), which is hereby incorporated by reference in its entirety). Pathway analysis and enrichment plots of the top 100 genes that were the most regulated by transcription and/or translation were generated using DAVID
(Huang da et al., "Systematic and Integrative Analysis of Large Gene Lists Using DAVID
Bioinformatics Resources," Nat. Protoc. 4(1):44-57 (2009), which is hereby incorporated by reference in its entirety) and Metascape. Prediction of transcription factors of the same list of 100 genes was performed using Enrichr (Chen et al., "Enrichr: Interactive and Collaborative HTML5 Gene List Enrichment Analysis Tool," BMC Bioinformatics 14:128 (2013), which is hereby incorporated by reference in its entirety) (TRANSFAC and JASPER PWM program) and PASTAA
(Roider et al., "Predicting Transcription Factor Affinities to DNA from a Biophysical Model,-Bioilfformatics 23(2):134-41 (2007), which is hereby incorporated by reference in its entirety) online tool. Genes enriched in TFH cells was determined from GSE16697 (Johnston et al., "Bc16 and Blimp-1 are Reciprocal and Antagonistic Regulators of T Follicular Helper Cell

-81 -Differentiation," Science 325(5943):1006-1010 (2009), which is hereby incorporated by reference in its entirety) and similar genes between datasets were determined using Venny.
Traumatic Injury Animal Model (Example 8) [0210] Three month old male mice, unless otherwise noted, were administered an intramuscular injection of 50 IA of filtered 1.2% BaC17 in sterile saline with control or with lentivirus AUF1 vector (Ix108 genome copy number/ml) (total volume 100 til) into the left tibialis anterior (TA) muscle. The right TA muscle remained uninjured as a control. Mice were sacrificed at 3 or 7 days post-injection. Muscles were weighed and frozen in OCT for immunofluorescence staining or put in Trizol for mRNA extraction.
Example 1 ¨ Skeletal Muscle AUF1 Expression is Downregulated with Age [0211]
Because mice deleted in the cuff/ gene undergo an accelerated loss of muscle mass (Chenette et al., "Targeted mRNA Decay by RNA Binding Protein AUF1 Regulates Adult Muscle Stem Cell Fate, Promoting Skeletal Muscle Integrity," Cell Rep.
16(5):1379-1390 (2016); 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 Pont et al., "mRNA Decay Factor AUF1 Maintains Normal Aging, Telomere Maintenance, and Suppression of Senescence by Activation of Telomerase Transcription," Molecular Cell 47(1):5-15 (2012), which are hereby incorporated by reference in their entreity), whether reduced expression of AUF1 with age occurs in wild type animals and is involved in age-related muscle atrophy was investigated. The expression of AUF1 in limb skeletal muscles of young (3 month), middle-aged (12 month) and older mice (18 month) was analyzed. Compared to 3 month young mice, aufl mRNA expression was strongly downregulated by 12 months of age in non-exercised animals, shown in the tibialis anterior (TA), gastrocnemius, extensor digitorum longus (EDL) and soleus muscles (FIG.
7A). In all studies test mRNAs were normalized to gapdh or tbp mRNAs which were unchanged in abundance regardless of AUF1 expression. As shown in the TA muscle, AUF1 protein levels tracked mRNA levels, demonstrating reduction by 60% at 12 months and 80% at 18 months (FIG. 7B). Reduced skeletal muscle AUF1 expression with age in non-exercised animals was associated with a significant loss of muscle mass in limb muscles, shown in the TA, EDL, gastrocnemius and soleus muscles in 12 and 18 month old mice compared to 3 month old animals (FIG. 7C). Importantly, by 18 months of age, loss of muscle mass began to plateau from 12 month values. The TA muscle was reduced in mass by almost 50%, the EDL by 30%, the

- 82 -soleus by almost 50% and the gastrocnemius by 25%. These data clearly show that by 12-18 months of age, sedentary mice have undergone a significant reduction in skeletal muscle mass consistent with muscle loss and atrophy typically observed in the absence of exercise and with aging.
Example 2 ¨ AUF1 Skeletal Muscle Gene Transfer Enhances Exercise Endurance in Middle-Aged and Old Mice [0212]
Whether loss of skeletal muscle mass with age in mice is a result of reduced expression of AUF1 in skeletal muscle was investigated. An AAV8 (adeno-associated virus type 8) vector was developed to deliver and selectively express AUF1 in skeletal muscle. AAV
vectors express AUF1 and GFP (AUF1-GFP, with GFP translated from the same mRNA
by the HCV IRES), or as a control only GFP. Expression of both genes is controlled by the creatine kinase tMCK promoter that is selectively active in skeletal muscle cells 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). Mice ages 3 and 12 months were administered a single retro-orbital injection of either AAV AUF1-GFP or control AAV GFP vectors (2.0 x 1011 genome copies). When analyzed starting at 40 days post-administration of AAV vectors, as shown in 12 month old mice, both GFP and AAV GFP control vector-treated animals displayed similar vector transduction and retention rates, shown by TA muscle GFP staining (FIGs. 1A-1B). cliff] mRNA
expression in skeletal muscle was increased at this time over that of endogenous levels by administration, on average 2.5-fold in EDL, 6-fold in TA, 2.5-fold in gastrocnemius and slightly in soleus muscle which has a high endogenous level, as shown later (FIG. 1C).
AUF1 protein levels in gene transferred animals in skeletal muscle, as shown in the TA
muscle, demonstrated 4-6 fold increased expression over endogenous levels, corresponding to mRNA
levels (FIG. 7D).
There was no evidence for increased expression of AUFI in non-muscle tissues compared to control mice (kidney, lung, spleen, liver) (FIG. 7E), demonstrating strong tissue specificity for skeletal muscle expression of AUF1 by the tMCK promoter. Importantly, Pax7 expression, a key marker for activation of muscle satellite cells and proliferating myoblasts, was also increased 3-4 fold with AAV AUF1-GFP administration (FIG. 7F). Correspondingly, markers of muscle atrophy such as trim63 and fbro32 (Nilwik et al., "The Decline in Skeletal Muscle Mass with Aging is Mainly Attributed to a Reduction in type II muscle Fiber Size," Exp.
Geronlol.
48(5):492-498 (2013), which is hereby incorporated by reference in its entirety), were downregulated 3-fold in the TA muscle of animals administered with AAV AU1F1-GFP (FIG.

- 83 -7G). Collectively, these data indicate that only moderate levels of AUF1 gene transfer into skeletal muscle was sufficient to reduce markers of muscle atrophy coincident with activation of satellite cells and myoblasts.
102131 It was therefore investigated whether AUF1 gene transfer can increase physical endurance in middle aged and older sedentary mice, using a number of well-established criteria.
Twelve month old sedentary mice were administered AAV8 AUF1-GFP or control AAV8 GFP, then tested at 40 days post-administration. AUF1 supplemented mice showed a ¨50%
improvement in grid hanging time (FIG. ID), a measure of limb-girdle skeletal muscle strength and endurance. When tested by treadmill, AAV AUF1-GFP mice displayed 25%
higher maximum speed (FIG. 1E) and 50% increase in work performance (FIG. 1F) compared to AAV
GFP control mice, as well as 25% greater time to exhaustion and 30% increased distance to exhaustion (FIG. 1G, FIG. 1H). When compared to 3 month old mice receiving control AAV
GFP, 12 month old mice gained equivalent physical endurance capacity to the level of young mice (FIGs. 1E-1H). Physical endurance was also tested 6 months post AAV-AUF1 injection of 12 month old mice that were 18 months at the time and kept non-exercised until the time of testing. Maximum speed (FIG. 1I), work performed (FIG. 1J), as well as time and distance to exhaustion (FIG. 1K, FIG. 1L) were all significantly higher in AUF1-AAV
treated animals, similar to 12 month old mice at 40 days post-treatment. These results demonstrate that the enhancement of exercise endurance in older mice with muscle loss and atrophy by supplementation with AUF1 is durable at 6 months post-treatment, with no evidence for diminution. It was therefore next investigated whether the biological and molecular characteristics of AUF1 restored skeletal muscle.
Example 3 ¨ AUF1 Gene Therapy Increases Muscle Mass and Greater Slow-Twitch than Fast-Twitch Myofibers [0214] Skeletal muscles vary in slow- and fast-twitch myofiber composition (Type I or II, respectively). TA, EDL, and gastrocnemius muscles are composed mostly of Type II fast-twitch myofibers (nearly 99% fast, 1% slow), whereas the soleus muscle is highly enriched in Type I slow-twitch myofibers (nearly 40% slow, 60% fast) (Augusto et at., "Skeletal Muscle Fiber Types in C57BL6J mice," J. Alorphot Sci. 21(2):89-94 (2004), which is hereby incorporated by reference in its entirety). Analysis of the gastrocnemius and TA muscles showed that 12 month sedentary old mice gained an average total increase of ¨20% in muscle mass relative to body weight in animals administered AAV AUF1-GFP compared to AAV GFP
controls (FIG. 2A, FIG. 2B). Increased muscle fiber size (myofiber cross-sectional area, CSA)

- 84 -and number are established hallmarks of muscle regeneration (Schiaffino &
Reggiani, "Fiber Types in Mammalian Skeletal Muscles," Physiol. Rev. 91(4):1447-1531 (2011) and Yin et al., "Satellite Cells and the Muscle Stem Cell Niche," Physiol. Rev. 93(1):23-67 (2013), which are hereby incorporated by reference in their entirety). Compared to GFP control mice, as shown in the TA and gastrocnemius muscles of 12 month old AAV AUF1-GFP mice, there was a significant increase in the percentage of larger myofibers, which was particularly pronounced for larger myofibers (>3200 tm2) (FIGs. 2C-2F). Increased myofiber size can be indicative of vigorous and mature muscle regeneration. It was also investigated whether supplemental AUF1 expression promotes slow-twitch, fast-twitch or both types of myofibers. AUF1 supplementation increased the number and size of slow-twitch myofibers per field by nearly 60%
compared to fast-twitch fibers, as shown in the gastrocnemius muscle (FIGs. 2G-2H). In the soleus muscle, which is composed primarily of slow-twitch muscle, the myofiber area was similarly increased with AUF1 supplementation (FIG. 21, FIG. 2J).
[0215] Expression levels of different myosin type mRNAs also support that AUF1 gene transfer resulted in real gain in skeletal muscle mass. The major slow-twitch myosin mRNA, myh7, was increased 6-fold in gastrocnemius and 2-fold in soleus muscle with AUF1 gene transfer (FIG. 3A, FIG. 3B), whereas fast myosin mRNAs such as myh 1 , rnyh2 and myh4 were not statistically changed (FIG. 3C, FIG. 3D).
[0216] Further evidence was obtained for increased muscle generation by AUF1 is supported by measuring the mRNA levels of several genes whose expression are hallmarks of increased myofiber regeneration, oxidative processes and mitochondrial biogenesis. Slow-twitch myofibers in particular are enriched in oxidative mitochondria (Schiaffino &
Reggiani, "Fiber Types in Mammalian Skeletal Muscles," Physiol. Rev. 91(4):1447-1531 (2011), which is hereby incorporated by reference in its entirety). The focus of these studies was on the gastrocnemius muscle because it demonstrated a median response to AUF1 gene therapy and it is not biased toward enrichment of slow-twitch myofibers. While AUF1 gene transfer had no effect on gastrocnemius mRNA levels of non-mitochondrial genes such as ppara (peroxisome proliferator-activated receptor alpha) or sic] (Sineoculis homeobox homolog 1), it increased levels of mitochondrial mRNAs for tfam (mitochondria transcription factor A) by 4-fold, acadvl (acyl-CoA dehydrogenase very long chain) by 6-fold, nrfl by 3-fold and nrf2 by 2-fold (nuclear respiratory factor) (FIGs. 3E-3H). The ratio of mitochondrial to nuclear DNA
was also increased in the gastrocnemius with AUF1 gene transfer, indicative of increased mitochondrial content at both 40 days and 6 months post-gene transfer (FIG. 31, FIG. 3J). Collectively, these results show that AUF1 promotes transition from fast to slow twitch myofiber.

- 85 -Example 4 ¨ AUF1 Stimulates Slow-Twitch Muscle Development in Part by Increasing PGCla Expression [0217] Increased levels slow-twitch Type I muscle fibers are particularly sought for combating muscle loss with age because it is associated with increased muscle endurance. A key feature of slow muscle is that it confers exercise endurance because slow-twitch myofibers have much higher oxidative capacity than fast-twitch fibers (Cartee et al., "Exercise Promotes Healthy Aging of Skeletal Muscle," Cell Metab. 23(6):1034-1047 (2016) and Yoo et al., "Role of Exercise in Age-Related Sarcopenia," I Exerc. Rehabil. 14(4):551-558 (2018), which are hereby incorporated by reference in their entirety). Therefore, the level of AUF1 expression in different muscles with varying proportions of slow- and fast myofibers was characterized. There was a notable 2-4 fold higher level of expression of aufl mRNA and AUF1 protein levels in the soleus muscle of 3 month and sedentary 12 month old untreated mice compared to other muscle types with fewer slow-twitch myofibers (FIG. 4A, FIG. 4B). Accordingly, of the lower limb skeletal muscles, the soleus muscle is the most endurant, the most enriched in slow-twitch myofibers (Schiaffino & Reggiani, "Fiber Types in Mammalian Skeletal Muscles," Physiol.
Rev.
91(4):1447-1531 (2011) and Augusto et al., "Skeletal Muscle Fiber Types in C57BL6J mice," J.
Morphol. Sci. 21(2):89-94 (2004), which are hereby incorporated by reference in their entirety), and expresses much higher levels of myh7 (FIG. 4C), the main slow-twitch myofiber myosin.
Therefore, the role of AUF1 in expression of different levels of myosin mRNAs was assessed by deletion of AUF1 in C2C12 mouse myoblasts. Deletion of AUF1 increased the expression of fast-twitch nlyh2 mRNA levels, while slow myosin mRNAs, such as ugh 7 or ntyI2, were decreased (FIG. 8A), consistent with AUF1 greater specification of slow-twitch myofiber development. Importantly, expression of the myocyte enhancer factor 2 (nief2c) gene, a key transcriptional regulator of overall skeletal muscle development, was also increased by AUF1 supplementation (FIG. 8B). MEF2c can activate or repress different myogenic transcriptional programs and its increased expression is also consistent with increased generation of Type slow-twitch muscle (Lin et al., "Transcriptional Co-Activator PGC-1 Alpha Drives the Formation of Slow-Twitch Muscle Fibres,- Nature 418 (6899):797-801 (2002), which is hereby incorproated by reference in its entirety), suggesting involvement in AUF1-mediated specification of slow-twitch muscle.
[0218] The MEF2c protein stimulates expression of PGCla (Peroxisome proliferator-activated receptor gamma coactivator 1 alpha) which drives the specification and development of slow-twitch myofibers (Lin et al., "Transcriptional Co-Activator PGC-1 Alpha Drives the

- 86 -Formation of Slow-Twitch Muscle Fibres," Nature 418 (6899):797-801 (2002), which is hereby incorproated by reference in its entirety). Deletion of the mill gene in C2C12 myoblasts induced to differentiate to myotubes decreased pgcla mRNA levels by half and protein levels by 4-fold (FIG. 4D), suggesting that AUF1 acts to increase PGC ice protein and mRNA
expression.
Accordingly, AAV8-AUF1 gene transfer in mice showed that pgcla mRNA levels were increased 2-3 fold in the gastrocnemius and EDL muscles, and trended toward upregulation in the TA muscle in 12 month old mice (FIG. 4E). AUF1 gene transfer in 18 month old sedentary mice also strongly increasedpgc/a mRNA levels ¨2.5-fold, as shown in the gastrocnemius muscle (FIG. 4E), which corresponded to an average 5-fold increase in PGCla protein levels (FIG. 4F).
[0219] The pgcice mRNA contains a 3' UTR with multiple ARE
motifs that could be potential AUF1-binding sites (Lai et al., "Effect of Chronic Contractile Activity on mRNA
Stability in Skeletal Muscle," Am. J. Physiol Cell. Physiol. 299(1):C155-163 (2010), which is hereby incorporated by reference in its entirety). Therefore, AUF1 was immunoprecipitated from WT C2C12 myoblasts 48 hours after differentiation when AUF1 is expressed, with control IgG or anti-AUF1 antibodies, followed by qRT-PCR to quantify the levels of bound pgcla mRNA (FIG. 4G). AUF1 bound strongly to the pgcla mRNA in differentiating C2C12 cells.
The effect of AUF1 expression on the pgcla mRNA half-life was then determined using WT and AUF1 KO C2C12 cells by addition of actinomycin D to block new transcription (FIG. 4H).
Surprisingly, in the absence of AUF1, pgcla mRNA displayed an almost 3-fold reduced stability. The pgcla mRNA therefore belongs to the class of ARE-mRNAs that are stabilized rather than destabilized by AUF1, accounting in part for increased levels of PGClcc protein and increased specification of slow-twitch fiber formation by AUF1. Therefore, the impact of AUF1 expression specifically on slow-twitch muscle loss and atrophy was investigated.
Example 5 ¨ Loss of AUF1 Expression Selectively Accelerates Atrophy of Slow-Twitch Muscle in Young Mice [0220] To better understand the role of AUF1 gene therapy in the formation and maintenance of slow-twitch myofibers, slow-twitch myofibers in WT and A UF1 KO
mice were investigated at 3 months of age, before the onset of dystrophy (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 incorproated by reference in its entirety). At 3 months, WT and ctufl KO mice have similar body weights (FIG.
5A). While deletion of aufl did not change the size, color (mitochondrial density, myoglobin

- 87 -content) or weight of the TA, EDL or gastrocnemius muscles, it did reduce the size and weight of the soleus muscle by half at 3 months, which was much paler, indicative of loss of mitochondrial and myoglobin-rich Type I myofibers (FIG. 5B; FIG. 9A). The proportion and number per field of slow myosin myofibers in the AUF1 KO mouse soleus muscle was reduced 40-50% (FIGs. 5C-5E; FIG. 9B). In contrast, both the proportion and number of fast-myosin-expressing myofibers was increased by 25% or more in the absence of AUF 1 expression (FIG.
5C, FIG. 5F, FIG. 5G; FIG. 9B). Reduced expression of slow myosin was also seen in the gastrocnemius muscle with aull deletion in cuff] KO mice (FIGs. 9C-9E). In addition, the mean CSA was reduced by 2-fold in slow-twitch myofibers, as shown in the soleus and gastrocnemius muscles, but was unchanged in fast-twitch myofibers (FIG. 5H; FIG. 9F).
Consistent with these data, AUF1 KO mice at 3 months expressed 3-4 fold lower levels of PGC la protein than WT
mice, as shown in the gastrocnemius and soleus muscles (FIG. 9G). AUF1 therefore specifies regeneration and maintenance of slow-twitch muscle.
Example 6 ¨ Loss of AUF1 in Older Mice Accelerates Atrophy and Loss of both Slow-Twitch and Fast-Twitch Muscle [0221]
At 6 months of age, auf I KO mice show a 20% loss of body weight, which is largely a result of loss of skeletal muscle mass (FIG. 6A). Unlike 3 month old mice where the slow-twitch rich soleus muscle was the only muscle showing significant atrophy in the absence of AUF1 expression, in 6 month old mice both fast-twitch rich and slow-twitch rich muscles demonstrate significant atrophy. The size and weight of the TA, EDL and gastrocnemius muscles were reduced by ¨25% in aufl KO compared to WT animals, and the soleus muscle was reduced by almost 50% (FIG. 6B). In addition, aufl KO mouse skeletal muscles were paler than control WT mice, consistent with greater loss of mitochondri al-dense, slow-twitch myofibers (FIG. 6C). Accordingly, the mean CSA of both slow- and fast-twitch myofibers, as shown in the soleus and gastrocnemius muscles, showed a striking reduction at 6 months in auf/ KO mice compared to WT, indicative of overall myofiber atrophy (FIG. 6D, FIG. 6E). As seen in young mice, AUF1 deficiency reduced by half the percentage and number of slow-twitch myofibers per field in the soleus and gastrocnemius muscles (FIGs. 6F-6I). Thus, while AUF1 specifies development of slow-twitch muscle, its additional activities are essential for maintenance and regeneration of both slow- and fast-twitch muscle, consistent with the ability of AUF1 gene transfer to promote increased overall muscle mass and function in sedentary animals that have undergone muscle loss and atrophy during aging.

- 88 -Discussion of Examples 1 ¨ 6 [0222] This work reports three important sets of findings: (1) AUF1 expression in skeletal muscle is lost with aging in sedentary mice, which contributes to the development of age-related muscle atrophy; (2) AUF1 gene therapy is a promising therapeutic intervention to delay or reverse the loss of muscle mass and strength with age; and (3) AUF1 is required to form both slow and fast myofiber, but also promotes transition from fast to slow muscle phenotype by increasing PGCla levels through stabilization of its mRNA. AUF I generally promotes rapid decay of ARE-containing mRNAs but can stabilize a subset of other 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). During muscle regeneration, AUF1 therefore regulates satellite cell maintenance and differentiation in part by programming each stage of myogenesis through selective degradation of short-lived myogenic checkpoint ARE-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):1379-1390 (2016) and 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), which are hereby incorporated by reference in their entirety). In addition, as shown here, by increasing AUF1 expression levels in sedentary mice using gene transfer, AUF1 increases myosin and oxidative mitochondrial gene expression that promotes slow myofiber formation and oxidative phenotype. There is also evidence for reduced AUF1 expression in human skeletal muscle with aging (Masuda et al., "Tissue- and Age-Dependent Expression of RNA-Binding Proteins that Influence mRNA Turnover and Translation," Aging (Albany NY) 1:681-698 (2009), which is hereby incorporated by reference in its entirety), although the general inability to obtain serial age-related but otherwise normal muscle specimens limits the ability to expand this finding.
[0223] Gene therapy of skeletal muscle with AUF1 by AAV8-AUF1 significantly promoted new muscle mass and exercise endurance in middle aged non-exercised mice that had significant muscle loss and atrophy. Notably, in a rat model designed to characterize skeletal muscle markers of increased physical exercise endurance, two major factors that were found to be increased in expression were AUF1 and PGCla (Lai et al., "Effect of Chronic Contractile Activity on mRNA Stability in Skeletal Muscle," Am. J. Physiol. Cell. Physiol.
299(1):C155-163 (2010), which is hereby incorporated by reference in its entirety). Moreover, an exercise study in mice found that while one week of exercise induced increased levels of PGCla, after four weeks of exercise AUF1 increased as much as 50% without changes in other ARE-binding proteins

- 89 -(Matravadia et al., "Exercise Training Increases the Expression and Nuclear Localization of mRNA Destabilizing Proteins in Skeletal Muscle,- Am. I Physiol. Regul Integr.
Comp. Physiol.
305(7):R822-831 (2013), which is hereby incorporated by refernece by its entirety).
[0224] Interestingly, pgc I a, tfam and nrf2 mRNAs all contain AREs in their 3'UTRs, which may be subject to regulation by ARE-binding proteins, including AUF1 (D' Souza et al., "mRNA Stability as a Function of Striated Muscle Oxidative Capacity," AM. J.
Physiol. Regul.
Integr. Comp. Physiol. 303(4):R408-417 (2012), which is hereby incorporated by reference in its entirety). While perplexing at the time, AUF1 was then only known to cause ARE-mRNA
decay, not stabilization. These findings, when combined with the results disclosed herein, suggests that AUF1 programs a feed-forward mechanism to promote muscle regeneration through stabilization ofpgc/amRNA and, through other AUF1 activities as well (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) and 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), which are hereby incorporated by reference in their entirety).
Consistent with this conclusion, the AUF1 KO mice used herein present at a young age a reduction of slow twitch myofiber size and a decreased level of PGC la expression.
[0225] That AUF1 muscle supplementation increased PGCla protein levels is important.
PGC la activates expression of downstream factors such as NRFs and Tfam that promote mitochondrial biogenesis, which are essential for the formation of slow-twitch muscle fibers, reduced fatigability of muscle and greater oxidative metabolism (Lin et al., "Transcriptional Co-Activator PGC-1 Alpha Drives the Formation of Slow-Twitch Muscle Fibres,"
Nature 418(6899):797-801 (2002), which is hereby incorproated by reference in its entirety). These findings, along with enhanced mitochondrial DNA content observed with AUF1 supplementation, suggest that AUF1 is responsible for key activities in slow-twitch myofiber maintenance and increased exercise endurance in mice. Previous studies have shown the benefit of increased PGCla expression in muscle damage repair and angiogenesis (Wiggs, M. P., "Can Endurance Exercise Preconditioning Prevention Disuse Muscle Atrophy?,- Front.
Physiol. 6:63 (2015); Wing et al., "Proteolysis in Illness-Associated Skeletal Muscle Atrophy: From Pathways to Networks," Crit. Rev. Clin. Lab. Sci. 48(2):49-70 (2011); Bost & Kaminski, "The Metabolic Modulator PGC-lalpha in Cancer," Am. J. Cancer Res. 9(2):198-211 (2019), Dos Santos et al., "The Effect of Exercise on Skeletal Muscle Glucose Uptake in type 2 Diabetes:
An Epigenetic Perspective," Metabolism 64(12):1619-1628 (2015); Haralampieva et al., "Human Muscle

- 90 -Precursor Cells Overexpressing PGC-lalpha Enhance Early Skeletal Muscle Tissue Formation,"
Cell Transplant 26(6):1103-1114 (2017); and Janice Sanchez et al., "Depletion of HuR in Murine Skeletal Muscle Enhances Exercise Endurance and Prevents Cancer-Induced Muscle Atrophy," Nat. Commun. 10(1):4171 (2019), which are hereby incorporated by reference in their entriety).
[0226] AUF1 skeletal muscle gene transfer is therefore beneficial in countering muscle loss and atrophy because it is required to enable multiple key steps in myogenesis. AUF1 stimulates greater muscle development and physical exercise capacity in aging sedentary muscle, which in turn likely further stimulates AUF1 expression as a result of exercise itself Moreover, the effects of AUF1 gene transfer appear to be long-lasting. Improved exercise endurance in the studies disclosed herein was found to be sustained for at least 6 months beyond the time of gene transfer (the last time point tested) with no evidence for reduction in AUF1 expression or efficacy. In this regard, AUF1 supplementation also increased levels of Pax7+
activated satellite cells and myoblasts, suggesting gene transfer into muscle stem cells and an active myogenesis process.
[0227] Apart from AUF1, other ARE RNA-binding proteins have also been shown to be involved in the myogenesis process. Of particular relevance to the studies disclosed herein, HuR
was recently found to destabilize pgela mRNA, leading to the formation of type II myofibers (Janice Sanchez et al., "Depletion of HuR in Murine Skeletal Muscle Enhances Exercise Endurance and Prevents Cancer-Induced Muscle Atrophy," Nat. Commun. 10(1):4171 (2019), which is hereby incorporated by reference in its entriety). It is noteworthy that AUF1 and HuR
often have opposite effects on ARE-mRNA stability, in accord with the findings disclosed herein, and both are essential for the maintenance of myofiber specification.
AUF1 can also interact with HuR although the potential functional consequence is unknown, and AUF1 can also compete for binding to AREs with TIA-1, which blocks AUF1-mediated mRNA decay ARE-mRNA translation (Pullmann et al., "Analysis of Turnover and Translation Regulatory RNA-Binding Protein Expression Through Binding to Cognate mRNAs,"Mol. Cell Biol.
27(18):6265-6278 (2007), which is hereby incorporated by reference in its entirety).
Clearly, the role of ARE-binding proteins in myogenesis is complex and further investigation into their combined activities is needed to better understand this complexity. How muscle homeostasis is regulated by AUF1 with the other ARE-binding proteins remains to be discovered.
[0228] Finally, it is important to note that while AUF1 specifies Type I slow-twitch myofiber development, it also promotes and reprograms the overall myogenesis regeneration program (Abbadi et al., "Muscle Development and Regeneration Controlled by AUF1-Mediated

-91 -Stage-Specific Degradation of Fate-Determining Checkpoint mRNAs," Proc. Natl.
Acad. Sci.
USA 116(23):11285-11290 (2019), which is hereby incorporated by reference in its entirety), evidenced by the fact that AUF1 skeletal muscle gene transfer did not result in abnormal muscle development, abnormal balance of muscle fiber types or muscle overgrowth.
Example 7 ¨ AUF1 Restores Skeletal Muscle Mass and Function in Duchenne Muscular Dystrophy (DMD) Mice [0229] To examine the effect of AUF1 gene therapy on skeletal muscle mass and function in a mouse model of Duchenne muscular dystrophy (DMD), the cDNA for full-length p45 AUF1 isoform, which carries out all AUF1 functions, was cloned into an AAV8 vector under the control of the tMCK promoter (AAV8-tMCK-AUF1-IRES-eGFP) (Vector Biolabs), with the AAV8-tMCK-1RES-eGFP 2RS5 enhancer sequences (3-Ebox) ligated to the truncated regulation region of the MCK (muscle creatine kinase) promoter, which provides high skeletal muscle specificity.
[0230] The transduction frequency of AAV8 AUF1-GFP and AAV8 GFP
control vectors was evaluated in mdx mice by tibialis and muscle GFP staining (FIG. 11A). No statistical differences in transduction efficiency was observed between control AAV8 GFP
and treatment AAV8 AUF1 GFP groups (FIG. 11B).
[0231] To determine whether AUF1 supplementation enhances muscle mass and/or endurance in indx mice, one month old C57B110 and mdx mice were administered GFP or control AAV8-GFP vectors at 2x1011 genome copies by retro-orbital injection (FIGs.
12A-12F and FIGs. 13A-13D). Mice were weighted and monitored for 2 months.

AUF1-GFP supplemented mdx mice had a significant increase in average body weight, as compared to control mdx mice (FIG. 12A). Moreover, AAV8 AUF1-GFP treated mdx mice demonstrated a 10% increase in tibialis anterior (TA) muscle mass and an 11%
increase in extensor digitorum longus (EDL) muscle mass (FIG. 12B), as compared to control treated mcbc mice. Compared to control AAV8 GFP treated mdx mice, AUF1 supplemented mdx mice showed a ¨40% improvement in grid hanging time, a measure of limb-girdle skeletal muscle strength and endurance (FIG. 12C). When tested by treadmill, AAV AUF1-GFP mdx mice displayed 16% higher maximum speed (FIG. 12D), a 35% greater time to exhaustion (FIG.
12E), and a 37% increased distance to exhaustion (FIG. 12F). These data demonstrate a substantial and statistically significant increase in exercise performance and endurance in mdx mice as a result of AUF1 gene transfer. In contrast to the mdx mice, there was no significant increase in body weight (FIG. 13A), treadmill time to exhaustion (FIG. 13B), maximum speed

- 92 -(FIG. 13C), or distance to exhaustion (FIG. 13D) in AAV8 AUF1-GFP treated WT
mice as compared to control AAV8 GFP treated mice of the same genetic background.
[0232] AUF1 overexpression in mdx mice also ameliorated the diaphragm dystrophic phenotype (FIGs. 15A-15B). The percent degenerative diaphragm muscle was reduced by 74% in AAV8 AUF1-GFP treated mdx mice as compared to control AAV8 GFP treated mdx mice (FIG. 15A). AUF1 gene transfer also significantly reduced diaphragm fibrosis (FIG. 15B) and macrophage infiltration (FIGs. 16A-16B) in AAV8 AUF1-GFP treated mdx mice, as compared to control AAV8 GFP treated mdx mice.
[0233] Histological signs of muscular dystrophy, including myofiber centro-nucleation and embryonic myosin heavy chain (eMHC) expression were tested. The percent of centro-nuclei and eMHC positive fibers found increased in mdx mice were highly downregulated upon AUF1 supplementation (FIGs 17A-17D). The size of centro-nuclei myofibers was also increased upon AUF1 supplementation (FIG. 17E).
[0234] Serological level of creatine kinase (CK) activity, a measure of sarcolemma leakiness used to aid diagnosis of DMD is found increased in control mdx mice, however CK
activity was highly decreased upon AUF1 supplementation in mdx mice (FIG. 14).
[0235] Utrophin expression was also assessed in vitro and in vivo. In vitro, only WT
C2C12 myoblasts differentiated into myotubes present an increase of utrophin mRNA and protein. AUF1 gene therapy strongly increased expression of utrophin and showed evidence for normalization of myofiber integrity in mdx mice, relative to control mdx mice receiving vector alone (FIGs. 18A-18C). AAV8 AUF1 gene transfer increased expression of satellite cell activation gene l'ax7 (FIG. 19A), key muscle regeneration genes pgc 1 a and mef2c (FIG.
19A), slow twitch determination genes (FIG. 19B), and mitochondrial DNA
content (FIG.
19C) in mdx mice, relative to control mdx mice receiving vector alone.
[0236] Genome-wide transcriptomic and translatomic studies were carried out to evaluate whether AUF1 activation of C2C12 activates myoblast muscle fiber development (FIG. 20).
These studies demonstrate that AUF1 supplementation (i) stimulates expression of major muscle development pathways and decreases expression of inflammatory cytokine, inflammation, cell proliferation, cell death, and anti-muscle regeneration pathways (FIGs. 21A-21B); (ii) upregulates pathways for major biological processes and molecular functions in muscle development and regeneration (FIGs. 22A-22B); (iii) decreases muscle inflammation, inflammatory cytokine, and signaling pathways that oppose muscle regeneration (FIGs. 23A-23B); and (iv) decreases expression of muscle genes associated with development of fibrosis (FIG. 24).

- 93 -Discussion of Example 7 Dystrophin Gene Therapy [0237] As described above, DMD is caused by mutations in the dystrophin gene, resulting in a near-absence of expression of the protein, which plays a key role in stabilization of muscle cell membranes (Bonilla et al., "Duchenne Muscular Dystrophy:
Deficiency of Dystrophin at the Muscle Cell Surface," Cell 54(4):447-452 (1988) and Hoffman et al., "Dystrophin: The Protein Product of the Duchenne Muscular Dystrophy Locus,"
Cell 51(6):919-928 (1987), which is hereby incorporated by reference in its entirety). Since the dystrophin gene is very large, it is impossible to reintroduce the entire gene by gene therapy. Thus, current gene therapy attempts involve introducing by gene transfer "mini" and "micro"
dystrophin genes, i.e., small pieces of the dystrophin gene packaged in AAV vectors. To date, none have been shown to be very effective and there is evidence that because mini and micro dystrophin genes are different than an individual's dystrophin gene, they evoke an immune response against the therapeutic gene. Since dystrophin is mutated in DMD, there is currently intense interest in finding ways to increase expression of the dystrophin homolog known as utrophin that has overlapping function. To date, this has not been achieved at therapeutic levels that can be shown to be effective.
DMD mdx Mouse Model [0238] The most widely used DMD niclic mouse (C57BL/10 background) has a spontaneous genetic mutation resulting in a nonsense mutation (premature stop codon) in exon 23 of the very large dystrophin mRNA, similar to the occurrence in roughly 13%
of DMD males (Bulfield et al., "X Chromosome-Linked Muscular Dystrophy (mdx) in the Mouse,"
Proc. Natl.
Acad. Sci. USA 81(4):1189-1192 (1984), which is hereby incorporated by refernce in its entirety). This mdx mouse model has been used extensively for DMD
investigations and therapeutics research, and is considered the "gold standard" animal model for study of DMD.
The C57BL/10 mdx mice are as susceptible to physical muscle damage as are humans and reflects human disease in certain tissues (diaphragm, cardiac muscles), although they are less susceptible to damage in skeletal muscle (Moens et al., "Increased Susceptibility of EDL
Muscles from mdx Mice to Damage Induced by Contractions with Stretch," J.
Muscle Res. Cell.
Moth. 14(4):446-451 (1993), which is hereby incorporated by refernce in its entirety). As in humans, the disease progresses in skeletal muscle with age in mdx mice (Moens et al., "Increased Susceptibility of EDL Muscles from mdx Mice to Damage Induced by Contractions with Stretch," J. Muscle Res. Cell. Moth. 14(4):446-451 (1993), which is hereby incorporated by reference in its entirety). Equally important, the diaphragm as a target for myo-pathogenesis in

- 94 -inch mice has been shown to very precisely reproduce the level and rate of damage seen in humans and is an excellent readout for effectiveness of therapeutic intervention (Stedman et al., "The mdx Mouse Diaphragm Reproduces the Degenerative Changes of Duchenne Muscular Dystrophy," Nature 352(6335):536-539 (1991), which is hereby incorporated by reference in its entirety), and will be studied here.
[0239] Importantly, both mdx mice and DMD patients deplete their satellite cells after cycles of necrosis and regeneration of myofibers which promotes disease progression (Manning & O'Malley, "What has the mdx Mouse Model of Duchenne Muscular Dystrophy Contributed to our Understanding of this Disease?" J. Muscle Res. Cell Motif. 36(2):155-167 (2015) and Coley et al., "Effect of Genetic Background on the Dystrophic Phenotype in mdx Mice," Hum. Mol.
Genet. 25(1).130-145 (2016), which are hereby incorporated by reference in their entirety).
Moreover, mdx mice and DMD patients both develop an inflammatory response that increases with disease progression (Manning & O'Malley, "What has the mdx Mouse Model of Duchenne Muscular Dystrophy Contributed to our Understanding of this Disease?" J.
Muscle Res. Cell Moth. 36(2):155-167 (2015) and Coley et al., "Effect of Genetic Background on the Dystrophic Phenotype in mdx Mice,- Hum. Mol. Genet. 25(1):130-145 (2016), which are hereby incorporated by reference in their entirety).
[0240] Despite the fact that skeletal muscle dystrophic disease is generally milder in the mcbc mouse than in humans, it still provides a predictive model for pharmacologic response, particularly when coupled with progression of disease in diaphragm. Thus, the mcbc mouse provides a reliable, well-established and predictive model in which to follow disease progression and treatment response in animals that has been proven to be useful in development of strategies for interventional agents for DMD clinical trial (Fairclough et al., "Davies, Pharmacologically Targeting the Primary Defect and Downstream Pathology in Duchenne Muscular Dystrophy,"
Cum Gene Ther. 12(3):206-244 (2012) and Stedman et al., "The mdx Mouse Diaphragm Reproduces the Degenerative Changes of Duchenne Muscular Dystrophy," Nature 352(6335):536-539 (1991), which are hereby incorporated by reference in their entirety).
Moreover, studies have also shown that allowing marx mice to participate in voluntary exercise (wheel running, treadmill) increases skeletal muscle disease due to the introduction of micro-tears from physical stress, similar to human (Smythe et al., "Voluntary Wheel Running in Dystrophin-Deficient (mdx) Mice: Relationships Between Exercise Parameters and Exacerbation of the Dystrophic Phenotype," PLoS Curr. 3:RRN1295 (2011); Nakae et al., "Quantitative Evaluation of the Beneficial Effects in the mdx Mouse of Epigallocatechin Gallate, an Antioxidant Polyphenol from Green Tea," Histochem. Cell Biol. 137(6):811-27 (2012); and

- 95 -Archer et at., "Persistent and Improved Functional Gain in mdx Dystrophic Mice after Treatment with L-Arginine and Deflazacort,- FASEB I 20(6):738-740 (2006), which are hereby incorporated by reference in their entirety). Thus, there are readily available methods for producing a representative human skeletal muscle form of disease in mdx mice that constitute a model for therapeutic assessment and clinical development.
AUF1 Gene Therapy [0241] The results of Example 7 demonstrate that muscle cell-specific AUF1 gene therapy restores skeletal muscle mass and function in a mouse model of Duchenne muscular dystrophy. In particular, evaluation of muscle cell-specific gene therapy in the DMD mdx mdoel provided evidenced that AAV8 vectored AUF1 gene therapy. (1) efficiently transduced skeletal muscle including cardiac diaphragm and to provide long-duration AUF1 expression without evidence of loss of expression over 6 months (the longest time point tested);
(2) activated high levels of satellite cells and myoblasts; (3) significantly increased skeletal muscle mass and normal muscle fiber formation; (4) significantly enhanced exercise endurance;
(5) strongly reduced biomarkers or muscle atrophy and muscle cell death in DMD mice; (6) strongly reduced inflammatory immune cell invasion in skeletal muscle including diaphragm; (7) strongly reduced muscle fibrosis and necrosis in skeletal muscle including diaphragm; (8) strongly increased expression of endogenous utrophin in DMD muscle cells while suppressing expression of embryonic dystrophin, a marker of muscle degeneration in DMD; (9) increased normal expression of a large group of genes all of which are involved in muscle development and regeneration, and to suppress genes involved in muscle cell fibrosis, death and muscle-expressed inflammatory cytokines; and (10) did not increase muscle mass, endurance or activate satellite cells in normal skeletal muscle. No aberrant effects of AUF1 skeletal muscle specific gene therapy were observed.
Example 8 ¨ AUF1 Gene Therapy Accelerates Skeletal Muscle Regeneration In Muscle-Injured Mice [0242] A mouse model of BaC12 induced necrosis (Garry et al., "Cardiotoxin Induced Injury and Skeletal Muscle Regeneration," Methods !Vol. Biol. 1460:61-71 (2016) and Tierney et al., "Inducing and Evaluating Skeletal Muscle Injury by Notexin and Barium Chloride," Methods Mol. Biol. 1460:53-60 (2016), which are hereby incorporated by reference in their entirety) was used to examine whether AUF I gene therapy accelerates skeletal muscle regeneration.
[0243] In this study, three month old male mice were administered an intramuscular injection of 50 IA of filtered 1.2% BaC12 in sterile saline with control lentivirus vector or with

- 96 -lentivirus p45 AUF1 vector (Abbadi et al., "Muscle Development and Regeneration Controlled by AUF1-mediated Stage-specific Degradation of Fate-determining Checkpoint mRNAs,- Proc.
Nat'l. Acad. Sci. USA 116:11285-90 (2019), which is hereby incorporated by reference in its entirety) into the left tibialis anterior (TA) muscle. The right TA muscle remained uninjured as a control.
[0244] Muscle atrophy was determined by weight of excised TA
muscle. In mice sacrificed at 7 days post-injection, TA injury reduced TA weight by 27% which was restored to near-uninjured levels by concurrent AUF1 gene therapy (FIG. 25A). p45 AUF I
gene transfer increased AUF1 expression by several fold in lentivirus transduced muscle (FIG. 25B), which was associated with reduced expression of TRIM63 and Fbxo32, two established biomarkers of muscle atrophy, that were strongly increased following muscle injury but reduced to near non-injured levels with AUF1 gene transfer (FIG. 25D). Strong muscle regeneration correlated with strong activation of the PAX7, gene consistent with satellite cell activation in the TA muscle (FIG. 25C). p45 AUF1 gene transfer also significantly enhanced expression of muscle regeneration factors (MRFs) such as MyoD and myogenin (FIG. 26A), myh8 (FIG.
26B), myh7 (FIG. 26C), and myh4 (FIG. 26D).
[0245] Images of muscle fibers provide further evidence for accelerated but normal muscle regeneration of myofibers in animals administered lentiviral AUF1 that was not seen in control vector mice. A disrupted myofiber architecture and high level of central nuclei in the vector alone TA muscle was observed compared to lenti-AUF1 supplementation (FIG. 27A).
Likewise, injured TA muscle receiving sham gene therapy sustained a 20% loss in mass by day 3 following injury, which only very slightly improved by day 7 (FIG. 27B). In contrast, injured TA muscle receiving AUF1 gene therapy showed a trend to less atrophy by day 3, which was almost fully recovered by day 7, demonstrating near normal mass (FIG. 27B).
Accelerated muscle regeneration produced mature myofibers, as shown by the striking increase in CSA and reduced central nuclei per myofiber (FIGs. 27C-27D).
[0246] Finally, using an inducible AUF1 conditional knockout mouse (FIGs. 28A-28D) developed as party of the technology described herein, selective AUF1 deletion only in skeletal muscle demonstrated the essential requirement for AUF1 expression to promote regeneration of muscle following traumatic injury (FIG. 28E), and the ability to protect muscle from extensive injury when delivered as AAV8 AUF1 gene therapy (FIG. 28E). In particular, TA
muscle from mice injured by 1.2% BaC12 injection were evaluated for muscle atrophy at 7 days injection. TA
muscle of AUF1Flox/Flox x PAX7c"ERT2 mice expressing AUF1 and WT mice expressing AUF1 (not induced for cre) showed 16-18% atrophy that was not statistically different (FIG. 28E). In

- 97 -contrast, deletion of the AUF1 gene caused strongly increased atrophy of the TA muscle, doubling atrophy levels to 35% (FIG. 28E). However, animals deleted for the AUFI gene but prophylactically administered AAV8 AUFI gene therapy demonstrated dramatically reduced levels of TA muscle atrophy, averaging ¨3% (FIG. 28E). AUF1 deleted mice were tested at 5 months for grip strength, a measure of limb-girdle skeletal muscle strength and endurance.
AUF I deleted mice showed a ¨50% reduction in grip strength (FIG. 28F).
[0247]
Collectively, these data demonstrate that AUF1 is essential for maintenance of muscle strength and muscle regeneration following injury, and that AUF1 gene therapy provides a remarkable ability to promote muscle regeneration and protect muscle from extensive damage despite traumatic injury.
Discussion of Example 8 [0248]
Large, severe, or traumatic muscle injuries can result in volumetric muscle loss (VML) in which the conventional muscle repair mechanisms of the body that innately repair and regenerate muscle are overwhelmed, resulting in permanent muscle injury, poor ability to repair muscle, muscle loss, and functional impairment (Grogan et al., "Volumetric Muscle Loss,"
J. Am. Acad. Orthop. Surg. 19(Suppl 1):S35-7 (2011); Sicherer et al., "Recent Trends in Injury Models to Study Skeletal Muscle Regeneration and Repair," Bioengineering (Basel) 7 (2020);
Qazi et al., "Cell Therapy to Improve Regeneration of Skeletal Muscle Injuries," J. Cachexia Sarcopenia Muscle 10:501-16 (2019); and Garg et al., "Volumetric Muscle Loss:
Persistent Functional Deficits Beyond Frank Loss of Tissue," J. Or/hop. Res. 33:40-6 (2015), which are hereby incorporated by reference in their entirety). Traumatic skeletal muscle injuries are the most common injuries whether in military service, sports or just accidents in everyday life (Copland et al., "Evidence-Based Treatment of Hamstring Tears," Curr. Sports /vied. Rep. 8:308-14 (2009), which is hereby incorporated by reference in its entirety).
Traumatic injuries typically result in muscle necrosis and chronic inflammation, and if they proceed to ViVIL, they can irreparably deplete muscle by 20% or more, which is replaced by fibrotic scar tissue and sets in and persistently long-term disability (Copland et al., "Evidence-Based Treatment of Hamstring Tears," Curr. Sports Med. Rep. 8:308-14 (2009) and Jarvinen et al., "Muscle Injuries: Biology and Treatment,- Am. J. Sports Med. 33:745-64 (2005), which are hereby incorporated by reference in their entirety). In fact, open bone fractures resulting from accidents or military injuries, of which there are more than 150,000 a year in the civilian population alone in the United States, are responsible for the majority (65%) of severe and poorly healing muscle injuries, in many cases resulting in permanent functional disabilities in as much as 8% of the

- 98 -population (Owens et at., "Characterization of Extremity Wounds in Operation Iraqi Freedom and Operation Enduring Freedom,- J. Orthop. Trauma 21:254-7 (2007); Corona et al., "Volumetric Muscle Loss Leads to Permanent Disability Following Extremity Trauma,"
RehabiL Res. Dev. 52:785-92 (2015); and Court-Brown et al., "The Epidemiology of Tibial Fractures," J. Bone ,Joint Surg. Br. 77:417-21 (1995), which are hereby incorporated by reference in their entirety).
[0249] With skeletal muscle injury, normally quiescent muscle satellite cells are released from their niche in the basal lamina, become activated and begin proliferating (Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function,"
Development 142:1572-81 (2015), which is hereby incorporated by reference in its entirety). Typically, activation of quiescent satellite cells results from micro-damage to muscle fibers (Murphy et al., "Satellite Cells, Connective Tissue Fibroblasts and their Interactions are Crucial for Muscle Regeneration," Development 138:3625-37 (2011); Carlson et al., "Loss of Stem Cell Regenerative Capacity within Aged Niches," Aging Cell 6:371-82 (2007); Collins et al., "Stem Cell Function, Self-Renewal, and Behavioral Heterogeneity of Cells from the Adult Muscle Satellite Cell Niche,- Cell 122:289-301 (2005); Gopinath et al., "Stem Cell Review Series:
Aging of the Skeletal Muscle Stem Cell Niche," Aging Cell 7:590-8 (2008);
Seale et al., "A New Look at the Origin, Function, and "Stem-Cell" Status of Muscle Satellite Cells," Dev Biol 218:115-24 (2000); and Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function,- Development 142:1572-81(2015), which are hereby incorporated by reference in their entirety) but with extensive damage there is chronic release and activation of satellite cells which can become functionally exhausted and even depleted in such circumstances.
[0250] Satellite cells are a small population of muscle cells comprising ¨2-4% of adult skeletal muscle cells. Only a small number of satellite cells self-renew and return to quiescence, while the rest differentiate into muscle progenitor cells called myoblasts.
Myoblasts undergo myogenesis (muscle development), a program that includes fusing with existing damaged muscle fibers (myofibers), thereby repairing and regenerating new muscle (Gunther et al., "Myf5-Positive Satellite Cells Contribute to Pax7-Dependent Long-Term Maintenance of Adult Muscle Stem Cells," Cell Stem Cell 13:590-601 (2013), which is hereby incorporated by reference in its entirety). However, traumatic muscle injury can easily exceed the ability of the myogenesis program to repair injured muscle fibers.
[0251] The newly generated myofibers fall into one of two categories: slow-twitch (Type I) or fast-twitch (Type II) fibers, defined according to their speed of movement, type of metabolism, and myosin gene expression. Type II myofibers are the first to atrophy in response

- 99 -to traumatic damage, whereas slow-twitch myofibers are more resilient (Arany, Z. "PGC-1 Coactivators and Skeletal Muscle Adaptations in Health and Disease," Curr.
Op/n. Genet. Dev.
18:426-34 (2008) and Wang et al., "Mechanisms for Fiber-Type Specificity of Skeletal Muscle Atrophy," Curr. Op/n. Chit. Nutr. _IVIetab. Care 16:243-50 (2013), which are hereby incorporated by reference in their entirety). The ability to stimulate skeletal muscle regeneration in general, and to selectively promote more resilient slow-twitch muscle in particular, has been a long-standing goal of regenerative muscle biology and clinical practice, as it could potentially be an effective therapy for traumatic muscle injury and various forms of muscular dystrophies (Ljubicic et al., "The Therapeutic Potential of Skeletal Muscle Plasticity in Duchenne Muscular Dystrophy: Phenotypic Modifiers as Pharmacologic Targets," FASEB J. 28:548-68 (2014), which is hereby incorporated by reference in its entirety). As satellite cells age, or with traumatic muscle injuries that result in chronic cycles of necro-regeneration, satellite cells lose their regenerative capacity and are difficult to reactivate (Bernet et al., "p38 MAPK Signaling Underlies a Cell-Autonomous Loss of Stem Cell Self-Renewal in Skeletal Muscle of Aged Mice," Nat. Med. 20:265-71 (2014); Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function," Development 142:1572-81 (2015);
Kudryashova et al., "Satellite Cell Senescence Underlies Myopathy in a Mouse Model of Limb-Girdle Muscular Dystrophy 2H," J. Cl/n. Invest. 122:1764-76 (2012); and Silva et al., "Inhibition of Stat3 Activation Suppresses Caspase-3 and the Ubiquitin-Proteasome System, Leading to Preservation of Muscle Mass in Cancer Cachexia," J. Biol. Chem. 290: 1177-87 (2015), which are hereby incorporated by reference in their entirety).
[0252] The cycles of muscle degeneration and regeneration in large or traumatic injuries can lead to functional exhaustion and even loss of muscle stem cells that are essential for muscle regeneration and repair (Carlson et al., -Loss of Stem Cell Regenerative Capacity within Aged Niches," Aging Cell 6:371-82 (2007); Shefer et al., "Satellite-Cell Pool Size does Matter:
Defining the Myogenic Potency of Aging Skeletal Muscle," Dev. Biol. 294:50-66 (2006); Bernet et al., "p38 MAPK Signaling Underlies a Cell-Autonomous Loss of Stem Cell Self-Renewal in Skeletal Muscle of Aged Mice," Nat. Med. 20:265-71 (2014); and Dumont et al., "Intrinsic and Extrinsic Mechanisms Regulating Satellite Cell Function," Development 142:1572-81 (2015), which are hereby incorporated by reference in their entirety), resulting in severe loss of muscle regenerative capacity, permanent muscle loss and chronic disability (Brack, A.
S., "Pax7 is Back," Skelet Muscle 4:24 (2014), which is hereby incorporated by reference in its entirety).
Consequently, there are few therapeutic options to increase de novo muscle regeneration, mass and strength available for individuals with severe skeletal muscle injuries, and little evidence

- 100 -that any approaches are very particularly effective (Corona et al., "Pathophysiology of Volumetric Muscle Loss Injury,- Cells Tissues Organs 202:180-88 (2016), which is hereby incorporated by reference in its entirety).
[0253] Physical rehabilitation approaches have not been found to be effective in increasing existing muscle mass, muscle regeneration or strength in individuals who have VML
injuries (Garg et al., "Volumetric Muscle Loss: Persistent Functional Deficits Beyond Frank Loss of Tissue,- J. Orthop. Res. 33:40-6 (2015) and Mase et al., "Clinical Application of an Acellular Biologic Scaffold for Surgical Repair of a Large, Traumatic Quadriceps Femoris Muscle Defect," Orthopedics 33:511(2010), which are hereby incorporated by reference in their entirety). Muscle regeneration approaches that are focused on attenuating the underlying inflammatory response resulting from injury fail to promote effective regeneration of new muscle mass or strength (Corona et al., "Pathophysiology of Volumetric Muscle Loss Injury,"
Cells Tissues Organs 202:180-88 (2016) and Qazi et al., "Cell Therapy to Improve Regeneration of Skeletal Muscle Injuries," J. Cachexia Sarcopenia Muscle 10:501-16 (2019), which are hereby incorporated by reference in their entirety).
[0254] Surgical treatments for individuals with chronic muscle injury are also not very effective and have significant limitations. Surgical intervention normally involves surgical reconstruction of injured muscle using autologous muscle transplant and engraftment from healthy muscle elsewhere in the body, which has a high rate of graft degeneration and failure, re-injury, and itself can cause traumatic injury of the resident healthy donor muscle and loss of function (Whiteside, L. A., "Surgical Technique: Gluteus Maximus and Tensor Fascia Lata Transfer for Primary Deficiency of the Abductors of the Hip," Cl/n. Orthop.
Re/at. Res. 472:645-53 (2014); Dziki et at, "An Acellular Biologic Scaffold Treatment for Volumetric Muscle Loss:
Results of a 13-Patient Cohort Study," A'PJ Regen. Med. 1:16008 (2016); Sicari et al., "An Acellular Biologic Scaffold Promotes Skeletal Muscle Formation in Mice and Humans with Volumetric Muscle Loss," Sci. Transl. Med. 6:234ra58 (2014); Hurtgen et al., "Autologous Minced Muscle Grafts Improve Endogenous Fracture Healing and Muscle Strength after Musculoskeletal Trauma," Physiol. Rep. 5 (2017); and Qazi et al., "Cell Therapy to Improve Regeneration of Skeletal Muscle Injuries," J. Cachexia Sarcopenia Muscle 10:501-16 (2019), which are hereby incorporated by reference in its entirety). Other surgical approaches that use experimental scaffolds and muscle organoids to promote increased muscle regeneration are technically complex and have also not shown consistent efficacy in model systems (Gholobova et al., "Vascularization of Tissue-Engineered Skeletal Muscle Constructs,"
Biomaterials 235:119708 (2020) and Sicherer et al., "Recent Trends in Injury Models to Study Skeletal

-101 -Muscle Regeneration and Repair," Bioengineering (Basel) 7 (2020), which are hereby incorporated by reference in their entirety).
[0255] Molecular approaches to treat skeletal traumatic injuries generally consist of growth factor therapies, including intramuscular administration or release from implanted biomaterials of hepatocyte growth factor (HGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) among others (Syverud et al., "Growth Factors for Skeletal Muscle Tissue Engineering,- Cells Tissues Organs 202:169-79 (2016); Pawlikowski et al., "Regulation of Skeletal Muscle Stem Cells by Fibroblast Growth Factors," Dev. Dyn. 246:359-67 (2017); Menetrey et al., "Growth Factors Improve Muscle Healing in vivo," J. Bone Joint Sttrg. Br. 82:131-7 (2000); Rodgers et al., "mTORC1 Controls the Adaptive Transition of Quiescent Stem Cells from GO to G(Alert)," Nature 510.393-6 (2014); Allen et al., "Hepatocyte Growth Factor Activates Quiescent Skeletal Muscle Satellite Cells in vitro," J. Cell Physiol. 165:307-12 (1995); Miller et al., "Hepatocyte Growth Factor Affects Satellite Cell Activation and Differentiation in Regenerating Skeletal Muscle," Am. J.
Physiol. Cell Physiol. 278:C174-81 (2000); Grasman et al., "Bi omim eti c Scaffolds for Regeneration of Volumetric Muscle Loss in Skeletal Muscle Injuries," Acta Biomater. 25:2-15 (2015); and Cezar et al., "Timed Delivery of Therapy Enhances Functional Muscle Regeneration," Adv. Healthc. Mater. 6 (2017), which are hereby incorporated by reference in their entirety). These approaches suffer from the limitation of administration of a single muscle growth promoting factor, and that these factors are short-lived, whereas muscle regeneration is complex and requires many factors that must act in concert with each other in a precise spatial and temporal manner over time to effect muscle repair and regeneration. It is therefore not surprising that administration of growth factors, even in combinations, have not shown significant muscle regenerative effects even in experimental models of traumatic muscle injury (Pumberger et al., "Synthetic Niche to Modulate Regenerative Potential of MSCs and Enhance Skeletal Muscle Regeneration," Biomaterials 99:95-108 (2016), which is hereby incorporated by reference in its entirety).
[0256] Most cellular therapies attempt to repopulate muscle regenerative stem (satellite) cells, and reduce necro-inflammation by using transplanted muscle satellite cells or other cells of myogenic origin. However, there are significant impediments to this approach.
First, the cells employed must be freshly isolated allogeneic, which means harvesting them from existing surgically removed healthy muscle, in the case of individuals with traumatic and VML injuries.
Second, the stem and myogenic cells need to be cultured and expanded, which is technically difficult and not scalable given the magnitude of unmet need. Thus, autologous muscle cell

- 102 -therapies are not clinically feasible for treatment of the majority of patients in need (Qazi et al., "Cell Therapy to Improve Regeneration of Skeletal Muscle Injuries,- I Cacheria Sareopenia Muscle 10:501-16 (2019), which is hereby incorporated by reference in its entirety).
[0257] The therapeutic options currently available for the treatment of large and/or traumatic muscle injury (e.g., cell therapies, surgical therapies, growth factor and hormonal therapies, molecular therapies, and gene therapies) aim to increase muscle regeneration, muscle mass, and muscle strength for severe skeletal muscle injuries. However, most of the available treatment options work only very poorly, if at all. The results of Example 8 demonstrate that AUF1 gene therapy (e.g., by lentivirus vector delivery directly to muscle or systemic delivery of AUF1 by AAV8 vector) is effective to: (1) activate muscle stem (satellite) cells; (2) reduce expression of established biomarkers of muscle atrophy; (3) accelerated the regeneration of mature muscle fibers (myofibers); (4) enhanced expression of muscle regeneration factors; (5) strongly accelerate the regeneration of injured muscle; (6) increase regeneration of both major types of muscle (i.e., slow-twitch (Type I) or fast-twitch (Type II) fibers);
and restore muscle mass, muscle strength, and create normal muscle.
[0258] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims (47)

WHAT IS CLAIMED IS:

1. 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, wherein the nucleic acid molecule is heterologous to and operatively coupled to the muscle cell-specific promoter.

2. The adeno-associated viral (AAV) vector according to claim 1, wherein the adeno-associated viral (AAV) vector is an adeno-associated virus type 8 vector.

3. The adeno-associated viral (AAV) vector according to claim 1 or claim 2, wherein the muscle cell-specific promoter is selected from the group consisting of a muscle creatine kinase (MCK) promoter, a C5-12 promoter, a CK6-CK9 promoter, a dMCK
promoter, a tMCK promoter, a smooth muscle 22 (SM22) promoter, a myo-3 promoter, a Spc512 promoter, a creatine kinase (CK) 8 promoter, a creatine kinase (CK) 8e promoter, a U6 promoter, a promoter, a desmin promoter, a Pitx3 promoter, a skeletal alpha-actin promoter, a MFICK7 promoter, and a Sp-301 promoter.

4. The adeno-associated viral (AAV) vector according to any one of claims 1-3, wherein the muscle cell-specific promoter is a muscle creatine-kinase (tMCK) promoter.

5. The adeno-associated viral (AAV) vector according to any one of claims 1-4, wherein the nucleic acid molecule encodes one or more of p37Aur 1, p40AUF
17 p42AuFl7 or p45AuFl.

6. The adeno-associated viral (AAV) vector according to claim 5, wherein the nucleic acid molecule encodes p37AuFl.

7. The adeno-associated viral (AAV) vector according to claim 5, wherein the nucleic acid molecule encodes p40"Fl.

8. The adeno-associated viral (AAV) vector according to claim 5, wherein the nucleic acid molecule encodes p42"Fl.

9. The adeno-associated viral (AAV) vector according to claim 5, wherein the nucleic acid molecule encodes p45AUFl.

10. The adeno-associated viral (AAV) vector according to any one of claims 1-9 further comprising:
a nucleic acid molecule encoding a reporter protein.

11. The adeno-associated viral (AAV) vector according to any one of claims 1-10 further comprising:
a nucleic acid molecule encoding a purine-rich element binding protein 13 (Pur13) inhibitor.

12. The adeno-associated viral (AAV) vector according to claim 11, wherein the Pur13 inhibitor is selected from an RNA element, a polypeptide, or a small molecule.

13. The adeno-associated viral (AAV) vector according to claim 12, wherein the RNA element is selected from the group consisting of siRNA, shRNA, and miRNA.

14. A composition comprising the adeno-associated viral (AAV) vector according to any one of claims 1-13.

15. The composition according to claim 14 further comprising:
a buffer solution.

16. A pharmaceutical composition comprising:
the adeno-associated viral (AAV) vector according to any one of claims 1-15 and a pharmaceutically-acceptable carrier.

17. A method of promoting muscle regeneration, said method comprising:
contacting muscle cells with the adeno-associated viral (AAV) vector according to any one of claims 1-14 or the composition according to any one of claims 14-16 under conditions effective to express exogenous AUF1 in the muscle cells to increase muscle cell mass, increase muscle cell endurance, and/or reduce serum markers of muscle atrophy.

18. The method according to claim 17, wherein the method is carried out ex vivo.

19. The method according to claim 18 further comprising:

culturing the muscle cells ex vivo under conditions effective to express exogenous AUF1.

20. The method according to claim 17, wherein the method is carried out in vivo

21. The method according to any one of claims 17-20 further comprising:
contacting the muscle cells with a purine-rich element binding protein13 (Pur13) inhibitor.

22. The method according to claim 21, wherein the Pur(3 inhibitor is selected from a nucleic acid molecule, a polypeptide, or a small molecule.

23. The method according to claim 22, wherein the nucleic acid molecule is selected from the group consisting of siRNA, shRNA, and miRNA.

24. A method of treating degenerative skeletal muscle loss in a subject, said method comprising:
selecting a subject in need of treatment for skeletal muscle loss and administering to the selected subject the adeno-associated viral (AAV) vector according to any one of claims 1-13 or the composition according to any one of claims 14-16 under conditions effective to cause skeletal muscle regeneration in the selected subject.

25. The method according to claim 24, wherein said administering is carried out by intramuscular injection.

26. The method according to claim 24 or claim 25, wherein the subject has sarcopenia, myopathy, a muscle disorder mediated by functional AUF1 deficiency, or a muscle disorder not mediated by functional AUF1 deficiency.

27. The method according to any one of claims 24-26, wherein the subject has an adult-onset myopathy or muscle disorder.

28. The method according to any one of claims 24-27 further comprising:
administering a purine-rich element binding protein 13 (Pur13) inhibitor.

29. The method according to claim 28, wherein the Put-I3 inhibitor is a nucleic acid molecule, a polypeptide, or a small molecule.

30. The method according to claim 29, wherein the nucleic acid molecule is selected from the group consisting of siRNA, shRNA, and miRNA.

31. The method according to anyone of claims 24-26, wherein the subject has Duchenne Muscular Dystrophy (DMD).

32. The method according to anyone of claims 24-26, wherein the subject has traumatic muscle injury.

33. The method according to anyone of claims 24-33, wherein said administering is effective to upregulate endogenous utrophin protein expression in the selected subject.

34. The method according to anyone of claims 17-23, wherein said administering is effective to upregulate endogenous utrophin protein expression in said muscle cells.

35. A method of preventing traumatic muscle injury in a subject, the method comprising:
selecting a subject at risk of traumatic muscle injury and administering to the selected subject the adeno-associated viral (AAV) vector according to any one of claims 1-13; the composition according to any one of claims 14-16; or a lentiviral vector comprising a muscle cell specific promoter and a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein or a functional fragment thereof, wherein the nucleic acid molecule is heterologous to and operatively coupled to the muscle cell-specific promoter.

36. The method according to claim 35, wherein the lentiviral vector is administered.

37. The method according to claim 35, wherein the lentiviral vector encodes AUF1 isoform p37AuFl, p40AuFl, p42AuFl, or p45AuFl.

38. The method according to claim 37, wherein thelentiviral vector encodes AUF isoform p45 AUF1

39. The method according to claim 35, wherein said administering is carried out by intramuscular injection.

40. The method according to claim 35, wherein said administering prevents muscle atrophy and/or muscle loss following traumatic muscle injury to the selected subject

41. A method of treating traumatic muscle injury in a subject, the method compri sing:
selecting a subject having traumatic muscle injury and administering to the selected subject the adeno-associated viral (AAV) vector according to any one of claims 1-13; the composition according to any one of claims 14-16; or a lentiviral vector comprising a muscle cell specific promoter and a nucleic acid molecule encoding an AU-rich mRNA binding factor 1 (AUF1) protein or a functional fragment thereof, wherein the nucleic acid molecule is heterologous to and operatively coupled to the muscle cell-specific promoter.

42. The method according to claim 41, wherein the lentiviral vector is admini stered.

43. The method according to claim 41, wherein thelentiviral vector encodes AUF1 isoform p37AUF1, p4OAUH 137 42AUFl 7 or p45AuF1 .

44. The method according to claim 43, wherein the lentiviral vector encodes AUF isoform 4SAUll U

45. The method according to claim 41, wherein said administering is carried out by intramuscular injection.

46. The method according to claim 41, wherein said administering treats muscle atrophy and/or muscle loss in the selected subject.

47. The method according to claim 41, wherein said administering accelerates muscle gain as compared to when said administering is not carried out.