Adapted human dystrophins with enhanced actin-binding affinity for treating muscular dystrophies

Abstract

Disclosed herein are adapted dystrophin proteins (e.g., adapted mini- or micro-dystrophin proteins), nucleic acid molecules and vectors encoding the same, as well as methods of treating a muscular dystrophy (e.g., Duchenne or Becker muscular dystrophy), for example by gene therapy comprising the adapted dystrophin proteins, the nucleic acid molecules, or the vectors of the disclosure.

Description

ADAPTED HUMAN DYSTROPHINS WITH ENHANCED ACTIN-BINDING AFFINITY FOR TREATING MUSCULAR DYSTROPHIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Application No. 63 / 585,149, filed on September 25, 2023. The entire contents of the foregoing applications are incorporated herein by reference.REFERENCE TO ELECTRONICALLY FILED SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 24, 2024, is named 4140_064PC01_Sequencelisting_ST26 and is 480,050 bytes in size.FIELD OF DISCLOSURE

[0003] The present disclosure pertains to the medical field including gene therapy, for example, to the treatment of genetic diseases (e.g., muscular dystrophies) by gene therapy.BACKGROUND

[0004] The dystrophin gene is the largest known human gene, containing 79 exons and spanning > 2,200 kb, roughly 0.1% of the whole genome (Koenig M et al., Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell. 1987; 50:509-517). Human Dystrophin is a 427 kDa cytoskeletal protein that localizes to the cytoplasmic face of the sarcolemma and is enriched at costameres ( (i.e,. a component of striated muscle cells which connects the sarcomere of the muscle to the cell membrane (i.e. the sarcolemma)) in muscle fibers (Porter GA et al., Dystrophin colocalizes with beta-spectrin in distinct subsarcolemmal domains in mammalian skeletal muscle. J Cell Biol. 1992; 117:997-1005). Dystrophin protein has four main functional domains: an actin-binding amino-terminal domain (ABDI), a central rod domain, a cysteine-rich domain and a carboxy-terminus. ABDI contains 2 calponin homology domains (CHI and CH2)SUBSTITUTE SHEET (RULE 26)(Korenbaum E and Rivero F, Calponin homology domains at a glance. J Cell Sci. 2002; 115:3543- 3545). This conventional CH-actin binding domain binds directly to F actin, linking dystrophin to the subsarcolemmal actin network (Wein N et al., Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice. Nat Med. 2014; 20:992-1000.). Dystrophin’s central rod domain contains 24 spectrin repeats and harbors a second actin-binding motif (ABD2). ABD2 collaborates with ABDI to form a strong lateral association with actin filaments (Rybakova IN et al., A new model for the interaction of dystrophin with F-actin. J Cell Biol. 1996; 135:661-672).

[0005] The 24 spectrin repeats are interrupted by four short proline-rich spacers, called "hinges". Hinge 4 is at the end of the rod domain and contains a WW domain. The WW domain along with two neighboring EF-hands binds the carboxy-terminus of [3-dystroglycan, anchoring the dystrophin at sarcolemma (Rentschler S et al., The WW domain of dystrophin requires EF- hands region to interact with beta-dystroglycan. Biol Chem. 1999; 380:431-442). The two EF- hands are located in the cysteine-rich domain, which resides between the central rod and C- terminus. The cysteine rich domain also contains a zinc finger (ZZ) domain that binds to calmodulin in a calcium-dependent manner (Anderson JT et al., Ca2+-calmodulin binds to the carboxyl-terminal domain of dystrophin. J Biol Chem. 1996; 271:6605-6610). The cysteine rich domain has also been shown to bind to ankyrin-B, an adaptor proteins that is required for retaining dystrophin at the sarcolemma (Ayalon G et al., An ankyrin-based mechanism for functional organization of dystrophin and dystroglycan. Cell. 2008; 135:1189-1200). The carboxy- terminal (CT) domain provides binding sites for dystrobrevin and syntrophins, mediating their sarcolemma localization (Sadoulet-Puccio HM et al., Dystrobrevin and dystrophin: an interaction through coiled-coil motifs. Proc Natl Acad Sci U S A. 1997; 94:12413-12418).

[0006] The full length dystrophin proteins consists of, from the amino-terminus to the carboxy-terminus: an actin-binding domain 1 (ABDI), a central rod domain consisting of a first hinge (Hl), 3 spectrin-like repeats (R1 to R3), a second hinge (H2), 16 spectrin-like repeats (R4 to R19), a third hinge (H3), 5 spectrin-like repeats (R20 to R4), a fourth hinge (H4), a cysteine rich domain (CR), and a carboxy-terminus (Chamberlain et al., Microdystrophin Expression as a Surrogate Endpoint for Duchenne Muscular Dystrophy Clinical Trials. Hum Gene Ther. 2023 May;34(9-10):404-415).SUBSTITUTE SHEET (RULE 26)

[0007] Dystrophin is an essential component of the dystrophin complex, a complex that has been hypothesized to act as a membrane stabilizer during muscle contraction to prevent contraction-induced damage (Danialou G et al., Dystrophin-deficient cardiomyocytes are abnormally vulnerable to mechanical stress-induced contractile failure and injury. FASEBJ. 2001; 15:1655-1657, Petrof BJ et al., Dystrophin protects the sarcolemma from stresses developed during muscle contraction. Proc Natl Acad Sci U S A. 1993; 90:3710-3714). In addition to its structural role, dystrophin complex is also thought to mediate cellular signaling such as mechanical force transduction and cell adhesion.

[0008] Muscular dystrophy is a collection of inherited diseases characterized by skeletal muscle weakness and degeneration. Muscular dystrophies are progressive disorders because over time healthy muscle fibers are lost and replaced by fibrosis and fat, making muscle tissues less able to generate force for everyday activity. As muscle wasting ensues, patients experience weakness, although muscle groups may be targeted differently in specific forms of muscular dystrophy. Respiratory failure, resulting from the weakening of breathing muscles, may limit lifespan in muscular dystrophy unless mechanical support is instituted. In some forms of muscular dystrophy, the heart is also affected resulting in cardiac complications including heart failure and irregular heart rhythms.

[0009] Duchenne muscular dystrophy (DMD) is the most common and lethal muscular dystrophy. DMD is caused by recessive mutations in the dystrophin gene on X chromosome. This fatal disease affects approximately 1 :3,500 to 6,000 live male births (Grossklauss LF, Distrofia Muscular de Duchenne. In: Oliveira AS. Reabilitaqao em doenqas neuromusculares: guia terapeutico pratico. 2014, Sao Paulo: Atheneu) and 1 :50,000,000 live female births (Webb CL, Parents' perspectives on coping with Duchenne muscular dystrophy. Child Care Health Dev. 2005 Jul;31(4):385-96; Pena FR et al., The contribution of physiotherapy for the well-being and participation of two students with Duchenne Muscular Dystrophy in regular school. Rev Bras Educ Espec 2008; 14:447-62). DMD is caused genetic variants in the dystrophin (DMD) gene caused by deletions (about 65% of cases) (Den Dunnen JT et al., Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am J Hum Genet 1989;45:835-47), specific sequence variants (about 26% of cases)SUBSTITUTE SHEET (RULE 26)(Deburgrave N et al., Protein- and mRNA-based phenotype- genotype correlations in DMD / BMD with point mutations and molecular basis for BMD with nonsense and frameshift mutations in the DMD gene. Hum Mutat 2007;28: 183-95), duplications (about 7% of cases) (White SJ et al., Duplications in the DMD gene. Hum Mutat 2006;27:938-45) and other unidentified causes (about 2% of cases). Becker muscular dystrophy (BMD) is also caused by genetic variants in the DMD gene that encodes dystrophin. Individuals with BMD share similar signs and symptoms with DMD but with later onset and more varied time course. Like DMD, the heart can be affected inBMD. Other muscular dystrophies are not associated with genetic variants in the DMD gene, for example Limb-girdle muscular dystrophy 2 (LGMD2) is associated with genetic variants in a number of genes that encode proteins that directly associate with dystrophin, forming integral parts of the dystrophin glycoprotein complex (DGC).

[0010] There are at least nineteen forms of LGMD, and the forms are classified by their associated genetic defects.Type Pattern of Inheritance Gene or ChromosomeLGMDIA Autosomal dominant Myotilin geneLGMDIB Autosomal dominant Lamin A / C geneLGMDIC Autosomal dominant Caveolin geneLGMDID Autosomal dominant Chromosome 7LGMDIE Autosomal dominant Desmin geneLGMDIF Autosomal dominant Chromosome 7LGMDIG Autosomal dominant Chromosome 4LGMD2A Autosomal recessive Calpain-3 geneLGMD2B Autosomal recessive Dysferlin geneLGMD2C Autosomal recessive Gamma-sarcoglycangeneLGMD2D Autosomal recessive Alpha-sarcoglycan geneLGMD2E Autosomal recessive Beta-sarcoglycan geneLGMD2F Autosomal recessive Delta-sarcoglycan geneLGMD2G Autosomal recessive Telethonin geneLGMD2H Autosomal recessive TRIM32LGMD2I Autosomal recessive FKRP geneLGMD2J Autosomal recessive Titin geneLGMD2K Autosomal recessive POMTI geneSUBSTITUTE SHEET (RULE 26)LGMD2L Autosomal recessive Fukutin gene

[0011] Despite their high prevalence in the population an effective treatment for muscular dystrophies, and in particular for DMD and BMD, is still lacking.

[0012] Clinical trials of myoblast transplantation have met with little success, owing to the poor survival of the transplanted cells (Gussoni, E et al., (1997) Nat. Med. 3, 970 -977). Gene therapy to deliver dystrophin constructs to rescue the linkage between the actin cytoskeleton and the sarcolemmal dystroglycan complex, has been extensively studied in animal models as an alternative strategy. Somatic gene transfer using both nonviral DNA vectors carrying dystrophin cDNA (Acsadi, G et al., (1991) Nature (London) 352, 815- 818) and RNA / DNA oligonucleotides (Rando, TA et al., (2000) Proc. Natl. Acad. Set. USA 97, 5363-5368) achieved transgene expression but with very limited efficiency. Adenovirus-based vectors have been successfully tested in dystrophic animal models (Ragot, T et al., (1993) Nature (London) 361, 647- 650; Howell, JM et al., (1998) Hum. Gene Ther. 9, 629 - 634). Nonetheless, the immunogenicity and inefficiency of infecting mature muscle cells remain major hurdles to overcome before the vectors can be safely used in humans. Adeno-associated virus (AAV) vectors are the only viral vector system that is based on a nonpathogenic and replication defective virus (Muzyczka N (1992) Curr. Top. Microbiol. Immunol. 158, 97-129). AAV vectors have been successfully used to establish efficient and long-term gene expression in vivo in a variety of tissues without significant immune response or toxicity (Kaplitt et al., (1994) Nat. Genet. 8, 148 -154; Xiao X et al., (1996) J. Virol. 70, 8098 - 8108; Kessler, PD et al.,. (1996) Proc. Natl. Acad. Sci. USA 93, 14082-14087; Xiao W et al., (1998) J. Virol, 'll, 10222-10226). Unlike other viral and nonviral vectors, AAV readily bypasses extracellular barriers because of its small viral particle size (20 nm) that facilitates efficient transduction of muscle myofibers of various maturity (Pruchnic R et al., (2000) Hum. Gene Ther. 11, 521-536). Currently, AAV vectors offer the best gene transfer efficiency and longevity among all viral and nonviral vectors tested in muscle tissues. The unparalleled efficiency and safety have led to an increasing interest in AAV-mediated gene therapy for genetic muscle disorders (Greelish JP et al., (1999) Nat. Med. 5, 439 - 443; Xiao X et al., (2000) J. Virol. 74, 1436 -1442; Cordier L et al., (2000) Mol. Ther. 1, 119 -129) as well as for metabolic diseases. A major barrier for achieving dystrophin restoration using AAV-mediated gene therapy is the large size of the dystrophin gene and the limited capacity of AAVs. The most commonly used adeno-associated viruses for human gene therapy are restricted to less than 4-5 kb as total genome content (Athanasopoulos T et al., Recombinant adeno-associated viral (rAAV) vectors as therapeutic toolsSUBSTITUTE SHEET (RULE 26)for Duchenne muscular dystrophy (DMD). Gene Ther. 2004; l l(Suppl 1): S109— 121), since the dystrophin gene alone spans > 2,200 kb, roughly 0.1% of the whole genome (Koenig M et al., Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell. 1987; 50:509-517) and the dystrophin cDNA is about 14 kb (W ang B et al., Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. Proc Natl Acad Sci USA. 2000 Dec 5;97(25): 13714-9), it is virtually impossible to insert the whole dystrophin gene into an AAV vector. This limitation stimulated the development of mini- and micro-dystrophins, truncated forms of dystrophin that can be encoded by a nucleic acid molecule with a size suitable to be inserted into an AAV vector to be used for gene therapy. However, mini- and micro-dystrophins only partially recapitulate the actin-binding activity of full-length dystrophin because the internal sequence deletions necessary to fit the constructs into AAV necessarily removes dystrophin’s second actin-binding domain (ABD2) located within spectrinlike repeats 11-17 (Amann, KJ et al., (1998). A cluster ofbasic repeats in the dystrophin rod domain binds F- actin through an electrostatic interaction. J. Biol. Chem. 275, 28419-28423). In vitro studies show that removing ABD2 causes a 30-fold reduction in actin binding affinity of mini- / micro-dystrophin compared to full-length dystrophin (Rybakova IN et al., (1996). A new model for the interaction of dystrophin with F-actin. J. Cell Biol. 135, 661-672; Rybakova, IN et al., (2006). Dystrophin and utrophin bind actin filaments through distinct modes of contact. J. Biol. Chem. 281, 9996-10001; Henderson DM et al., (2012). The carboxy-terminal third of dystrophin enhances actin binding activity. J. Mol. Biol. 416, 414-424). Removing ABD2 also abolishes key biophysical properties exhibited by full-length dystrophin including effects on actin structural dynamics and actin resilience (Lin AY et al., (2012). Impacts of dystrophin and utrophin domains on actin structural dynamics: implications for therapeutic design. J. Mol. Biol. 420, 87-98) both of which result in part from reduced actin-binding affinity. Thus, new strategies are urgently needed.BRIEF SUMMARY

[0013] In some aspects, provided herein are polynucleotides encoding polypeptides comprising a dystrophin protein actin-binding domain 1 modified to achieve an increased actin- binding affinity (GoA-DysABD1), or an actin-binding fragment thereof, wherein: 1) the GoA- DysABD1, or an actin-binding fragment thereof, has at least one amino acid substitution compared to a same dystrophin protein actin-binding domain 1, or an actin-binding fragment thereof, absentSUBSTITUTE SHEET (RULE 26)the modification to achieve the increased actin-binding affinity (NotGoA-DysABD1); and 2) wherein in a fluorescence resonance energy transfer (FRET) assay the GoA-DysABD1, or an actin- binding fragment thereof, binds to actin with a dissociation constant (Kd) reduced of at least 20%, at least 50%, at least 80%, or at least 99.9% compared to a dissociation constant of the NotGoA- DysABD1 in a same FRET assay.

[0014] In some aspects, in the FRET assay, the actin is a phalloidin-stabilized F-actin. In some aspects, an Alexa-568 fluorophore is attached to actin (actin-Alexa-568). In some aspects, the Alexa-568 fluorophore is attached to residue C374 of actin. In some aspects, in the FRET assay an mClover3 fluorophore is attached to the GoA-DysABD1 or to the NotGoA-DysABD1. In some aspects, the mClover3 fluorophore is attached to the carboxy -terminus of the GoA-DysABD1 (GoA-DysABD1-mClover3) or to the carboxy-terminus of the NotGoA-DysABD1 (NotGoA- DysABD1 -mClover3). In some aspects, sub-micromolar amounts of GoA-DysABD1-mClover3 or of NotGoA-DysABD1-mClover3 are used in the FRET assay. In some aspects, in the FRET assay, the amount of actin-Alexa-568 is increased over time. In some aspects, in the FRET assay, a fluorescence decay lifetime of the GoA-DysABD l-mClover3 or of the NotGoA-DysABD1- mClover3 following a sub-nanosecond excitation pulse is measured. In some aspects, the FRET assay is a time-resolved FRET assay.

[0015] In some aspects, provided herein are polynucleotides encoding polypeptides comprising a dystrophin protein actin-binding domain 1 modified to achieve an increased actin- binding affinity (GoA-DysABD1), or an actin-binding fragment thereof, compared to a same dystrophin protein actin-binding domain 1, or an actin-binding fragment thereof, absent the modification to achieve the increased actin-binding affinity (NotGoA-DysABD1), wherein the NotGoA-DysABD1, from N-terminus to C-terminus, comprises an amino acid sequence of SEQ ID NO: 2, wherein: XI 18 is W orR; XI33 is Q or P; XI 65 is D or V; and wherein the modification to achieve the increased actin-binding affinity is at least one amino acid substitution at one or more of residues D9, E12, Q17, K19, T20, F21, S30, Q35, H36, N75, K79, L81, L84, N86, K105, T107, 1114, 1115, V120, M128, W143, R145, Q153, T161, A168, L169, V187, A192, A199, 1202, L207, G208, 1209, L212, D214, E216, or K226. In some aspects, the NotGoA-DysABD1, from N- terminus to C-terminus, comprises an amino acid sequence of SEQ ID NO: 1.

[0016] In some aspects, the polynucleotides disclosed herein are human or are codon- optimized for expression in humans.SUBSTITUTE SHEET (RULE 26)

[0017] In some aspects, the GoA-DysABD1 comprised in the polypeptides encoded by the polynucleotides disclosed herein, or an actin-binding fragment thereof, comprises 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, 8 amino acid substitutions, 9 amino acid substitutions, or 10 amino acid substitutions compared to the NotGoA- DysABD1, or an actin-binding fragment thereof In some aspects, the GoA-DysABD1, or an actin- binding fragment thereof, comprise 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, or 4 amino acid substitutions compared to the NotGoA-DysABD1, or an actin- binding fragment thereof.

[0018] In some aspects, the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (E12D), A at residue 17 (Q17A), E at residue 19 (K19E), D at residue19 (K19D), A at residue 20 (T20A), Q at residue 20 (T20Q), N at residue 20 (T20N), S at residue20 (T20S), M at residue 20 (T20M), V at residue 20 (T20V), L at residue 20 (T20L), I at residue 20 (T20I), L at residue 21 (F21L), A at residue 21 (F21A), V at residue 21 (F21V), I at residue 21 (F21I), Q at residue 30 (S30Q), S at residue 30 (S30N), T at residue 30 (S30T), M at residue 30 (S30M), R at residue 35 (Q35R), K at residue 35 (Q35K), R at residue 36 (H36R), K at residue 36 (H36K), R at residue 75 (N75R), K at residue 75 (N75K), N at residue 79 (K79N), Q at residue 79 (K79Q), S at residue 79 (K79S), T at residue 79 (K79T), M at residue 79 (K79M), P at residue 81 (L81P), S at residue 84 (L84S), N at residue 84 (L84N), Q at residue 84 (L84Q), T at residue 84 (L84T), M at residue 84 (L84M), G at residue 86 (N86G), N at residue 105 (K105N), Q at residue 105 (K105Q), S at residue 105 (K105S), T at residue 105 (K105T), M at residue 105 (K105M), G at residue 107 (T107G, L at residue 114 (I114L), E at residue 114 (I114E), A at residue 114 (I114A), V at residue 114 (Il 14V), D at residue 114 (Il 14D), S at residue 115 (1115 S), N at residue 115 (I115N), Q at residue 115 (I115Q), T at residue 115 (I115T), M at residue 115 (I115M), P at residue 120 (V120P), A at residue 120 (VI 20 A), L at residue 120 (V120L), I at residue 120 (V120I), K at residue 128 (M128K), R at residue 128 (M128R), R at residue 143 (W143R), K at residue 143 (W143K), V at residue 145 (R145V), A at residue 145 (R145A),L at residue 145 (R145L), I 145(R145I), H at residue 153 (Q153H), Y at residue 153 (Q153Y), W at residue 153 (Q153W), F at residue 153 (Q153F), I at residue 156 (VI 561), A at residue 156 (V156A), L at residue 156 (V156L); H at residue 161 (T161H), Y at residue 161 (T161Y), W at residue 161 (T161W), F at residue 161 (T161F), V at residue 168 (A168V), I at residue 168 (A168I), L at residue 168 (A168L), S at residue 169 (L169S), N at residue 169 (L169N), Q at residue 169SUBSTITUTE SHEET (RULE 26)(L169Q), T at residue 169 (L169T), M at residue 169 (L169M), G at residue 187 (V187G), L at residue 192 (A192L), V at residue 192 (A192V), I at residue 192 (A192I), V at residue 199 (A199V), L at residue 199 (A199L), I at residue 199 (A199I), G at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue 207 (L207S), Q at residue 207 (L207Q), T at residue 207 (L207T), M at residue 207 (L207M), A at residue 207 (L207A), I at residue 207 (L207I), Y at residue 207 (L207Y), W at residue 207 (L207W), F at residue 207 (L207F), E at residue 207 (L207E), P at residue 208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (I209V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue 209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), H at residue 209 (I209H), Y at residue 209 (I209Y), A at residue 209 (I209A), N at residue 209 (I209N), T at residue 209 (I209T), M at residue 209 (I209M), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), R at residue 216 (E216R), E at residue 226 (K226E), or D at residue 226 (K226D), or any combination thereof.

[0019] In some aspects, the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (E12D), A at residue 17 (Q17A), E at residue 19 (K19E), A at residue 20 (T20A), Q at residue 20 (T20Q), L at residue 21(F21L), A at residue 21 (F21A), Q at residue 30 (S30Q), R at residue 35 (Q35R), R at residue 36 (H36R), R at residue 75 (N75R), N at residue 79 (K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 86 (N86G), N at residue 105 (K105N), G at residue 107 (T107G), L at residue 114 (I114L), E at residue 114 (I114E), S at residue 115 (I115S), P at residue 120 (V120P), A at residue 120 (V120A), K at residue 128 (M128K), R at residue 143 (W143R), V at residue 145 (R145V), H at residue 153 (Q153H), I at residue 156 (V156I), H at residue 161 (T161H), V at residue 168 (A168V), I at residue 168 (A168I), S at residue 169 (L169S), G at residue 187 (V187G), , L at residue 192 (A192L), V at residue 199 (Al 99V), I at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue 207 (L207S), P at residue 208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (I209V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue 209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), P at residue 212SUBSTITUTE SHEET (RULE 26)(L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226(K226E), or any combination thereof.

[0020] In some aspects, the at least one amino acid substitution consists of one amino acid substitution. In some aspects, the one amino acid substitution is selected from Q at residue 20 (T20Q), Q at residue 30 (S30Q), N at residue 79 (K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 107 (T107G), L at residue 114 (Il 14L), E at residue 114 (Il 14E), S at residue 115 (Il 15S), P at residue 120 (V120P), R at residue 143 (W143R), H at residue 153 (Q153H), I at residue 156 (V156I), H at residue 161 (T161H), I at residue 168 (A168I), G at residue 187 (V187G), L at residue 192 (A192L), V at residue 199 (A199V), I at residue 202 (I202G), V at residue 207 (L207V), P at residue 208 (G208P), K at residue 209 (I209K), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226 (K226E). In some aspects, the one amino acid substitution is selected from Q at residue 20 (T20Q), P at residue 120 (V120P), I at residue 156 (V156I), P at residue 212 (L212P), P at residue 214 (D214P), or E at residue 226 (K226E). In some aspects, the polypeptides encoded by the polynucleotides disclosed herein comprise an amino acid sequence of any one of SEQ ID NOs: 5-30. In some aspects, the polynucleotides disclosed herein comprise a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 144-169.

[0021] In some aspects, the at least one amino acid substitution consists of two amino acid substitutions. In some aspects, the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P-K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); N at residue 207 and L at residue 209 (L207N-I209L); R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); D at residue 207 and V at residue 209 (L207D-I209V); G at residue 187 and P at residue 212 (V187G-L212P); Q at residue 20 and I at residue 156 (T20Q-V156I); Q at residue 20 and P at residue 214 (T20Q-D214P); P at residue 120 and E at residue 226 (V120P-K226E); I at residue 156 and P at residue 214 (V156I- D214P); P at residue 120 and G at residue 187 (V120P-V187G); P at residue 212 and P at residueSUBSTITUTE SHEET (RULE 26)214 (L212P-D214P); P at residue 214 and E at residue 226 (D214P-K226E); G at residue 187 and E at residue 226 (V187G-K226E); Q at residue 20 andP at residue 120 (T20Q-V120P); l at residue 156 and P at residue 212 (V156I-L212P); P at residue 120 and I at residue 156 (V120P-V156I); Q at residue 20 and P at residue 212 (T20Q-L212P); P at residue 120 and P at residue 214 (V120P- D214P); G at residue 187 and P at residue 214 (V187G-D214P); Q at residue 20 and G at residue 187 (T20Q-V187G); P at residue 120 and P at residue 212 (V120P-L212P); Q at residue 20 and E at residue 226 (T20Q-K226E); I at residue 156 and G at residue 187 (V156I-V187G); I at residue 156 and E at residue 226 (V156I-K226E); P at residue 212 and E at residue 226 (L212P-K226E); A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P- K226E); or R at residue 35 and K at residue 207 (Q35R-L207K). In some aspects, the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P-K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); N at residue 207 and L at residue 209 (L207N-I209L); R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); or D at residue 207 and V at residue 209 (L207D-I209V). In some aspects, the two amino acid substitutions are selected from is A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P-K226E); or R at residue 35 and K at residue 207 (Q35R-L207K). In some aspects, the polypeptides encoded by the polynucleotides disclosed herein comprise an amino acid sequence of any one of SEQ ID NOs: 31-69. In some aspects, the polynucleotides disclosed herein comprise a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 170-208.

[0022] In some aspects, the at least one amino acid substitution consists of three amino acid substitutions. In some aspects, the three amino acid substitutions are selected from G at residue 187, P at residue 214, E at residue 226 (V187G-D214P-K226E); Q at residue 20, P at residue 120, and G at residue 187 (T20Q-V120P-V187G); Q at residue 20, P at residue 120, and E at residue 226 (T20Q-V120P-K226E); Q at residue 20, G at residue 187, and E at residue 226 (T20Q-V187G- K226E); P at residue 120, I at residue 156, and P at residue 214 (V120P-V156I-D214P); P atSUBSTITUTE SHEET (RULE 26)residue 212, P at residue 214, and E at residue 226 (L212P-D214P-K226E); P at residue 120, G at residue 187, and E at residue 226 (V120P-V187G-K226E); P at residue 120, P at residue 212, and P at residue 214 (V120P-L212P-D214P); I at residue 156, P at residue 212, and P at residue 214 (V156I-L212P-D214P); Q at residue 20, I at residue 156, and P at residue 212 (T20Q-V156I- L212P); I at residue 156, G at residue 187, and E at residue 226 (V156I-V187G-K226E); Q at residue 20, P at residue 214, and E at residue 226 (T20Q-D214P-K226E); P at residue 120, P at residue 214, and E at residue 226 (V120P-D214P-K226E); Q at residue 20, P at residue 120, and P at residue 214 (T20Q-V120P-D214P); Q at residue 20, P at residue 120, and I at residue 156 (T20Q-V120P-V156I); Q at residue 20, G at residue 187, and P at residue 214 (T20Q-V187G- D214P); P at residue 120, l at residue 156, andP at residue 212 (V120P-V156I-L212P); l at residue 156, P at residue 214, and E at residue 226 (V156I-D214P-K226E); G at residue 187, P at residue 212, and E at residue 226 (V187G-L212P-K226E); P at residue 120, G at residue 187, and P at residue 214 (V120P-V187G-D214P); Q at residue 20, 1 at residue 156, and G at residue 187 (T20Q- V156I-V187G); I at residue 156, G at residue 187, and P at residue 214 (V156I-V187G-D214P); Q at residue 20, and I at residue 156, E226 (T20Q-V156I-K226E); Q at residue 20, P at residue 212, and E at residue 226 (T20Q-L212P-K226E); Q at residue 20, P at residue 120, and P at residue 212 (T20Q-V120P-L212P); Q at residue 20, G at residue 187, and P at residue 212 (T20Q-V187G- L212P); P at residue 120, I at residue 156, and E at residue 226 (V120P-V156I-K226E); P at residue 120, 1 at residue 156, and G at residue 187 (V120P-V156I-V187G); P at residue 120, P at residue 212, and E at residue 226 (V120P-L212P-K226E); P at residue 120, G at residue 187, and P at residue 212 (V120P-V187G-L212P); G at residue 187, P at residue 212, and P at residue 214 (V187G-L212P-D214P); I at residue 156, G at residue 187, and P at residue 212 (V156I-V187G- L212P); Q at residue 20, 1 at residue 156, and P at residue 214 (T20Q-V156I-D214P); I at residue 156, P at residue 212, and E at residue 226 (V156I-L212P-K226E); Q at residue 20, P at residue 212, and P at residue 214 (T20Q-L212P-D214P); A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N-V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L- I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R-A168V). In some aspects, the three amino acid substitutions are selected from A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N-V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L-I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R- A168V). In some aspects, the polypeptides encoded by the polynucleotides disclosed herein comprise an amino acid sequence of any one of SEQ ID NOs: 70-107. In some aspects, theSUBSTITUTE SHEET (RULE 26)polynucleotides disclosed herein comprise a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 209-247.

[0023] In some aspects, the at least one amino acid substitution consists of four amino acid substitutions. In some aspects, the four amino acid substitutions are selected from Q at residue 20, P at residue 120, Gat residue 187, andP at residue 212 (T20Q-V120P-V187G-L212P); Q at residue 20, P at residue 120, P at residue 212, and P at residue 214 (T20Q-V120P-L212P-D214P); Q at residue 20, 1 at residue 156, G at residue 187, and P at residue 214 (T20Q-V156I-V187G-D214P); Q at residue 20, P at residue 212, P at residue 214, and E at residue 226 (T20Q-L212P-D214P- K226E); P at residue 120, 1 at residue 156, G at residue 187, and P at residue 212 (V120P-V156I- V187G-L212P); I at residue 156, G at residue 187, P at residue 212, and E at residue 226 (V156I- V187G-L212P-K226E); P at residue 120, 1 at residue 156, P at residue 212, and E at residue 226 (V120P-V156I-L212P-K226E); Q at residue 20, G at residue 187, P at residue 212, and P at residue 214 (T20Q-V187G-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and E at residue 226 (V120P-V187G-L212P-K226E); Q at residue 20, I at residue 156, P at residue 212, and E at residue 226 (T20Q-V156I-L212P-K226E); Gat residue 187, P at residue 212, P at residue 214, and E at residue 226 (V187G-L212P-D214P-K226E); Q at residue 20, P at residue 120, G at residue 187, and E at residue 226 (T20Q-V120P-V187G-K226E); Q at residue 20, P at residue 120, I at residue 156, and P at residue 212 (T20Q-V120P-V156I-L212P); Q at residue 20, P at residue 120, P at residue 214, and E at residue 226 (T20Q-V120P-D214P-K226E); Q at residue 20, I at residue 156, G at residue 187, and P at residue 212 (T20Q-V156I-V187G-L212P); I at residue 156, G at residue 187, P at residue 212, and P at residue 214 (V156I-V187G-L212P- D214P); P at residue 120, 1 at residue 156, G at residue 187, and E at residue 226 (V120P-V156I- V187G-K226E); P at residue 120, 1 at residue 156, P at residue 212, and P at residue 214 (V120P- V156I-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and P at residue 214 (V120P-V187G-L212P-D214P); Q at residue 20, G at residue 187, P at residue 214, and E at residue 226 (T20Q-V187G-D214P-K226E); Q at residue 20, 1 at residue 156, P at residue 212, and P at residue 214 (T20Q-V156I-L212P-D214P); I at residue 156, P at residue 212, P at residue 214, and E at residue 226 (V156I-L212P-D214P-K226E); Q at residue 20, P at residue 120, Gat residue 187, and P at residue 214 (T20Q-V120P-V187G-D214P); Q at residue 20, P at residue 120, I at residue 156, and G at residue 187 (T20Q-V120P-V156I-V187G); I at residue 156, G at residue 187, P at residue 214, and E at residue 226 (V156I-V187G-D214P-K226E); Q at residue 20, P atSUBSTITUTE SHEET (RULE 26)residue 120, 1 at residue 156, and E at residue 226 (T20Q-V120P-V156I-K226E); P at residue 120, I at residue 156, P at residue 214, and E at residue 226 (V120P-V156I-D214P-K226E); Q at residue 20, 1 at residue 156, G at residue 187, and E at residue 226 (T20Q-V156I-V187G-K226E); Q at residue 20, P at residue 120, P at residue 212, and E at residue 226 (T20Q-V120P-L212P- K226E); P at residue 120, G at residue 187, P at residue 214, and E at residue 226 (V120P-V 187G- D214P-K226E); P at residue 120, P at residue 212, P at residue 214, and E at residue 226 (V120P- L212P-D214P-K226E); Q at residue 20, G at residue 187, P at residue 212, and E at residue 226 (T20Q-V187G-L212P-K226E); P at residue 120, I at residue 156, G at residue 187, and P at residue 214 (V120P-V156I-V187G-D214P); Q at residue 20, I at residue 156, P at residue 214, and E at residue 226 (T20Q-V156I-D214P-K226E); Q at residue 20, P at residue 120, 1 at residue 156, and P at residue 214 (T20Q-V120P-V156I-D214P); or A at residue 17, A at residue 20, P at residue 212, and E at residue 226 (Q17A-T20A-L212P-K226E). In some aspects, the four amino acid substitutions are A at residue 17, A at residue 20, P at residue 212, and E at residue 226 (Q17A-T20A-L212P-K226E). In some aspects, the polypeptides encoded by the polynucleotides disclosed herein comprise an amino acid sequence at of any one of SEQ ID NOs: 108-143. In some aspects, the polynucleotides disclosed herein comprise a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 248-282.

[0024] In some aspects, the polypeptides encoded by the polynucleotides disclosed herein further comprise one or more additional portions of a dystrophin protein. In some aspects, the polypeptides encoded by the polynucleotides disclosed herein further comprise: at least one dystrophin protein spectrin-like repeat located downstream of the ABDI domain; and at least one cysteine-rich domain located downstream of the dystrophin protein spectrin-like repeat.

[0025] In some aspects, the dystrophin protein spectrin-like repeat is a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein spectrin-like repeat 4, a dystrophin protein spectrin-like repeat 5, a dystrophin protein spectrin-like repeat 6, a dystrophin protein spectrin-like repeat 7, a dystrophin protein spectrin-like repeat 8, a dystrophin protein spectrin-like repeat 9, a dystrophin protein spectrin-like repeat 10, a dystrophin protein spectrin-like repeat 11, a dystrophin protein spectrinlike repeat 12, a dystrophin protein spectrin-like repeat 13, a dystrophin protein spectrin-like repeat 14, a dystrophin protein spectrin-like repeat 15, a dystrophin protein spectrin-like repeat 16, a dystrophin protein spectrin-like repeat 17, a dystrophin protein spectrin-like repeat 18, a dystrophinSUBSTITUTE SHEET (RULE 26)protein spectrin-like repeat 19, a dystrophin protein spectrin-like repeat 20, a dystrophin protein spectrin-like repeat 21, a dystrophin protein spectrin-like repeat 22, a dystrophin protein spectrinlike repeat 23, or a dystrophin protein spectrin-like repeat 24

[0026] In some aspects, the spectrin-like repeat 1 comprises amino acids 339-447 of SEQ ID NO: 4; the spectrin-like repeat 2 comprises amino acids 448-556 of SEQ ID NO: 4; the spectrinlike repeat 3 comprises amino acids 559-667 of SEQ ID NO: 4; the spectrin-like repeat 4 comprises amino acids 719-828 of SEQ ID NO: 4; the spectrin-like repeat 5 comprises amino acids 830-934 of SEQ ID NO: 4; the spectrin-like repeat 6 comprises amino acids 943-1045 of SEQ ID NO: 4; the spectrin-like repeat 7 comprises amino acids 1048-1154 of SEQ ID NO: 4; the spectrin-like repeat 8 comprises amino acids 1157-1263 of SEQ ID NO: 4; the spectrin-like repeat 9 comprises amino acids 1266-1367 of SEQ ID NO: 4; the spectrin-like repeat 10 comprises amino acids 1368- 1463 of SEQ ID NO: 4; the spectrin-like repeat 11 comprises amino acids 1468-1568 of SEQ ID NO: 4; the spectrin-like repeat 12 comprises amino acids 1571-1676 of SEQ ID NO: 4; the spectrinlike repeat 13 comprises amino acids 1679-1778 of SEQ ID NO: 4; the spectrin-like repeat 14 comprises amino acids 1779-1874 of SEQ ID NO: 4; the spectrin-like repeat 15 comprises amino acids 1877-1979 of SEQ ID NO: 4; the spectrin-like repeat 16 comprises amino acids 1992-2101 of SEQ ID NO: 4; the spectrin-like repeat 17 comprises amino acids 2104-2208 of SEQ ID NO: 4; the spectrin-like repeat 18 comprises amino acids 2211-2318 of SEQ ID NO: 4; the spectrin-like repeat 19 comprises amino acids 2319-2423 of SEQ ID NO: 4; the spectrin-like repeat 20 comprises amino acids 2475-2577 of SEQ ID NO: 4; the spectrin-like repeat 21 comprises amino acids 2580-2686 of SEQ ID NO: 4; the spectrin-like repeat 22 comprises amino acids 2689-2802 of SEQ ID NO: 4; the spectrin-like repeat 23 comprises amino acids 2808-2930 of SEQ ID NO: 4; the spectrin-like repeat 24 comprises amino acids 2935-3040 of SEQ ID NO: 4. In some aspects, the cysteine-rich domain comprises amino acids 3113-3360 of SEQ ID NO: 4.

[0027] In some aspects, the polypeptides encoded by the polynucleotides disclosed herein further comprise at least one dystrophin protein hinge region located downstream of the ABDI and upstream of the cysteine-rich domain. In some aspects, the dystrophin protein hinge region is a dystrophin protein hinge region 1, a dystrophin protein hinge region 2, a dystrophin protein hinge region 3, or a dystrophin protein hinge region 4. In some aspects, the hinge region 1 comprises amino acids 247-338 of SEQ ID NO: 4; the hinge region 2 comprises amino acids 668-718 of SEQ ID NO: 4; the hinge region 3 comprises amino acids 2424-2474 of SEQ ID NO: 4; the hinge region 4 comprises amino acids 3041-3112 of SEQ ID NO: 4.SUBSTITUTE SHEET (RULE 26)

[0028] In some aspects, the polypeptides encoded by the polynucleotides disclosed herein further comprise a dystrophin protein carboxy-terminal domain (CTD) located downstream of the cysteine-rich domain. In some aspects, the dystrophin protein carboxyl-terminus comprises amino acids 3361-3685 of SEQ ID NO: 4.

[0029] In some aspects, the polypeptides encoded by the polynucleotides disclosed herein comprise: the GoA-DysABD1; dystrophin protein spectrin-like repeats 1, 2, 3, and 24; dystrophin protein hinge regions 1, 2, and 4; and a dystrophin protein cysteine-rich domain. In some aspects, the polypeptides encoded by the polynucleotides disclosed herein from amino-terminus to carboxyl-terminus consists of: the GoA-DysABD1; a dystrophin protein hinge region 1; a dystrophin protein spectrin-like repeat 1; a dystrophin protein spectrin-like repeat 2; a dystrophin protein spectrin-like repeat 3; a dystrophin protein hinge region 2; a dystrophin protein spectrinlike repeat 24; a dystrophin protein hinge region 4; and a dystrophin protein cysteine-rich domain.

[0030] In some aspects, the polypeptides encoded by the polynucleotides disclosed herein comprise: the GoA-DysABD1; dystrophin protein spectrin-like repeats 1, 2, 22, 23, and 24; dystrophin protein hinge regions 1, 3, and 4; and a dystrophin protein cysteine-rich domain. In some aspects, the polypeptides encoded by the polynucleotides disclosed herein from aminoterminus to carboxyl-terminus consists of: the GoA-DysABD1; a dystrophin protein hinge region 1; a dystrophin protein spectrin-like repeat 1; a dystrophin protein spectrin-like repeat 22; a dystrophin protein spectrin-like repeat 23 ; a dystrophin protein spectrin-like repeat 24; a dystrophin protein hinge region 4; and a dystrophin protein cysteine-rich domain.

[0031] In some aspects, the polypeptides encoded by the polynucleotides disclosed herein comprise: the GoA-DysABD1; dystrophin protein spectrin-like repeats 1, 16, 17, 23, and 24; dystrophin protein hinge regions 1, and 4; and a dystrophin protein cysteine-rich domain. In some aspects, the polypeptides encoded by the polynucleotides disclosed herein from amino-terminus to carboxyl-terminus consists of: the GoA-DysABD1; a dystrophin protein hinge region 1; a dystrophin protein spectrin-like repeat 1; a dystrophin protein spectrin-like repeat 16; a dystrophin protein spectrin-like repeat 17; a dystrophin protein spectrin-like repeat 23; a dystrophin protein spectrin-like repeat 24; a dystrophin protein hinge region 4; and a dystrophin protein cysteine-rich domain.

[0032] In some aspects, the polypeptides encoded by the polynucleotides disclosed herein comprise: the GoA-DysABD1; dystrophin protein spectrin-like repeats 1, 2, 3, and 24; dystrophin protein hinge regions 1, 3, and 4; a dystrophin protein cysteine-rich domain; and a dystrophinSUBSTITUTE SHEET (RULE 26)protein carboxyl-terminus. In some aspects, the polypeptides encoded by the polynucleotides disclosed herein from amino-terminus to carboxyl-terminus consists of: the GoA-DysABD1; a dystrophin protein hinge region 1; a dystrophin protein spectrin-like repeat 1; a dystrophin protein spectrin-like repeat 2; a dystrophin protein spectrin-like repeat 3; a dystrophin protein hinge region 3; a dystrophin protein spectrin-like repeat 24; a dystrophin protein hinge region 4; a dystrophin protein cysteine-rich domain; and a dystrophin protein CTD.

[0033] In some aspects, the polypeptides encoded by the polynucleotides disclosed herein are capable of linking a subsarcolemmal cytoskeleton with the extracellular matrix. In some aspects, the polypeptides encoded by the polynucleotides disclosed herein are capable of recruiting a dystrophin-associated protein complex.

[0034] In some aspects, the polynucleotides disclosed herein are DNA polynucleotides, an RNA polynucleotides, or a combination thereof.

[0035] In some aspects, provided herein are expression cassettes comprising the polynucleotides disclosed herein. In some aspects, the expression cassettes disclosed herein further comprise a promoter, a Kozak sequence, an enhancer, a silencer, a polyA tail, a poly adenylation signal, a 3'-UTR, a 5'-UTR, an intronic sequence, a nucleotide sequence encoding a molecular tag, a nucleotide sequence encoding a self-cleaving peptide, a linker, a filler sequence, or any combination thereof. In some aspects, the promoter is a constitutive promoter, a regulatable promoter, an ubiquitous promoter, or a tissue-specific promoter. In some aspects, the promoter is a constitutive promoter. In some aspects, the promoter is selected from the group consisting of a CBA promoter, a CMV promoter, an EFla promoter, or a CAG promoter. In some aspects, the promoter is a muscle tissue-specific promoter. In some aspects, the muscle tissue-specific promoter is selected from the group consisting of a MHCK7 promoter, a CK8e promoter, a MCK promoter, a dMCK promoter, a tMCK promoter, a DES promoter, a HSA promoter, a SPc5-12 promoter, a SP-301 promoter, a MHC promoter, a Sk-CRM promoter, a Sk-CRM4 promoter. In some aspects, the muscle tissue-specific promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 283. In some aspects, the intronic sequence is a CAG intron, an SV40 intron, MVM intron, or a human beta-globin intron, or any combination thereof. In some aspects, the intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 287-289. In some aspects, the poly(A) sequence is selected from a bGHpA, a hGHpA, a SV40pA, or a synthetic pA. In some aspects, theSUBSTITUTE SHEET (RULE 26)poly(A) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 290.

[0036] In some aspects, provided herein are vectors comprising the polynucleotides or the expression cassettes disclosed herein. In some aspects, the vectors disclosed herein are a non-viral vector or a viral vector. In some aspects, the viral vectors are AAV vectors. In some aspects, vectors disclosed herein further comprise a nucleotide sequence encoding a first inverted terminal repeat (ITR) and a second ITR. In some aspects, the first ITR is at the 5' of the polynucleotide and a second ITR is at the 3' of the polynucleotide. In some aspects, the first and the second ITR are of a same or of a different serotype. In some aspects, the serotype of the ITRs is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVRH10, AAV11, or AAV12 serotype. In some aspects, the first and the second ITR comprise a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 294-295. In some aspects, the vectors disclosed herein further comprise a nucleotide sequence encoding a selectable marker.

[0037] In some aspects, provided herein are recombinant adeno-associated virus (rAAV) particles, comprising the vectors disclosed herein and a capsid. In some aspects, the serotype of the capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV6P1, AAV7, AAV8, AAV8P1, AAV9, AAVrhlO, AAVrhlOPl, AAVS10P4, AAVpol, AAV11, AAV12, AAV-DJ, AAV-DJ / 8, AAV-PHP.Eb, AAV-PHP S, AAV-PHP.B, AAV2-retro, AAV2- QuadYF, AAV2.7m8, AAVS1, AAVS10, AAVrh74, AAVS1, AAVS10, AAVH15, AAVMYO (AAV9P1), AAVMYO 2 (AAVS1P1), AAVMY03 (AAVS10P1), AAV9-RGD, MyoAAVlA, MyoAAV 1C, MyoAAV IE, MyoAAV 2 A, MyoAAV 2E, MyoAAV 3 A, MyoAAV 4 A, MyoAAV 4C, or MyoAAV 4E. In some aspects, the serotype of the capsid is selected from AAV1, AAV8, AAV9, AAVrh74, AAVS1, AAVS10, AAVH15, AAVMYO (AAV9P1), AAVMYO 2 (AAVS1P1), AAVMYO3 (AAVS10P1), AAV9-RGD, MyoAAVlA, MyoAAV 1C, MyoAAV IE, MyoAAV 2A, MyoAAV 2E, MyoAAV 3 A, MyoAAV 4A, MyoAAV 4C, or MyoAAV 4E. In some aspects, the serotype of the capsid is AAVrh74 serotype.

[0038] In some aspects, provided herein are polypeptides encoded by the polynucleotides disclosed herein, by the expression cassettes disclosed herein, by the vectors disclosed herein, or by the rAAV particles disclosed herein.

[0039] In some aspects, provided herein are engineered dystrophin protein actin-binding domains 1 (GoA-DysABD1), or an actin-binding fragment thereof, encoded by the polynucleotidesSUBSTITUTE SHEET (RULE 26)disclosed herein, by the expression cassettes disclosed herein, by the vectors disclosed herein, or by the rAAV particles disclosed herein.

[0040] In some aspects, provided herein are engineered dystrophin proteins or an actin- binding fragment thereof, comprising the polypeptides or the GoA-DysABD1 encoded by the polynucleotides disclosed herein, by the expression cassettes disclosed herein, by the vectors disclosed herein, or by the rAAV particles disclosed herein.

[0041] In some aspects, provided herein are polypeptides comprising a dystrophin protein actin-binding domain 1 modified to achieve an increased actin-binding affinity (GoA-DysABD1), or an actin-binding fragment thereof, wherein: 1) the GoA-DysABD1, or an actin-binding fragment thereof, has at least one amino acid substitution compared to a same dystrophin protein actin- binding domain 1, or an actin-binding fragment thereof, absent the modification to achieve the increased actin-binding affinity (NotGoADysABD1); and 2) wherein in a fluorescence resonance energy transfer (FRET) assay the GoA-DysABD1, or an actin-binding fragment thereof, binds to actin with a dissociation constant (Kd) reduced at least 20%, 50%, 80%, or 99.9% compared to a dissociation constant of the NotGoADysABD1 in a same FRET assay.

[0042] In some aspects, in the FRET assay, the actin is a phalloidin-stabilized F-actin. In some aspects, an Alexa-568 fluorophore is attached to actin (actin-Alexa-568). In some aspects, the Alexa-568 fluorophore is attached to residue C374 of actin.

[0043] In some aspects, in the FRET assay, an mClover3 fluorophore is attached to the GoA-DysABD1 or to the NotGoADysABD1. In some aspects, the mClover3 fluorophore is attached to a carboxy-terminus of the GoA-DysABD1 (GoA-DysABD1-mClover3) or to a carboxy-terminus of the NotGoADysABD1 (NotGoA-DysABD1-mClover3). In some aspects, sub-micromolar amounts of GoA-DysABD1 -mClover3 or of NotGoA-DysABD1-mClover3 are used.

[0044] In some aspects, in the FRET assay, the amount of actin-Alexa-568 is increased over time. In some aspects, in the FRET assay, a fluorescence decay lifetime of the GoA- DysABD1 -mClover3 or of the NotGoA-DysABD1-mClover3 following a sub-nanosecond excitation pulse is measured. In some aspects, the FRET assay is a time-resolved FRET assay.

[0045] In some aspects, provided herein are polypeptides comprising a dystrophin protein actin-binding domain 1 modified to achieve an increased actin-binding affinity (GoA-DysABD1), or an actin-binding fragment thereof, compared to a same dystrophin protein actin-binding domain 1, or an actin-binding fragment thereof, absent the modification to achieve the increased actin-SUBSTITUTE SHEET (RULE 26)binding affinity (NotGoADysABD1), wherein the NotGoA-DysABD1, from N-terminus to C- terminus, comprises an amino acid sequence of SEQ ID NO: 2, wherein: XI 18 is W or R; X133 is Q or P; X165 is D or V; and wherein the modification to achieve the increased actin-binding affinity is at least one amino acid substitution at one or more of residues D9, E12, Q17, K19, T20, F21, S30, Q35, H36, N75, K79, L81, L84, N86, K105, T107, 1114, 1115, V120, M128, W143, R145, Q153, T161, A168, L169, V187, A192, A199, 1202, L207, G208, 1209, L212, D214, E216, or K226. In some aspects, the NotGoADysABD1, from N-terminus to C-terminus, comprises an amino acid sequence of SEQ ID NO: 1.

[0046] In some aspects, the GoA-DysABD1, or an actin-binding fragment thereof, comprised in the polypeptides disclosed herein comprise 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, 8 amino acid substitutions, 9 amino acid substitutions, 10 amino acid substitutions compared to the NotGoADysABD1, or an actin-binding fragment thereof. In some aspects, the GoA-DysABD1, or an actin-binding fragment thereof, comprise 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions compared to the NotGoADysABD1, or an actin-binding fragment thereof.

[0047] In some aspects, the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (E12D), A at residue 17 (Q17A), E at residue 19 (K19E), D at residue19 (K19D), A at residue 20 (T20A), Q at residue 20 (T20Q), N at residue 20 (T20N), S at residue20 (T20S), M at residue 20 (T20M), V at residue 20 (T20V), L at residue 20 (T20L), I at residue 20 (T20I), L at residue 21 (F21L), A at residue 21 (F21A), V at residue 21 (F21V), I at residue 21 (F21I), Q at residue 30 (S30Q), S at residue 30 (S30N), T at residue 30 (S30T), M at residue 30 (S30M), R at residue 35 (Q35R), K at residue 35 (Q35K), R at residue 36 (H36R), K at residue 36 (H36K), R at residue 75 (N75R), K at residue 75 (N75K), N at residue 79 (K79N), Q at residue 79 (K79Q), S at residue 79 (K79S), T at residue 79 (K79T), M at residue 79 (K79M), P at residue 81 (L81P), S at residue 84 (L84S), N at residue 84 (L84N), Q at residue 84 (L84Q), T at residue 84 (L84T), M at residue 84 (L84M), G at residue 86 (N86G), N at residue 105 (K105N), Q at residue 105 (K105Q), S at residue 105 (K105S), T at residue 105 (KI05T), M at residue 105 (K105M), G at residue 107 (T107G, L at residue 114 (I114L), E at residue 114 (I114E), A at residue 114 (I114A), V at residue 114 (Il 14V), D at residue 114 (Il 14D), S at residue 115 (1115 S), N at residue 115 (I115N), Q at residue 115 (Il 15Q), T at residue 115 (I115T), M at residue 115 (I115M), P at residue 120 (V120P), A at residue 120 (VI 20 A), L at residue 120 (V120L), I at residue 120SUBSTITUTE SHEET (RULE 26)(V120I), K at residue 128 (M128K), R at residue 128 (M128R), R at residue 143 (W143R), K at residue 143 (W143K), V at residue 145 (R145V), A at residue 145 (R145A),L at residue 145 (R145L), I 145(R145I), H at residue 153 (Q153H), Y at residue 153 (Q153Y), W at residue 153 (Q153W), F at residue 153 (Q153F), I at residue 156 (V156I), A at residue 156 (V156A), L at residue 156 (V156L); H at residue 161 (T161H), Y at residue 161 (T161Y), W at residue 161 (T161W), F at residue 161 (T161F), V at residue 168 (A168V), I at residue 168 (A168I), L at residue 168 (A168L), S at residue 169 (L169S), N at residue 169 (L169N), Q at residue 169 (L169Q), T at residue 169 (L169T), M at residue 169 (L169M), G at residue 187 (V187G), , L at residue 192 (A192L), V at residue 192 (A192V), I at residue 192 (A192I), V at residue 199 (A199V), L at residue 199 (A199L), I at residue 199 (A199I), G at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue 207 (L207S), Q at residue 207 (L207Q), T at residue 207 (L207T), M at residue 207 (L207M), A at residue 207 (L207A), I at residue 207 (L207I), Y at residue 207 (L207Y), W at residue 207 (L207W), F at residue 207 (L207F), E at residue 207 (L207E), P at residue 208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (I209V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue 209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), H at residue 209 (I209H), Y at residue 209 (I209Y), A at residue 209 (I209A), N at residue 209 (I209N), T at residue 209 (I209T), M at residue 209 (I209M), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), R at residue 216 (E216R), E at residue 226 (K226E), or D at residue 226 (K226D), or any combination thereof.

[0048] In some aspects, the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (E12D), A at residue 17 (Q17A), E at residue 19 (K19E), A at residue 20 (T20A), Q at residue 20 (T20Q), L at residue 21(F21L), A at residue 21 (F21A), Q at residue 30 (S30Q), R at residue 35 (Q35R), R at residue 36 (H36R), R at residue 75 (N75R), N at residue 79 (K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 86 (N86G), N at residue 105 (K105N), G at residue 107 (T107G), L at residue 114 (I114L), E at residue 114 (I114E), S at residue 115 (I115S), P at residue 120 (V120P), A at residue 120 (V120A), K at residue 128 (M128K), R at residue 143 (W143R), V at residue 145 (R145V), H at residue 153 (Q153H), I at residue 156 (V156I), H at residue 161 (T161H), V at residue 168 (A168V), I at residue 168 (A168I), S at residue 169 (L169S), G at residue 187 (V187G), , L at residue 192 (A192L), V atSUBSTITUTE SHEET (RULE 26)residue 199 (Al 99V), I at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue 207 (L207S), P at residue 208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (I209V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue 209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226(K226E), or any combination thereof.

[0049] In some aspects, the at least one amino acid substitution consists of one amino acid substitution. In some aspects, the one amino acid substitution is selected from Q at residue 20 (T20Q), Q at residue 30 (S30Q), N at residue 79 (K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 107 (T107G), L at residue 114 (Il 14L), E at residue 114 (Il 14E), S at residue 115 (Il 15S), P at residue 120 (V120P), R at residue 143 (W143R), H at residue 153 (Q153H), I at residue 156 (V156I), H at residue 161 (T161H), I at residue 168 (A168I), G at residue 187 (V187G), , L at residue 192 (A192L), V at residue 199 (A199V), I at residue 202 (I202G), V at residue 207 (L207V), P at residue 208 (G208P), K at residue 209 (I209K), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226 (K226E). In some aspects, the one amino acid substitution is Q at residue 20 (T20Q), P at residue 120 (V120P), I at residue 156 (V156I), P at residue 212 (L212P), P at residue 214 (D214P), E at residue 226 (K226E). In some aspects, the polypeptides disclosed herein comprise an amino acid sequence of SEQ ID NOs: 5-30. In some aspects, the polypeptides disclosed herein are encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 144-169.

[0050] In some aspects, the at least one amino acid substitution consists of two amino acid substitutions. In some aspects, the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P-K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); N at residue 207 and L at residue 209 (L207N-I209L);SUBSTITUTE SHEET (RULE 26)R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); D at residue 207 and V at residue 209 (L207D-I209V); G at residue 187 and P at residue 212 (V187G-L212P); Q at residue 20 and I at residue 156 (T20Q-V156I); Q at residue 20 and P at residue 214 (T20Q-D214P); P at residue 120 and E at residue 226 (V120P-K226E); I at residue 156 and P at residue 214 (V156I- D214P); P at residue 120 and G at residue 187 (V120P-V187G); P at residue 212 and P at residue 214 (L212P-D214P); P at residue 214 and E at residue 226 (D214P-K226E); G at residue 187 and E at residue 226 (V187G-K226E); Q at residue 20 andP at residue 120 (T20Q-V120P); l at residue 156 and P at residue 212 (V156I-L212P); P at residue 120 and I at residue 156 (V120P-V156I); Q at residue 20 and P at residue 212 (T20Q-L212P); P at residue 120 and P at residue 214 (V120P- D214P); G at residue 187 and P at residue 214 (V187G-D214P); Q at residue 20 and G at residue 187 (T20Q-V187G); P at residue 120 and P at residue 212 (V120P-L212P); Q at residue 20 and E at residue 226 (T20Q-K226E); I at residue 156 and G at residue 187 (V156I-V187G); I at residue 156 and E at residue 226 (V156I-K226E); P at residue 212 and E at residue 226 (L212P-K226E); A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P- K226E); or R at residue 35 and K at residue 207 (Q35R-L207K). In some aspects, the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P-K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); N at residue 207 and L at residue 209 (L207N-I209L); R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); or D at residue 207 and V at residue 209 (L207D-I209V). In some aspects, the two amino acid substitutions are selected from A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P-K226E); or R at residue 35 and K at residue 207 (Q35R-L207K). In some aspects, the polypeptides disclosed herein comprise an amino acid sequence of SEQ ID NOs: 31-69. In some aspects, the polypeptides disclosed herein are encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 170-208.SUBSTITUTE SHEET (RULE 26)

[0051] In some aspects, the at least one amino acid substitution consists of three amino acid substitutions. In some aspects, the three amino acid substitutions are selected from G at residue 187, P at residue 214, E at residue 226 (V187G-D214P-K226E); Q at residue 20, P at residue 120, and G at residue 187 (T20Q-V120P-V187G); Q at residue 20, P at residue 120, and E at residue 226 (T20Q-V120P-K226E); Q at residue 20, G at residue 187, and E at residue 226 (T20Q-V187G- K226E); P at residue 120, I at residue 156, and P at residue 214 (V120P-V156I-D214P); P at residue 212, P at residue 214, and E at residue 226 (L212P-D214P-K226E); P at residue 120, G at residue 187, and E at residue 226 (V120P-V187G-K226E); P at residue 120, P at residue 212, and P at residue 214 (V120P-L212P-D214P); I at residue 156, P at residue 212, and P at residue 214 (V156I-L212P-D214P); Q at residue 20, I at residue 156, and P at residue 212 (T20Q-V156I- L212P); I at residue 156, G at residue 187, and E at residue 226 (V156I-V187G-K226E); Q at residue 20, P at residue 214, and E at residue 226 (T20Q-D214P-K226E); P at residue 120, P at residue 214, and E at residue 226 (V120P-D214P-K226E); Q at residue 20, P at residue 120, and P at residue 214 (T20Q-V120P-D214P); Q at residue 20, P at residue 120, and I at residue 156 (T20Q-V120P-V156I); Q at residue 20, G at residue 187, and P at residue 214 (T20Q-V187G- D214P); P at residue 120, l at residue 156, andP at residue 212 (V120P-V156I-L212P); l at residue 156, P at residue 214, and E at residue 226 (V156I-D214P-K226E); G at residue 187, P at residue 212, and E at residue 226 (V187G-L212P-K226E); P at residue 120, G at residue 187, and P at residue 214 (V120P-V187G-D214P); Q at residue 20, 1 at residue 156, and G at residue 187 (T20Q- V156I-V187G); I at residue 156, G at residue 187, and P at residue 214 (V156I-V187G-D214P); Q at residue 20, and I at residue 156, E226 (T20Q-V156I-K226E); Q at residue 20, P at residue 212, and E at residue 226 (T20Q-L212P-K226E); Q at residue 20, P at residue 120, and P at residue 212 (T20Q-V120P-L212P); Q at residue 20, G at residue 187, and P at residue 212 (T20Q-V187G- L212P); P at residue 120, I at residue 156, and E at residue 226 (V120P-V156I-K226E); P at residue 120, 1 at residue 156, and G at residue 187 (V120P-V156I-V187G); P at residue 120, P at residue 212, and E at residue 226 (V120P-L212P-K226E); P at residue 120, G at residue 187, and P at residue 212 (V120P-V187G-L212P); G at residue 187, P at residue 212, and P at residue 214 (V187G-L212P-D214P); I at residue 156, G at residue 187, and P at residue 212 (V156I-V187G- L212P); Q at residue 20, 1 at residue 156, and P at residue 214 (T20Q-V156LD214P); I at residue 156, P at residue 212, and E at residue 226 (V156I-L212P-K226E); Q at residue 20, P at residue 212, and P at residue 214 (T20Q-L212P-D214P); A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N-V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L-SUBSTITUTE SHEET (RULE 26)I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R-A168V). In some aspects, the three amino acid substitutions are selected from A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N-V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L-I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R- A168V). In some aspects, the polypeptides disclosed herein comprise an amino acid sequence of SEQ ID NOs: 70-107. In some aspects, the polypeptides disclosed herein are encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 209-246.

[0052] In some aspects, the at least one amino acid substitution consists of four amino acid substitutions. In some aspects, the four amino acid substitutions are selected from Q at residue 20, P at residue 120, Gat residue 187, andP at residue 212 (T20Q-V120P-V187G-L212P); Q at residue 20, P at residue 120, P at residue 212, and P at residue 214 (T20Q-V120P-L212P-D214P); Q at residue 20, 1 at residue 156, G at residue 187, and P at residue 214 (T20Q-V156I-V187G-D214P); Q at residue 20, P at residue 212, P at residue 214, and E at residue 226 (T20Q-L212P-D214P- K226E); P at residue 120, 1 at residue 156, G at residue 187, and P at residue 212 (V120P-V156I- V187G-L212P); I at residue 156, G at residue 187, P at residue 212, and E at residue 226 (V156I- V187G-L212P-K226E); P at residue 120, 1 at residue 156, P at residue 212, and E at residue 226 (V120P-V156I-L212P-K226E); Q at residue 20, G at residue 187, P at residue 212, and P at residue 214 (T20Q-V187G-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and E at residue 226 (V120P-V187G-L212P-K226E); Q at residue 20, I at residue 156, P at residue 212, and E at residue 226 (T20Q-V156I-L212P-K226E); Gat residue 187, P at residue 212, P at residue 214, and E at residue 226 (V187G-L212P-D214P-K226E); Q at residue 20, P at residue 120, G at residue 187, and E at residue 226 (T20Q-V120P-V187G-K226E); Q at residue 20, P at residue 120, I at residue 156, and P at residue 212 (T20Q-V120P-V156I-L212P); Q at residue 20, P at residue 120, P at residue 214, and E at residue 226 (T20Q-V120P-D214P-K226E); Q at residue 20, I at residue 156, G at residue 187, and P at residue 212 (T20Q-V156I-V187G-L212P); I at residue 156, G at residue 187, P at residue 212, and P at residue 214 (V156I-V187G-L212P- D214P); P at residue 120, 1 at residue 156, G at residue 187, and E at residue 226 (V120P-V156I- V187G-K226E); P at residue 120, 1 at residue 156, P at residue 212, and P at residue 214 (V120P- V156I-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and P at residue 214 (V120P-V187G-L212P-D214P); Q at residue 20, G at residue 187, P at residue 214, and E atSUBSTITUTE SHEET (RULE 26)residue 226 (T20Q-V187G-D214P-K226E); Q at residue 20, 1 at residue 156, P at residue 212, and P at residue 214 (T20Q-V156I-L212P-D214P); I at residue 156, P at residue 212, P at residue 214, and E at residue 226 (V156I-L212P-D214P-K226E); Q at residue 20, P at residue 120, Gat residue 187, and P at residue 214 (T20Q-V120P-V187G-D214P); Q at residue 20, P at residue 120, I at residue 156, and G at residue 187 (T20Q-V120P-V156I-V187G); I at residue 156, G at residue 187, P at residue 214, and E at residue 226 (V156I-V187G-D214P-K226E); Q at residue 20, P at residue 120, 1 at residue 156, and E at residue 226 (T20Q-V120P-V156I-K226E); P at residue 120, I at residue 156, P at residue 214, and E at residue 226 (V120P-V156I-D214P-K226E); Q at residue 20, 1 at residue 156, G at residue 187, and E at residue 226 (T20Q-V156I-V187G-K226E); Q at residue 20, P at residue 120, P at residue 212, and E at residue 226 (T20Q-V120P-L212P- K226E); P at residue 120, G at residue 187, P at residue 214, and E at residue 226 (V120P-V 187G- D214P-K226E); P at residue 120, P at residue 212, P at residue 214, and E at residue 226 (V120P- L212P-D214P-K226E); Q at residue 20, G at residue 187, P at residue 212, and E at residue 226 (T20Q-V187G-L212P-K226E); P at residue 120, I at residue 156, G at residue 187, and P at residue 214 (V120P-V156I-V187G-D214P); Q at residue 20, I at residue 156, P at residue 214, and E at residue 226 (T20Q-V156I-D214P-K226E); Q at residue 20, P at residue 120, 1 at residue 156, and P at residue 214 (T20Q-V120P-V156I-D214P); or A at residue 17, A at residue 20, P at residue 212, and E at residue 226 (Q17A-T20A-L212P-K226E). In some aspects, the four amino acid substitutions are A at residue 17, A at residue 20, P at residue 212, and E at residue 226 (Q17A-T20A-L212P-K226E). In some aspects, the polypeptides disclosed herein comprise an amino acid sequence at of SEQ ID NO: 108-143. In some aspects, the polypeptides disclosed herein are encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 248-282.

[0053] In some aspects, the polypeptides disclosed herein further comprise one or more additional portions of a dystrophin protein. In some aspects, the polypeptides disclosed herein further comprise: at least one dystrophin protein spectrin-like repeat located downstream of the ABD 1 domain; and at least one cysteine-rich domain located downstream of the dystrophin protein spectrin-like repeat.

[0054] In some aspects, the dystrophin protein spectrin-like repeat is a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein spectrin-like repeat 4, a dystrophin protein spectrin-like repeat 5, aSUBSTITUTE SHEET (RULE 26)dystrophin protein spectrin-like repeat 6, a dystrophin protein spectrin-like repeat 7, a dystrophin protein spectrin-like repeat 8, a dystrophin protein spectrin-like repeat 9, a dystrophin protein spectrin-like repeat 10, a dystrophin protein spectrin-like repeat 11, a dystrophin protein spectrinlike repeat 12, a dystrophin protein spectrin-like repeat 13, a dystrophin protein spectrin-like repeat 14, a dystrophin protein spectrin-like repeat 15, a dystrophin protein spectrin-like repeat 16, a dystrophin protein spectrin-like repeat 17, a dystrophin protein spectrin-like repeat 18, a dystrophin protein spectrin-like repeat 19, a dystrophin protein spectrin-like repeat 20, a dystrophin protein spectrin-like repeat 21, a dystrophin protein spectrin-like repeat 22, a dystrophin protein spectrinlike repeat 23, or a dystrophin protein spectrin-like repeat 24.

[0055] In some aspects, the spectrin-like repeat 1 comprises amino acids 339-447 of SEQ ID NO: 4; the spectrin-like repeat 2 comprises amino acids 448-556 of SEQ ID NO: 4; the spectrinlike repeat 3 comprises amino acids 559-667 of SEQ ID NO: 4; the spectrin-like repeat 4 comprises amino acids 719-828 of SEQ ID NO: 4; the spectrin-like repeat 5 comprises amino acids 830-934 of SEQ ID NO: 4; the spectrin-like repeat 6 comprises amino acids 943-1045 of SEQ ID NO: 4; the spectrin-like repeat 7 comprises amino acids 1048-1154 of SEQ ID NO: 4; the spectrin-like repeat 8 comprises amino acids 1157-1263 of SEQ ID NO: 4; the spectrin-like repeat 9 comprises amino acids 1266-1367 of SEQ ID NO: 4; the spectrin-like repeat 10 comprises amino acids 1368- 1463 of SEQ ID NO: 4; the spectrin-like repeat 11 comprises amino acids 1468-1568 of SEQ ID NO: 4; the spectrin-like repeat 12 comprises amino acids 1571-1676 of SEQ ID NO: 4; the spectrinlike repeat 13 comprises amino acids 1679-1778 of SEQ ID NO: 4; the spectrin-like repeat 14 comprises amino acids 1779-1874 of SEQ ID NO: 4; the spectrin-like repeat 15 comprises amino acids 1877-1979 of SEQ ID NO: 4; the spectrin-like repeat 16 comprises amino acids 1992-2101 of SEQ ID NO: 4; the spectrin-like repeat 17 comprises amino acids 2104-2208 of SEQ ID NO: 4; the spectrin-like repeat 18 comprises amino acids 2211-2318 of SEQ ID NO: 4; the spectrin-like repeat 19 comprises amino acids 2319-2423 of SEQ ID NO: 4; the spectrin-like repeat 20 comprises amino acids 2475-2577 of SEQ ID NO: 4; the spectrin-like repeat 21 comprises amino acids 2580-2686 of SEQ ID NO: 4; the spectrin-like repeat 22 comprises amino acids 2689-2802 of SEQ ID NO: 4; the spectrin-like repeat 23 comprises amino acids 2808-2930 of SEQ ID NO: 4; the spectrin-like repeat 24 comprises amino acids 2935-3040 of SEQ ID NO: 4. In some aspects, the cysteine-rich domain comprises amino acids 3113-3360 of SEQ ID NO: 4.

[0056] In some aspects, the polypeptides disclosed herein further comprise at least one dystrophin protein hinge region located downstream of the ABDI and upstream of the cysteine-SUBSTITUTE SHEET (RULE 26)rich domain. In some aspects, the dystrophin protein hinge region is a dystrophin protein hinge region 1, a dystrophin protein hinge region 2, a dystrophin protein hinge region 3, or a dystrophin protein hinge region 4. In some aspects, the hinge region 1 comprises amino acids 247-338 of SEQ ID NO: 4; the hinge region 2 comprises amino acids 668-718 of SEQ ID NO: 4; the hinge region 3 comprises amino acids 2424-2474 of SEQ ID NO: 4; the hinge region 4 comprises amino acids 3041-3112 of SEQ ID NO: 4.

[0057] In some aspects, the polypeptides disclosed herein further comprise a dystrophin protein carboxy-terminal domain (CTD) located downstream of the cysteine-rich domain. In some aspects, the dystrophin protein carboxyl-terminus comprises amino acids 3361-3685 of SEQ ID NO: 4.

[0058] In some aspects, the polypeptides disclosed herein comprise: the GoA-DysABD1; dystrophin protein spectrin-like repeats 1, 2, 3, and 24; dystrophin protein hinge regions 1, 2, and 4; and a dystrophin protein cysteine-rich domain. In some aspects, the polypeptides disclosed herein from amino-terminus to carboxyl-terminus consists of: the GoA-DysABD1; a dystrophin protein hinge region 1; a dystrophin protein spectrin-like repeat 1; a dystrophin protein spectrinlike repeat 2; a dystrophin protein spectrin-like repeat 3; a dystrophin protein hinge region 2; a dystrophin protein spectrin-like repeat 24; a dystrophin protein hinge region 4; and a dystrophin protein cysteine-rich domain.

[0059] In some aspects, the polypeptides disclosed herein comprise: the GoA-DysABD1; dystrophin protein spectrin-like repeats 1, 2, 22, 23, and 24; dystrophin protein hinge regions 1, 3, and 4; and a dystrophin protein cysteine-rich domain. In some aspects, the polypeptides disclosed herein from amino-terminus to carboxyl-terminus consists of: the GoA-DysABD1; a dystrophin protein hinge region 1; a dystrophin protein spectrin-like repeat 1; a dystrophin protein spectrinlike repeat 22; a dystrophin protein spectrin-like repeat 23; a dystrophin protein spectrin-like repeat 24; a dystrophin protein hinge region 4; and a dystrophin protein cysteine-rich domain.

[0060] In some aspects, the polypeptides disclosed herein comprise: the GoA-DysABD1; dystrophin protein spectrin-like repeats 1, 16, 17, 23, and 24; dystrophin protein hinge regions 1, and 4; and a dystrophin protein cysteine-rich domain. In some aspects, the polypeptides disclosed herein from amino-terminus to carboxyl-terminus consists of: the GoA-DysABD1; a dystrophin protein hinge region 1; a dystrophin protein spectrin-like repeat 1; a dystrophin protein spectrinlike repeat 16; a dystrophin protein spectrin-like repeat 17; a dystrophin protein spectrin-like repeatSUBSTITUTE SHEET (RULE 26)23; a dystrophin protein spectrin-like repeat 24; a dystrophin protein hinge region 4; and a dystrophin protein cysteine-rich domain.

[0061] In some aspects, the polypeptides disclosed herein comprise: the GoA-DysABD1; dystrophin protein spectrin-like repeats 1, 2, 3, and 24; dystrophin protein hinge regions 1, 3, and 4; a dystrophin protein cysteine-rich domain; and a dystrophin protein carboxyl-terminus. In some aspects, the polypeptides disclosed herein from amino-terminus to carboxyl-terminus consists of: the GoA-DysABD1; a dystrophin protein hinge region 1; a dystrophin protein spectrin-like repeat 1; a dystrophin protein spectrin-like repeat 2; a dystrophin protein spectrin-like repeat 3; a dystrophin protein hinge region 3; a dystrophin protein spectrin-like repeat 24; a dystrophin protein hinge region 4; a dystrophin protein cysteine-rich domain; and a dystrophin protein carboxyl- terminus.

[0062] In some aspects, the polypeptides disclosed herein are capable of linking a subsarcolemmal cytoskeleton with the extracellular matrix. In some aspects, the polypeptides disclosed herein are capable of recruiting a dystrophin-associated protein complex.

[0063] In some aspects, provided herein are dystrophin protein actin-binding domains consisting of the polypeptide disclosed herein. In some aspects, provided herein are engineered dystrophin protein comprising the polypeptides disclosed herein or the GoA-DysABD1 comprised in the polypeptides disclosed herein.

[0064] In some aspects, provided herein are pharmaceutical formulations comprising the polynucleotides, the expression cassettes, the vectors, the rAAV particles, the polypeptides, the GoA-DysABD1, or the engineered dystrophin proteins, disclosed herein, or any combination thereof and at least one pharmaceutically acceptable excipient or adjuvant.

[0065] In some aspects, provided herein are methods of increasing the amount of functional dystrophin protein in a cell comprising contacting the cell with the polynucleotides, the expression cassettes, the vectors, the rAAV particles, the polypeptides, the GoA-DysABD1, the engineered dystrophin proteins, or the pharmaceutical formulations, disclosed herein, or any combination thereof. In some aspects, the cell is a muscle cell. In some aspects, the cell is a human cell. In some aspects, the cell is comprised in a subject. In some aspects, the subjects suffers from a muscular dystrophy. In some aspects, the muscular dystrophy is a dystrophin-deficient muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, the amount of functional dystrophin protein in the cell is increased of at least 0.1 -fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-SUBSTITUTE SHEET (RULE 26)fold compared to a same cell not administered the polynucleotides, the expression cassettes, the vectors, the rAAV particles, the polypeptides, the GoA-DysABD1, the engineered dystrophin proteins, the pharmaceutical formulations, disclosed herein, any combination thereof.

[0066] In some aspects, provided herein are methods of treating a muscular dystrophy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polynucleotides, the expression cassettes, the vectors, the rAAV particles, the polypeptides, the GoA-DysABD1, the engineered dystrophin proteins, or the pharmaceutical formulations, disclosed herein, or any combination thereof. In some aspects, the muscular dystrophy is a dystrophin-deficient muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy. In some aspects, a degeneration of a muscle of the subject is reduced of at least 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, or 10-fold. In some aspects, the muscle is a smooth muscle, a skeletal muscle, a cardiac muscle, or any combination thereof. In some aspects, a symptom of the muscular dystrophy in the subject is reduced. In some aspects, the muscular dystrophy in the subject is ameliorated. In some aspects, the muscular dystrophy in the subject is treated. In some aspects, the subject is a human.BRIEF DESCRIPTION OF DRAWINGS

[0067] FIG. 1 shows a schematic of the ptd68 ABDI expression constructs used in this study. 6His-Sumo: six histidine sumo tag attached to the N-terminus of all constructs, is removed by incubation with ULP1 digestion. ABDI : Dystrophin or Utrophin actin-binding domain 1. mClover3: GFP fluorescent protein variant attached to ABD1s for measurement of actin-binding by FRET. Position of translation start, unique multiple cloning sites flanking tags and ABD1s, and tandem stop codons to terminate translation are indicated.

[0068] FIGs. 2A-F show binding of ABDI proteins to actin measured by actin-high-speed cosedementation (HSC). FIGs. 2A-D: Fluorescence total protein staining of SDS-PAGE gels containing HSC supernatants (S) and pellets (P). Samples contain constant amounts of indicated ABDI proteins and increasing amounts of phalloidin stabilized F-actin. FIGs. 2E-F: Quantitation of the fraction of indicated Dys-ABD1 (FIG. 2E) and Utr-ABD1 (FIG. 2F) proteins sedimenting with actin calculated according to the equation F = P / (P+S) where F is the fraction ABDI bound to actin, P and S are the amount of ABDI protein present in the pellet and supernatant respectively.SUBSTITUTE SHEET (RULE 26)S and P are determined by the total fluorescence by LICOR imaging of the ABD 1 containing band. Lines represent hyperbolic binding model fits to the respective data.

[0069] FIGs. 3A-C show time-resolved FRET assay measuring binding of mClover3 tagged ABDI proteins to Alexa-568 labeled phalloidin stabilized F-actin. FIG. 3A: schematic of the experiment. FIG. 3B: Examples of time-resolved fluorescence waveforms reporting differences in actin-binding. FIG. 3C: Fluorescence lifetime in a time-resolved fluorescence measurement, determined by fitting individual decay curves with a single exponential function convolved with the measured instrument response function (Muretta 2010 Rev. Sci. Inst). Decreasing lifetime is proportional to increasing FRET calculated according to the energy transfer equation E = 1-DA / D where DA is the fluorescence lifetime of the donor probe in the presence of the acceptor probe and D is the lifetime of the donor probe in the absence of the acceptor probe. (C) FRET measured for N- and C-terminally tagged Dys-ABD1 proteins showing that the C- terminal mClover3 tag attachment exhibits the largest relative change in FRET at saturating [Actin] and that the mClover3 probe does not interact with actin when it is not attached to ABDI proteins.

[0070] FIGs. 4A-B show measurement of actin-binding of GoA candidates examined by HSC (FIG. 4A) or FRET (FIG. 4B). Data points represent Mean of N experiments, error bars represent Standard Deviation of N replicate experiments. Lines indicate non-constrained single hyperbolic binding model used to determine maximum HSC (Bmax) and FRET (Emax) and apparent dissociation constants (Kd).

[0071] FIGs. 5A-B show measurement of actin-binding by T20Q identified by in silico modeling examined by HSC (FIG. 5A) or FRET (FIG. 5B). Data points represent Mean of N experiments, error bars represent Standard Deviation of N replicate experiments. Lines indicate non-constrained single hyperbolic binding model used to determine maximum HSC (Bmax) and FRET (Emax) and apparent dissociation constants (Kd).

[0072] FIG. 6 shows self-sedimentation of ABDI proteins. Bars represent Mean of N HSC experiments. Error bars represent Standard Deviation of N HSC experiments.

[0073] FIGs. 7A-B show molecular weight standard calibration of size-exclusion chromatography of ABDI proteins. FIG. 7A: Bio- Rad SEC molecular weight standards used to calibrate SEC elution. FIG. 7B: Elution profiles of Bio-Rad Molecular Weight standard, Dys- ABD1 -mClover3, or Utr-ABD1-mClover3, as indicated. Relative abundance of ABDI protein species determined by integration of the UV 280 nm absorbance after peak fitting using Bio-Rad chromatogram analysis software provided with the NGC instrument used in this study.SUBSTITUTE SHEET (RULE 26)

[0074] FIGs. 8A-D show size-exclusion chromatography of select ABDI proteins. FIG. 8A: Representative chromatograms of Dys-ABD1-mClover3 (WT-ABD1) or Dys-ABD1- mClover3 containing two GoA candidate mutations L212P-K226E, as indicated. FIG. 8B: Actin- binding measured by HSC of Dys-ABD1-mClover3 (WT) before SEC or after SEC showing removal of the cleaved His-Sumo tag does not alter actin-binding. FIGs. 8C-D: Pilot studies of actin-binding measured by HSC of L212P or K226E single GoA mutations comparing high- molecular weight and low-molecular weight protein binding to actin showing that the high and low molecular weight species bind actin similarly.

[0075] FIG. 9 shows phage-display workflow. Decision point steps were followed from top to bottom of the workflow diagram.

[0076] FIGs. 10A-B show validation of key steps in actin-binding phage-display selection. FIG. 10A: Anti-actin antibody cross-reactivity detected by ELISA of 50 micromolar biotinylated F-actin bound to NUNC immuno-tube surfaces coated with indicated concentrations of streptavidin (SA). Solid line is a fit to a single site hyperbolic binding model similar to Eq 1. Dotted lines are 95% confidence intervals for the fit. FIG. 10B: Anti-actin ELISA dependence on actin concentration of surfaces coated with 0.1 micrograms / ml streptavidin.

[0077] FIG. 11 shows the gradient purification of ABDI -phage. Anti -His western blotting of 1.3 ml fractions from 9 ml discontinuous gradient of optiprep (1 ml 50%, 1 ml 40%, 1.2 ml 25%, 1.8 ml 15%) overlaid with 1.5 ml of WT-hDys-ABD1 displaying phage (2 x 1013particles / ml) centrifuged overnight at 390,000 ref for 80 minutes. Fraction 4 corresponds to the maximum anti- His reactivity and a phage particle concentration of 2 x 1013 / ml.

[0078] FIG. 12 shows anti-actin ELISA dependence on actin concentration of surfaces coated with 0.1 micrograms / ml streptavidin.

[0079] FIG. 13 shows PCR reactions using primers spanning the ABDI reading frame and template DNA from single TGI cell colonies infected with WT-hDys or WT-hUtr displaying phage.

[0080] FIGs. 14A-B show relative abundance of WT-hDys-ABD1 DNA (bars denoted with *) and WT-hUtr- ABDI DNA (bars not denoted) detected by quantitative PCR of plasmids prepared from TGI glycerol stocks after 1, 2, and 3 rounds of biopanning. Data represent the mean + / - standard deviation of a single biological replicate performed in triplicate (FIG. 14A), and the relative WT-hUtr-DNA content detected by quantitative PCR as performed for panel D after a single round of biopanning performed on mixtures of phage containing 2x, lOx, or lOOx WT-hDysSUBSTITUTE SHEET (RULE 26)phage to WT-hUtr phage. Data represent the mean + / - standard deviation of 3 independent biological replicates (FIG. 14B).

[0081] FIGs. 15A-D show next generation sequence (NGS) characterization of ABDI sitesaturation mutagenesis. FIG. 15A: Short-read amplicon Illumina sequencing using primers that amplified indicated regions of the ABDI reading frame. Symbols indicate the frequency of each amino acid I 10,000 across reading frame. WT residue at each position is amino acid corresponds to symbols near 9,000 / 10,000 reads. * indicates regions that did not sequence. FIG. 15B: Long- read Pac-Bio sequencing using primers that span the ABDI reading frame. Symbols indicate the frequency of amino acids at each position as in FIG. 15A. FIGs. 15C-D: Median frequency + / - median absolute deviation (MAD) of amino acids across the ABDI reading frame in the short-read sequencing from FIG. 1A (FIG. 15C) and in the long-read sequencing from FIG. 15B (FIG. 15D).

[0082] FIG. 16 shows the median amino-acid substitution frequency in the ABDI sitesaturation mutagenesis library from FIGs. 15A-B.

[0083] FIGs. 17A-E show actin-binding ELISA assay. Anti-HA reactivity for conditioned media from phage-display output clones incubated on actin-coated surfaces (FIG. 17A) or blocked surfaces not coated with actin (FIG. 17D). Total ABDI -HA present in the conditioned media detected by anti-HA ELISA (FIG. 17B). Specific binding (Actin - Actin Plate Background / Total - Total Plate Background) computed for each clone. Actin Plate Background determined from the mean of LB alone controls on each plate ("LB" bar). WT-hDys ("hDys" bar) and WT-hUtr ("hUtr" bar) controls included on each plate (FIG. 17C). Correlation between data in panel A and panel B. Select ABDI sequence variants indicated in panel A and E. Clones above the black line bind better than WT-hDys. Clones above the light gray line bind better than WT-hUtr (FIG. 17E).

[0084] FIGs. 18A-B show actin-binding of ABDI variants selected by short-read next generation sequencing of round 5 phage-display biopanning output. (A) Fraction ABDI protein bound to actin measured by high-speed actin cosedimentation (HSC), quantified by fluorescence imaging of HSC supernatants and actin pellets by SDS-PAGE (FIG. 18A). Actin-binding quantified by FRET between mClover3 tagged hDys- ABDI (donor) and Alexa-568 labeled F-actin (acceptor). WT-hDys-mClover3 and WT-hUtr-mClover3 (FIG. 18B).

[0085] FIG. 19 shows actin-binding parameters of variants selected at round 5 by NGS and tested by N = 3 HSC, N = 3 FRET and N = 3 DSF-GTP. For N = 3 experiments, parameter values are the mean value of 3 independently fit data sets + / - standard deviation of those values. Measurements for HSC and DSF-GTP are from the same 3 independent protein preparations.SUBSTITUTE SHEET (RULE 26)FRET measurements are from additional independent preparations. For HSC experiments Kd values were determined fitting Eq. 5 with Bmax unconstrained or constrained to 1.0.

[0086] FIGs. 20A-B show characterization of biopanning output. FIG. 20A: PCR using 4 different primer pairs spanning the ABDI reading frame using WT-hDys template (WT) or plasmids rescued from round 5 - 10 biopanning output (R5 - RIO respectively). Full length ABDI and Truncated ABDI species indicated by arrows. FIG. 20B: Actin-binding ELISA of R6, R8, and RIO biopanning output.

[0087] FIGs. 21A-B show FRET-based prioritization of phage-display candidates. FIG. 21A: Kd values from single biological replicate actin-binding FRET experiments for candidates selected by ELISA. Individual traces for each candidate shown in FIG. 22A-PP. Bars are the fit parameter + / - 95% confidence interval for the fit parameters. FIG. 21B: Correlation plot showing Kd values and Emax values from fit lines in FIG. 22A-PP (hUtr: control; T20Q: Aim 1 control).

[0088] FIGs. 22A-PP show triaging FRET N = 1 individual actin-binding curves. Data from N = 1 independent biological experiment. Each sample contains at least 2 technical replicates. Symbols represent FRET measured at indicated actin-concentrations. Lines represent fits to equation Eq. 4.

[0089] FIGs. 23A-C show actin-binding FRET of N = 3 candidates. FIG. 23A: Actin- dependence of FRET of candidates. Symbols are single data points, lines are fits to Eq. 5. Data are from individual traces in FIGs. 29A-N. FIG. 23B: Kd values for data in A. Bars are fit values for lines in panel A, Error bars are 95% confidence intervals for the fit lines in A. FIG. 23C: Correlation plot showing Kd values and Emax values from fit lines in FIGs. 29A-N. hUtr: control.

[0090] FIG. 24 shows high-speed cosedimentation of candidates. Symbols are the mean + / - standard deviation of 3 independent biological replicates.

[0091] FIG. 25 shows actin-binding parameters N = 3 HSC, N = 3 FRET and N = 3 DSF- GTP. For N = 3 experiments, parameter values are the mean value of 3 independently fit data sets + / - standard deviation of those values. Measurements for HSC and DSF-GTP are from the same 3 independent protein preparations. FRET measurements are from additional independent preparations. For HSC experiments, Kd values were determined fitting Eq. 5 with Bmaxunconstrained or constrained to 1.0.

[0092] FIGs. 26A-H show SDS-PAGE analysis of ABD1-mClover3 proteins used in N = 3 HSC experiments depicted in FIG. 24. FIGs. 26A-B: Coomassie fluorescence imaging detected at 700 nm using a Li-Cor imager. Each lane contains material from a different biological sample.SUBSTITUTE SHEET (RULE 26)FIGs. 26C-H: Fluorescence intensity profiles of material in each lane for the indicated ABDI variants. ABD1-mClover3 and Sumo bands indicated.

[0093] FIGs. 27A-B show quantitation of ABDI protein species in HSC samples based on analysis of data in FIG. 26A-H. FIG. 27A: Total protein in each lane. FIG. 27B: Relative content of ABDI derived species. ABDI + Sumo / Total protein, ABDI I Total Protein, ABDI / (ABDI + Sumo) for indicated sequence variants tested by HSC in FIG. 24.

[0094] FIGs. 28A-E show binding equilibrium simulations and error analysis. FIG. 28A: HSC data from FIG. 24. Symbols are mean + / - standard deviation from 3 independent biological replicate experiments. Lines are fits to Eq. 5. Dotted lines are the results of numerical simulations to the binding equilibrium described by a single binding equilibrium described by in Eq. 6. FIG. 28B: Kd values determined by numerical integration assuming indicated concentrations of total ABDI (x axis). FIGs. 28C-E: Chi squared error surfaces for fitting data in A with indicated total ABDI concentrations. The minimum of the error surfaces indicate the Kd value of each fit performed over a range of assumed ABDI concentrations (1 pM to 12 pM).

[0095] FIGs. 29A-N show actin-binding measured by FRET of N = 3 biological replicates (N = 1 data set with 2 additional replicates). Each replicate consists of at least 2 technical replicates. Symbols represent FRET measured at indicated actin-concentrations. Lines represent fits to equation Eq. 4.

[0096] FIG. 30 shows a summary of the results of the study.

[0097] FIGs. 31A-C show functional outcomes associated with treatment of the DMD mouse model with GO A- ABDI mutant microdystrophin proteins.DETAILED DESCRIPTION OF THE DISCLOSURE

[0098] In some aspects, disclosed herein are polypeptides comprising dystrophin protein actin-binding domains (DysABDs) modified to achieve increased actin-binding affinity, herein also referred to as gain-of-affinity DysABDs, or "GoA-DysABDs," actin-binding fragments thereof, and dystrophin proteins comprising the same, herein also referred to as "GoA-Dys". The disclosure also provides for polynucleotides (e.g., expression cassettes) encoding such polypeptides; vectors comprising such polynucleotides (e.g., AAV vectors comprising such expression cassettes); methods of making those vectors; recombinant AAV (rAAV) particles comprising such vectors; pharmaceutical compositions comprising the polypeptides, the polynucleotides, the vectors, and / or the rAAV particles disclosed herein; and methods of using theSUBSTITUTE SHEET (RULE 26)polypeptides, the polynucleotides, the vectors, the rAAV particles, and / or the pharmaceutical compositions disclosed herein.1. Definitions

[0099] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0100] It is to be noted that, as used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or enumerated component; for example, "a nucleic acid sequence," is understood to represent one or more nucleic acid sequences, unless stated otherwise. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

[0101] Furthermore, "and / or", where used herein, is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0102] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of are also provided.

[0103] As used herein, the term "about" refers to a value that is within 10% above or below the value being described.

[0104] The term "at least" prior to a value or series of values is understood to include the values adjacent to the term "at least," and all subsequent values (numbers, integers, or fractions) that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer, e.g., "at least 18 nucleotides of a 21- nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When "at least" is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, 5.18% without consideration of the number of significant figures).SUBSTITUTE SHEET (RULE 26)

[0105] As used herein, "no more than" or "less than" is understood as the value adjacent to the phrase and logical lower values (numbers, integers, or fractions), as logical from context, to zero. When "no more than" is present before a series of values or a range, it is understood that "no more than" can modify each of the value in the series or range.

[0106] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0107] The term "derived from," as used herein, refers to a component that is isolated from or made using a specified molecule or organism, or information (e.g., amino acid or nucleic acid sequence) from the specified molecule or organism.

[0108] "Nucleic acid," "polynucleotide," and "oligonucleotide," are used interchangeably in the present application. These terms refer only to the primary structure of the molecule. Thus, these terms include double- and single-stranded DNA, as well as double- and single-stranded RNA (e.g., messenger RNAs (mRNAs), genomic DNAs, plasmid DNAs (pDNAs), or complementary DNAs (cDNAs)). The terms "nucleic acid," "polynucleotide," and "oligonucleotide," as used herein, are defined as it is generally understood by the person skilled in the art as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides can also be referred to as nucleic acid molecules or oligomers. Polynucleotides can be made recombinantly, enzymatically, or synthetically, e.g., by solid-phase chemical synthesis followed by purification. When referring to a sequence of the polynucleotide or nucleic acid, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides.

[0109] As used herein, the term "nucleotide" refers to monomeric units of nucleic acid polymers (e.g., deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)). Naturally occurring nucleotides are composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and phosphate group consisting of one to three phosphates. A nucleotide can also be a modified nucleotide, which is a nucleotide comprising a modified nucleobase, a modified sugar moiety, a modified backbone, or any combination thereof. As used herein, the term "nucleobases", also known as "nitrogenous bases" or "bases", refers to biological compounds that form nucleosides, which, in turn, are components of nucleotides. A "nucleoside" comprises a nucleobase and a sugar moiety. A "sugar moiety" may comprise ribose or deoxyribose, as presentSUBSTITUTE SHEET (RULE 26)in naturally occurring nucleotides. As used herein, the term "backbone," and "backbone structure", refer to the connection between monomers of a nucleic acid. In naturally occurring oligonucleotides, the backbone comprises a 3'-5' phosphodiester linkage connecting sugar moieties of the oligomer.

[0110] As used herein, the term "coding sequence" or a sequence "encoding" refers to a particular molecule which is a nucleic acid that is transcribed (in the case of DNA) or translated (in the case of RNA) into protein, in vitro or in vivo, when operably linked to an appropriate regulatory sequence, such as a promoter. The boundaries of the coding sequence are determined by a start codon at the 5 ' (amino) terminus and a translation stop codon at the 3 ' (carboxy) terminus. Although a "stop codon" (e.g., TAG, TGA, or TAA) is not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the coding sequence. [OHl] As used herein, the term "exon" refers to coding sections of a DNA molecule, or of an RNA molecule which is transcribed from a DNA molecule that are translated into protein. Exons can be separated by intervening sections of DNA that do not code for proteins, known as "introns". Therefore, the term "intron", as used herein, refers to a segment of nucleic acid that is transcribed and is present in the "pre-mRNA" but excised by the splicing machinery and therefore not present in the mature mRNA transcript. Following transcription, new, immature strands of messenger RNA, called "pre-mRNA", may contain both introns and exons. These pre-mRNA molecules go through a modification process in the nucleus called splicing during which the noncoding introns are cut out and only the coding exons remain in the "mature mRNA1. Splicing produces a mature messenger RNA molecule that is then translated into a protein. The term "first exon" refers to a coding sequence or sequence of nucleic acid that encodes a polypeptide or polypeptide region and the term "second exon" refers to a different second coding sequence or sequence of nucleic acid that encodes a second polypeptide region. Where the two exons are separated by an intervening intron in the pre-mRNA, the splicing machinery operates to remove the intervening intron and join the two exons in the mature mRNA.SUBSTITUTE SHEET (RULE 26)

[0112] The term "polyadenylation signal" refers to a nucleic acid sequence present in the RNA transcript that allows for the transcript, when in the presence of the enzyme polyadenyl transferase, to be polyadenylated.

[0113] The term "promoter," as used herein in, refers to a sequence sufficient to direct transcription, in a cell. A promoter is intended as a DNA region to which RNA polymerases bind and that directs the enzyme to transcribe an operably linked DNA sequence. A DNA sequence is operably linked to a promoter if the promoter is capable of directing transcription of that DNA sequence. Promoters for use in the invention include prokaryotic, eukaryotic (e.g., mammalian or yeast), and viral promoters, e g., the CMV (mammalian cytomegalovirus) promoter, the CAG promoter (also known as CBA promoter; CMV early enhancer / chicken p actin promoter), the UbC (polyubiquitin C gene) promoter, or the CBh (an engineered CBA promoter in which the SV40 intron is replaced with a hybrid intron composed of a 5' donor splice site from the chicken P-actin 5' UTR and a 3' acceptor splice site from MVM). A promoter can be a "constitutive" promoter that is a promoter that, when operably linked to a polynucleotide encoding a gene product, results in the production of a gene product in the cell under most or all conditions of the cell. A promoter can be a "regulatable" promoter that is a promoter whose activity is affected by a cis or trans acting factor (e.g., an inducible promoter, such as an external signal or agent). The term "inducible" promoter means that when the promoter is operably linked to a polynucleotide encoding a specified gene product, it results in the production of a gene in the cell basically only when the inducer corresponding to the promoter is present in the cell. A promoter can be a "ubiquitous" promoter that is a promoter that is active in a wide range of cells, tissues and cell cycles, or a "tissue-specific" promoter, that is a promoter that has activity only or mostly in certain cell types, i.e., drives the expression of the operably linked nucleotide sequence only or mostly in certain cell types. A promoter can be a "bidirectional" promoter, which is a promoter that is an intergenic region between two divergent genes located on complementary strands of the DNA, and drives their coordinated transcription in opposite directions.

[0114] As used herein, the term "regulatory sequence" refers to a nucleic acid sequence capable of regulating the expression of a nucleic acid sequence operably linked to said regulatory sequence, non-limiting examples of regulatory sequences are enhancers (a DNA sequence that increases the level of transcription of an operably linked gene), and silencers (a DNA sequence that decreases the level of transcription of an operably linked gene). The term "regulatory sequence" also refers to nucleic acid sequence in RNA transcripts capable of regulating, for example, theSUBSTITUTE SHEET (RULE 26)processing or the expression of said transcripts. Non-limiting examples of regulatory sequences that can be in RNA transcripts are nucleotide sequences that regulate localization or splicing of said RNA transcripts. The term "regulatory sequence" further refers to an amino acid sequence capable of regulating, for example, the localization (e.g., subcellular localization signals, such as nuclear localization signals), or the stability (e.g., degradation signals) of a protein.

[0115] The terms "operatively linked," "operatively inserted," "operatively positioned," "under control" means, with reference to two or more nucleic acid sequences, that the nucleic acid sequences are arranged in such a way that one of the two or more nucleic acid sequences can mediate a function that is exerted upon at least one of the other two or more nucleic acid sequences. For example, a regulatory nucleic acid sequence (e.g., a promoter, an enhancer, or a silencer) can be "operatively linked," to a coding nucleic acid sequence, that is the regulatory nucleic acid sequence is in the correct location and orientation in relation to the coding nucleic acid sequence to control expression of the coding nucleic acid sequence (e.g., via control of RNA polymerase initiation). Wherein a regulatory nucleic acid sequence (e.g., a promoter, an enhancer, or a silencer) is "operatively linked," to a coding region, the coding region is "under transcriptional control" of the regulatory nucleic acid sequence (e.g., a promoter, an enhancer, or a silencer).

[0116] The term "operably linked" means that a nucleic acid sequence and a regulatory sequence(s) are arranged in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). The term "operably inserted" means that a nucleic acid sequence of interest is positioned adjacent a regulatory nucleic acid sequence which directs transcription and translation of the nucleic acid sequence of interest (i.e., facilitates the production of, e.g., a polypeptide encoded by a DNA of interest).

[0117] As used herein, the term "RNA" relates to a nucleic acid molecule that comprises ribonucleotide residues. In some aspects, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'- position of a b-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standardSUBSTITUTE SHEET (RULE 26)nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered analogs of naturally-occurring RNA. The term "mRNA," as used herein, refers to a single stranded RNA that encodes the amino acid sequence of one or more peptide (e.g., oligopeptide, or polypeptide) or protein. The term "mRNA," as used herein includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. An mRNA molecule may also contain a 5' untranslated region (5'-UTR), and / or a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template where DNA refers to a nucleic acid that contains deoxyribonucleotides.

[0118] As used herein the term "splicing" refers to the process by which introns are removed from primary transcripts (pre-mRNA) and exons are joined to form the mature mRNA. Introns are removed by the pre-mRNA by cleavage at conserved sequences called "splice sites", or "splicing sites". These sites are located at the 5' and 3' ends of introns. Most commonly, the RNA sequence that is removed begins with the dinucleotide GU at its 5' end, and ends with AG at its 3' end. These consensus sequences are known to be critical, because changing one of the conserved nucleotides may result in the inhibition of splicing. Another important sequence occurs at what is called the branch point, located anywhere from 18 to 40 nucleotides upstream from the 3' end of an intron. The branch point always contains an adenine, but it is otherwise loosely conserved. A typical sequence is YNYYRAY, where Y indicates a pyrimidine, N denotes any nucleotide, R denotes any purine, and A denotes adenine. Rarely, splice site sequences are found that begin with the dinucleotide AU and end with AC, these are spliced through a similar mechanism.

[0119] Splicing occurs in several steps and is catalyzed by small nuclear ribonucleoproteins (snRNPs, commonly pronounced "snurps"). First, the pre-mRNA is cleaved at the 5' end of the intron following the attachment of a snRNP called U1 to its complementary sequence within the intron. The cut end then attaches to the conserved branch point region downstream through pairing of guanine and adenine nucleotides from the 5' end and the branch point, respectively, to form a looped structure known as a lariat. The bonding of the guanine and adenine bases takes place via a chemical reaction known as transesterification, in which a hydroxyl (OH) group on a carbon atom of the adenine attacks the bond of the guanine nucleotide at the splice site. The guanine residue is thus cleaved from the RNA strand and forms a new bond with the adenine.

[0120] Next, the snRNPs U2 and U4 / U6 appear to contribute to positioning of the 5' end and the branch point in proximity. With the participation of U5, the 3' end of the intron is broughtSUBSTITUTE SHEET (RULE 26)into proximity, cut, and joined to the 5' end. This step occurs by transesterification; in this case, an OH group at the 3' end of the exon attacks the phosphodiester bond at the 3' splice site. The adjoining exons are covalently bound, and the resulting lariat is released with U2, U5, and U6 bound to it. In addition to consensus sequences at their splice sites, eukaryotic genes with long introns also contain exonic splicing enhancers (ESEs). These sequences, which help position the splicing apparatus, are found in the exons of genes and bind proteins that help recruit splicing machinery to the correct site. Most splicing occurs between exons on a single RNA transcript, but occasionally trans-splicing occurs, in which exons on different pre-mRNAs are ligated together.

[0121] The splicing process occurs in cellular machines called spliceosomes, in which the snRNPs are found along with additional proteins. The primary variety of spliceosome is one of the most plentiful structures in the cell, and recently, a secondary type of spliceosome has been identified that processes a minor category of introns. These introns are referred to as U12-type introns because they depend upon the action of a snRNP called U12 (the common introns described above are called U2-type introns). The role of U12-type introns is not yet defined, but their persistence throughout evolution and conservation between homologous genes of widely divergent species suggests an important functional basis.

[0122] As used herein, the term "alternative splicing" refers to a deviation from the constitutive splicing in which introns are removed and exon are in the order in which they appear in a gene. In alternative splicing certain exons are skipped resulting in various forms of mature mRNA from a single pre-RNA transcript. Weaker splicing signals at alternative splice sites, shorter exon length or higher sequence conservation surrounding orthologous alternative exons influence the exons that are ultimately included in the mature mRNA. Three possible mechanisms: exon shuffling, exonization of transposable elements and constitutively spliced exons, have been proposed for the origin of alternative splicing. Alternative splicing is the mechanism that accounts for the discrepancy between the number of protein-coding genes (-25,000) in humans and the >90,000 different proteins that are actually generated.

[0123] The term "expression" as used herein refers to a process by which a gene produces a biochemical, for example, an RNA transcript or a protein. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knock-in, as well as both transient expression and stable expression. It may include, without limitation, transcription of the gene into messenger RNA (mRNA), and the translation of such mRNA into protein(s). Expression of a gene produces a "gene product." As used herein, a gene product canSUBSTITUTE SHEET (RULE 26)be either a nucleic acid, e.g., a messenger RNA, or a non-coding RNA, produced by transcription of a gene, or a protein, which is translated from an mRNA transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., mRNAs which are processed, for example, by capping, splicing, and / or polyadenylation, or peptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like. Thus, term "protein expression" refers to the process by which a nucleic acid sequence undergoes transcription (DNA) translation (RNA) such that detectable levels of the amino acid sequence or protein are expressed.

[0124] As used herein, the terms "protein," "polypeptide," and "peptide," are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another. The amino acids may be natural or synthetic, and can contain chemical modifications such as disulfide bridges, substitution of radioisotopes, phosphorylation, substrate chelation (e.g., chelation of iron or copper atoms), glycosylation, acetylation, formylation, amidation, biotinylation, and a wide range of other modifications. A polypeptide may be attached to other molecules, for instance molecules required for function. Examples of molecules which may be attached to a polypeptide include, without limitation, cofactors, polynucleotides, lipids, metal ions, phosphate, etc. A polypeptide is comprised of approximately twenty, standard naturally occurring amino acids, although natural and synthetic amino acids, which are not members of the standard twenty amino acids, may also be used. The standard twenty amino acids include alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine, (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Vai, V). These letters as used in terms of a " polypeptide sequence" or "amino acid sequence" are an alphabetical representation of a polypeptide molecule. A polypeptide may be naturally occurring, recombinant, or synthetic, or any combination of these. A polypeptide may also comprise a fragment of a naturally occurring polypeptide. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0125] A polypeptide as disclosed herein can be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more,SUBSTITUTE SHEET (RULE 26)1,000 or more, or 2,000 or more amino acids. Polypeptides can have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three- dimensional structure are referred to as folded, and polypeptides that do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded. Non-limiting examples of polypeptide include polypeptide fragments, denatured / unstructured polypeptide, polypeptide having a primary, a polypeptide, a polypeptide, or a polypeptide or aggregated structure, etc. A polypeptide may be a single molecule or may be a multi-molecular complex. There is expressly no requirement that a polypeptide must contain an intended function; a polypeptide can be functional, non-functional, function for unexpected / unintended purposes, or have unknown function.

[0126] As used herein, the term "domain" when referred to a polypeptide means a distinct functional and / or structural portion of a polypeptide. Usually domains are responsible for a particular function or interaction, contributing to the overall role of a polypeptide. Domains may exist in a variety of biological contexts, where similar domains can be found in polypeptide with different functions. For example, an "actin-binding domain" or "ABD" is to be understood as a portion of a polypeptide that is involved in the binding of the polypeptide with actin. For example, an ABD can comprise one or more amino acids that directly bind to actin.

[0127] As used herein, the term "truncated" when referred to a protein means a protein that is lacking one or more portion with the respect to the full length protein. A protein can lack, for example, an amino-terminal portion, a carboxy-terminal portion, any portion in between the two termini, or any combination thereof.

[0128] By "isolated" molecule (e.g., a protein or a nucleic acid molecule) is intended a molecule, which has been removed from its native environment. An isolated molecule includes molecules produced and maintained (e.g, upon purification) in vitro, or recombinant molecules maintained in host cells or purified (partially or substantially) from the host cell and maintained in solution. Non-limiting examples of isolated molecules include proteins or nucleic acid molecules extracted from a cell, produced synthetically (i.e., by chemical synthesis), produced by enzymatic digestion; DNA molecules produced by PCR, produced by in vitro retro-transcription (cDNA), by molecular cloning in a vector (e.g., a viral or a non-viral vector); RNA transcripts produced by in vitro transcription or produced in host cells (e.g., a heterologous host cells) and maintained in the host cell or purified (partially or substantially) from the host cell and maintained outside of the cell; proteins produced by in vitro translation or produced in host cells (e.g., a heterologous hostSUBSTITUTE SHEET (RULE 26)cells) and maintained in the host cell or purified (partially or substantially) from the host cell and maintained outside of the cell.

[0129] As used herein, "purify," "purified," "purification" means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.

[0130] As used herein, the term "variant" refers to an alternative molecule (e.g., an alternative genomic sequence, transcript, or protein) that differs from the corresponding wild type molecule. As used herein, the term "wild type," refers to a strain, a molecule (e.g., a nucleic acid (e.g., a gene or a transcript) or a protein) or characteristic, which prevails among individuals in natural conditions, and it is distinct from a variant of the strain, molecule (e.g., nucleic acid (e.g., gene or transcript) or protein), or characteristic. A variant may differ from the wild type molecule in any region of the molecule, for example, in any domain of a protein, in any regulatory region of a protein or nucleic acid molecule, or in any coding or non-coding region of a nucleic acid molecule, or any combination thereof. A variant may be present in any percentage of the population (e.g., <0,001%, or >10%). A variant may or may not cause phenotypic changes, and may or may not cause a disorder, disease, syndrome, or condition. A variant can be present in all the cells of an individual, or in some of the cells of the individual (e.g., in some cell lineages of the individual).

[0131] A variant genomic sequence or transcript can comprise one or more nucleotide differences with respect to the wild type sequence, such as indels (i.e., insertions and / or deletion), nucleotide expansions, transversions, translocations, inversions, chromosomal structure alterations, gene fusions, chromosome fusions, truncations, amplifications, duplications, chromosomal lesions, or any combination thereof. As a result, a variant nucleotide sequence can be altered at a single nucleotide, which may be added, deleted, or substituted, or at more than one nucleotide. Further the structure of a chromosome can be altered, with regions being flipped, deleted, duplicated, truncated, or translocated. The expression of a gene can also be amplified (or reduced) through increased (or decreased) copy number of a gene; this scenario is defined as "copy number variation."

[0132] A variant genomic sequence can arise, for example, from unrepaired DNA damage, replication errors, or mobile genetic elements. A variant genomic sequence can be inherited from parents (i.e. , may be present in the germline of one or both parents) or can be acquired over the life of an individual (i.e., appear at any time over the life of an individual in the somatic or germ line of the individual). It is also to be understood that "a copy of a gene that is not a wild type copy of the gene," is herein intended to refer to a variant of the gene. It is understood, that a gene (e.g., theSUBSTITUTE SHEET (RULE 26)DMD gene) is to be intended as the whole genomic region, i.e., comprising coding regions, noncoding regions, regulatory regions, or any combination thereof.

[0133] It is to be understood that the term a "variant" further encompasses proteins encoded by variant genomic sequences or transcripts.

[0134] A variant transcript can also be encoded and / or produced from the same genomic region from which the wild type molecule is encoded and / or produced, but yet differ from the wild type molecule encoded and / or produced from the same genomic region as a results of differences introduced during the transcription process and / or during RNA processing (e.g., may be the product of alternative splicing).

[0135] Additionally, a variant protein can be encoded or produced from the same genomic region or transcript as the wild type protein, but yet differ from the wild type protein as a results of differences introduced during the translation process.

[0136] As used herein, the term "recombinant DNA / RNA technology" refers to the manipulation of nucleic acid sequences outside of an organism. This technology comprises, but is not limited to, combining nucleic acid sequences (e.g., coding sequences, regulatory elements (e.g., promoters, enhancers, silencers, termination sequences), linkers (e.g., spacers, internal ribosome entry sites, cleavage sites)) derived from a variety of sources, inserting nucleic acid sequences from a variety of sources in appropriate vectors (e.g., delivery vectors, expression vectors, integrating vectors), modifying or altering nucleotide sequences (e.g., by mutagenesis, insertion of modified nucleotides, 5'-capping, polyadenylation), synthesizing artificial nucleotide sequence. A variety of techniques described in the literature (e.g., molecular cloning, polymerase chain reaction (PCR), digestion with restriction enzymes, in vitro ligation, mutagenesis, site-directed mutagenesis, prokaryotic and eukaryotic cell transformation or transduction, in vitro DNA / RNA synthesis, in vitro RNA-5'-capping, in vitro RNA-polyadenylation, complementary DNA (cDNA) synthesis, nucleic acid isolation, and the like) can be used to manipulate nucleic acid sequences outside an organism (see for example Green & Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-3, 4thedition).

[0137] As used herein, the term "recombinant" or "engineered" refers to any nucleic acid (e.g., DNA, or RNA), peptide (e.g., oligopeptide, polypeptide, protein, or protein domain), cell, or organism, which is made by manipulation of nucleic acid or amino acid sequences. For example, "engineered DNA" molecules are DNA molecules that are manipulated outside of (e.g., via recombinant DNA / RNA technology) or inside an organism (e.g. , via transgenesis), to obtain a non-SUBSTITUTE SHEET (RULE 26)naturally occurring DNA molecule. An "engineered DNA" molecule can comprise, for example, one or more nucleotide variations compared to the DNA molecule from which it is derived. The "engineered DNA" molecule can be transcribed into RNA, which is referred to as an "engineered RNA" molecule. The "engineered RNA" molecule can be translated into a polypeptide or a polypeptide domain, which is referred to as an "engineered polypeptide" or an "engineered polypeptide domain." Thus, an "engineered polypeptide" or an "engineered polypeptide domain" can be any polypeptide or polypeptide domain, which is encoded by nucleic acids manipulated via recombinant DNA / RNA technology, additionally, an "engineered polypeptide" or an "engineered polypeptide domain" can be produce by chemical synthesis. An "engineered polypeptide" or an "engineered polypeptide domain" can be for example a truncated polypeptide or polypeptide domain, or a polypeptide or polypeptide domain comprising one or more amino acid substitutions compared to a same polypeptide or polypeptide domain that is not engineered.

[0138] An engineered polypeptide or polypeptide domain can be manipulated without the purpose of achieving a desired altered function of the polypeptide or polypeptide domain, or with the purpose of achieving a desired altered function of the polypeptide or polypeptide domain. For example, an engineered polypeptide or polypeptide domain can be modified to increase a function or characteristic of the polypeptide or polypeptide domain (e.g., to increase the binding affinity of the engineered polypeptide or polypeptide domain for a specific target (e.g., a second polypeptide or polypeptide domain)). These modifications are referred to as gain-of-function / characteristic modifications and result in gain-of-function / characteristic polypeptides or polypeptide domains. For example, a polypeptide or polypeptide domain can be modified to achieve increased binding affinity to a specific target (e.g., a second polypeptide or polypeptide domain) (i.e., the function / characteristic of the polypeptide or polypeptide domain that is increased by the modification is the binding affinity to a specific target of the polypeptide or polypeptide domain), these modified polypeptides or polypeptide domains are referred to as gain-of-affinity or GoA- polypeptides or GoA-polypeptide domains. Alternatively, a polypeptide or polypeptide domain can be modified to decrease a function or characteristic of the polypeptide or polypeptide domain. These modification are referred to as loss-of-function / characteristic modification, resulting in loss- of-function / characteristic polypeptide or polypeptide domains).

[0139] As used herein the term "amino acid substitution" refers to the replacement of one amino acid in a protein with a different amino acid. An "amino acid substitution" can be naturally occurring or experimentally induced. For example, an "amino acid substitution" can be introducedSUBSTITUTE SHEET (RULE 26)in a protein via manipulation of the nucleic acid molecule encoding for the protein by "recombinant DNA / RNA technology." Typically, amino acid substitution are indicated as XNY, wherein X indicates the amino acid present at position N before the amino acid substitution, and Y in indicates the amino acid present at position N after the amino acid substitution. Thus, for example T20A indicates that a T was present at potion 20 before the amino acid substitution, and an A is present at the same position 20 after the amino acid substitution.

[0140] As used herein, the term "genetic locus, " refers to the physical site or location within a genome of a specific DNA sequence, for example a gene.

[0141] As used herein, the term "transgene" refers to a gene (e.g., micro-dystrophin gene) or a nucleic acid molecule that is introduced into a cell. An example of a transgene is a nucleic acid encoding a therapeutic protein. In some aspects, the gene can be present, but in some cases, the gene is not expressed or is expressed at an insufficient level in the cell. In this context, "insufficient" means that although said gene is expressed in the cell, a condition and / or disease could still be developed. In certain aspects, the transgene allows for the increased expression or over-expression of the gene. The transgene can comprise sequences that are native to the cell, sequences that do not naturally occur in the cell, or combinations of both. In certain aspects, the transgene can comprise a sequence that can be operably linked to appropriate regulatory sequences for expression of the gene in the cell. In some aspects, the transgene is integrated into the host cell's genome. In some aspects, the transgene is not integrated into the host cell's genome.

[0142] The term "vector" as used herein includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may comprise specific functional sequences needed for insertion and / or expression of the desired DNA fragments. A "vector" can be any vehicle for the cloning of and / or transfer of a nucleic acid into a host cell, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc. The term "vector" includes both viral and nonviral vehicles for introducing theSUBSTITUTE SHEET (RULE 26)nucleic acid into a cell in vitro, ex vivo or in vivo. In some aspects, insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that may or not have complementary cohesive termini. Vectors can be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and / or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes described in the literature include: genes providing resistance to neomycin, ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters described in the literature include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), P-galactosidase (LacZ), P-glucuronidase (Gus), and the like. Selectable markers can also be considered to be reporters.

[0143] As used herein, the term "adeno-associated vector" or "AAV vector" refers to a vector comprising one or more polynucleotides of interest (e.g., transgenes, such as microdystrophin) that are flanked by AAV terminal repeat sequences (ITRs). AAV is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York); Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. "AAV Cap" means AAV Cap proteins, VP1, VP2, and VP3 and analogs thereof. "AAV Rep" means AAV Rep proteins and analogs thereof. "Serotype," with respect to a vector or virus capsid, is defined by a distinct immunological profile based on the capsid protein sequences and capsid structure. Non-limiting examples of AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV6P1, AAV7, AAV8, AAV8P1, AAV9, AAVrhlO, AAVrhlOPl, AAVS10P4, AAVpol, AAV11, AAV12, AAV-DJ, AAV-DJ / 8, AAV-PHP.Eb, AAV-PHP.S, AAV-PHP.B, AAV2-retro, AAV2-QuadYF, AAV2.7m8, AAVS1, AAVS10, AAVrh74, AAVS1, AAVS10, AAVH15, AAVMYO (AAV9P1), AAVMYO 2 (AAVS1P1), AAVMY03 (AAVS10P1), AAV9-RGD (e g.,SUBSTITUTE SHEET (RULE 26)MyoAAVlA, MyoAAV 1C, MyoAAV IE, MyoAAV 2 A, MyoAAV 2E, MyoAAV 3 A, MyoAAV 4A, MyoAAV 4C, and MyoAAV 4E).

[0144] As used herein, "flanked," with respect to a sequence that is flanked by other elements, indicates the presence of one or more the elements upstream and / or downstream, i.e., 5' and / or 3', relative to the sequence. The term "flanked" is not intended to indicate that the sequences are necessarily contiguous. For example, there may be intervening sequences between the nucleic acid encoding the transgene and a flanking element. A sequence (e.g., a transgene) that is "flanked" by two other elements (e g., ITRs), indicates that one element is located 5' to the sequence and the other is located 3' to the sequence; however, there may be intervening sequences between.

[0145] As used herein, the terms "AAV virion," "AAV viral particle," or "AAV particle" refer to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. The particle can comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell). An AAV particle comprising a heterologous polynucleotide can be also referred to as a "recombinant AAV particle," "recombinant AAV vector," "rAAV particle," or "rAAV vector."

[0146] As used herein, the term "transfection" of a cell refers to the introduction of genetic material into a cell by means of a non-viral vector for the purpose of genetically modifying the cell.

[0147] As used herein, the term "transduction" of a cell refers to the introduction of genetic material into a cell by means of a viral vector for the purpose of genetically modifying the cell. For example, the coding region of a gene or of a portion thereof (e.g., a micro-dystrophin) can be administrated / delivered to a recipient cell either in vivo or in vitro, via an AAV particle resulting in the expression of the gene or portion thereof (e.g., a micro-dystrophin) in the recipient cell.

[0148] As used herein, the term "cell" or "cells" refers not only to the particular subject cell, but also to the progeny or to the potential progeny of such cell(s). The scope of the term as used herein also encompasses the progeny that may or may not in fact be identical to the parent cell because certain modifications may occur in succeeding generations due to either mutation or environmental influences. A cell can be, for example, a "muscle cell," which is a cell derived from a muscle of any kind (for example, skeletal muscle and smooth muscle, e.g. from the digestive tract, urinary bladder, blood vessels or cardiac tissue), as well as a cell differentiated in vitro to possess the characteristics of a cell derived from a muscle of any kind. Such muscle cells may beSUBSTITUTE SHEET (RULE 26)differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts. A cell group of muscle cells can for a "muscle tissue."

[0149] The term "muscle specific control element" refers to a nucleotide sequence that regulates expression of a coding sequence that is specific for expression in muscle tissue. These control elements include enhancers and promoters. Exemplary muscle specific control elements include, but are not limited to, the MCKH7 promoter, the MCK promoter and the MCK enhancer.

[0150] As used herein, the term "binding affinity" refers to the strength of the interaction between two (or more than two) molecules that bind reversibly (i.e., interact). The binding affinity can be translated into physico-chemical terms in the dissociation constant (Kd). The dissociation constant is a measure of the tendency of a complex MxNyto reversibly dissociate (separate) into its components M and N (MxNy^xM+yN). The dissociation constant is denoted Kd and is calculated by the formula:

[0151] where [M], [N], are the molar concentrations of the componentM, N, and [MxNy] MxNy is the molar concentration of the complex. A low Kd value indicates a high affinity betweenM and N, and a high Kd value indicates a low affinity between M and N.

[0152] As used herein, the term "Forster (or Fluorescence) Resonance Energy Transfer" or "FRET" refers to a technique used to infer the distance between two fluorophores, called donor and acceptor, which is based on the Forster energy transfer phenomenon. The Forster energy transfer phenomenon describes the process by which an excited donor fluorophore transfers energy (not an electron) to an acceptor fluorophore through a non-radiative process. A donor is excited by a photon and then relaxes to its lowest excited state, if the acceptor is not too far, the energy released when the electron of the donor returns to the ground state may simultaneously excite the acceptor. This non-radiative process is referred to as "resonance". After excitation, the excited acceptor emits a photon and returns to the ground state. Forster resonance energy transfer (FRET)-based sensors have been extensively used to infer the distance between two molecules. Briefly, a first molecule of interest is attached to a first fluorophore (the donor) and a second molecule of interest is attached to a second fluorophore (the acceptor). The donor is then excited with light at the appropriate wave length, as the donor returns to its lowest excited state it transfers energy via resonance to the acceptor, if the acceptor is close enough, thus the acceptor is in turn excited and while it then returns to its lowest excited state emits photons that can be experimentally detected. TheSUBSTITUTE SHEET (RULE 26)fluorophores to be attached to the molecules of interest must be selected based on the overlap of their absorbance and emission spectra, the emission spectrum of the donor fluorophore must overlap the absorption spectrum of the acceptor fluorophore. The amount of energy emitted by the acceptor is a measure of the proximity of the two fluorophores, and therefore of the proximity of the two molecules of interest. FRET can take place only when the two fluorophores (therefore the two molecules of interest) are situated at distances lower than 10 nm. In the case of two proteins labelled with donor and acceptor fluorophores, this implies that FRET occurs only if and when the two proteins interact ((i.e,. bind) with each other. FRET has therefore been widely exploited to study protein-protein binding and thus protein-protein binding affinity. For example, given a protein A and a protein B, their binding affinity for a protein X can be measured by measuring their respective FRET when bound to a same donor fluorophore in the presence of X bound to an acceptor fluorophore. If A has a higher binding affinity to X than B, there is more FRET between the donor attached to A and the acceptor attached to X, than FRET between the donor attached to B and the acceptor attached to X, since A would more likely be bound to X than B given the higher affinity of A for X.

[0153] As used herein, the term "actin co-sedimentation assay" or "HSC" is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains to actin. The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with actin, ultracentrifugation to pellet actin, and analysis of the protein co-sedimenting with actin. If a protein binds to actin it co-sediments with actin during the ultracentrifugation to pellet actin. Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

[0154] As used herein, the term "mini-dystrophin" refers to a truncated dystrophin protein comprising more than four spectrin repeats.

[0155] As used herein, the term "micro-dystrophin" refers to a truncated dystrophin protein comprising four or fewer spectrin repeats.

[0156] Non-limiting examples of mini- and micro-dystrophins are: DysAR4-R23 / ACTD (SRP-9001, pDysH2), DysA17-48, DysAH2-R19, DysAH2-R15, DysAR2-23, AR2-15 / AR18-22 / ACTD (pDys5), AR3-19 / AR20-21 / ACTD, AR2-15 / AR18-19 / AR20-23 / ACTD, Dys3978, Dys3849, minidystrophin d3990, RGX-202, mDys5R (SGT-001), and DYSF.

[0157] As used herein, the term or "pharmaceutical formulation" or "pharmaceutical composition" refers to an admixture comprising an effective amount of a therapeutically and / orSUBSTITUTE SHEET (RULE 26)prophylactic effective agent and at least one pharmaceutically acceptable excipient (e.g., carrier, diluent, stabilizer, or any combination thereof) or adjuvant. Examples of pharmaceutically acceptable excipients are, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, colorants, amino acids, stabilizers, bulking agents, surfactants, antimicrobials, preservatives, metal ions, chelators, cyclodextrin-based excipients, polyanions, polycations, salts, solubilizers, detergents, compatible solid or liquid fillers, encapsulating substances, or any combination thereof, which are suitable for administration to a subject. Specific examples of excipient include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxy-propylene copolymers, or any combination thereof. Examples of pharmaceutically acceptable carriers are, but are not limited to, lipids, polymers, polysaccharides, peptides, proteins, lipidoids, and any combination thereof. In some aspects, the lipid is selected from the group consisting of: cationic lipids, non-cationic lipids, steroid lipids, ionizable lipids, PEG-conjugated lipids, and any combination thereof. In some aspects, the fusion proteins, the expression cassettes, and the vectors disclosed herein are complexed, or packaged in a liposome, a nanoliposome, a lipid nanoparticle, a lipoplex, a micell, a nanomicell, a nanoemulsion, an oil-in-water emulsions, a PEG-conjugated lipid nanoparticle, a polymeric nanoparticle, a lipid-polymer hybrid nanoparticle, a polysaccharidic nanocarrier, an RNA / DNA- peptide nanoparticle, an RNA / DNA -peptide nanocomplex, a biomimetic nanovesicle, a lipidoid- RNA / DNA complex, a virus-like particle, dendrimer nanoparticle, a nanogel, a metallic nanoparticle, a gold nanoparticle (AuPNs), a magnetic nanoparticle, a theranostic nanoparticle, or any combination thereof. In some aspects, the combination of the fusion proteins, the expression cassettes, and the vectors disclosed herein with one or more of the carriers described herein facilitates, enhances or enables administration of the fusion proteins, the expression cassettes, and the vectors disclosed herein to the subject. In some aspects, the combination of a therapeutic agent with one or more of the carriers described herein facilitates, enhances or enables the delivery of the therapeutic agent to a target cell.

[0158] Pharmaceutically acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985). Pharmaceutical excipients can be selected with regard to the intended route of administration and standard pharmaceutical practice.SUBSTITUTE SHEET (RULE 26)

[0159] As used herein, a "therapeutic agent" refers to a chemical compound, a peptide, a protein, a lipid, a carbohydrate, a nucleic acid, or any other molecule or compound capable of treating, ameliorating, or reducing the symptoms of a disease, a disorder, a condition, or a syndrome, or otherwise having a beneficial effect, upon administration to a subject suffering from the disease, disorder, condition, or syndrome.

[0160] As used herein, the term "administration" refers to the administration of a composition or substance to a subject or system. Administration to an animal subject (e.g., to a mammal) can be by any appropriate route. "Administering" refers to the physical introduction of a composition or substance, which may comprising a therapeutic agent, to a subject, using any of the various methods and delivery systems known to those skilled in the art. Examples of routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracap sul ar, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Administration can also be via a non-parenteral route, for example, orally. Other non-parenteral routes include atopical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, aborally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0161] As used herein, the terms "treat," "treated," and "treating" mean both therapeutic and prophylactic treatment or preventative measures wherein the object is to reverse, alleviate, ameliorate, lessen, inhibit, slow down progression, development, severity or recurrence of an undesired symptom, complication, condition, biochemical indicia of a disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. In some aspects,SUBSTITUTE SHEET (RULE 26)treatment includes eliciting a clinically significant response without excessive levels of side effects. In some aspects, treatment includes prolonging survival as compared to expected survival if not receiving treatment. As used herein, the term "amelioration" or "ameliorating" refers to a lessening of severity of at least one indicator of a condition or disease. As used herein, the term "preventing" or "prevention" refers to delaying or forestalling the onset, development or progression of a condition or disease for a period of time, including weeks, months, or years. As used herein, the term "prophylactic" (e.g., "prophylactic agent", "prophylactic treatment", "prophylactically effective amount"), refers to any complete or partial prevention of a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect and / or symptom attributable to the disease.

[0162] As used herein, the term "gene therapy" refers to the administration into an individual's cells and / or tissues of an exogenous molecule (e.g., a nucleic acid sequence (e g., a polynucleotide comprising a promoter operably linked to a nucleic acid encoding a gene product capable of interfering with the genomic sequence of the individual's cells and / or tissues)) to treat, reduce the symptoms of, or reduce the likelihood of a disease, disorder, syndrome, or condition. An exogenous molecule or sequence is understood to be molecule or sequence not normally occurring in the cell, tissue and / or individual to be treated. Both acquired and congenital diseases are amenable to gene therapy.

[0163] As used herein, the term "subject" refers to any organism to which a composition or a substance (e.g., a nucleotide molecule) can be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal. A subject can seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be under care by a trained professional for a particular disease or condition.

[0164] As used herein, the term "expression cassette" refers to a nucleic acid molecule, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a competent host cell, such that a particular gene product (e.g., RNA or protein) is expressed. Expression of any gene product may be dependent upon presence of cellular factors or additional gene products from other expression cassettes. An expression cassette may be part of a vector, such as a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression cassette includes a polynucleotide to be transcribed, operably linked to a promoter.SUBSTITUTE SHEET (RULE 26)

[0165] As used herein, the term "genetic construct" or "construct" refers to a nucleic acid molecule (RNA, DNA, or a combination thereof) made recombinanlty or syntheiticallycomprising one or more nucleotide sequences, such as a nucleotide sequence encoding (i.e., a coding sequence) a particular gene product (e.g., RNA or protein), and comprising initiation and termination signals; a regulatory element, such as a promoter; and / or a polyadenylation signal. The one or more nucleotide sequences comprised in a genetic construct can be operably linked, such that they are capable of directing expression of the coding sequence in a cell, such as a cell maintained in culture (in vitro), or a cell comprised in an individual (in vivo), to which the genetic construct is administered. A genetic construct can be, for example, an expression cassette, or a vector, e.g., a vector comprising an expression cassette.

[0166] As used herein, the terms "percent identity" and / or "percent identical," as applicable to a particular polynucleotide or amino acid sequence, refer to the proportion of identical residues between this particular reference sequence and another sequence, as calculated by a pairwise alignment using the Needleman-Wunsch algorithm using a generally available alignment program, e.g., the Needle (EMBOSS) program.

[0167] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 5th ed., 2013, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, 2006, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0168] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0169] Various aspects of the invention are described in further detail in the following subsections.SUBSTITUTE SHEET (RULE 26)2. Gain-of-Affinity Dystrophin Proteins Actin-Binding Domains and Dystrophin Proteins comprising the same

[0170] In some aspects, disclosed herein are polynucleotides encoding polypeptides comprising dystrophin protein actin-binding domains (DysABDs) modified to achieve increased actin-binding affinity, herein also referred to as gain-of-affinity DysABDs, or "GoA-DysABDs," actin-binding fragments thereof, and dystrophin proteins comprising the same, herein also referred to as "GoA-Dys".

[0171] In some aspects, disclosed herein are polypeptides comprising dystrophin protein actin-binding domains (DysABDs) modified to achieve increased actin-binding affinity, herein also referred to as gain-of-affinity DysABDs, or "GoA-DysABDs," actin-binding fragments thereof, and dystrophin proteins comprising the same, herein also referred to as "GoA-Dys".

[0172] In some aspects, disclosed herein are dystrophin protein actin-binding domains (DysABDs) modified to achieve increased actin-binding affinity, herein also referred to as gain- of-affinity DysABDs, or "GoA-DysABDs," actin-binding fragments thereof, and dystrophin proteins comprising the same, herein also referred to as "GoA-Dys". In some aspects, the GoA- DysABDs, or actin-binding fragments thereof, have increased actin-binding affinity compared to a same DysABD, or an actin-binding fragment thereof, prior to being modified to achieve the increased actin-binding affinity, (i.e., a same DysABD, or an actin-binding fragment thereof, absent the modification to achieve the increased actin-binding affinity).

[0173] The same DysABD prior to being modified to achieve the increased actin-binding affinity (i.e., a same DysABD absent the modification to achieve the increased actin-binding affinity) is herein also referred to as "not gain-of-affinity DysABD," or "NotGoA-DysABD." Additionally, a same dystrophin protein comprising the NotGoA-DysABD is herein also referred to as a "NotGoA-Dys."

[0174] It is to be understood that "a DysABD prior to being modified to achieve the increased actin-binding affinity," "a same DysABD absent the modification to achieve the increased actin-binding affinity," or "a same NotGoA-DysABDs," is a DysABDs comprising a same amino acid sequence of the GoA-DysABD except for a specific modification (e.g., a specific amino acid substitution) that is present in the GoA-DysABD and absent in the NotGoA-DysABD, this specific modification increases the actin-binding affinity of the GoA-DysABD compared to the NotGoA-DysABD. It is also to be understood that "a same NotGoA-Dys" is a dystrophin protein comprising a same amino acid sequence of the GoA-Dys except for the specific modification (e.g., a specific amino acid substitution) that is present in the GoA-DysABDSUBSTITUTE SHEET (RULE 26)comprised in the GoA-Dys and absent in the NotGoA-DysABD comprised in the NotGoA-Dys, this specific modification increases the actin-binding affinity of the GoA-DysABD, and therefor of the GoA-Dys comprising the same, compared to the NotGoA-Dys, which comprises the NotGoA-DysABD.

[0175] Additionally, it is to be understood that when referring to a GoA-DysABD actin- binding fragment (i.e., "a GoA-DysABD, or an actin-binding fragment thereof ') such actin-binding fragment is intended to comprise the specific modification that distinguishes the GoA-DysABD from the NotGoA-DysABD. Analogously, when referring to a NotGoA-DysABD actin-binding fragment (i.e., "a NotGoA-DysABD, or an actin-binding fragment thereof1) such actin-binding fragment is intended to not comprise the specific modification that distinguishes the NotGoA- DysABD from the GoA-DysABD.

[0176] For example, a GoA-DysABD comprising an amino acid sequence ofSEQ ID NO: 14MLWWEEVEDC YEREDVQKKTFTKWVNAQF SKFGKQHIENLF SDLQDGR RLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGST DIVDGNHKLTLGLIWNIILHWQPKNVMKNIMAGLQQTNSEKILLSWVRQS TRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLE HAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIE has increased actin-binding affinity compared to a same NotGoA-DysABD comprising an amino acid sequence ofSEQ ID NO: 1MLWWEEVEDC YEREDVQKKTFTKWVNAQF SKFGKQHIENLF SDLQDGR RLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGST DIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLSWVRQS TRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLE HAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIE wherein SEQ ID NO: 14 differs from SEQ ID NO: 1 only at position 120, and wherein the substitution of the V with the P at position 120 is the specific modification (i.e., amino acid substitution) that is present in the GoA-DysABD (SEQ ID NO: 14) and absent in the NotGoA- DysABD (SEQ ID NO: 1), this specific modification increases the actin-binding affinity of the GoA-DysABD compared to the NotGoA-DysABD.SUBSTITUTE SHEET (RULE 26)Dystrophin Proteins Actin-Binding Domains

[0177] In some aspects, the polypeptides disclosed herein comprise an actin-binding domain 1 (ABDI), or an actin-binding fragment thereof, an actin-binding domain 2 (ABD2), or an actin-binding fragment thereof, or a combination thereof.

[0178] In some aspects, the polypeptides disclosed herein comprise an ABDI, or an actin- binding fragment thereof, which is a GoA-DysABD1, or an actin-binding fragment thereof, and an ABD2, or an actin-binding fragment thereof, which is not a GoA-DysABD2 (i.e., the ABD2, or an actin-binding fragment thereof, has not been manipulated with the purpose of altering the actin- binding affinity of the ABD2).

[0179] In some aspects, polypeptides disclosed herein comprise ABDI, or an actin-binding fragment thereof, which is a GoA-DysABD1, or an actin-binding fragment thereof, and lack an ABD2, or an actin-binding fragment thereof.

[0180] In some aspects, the polypeptides disclosed herein comprise an ABD2, or an actin- binding fragment thereof, which is a GoA-DysABD2, or an actin-binding fragment thereof, and an ABDI, or an actin-binding fragment thereof, which is not a GoA-DysABD1 ((i.e,. the ABDI, or an actin-binding fragment thereof, has not been manipulated with the purpose of altering the actin- binding affinity of the ABDI).

[0181] In some aspects, polypeptides disclosed herein comprise ABD2, or an actin-binding fragment thereof, which is a GoA-DysABD2, or an actin-binding fragment thereof, and lack an ABD 1 , or an actin-binding fragment thereof.

[0182] In some aspects, the NotGoA-DysABD has an amino acid sequence of:MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKE KGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHX118QVKNVMKNI MAGLQX133TNSEKILLSWVRQSTRNYPQVNVINFTTSWSX165GLALNALIHSHRPDLFDWNSV VCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIE (SEQ ID NO: 2) whereinXI 18 is W or R; X133 is Q or P; X165 is D or V.

[0183] In some aspects, the NotGoA-DysABD has an amino acid sequence of:X1X2X3X4X5X6X7X8X9X10X11EREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLD LLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILH X118QVKNVMKNIMAGLQX133TNSEKILLSWVRQSTRNYPQVNVINFTTSWSX165GLALNALISUBSTITUTE SHEET (RULE 26)HSHRPDLFDWNSWCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQV LPQQVSIE (SEQ ID NO: 293) whereinXI is M or absent; X2 is L or absent; X3 is W or absent; X4 is W or absent; X5 is M, E, or absent ; X6 is S, E, or absent; X7 is V, E, or absent; X8 is V, E, or absent; X9 is S, M, or D; XI 0 is C, E, or S; XI 1 is D or Y; XI 18 is W or R; X133 is Q or P; X165 is D or V.

[0184] In some aspects, the NotGoA-DysABD has an amino acid sequence of:MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKE KGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAG LQQTNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSWCQQSATQR LEHAFNIARYQLGIEKLLDPEDVDTTYPDKKSILMYITSLFQVLPQQVSIE (SEQ ID NO: 1)

[0185] Alternatively, the NotGoA-DysABD can have the amino acid sequence of an ABD of an engineered dystrophin protein, or can be encoded by a polynucleotide encoding an ABD of an engineered dystrophin protein.

[0186] For example, an amino acid sequence of, or a polynucleotide encoding a, naturally occurring dystrophin protein or dystrophin protein ABD can be first engineered to achieve optimized (e.g., increased) expression is certain organisms or cell types, or to achieve optimized (e.g, increased) half-life, or to achieve any other desired outcome, and then further modified to achieve increased actin-binding affinity as disclosed herein. For example, a nucleic acid sequence encoding a naturally occurring dystrophin protein ABDI (DysABD1) can be derived from the genome of a cell, for example by retro-transcription of the mRNA extracted from said cell. The sequence of the resulting cDNA (i.e., cDNA encoding the naturally occurring DysABD1) can be modified for example to achieve increased expression in a specific cell type or organism. Subsequently, the cDNA can be further modified to introduce one or more of the modifications disclosed herein, which will result in increased actin-binding affinity of the DysABD1.

[0187] It is to be understood that the opposite is also contemplated, that is a dystrophin protein ABD can be first engineered to achieve increased actin-binding as disclosed herein and then further modified to achieve optimized (e.g, increased) expression is certain organisms or cell types, or to achieve optimized (e.g, increased) half-life, or to achieve any other desired outcome.

[0188] In some aspects, the polypeptides disclosed herein comprise one or more acting binding domains, and at least one of the acting binding domains is a GoA-DysABD.SUBSTITUTE SHEET (RULE 26)Dystrophin Proteins Actin-Binding Domains: Modifications

[0189] In some aspects, the GoA-DysABDs, or actin-binding fragments thereof, disclosed herein comprise at least one amino acid substitution compared to a same NotGoA-DysABD. In some aspects a dystrophin protein actin-binding domain 1 modified to achieve an increased actin- binding affinity (GoA-DysABD1), or an actin-binding fragment thereof, disclosed herein, has at least one amino acid substitution compared to a same dystrophin protein actin-binding domain 1, or an actin-binding fragment thereof, absent the modification to achieve the increased actin-binding affinity (NotGoA-DysABD 1).

[0190] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof, disclosed herein comprise 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, 8 amino acid substitutions, 9 amino acid substitutions, or 10 amino acid substitutions compared to a same NotGoA-DysABD (e g., a NotGoA-DysABD 1), or an actin-binding fragment thereof.

[0191] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof, disclosed herein comprise 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, or 4 amino acid substitutions compared to a same NotGoA-DysABD (e.g., a NotGoA-DysABD 1), or an actin-binding fragment thereof.

[0192] In some aspects, the at least one amino acid substitution is at one or more of residues D9, E12, Q17, K19, T20, F21, S30, Q35, H36, N75, K79, L81, L84, N86, K105, T107, Il 14, Il 15, V120, M128, W143, R145, Q153, T161, A168, L169, V187, A192, A199, 1202, L207, G208, 1209, L212, D214, E216, or K226 of any one of SEQ ID NOs: 1, 2, or 293.

[0193] In some aspects, the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (E12D), A at residue 17 (Q17A), E at residue 19 (K19E), D at residue19 (K19D), A at residue 20 (T20A), Q at residue 20 (T20Q), N at residue 20 (T20N), S at residue20 (T20S), M at residue 20 (T20M), V at residue 20 (T20V), L at residue 20 (T20L), I at residue 20 (T20I), L at residue 21 (F21L), A at residue 21 (F21A), V at residue 21 (F21V), I at residue 21 (F21I), Q at residue 30 (S30Q), S at residue 30 (S30N), T at residue 30 (S30T), M at residue 30 (S30M), R at residue 35 (Q35R), K at residue 35 (Q35K), R at residue 36 (H36R), K at residue 36 (H36K), R at residue 75 (N75R), K at residue 75 (N75K), N at residue 79 (K79N), Q at residue 79 (K79Q), S at residue 79 (K79S), T at residue 79 (K79T), M at residue 79 (K79M), P at residue 81 (L81P), S at residue 84 (L84S), N at residue 84 (L84N), Q at residue 84 (L84Q), T at residue 84SUBSTITUTE SHEET (RULE 26)(L84T), M at residue 84 (L84M), G at residue 86 (N86G), N at residue 105 (K105N), Q at residue 105 (K105Q), S at residue 105 (K105S), T at residue 105 (K105T), M at residue 105 (K105M), G at residue 107 (T107G, L at residue 114 (I114L), E at residue 114 (I114E), A at residue 114 (I114A), V at residue 114 (Il 14V), D at residue 114 (Il 14D), S at residue 115 (1115 S), N at residue 115 (I115N), Q at residue 115 (I115Q), T at residue 115 (I115T), M at residue 115 (I115M), P at residue 120 (V120P), A at residue 120 (VI 20 A), L at residue 120 (V120L), I at residue 120 (V120I), K at residue 128 (M128K), R at residue 128 (M128R), R at residue 143 (W143R), K at residue 143 (W143K), V at residue 145 (R145V), A at residue 145 (R145A),L at residue 145 (R145L), I 145(R145I), H at residue 153 (Q153H), Y at residue 153 (Q153Y), W at residue 153 (Q153W), F at residue 153 (Q153F), I at residue 156 (V156I), A at residue 156 (V156A), L at residue 156 (V156L); H at residue 161 (T161H), Y at residue 161 (T161Y), W at residue 161 (T161W), F at residue 161 (T161F), V at residue 168 (A168V), I at residue 168 (A168I), L at residue 168 (A168L), S at residue 169 (L169S), N at residue 169 (L169N), Q at residue 169 (L169Q), T at residue 169 (L169T), M at residue 169 (L169M), G at residue 187 (V187G) , L at residue 192 (A192L), V at residue 192 (A192V), I at residue 192 (A192I), V at residue 199 (A199V), L at residue 199 (A199L), I at residue 199 (A199I), G at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue 207 (L207S), Q at residue 207 (L207Q), T at residue 207 (L207T), M at residue 207 (L207M), A at residue 207 (L207A), I at residue 207 (L207I), Y at residue 207 (L207Y), W at residue 207 (L207W), F at residue 207 (L207F), E at residue 207 (L207E), P at residue 208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (I209V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue 209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), H at residue 209 (I209H), Y at residue 209 (I209Y), A at residue 209 (I209A), N at residue 209 (I209N), T at residue 209 (I209T), M at residue 209 (I209M), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), R at residue 216 (E216R), E at residue 226 (K226E), or D at residue 226 (K226D), or any combination thereof.

[0194] In some aspects, the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (E12D), A at residue 17 (Q17A), E at residue 19 (K19E), A at residue 20 (T20A), Q at residue 20 (T20Q), L at residue 21(F21L), A at residue 21 (F21A), Q at residue 30 (S30Q), R at residue 35 (Q35R), R at residue 36 (H36R), R at residue 75 (N75R), N at residue 79SUBSTITUTE SHEET (RULE 26)(K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 86 (N86G), N at residue 105 (K105N), G at residue 107 (T107G), L at residue 114 (I114L), E at residue 114 (I114E), S at residue 115 (I115S), P at residue 120 (V120P), A at residue 120 (V120A), K at residue 128 (M128K), R at residue 143 (W143R), V at residue 145 (R145V), H at residue 153 (Q153H), I at residue 156 (V156I), H at residue 161 (T161H), V at residue 168 (A168V), I at residue 168 (A168I), S at residue 169 (L169S), G at residue 187 (V187G), , L at residue 192 (A192L), V at residue 199 (Al 99V), I at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue 207 (L207S), P at residue 208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (I209V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue 209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226(K226E), or any combination thereof.

[0195] In some aspects, the at least one amino acid substitution consists of one amino acid substitution ((i.e,. the amino acid sequence of the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof, differs from the amino acid sequence of the NotGoA-DysABD (e.g., a NotGoA-DysABD 1), or an actin-binding fragment thereof, only at 1 position).

[0196] In some of these aspects, the one amino acid substitution is selected from Q at residue 20 (T20Q), Q at residue 30 (S30Q), N at residue 79 (K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 107 (T107G), L at residue 114 (Il 14L), E at residue 114 (Il 14E), S at residue 115 (I115S), P at residue 120 (V120P), R at residue 143 (W143R), H at residue 153 (Q153H), I at residue 156 (VI 561), H at residue 161 (T161H), I at residue 168 (A168I), Gat residue 187 (V187G), L at residue 192 (A192L), V at residue 199 (A199V), I at residue 202 (I202G), V at residue 207 (L207V), P at residue 208 (G208P), K at residue 209 (I209K), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226 (K226E).

[0197] In some of these aspects, the one amino acid substitution is selected from Q at residue 20 (T20Q), P at residue 120 (V120P), I at residue 156 (V156I), P at residue 212 (L212P), P at residue 214 (D214P), or E at residue 226 (K226E).

[0198] In some aspects, the polypeptides comprise an amino acid sequence of any one of SEQ ID NOs: 5-30. In some aspects, the polypeptides are encoded by polynucleotides comprisingSUBSTITUTE SHEET (RULE 26)a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 144-169.

[0199] In some aspects, the polypeptides comprise an amino acid sequence of any one of SEQ ID NOs: 5, 14, 17, 27, 28, or 30. In some aspects, the polypeptides are encoded by polynucleotides comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 144, 153, 156, 166, 167, or 169.

[0200] In some aspects, the at least one amino acid substitution consists of two amino acid substitutions (i.e., the amino acid sequence of the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof, differs from the amino acid sequence of the NotGoA-DysABD (e.g., a NotGoA-DysABD 1), or an actin-binding fragment thereof, only at 2 positions).

[0201] In some of these aspects, the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P- K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); N at residue 207 and L at residue 209 (L207N-I209L); R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); D at residue 207 and V at residue 209 (L207D-I209V); G at residue 187 and P at residue 212 (V187G-L212P); Q at residue 20 and I at residue 156 (T20Q-V156I); Q at residue 20 and P at residue 214 (T20Q- D214P); P at residue 120 and E at residue 226 (V120P-K226E); I at residue 156 and P at residue 214 (V156I-D214P); P at residue 120 and G at residue 187 (V120P-V187G); P at residue 212 and P at residue 214 (L212P-D214P); P at residue 214 and E at residue 226 (D214P-K226E); G at residue 187 and E at residue 226 (V187G-K226E); Q at residue 20 and P at residue 120 (T20Q- V120P); I at residue 156 and P at residue 212 (V156I-L212P); P at residue 120 and I at residue 156 (V120P-V156I); Q at residue 20 and P at residue 212 (T20Q-L212P); P at residue 120 and P at residue 214 (V120P-D214P); G at residue 187 and P at residue 214 (V187G-D214P); Q at residue 20 and G at residue 187 (T20Q-V187G); P at residue 120 and P at residue 212 (V120P- L212P); Q at residue 20 and E at residue 226 (T20Q-K226E); I at residue 156 and G at residue 187 (V156I-V187G); I at residue 156 and E at residue 226 (V156I-K226E); P at residue 212 and E atSUBSTITUTE SHEET (RULE 26)residue 226 (L212P-K226E); A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P-K226E); or R at residue 35 and K at residue 207 (Q35R-L207K).

[0202] In some of these aspects, the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P- K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); N at residue 207 and L at residue 209 (L207N-I209L); R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); or D at residue 207 and V at residue 209 (L207D-I209V).

[0203] In some of these aspects, the two amino acid substitutions are selected from is A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P- K226E); or R at residue 35 and K at residue 207 (Q35R-L207K),

[0204] In some aspects, the polypeptides comprise an amino acid sequence of any one of SEQ ID NOs: 31-69. In some aspects, the polypeptides are encoded by polynucleotides comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 170-208.

[0205] In some aspects, the polypeptides comprise an amino acid sequence of SEQ ID NOs: 53, 67, or 69. In some aspects, the polypeptides are encoded by polynucleotides comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 192, 206, or 208.

[0206] In some aspects, the at least one amino acid substitution consists of three amino acid substitutions (i.e., the amino acid sequence of the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof, differs from the amino acid sequence of the NotGoA-DysABD (e.g., a NotGoA-DysABD 1), or an actin-binding fragment thereof, only at 3 positions).

[0207] In some of these aspects, the three amino acid substitutions are selected from G at residue 187, P at residue 214, E at residue 226 (V187G-D214P-K226E); Q at residue 20, P at residue 120, and G at residue 187 (T20Q-V120P-V187G); Q at residue 20, P at residue 120, and E at residue 226 (T20Q-V120P-K226E); Q at residue 20, G at residue 187, and E at residue 226 (T20Q-V187G-K226E); P at residue 120, I at residue 156, and P at residue 214 (V120P-V156I-SUBSTITUTE SHEET (RULE 26)D214P); P at residue 212, P at residue 214, and E at residue 226 (L212P-D214P-K226E); P at residue 120, G at residue 187, and E at residue 226 (V120P-V187G-K226E); P at residue 120, P at residue 212, and P at residue 214 (V120P-L212P-D214P); I at residue 156, P at residue 212, and P at residue 214 (V156I-L212P-D214P); Q at residue 20, I at residue 156, and P at residue 212 (T20Q-V156I-L212P); I at residue 156, G at residue 187, and E at residue 226 (V156I-V187G- K226E); Q at residue 20, P at residue 214, and E at residue 226 (T20Q-D214P-K226E); P at residue 120, P at residue 214, and E at residue 226 (V120P-D214P-K226E); Q at residue 20, P at residue 120, and P at residue 214 (T20Q-V120P-D214P); Q at residue 20, P at residue 120, and I at residue 156 (T20Q-V120P-V156I); Q at residue 20, G at residue 187, and P at residue 214 (T20Q-V187G- D214P); P at residue 120, l at residue 156, andP at residue 212 (V120P-V156I-L212P); l at residue 156, P at residue 214, and E at residue 226 (V156I-D214P-K226E); G at residue 187, P at residue 212, and E at residue 226 (V187G-L212P-K226E); P at residue 120, G at residue 187, and P at residue 214 (V120P-V187G-D214P); Q at residue 20, 1 at residue 156, and G at residue 187 (T20Q- V156I-V187G); I at residue 156, G at residue 187, and P at residue 214 (V156I-V187G-D214P); Q at residue 20, and I at residue 156, E226 (T20Q-V156I-K226E); Q at residue 20, P at residue 212, and E at residue 226 (T20Q-L212P-K226E); Q at residue 20, P at residue 120, and P at residue 212 (T20Q-V120P-L212P); Q at residue 20, G at residue 187, and P at residue 212 (T20Q-V187G- L212P); P at residue 120, I at residue 156, and E at residue 226 (V120P-V156I-K226E); P at residue 120, 1 at residue 156, and G at residue 187 (V120P-V156I-V187G); P at residue 120, P at residue 212, and E at residue 226 (V120P-L212P-K226E); P at residue 120, G at residue 187, and P at residue 212 (V120P-V187G-L212P); G at residue 187, P at residue 212, and P at residue 214 (V187G-L212P-D214P); I at residue 156, G at residue 187, and P at residue 212 (V156I-V187G- L212P); Q at residue 20, 1 at residue 156, and P at residue 214 (T20Q-V156LD214P); I at residue 156, P at residue 212, and E at residue 226 (V156I-L212P-K226E); Q at residue 20, P at residue 212, and P at residue 214 (T20Q-L212P-D214P); A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N-V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L- I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R-A168V).

[0208] In some of these aspects, the three amino acid substitutions are selected from A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N-V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L-I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R-A168V).SUBSTITUTE SHEET (RULE 26)

[0209] In some aspects, the polypeptides comprise an amino acid sequence of any one of SEQ ID NOs: 70-107. In some aspects, the polypeptides are encoded by polynucleotides comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 209- 246.

[0210] In some aspects, the polypeptides comprise an amino acid sequence of SEQ ID NOs: 105, 106, or 107. In some aspects, the polypeptides are encoded by polynucleotides comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 244, 245, or 246.

[0211] In some aspects, the at least one amino acid substitution consists of four amino acid substitutions ((i.e,. the amino acid sequence of the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof, differs from the amino acid sequence of the NotGoA-DysABD (e.g., a NotGoA-DysABD 1), or an actin-binding fragment thereof, only at 4 positions).

[0212] In some of these aspects, the four amino acid substitutions are selected from Q at residue 20, P at residue 120, G at residue 187, and P at residue 212 (T20Q-V120P-V187G-L212P); Q at residue 20, P at residue 120, P at residue 212, and P at residue 214 (T20Q-V120P-L212P- D214P); Q at residue 20, 1 at residue 156, G at residue 187, and P at residue 214 (T20Q-V156I- V187G-D214P); Q at residue 20, P at residue 212, P at residue 214, and E at residue 226 (T20Q- L212P-D214P-K226E); P at residue 120, 1 at residue 156, G at residue 187, and P at residue 212 (V120P-V156I-V187G-L212P); I at residue 156, G at residue 187, P at residue 212, and E at residue 226 (V156I-V187G-L212P-K226E); P at residue 120, I at residue 156, P at residue 212, and E at residue 226 (V120P-V156I-L212P-K226E); Q at residue 20, G at residue 187, P at residue 212, and P at residue 214 (T20Q-V187G-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and E at residue 226 (V120P-V187G-L212P-K226E); Q at residue 20, I at residue 156, P at residue 212, and E at residue 226 (T20Q-V156I-L212P-K226E); G at residue 187, P at residue 212, P at residue 214, and E at residue 226 (V187G-L212P-D214P-K226E); Q at residue 20, P at residue 120, G at residue 187, and E at residue 226 (T20Q-V120P-V187G-K226E); Q at residue 20, P at residue 120, 1 at residue 156, and P at residue 212 (T20Q-V120P-V156I-L212P); Q at residue 20, P at residue 120, P at residue 214, and E at residue 226 (T20Q-V120P-D214P- K226E); Q at residue 20, 1 at residue 156, G at residue 187, and P at residue 212 (T20Q-V156I- V187G-L212P); I at residue 156, G at residue 187, P at residue 212, and P at residue 214 (V156I- V187G-L212P-D214P); P at residue 120, 1 at residue 156, G at residue 187, and E at residue 226SUBSTITUTE SHEET (RULE 26)(V120P-V156I-V187G-K226E); P at residue 120, I at residue 156, P at residue 212, and P at residue 214 (V120P-V156I-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and P at residue 214 (V120P-V187G-L212P-D214P); Q at residue 20, G at residue 187, P at residue 214, and E at residue 226 (T20Q-V187G-D214P-K226E); Q at residue 20, I at residue 156, P at residue 212, and P at residue 214 (T20Q-V156I-L212P-D214P); I at residue 156, P at residue 212, P at residue 214, and E at residue 226 (V156I-L212P-D214P-K226E); Q at residue 20, P at residue 120, G at residue 187, and P at residue 214 (T20Q-V120P-V187G-D214P); Q at residue 20, P at residue 120, l at residue 156, and G at residue 187 (T20Q-V120P-V156I-V187G); l at residue 156, G at residue 187, P at residue 214, and E at residue 226 (V156I-V187G-D214P-K226E); Q at residue 20, P at residue 120, 1 at residue 156, and E at residue 226 (T20Q-V120P-V156I-K226E); P at residue 120, 1 at residue 156, P at residue 214, and E at residue 226 (V120P-V156I-D214P- K226E); Q at residue 20, I at residue 156, G at residue 187, and E at residue 226 (T20Q-V156I- V187G-K226E); Q at residue 20, P at residue 120, P at residue 212, and E at residue 226 (T20Q- V120P-L212P-K226E); P at residue 120, G at residue 187, P at residue 214, and E at residue 226 (V120P-V187G-D214P-K226E); P at residue 120, P at residue 212, P at residue 214, and E at residue 226 (V120P-L212P-D214P-K226E); Q at residue 20, G at residue 187, P at residue 212, and E at residue 226 (T20Q-V187G-L212P-K226E); P at residue 120, l at residue 156, Gat residue 187, and P at residue 214 (V120P-V156I-V187G-D214P); Q at residue 20, 1 at residue 156, P at residue 214, and E at residue 226 (T20Q-V156I-D214P-K226E); Q at residue 20, P at residue 120, I at residue 156, and P at residue 214 (T20Q-V120P-V156I-D214P); or A at residue 17, A at residue 20, P at residue 212, and E at residue 226 (Q17A-T20A-L212P-K226E).

[0213] In some of these aspects, the four amino acid substitutions are A at residue 17, A at residue 20, P at residue 212, and E at residue 226 (Q17A-T20A-L212P-K226E).

[0214] In some aspects, the polypeptides comprise an amino acid sequence of any one of SEQ ID NOs: 108-143. In some aspects, the polypeptides are encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 248- 282.

[0215] In some aspects, the polypeptides comprise an amino acid sequence of SEQ ID NO: 143. In some aspects, the polypeptides are encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 282.SUBSTITUTE SHEET (RULE 26)

[0216] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 20% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA- DysABD1), or an actin-binding fragment thereof, (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-Dys ABDI), or an actin-binding fragment thereof), in a same fluorescence resonance energy transfer (FRET) assay.

[0217] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 30% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA- DysABD 1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.

[0218] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 40% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.

[0219] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 50% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.

[0220] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 60% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA-SUBSTITUTE SHEET (RULE 26)DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.

[0221] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 70% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.

[0222] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 80% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.

[0223] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 90% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.

[0224] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 95% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.SUBSTITUTE SHEET (RULE 26)

[0225] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 99% compared to a Kd of a same NotGoA-DysABD (e g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-Dys ABDI), or an actin-binding fragment thereof), in a same FRET assay.

[0226] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 99.9% compared to a Kd of a same NotGoA-DysABD (e.g., a NotGoA- DysABD 1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.

[0227] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a dissociation constant Kd reduced of at least 100% compared to a Kd of a same NotGoA-DysABD (e.g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.

[0228] It is to be understood that a GoA-DysABD (e.g., a GoA-DysABD1), or an actin- binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), is intended to bind to actin with a dissociation constant Kd reduced of at least, e.g., 50% compared to a Kd of a same NotGoA-DysABD (e.g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay, when the GoA-DysABD1 (e.g., a GoA-DysABD1), or an actin-binding fragment thereof (or a same GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin- binding fragment thereof), binds to actin with a Kd of, e.g., 50 pM, and a same NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof (or a same NotGoA-DysSUBSTITUTE SHEET (RULE 26)comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), binds to actin with a Kd of 100 pM as measured in a same FRET assay.

[0229] It is also to be understood that "a same FRET assay" is a FRET assay performed using the same reagents, conditions and parameters (as described herein) as another FRET assay. The FRET assay used to measure the Kd of the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA- DysABD1), or an actin-binding fragment thereof), and the RET assay used to measure the Kd of the same NotGoA-DysABD (e g., a NotGoA-DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e g., a NotGoA-DysABD1), or an actin- binding fragment thereof), are thus performed using the same reagents, conditions and parameters. The FRET assay used to measure the Kd of the GoA-DysABD (e.g., a GoA-DysABD 1), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA- DysABD1), or an actin-binding fragment thereof), and the RET assay used to measure the Kd of the a same NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), can be performed simultaneously or sequentially, as long as are performed using the same reagents, conditions and parameters.

[0230] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein bind to actin with a Kd of about 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM, 160 pM, 150 pM, 140 pM, 130 pM, 120 pM, 110 pM, 100 pM, 95 pM, 90 pM, 85 pM, 80 pM, 75 pM, 70 pM, 65 pM, 60 pM, 55 pM, 50 pM, 45 pM, 40 pM, 35 pM, 30 pM, 25 pM, 20 pM, 15 pM, 10 pM, 9 pM, 8 pM, 7 pM, 6 pM, 5 pM, 4 pM, 3 pM, 2 pM, 1 pM, 500 nM, 450 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM, 55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM as measured in a FRET assay.

[0231] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 99 pM and 0.1 pM as measured in a FRET assay.SUBSTITUTE SHEET (RULE 26)

[0232] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 80 pM and 0.1 pM as measured in a FRET assay.

[0233] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 70 pM and 0.1 pM as measured in a FRET assay.

[0234] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 60 pM and 0.1 pM as measured in a FRET assay.

[0235] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 50 pM and 0.1 pM as measured in a FRET assay.

[0236] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 40 pM and 0.1 pM as measured in a FRET assay.

[0237] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 30 pM and 0.1 pM as measured in a FRET assay.

[0238] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 20 pM and 0.1 pM as measured in a FRET assay.

[0239] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 10 pM and 0.1 pM as measured in a FRET assay.SUBSTITUTE SHEET (RULE 26)

[0240] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 5 pM and 0.1 pM as measured in a FRET assay.

[0241] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein binds to actin with a Kd between 1 pM and 0.1 pM as measured in a FRET assay.

[0242] In some aspects, the GoA-DysABDs (e.g., a GoA-DysABD1), or actin-binding fragments thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), disclosed herein has an actin-binding affinity that is increased on at least 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21- fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32- fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43- fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, 50-fold, compared to a Kd of a sameNotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), in a same FRET assay.Fluorescence Resonance Energy Transfer (FRET) Assay: reagents, conditions and parameters

[0243] In some aspects, in the FRET assay a first fluorophore is attached to the GoA- DysABD (e g., a GoA-DysABD1), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), and a second fluorophore is attached to actin. In some aspects, an mClover3 fluorophore is attached to the GoA- DysABD (e g., a GoA-DysABD1), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), and an Alexa- 568 fluorophore is attached to actin. In some aspects, the mClover3 fluorophore is attached to an amino-terminal of the GoA-DysABD (e.g., a GoA-DysABD1, mClover3 -GoA-DysABD1), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA- DysABD1), or an actin-binding fragment thereof). In some aspects, the mClover3 fluorophore is attached to a carboxy-terminal of the GoA-DysABD (e.g., a GoA-DysABD1, GoA-DysABD1-SUBSTITUTE SHEET (RULE 26)mClover3), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD 1), or an actin-binding fragment thereof).

[0244] In some aspects, in the FRET assay a first fluorophore is attached to the NotGoA- DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof (or a same NotGoA- Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), and a second fluorophore is attached to actin. In some aspects, an mClover3 fluorophore is attached to the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment therof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), and an Alexa-568 fluorophore is attached to actin. In some aspects, the mClover3 fluorophore is attached to an amino-terminal of the NotGoA-DysABD (e g., a NotGoA-DysABD1, mClover3-NotGoA-DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin- binding fragment thereof). In some aspects, the mClover3 fluorophore is attached to a carboxyterminal of the NotGoA-DysABD (e.g., a NotGoA-DysABD1, NotGoA-DysABD l-mClover3), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e g., a NotGoA-DysABD1), or an actin-binding fragment thereof).

[0245] In some aspects, in the FRET assay the actin is a phalloidin-stabilized F-actin. In some aspects, the Alexa-568 fluorophore is attached to residue C374 of the phalloidin-stabilized F-actin.

[0246] In some aspects, the FRET assay is a time-resolved FRET assay (see, for example Guhathakurta P et al., High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin-myosin structure and function. J Biol Chem. 2018 Aug 3;293(31): 12288-12298.).

[0247] In some aspects, sub-micromolar amounts of the GoA-DysABD (e g., a GoA- DysABD1), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), attached to the first fluorophore are used. In some aspects, sub -micromol ar amounts of the NotGoA-DysABD (e.g., a NotGoA- DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), attached to the first fluorophore are used.

[0248] In some aspects, sub-micromolar amounts of the GoA-DysABD (e g., a GoA- DysABD1), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABDSUBSTITUTE SHEET (RULE 26)(e.g., a GoA-DysABD1), or an actin-binding fragment thereof), attached to the mClover3 fluorophore are used. In some aspects, sub-micromolar amounts of the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof (or a same NotGoA-Dys comprising the NotGoA-DysABD (e g., aNotGoA-DysABD1), or an actin-binding fragment thereof), attached to the mClover3 fluorophore are used.

[0249] In some aspects, sub-micromolar amounts of GoA-DysABD1-mClover3 or of NotGoA-DysABD l-mClover3 are used.

[0250] In some aspects, the amount of actin attached to the second fluorophore is increased over time. In some aspects, the amount of actin attached to the Alexa-568 fluorophore is increased over time.

[0251] In some aspects, the fluorescence decay lifetime of the GoA-DysABD (e.g., a GoA- DysABD1), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), attached to the first fluorophore following a sub-nanosecond excitation pulse is measured. In some aspects, the fluorescence decay lifetime of the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof (or a NotGoA-Dys comprising the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof), attached to the first fluorophore following a sub -nanosecond excitation pulse is measured.

[0252] In some aspects, the fluorescence decay lifetime of the GoA-DysABD (e.g., a GoA- DysABD1), or an actin-binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), attached to the mClover3 fluorophore following a sub-nanosecond excitation pulse is measured. In some aspects, the fluorescence decay lifetime of the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin- binding fragment thereof (or a NotGoA-Dys comprising the NotGoA-DysABD (e g., a NotGoA- DysABD1), or an actin-binding fragment thereof), attached to the mClover3 fluorophore following a sub -nanosecond excitation pulse is measured.

[0253] In some aspects, the fluorescence decay lifetime of the GoA-DysABD l-mClover3 or of the NotGoA-DysABD l-mClover3 following a sub-nanosecond excitation pulse is measured.

[0254] In some aspects, the Kd of the GoA-DysABD (e.g., a GoA-DysABD 1), or an actin- binding fragment thereof (or a GoA-Dys comprising the GoA-DysABD (e.g., a GoA-DysABD1), or an actin-binding fragment thereof), and of the NotGoA-DysABD (e.g., a NotGoA-DysABD1), or an actin-binding fragment thereof (or a NotGoA-Dys comprising the NotGoA-DysABD (e.g., aSUBSTITUTE SHEET (RULE 26)NotGoA-DysABD1), or an actin-binding fragment thereof), can be additionally measured with a high-speed actin-cosedimentation (HSC) assay.

[0255] The GoA-DysABD (e.g., a GoA-DysABD1) or the GoA-Dys of the disclosure may be produced by any of the suitable means disclosed in the published literature. For example, the GoA-DysABDs (e.g., a GoA-DysABD1) or the GoA-Dys of the disclosure may be produced by chemical synthesis or by recombinant DNA technology. If produced by recombinant DNA technology the GoA-DysABDs (e.g., a GoA-DysABD1) or the GoA-Dys of the disclosure may be produced in any suitable cell line (e.g., a prokaryotic or an eukaryotic (e.g., a mammalian) cell line). Upon chemical synthesis or production in recombinant cells, the GoA-DysABDs (e g., a GoA-DysABD1) or the GoA-Dys of the disclosure may be purified any of the suitable means disclosed in the published literature and maintained in a purified form. Alternatively, the GoA- DysABDs (e.g., a GoA-DysABD1) or the GoA-Dys of the disclosure can be maintained in the recombinant cells.

[0256] Alternatively, the GoA-DysABDs (e.g., a GoA-DysABD1) or the GoA-Dys of the disclosure can be expressed into an organism by introducing a nucleotide sequence encoding the GoA-DysABDs (e g., a GoA-DysABD1) or the GoA-Dys of the disclosure into a cell (e.g., into the genome of a cell as a transgene) of the organism. For example, a nucleotide sequence encoding the GoA-DysABD or dystrophin protein comprising the same can be inserted into a vector (e.g., a viral vector such as an AAV vector), and a cell within the organism can be contacted with the vector (e.g., with an rAAV particle comprising an AAV vector genome encoding the GoA- DysABD (e.g., a GoA-DysABD1) or dystrophin protein comprising the same), to achieve expression of the GoA-DysABD (e.g., a GoA-DysABD1) or dystrophin protein comprising the same into the cell of the organism. Vectors comprising a polynucleotide encoding the GoA- DysABDs (e.g., a GoA-DysABD1) or the GoA-Dys of the disclosure are also provided herein, as well as methods of making the vectors and using the same.Dystrophin Proteins

[0257] A NotGoA-DysABD (e.g., a NotGoA-DysABD1) can have the amino acid sequence of an actin-binding domain (ABD) of a naturally occurring dystrophin protein, or can be encoded by a polynucleotide having a nucleotide sequence encoding an ABD of a naturally occurring dystrophin protein. In some aspects the ABD is an ABDI, an ABD2, or a combination thereof. In some aspects the ABD is an ABDI.SUBSTITUTE SHEET (RULE 26)

[0258] The naturally occurring dystrophin protein can be a wild type dystrophin protein or a variant dystrophin protein. A naturally occurring dystrophin protein can have an amino acid sequence of a wild type, an isoform, or a variant of the human dystrophin protein A naturally occurring dystrophin protein can be encoded by a nucleotide sequence of a wild type transcript or of a transcript variant of the human dystrophin gene. A naturally occurring dystrophin protein can be encoded by a nucleotide sequence of a wild type or a variant of the human dystrophin gene.

[0259] The human dystrophin protein is encoded by the Dystrophin DMD gene (also known as BMD; CMD3B; MRX85; DXS142; DXS164; DXS206; DXS230; DXS239; DXS268; DXS269; DXS270; DXS272) (e.g., reference sequence NC_000023.l l (GRCh38.pl 4 (GCF_000001405.40) 31119222-33339388), NC_060947.1 (T2T-CHM13v2.0(GCF_009914755.1) 30717372-32937500). A number of transcript variants of the human dystrophin gene are known, for example XM_006724469.4 (dystrophin transcript variant X2), NM_000109.4 (dystrophin transcript variant Dp427c), NM_004009.3 (dystrophin transcript variant Dp427pl),XM_011545467.2 (dystrophin transcript variant X5), XM_006724473.3 (dystrophin transcript variant X6), XM_006724474.4 (dystrophin transcript variant X7), XM_006724475.3 (dystrophin transcript variant X8), XM_006724470.4 (dystrophin transcript variant X3), XM_017029328.2 (dystrophin transcript variant X4), XM_006724468.3 (dystrophin transcript variant XI), or NM_004006.3 (dystrophin transcript variant Dp427m, or Pl 1532-1), encoding XP_006724532.1 (dystrophin isoform X2), NP_000100.3 (dystrophin isoform Dp427c), NP_004000.1 (dystrophin isoform Dp427pl), XP_011543769.1 (dystrophin isoform X5), XP_006724536.1 (dystrophin isoform X6), XP_006724537.1 (dystrophin isoform X7), XP_006724533.1 (dystrophin isoform X3), XP_006724531.1 (dystrophin isoform XI), , XP_006724538.1 (dystrophin isoform X8), XP_016884817.1 (dystrophin isoform X4), or NP_003997.2 (dystrophin isoform Dp427m, or Pl 1532-1). Table 1 shows non-limiting examples of Reference Sequences of DMD transcript variants and Dystrophin Protein Isoform.

[0260] Table 1. DMD transcript variants and Dystrophin Protein IsoformReference Sequences.SUBSTITUTE SHEET (RULE 26)

[0261] Additionally, exemplary variants of the human dystrophin protein are VAR_065764 [W118R], VAR_005148 [Q133P] (rsl800256), VAR_023538 [D165V], VAR_036353, VAR_005151 (rsl800266), VAR_057642 (rs34155804), VAR_057643 (rs5972599), VAR_005153 (rsl800259), VAR_062110 (rs34563188), VAR_057644(rsl6998350), VAR_005155 (rsl800260), VAR_005156 (rs228406), VAR_057645 (rs3827462), VAR_005157 (rsl800262), VAR_036354, VAR_005158 (rsl800269), VAR_005159 (rsl800270), VAR_005160 (rsl800263), VAR_057646 (rs28715870), VAR_005161 (rsl057872), VAR_036355, VAR_023542 (rsl6990264), VAR_005162 (rsl801187), VAR_005163 (rsl801186), VAR_057647 (rsl6990169), VAR_005164 (rsl800273), VAR_036356, VAR_005165, VAR_023543, VAR_005167 (rsl800275), VAR_005168 (rs41305353), VAR_005169 (rsl800278), and VAR_005171 (rsl800280) .

[0262] Table 2 shows non-limiting examples of Reference Sequences of human Dystrophin Protein variants, wherein the amino acids positions are to be understood as referring to the amino acid positions of the UniProt reference sequence Pl 1532.SUBSTITUTE SHEET (RULE 26)SUBSTITUTE SHEET (RULE 26)Dystrophin Proteins Additional Portions

[0263] A full length human dystrophin protein consists of, from the amino-terminus to the carboxy-terminus: an actin-binding domain 1 (ABDI), a central rod domain consisting of a first hinge (Hl), 3 spectrin-like repeats (R1 to R3), a second hinge (H2), 16 spectrin-like repeats (R4 to R19), a third hinge (H3), 5 spectrin-like repeats (R20 to R4), a fourth hinge (H4), a cysteine rich domain (CR), and a carboxy-terminus (Chamberlain et al., Microdystrophin Expression as a Surrogate Endpoint for Duchenne Muscular Dystrophy Clinical Trials. Hum Gene Ther. 2023 May;34(9-10):404-415).

[0264] In some aspects, the polypeptides disclosed herein further comprise one or more additional portions of a dystrophin protein. In some aspects, the polypeptides disclosed herein further comprise one or more dystrophin protein spectrin-like repeat or a variant or fragment thereof, one or more dystrophin protein hinge region or a variant or fragment thereof, one or more dystrophin protein cysteine-rich domain or a variant or fragment thereof, and / or one or more dystrophin protein carboxyl-terminus or a variant or fragment thereof.

[0265] In some aspects, the polypeptides disclosed herein further comprise at least one dystrophin protein spectrin-like repeat located downstream to the GoA-DysABD1 domain.

[0266] The at least one dystrophin protein spectrin-like repeat can be a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein spectrin-like repeat 4, a dystrophin protein spectrin-like repeat 5, a dystrophin protein spectrin-like repeat 6, a dystrophin protein spectrin-like repeat 7, a dystrophin protein spectrin-like repeat 8, a dystrophin protein spectrin-like repeat 9, a dystrophin protein spectrin-like repeat 10, a dystrophin protein spectrin-like repeat 11, a dystrophin protein spectrinlike repeat 12, a dystrophin protein spectrin-like repeat 13, a dystrophin protein spectrin-like repeat 14, a dystrophin protein spectrin-like repeat 15, a dystrophin protein spectrin-like repeat 16, a dystrophin protein spectrin-like repeat 17, a dystrophin protein spectrin-like repeat 18, a dystrophin protein spectrin-like repeat 19, a dystrophin protein spectrin-like repeat 20, a dystrophin protein spectrin-like repeat 21, a dystrophin protein spectrin-like repeat 22, a dystrophin protein spectrinlike repeat 23, a dystrophin protein spectrin-like repeat 24, or any combination thereof.

[0267] In some aspects, the polypeptides disclosed herein further comprise at least one dystrophin protein cysteine-rich domain located downstream to the dystrophin protein spectrinlike repeat.SUBSTITUTE SHEET (RULE 26)

[0268] In some aspects, the polypeptides disclosed herein further comprise at least one dystrophin protein hinge region located downstream to the GoA-DysABD1 and upstream to the cysteine-rich domain.

[0269] The at least one dystrophin protein hinge region can be a dystrophin protein hinge region 1, a dystrophin protein hinge region 2, a dystrophin protein hinge region 3, or a dystrophin protein hinge region 4.

[0270] It is to be understood that each dystrophin protein hinge region can be located upstream or downstream to a dystrophin protein spectrin-like repeat, and vice versa.

[0271] For example, a polypeptide disclosed herein can comprise from amino-terminus to carboxy-terminus: a GoA-DysABD1, a dystrophin protein hinge region 1, a dystrophin protein spectrin-like repeat 1, and a dystrophin protein hinge region 4; or a GoA-DysABD1, a dystrophin protein hinge region 1, a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrinlike repeat 16, and a dystrophin protein hinge region 4.

[0272] In some aspects, the polypeptides disclosed herein further comprise at least one dystrophin protein carboxy-terminal domain (CTD) located downstream to the cysteine-rich domain.

[0273] As used herein the terms "downstream" and "upstream" are used as descriptors of the reciprocal position of two or more sequences. The terms can be used to describe the reciprocal position of both amino acid and nucleotide sequences.

[0274] When referring to amino acid sequences the term "downstream" indicates that a sequence is located carboxy-terminal to another sequence, and the term "upstream" indicates that a sequence is located amino-terminal of another sequence. For example, in a polypeptide of formula:N-terminus_Z-X-Y -W_C -terminus wherein Z, X, Y and W correspond to four amino acid sequences comprised in the same polypeptide; for example, X is located upstream of Y, and Y is located downstream of X.

[0275] Similarly, when referring to nucleotide sequences the term "downstream" indicates that a sequence is located at the 3' of another sequence, and the term "upstream" indicates that a sequence is located at the 5' of another sequence. For example, in a polynucleotide of formula:5'-Z-X-Y-W-3'SUBSTITUTE SHEET (RULE 26)wherein Z, X, Y and W correspond to four nucleotide sequences comprised in the same polynucleotide; for example, X is located upstream of Y, and Y is located downstream of X.

[0276] Thus, a polypeptide comprising, for example:(i) a dystrophin protein spectrin-like repeat located downstream to a GoA- DysABD1 domain;(ii) a dystrophin protein hinge region located downstream to a GoA-DysABD1 domain and upstream to the dystrophin protein spectrin-like repeat;(iii) a dystrophin protein cysteine-rich domain located downstream to the dystrophin protein spectrin-like repeat; and(iv) a dystrophin protein CTD located downstream to the cysteine-rich domain; it is to be understood to comprise, from amino-terminus to carboxy-terminus: a GoA-DysABD1 - a dystrophin protein hinge region - a dystrophin protein spectrin-like repeat - dystrophin protein cysteine-rich domain - a dystrophin protein CTD.

[0277] In some aspects, the polypeptides disclosed herein comprise at least a dystrophin protein cysteine-rich domain or a variant or fragment thereof. In some aspects, the polypeptides disclosed herein comprise at least a dystrophin protein cysteine-rich domain or a variant or fragment thereof, and at least four dystrophin protein spectrin-like repeats.

[0278] In some aspects, the polypeptides disclosed herein comprise:(i) a GoA-DysABD1;(ii) dystrophin protein spectrin-like repeats 1, 2, 3, and 24;(iii) dystrophin protein hinge regions 1, 2, and 4; and(iv) a dystrophin protein cysteine-rich domain.

[0279] In some aspects, the polypeptides disclosed herein, from amino-terminus to carboxyl-terminus comprise:(i) a GoA-DysABD 1 ;(ii) a dystrophin protein hinge region 1 ;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 2;(v) a dystrophin protein spectrin-like repeat 3;(vi) a dystrophin protein hinge region 2;SUBSTITUTE SHEET (RULE 26)(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4; and(ix) a dystrophin protein cysteine-rich domain.

[0280] In some aspects, the polypeptides disclosed herein, from amino-terminus to carboxyl-terminus consists of:(i) a GoA-DysABD 1 ;(ii) a dystrophin protein hinge region 1 ;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 2;(v) a dystrophin protein spectrin-like repeat 3;(vi) a dystrophin protein hinge region 2;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4; and(ix) a dystrophin protein cysteine-rich domain.

[0281] In some aspects, the polypeptides disclosed herein comprise:(i) a GoA-DysABD 1 ;(ii) dystrophin protein spectrin-like repeats 1, 2, 22, 23, and 24;(iii) dystrophin protein hinge regions 1, 3, and 4; and(iv) a dystrophin protein cysteine-rich domain.

[0282] In some aspects, the polypeptides disclosed herein, from amino-terminus to carboxyl-terminus comprise:(i) the GoA-DysABD 1 ;(ii) a dystrophin protein hinge region 1 ;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 22;(v) a dystrophin protein spectrin-like repeat 23;(vi) a dystrophin protein spectrin-like repeat 24;(vii) a dystrophin protein hinge region 4; and(viii) a dystrophin protein cysteine-rich domain.

[0283] In some aspects, the polypeptides disclosed herein, from amino-terminus to carboxyl-terminus consist of:(i) the GoA-DysABD 1 ;SUBSTITUTE SHEET (RULE 26)(ii) a dystrophin protein hinge region 1 ;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 22;(v) a dystrophin protein spectrin-like repeat 23;(vi) a dystrophin protein spectrin-like repeat 24;(vii) a dystrophin protein hinge region 4; and(viii) a dystrophin protein cysteine-rich domain.

[0284] In some aspects, the polypeptides disclosed herein comprise:(i) the GoA-DysABD 1 ;(ii) dystrophin protein spectrin-like repeats 1, 16, 17, 23, and 24;(iii) dystrophin protein hinge regions 1, and 4; and(iv) a dystrophin protein cysteine-rich domain.

[0285] In some aspects, the polypeptides disclosed herein, from amino-terminus to carboxyl-terminus comprise:(i) the GoA-DysABD 1 ;(ii) a dystrophin protein hinge region 1 ;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 16;(v) a dystrophin protein spectrin-like repeat 17;(vi) a dystrophin protein spectrin-like repeat 23;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4; and(ix) a dystrophin protein cysteine-rich domain.

[0286] In some aspects, the polypeptides disclosed herein, from amino-terminus to carboxyl-terminus consist of:(i) the GoA-DysABD 1 ;(ii) a dystrophin protein hinge region 1 ;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 16;(v) a dystrophin protein spectrin-like repeat 17;(vi) a dystrophin protein spectrin-like repeat 23;(vii) a dystrophin protein spectrin-like repeat 24;SUBSTITUTE SHEET (RULE 26)(viii) a dystrophin protein hinge region 4; and(ix) a dystrophin protein cysteine-rich domain.

[0287] In some aspects, the polypeptides disclosed herein comprise:(i) the GoA-DysABD 1 ;(ii) dystrophin protein spectrin-like repeats 1, 2, 3, and 24;(iii) dystrophin protein hinge regions 1, 3, and 4;(iv) a dystrophin protein cysteine-rich domain; and(v) a dystrophin protein carboxyl-terminus.

[0288] In some aspects, the polypeptides disclosed herein, from amino-terminus to carboxyl-terminus comprise:(i) the GoA-DysABD 1;(ii) a dystrophin protein hinge region 1;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 2;(v) a dystrophin protein spectrin-like repeat 3;(vi) a dystrophin protein hinge region 3;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4;(ix) a dystrophin protein cysteine-rich domain; and(x) a dystrophin protein CTD.

[0289] In some aspects, the polypeptides disclosed herein, from amino-terminus to carboxyl-terminus consist of:(i) the GoA-DysABD 1 ;(ii) a dystrophin protein hinge region 1 ;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 2;(v) a dystrophin protein spectrin-like repeat 3;(vi) a dystrophin protein hinge region 3;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4;(ix) a dystrophin protein cysteine-rich domain; and(x) a dystrophin protein CTD.SUBSTITUTE SHEET (RULE 26)

[0290] In some aspects, the polypeptides disclosed herein comprise an amino acid sequence of a dystrophin protein, for example of a truncated dystrophin protein, wherein at least one ABD is a GoA-DysABD (e.g., a GoA-DysABD1).

[0291] Several isoforms and variant dystrophin proteins, as well as truncated dystrophin proteins, such as microdystrophins or minidystrophins, are known in the art. It is to be understood that any DysABD (e.g., a DysABD1) of any isoform or variant dystrophin protein, as well as of any truncated dystrophin protein, such as a microdystrophin or a minidystrophin known in the art, can be substituted with a GoA-DysABD (e.g., a GoA-DysABD1) disclosed herein, to obtain a dystrophin protein, for example a truncated dystrophin protein, having increased actin binding affinity.

[0292] Exemplary truncated dystrophin proteins (e.g., microdystrophins and minidystrophins) are:(a) DysAR4-R23 / ACTD: comprising a DysABD1 and dystrophin protein spectrin-like repeats 1, 2, 3, and 24, dystrophin protein hinge regions 1, 2, and 4, and a dystrophin protein cysteine-rich domain;(b) DysAR3-R21+H3 / AC: comprising a DysABD1 and dystrophin protein spectrin-like repeats 1, 2, 22, 23, and 24, dystrophin protein hinge regions 1, 3, and 4, and a dystrophin protein cysteine-rich domain;(c) DysRl-R22+R16R17 / AC: comprising a DysABD1 and dystrophin protein spectrin-like repeats 1, 16, 17, 23, and 24, dystrophin protein hinge regions 1, and 4, and a dystrophin protein cysteine-rich domain;(d) DysAH2-R23+H3: comprising a DysABD1 and dystrophin protein spectrin-like repeats 1, 2, 3, and 24, dystrophin protein hinge regions 1, 3, and 4, a dystrophin protein cysteine-rich domain, and a dystrophin protein carboxyl-terminus.

[0293] Each DysABD1 in each one of the above truncated dystrophin proteins can be substituted with a GoA-DysABD1 disclosed herein to obtain a truncated dystrophin protein having increased actin-binding affinity.

[0294] Other non-limiting examples of truncated dystrophin proteins (e.g., microdystrophins and minidystrophins) are: DysA17-48, DysAH2-R19, DysAH2-R15, DysAR2-23, DysAR2-15 / AR18-22 / ACTD, DysAR3-19 / AR20-21 / ACTD, DysAR2-15 / AR18-19 / AR20-23 / ACTD, Dys3978, Dys3849, Dys-d3990, or DYSF. Each DysABD1 in each one of the above truncatedSUBSTITUTE SHEET (RULE 26)dystrophin proteins can be substituted with a GoA-DysABD1 disclosed herein to obtain a truncated dystrophin protein having increased actin-binding affinity.

[0295] The structure of dystrophin proteins is well known in the publish literature, and a person skilled in the art can easily identify the amino acid residues corresponding to each and every one of the dystrophin protein portions (i.e., dystrophin protein spectrin -like repeats, dystrophin protein hinge regions, dystrophin protein cysteine-rich domain, dystrophin protein CTD) disclosed herein.

[0296] For example, in some aspects:

[0297] a dystrophin protein can comprise, from amino-terminus to carboxy-terminus, an amino acid sequence of:ABDI - (aal -246 ) -AIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVTTSD PTRSPFPSQHLEAPEDKSFGSSX334MESEVNLDRYQTALEEVLSWLLSAEDTLQAX365GEISN DVEWKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGX409GKLSEDEETEVQEQMNLLNSRWE CLRVASMEKQSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQ EDLEQEQVRVNSLTHMWWDESSGDHATAALEEQLKVLGDRWANICRWTEDRWVLLQDILLKWQ RLTEEQCLFSX573WLSEKEDAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSX6 23KQDLLSTLKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQIX666QAVTTTQPSLTQTTVME TVTTVTTREQILVKHAQEELPPPPPQKKRQIX715VDSEIRKRLDVDITELHSWITRSEAVLQSP EFAIFRKEGNFSDLKEKVNAIEREKAEKFRKLQDASRSX784QALVEQMVNEGVNADSIKQASEQ LNSRWIEFCQLLSERLNWLEYQNNIIAFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKI CKDEVNRLSX882LQPQIERLKIQSIALKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREKELQT IFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSLQEQQSGLYYL STTVKEMSKKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQMNKLRKIQNHIQTLKKWM AEVDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTIQPSLNSVNEGGQKIKNEAEPEFASRLET ELKELNX1136QWDHMCQQVYARKEALKGGLEKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTP DELQKAX1197EEMKRAKEEAQQKEAKVKLLTX1219SVNSVIAQAPPVAQEALKKELETLTX12 45NYQWLCTRLNGKCKTLEEVWACWHELLSYLEKX1278NKWLNEVEFKLKTTENIPGGAEEISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMDELINEELETFNSRWRELHEEAVRRQKLLEQSI QSAQETEX1377SLHLIQESLTX1388IDKQLAAYIADKVDAAQMPQEAQKIQSDLTSHEISLEE MKKHNQGKEAAQRVLSQIDVAQKKLQDVSMKFRLFQKPANFEX1469X147 OLQESKMILDEVKM HLPALETKSVEQEWQSQLNHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTENPKELDERVTAL KLHYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEVAWSUBSTITUTE SHEET (RULE 26)GKATQKE IEKQKVHLKS ITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLX1672 LL LEYQKHMETFDQNVDHITKWIIQADTLLDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRDQAA NLMANX1745GDHCRKLVEPQISELNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAE IQQGVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQRITDERKX1844EEIKIKQQLLQTKHNAL KDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIKEIDRELQKKKEEL NAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIESKFAQFRRLNFAQIHTVREETMMVMTEDMPL EISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIH SKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRX2108FHYDIKIFN QWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQX2155QTWRTLNX2164TGEEIIQQSS KTDASILQEKLGSLNLX2191WQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIAS IPLEPGKEQQLKEKLEQVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTX2 299LQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEG PFDVX2366ETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQELRA KQPDLAPGLTTIGASPTQTVTLVTQPWTKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLS LLDQVIKSQRVMVGDLEDINEMIIKQKATMQDLEQRRPQLEELITTKAQNLKNKTSNQEARTIITD RIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLGQARAKLESWKEGPYTVDAIQ KKITETKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHMITENINASWRSIHKRVSEREA ALEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGEIEA HTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEASSDQWKRLHL SLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQP LEGLEKLYQEPRELPPEERAQNVTRLLRKQAEX2910VX2912TEWEKLNLHSADWQRKIDETLE RLX2937ELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHV NDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGP WERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLS LSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGR TGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFG GSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECP IIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRY FAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENS NGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEE ENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILE DHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPMLLRWGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM (SEQ ID NO: 3)SUBSTITUTE SHEET (RULE 26)whereinABD l-(aa 1-246) corresponds to any one of SEQ ID NOs: 1, 2, or 293; wherein the amino terminal A corresponds to amino acid residue 247; and whereinX334 is L or F; X365 is Q or H; X409 is T or S; X573 is A or V; X623 is L or I; X666 is S or L; X715 is T or S; X784 is A or G; X882 is D or G; XI 136 is T or S; XI 197 is V or F; X1219 is E or Q; X1245 is T or I; X1278 is A or P; X1377 is K or N; X1388 is F or V; X1469 is Q or L; X1470 is R or H; X1672 is N or K; X1745 is R or H; X1844 is R or S; X2108 is R or C; X2155 is R or W; X2164 is A or V; X2191 is R or W; X2299 isN or T; X2366 is K or Q; X2910 is E or V; X2912 is N or D; and X2937 is Q or R;.

[0298] With reference to the above exemplary dystrophin protein amino acid sequence, each dystrophin protein portion can be described, for example, as:(i) spectrin-like repeat 1 : amino acids 339-447 of SEQ ID NO: 3;(ii) spectrin-like repeat 2: amino acids 448-556 of SEQ ID NO: 3;(iii) spectrin-like repeat 3: amino acids 559-667 of SEQ ID NO: 3;(iv) spectrin-like repeat 4: amino acids 719-828 of SEQ ID NO: 3;(v) spectrin-like repeat 5: amino acids 830-934 of SEQ ID NO: 3;(vi) spectrin-like repeat 6: amino acids 943-1045 of SEQ ID NO: 3;(vii) spectrin-like repeat 7: amino acids 1048-1154 of SEQ ID NO: 3;(viii) spectrin-like repeat 8: amino acids 1157-1263 of SEQ ID NO: 3;(ix) spectrin-like repeat 9: amino acids 1266-1367 of SEQ ID NO: 3;(x) spectrin-like repeat 10: amino acids 1368-1463 of SEQ ID NO: 3;(xi) spectrin-like repeat 11: amino acids 1468-1568 of SEQ ID NO: 3;(xii) spectrin-like repeat 12: amino acids 1571-1676 of SEQ ID NO: 3;(xiii) spectrin-like repeat 13: amino acids 1679-1778 of SEQ ID NO: 3;(xiv) spectrin-like repeat 14: amino acids 1779-1874 of SEQ ID NO: 3;(xv) spectrin-like repeat 15: amino acids 1877-1979 of SEQ ID NO: 3;(xvi) spectrin-like repeat 16: amino acids 1992-2101 of SEQ ID NO: 3;(xvii) spectrin-like repeat 17: amino acids 2104-2208 of SEQ ID NO: 3;(xviii) spectrin-like repeat 18: amino acids 2211-2318 of SEQ ID NO: 3;(xix) spectrin-like repeat 19: amino acids 2319-2423 of SEQ ID NO: 3;(xx) spectrin-like repeat 20: amino acids 2475-2577 of SEQ ID NO: 3;SUBSTITUTE SHEET (RULE 26)(xxi) spectrin-like repeat 21 : amino acids 2580-2686 of SEQ ID NO: 3;(xxii) spectrin-like repeat 22: amino acids 2689-2802 of SEQ ID NO: 3;(xxiii) spectrin-like repeat 23: amino acids 2808-2930 of SEQ ID NO: 3;(xxiv) spectrin-like repeat 24: amino acids 2935-3040 of SEQ ID NO: 3;(xxv) hinge region 1: amino acids 247-338 of SEQ ID NO: 3;(xxvi) hinge region 2: amino acids 668-718 of SEQ ID NO: 3;(xxvii) hinge region 3: amino acids 2424-2474 of SEQ ID NO: 3;(xxviii) hinge region 4: amino acids 3041-3112 of SEQ ID NO: 3;(xxix) cysteine-rich domain: amino acids 3113-3360 of SEQ ID NO: 3;(xxx) dystrophin protein CTD: amino acids 3361-3685 of SEQ ID NO: 3.

[0299] For example, in other aspects:

[0300] a dystrophin protein can comprise, from amino-terminus to carboxy-terminus, an amino acid sequence of:ABDI - (aal -246 ) -AIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRFKSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVEWKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEK QSNLHRVLMDLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVR VNSLTHMWWDESSGDHATAALEEQLKVLGDRWANICRWTEDRWVLLQDILLKWQRLTEEQCLF SAWLSEKEDAVNKIHTTGFKDQNEMLSSLQKLAVLKADLEKKKQSMGKLYSLKQDLLSTLKNKSV TQKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTVMETVTTVTTREQILVKHAQE ELPPPPPQKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQSPEFAIFRKEGNFSDLKEKVNAI EREKAEKFRKLQDASRSAQALVEQMVNEGVNADSIKQASEQLNSRWIEFCQLLSERLNWLEYQNN IIAFYNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKICKDEVNRLSDLQPQIERLKIQSIA LKEKGQGPMFLDADFVAFTNHFKQVFSDVQAREKELQTIFDTLPPMRYQETMSAIRTWVQQSETK LSIPQLSVTDYEIMEQRLGELQALQSSLQEQQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIE GRWKKLSSQLVEHCQKLEEQMNKLRKIQNHIQTLKKWMAEVDVFLKEEWPALGDSEILKKQLKQC RLLVSDIQTIQPSLNSVNEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQQVYARKEALKGGL EKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTESV NSVIAQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSYLEKANKWLNEVE FKLKTTENIPGGAEEISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMDELINEELETFNSRW RELHEEAVRRQKLLEQSIQSAQETEKSLHLIQESLTFIDKQLAAYIADKVDAAQMPQEAQKIQSD LTSHEISLEEMKKHNQGKEAAQRVLSQIDVAQKKLQDVSMKFRLFQKPANFEQRLQESKMILDEVSUBSTITUTE SHEET (RULE 26)KMHLPALETKSVEQEWQSQLNHCVNLYKSLSEVKSEVEMVIKTGRQIVQKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPSNLDSEVAWGKATQKEIEKQKVHLKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLLLEYQKHMETFDQNVDHITKWIIQADTLLDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRDQAANLMANRGDHCRKLVEPQISELNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEIQQGVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQRITDERKREEIKIKQQLLQTKHNALKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIKEIDRELQKKKEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIESKFAQFRRLNFAQIHTVREETMMVMTEDMPLEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSATPVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKIFNQWLTEAEQFLRKTQIPENWEHAKYKWYLKELQDGIGQRQTWRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLNEFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQTNLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEGPFDVKETEIAVQAKQPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTIGASPTQTVTLVTQPWTKETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVMVGDLEDINEMIIKQKATMQDLEQRRPQLEELITAAQNLKNKTSNQEARTIITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLGQARAKLESWKEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHMITENINASWRSIHKRVSEREAALEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGEIEAHTDVYHNLDENSQKILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGDFPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEWEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGEIAPLKENVSHVNDLARQLTTLGIQLSPYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAISPNKVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPMDILQIINCLTTIYDRLEQEHNNLVNVPLCVDMCLNWLLNVYDTGRTGRIRVLSFKTGIISLCKAHLEDKYRYLFKQVASSTGFCDQRRLGLLLHDSIQIPRQLGEVASFGGSNIEPSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLHRVAAAETAKHQAKCNICKECPIIGFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVLKNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASSPQLSHDDTHSRIEHYASRLAEMENSNGSYLNDSISPNESIDDEHLLIQHYCQSLNQDSPLSQPRSPAQILISLESEERGELERILADLEEENRNLQAEYDRLKQQHEHKGLSPLPSPPEMMPTSPQSPRDAELIAEAKLLRQHKGRLEARMQILEDHNKQLESQLHRLRQLLEQPQAEAKVNGTTVSSPSTSLQRSDSSQPSUBSTITUTE SHEET (RULE 26)MLLRWGSQTSDSMGEEDLLSPPQDTSTGLEEVMEQLNNSFPSSRGRNTPGKPMREDTM (SEQID NO: 4) whereinABD l-(aa 1-246) corresponds to any one of SEQ ID NOs: 1, 2, or 293; and wherein the amino terminal A corresponds to amino acid residue 247

[0301] With reference to the above exemplary dystrophin protein amino acid sequence, each dystrophin protein portion can be described, for example, as:(i) spectrin-like repeat 1 : amino acids 339-447 of SEQ ID NO: 4;(ii) spectrin-like repeat 2: amino acids 448-556 of SEQ ID NO: 4;(iii) spectrin-like repeat 3 : amino acids 559-667 of SEQ ID NO: 4;(iv) spectrin-like repeat 4: amino acids 719-828 of SEQ ID NO: 4;(v) spectrin-like repeat 5: amino acids 830-934 of SEQ ID NO: 4;(vi) spectrin-like repeat 6: amino acids 943-1045 of SEQ ID NO: 4;(vii) spectrin-like repeat 7: amino acids 1048-1154 of SEQ ID NO: 4;(viii) spectrin-like repeat 8: amino acids 1157-1263 of SEQ ID NO: 4;(ix) spectrin-like repeat 9: amino acids 1266-1367 of SEQ ID NO: 4;(x) spectrin-like repeat 10: amino acids 1368-1463 of SEQ ID NO: 4;(xi) spectrin-like repeat 11: amino acids 1468-1568 of SEQ ID NO: 4;(xii) spectrin-like repeat 12: amino acids 1571-1676 of SEQ ID NO: 4;(xiii) spectrin-like repeat 13: amino acids 1679-1778 of SEQ ID NO: 4;(xiv) spectrin-like repeat 14: amino acids 1779-1874 of SEQ ID NO: 4;(xv) spectrin-like repeat 15: amino acids 1877-1979 of SEQ ID NO: 4;(xvi) spectrin-like repeat 16: amino acids 1992-2101 of SEQ ID NO: 4;(xvii) spectrin-like repeat 17: amino acids 2104-2208 of SEQ ID NO: 4;(xviii) spectrin-like repeat 18: amino acids 2211-2318 of SEQ ID NO: 4;(xix) spectrin-like repeat 19: amino acids 2319-2423 of SEQ ID NO: 4;(xx) spectrin-like repeat 20: amino acids 2475-2577 of SEQ ID NO: 4;(xxi) spectrin-like repeat 21 : amino acids 2580-2686 of SEQ ID NO: 4;(xxii) spectrin-like repeat 22: amino acids 2689-2802 of SEQ ID NO: 4;(xxiii) spectrin-like repeat 23: amino acids 2808-2930 of SEQ ID NO: 4;(xxiv) spectrin-like repeat 24: amino acids 2935-3040 of SEQ ID NO: 4;(xxv) hinge region 1: amino acids 247-338 of SEQ ID NO: 4;SUBSTITUTE SHEET (RULE 26)(xxvi) hinge region 2: amino acids 668-718 of SEQ ID NO: 4;(xxvii) hinge region 3: amino acids 2424-2474 of SEQ ID NO: 4; (xxviii) hinge region 4: amino acids 3041-3112 of SEQ ID NO: 4;(xxix) cysteine-rich domain: amino acids 3113-3360 of SEQ ID NO: 4;(xxx) dystrophin protein CTD: amino acids 3361-3685 of SEQ ID NO: 4.

[0302] For example, in other aspects, a dystrophin protein can comprise, from aminoterminus to carboxy-terminus, an amino acid sequence of any one of XP_006724532.1 (dystrophin isoform X2), NP_000100.3 (dystrophin isoform Dp427c), NP_004000.1 (dystrophin isoform Dp427pl), XP_011543769.1 (dystrophin isoform X5), XP_006724536.1 (dystrophin isoform X6), XP_006724537.1 (dystrophin isoform X7), XP_006724533.1 (dystrophin isoform X3), XP_006724531.1 (dystrophin isoform XI), , XP_006724538.1 (dystrophin isoform X8), XP_016884817.1 (dystrophin isoform X4), NP_003997.2 (dystrophin isoform Dp427m, or Pl 1532-1), VAR_065764 [W118R], VAR_005148 [Q133P] (rsl800256), VAR_023538 [D165V], VAR_036353, VAR_005151 (rsl800266), VAR_057642 (rs34155804), VAR_057643 (rs5972599), VAR_005153 (rsl800259), VAR_062110 (rs34563188), VAR_057644(rsl6998350), VAR_005155 (rsl800260), VAR_005156 (rs228406), VAR_057645 (rs3827462), VAR_005157 (rsl800262), VAR_036354, VAR_005158 (rsl800269), VAR_005159(rsl800270), VAR_005160 (rsl800263), VAR_057646 (rs28715870), VAR_005161 (rsl057872), VAR_036355, VAR_023542 (rsl6990264), VAR_005162 (rsl801187), VAR_005163 (rsl801186), VAR_057647 (rsl6990169), VAR_005164 (rsl800273), VAR_036356,VAR_005165, VAR_023543, VAR_005167 (rsl800275), VAR_005168 (rs41305353), VAR_005169 (rsl800278), or VAR_005171 (rsl800280).

[0303] In some aspects, the polypeptides disclosed herein are capable of linking a subsarcolemmal cytoskeleton with the extracellular matrix. In some aspects, the polypeptides disclosed herein are capable of recruiting a dystrophin-associated protein complex.

[0304] In some aspects, the DysABDs disclosed herein are derived from human dystrophin protein (i.e., the DysABDs are human dystrophin protein ABDs).

[0305] In some aspects, the dystrophin proteins disclosed herein (e.g., truncated dystrophin proteins disclosed herein) are derived from human dystrophin proteins (z'.e., the dystrophin protein are human dystrophin proteins).SUBSTITUTE SHEET (RULE 26)3. Polynucleotides encoding Adapted Dystrophin Proteins and Adapted Dystrophin Proteins Actin-Binding Domains

[0306] In some aspects, disclosed herein are polynucleotides comprising a nucleotide sequence encoding the polypeptides disclosed herein. In some aspects, the polynucleotides consists of about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 5.5 kb, 6 kb. In some aspects, the polynucleotides have a size that is suitable for cloning in a vector. In some aspects, the polynucleotides have a size that is suitable for cloning in a viral vector. In some aspects, the polynucleotides have a size that is suitable for cloning in an AAV vector.

[0307] In some aspects, the polynucleotides disclosed herein further comprise additional nucleotide sequences. In some aspects, the polynucleotides disclosed herein can be expression cassettes encoding the polypeptides disclosed herein.

[0308] In some aspects, the polynucleotides disclosed herein further comprise a promoter, a Kozak sequence, an enhancer, a silencer, a polyA tail, a poly adenylation signal, a 3'-UTR, a 5'- UTR, an intronic sequence, a nucleotide sequence encoding a molecular tag, a nucleotide sequence encoding a selectable marker, a nucleotide sequence encoding a self-cleaving peptide, a linker, a fdler sequence, any combination thereof, or any other nucleotide sequence that may facilitate the expression of the nucleotide sequences encoding the polypeptides disclosed herein. Any further nucleotide sequences present in the polynucleotide is in the appropriate position required to exert its function. For example, the nucleotide sequence of a promoter is operably linked to the nucleotide sequence encoding the polypeptides disclosed herein.

[0309] The promoter can be a constitutive, a regulatable, an inducible, an ubiquitous, or a tissue-specific.

[0310] In some aspects, the promoter is a constitutive promoter. Non-limiting example of constitutive promoters are a CBA promoter, a CMV promoter, an EFla promoter, or a CAG promoter.

[0311] In some aspects, the promoter is a tissue-specific promoter. In some aspects, the promoter drives expression of the therapeutic protein, e.g., a dystrophin or a micro-dystrophin, in the brain, muscle, kidney, lung, testis, or any combination thereof. In some aspects, the tissuespecific promoter is a muscle tissue-specific promoter. Non-limiting example of tissue-specific promoters are a MHC promoter, a a-MHC promoter, a MLC-2 promoter, a cTnC promoter, a MHCK7 promoter (SEQ ID NO: 283), MHCK promoter, a Enh358MCK promoter, a CK promoter (e.g., hCK (SEQ ID NO: 284) or hCK Plus (SEQ ID NO: 285) a CK8e promoter, a MCK promoter, a dMCK promoter, a tMCK promoter (SEQ ID NO: 286), a DES promoter, a HSA promoter, aSUBSTITUTE SHEET (RULE 26)SPc5-12 promoter, a SP-301 promoter, a MHC promoter, a Sk-CRM promoter, a Sk-CRM4 promoter, or C5-12 promoter. In some aspects, the tissue-specific promoter comprises a tMCK promoter. In some aspects, the tissue-specific promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 286. In some aspects, the tissue-specific promoter comprises a MHCK7 promoter. In some aspects, the tissue-specific promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 283.

[0312] In some aspects, the enhancer is selected from a human skeletal actin gene element, a cardiac actin gene element, a myocyte-specific enhancer binding factor MEF (e.g., MEF2), a MyoD enhancer element, a cardiac enhancer factor (CEF) site, murine creatine kinase enhancer element, skeletal fast-twitch troponin C gene element, slow-twitch cardiac troponin C gene element, the slow-twitch troponin I gene element, hypozia-inducible nuclear factors, steroid- inducible element, glucocorticoid response element (GRE) and any combination thereof. In some aspects, the enhancer is a MEF2 enhancer. In some aspects, the enhancer is a MyoD enhancer. In some aspects, the enhancer is a MCK enhancer. In some aspects, the enhancer is a CEF site. In some aspects, the enhancer is a SV40 enhancer. In some aspects, the enhancer is a c-Myc enhancer.

[0313] In some aspects, the intron is a CAG intron, an SV40 intron, MVM intron, or a human beta-globin intron, or any combination thereof. In some aspects, the intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 287-289.

[0314] In some aspects, the poly(A) sequence is selected from an actin poly(A), a bGHpA, a hGHpA, a SV40pA, or a synthetic pA. In some aspects, the poly(A) sequence comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 290.

[0315] In some aspects, the polynucleotides further comprise a nucleotide sequence encoding a first inverted terminal repeat (ITR) and a second ITR. In some aspects, the first inverted terminal repeat (ITR) and a second ITR are of a same serotype. In some aspects, the first inverted terminal repeat (ITR) and a second ITR are of a different serotype. In some aspects, the serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVRH10, AAV1 1, or AAV12 serotype. In some aspects, the serotype is AAV2 serotype.SUBSTITUTE SHEET (RULE 26)

[0316] In some aspects, the polynucleotides disclosed herein are isolated polynucleotides. In some aspects, the polynucleotides disclosed herein are comprised in a vector.

[0317] Vectors comprising the polynucleotides (e.g., expression cassette) disclosed herein are also provided for herein. In some aspects, the vectors are non-viral vectors. In some aspects, the vectors are viral vectors (i.e., viral genomes). In some aspects, the viral vectors are AAV vectors, (i.e., AAV viral genomes).

[0318] Viral particles comprising the vectors (i.e., viral genomes, such as AAV viral genomes) disclosed herein are also provided for herein.

[0319] In some aspects, the viral particles disclosed herein are recombinant adeno- associated virus (rAAV) particles. In some aspects, the rAAV particles disclosed herein further comprise a capsid.

[0320] In some aspects, the capsid serotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV6P1, AAV7, AAV8, AAV8P1, AAV9, AAVrhlO, AAVrhlOPl, AAVS10P4, AAVpol, AAV11, AAV12, AAV-DJ, AAV-DJ / 8, AAV- PHP.Eb, AAV-PHP.S, AAV-PHP.B, AAV2-retro, AAV2-QuadYF, AAV2.7m8, AAVS1, AAVS10, AAVrh74, AAVS1, AAVS10, AAVH15, AAVMYO (AAV9P1), AAVMYO 2 (AAVS1P1), AAVMYO3 (AAVS10P1), AAV9-RGD (e g., MyoAAVlA, MyoAAV 1C, MyoAAV IE, MyoAAV 2 A, MyoAAV 2E, MyoAAV 3 A, MyoAAV 4 A, MyoAAV 4C, and MyoAAV 4E). In some aspects, the capsid serotype is selected from the group consisting of AAV1, AAV8, AAV9, AAVrh74, AAVS1, AAVS10, AAVH15, AAVMYO (AAV9P1), AAVMYO 2 (AAVS1P1), AAVMYO3 (AAVS10P1), AAV9-RGD (e g., MyoAAVlA, MyoAAV 1C, MyoAAV IE, MyoAAV 2 A, MyoAAV 2E, MyoAAV 3 A, MyoAAV 4 A, MyoAAV 4C, and MyoAAV 4E). In some aspects, the capsid serotype is AAVrh74.

[0321] In some aspects, the capsid is encoded by a polynucleotide comprising a nucleic acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 291.

[0322] In some aspects, the capsid comprises a polypeptide comprising an amino acid sequence having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, atSUBSTITUTE SHEET (RULE 26)least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 292.

[0323] The nucleic acid molecules and the vectors (e.g, AAV vectors) of the disclosure may be produced by any of the suitable means disclosed in the published literature. For example, the nucleic acid molecules may be produced by chemical synthesis or by recombinant DNA technology. Methods for producing recombinant AAV vectors are disclosed in the published literature (e.g., Kimura, T., Ferran, B., Tsukahara, Y. et al. Production of adeno-associated virus vectors for in vitro and in vivo applications. Sci Rep 9, 13601 (2019); Viral Vectors for Gene Therapy: Methods and Protocols, Manfredsson F.P, Benskey M.J., Springer Link (2019)).

[0324] The genetic code is well known in the published literature, and a person skilled in the art would be readily able to produce a nucleotide sequence encoding a defined amino acid sequence. Thus, a person skilled in the art, provided the amino acids sequence of, e.g., SEQ ID NOs: 1-4, would be readily able to produce a nucleotide sequence encoding the same.

[0325] A person skilled in the art would know the that the four nucleotide bases of DNA: adenine (A), cytosine (C), guanine (G) and thymine (T) — can be used in various ways to spell out three-letter "codons" that specify which amino acid is needed at each position within a protein, and provided any amino acid sequence would be readily able to produce a nucleotide sequence encoding the same. A person skilled in the art would also know that a nucleotide sequence may vary due to the degeneracy of the genetic code. An exemplary representation of the genetic code is:SUBSTITUTE SHEET (RULE 26)

[0326] Nevertheless, exemplary nucleotide sequences encoding the polypeptides of the disclosure are provided in the Sequence Table provided in the present disclosure.4. Pharmaceutical Formulations

[0327] In some aspects, provided herein are pharmaceutical formulations comprising the adapted dystrophin proteins, the nucleic acid molecules, or the vectors disclosed herein, and a pharmaceutically acceptable excipient, or pharmaceutically acceptable adjuvants, or any combination thereof.

[0328] In some aspects, the combination of the adapted dystrophin proteins, the nucleic acid molecules, or the vectors disclosed herein with one or more of the carriers described herein facilitates, enhances or enables administration of the adapted dystrophin proteins, the nucleic acid molecules, or the vectors disclosed herein to the subject. In some aspects, the combination of the adapted dystrophin proteins, the nucleic acid molecules, or the vectors disclosed herein with one or more of the carriers described herein facilitates, enhances or enables the delivery of the adapted dystrophin proteins, the nucleic acid molecules, or the vectors disclosed herein to a target cell. In some aspects, after administration to the subject of the fusion proteins, the adapted dystrophin proteins, the nucleic acid molecules, or the vectors disclosed herein, at least a portion of the adapted dystrophin proteins, the nucleic acid molecules, or the vectors disclosed herein are delivered to a target cell.5. Method of treatment and use

[0329] In some aspects, disclosed herein are methods of increasing the amount of functional dystrophin protein in a cell comprising contacting the cell with the adapted dystrophin proteins, the nucleic acid molecules, the vectors, or the pharmaceutical formulations disclosed herein.

[0330] In some aspects, the cell is a muscle cell. In some aspects, the muscle cell is derived from skeletal muscle or smooth muscle, e.g. from the digestive tract, urinary bladder, blood vessels or cardiac tissue. In some aspects, the muscle cell is a myoblast, a myocyte, a myotube, a cardiomyocyte, or a cardiomyoblast.

[0331] In some aspects, the cell is a mammalian cell. In some aspects, the cell is comprised in a subject. In some aspects, the subject suffers from a diseases, disorders, syndromes orSUBSTITUTE SHEET (RULE 26)conditions that can benefit from an increase in dystrophin proteins expression and / or activity. In some aspects, the subject suffers from muscular dystrophy. In some aspects, the muscular dystrophy is dystrophin-deficient muscular dystrophy In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

[0332] In some aspects, the amount of functional dystrophin protein in the cell is increased of at least 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a same cell not administered the adapted dystrophin proteins, the nucleic acid molecules, the vectors, or the pharmaceutical formulations disclosed herein.

[0333] In some aspects, disclosed herein are methods of treating a subject suffering from a diseases, disorders, syndromes or conditions that can benefit from an increase in dystrophin proteins expression and / or activity. In some aspects, disclosed herein are methods of treating a subject suffering from a muscular dystrophy. In some aspects, the muscular dystrophy is dystrophin-deficient muscular dystrophy. In some aspects, the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

[0334] In some aspects, the methods disclosed herein comprise administering to the subject a therapeutically effective amount of the adapted dystrophin proteins, the nucleic acid molecules, the vectors, or the pharmaceutical formulations disclosed herein. The adapted dystrophin proteins, the nucleic acid molecules, the vectors, or the pharmaceutical formulations disclosed herein may be administered to a subject in a single dose, or a multiple dose. In some aspects, the multiple dose comprises two, three, or four or more doses. In some aspects, the adapted dystrophin proteins, the nucleic acid molecules, the vectors, or the pharmaceutical formulations disclosed herein are administered to the subject at regular intervals, for example, weekly, biweekly, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, or yearly. In some aspects, the first set of doses (e.g., two, three, four, five, six, seven, eight or ten doses) are administered monthly, with further doses administered less frequently (e.g., every 3 months, every 6 months or yearly).

[0335] Administration can be by any suitable route, for example, administration can be systemic (e.g., intravenous) or local (e.g., to a muscle), or both.

[0336] In some aspects, the degeneration of a muscle of the subject is reduced of at least 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold after administration of the adapted dystrophin proteins, the nucleic acid molecules, the vectors, or the pharmaceutical formulations disclosed herein. In some aspects, the muscle is a smooth muscle,SUBSTITUTE SHEET (RULE 26)a skeletal muscle, a cardiac muscle, or any combination thereof. In some aspects, a symptom of the muscular dystrophy in the subject is reduced after administration of the adapted dystrophin proteins, the nucleic acid molecules, the vectors, or the pharmaceutical formulations disclosed herein. In some aspects, diseases, disorders, syndromes or conditions (e.g, a muscular dystrophy such as Duchenne muscular dystrophy or Becker muscular dystrophy) is ameliorated or treated after administration of the adapted dystrophin proteins, the nucleic acid molecules, the vectors, or the pharmaceutical formulations disclosed herein. In some aspects, the therapeutically effective amount of the adapted dystrophin proteins, the nucleic acid molecules, the vectors, or the pharmaceutical formulations disclosed herein, is reduced compared to a therapeutically effective amount of a same wild type dystrophin protein, of a nucleic acid molecule encoding a same wild type dystrophin protein, of a vector comprising a nucleic acid molecule encoding a same wild type dystrophin protein, or of a pharmaceutical formulation thereof.EXAMPLESExample 1: Optimization of actin-binding assaysConstruction of pdt68-Dys-ABD1-mClover3:

[0337] N- and C-terminally mClover3 tagged Dys-ABD1 (mClover3-Dys-ABD1wt and Dys-ABD1wt-mClover3, respectively) and Utrophin-ABD1 (mClover3-Utr-ABD1 and Utr- ABD1-mClover3, respectively) cDNAs were generated by commercial DNA synthesis and subcloned into the ptd68 6xHis-Sumo plasmid backbone (ptd68 6xHis-Sumo- mClover3-Dys-ABD1, ptd68 6xHis-Sumo-Dys-ABD1-mClover3, ptd68 6xHis-Sumo- mClover3-Utr-ABD1, and ptd68 6xHis-Sumo-Utr-ABD1-mClover3) (FIG. 1). The amino acid sequence indicated in the present examples as ABD1wt corresponds to the amino acid sequence of SEQ ID NO: 1. The resulting plasmids were verified by Sanger sequencing across the protein reading frame. Expression of these proteins in the BL21 E. coli strain under IPTG induction was optimized. Proteins obtained from culture lysates were purified using NiNTA affinity resin, dialyzed into Phosphate Buffered Saline, and then the Sumo tag was cleaved by addition of ULP1 protease. The Sumo tag is recognized and proteolyzes by the ULP1 protease (Avery, A.W., et al., Structural basis for high-affinity actin binding revealed by a beta-III-spectrin SCA5 missense mutation. Nat Commun, 2017. 8(1): p. 1350).SUBSTITUTE SHEET (RULE 26)

[0338] As a proof of principle for subsequent studies, a first adapted dystrophin protein (Dys-ABD1L212P) was also generated. The Dys-ABD1L212P was based on the L253P mutation found in p3-spectrin, a variant in the tandem CH ABD of P-III-spectrin, a more distant homolog of the dystrophin ABDI, which increases actin-binding affinity by 1000-fold (Avery, A.W., Crain, J., Thomas, D.D., and Hays, T.S. (2016). A human P-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affmity F-actin binding. Set. Rep. 6, 21375).

[0339] The resulting proteins (mClover3-Dys-ABD1wt, Dys-ABD1wt-mClover3, mClover3-Dys-ABD1L212P, Dys-ABD1L212P-mClover3, mClover3-Utr-ABD1 and Utr-ABD1- mClover3) were used to optimize two actin-binding assays:HSC assay Optimization:

[0340] (i) An High-speed actin-cosedementation (HS actin-C or HSC) assay, quantified bySDS-PAGE gel densitometry (FIGs. 2A-D) were conducted. Samples were equilibrated with increasing concentrations of phalloidin stabilized F-Actin in F-Buffer (10 mM Tris, pH 7.5, 2 mM MgCb) at a total volume of 100 microliters and sedimented by ultracentrifugation at 100,000 x g for 20 minutes at fixed temperature. The supernatants were removed and pellets were resuspended in sample buffer for SDS-PAGE and total protein staining. The ABDI containing bands were quantitated for each sample, the ratio of mCl over-tagged protein in the pellet to total protein was calculated and plotted against the sample's actin concentration (FIGs. 2E-F).TR-FRET assay Optimization:

[0341] (ii) A time-resolved fluorescence energy transfer (TR-FRET) assay measuring energy transfer from the ABDI attached mClover3 to an Alexa-568 covalently attached to C374 on phalloidin-stabilized F-actin (FIG. 3A). Sub-micromolar concentrations of mClover3 tagged Dys-ABD1 (mClover3-Dys-ABD1wt (NTWT), Dys-ABD1wt-mClover3 (CTWT), mClover3- Dys-ABD1L212P (NTL21P), Dys-ABD1L212P-mClover3 (CTL212P)), proteins were equilibrated in F-Buffer with increasing concentrations of Alexa-568 labeled and the average time- resolved fluorescence decay lifetime of the mClover3 donor probe following a sub -nanosecond excitation pulse was measured to determine the degree of binding between actin and the Dys-ABD 1 (FIGs. 3B-C).

[0342] Results from these tests allowed to optimize the protein constructs (for example byadjusting the position of the mClover3 tag). The Dys-ABD1 and Utr-ABD1 constructs exhibited expected actin-binding affinities, and the L212P construct increased Dys-ABD1 binding to actin.SUBSTITUTE SHEET (RULE 26)

[0343] The position of the mClover3 tag on Dys-ABD1 did not affect the actin-binding affinity measured by HSC or by FRET, however, the C-terminal mClover3 Dys-ABD1 exhibited the greatest magnitude of FRET change at saturating actin concentrations, suggesting that the C- terminus of the Dystrophin positions the mClover3 closer to the Alexa-568 acceptor on actin residue C374.

[0344] The mClover3 tag weakened actin binding of Utr when attached to the protein’s N- terminus.

[0345] Removing the Sumo tag by Size Exclusion Chromatography did not significantly change actin-binding.

[0346] The HSC and TR-FRET assays detected changes in actin binding with precision sufficient to evaluate differences in the actin-binding affinity of adapted proteins as indicated by statistical analysis of the differences between Dys-ABD1 and Utr- ABDI constructs and by the statistical difference between samples of distinct actin concentrations. The position of the mClover3 tag impacted the measured affinity, indicated by reduced mClover3-Utr binding compared to Utr-mClover3. The position of the mClover3 tag also impacted the magnitude of FRET at saturating actin. Based on these results the C-terminal tagged mClover3 design was used in all the following experiments. The L212P mutation increased actin-binding affinity. Thus, mutations can cause GoA in Dys-ABD1. Cleavage of the Sumo tag increased actin-binding but removal of the cleaved tag from the samples by size exclusion chromatography was not required for precise measurement of differences in actin-binding affinity. Thus, for subsequent mutation screening, the cleaved 6xHis-Sumo tag was left in the sample, minimizing sample processing time and was removed by size exclusion chromatography only after identifying a mutation as a GoA candidate, prior to the more detailed biophysical characterizations described below.Example 2: Identification and testing of gain-of-affinity (GoA) Dys candidates

[0347] A first set of 8 candidate GoA variations were identified in homologous proteins including P-3 -spectrin, a-actinin-4, and filamin tandem calponin homology domains, and were introduced individually into the ptd68-Dys-ABD1wt-mClover3 plasmid using incorporation of synthetic gene-blocks via ligation independent cloning. Plasmids were verified by Sanger sequencing across the entire ABDI reading frame to ensure the amino acid sequence of the resulting protein.

[0348] Plasmids were transformed into BL21 E. coli strain cells and individual transformants were screened for Dys-ABD1 -mClover3 expression upon IPTG induction.SUBSTITUTE SHEET (RULE 26)Representative clones with high protein expression levels were cultured between 0.25 L to 8 L scale depending on the total protein needs. Cell cultures were induced with IPTG, harvested by centrifugation, lysed in lysis buffer with mild sonication, clarified by centrifugation, and then subjected to NiNTA affinity chromatography as described above, to obtain preparations of the ABD 1 -mClover3 proteins.

[0349] Several of the adapted protein tested caused GoA by both HSC and FRET assays. The most promising of these were L212P and K226E, which were then combined in an adapted protein to determine if they would act synergistically to yield a greater GoA than either individually. In addition, a second adapted protein comprising second double substitution, Dys- ABD1 Q17A-T20A, was tested. An adapted protein comprising a quadruple substitution, combining the two double site substitutions, Q17A-T20A-L212P-K226E was also tested. The actin-binding, of each adapted protein was measure. Notably, adapted protein comprising the double substitutions bound actin better than the adapted protein comprising single substitutions and the adapted protein comprising quadruple substitutions better than the adapted protein comprising double substitutions, indicating that the substitutions acted synergistically (FIGs. 4A- B and Table 1).Table 1: single and double substitutions assay.C-terminal construct Aim Kd (gM) Bmax Bmax = 1.0 N Kd( |1M) Emax NDys Abdl WT 1 75.7 0.441 311 12.0 >100 0.196 7Dys Abdi W143R 1 167.8 0.597 358.2 3.0 81.0 0.274 3Dys Abdi Q133P 1 106.6 0.328 560.8 3.0 >100 0.206 3Dys Abdi E216K 1 76.1 0.506 255.9 3.0 36.8 0.327 3Dys Abdi S30Q 1 65.5 0.275 540.5 3.0 >100 0.197 3Dys Abdi Q17A T20A 1 57.0 0.760 105.2 3.0 6.4 0.251 3Dys Abdi W24R 1 53.7 0.768 98.44 1.0 46.3 0.336 3Dys Abdi L212P 1 38.2 0.967 42.05 5.0 2.0 0.195 6Dys Abdi K226E 1 22.7 0.801 44.97 3.0 4.6 0.244 3 hUTR Q33A_T36A 1 15.4 0.984 16.38 3.0 0.8 0.351 4Dys Abdi QUAD 1 15.1 0.566 93.06 3.0 0.7 0.159 4Dys Abdi L212P K226E 1 7.4 0.837 17.7 3.0 0.2 0.133 5 hUtr Abdl WT 1 9.2 0.969 10.74 3.0 1.3 0.328 8

[0350] Several additional potential GoA-substitutions were tested in silico. The initial tests predicted that T20Q would exhibit enhanced actin-binding affinity. This substitution was then introduced into the Dys-ABD1- mClover3 plasmid and the actin-binding affinity of the resulting adapted protein was tested (FIGs. 5A-B and Table 2). The adapted protein comprising the T20QSUBSTITUTE SHEET (RULE 26)substitution outperformed all of adapted proteins described above (i.e., comprising the single, double, and quadruple site substitutions described above).Table 2: T20Q substitution assay.Example 3: Biophysical characterization of gain-of-function (GoA) Dys candidates

[0351] A fluorescence-based thermal stability assay was developed to measure the stability of each of the adapted Dys-ABD1 proteins to determine the degree to which the substitutions change the proteins' folding energetics. In this assay, the fluorescence of the mClover3 tag was monitored as samples were heated in a qPCR machine. The fluorescence reports folding of the tag, which is sensitive to the dynamics and stability of the proteins that are attached to it. Two melting transitions were observed in most samples. Control experiments were performed on mClover3 alone and showed that the lower temperature transition corresponded to melting of the Dys-ABD1 protein while the high temperature transition corresponded to the melting of the mClover3. (Table 3).Table 3: Melting temperature of adapted Dys-ABD1.SUBSTITUTE SHEET (RULE 26)

[0352] Increases in actin-binding affinity correlated with decreasing thermal stability. This analysis showed that the most promising adapted protein identified (comprising the T20Q substitution) exhibit well defined melting profiles indicating that the proteins are well formed and not non-specifically aggregated.

[0353] The aggregation state of each adapted protein was evaluated by measuring their self-sedimentation in the high-speed centrifugation assay performed in the absence of actin. Though not a direct measure of a protein molecular weight, this analysis showed that more than 95% of the adapted protein from each preparation remained in solution in the absence of actin after centrifugation (FIG. 6). Thus, any aggregates present in the sample were too small to sediment and the sedimentation measured in the actin-binding HSC assay was dependent on interaction with actin. Later experiments measured the apparent molecular weight of each GoA adapted protein using size exclusion chromatography (FIGs. 7A-B, Table 4, FIGs. 8A-D). This analysis showed that GoA adapted protein elute from the size exclusion column in lower molecular weight species (consistent with monomers) and higher molecular weight species (consistent with oligomers). The exact molecular nature of the oligomers is not yet known.

[0354] Control experiments showed these high and low molecular weight species of the GoA adapted protein examined thus far bind actin similarly (FIGs. 8A-D) and that the high- molecular weight species, when isolated by SEC, do not self-sediment during the actin- cosedimentation assay.Table 4: Relative abundance of high and low MW species.Example 4: Denovo engineering of novel ABDI derived variants with GoA actin-binding: optimization of the experimental proceduresPhage-display control plasmids

[0355] Three phage-display control plasmids containing WT-hDys-ABD1, L212P-hDys-ABD1, or WT-hUtr-ABD1 reading frames inserted into the display site of the pADL22c phagemidSUBSTITUTE SHEET (RULE 26)were generated. The constructs were verified by Sanger Sequencing and used to produce replication deficient Ml 3 phage particles following methods outlined by Kontermann et al. (Kontermann, R D S ., Antibody Engineering Vol. 1. 2010) and then used for design and validation of the actin-binding phage-display workflow (FIG. 9).Biotinylated actin binds streptavidin-coated immuno surfaces

[0356] Biotinylated F-actin was prepared by reacting maleimide-biotin with cysteine 374 on F-actin following protocols used for labeling F-actin with Alexa-568 described above, and then conditions for capturing biotinylated F-actin on high-binding Nunc immuno-tubes and immunoplates coated with streptavidin were tested (FIGs. 10A-B). Binding of the actin to the immunotube surface was verified by ELISA using an anti-actin primary antibody and a HRP conjugated secondary antibody followed by incubation with the HRP substrate TMB. The reaction was quantitated by absorbance spectroscopy measuring accumulation of the HRP reaction product. The dependence of actin capture on the concentration of streptavidin used to coat the plates was also tested, and concentrations above 0.1 micrograms / ml were sufficient to maximize actin capture (FIG. 10A). For subsequent biopanning experiments, immuno-tubes coated with 0.1 mg / ml streptavidin, blocked with 2% BSA, and then loaded with indicated amounts of biotinylated F- actin were utilized. Control tubes contained streptavidin and blocking agent but not actin. The anti- actin ELISA signal was typically 5-10 times greater than the signal from tubes containing streptavidin without actin (FIG. 10B)Assay to determine ABDI protein presence on phage-particles

[0357] The ABDI protein content of phage particles purified in an iodoxinol (optiprep) density gradients was analyzed (FIG. 11). Phage particles (2 x 1013) were loaded in 1.5 ml at the top of a preformed density gradient and subjected to sedimentation. The phage particles resolved into a single band in the gradient. Fractions across the gradient were collected and subjected to SDS-PAGE and western blotting for the His-tagged ABDI protein. Anti-His antibodies crossreacted with a protein species that migrates at 75 kDa, consistent with the molecular weight of hDys-ABD1-g3 fusion protein. This fraction contained 2 x 1013phage particles, determined by UV absorbance spectroscopy as described in Kontermann et al. (Kontermann, R.D.S., Antibody Engineering Vol. 1. 2010). Fractions that did not contain the ABDI cross-reacting species did not contain phage measurable by UV absorbance. Thus, ABDI was displayed on the surface of phage particles.SUBSTITUTE SHEET (RULE 26)Assay to determine ABDI phage-binding to actin-coated immuno-tubes

[0358] The binding of ABDI displaying phage-particles to surface immobilized actin was then validated. Actin-coated immuno-tubes were incubated with either WT-hDys-ABD1, WT- hUtr-ABD1, or L212P-hDys-ABD1-displaying phage-particles. The phage were diluted to equivalent concentrations in phosphate buffered saline pH 7.5 containing 2% BSA and 0.05% Tween and incubated for 1 hour in 96 well Nunc Immuno-plates that were coated with 0.1 micrograms / ml streptavidin, loaded with 50 micromolar biotin-actin, and blocked with 2% BSA. Unbound phage were removed by pipetting and the tubes washed 3 times with phosphate-buffered saline (PBS). Bound phage was measured by anti -HA ELISA detecting the HA epitope tag located at the C-terminus of the displayed ABDI. The hUtr-ABD1 and L212P-hDys-ABD1 phage bound to the actin-coated surfaces but not surfaces that lacked actin (FIG. 12). The WT-hDys-ABD1 phage were not distinguishable from background, consistent with weak actin-binding of the WT- hDys-ABD1 protein shown above.Assay to if actin-selected phage could infect TGI E. coli

[0359] Actin-bound phage were eluted via incubation with 100 mM Tri ethylamine for 15 minutes followed by neutralization with 1 M Tris pH 7.5. The eluate was added to cultures of TGI E. coli and incubated overnight. The phage plasmid was rescued from the overnight cultures by plasmid purification and subjected to PCR using primers that span the ABDI reading frame (FIG. 13). This showed that recovered plasmids contained the respective reading frames, thus the phagemids were being eluted and following elution, retained infectivity.Strong-actin-binding ABDI control sequences were enriched by phage-display

[0360] The ability of actin-binding phage-display selection to enrich for higher-affinity actin-binding phage particles was evaluated by diluting hUtr-ABD1 phage with 2-fold excess hDys-ABD1 phage and then subjecting the mixture to three rounds of biopanning on actin-coated immuno-tubes. At each round, plasmid DNA from the TGI glycerol stocks was recovered and the DNA was subjected to qPCR using primers specific for hUtr-ABD1 or hDys-ABD1 to quantitate the abundance of the two sequences. After three rounds of selection, the hUtr-ABD1 DNA represented 99% of the total recovered DNA (FIG. 14A), thus showing a selective advantage of hUtr-ABD1, which, as shown above, exhibits higher actin-binding affinity compared to hDys- ABD1. The ability of a single round of biopanning to enrich for higher affinity hUtr-ABD1SUBSTITUTE SHEET (RULE 26)binding, was also evaluated. Enrichment was found in mixtures containing as little as 100 times more hDys-ABD1 compared to hUtr-ABD1 phage (FIG. 14B). The day-to-day variability in single rounds of biopanning was high, indicated by the standard deviation error bars of 3 independent biological replicate experiments. This variability highlights why repeated rounds of biopanning are needed to select for enhanced binding activity. Collectively, these experiments showed that the biopanning workflow was able to select for enhanced binders and was ready to deploy for screening ABDI sequence variant libraries.Generation of pADL22c hDys-ABD1 sequence variant phage library

[0361] A library of ABDI sequence variants was generated using multi-fragment overlapping PCR with primers containing degenerate NNN codons positioned at each codon of the ABDI reading frame. Fragments containing mutations in CHI were shuffled by PCR with fragments containing mutations in CHI to generate a library with mutations in both domains. The resulting PCR products were inserted into the pADL22c plasmid using ligation independent Gibson assembly and transformed into TGI E. coli. The TGI cells were then infected with Ml 3 helper phage to generate replication deficient phage particles that infect TGI E. coli, but do not replicate without co-infection with Ml 3 helper phage. The phage particles were purified by precipitation with PEG, the precipitated pellet was resuspended in phosphate buffered saline (PBS), and then the material was stored at -80 C in PBS with 50% glycerol by volume.

[0362] The sequence diversity of the purified phage was examined by infecting TGI cells, culturing the infected cells in media containing ampicillin, and then isolating the pADL22c phagemid by plasmid purification. The isolated plasmids were subjected to short-read Illumina next generation sequencing and long-read PacBio next generation sequencing. This analysis showed site-saturation mutations across the ABDI reading frame (FIGs. 15A-D). The primers used for short-read analysis were not optimal, which resulted in several gaps in the regions sequenced (FIG. 15A). These regions were covered in the long read sequence. For subsequent short read analysis performed after each round of biopanning, we included additional primer sets to ensure sequence coverage across the entire reading frame.

[0363] The median frequency of amino acids across the ABDI reading frame was similar in both short and long-read analysis (FIGs. 15A-B). Furthermore, all amino acids were represented in the mutagenesis (FIGs. 15C-D). The mean frequency of substitutions determined from the shortread analysis was 10.2 + / - 7.7 (standard deviation) per 10,000. The mean frequency of substitutions in the long-read analysis was 2.0 + / - 2.4 (standard deviation) per 10,000 (FIG. 16). The theoreticalSUBSTITUTE SHEET (RULE 26)maximum number of site-saturation amino acid substitutions across the 245 residue ABDI sequence is 4900, 2.0 per 10,000. Thus, the library contained the expected density of substitutions. Sequences containing frame shifts were omitted from this analysis and because the short-read amplicons were shorter, the probability of them having a frame shift is lower than the long-read sequences. This explains the difference in the mean substitution frequency evaluated by both methods.Development of an ELISA phage-display triaging assay

[0364] A critical stage in phage-display is the triaging and prioritization of clones selected by biopanning. Several approaches for doing this were tested and a surface immobilized actin- binding ELISA assay was chosen due to assay reproducibility and evaluation of the robust Z-prime (rZ') assay performance statistic. In an assay with an rZ' value > 0.5, the assay response, defined as the absolute difference between the median negative and median positive control samples, is six times larger than the sum of their median absolute deviations.Eq. 1

[0365] Single colonies of TGI cells infected with negative control hDys-ABD1 or positive control hUtr-ABD1 phage were picked to inoculate 1 ml of 2xYT growth media supplemented with ampicillin in 96 well culture plates. Expression of the ABDI display protein was induced with 1 mM IPTG. The expressed protein was tagged with an N-terminal pelB leader secretion sequence which targets the protein for secretion into the periplasm for phage-particle assembly. In the absence of co-infecting Ml 3 helper phage, the secreted protein accumulates in the periplasm. The pelB leader sequence was cleaved during secretion. A significant amount of the secreted protein leaked from the periplasm into the culture media. The IPTG induced TGI cells were sedimented by centrifugation the conditioned media were tested for ABD 1 protein content and binding to actin- coated plates to determine relative actin-binding activity (FIGs. 17A-E). The media were also tested for ABDI binding to blocked plates that did not contain actin (FIGs. 17A-E). After incubating with conditioned media, the plates were washed 3 times with PBS and developed with anti-HA-HRP antibody using TMB HRP substrate. The hDys-ABD1 and hUtr-ABD1 proteins were expressed to similar levels (FIG. 17B). The background binding was also similar for each. Significantly more hUtr-ABD1 was present on actin-coated plates compared with hDys-ABD1 coated plates. The median robust Z prime of the difference in actin-binding between hDys-ABD1SUBSTITUTE SHEET (RULE 26)- I l l - and hUtr-ABD1 controls across 12 independent biological replicates of this assay was 0.62. Thus, the quality of the ELISA assay was sufficient to identify candidate sequences for subsequent testing and to prioritize clones for downstream work.

[0366] All these results taken together showed that:• WT-hDys-ABD1, WT-hUtr-ABD1, and L212P-hDys-ABD1, a variant that exhibits actin-binding gain-of-affinity, could be displayed on the phage coat.• Actin-binding selection could distinguish between weak (WT-hDys-ABD1), intermediate (L212P-hDys-ABD1) and strong (WT-hUtr-ABD1) phage binding to actin.• ABDI sequences with enhanced actin-binding selection could be enriched from a background pool of weak-binding candidates.• The diversity of the site-saturation mutagenesis library was sufficiently high to evaluate single-site variants by phage display.• Phagemid sequences could be selected for sequencing based on a surface immobilized actin-binding ELISA assay.

[0367] These experiments do not rule out the possibility that some sequences could be selected because they are more easily expressed, more properly folded, more easily displayed, or more prone to non-specific binding, than others, and thus could be enriched independent of their absolute actin-binding activity measured in orthogonal biochemical assays.Example 5: Denovo engineering of novel ABDI derived variants with GoA actin-binding: identification of GoA candidates

[0368] Actin-binding phage-display selection followed by amplification of the actin- binding phage particles was performed to identify potential actin-binding gain-of-affinity variants in the hDys-ABD1 sequence. 10 rounds of biopanning were performed starting with the sitesaturation mutagenesis hDys-ABD1 phage library generated as described above. The amplified phage output from each round was used as input for the next round of selection following workflows outlined in Kontermann et al. (Kontermann, R.D.S., Antibody Engineering Vol. 1. 2010). At each round, the output was stored as a TGI E. coll glycerol stock for later studies.Selection of variants from short-read NGS. At each round of biopanning

[0369] The frequency of amino acid substitutions in the ABDI reading frame was evaluated using short-read sequencing of amplicons prepared with four overlapping primer sets.SUBSTITUTE SHEET (RULE 26)At round 5, 10 of the most abundant sequences detected by short read sequencing were selected for cloning into the pdt68 expression plasmid and then expressed, purified, and tested in three independent actin-binding FRET and actin-binding high-speed actin-cosedimentation (HSC) experiments (FIGs. 18A-B). Results from these experiments showed that the phage-display workflow was identifying gain-of-affinity actin-binding sequence variants as each candidate exhibited enhanced actin-binding compared to WT-hDys-ABD1. The stability of each candidate was also tested using the DSF-GTP assay described above. The candidates exhibited lower thermal stability than WT-hDys-ABD1 -mClover3 protein, a consistent phenotype of all gain-of-affinity proteins tested in this work (FIG. 19).

[0370] Possibly the correlation between binding affinity and thermal stability reflected stabilization of an extended structure of the ABDI protein that can more easily bind actin.

[0371] In addition to candidates selected at round 5, several sequences from NGS of round 10 output were selected.ELISA-based selection of biopanning strains for sequencing

[0372] Biopanning output by PCR using primer sets that spanned the ABD 1 reading frame was examined (FIGs. 20A-B) This analysis showed that at round 6, a truncated sequence that spans the display site appears in the output and increases in abundance with each successive biopanning round. This suggested that the small sequence fragment retained actin-coated surface binding activity, while gaining a competitive advantage due to efficiency of expression This phenomena is known to occur in other phage-display workflows such as antibody engineering. We plated TGI glycerol stock outputs from round 6, 8, and 10 on antibiotic selection plates at densities sufficient to select single colonies for inoculating 1 ml of 2xYT growth media supplemented with ampicillin in 96 well culture plates. The plates were cultured for 4 hours and then brought to 1 mM IPTG to induce ABDI expression and cultured at 30C overnight. The culture plates were centrifuged to pellet TGI cells and aliquots of the conditioned media transferred to fresh plates for storage at -20C for later testing. Replicate plates were stored as glycerol stocks for later isolation of associated plasmids.

[0373] The conditioned media were diluted 5-fold in F-Buffer and incubated on immunoplates coated with actin and blocking solution (PBS with 2% BSA and 0.05% Tween), blocking solution only, or non-coated plates as described above. Each ELISA assay plates contained media from 24 clones in triplicate, 4 wells of positive and negative controls (WT-hUtr-ABD1 and WT- hDys-ABD1), 8 wells of non-induced culture controls, and 8 wells of culture media controls. AfterSUBSTITUTE SHEET (RULE 26)loading, the plates were washed three times with PBS and developed using anti-HA-HRP antibody and TMB HRP substrate. The background was calculated subtracting specific actin-binding activity of the individual clones (actin-binding - background binding / total expression) (FIG. 17 (data from round 6); FIG. 20B (specific actin-binding activity determined as described in FIG. 17, for round 6, 8, and 10 output)). Clones that exhibited specific actin-binding activity greater than the WT-hDys-ABD1 control were prioritized for Sanger Sequencing. Negative candidates were also selected for Sanger sequencing to verify that colonies that did not exhibit increased potential for actin-binding were distinct from those that did. The sequenced candidates were unique, indicating that sequence space was not saturate and that additional screening of the output could identify unique gain-of-affinity constructs that have not yet been discovered.

[0374] From this work, 20 candidate sequences were subcloned into the pdt68 plasmid, verified by Sanger Sequencing, transformed into BL21 cells and then tested for expression in 10 mL to 50 mL IPTG induced expression cultures. The proteins were purified using NiNTA affinity resin, desalted them into 50 mM Tris pH 8.0, 300 mM NaCl, with 1 mM DTT, and then the N- terminal 6xHis-Sumo tag was removed using digestion with ULP1 protease. Control experiments described above showed that the Sumo tag, when attached, decreased actin-binding affinity but once cleaved, the tag did not affect actin-binding. As described above, the cleaved tag was left in the sample to reduce the amount of processing required for sample preparation. The concentration of the purified proteins was measured using UV absorbance and the purified proteins were subjected to SDS-PAGE on 4%-20% Tris-glycine poly-acrylamide gels, and the gels were measured with Bio-Rad Bio-Safe Coomassie protein stain. The gels were visualized using a white light transmittance to verify the quality of the preparations. The material consisted predominantly of the expressed ABD1-mClover3 protein. This material was flash frozen in liquid nitrogen and stored at -80 C for subsequent testing of actin-binding affinity by FRET.Biopanning subset ABDI sequence variant libraries

[0375] The ABDI sequence library was constructed in blocks and the blocks were pooled to prepare the library used for the biopanning described above. A smaller library with less total diversity could be enriched for mutations in regions suspected to contribute to actin-binding, and could increase the chances of identifying gain-of-affinity variants in those regions. 3 rounds of biopanning on smaller libraries, composed of subsets of the site-saturation blocks were performed. Clones with enhanced actin-binding properties were selected by ELISA for sequencing and subcloned into pdt68 as described above. Twenty-two candidates were expressed and purified,SUBSTITUTE SHEET (RULE 26)subjected to ULP1 digestion and characterized by SDS-PAGE. The purified material was frozen and stored at -80C for subsequent testing of actin-binding affinity by FRET.Site-saturation screening of L207-I209

[0376] Biopanning subset ABDI sequence variant libraries identified residue 207 and 209 as potential gain-of-affinity hotspots. To test the impact of combining mutations at these sites, a site-saturation mutagenesis library with NNN codons at both sites was prepared and subjected to three rounds of biopanning selection. Candidate clones were selected as described above, subcloned into pdt68 and tested for expression. From this work, 13 candidates were selected for expression, purification and subsequent testing by actin-binding assays.

[0377] These results taken together showed that actin-binding phage-display selected ABDI sequence candidates that bind actin with increased binding properties, 69 ABDI sequence variants were identified for subsequent testing and characterization.Example 6: Denovo engineering of novel ABDI derived variants with GoA actin-binding: characterization of GoA candidates

[0378] The FRET described above was used to measure actin-binding of the ABDI candidates identified above. Aliquots of the purified hDys-ABD1 proteins were thawed on ice, clarified by high-speed centrifugation to remove aggregates present after thawing, and standardized to a concentration of 2 micromolar in actin-binding buffer (F -buffer, 10 mM Tris pH 7.5 at 25 C, 2 mM MgCh, 1 mM DTT). The standardized samples were diluted to 100 nanomolar and mixed with increasing concentrations of Alexa-568 labeled F-actin in actin-binding buffer. Actin was labeled as described in above based on protocols in Guhathkurta et al. (Guhathakurta, P., E. Prochniewicz, and D.D. Thomas, Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain. Proc Natl Acad Sci U S A, 2015. 112(15): p. 4660-5). The samples were dispensed in 12 microliter reaction volumes in low-volume black bottom 384 well plates and incubated in the dark at room temperature for 20 to 30 minutes prior to nanosecond-resolved fluorescent lifetime measurements.

[0379] In the actin-binding FRET assay, the fluorescence lifetime of the donor probe (mClover3) reports binding of ABDI proteins to the Alexa-568 labeled actin filaments via energy transfer, which decreases the lifetime of the donor probe in proportion to the relative number of ABDI molecules that are bound to actin. The average lifetime of the time-resolved fluorescence decay waveforms was determined by fitting a single exponential decay function following methodsSUBSTITUTE SHEET (RULE 26)described in Muretta et al. (Muretta, J.M , et al., High-performance time-resolved fluorescence by direct waveform recording. Rev Sci Instrum, 2010. 81(10): p. 103101). The actin concentration dependence of the lifetime was analyzed by fitting the datasets with a single site binding hyperbolic function (Eq. 2) to determine the lifetimes of the donor (TD) and FRET (TDA) photophysical states and the apparent Kd for the actin-dependent increase in FRET. The donor and FRET lifetimes was used to compute energy transfer according to Eq. 3 then the actin dependence of energy transfer parameter, E, was fit to a single site hyperbolic function (Eq. 4) (FIGs. 21A-B, FIGs. 22A-PP, and Table 5).Table 5: Actin-binding Kd and Emax values from the fitsSUBSTITUTE SHEET (RULE 26)

[0380] Nine candidates predicted to bind actin exhibited Emaxvalues less than WT-hDys. For example, D9G-N86G, T107G, and I114R were each predicted to bind better than WT-hDys- ABD1 based on the ELISA assay (FIG. 17), but did not. L106P was also predicted to bind better than WT but was dropped from the workflow because the pdt68 plasmid containing this variant failed to transform into BL21 cells.Eq. 2Eq. 3Eq. 4

[0381] 12 candidates from the N = 1 FRET experiment that exhibited Emax values >0.15 were selected for N = 3 biological replicates of expression, purification, and actin-binding FRET. The candidates were selected based on their apparent Ka values in the N = 1 measurements and on their behaviors during purification. Several candidates that performed well in the N = 1 tests were not pursued in N = 3 due to low protein expression, low solubility or propensity for aggregation during purification. Each sample was prepared as above, evaluated by SDS-PAGE for relative purity and cleavage of the 6xHis-Sumo tag and then stored at -80 C prior to testing by FRET. The samples were subjected to actin-binding FRET measurements and the data analyzed as described above. For each biological replicate, at least 2 technical replicates were performed for each biological replicate. The variants exhibited robust actin-binding activities consistent with the N =SUBSTITUTE SHEET (RULE 26)1 FRET measurements. Energy transfer increased hyperbolically with increasing actin (FIGs. 23A- B and Table 5).

[0382] 6 candidates from these experiments were selected for 3 additional biological replicates of protein expression, purification, and testing by high-speed F-actin cosedimentation (FIG. 24) and thermal stability testing using DSF-GTP. The HSC and DSF-GTP assays were developed and optimized as described above. The apparent actin-binding Kd of each sample was determined by fitting Eq. 5 to the individual data sets. The performance of the HSC experiment was exceptional, indicated by the standard deviations of the measured fraction of ABDI molecules bound to actin at each actin concentration tested and by the standard deviation of the apparent Kd and Bmax values determined by fitting Eq. 5 to each dataset independently (FIG. 25).Eq. 5Considerations for the impact of recombinant ABDl-mClover3 purity and errors in measured protein concentration on the apparent actin-binding affinity and prioritization of gain-of-affinity candidates.

[0383] The removal of the 6xHis-Sumo tag fragment from ABDI protein preparations after ULP1 digestion was shown not to impact measurements of actin-binding affinity. SDS-PAGE analysis of the distribution of protein species in gain-of-affinity ABDI candidate samples tested by HSC shows the presence of the ABDI protein, the sumo-tag, and minor contaminants (FIGs. 26A-H). Measurements of total protein concentration were used to determine the concentration of ABD1-mClover3 used in each experiment. Thus, the relative purify of the sample could impact the concentration of ABDI in each experiment. The relative ABDI content and total ABDI derived content (ABDI + Sumo) of each sample were directly quantitated using Li-Cor fluorescence imaging of the N = 3 HSC samples (FIGs. 26A-H). Each sample consisted of more than 85% total ABDI derived protein (FIGs. 27A-B). The relative proportion of ABDI and Sumo tag varied between 20% and 60% of the total protein staining and the relative proportion of ABDI to total ABDI derived protein varied between 30% and 70%. Thus, the ABD derived material (ABDI + Sumo) account for m...

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide encoding a polypeptide comprising a dystrophin protein actin-binding domain 1 modified to achieve an increased actin-binding affinity (GoA-DysABD1), or an actin-binding fragment thereof, wherein: 1) the GoA-DysABD1, or an actin-binding fragment thereof, has at least one amino acid substitution compared to a same dystrophin protein actin-binding domain 1, or an actin-binding fragment thereof, absent the modification to achieve the increased actin-binding affinity (NotGoA-DysABD1); and 2) wherein in a fluorescence resonance energy transfer (FRET) assay the GoA-DysABD1, or an actin-binding fragment thereof, binds to actin with a dissociation constant (Kd) reduced of at least 20% compared to a dissociation constant of the NotGoA-DysABD1 in a same FRET assay.

2. The polynucleotide of claim 1, wherein the GoA-DysABD1 binds to actin with a Kd reduced of at least 50% compared to a dissociation constant of the NotGoA-DysABD1 in the same FRET assay.

3. The polynucleotide of claim 2, wherein the GoA-DysABD1 binds to actin with a Kd reduced of at least 80% compared to a dissociation constant of the NotGoA-DysABD1 in the same FRET assay.

4. The polynucleotide of claim 3, wherein the GoA-DysABD1 binds to actin with a reduced of at least 99.9% compared to a dissociation constant of the NotGoA-DysABD1 in the same FRET assay.

5. The polynucleotide of any one of claims 1-4, wherein in the FRET assay the actin is a phalloidin-stabilized F-actin.

6. The polynucleotide of any one of claims 1-5, wherein in the FRET assay an Alexa-568 fluorophore is attached to actin (actin-Alexa-568).

7. The polynucleotide of claim 6, wherein in the FRET assay the Alexa-568 fluorophore is attached to residue C374 of actin.

8. The polynucleotide of any one of claims 1-7, wherein in the FRET assay an mClover3 fluorophore is attached to the GoA-DysABD1 or to the NotGoA-DysABD1.SUBSTITUTE SHEET (RULE 26)9. The polynucleotide claim 8, wherein in the FRET assay the mClover3 fluorophore is attached to the carboxy-terminus of the GoA-DysABD1 (GoA-DysABD1-mClover3) or to the carboxy -terminus of the NotGoA-DysABD1 (NotGoA-DysABD1-mClover3).

10. The polynucleotide of claim 9, wherein in the FRET assay sub-micromolar amounts of GoA-DysABD1-mClover3 or of NotGoA-DysABD1 -mCl over 3 are used.

11. The polynucleotide of any one of claims 6-10, wherein in the FRET assay the amount of actin-Alexa-568 is increased over time.

12. The polynucleotide of any one of claims 9-11, wherein in the FRET assay a fluorescence decay lifetime of the GoA-DysABD1-mClover3 or of the NotGoA-DysABD1-mClover3 following a sub-nanosecond excitation pulse is measured.

13. The polynucleotide of any one of claims 1-12, wherein the FRET assay is a time-resolved FRET assay14. A polynucleotide encoding a polypeptide comprising a dystrophin protein actin-binding domain 1 modified to achieve an increased actin-binding affinity (GoA-DysABD1), or an actin-binding fragment thereof, compared to a same dystrophin protein actin-binding domain 1, or an actin-binding fragment thereof, absent the modification to achieve the increased actin-binding affinity (NotGoA-DysABD1), wherein the NotGoA-DysABD1, from N-terminus to C-terminus, comprises an amino acid sequence ofMLWWEEVEDC YERED VQKKTFTKWVNAQF SKFGKQHIENLF SDL QDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNN VDLVNIGSTDIVDGNHKLTLGLIWNIILHX118QVKNVMKNIMAGL QX133TNSEKILLSWVRQSTRNYPQVNVINFTTSWSX165GLALNAL IHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDV DTTYPDKKSILMYITSLFQVLPQQVSIE (SEQ ID NO: 2) wherein:X118 is W or R;X133 is Q or P;X165 is D or V; wherein the modification to achieve the increased actin-binding affinity is at least one amino acid substitution at one or more of residues D9, E12, Q17, K19, T20, F21, S30, Q35,SUBSTITUTE SHEET (RULE 26)H36, N75, K79, L81, L84, N86, K105, T107, Il 14, Il 15, V120, M128, W143, R145, Q153, T161, A168, L169, V187, A192, A199, 1202, L207, G208, 1209, L212, D214, E216, or K226.

15. The polynucleotide of claim 14, wherein the NotGoA-DysABD 1 , from N-terminus to C- terminus, comprises an amino acid sequence ofMLWWEEVEDC YERED VQKKTFTKWVNAQF SKFGKQHIENLF SDL QDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNN VDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQ TNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPD LFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPD KKSILMYITSLFQVLPQQVSIE (SEQ ID NO: 1).

16. The polynucleotide of any one of claims 1-15, wherein the polynucleotide is human or is codon-optimized for expression in humans.

17. The polynucleotide of any one of claims 1-16, wherein the GoA-DysABD1, or an actin- binding fragment thereof, comprises 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, 8 amino acid substitutions, 9 amino acid substitutions, or 10 amino acid substitutions compared to the NotGoA-DysABD 1, or an actin-binding fragment thereof.

18. The polynucleotide of claim 17, wherein the GoA-DysABD1, or an actin-binding fragment thereof, comprises 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, or 4 amino acid substitutions compared to the NotGoA-DysABD 1, or an actin-binding fragment thereof.

19. The polynucleotide of any one of claims 14-18, wherein the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (E12D), A at residue 17 (Q17A), E at residue 19 (K19E), D at residue 19 (K19D), A at residue 20 (T20A), Q at residue 20 (T20Q), N at residue 20 (T20N), S at residue 20 (T20S), M at residue 20 (T20M), V at residue 20 (T20V), L at residue 20 (T20L), I at residue 20 (T20I), L at residue 21 (F2 IL), A at residue 21 (F21 A), V at residue 21 (F21V), I at residue 21 (F21I), Q at residue 30 (S30Q), S at residue 30 (S30N), T at residue 30 (S30T), M at residue 30 (S30M), R atSUBSTITUTE SHEET (RULE 26)residue 35 (Q35R), K at residue 35 (Q35K), R at residue 36 (H36R), K at residue 36 (H36K), R at residue 75 (N75R), K at residue 75 (N75K), N at residue 79 (K79N), Q at residue 79 (K79Q), S at residue 79 (K79S), T at residue 79 (K79T), M at residue 79 (K79M), P at residue 81 (L81P), S at residue 84 (L84S), N at residue 84 (L84N), Q at residue 84 (L84Q), T at residue 84 (L84T), M at residue 84 (L84M), G at residue 86 (N86G), N at residue 105 (K105N), Q at residue 105 (K105Q), S at residue 105 (K105S), T at residue 105 (K105T), M at residue 105 (K105M), G at residue 107 (T107G, L at residue 114 (Il 14L), E at residue 114 (Il 14E), A at residue 114 (Il 14A), V at residue 114 (Il 14V), D at residue 114 (Il 14D), S at residue 115 (Il 15S), N at residue 115 (Il 15N), Q at residue 115 (I115Q), T at residue 115 (I115T), M at residue 115 (I115M), P at residue 120 (V120P), A at residue 120 (V120A), L at residue 120 (V120L), I at residue 120 (V120I), K at residue 128 (M128K), R at residue 128 (M128R), R at residue 143 (W143R), K at residue 143 (W143K), V at residue 145 (R145V), A at residue 145 (R145A),L at residue 145 (R145L), I 145(R145I), H at residue 153 (Q153H), Y at residue 153 (Q153Y), W at residue 153 (Q153W), F at residue 153 (Q153F), I at residue 156 (VI 561), A at residue 156 (V156A), L at residue 156 (V156L); H at residue 161 (T161H), Y at residue 161 (T161Y), W at residue 161 (T161W), F at residue 161 (T161F), V at residue 168 (A168V), I at residue 168 (Al 681), L at residue 168 (A168L), S at residue 169 (L169S), N at residue 169 (L169N), Q at residue 169 (L169Q), T at residue 169 (L169T), M at residue 169 (L169M), G at residue 187 (V187G), L at residue 192 (A192L), V at residue 192 (A192V), I at residue 192 (A192I), V at residue 199 (A199V), L at residue 199 (A199L), I at residue 199 (A199I), G at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue207 (L207S), Q at residue 207 (L207Q), T at residue 207 (L207T), M at residue 207 (L207M), A at residue 207 (L207A), I at residue 207 (L207I), Y at residue 207 (L207Y), W at residue 207 (L207W), F at residue 207 (L207F), E at residue 207 (L207E), P at residue208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (I209V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), H at residue 209 (I209H), Y at residue 209 (I209Y), A at residue 209 (I209A), N at residue 209 (I209N), T at residue 209 (I209T), M at residue 209 (I209M), P at residue 212 (L212P), P at residue 214 (D214P),SUBSTITUTE SHEET (RULE 26)K at residue 216 (E216K), R at residue 216 (E216R), E at residue 226 (K226E), or D at residue 226 (K226D), or any combination thereof.

20. The polynucleotide of claim 19, wherein the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (El 2D), A at residue 17 (Q17A), E at residue 19 (K19E), A at residue 20 (T20A), Q at residue 20 (T20Q), L at residue 21(F21L), A at residue 21 (F21 A), Q at residue 30 (S30Q), R at residue 35 (Q35R), R at residue 36 (H36R), R at residue 75 (N75R), N at residue 79 (K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 86 (N86G), N at residue 105 (K105N), G at residue 107 (T107G), L at residue 114 (Il 14L), E at residue 114 (Il 14E), S at residue 115 (Il 15S), P at residue 120 (V120P), A at residue 120 (V120A), K at residue 128 (M128K), R at residue 143 (W143R), V at residue 145 (R145V), H at residue 153 (Q153H), I at residue 156 (VI 561), H at residue 161 (T161H), V at residue 168 (A168V), I at residue 168 (A168I), S at residue 169 (L169S), G at residue 187 (V187G), , L at residue 192 (A192L), V at residue 199 (A199V), I at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue 207 (L207S), P at residue 208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (1209 V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue 209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226(K226E), or any combination thereof.

21. The polynucleotide of claim 19 or 20, wherein the at least one amino acid substitution consists of one amino acid substitution.

22. The polynucleotide of claim 21, wherein the one amino acid substitution is selected from Q at residue 20 (T20Q), Q at residue 30 (S30Q), N at residue 79 (K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 107 (T107G), L at residue 114 (Il 14L), E at residue 114 (Il 14E), S at residue 115 (Il 15S), P at residue 120 (V120P), R at residue 143 (W143R), H at residue 153 (Q153H), I at residue 156 (V156I), H at residue 161 (T161H), I at residue 168 (A168I), G at residue 187 (V187G), L at residue 192 (A192L), V at residue 199 (A199V), I at residue 202 (I202G), V at residue 207 (L207V), P at residue 208SUBSTITUTE SHEET (RULE 26)(G208P), K at residue 209 (I209K), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226 (K226E).

23. The polynucleotide of claim 22, wherein the one amino acid substitution is selected from Q at residue 20 (T20Q), P at residue 120 (V120P), I at residue 156 (V156I), P at residue 212 (L212P), P at residue 214 (D214P), or E at residue 226 (K226E).

24. The polynucleotide of any one of claims 22-23, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 5-30.

25. The polynucleotide of claim 24, wherein the polynucleotide comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 144-169.

26. The polynucleotide of claim 19 or 20, wherein the at least one amino acid substitution consists of two amino acid substitutions.

27. The polynucleotide of claim 26, wherein the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P-K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); N at residue 207 and L at residue 209 (L207N-I209L); R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); D at residue 207 and V at residue 209 (L207D- I209V); G at residue 187 and P at residue 212 (V187G-L212P); Q at residue 20 and I at residue 156 (T20Q-V156I); Q at residue 20 and P at residue 214 (T20Q-D214P); P at residue 120 and E at residue 226 (V120P-K226E); I at residue 156 and P at residue 214 (V156I-D214P); P at residue 120 and G at residue 187 (V120P-V187G); P at residue 212 and P at residue 214 (L212P-D214P); P at residue 214 and E at residue 226 (D214P- K226E); G at residue 187 and E at residue 226 (V187G-K226E); Q at residue 20 and P at residue 120 (T20Q-V120P); I at residue 156 and P at residue 212 (V156I-L212P); P at residue 120 and I at residue 156 (V120P-V156I); Q at residue 20 and P at residue 212SUBSTITUTE SHEET (RULE 26)(T20Q-L212P); P at residue 120 and P at residue 214 (V120P-D214P); G at residue 187 and P at residue 214 (V187G-D214P); Q at residue 20 and G at residue 187 (T20Q-V187G); P at residue 120 and P at residue 212 (V120P-L212P); Q at residue 20 and E at residue 226 (T20Q-K226E); I at residue 156 and G at residue 187 (V156I-V187G); I at residue 156 and E at residue 226 (V156I-K226E); P at residue 212 and E at residue 226 (L212P-K226E); A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P-K226E); or R at residue 35 and K at residue 207 (Q35R-L207K).

28. The polynucleotide of claim 27, wherein the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P-K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); N at residue 207 and L at residue 209 (L207N-I209L); R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); or D at residue 207 and V at residue 209 (L207D- I209V).

29. The polynucleotide of claim 28, wherein the two amino acid substitutions are selected from is A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P-K226E); or R at residue 35 and K at residue 207 (Q35R-L207K).

30. The polynucleotide of any one of claims 27-29, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 31-69.

31. The polynucleotide of claim 30, wherein the polynucleotide comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 170-208.

32. The polynucleotide of claim 19 or 20, wherein the at least one amino acid substitution consists of three amino acid substitutions.SUBSTITUTE SHEET (RULE 26)33. The polynucleotide of claim 32, wherein the three amino acid substitutions are selected from G at residue 187, P at residue 214, E at residue 226 (V187G-D214P-K226E); Q at residue 20, P at residue 120, and G at residue 187 (T20Q-V120P-V187G); Q at residue 20, P at residue 120, and E at residue 226 (T20Q-V120P-K226E); Q at residue 20, G at residue 187, and E at residue 226 (T20Q-V187G-K226E); P at residue 120, 1 at residue 156, and P at residue 214 (V120P-V156I-D214P); P at residue 212, P at residue 214, and E at residue 226 (L212P-D214P-K226E); P at residue 120, G at residue 187, and E at residue 226 (V120P-V187G-K226E); P at residue 120, P at residue 212, and P at residue 214 (V120P- L212P-D214P); I at residue 156, P at residue 212, and P at residue 214 (V156I-L212P- D214P); Q at residue 20, 1 at residue 156, and P at residue 212 (T20Q-V156I-L212P); I at residue 156, G at residue 187, and E at residue 226 (V156I-V187G-K226E); Q at residue 20, P at residue 214, and E at residue 226 (T20Q-D214P-K226E); P at residue 120, P at residue 214, and E at residue 226 (V120P-D214P-K226E); Q at residue 20, P at residue 120, and P at residue 214 (T20Q-V120P-D214P); Q at residue 20, P at residue 120, and I at residue 156 (T20Q-V120P-V156I); Q at residue 20, G at residue 187, and P at residue 214 (T20Q-V187G-D214P); P at residue 120, I at residue 156, and P at residue 212 (V120P-V156I-L212P); I at residue 156, P at residue 214, and E at residue 226 (V156I- D214P-K226E); G at residue 187, P at residue 212, and E at residue 226 (V187G-L212P- K226E); P at residue 120, G at residue 187, and P at residue 214 (V120P-V187G-D214P); Q at residue 20, 1 at residue 156, and G at residue 187 (T20Q-V156I-V187G); I at residue 156, G at residue 187, and P at residue 214 (V156I-V187G-D214P); Q at residue 20, and I at residue 156, E226 (T20Q-V156I-K226E); Q at residue 20, P at residue 212, and E at residue 226 (T20Q-L212P-K226E); Q at residue 20, P at residue 120, and P at residue 212 (T20Q-V120P-L212P); Q at residue 20, G at residue 187, and P at residue 212 (T20Q- V187G-L212P); P at residue 120, I at residue 156, and E at residue 226 (V120P-V156I- K226E); P at residue 120, I at residue 156, and G at residue 187 (V120P-V156I-V187G); P at residue 120, P at residue 212, and E at residue 226 (V120P-L212P-K226E); P at residue 120, G at residue 187, and P at residue 212 (V120P-V187G-L212P); G at residue 187, P at residue 212, and P at residue 214 (V187G-L212P-D214P); I at residue 156, G at residue 187, and P at residue 212 (V156I-V187G-L212P); Q at residue 20, 1 at residue 156, and P at residue 214 (T20Q-V156I-D214P); I at residue 156, P at residue 212, and E at residue 226 (V156I-L212P-K226E); Q at residue 20, P at residue 212, and P at residue 214 (T20Q-SUBSTITUTE SHEET (RULE 26)L212P-D214P); A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N- V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L-I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R-A168V).

34. The polynucleotide of claim 33, wherein the three amino acid substitutions are selected from A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N-V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L-I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R-A168V).

35. The polynucleotide of any one of claims 33-34, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 70-107.

36. The polynucleotide of claim 35, wherein the polynucleotide comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 209-247.

37. The polynucleotide of claim 19 or 20, wherein the at least one amino acid substitution consists of four amino acid substitutions.

38. The polynucleotide of claim 37, wherein the four amino acid substitutions are selected from Q at residue 20, P at residue 120, G at residue 187, and P at residue 212 (T20Q-V120P- V187G-L212P); Q at residue 20, P at residue 120, P at residue 212, and P at residue 214 (T20Q-V120P-L212P-D214P); Q at residue 20, 1 at residue 156, G at residue 187, and P at residue 214 (T20Q-V156I-V187G-D214P); Q at residue 20, P at residue 212, P at residue 214, and E at residue 226 (T20Q-L212P-D214P-K226E); P at residue 120, 1 at residue 156, G at residue 187, and P at residue 212 (V120P-V156I-V187G-L212P); I at residue 156, G at residue 187, P at residue 212, and E at residue 226 (V156I-V187G-L212P-K226E); P at residue 120, 1 at residue 156, P at residue 212, and E at residue 226 (V120P-V156I-L212P- K226E); Q at residue 20, G at residue 187, P at residue 212, and P at residue 214 (T20Q- V187G-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and E at residue 226 (V120P-V187G-L212P-K226E); Q at residue 20, 1 at residue 156, P at residue 212, and E at residue 226 (T20Q-V156I-L212P-K226E); G at residue 187, P at residue 212, P at residue 214, and E at residue 226 (V187G-L212P-D214P-K226E); Q at residue 20, P at residue 120, G at residue 187, and E at residue 226 (T20Q-V120P-V187G-K226E); Q at residue 20, P at residue 120, I at residue 156, and P at residue 212 (T20Q-V120P-V156I-SUBSTITUTE SHEET (RULE 26)L212P); Q at residue 20, P at residue 120, P at residue 214, and E at residue 226 (T20Q- V120P-D214P-K226E); Q at residue 20, 1 at residue 156, G at residue 187, and P at residue 212 (T20Q-V156I-V187G-L212P); I at residue 156, Gat residue 187, P at residue 212, and P at residue 214 (V156I-V187G-L212P-D214P); P at residue 120, I at residue 156, G at residue 187, and E at residue 226 (V120P-V156I-V187G-K226E); P at residue 120, I at residue 156, P at residue 212, and P at residue 214 (V120P-V156I-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and P at residue 214 (V120P-V187G- L212P-D214P); Q at residue 20, G at residue 187, P at residue 214, and E at residue 226 (T20Q-V187G-D214P-K226E); Q at residue 20, I at residue 156, P at residue 212, and P at residue 214 (T20Q-V156I-L212P-D214P); I at residue 156, P at residue 212, P at residue 214, and E at residue 226 (V156I-L212P-D214P-K226E); Q at residue 20, P at residue 120, G at residue 187, and P at residue 214 (T20Q-V120P-V187G-D214P); Q at residue 20, P at residue 120, I at residue 156, and G at residue 187 (T20Q-V120P-V156I-V187G); I at residue 156, G at residue 187, P at residue 214, and E at residue 226 (V156I-V187G- D214P-K226E); Q at residue 20, P at residue 120, I at residue 156, and E at residue 226 (T20Q-V120P-V156I-K226E); P at residue 120, 1 at residue 156, P at residue 214, and E at residue 226 (V120P-V156I-D214P-K226E); Q at residue 20, I at residue 156, G at residue 187, and E at residue 226 (T20Q-V156I-V187G-K226E); Q at residue 20, P at residue 120, P at residue 212, and E at residue 226 (T20Q-V120P-L212P-K226E); P at residue 120, G at residue 187, P at residue 214, and E at residue 226 (V120P-V187G- D214P-K226E); P at residue 120, P at residue 212, P at residue 214, and E at residue 226 (V120P-L212P-D214P-K226E); Q at residue 20, G at residue 187, P at residue 212, and E at residue 226 (T20Q-V187G-L212P-K226E); P at residue 120, I at residue 156, G at residue 187, and P at residue 214 (V120P-V156I-V187G-D214P); Q at residue 20, I at residue 156, P at residue 214, and E at residue 226 (T20Q-V156I-D214P-K226E); Q at residue 20, P at residue 120, I at residue 156, and P at residue 214 (T20Q-V120P-V156L D214P); or A at residue 17, A at residue 20, P at residue 212, and E at residue 226 (Q17A- T20A-L212P-K226E).

39. The polynucleotide of claim 38, wherein the four amino acid substitutions are A at residue17, A at residue 20, P at residue 212, and E at residue 226 (Q17A-T20A-L212P-K226E).SUBSTITUTE SHEET (RULE 26)40. The polynucleotide of any one of claims 38-39, wherein the polypeptide comprises an amino acid sequence at of any one of SEQ ID NOs: 108-143.

41. The polynucleotide of claim 40, wherein the polynucleotide comprises a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 248-282.

42. The polynucleotide of any one of claims 1-41, wherein the polypeptide further comprises one or more additional portions of a dystrophin protein.

43. The polynucleotide of claim 42, wherein the polypeptide further comprises:(i) at least one dystrophin protein spectrin-like repeat located downstream of the ABD 1 domain; and(ii) at least one cysteine-rich domain located downstream of the dystrophin protein spectrin-like repeat.

44. The polynucleotide of claim 43, wherein the dystrophin protein spectrin-like repeat is a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein spectrin-like repeat 4, a dystrophin protein spectrin-like repeat 5, a dystrophin protein spectrin-like repeat 6, a dystrophin protein spectrin-like repeat 7, a dystrophin protein spectrin-like repeat 8, a dystrophin protein spectrin-like repeat 9, a dystrophin protein spectrin-like repeat 10, a dystrophin protein spectrin-like repeat 11, a dystrophin protein spectrin-like repeat 12, a dystrophin protein spectrin-like repeat 13, a dystrophin protein spectrin-like repeat 14, a dystrophin protein spectrin-like repeat 15, a dystrophin protein spectrin-like repeat 16, a dystrophin protein spectrin-like repeat 17, a dystrophin protein spectrin-like repeat 18, a dystrophin protein spectrin-like repeat 19, a dystrophin protein spectrin-like repeat 20, a dystrophin protein spectrin-like repeat 21, a dystrophin protein spectrin-like repeat 22, a dystrophin protein spectrin-like repeat 23, or a dystrophin protein spectrin-like repeat 24.

45. The polynucleotide of claim 44, wherein:(i) the spectrin-like repeat 1 comprises amino acids 339-447 of SEQ ID NO: 4;(ii) the spectrin-like repeat 2 comprises amino acids 448-556 of SEQ ID NO: 4;(iii) the spectrin-like repeat 3 comprises amino acids 559-667 of SEQ ID NO: 4;(iv) the spectrin-like repeat 4 comprises amino acids 719-828 of SEQ ID NO: 4;SUBSTITUTE SHEET (RULE 26)(v) the spectrin-like repeat 5 comprises amino acids 830-934 of SEQ ID NO: 4;(vi) the spectrin-like repeat 6 comprises amino acids 943-1045 of SEQ ID NO: 4;(vii) the spectrin-like repeat 7 comprises amino acids 1048-1154 of SEQ ID NO: 4;(viii) the spectrin-like repeat 8 comprises amino acids 1157-1263 of SEQ ID NO: 4;(ix) the spectrin-like repeat 9 comprises amino acids 1266-1367 of SEQ ID NO: 4;(x) the spectrin-like repeat 10 comprises amino acids 1368-1463 of SEQ ID NO: 4;(xi) the spectrin-like repeat 11 comprises amino acids 1468-1568 of SEQ ID NO: 4;(xii) the spectrin-like repeat 12 comprises amino acids 1571-1676 of SEQ ID NO: 4;(xiii) the spectrin-like repeat 13 comprises amino acids 1679-1778 of SEQ ID NO: 4;(xiv) the spectrin-like repeat 14 comprises amino acids 1779-1874 of SEQ ID NO: 4;(xv) the spectrin-like repeat 15 comprises amino acids 1877-1979 of SEQ ID NO: 4;(xvi) the spectrin-like repeat 16 comprises amino acids 1992-2101 of SEQ ID NO: 4;(xvii) the spectrin-like repeat 17 comprises amino acids 2104-2208 of SEQ ID NO: 4;(xviii) the spectrin-like repeat 18 comprises amino acids 2211-2318 of SEQ ID NO: 4;(xix) the spectrin-like repeat 19 comprises amino acids 2319-2423 of SEQ ID NO: 4;(xx) the spectrin-like repeat 20 comprises amino acids 2475-2577 of SEQ ID NO: 4;(xxi) the spectrin-like repeat 21 comprises amino acids 2580-2686 of SEQ ID NO: 4;(xxii) the spectrin-like repeat 22 comprises amino acids 2689-2802 of SEQ ID NO: 4; (xxiii) the spectrin-like repeat 23 comprises amino acids 2808-2930 of SEQ ID NO: 4;(xxiv) the spectrin-like repeat 24 comprises amino acids 2935-3040 of SEQ ID NO: 4.

46. The polynucleotide of any one of claims 43-45, wherein the cysteine-rich domain comprises amino acids 3113-3360 of SEQ ID NO: 4.

47. The polynucleotide of any one of claims 43-46, wherein the polypeptide further comprises at least one dystrophin protein hinge region located downstream of the ABDI and upstream of the cysteine-rich domain.

48. The polynucleotide of claim 47, wherein the dystrophin protein hinge region is a dystrophin protein hinge region 1, a dystrophin protein hinge region 2, a dystrophin protein hinge region 3, or a dystrophin protein hinge region 4.

49. The polynucleotide of claim 48, wherein:(i) the hinge region 1 comprises amino acids 247-338 of SEQ ID NO: 4;SUBSTITUTE SHEET (RULE 26)(ii) the hinge region 2 comprises amino acids 668-718 of SEQ ID NO: 4;(iii) the hinge region 3 comprises amino acids 2424-2474 of SEQ ID NO: 4;(iv) the hinge region 4 comprises amino acids 3041-3112 of SEQ ID NO: 4.

50. The polynucleotide of any one of claims 43-49, wherein the polypeptide further comprises a dystrophin protein carboxy-terminal domain (CTD) located downstream of the cysteine- rich domain.

51. The polynucleotide of claim 50, wherein the dystrophin protein carboxyl-terminus comprises amino acids 3361-3685 of SEQ ID NO: 4.

52. The polynucleotide of any one of claims 48-49, wherein the polypeptide comprises:(i) the Go A-Dy s ABD 1 ;(ii) dystrophin protein spectrin-like repeats 1, 2, 3, and 24;(iii) dystrophin protein hinge regions 1, 2, and 4; and(iv) a dystrophin protein cysteine-rich domain.

53. The polynucleotide of claim 52, wherein the polypeptide from amino-terminus to carboxyl- terminus consists of:(i) the Go A-Dy s ABD 1 ;(ii) a dystrophin protein hinge region 1;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 2;(v) a dystrophin protein spectrin-like repeat 3;(vi) a dystrophin protein hinge region 2;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4; and(ix) a dystrophin protein cysteine-rich domain.

54. The polynucleotide of any one of claims 48-49, wherein the polypeptide comprises:(i) the Go A-Dy s ABD 1 ;(ii) dystrophin protein spectrin-like repeats 1, 2, 22, 23, and 24;(iii) dystrophin protein hinge regions 1, 3, and 4; and(iv) a dystrophin protein cysteine-rich domain.SUBSTITUTE SHEET (RULE 26)55. The polynucleotide of claim 54, wherein the polypeptide from amino-terminus to carboxylterminus consists of:(i) the Go A-Dy s ABD 1 ;(ii) a dystrophin protein hinge region 1;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 22;(v) a dystrophin protein spectrin-like repeat 23;(vi) a dystrophin protein spectrin-like repeat 24;(vii) a dystrophin protein hinge region 4; and(viii) a dystrophin protein cysteine-rich domain.

56. The polynucleotide of any one of claims 48-49, wherein the polypeptide comprises:(i) the Go A-Dy s ABD 1 ;(ii) dystrophin protein spectrin-like repeats 1, 16, 17, 23, and 24;(iii) dystrophin protein hinge regions 1, and 4; and(iv) a dystrophin protein cysteine-rich domain.

57. The polynucleotide of claim 56, wherein the polypeptide from amino-terminus to carboxylterminus consists of:(i) the Go A-Dy s ABD 1 ;(ii) a dystrophin protein hinge region 1;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 16;(v) a dystrophin protein spectrin-like repeat 17;(vi) a dystrophin protein spectrin-like repeat 23;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4; and(ix) a dystrophin protein cysteine-rich domain.

58. The polynucleotide of any one of claims 50-51, wherein the polypeptide comprises:(i) the Go A-Dy s ABD 1 ;(ii) dystrophin protein spectrin-like repeats 1, 2, 3, and 24;(iii) dystrophin protein hinge regions 1, 3, and 4;(iv) a dystrophin protein cysteine-rich domain; andSUBSTITUTE SHEET (RULE 26)(v) a dystrophin protein carboxyl-terminus.

59. The polynucleotide of claim 58, wherein the polypeptide from amino-terminus to carboxyl- terminus consists of:(i) the Go A-Dy s ABD 1 ;(ii) a dystrophin protein hinge region 1;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 2;(v) a dystrophin protein spectrin-like repeat 3;(vi) a dystrophin protein hinge region 3;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4;(ix) a dystrophin protein cysteine-rich domain;(x) and a dystrophin protein CTD.

60. The polynucleotide of any one of claims 1-59, wherein the polypeptide is capable of linking a subsarcolemmal cytoskeleton with the extracellular matrix.

61. The polynucleotide of any one of claims 1-60, wherein the polypeptide is capable of recruiting a dystrophin-associated protein complex.

62. The polynucleotide of any one of claims 1-61, wherein the polynucleotide is a DNA polynucleotide, an RNA polynucleotide, or a combination thereof.

63. An expression cassette comprising the polynucleotide of any one of claims 1-61.

64. The expression cassette of claim 63, wherein the expression cassette further comprises a promoter, a Kozak sequence, an enhancer, a silencer, a polyA tail, a poly adenylation signal, a 3'-UTR, a 5 -UTR, an intronic sequence, a nucleotide sequence encoding a molecular tag, a nucleotide sequence encoding a self-cleaving peptide, a linker, a filler sequence, or any combination thereof.

65. The expression cassette of claim 64, wherein the promoter is a constitutive promoter, a regulatable promoter, an ubiquitous promoter, or a tissue-specific promoter.

66. The expression cassette of claim 65, wherein the promoter is a constitutive promoter.SUBSTITUTE SHEET (RULE 26)67. The expression cassette of claim 66, wherein the promoter is selected from the group consisting of a CBA promoter, a CMV promoter, an EFla promoter, or a CAG promoter.

68. The expression cassette of claim 65, wherein the promoter is a muscle tissue-specific promoter.

69. The expression cassette of claim 68, wherein the muscle tissue-specific promoter is selected from the group consisting of a MHCK7 promoter, a CK8e promoter, a MCK promoter, a dMCK promoter, a tMCK promoter, a DES promoter, a HSA promoter, a SPc5-12 promoter, a SP-301 promoter, a MHC promoter, a Sk-CRM promoter, a Sk-CRM4 promoter70. The expression cassette of claim 69, wherein the muscle tissue-specific promoter comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 283.

71. The expression cassette of any one of claims 64-70, wherein the intronic sequence is a CAG intron, an SV40 intron, MVM intron, or a human beta-globin intron, or any combination thereof.

72. The expression cassette of claim 71 wherein the intron comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 287-289.

73. The expression cassette of any one of claims 64-72, wherein the poly(A) sequence is selected from a bGHpA, a hGHpA, a SV40pA, or a synthetic pA.

74. The expression cassette of claim 73, wherein the poly(A) comprises a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 290.

75. A vector comprising the polynucleotide of any one of claims 1-60 or the expression cassette of any one of claims 61-74.

76. The vector of claim 75, wherein the vector is a non-viral vector or a viral vector.

77. The vector of claim 76, wherein the viral vector is an AAV vector.SUBSTITUTE SHEET (RULE 26)78. The vector of claim 77, wherein the vector further comprises a nucleotide sequence encoding a first inverted terminal repeat (ITR) and a second ITR.

79. The vector of claim 78, wherein the first ITR is at the 5' of the polynucleotide and a second ITR is at the 3' of the polynucleotide.

80. The vector of claims 78-79, wherein the first and the second ITR are of a same or of a different serotype.

81. The vector of claim 80, wherein serotype of the ITRs is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVRH10, AAV11, or AAV12 serotype.

82. The vector of claim 81, wherein the first and the second ITR comprise a nucleotide sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 294-295.

83. The vector of any one of claims 75-82, wherein the vector further comprises a nucleotide sequence encoding a selectable marker.

84. A recombinant adeno-associated virus (rAAV) particle, comprising the vector of any one of claims 75-83 and a capsid.

85. The rAAV particle of claim 84, wherein the serotype of the capsid is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6.2, AAV6P1, AAV7, AAV8, AAV8P1, AAV9, AAVrhlO, AAVrhlOPl, AAVS10P4, AAVpol, AAV11, AAV12, AAV-DJ, AAV- DJ / 8, AAV-PHP.Eb, AAV-PHP.S, AAV-PHP.B, AAV2-retro, AAV2-QuadYF, AAV2.7m8, AAVS1, AAVS10, AAVrh74, AAVS1, AAVS10, AAVH15, AAVMYO (AAV9P1), AAVMYO 2 (AAVS1P1), AAVMY03 (AAVS10P1), AAV9-RGD, MyoAAVlA, MyoAAV 1C, MyoAAV IE, MyoAAV 2 A, MyoAAV 2E, MyoAAV 3 A, MyoAAV 4A, MyoAAV 4C, or MyoAAV 4E .

86. The rAAV particle of claim 85, wherein the serotype of the capsid is selected from AAV1, AAV8, AAV9, AAVrh74, AAVS1, AAVS10, AAVH15, AAVMYO (AAV9P1), AAVMYO 2 (AAVS1P1), AAVMYO3 (AAVS10P1), AAV9-RGD, MyoAAVlA, MyoAAV 1C, MyoAAV IE, MyoAAV 2A, MyoAAV 2E, MyoAAV 3 A, MyoAAV 4A, MyoAAV 4C, or MyoAAV 4E.SUBSTITUTE SHEET (RULE 26)87. The rAAV particle of claim 86, wherein the serotype of the capsid is AAVrh74 serotype.

88. A polypeptide encoded by the polynucleotide of any one of claims 1-60, by the expression cassette of any one of claims 61-74, by the vector of any one of claims 75-83, or by the rAAV particle of any one of claims 84-87.

89. An engineered dystrophin protein actin-binding domain 1 (GoA-DysABD1), or an actin- binding fragment thereof, encoded by the polynucleotide of any one of claims 1-60, by the expression cassette of any one of claims 61-74, by the vector of any one of claims 75-83, or by the rAAV particle of any one of claims 84-87.

90. An engineered dystrophin protein or an actin-binding fragment thereof comprising the polypeptide of claim 88 or the GoA-DysABD1 of claim 89.

91. A polypeptide comprising a dystrophin protein actin-binding domain 1 modified to achieve an increased actin-binding affinity (GoA-DysABD1), or an actin-binding fragment thereof, wherein: 1) the GoA-DysABD1, or an actin-binding fragment thereof, has at least one amino acid substitution compared to a same dystrophin protein actin-binding domain 1, or an actin-binding fragment thereof, absent the modification to achieve the increased actin- binding affinity (NotGoA-DysABD1); and 2) wherein in a fluorescence resonance energy transfer (FRET) assay the GoA-DysABD1, or an actin-binding fragment thereof, binds to actin with a dissociation constant (Kd) reduced at least 20% compared to a dissociation constant of the NotGoADysABD1 in a same FRET assay.

92. The polypeptide of claim 91, wherein the GoA-DysABD1 binds to actin with a Kd reduced at least 50% compared to a dissociation constant of the NotGoADysABD1 in the same FRET assay.

93. The polypeptide of claim 92, wherein the GoA-DysABD1 binds to actin with a Kd reduced at least 80% compared to a dissociation constant of the NotGoADysABD1 in the same FRET assay.

94. The polypeptide of claim 93, wherein the GoA-DysABD1 binds to actin with a reduced at least 99.9% compared to a dissociation constant of the NotGoADysABD1 in the same FRET assay.SUBSTITUTE SHEET (RULE 26)95. The polypeptide of any one of claims 91-94, wherein in the FRET assay the actin is a phalloidin-stabilized F-actin.

96. The polypeptide of any one of claims 91-95, wherein in the FRET assay an Alexa-568 fluorophore is attached to actin (actin-Alexa-568).

97. The polypeptide of claim 96, wherein in the FRET assay the Alexa-568 fluorophore is attached to residue C374 of actin.

98. The polypeptide of any one of claims 91-97, wherein in the FRET assay an mClover3 fluorophore is attached to the GoA-DysABD1 or to the NotGoADysABD 1.

99. The polypeptide claim 98, wherein in the FRET assay the mClover3 fluorophore is attached to a carboxy-terminus of the GoA-DysABD1 (GoA-DysABD1-mClover3) or to a carboxyterminus of the NotGoADysABD 1 (NotGoA-DysABD1-mClover3).

100. The polypeptide of claim 99, wherein in the FRET assay sub-micromolar amounts of GoA- DysABD1 -mClover3 or of NotGoA-DysABD1-mClover3 are used.

101. The polypeptide of any one of claims 96-100, wherein in the FRET assay the amount of actin-Alexa-568 is increased over time.

102. The polypeptide of any one of claims 99-101, wherein in the FRET assay a fluorescence decay lifetime of the GoA-DysABD1 -mClover3 or of the NotGoA-DysABD1-mClover3 following a sub-nanosecond excitation pulse is measured103. The polypeptide of any one of claims 91-102, wherein the FRET assay is a time-resolved FRET assay.

104. A polypeptide comprising a dystrophin protein actin-binding domain 1 modified to achieve an increased actin-binding affinity (GoA-DysABD1), or an actin-binding fragment thereof, compared to a same dystrophin protein actin-binding domain 1, or an actin-binding fragment thereof, absent the modification to achieve the increased actin-binding affinity (NotGoADysABD 1), wherein the NotGoA-DysABD1, from N-terminus to C-terminus, comprises an amino acid sequence ofSUBSTITUTE SHEET (RULE 26)MLWWEEVEDC YERED VQKKTFTKWVNAQF SKFGKQHIENLF SDL QDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNN VDLVNIGSTDIVDGNHKLTLGLIWNIILHX118QVKNVMKNIMAGL QX133TNSEKILLSWVRQSTRNYPQVNVINFTTSWSX165GLALNAL IHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDV DTTYPDKKSILMYITSLFQVLPQQVSIE (SEQ ID NO: 2) wherein:X118 is W or R;X133 is Q or P;X165 is D or V; and wherein the modification to achieve the increased actin-binding affinity is at least one amino acid substitution at one or more of residues D9, E12, Q17, K19, T20, F21, S30, Q35, H36, N75, K79, L81, L84, N86, K105, T107, Il 14, Il 15, V120, M128, W143, R145, Q153, T161, A168, L169, V187, A192, A199, 1202, L207, G208, 1209, L212, D214, E216, or K226.

105. The polypeptide of claim 104, wherein the NotGoADysABD 1 , from N-terminus to C- terminus, comprises an amino acid sequence ofMLWWEEVEDC YERED VQKKTFTKWVNAQF SKFGKQHIENLF SDL QDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNN VDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQ TNSEKILLSWVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPD LFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVDTTYPD KKSILMYITSLFQVLPQQVSIE (SEQ ID NO: 1).

106. The polypeptide of any one of claims 91-105, wherein the GoA-DysABD1, or an actin- binding fragment thereof, comprises 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions, 5 amino acid substitutions, 6 amino acid substitutions, 7 amino acid substitutions, 8 amino acid substitutions, 9 amino acid substitutions, 10 amino acid substitutions compared to the NotGoADysABD1, or an actin- binding fragment thereof.SUBSTITUTE SHEET (RULE 26)107. The polypeptide of claim 106, wherein the GoA-DysABD1, or an actin-binding fragment thereof, comprises 1 amino acid substitution, 2 amino acid substitutions, 3 amino acid substitutions, 4 amino acid substitutions compared to the NotGoADysABD1, or an actin- binding fragment thereof.

108. The polypeptide of any one of claims 104-107, wherein the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (E12D), A at residue 17 (Q17A), E at residue 19 (K19E), D at residue 19 (K19D), A at residue 20 (T20A), Q at residue 20 (T20Q), N at residue 20 (T20N), S at residue 20 (T20S), M at residue 20 (T20M), V at residue 20 (T20V), L at residue 20 (T20L), I at residue 20 (T20I), L at residue 21 (F2 IL), A at residue 21 (F21 A), V at residue 21 (F21V), I at residue 21 (F21I), Q at residue 30 (S30Q), S at residue 30 (S30N), T at residue 30 (S30T), M at residue 30 (S30M), R at residue 35 (Q35R), K at residue 35 (Q35K), R at residue 36 (H36R), K at residue 36 (H36K), R at residue 75 (N75R), K at residue 75 (N75K), N at residue 79 (K79N), Q at residue 79 (K79Q), S at residue 79 (K79S), T at residue 79 (K79T), M at residue 79(K79M), P at residue 81 (L81P), S at residue 84 (L84S), N at residue 84 (L84N), Q at residue 84 (L84Q), T at residue 84 (L84T), M at residue 84 (L84M), G at residue 86(N86G), N at residue 105 (K105N), Q at residue 105 (K105Q), S at residue 105 (K105S),T at residue 105 (K105T), M at residue 105 (K105M), G at residue 107 (T107G, L at residue 114 (Il 14L), E at residue 114 (Il 14E), A at residue 114 (Il 14A), V at residue 114 (Il 14V), D at residue 114 (Il 14D), S at residue 115 (Il 15S), N at residue 115 (Il 15N), Q at residue 115 (I115Q), T at residue 115 (I115T), M at residue 115 (I115M), P at residue 120 (V120P), A at residue 120 (V120A), L at residue 120 (V120L), I at residue 120 (V120I), K at residue 128 (M128K), R at residue 128 (M128R), R at residue 143 (W143R), K at residue 143 (W143K), V at residue 145 (R145V), A at residue 145 (R145A),L at residue 145 (R145L), 1 145(R145I), H at residue 153 (Q153H), Y at residue 153 (Q153Y), W at residue 153 (Q153W), F at residue 153 (Q153F), I at residue 156 (VI 561), A at residue 156 (V156A), L at residue 156 (V156L); H at residue 161 (T161H), Y at residue 161 (T161Y), W at residue 161 (T161W), F at residue 161 (T161F), V at residue 168 (A168V), I at residue 168 (Al 681), L at residue 168 (A168L), S at residue 169 (L169S), N at residue 169 (L169N), Q at residue 169 (L169Q), T at residue 169 (L169T), M at residue 169 (L169M), G at residue 187 (VI 87G), , L at residue 192 (Al 92L), V at residue 192 (A192V), I at residue 192 (A192I), V at residue 199 (A199V), L at residue 199 (A199L), I at residueSUBSTITUTE SHEET (RULE 26)199 (A199I), G at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue207 (L207S), Q at residue 207 (L207Q), T at residue 207 (L207T), M at residue 207 (L207M), A at residue 207 (L207A), I at residue 207 (L207I), Y at residue 207 (L207Y), W at residue 207 (L207W), F at residue 207 (L207F), E at residue 207 (L207E), P at residue208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (I209V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), H at residue 209 (I209H),Y at residue 209 (I209Y), A at residue 209 (I209A), N at residue 209 (I209N), T at residue 209 (I209T), M at residue 209 (I209M), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), R at residue 216 (E216R), E at residue 226 (K226E), or D at residue 226 (K226D), or any combination thereof.

109. The polypeptide of claim 108, wherein the at least one amino acid substitution is selected from G at residue 9 (D9G), D at residue 12 (El 2D), A at residue 17 (Q17A), E at residue 19 (K19E), A at residue 20 (T20A), Q at residue 20 (T20Q), L at residue 21(F21L), A at residue 21 (F21 A), Q at residue 30 (S30Q), R at residue 35 (Q35R), R at residue 36 (H36R), R at residue 75 (N75R), N at residue 79 (K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 86 (N86G), N at residue 105 (K105N), G at residue 107 (T107G), L at residue 114 (Il 14L), E at residue 114 (Il 14E), S at residue 115 (Il 15S), P at residue 120 (V120P), A at residue 120 (V120A), K at residue 128 (M128K), R at residue 143 (W143R),V at residue 145 (R145V), H at residue 153 (Q153H), I at residue 156 (VI 561), H at residue 161 (T161H), V at residue 168 (A168V), I at residue 168 (A168I), S at residue 169 (L169S), G at residue 187 (V187G), , L at residue 192 (A192L), V at residue 199 (A199V), I at residue 202 (I202G), C at residue 207 (L207C), V at residue 207 (L207V), K at residue 207 (L207K), P at residue 207 (L207P), R at residue 207 (L207R), H at residue 207 (L207H), D at residue 207 (L207D), N at residue 207 (L207N), S at residue 207 (L207S), P at residue 208 (G208P), C at residue 209 (I209C), K at residue 209 (I209K), V at residue 209 (1209 V), F at residue 209 (I209F), R at residue 209 (I209R), W at residue 209 (I209W), L at residue 209 (I209L), S at residue 209 (I209S), Q at residue 209 (I209Q), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226(K226E), or any combination thereof.SUBSTITUTE SHEET (RULE 26)110. The polypeptide of claim 108 or 109, wherein the at least one amino acid substitution consists of one amino acid substitution.

111. The polypeptide of claim 110, wherein the one amino acid substitution is selected from Q at residue 20 (T20Q), Q at residue 30 (S30Q), N at residue 79 (K79N), P at residue 181 (L81P), S at residue 84 (L84S), G at residue 107 (T107G), L at residue 114 (Il 14L), E at residue 114 (Il 14E), S at residue 115 (Il 15S), P at residue 120 (V120P), R at residue 143 (W143R), H at residue 153 (Q153H), I at residue 156 (V156I), H at residue 161 (T161H), I at residue 168 (A168I), Gat residue 187 (V187G), , L at residue 192 (A192L), V at residue 199 (A199V), I at residue 202 (I202G), V at residue 207 (L207V), P at residue 208 (G208P), K at residue 209 (I209K), P at residue 212 (L212P), P at residue 214 (D214P), K at residue 216 (E216K), or E at residue 226 (K226E).

112. The polypeptide of claim 111, wherein the one amino acid substitution is Q at residue 20 (T20Q), P at residue 120 (V120P), I at residue 156 (V156I), P at residue 212 (L212P), P at residue 214 (D214P), E at residue 226 (K226E).

113. The polypeptide of any one of claims 111-112, wherein the polypeptide comprises an amino acid sequence of SEQ ID NOs: 5-30.

114. The polypeptide of claim 113, wherein the polypeptide is encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 144- 169.

115. The polypeptide of claim 108 or 109, wherein the at least one amino acid substitution consists of two amino acid substitutions.

116. The polypeptide of claim 115, wherein the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P-K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); NSUBSTITUTE SHEET (RULE 26)at residue 207 and L at residue 209 (L207N-I209L); R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); D at residue 207 and V at residue 209 (L207D- I209V); G at residue 187 and P at residue 212 (V187G-L212P); Q at residue 20 and I at residue 156 (T20Q-V156I); Q at residue 20 and P at residue 214 (T20Q-D214P); P at residue 120 and E at residue 226 (V120P-K226E); I at residue 156 and P at residue 214 (V156I-D214P); P at residue 120 and G at residue 187 (V120P-V187G); P at residue 212 and P at residue 214 (L212P-D214P); P at residue 214 and E at residue 226 (D214P- K226E); G at residue 187 and E at residue 226 (V187G-K226E); Q at residue 20 and P at residue 120 (T20Q-V120P); I at residue 156 and P at residue 212 (V156I-L212P); P at residue 120 and I at residue 156 (V120P-V156I); Q at residue 20 and P at residue 212 (T20Q-L212P); P at residue 120 and P at residue 214 (V120P-D214P); G at residue 187 and P at residue 214 (V187G-D214P); Q at residue 20 and G at residue 187 (T20Q-V187G); P at residue 120 and P at residue 212 (V120P-L212P); Q at residue 20 and E at residue 226 (T20Q-K226E); I at residue 156 and G at residue 187 (V156I-V187G); I at residue 156 and E at residue 226 (V156I-K226E); P at residue 212 and E at residue 226 (L212P-K226E); A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P-K226E); or R at residue 35 and K at residue 207 (Q35R-L207K).

117. The polypeptide of claim 116, wherein the two amino acid substitutions are selected from E at residue 19 and V at residue 145 (K19E-R145V); P at residue 212 and E at residue 226 (L212P-K226E); G at residue 9 and G at residue 86 (D9G-N86G); C at residue 207 and C at residue 209 (L207C-I209C); K at residue 128 and S at residue 169 (M128K-L169S); V at residue 207 and V at residue 209 (L207V-I209V); R at residue 35 and K at residue 207 (Q35R-L207K); P at residue 207 and F at residue 209 (L207P-I209F); S at residue 207 and R at residue 209 (L207S-I209R); C at residue 207 and S at residue 209 (L207C-I209S); N at residue 207 and L at residue 209 (L207N-I209L); R at residue 207 and Q at residue 209 (L207R-I209Q); H at residue 207 and R at residue 209 (L207H-I209R); D at residue 207 and W at residue 209 (L207D-I209W); or D at residue 207 and V at residue 209 (L207D- I209V).SUBSTITUTE SHEET (RULE 26)118. The polypeptide of claim 117, wherein the two amino acid substitutions are selected from A at residue 17 and Q at residue 20 (Q17A-T20Q); P at residue 212 and E at residue 226 (L212P-K226E); or R at residue 35 and K at residue 207 (Q35R-L207K).

119. The polypeptide of any one of claims 116-118, wherein the polypeptide comprises an amino acid sequence of SEQ ID NOs: 31-69.

120. The polypeptide of claim 119, wherein the polypeptide is encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 170- 208.

121. The polypeptide of claim 108 or 109, wherein the at least one amino acid substitution consists of three amino acid substitutions.

122. The polypeptide of claim 121, wherein the three amino acid substitutions are selected from G at residue 187, P at residue 214, E at residue 226 (V187G-D214P-K226E); Q at residue 20, P at residue 120, and G at residue 187 (T20Q-V120P-V187G); Q at residue 20, P at residue 120, and E at residue 226 (T20Q-V120P-K226E); Q at residue 20, Gat residue 187, and E at residue 226 (T20Q-V187G-K226E); P at residue 120, I at residue 156, and P at residue 214 (V120P-V156I-D214P); P at residue 212, P at residue 214, and E at residue 226 (L212P-D214P-K226E); P at residue 120, G at residue 187, and E at residue 226 (V120P-V187G-K226E); P at residue 120, P at residue 212, and P at residue 214 (V120P- L212P-D214P); I at residue 156, P at residue 212, and P at residue 214 (V156I-L212P- D214P); Q at residue 20, 1 at residue 156, and P at residue 212 (T20Q-V156I-L212P); I at residue 156, G at residue 187, and E at residue 226 (V156I-V187G-K226E); Q at residue 20, P at residue 214, and E at residue 226 (T20Q-D214P-K226E); P at residue 120, P at residue 214, and E at residue 226 (V120P-D214P-K226E); Q at residue 20, P at residue 120, and P at residue 214 (T20Q-V120P-D214P); Q at residue 20, P at residue 120, and I at residue 156 (T20Q-V120P-V156I); Q at residue 20, G at residue 187, and P at residue 214 (T20Q-V187G-D214P); P at residue 120, I at residue 156, and P at residue 212 (V120P-V156I-L212P); I at residue 156, P at residue 214, and E at residue 226 (V156I- D214P-K226E); G at residue 187, P at residue 212, and E at residue 226 (V187G-L212P- K226E); P at residue 120, G at residue 187, and P at residue 214 (V120P-V187G-D214P);SUBSTITUTE SHEET (RULE 26)Q at residue 20, 1 at residue 156, and G at residue 187 (T20Q-V156I-V187G); I at residue 156, G at residue 187, and P at residue 214 (V156I-V187G-D214P); Q at residue 20, and I at residue 156, E226 (T20Q-V156I-K226E); Q at residue 20, P at residue 212, and E at residue 226 (T20Q-L212P-K226E); Q at residue 20, P at residue 120, and P at residue 212 (T20Q-V120P-L212P); Q at residue 20, G at residue 187, and P at residue 212 (T20Q- V187G-L212P); P at residue 120, I at residue 156, and E at residue 226 (V120P-V156I- K226E); P at residue 120, I at residue 156, and G at residue 187 (V120P-V156I-V187G); P at residue 120, P at residue 212, and E at residue 226 (V120P-L212P-K226E); P at residue 120, G at residue 187, and P at residue 212 (V120P-V187G-L212P); G at residue 187, P at residue 212, and P at residue 214 (V187G-L212P-D214P); I at residue 156, G at residue 187, and P at residue 212 (V156I-V187G-L212P); Q at residue 20, 1 at residue 156, and P at residue 214 (T20Q-V156I-D214P); I at residue 156, P at residue 212, and E at residue 226 (V156I-L212P-K226E); Q at residue 20, P at residue 212, and P at residue 214 (T20Q- L212P-D214P); A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N- V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L-I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R-A168V).

123. The polypeptide of claim 122, wherein the three amino acid substitutions are selected from A at residue 21, N at residue 105, and A at residue 120 (F21A-K105N-V120A); D at residue 12, L at residue 21, and K at residue 209 (E12D-F21L-I209K); or R at residue 36, R at residue 75, and V at residue 168 (H36R-N75R-A168V).

124. The polypeptide of any one of claims 122-123 118, wherein the polypeptide comprises an amino acid sequence of SEQ ID NOs: 70-107.

125. The polypeptide of claim 124, wherein the polypeptide is encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NOs: 209- 246.

126. The polypeptide of claim 108 or 109, wherein the at least one amino acid substitution consists of four amino acid substitutions.

127. The polypeptide of claim 126, wherein the four amino acid substitutions are selected from Q at residue 20, P at residue 120, G at residue 187, and P at residue 212 (T20Q-V120P-SUBSTITUTE SHEET (RULE 26)V187G-L212P); Q at residue 20, P at residue 120, P at residue 212, and P at residue 214 (T20Q-V120P-L212P-D214P); Q at residue 20, 1 at residue 156, G at residue 187, and P at residue 214 (T20Q-V156I-V187G-D214P); Q at residue 20, P at residue 212, P at residue 214, and E at residue 226 (T20Q-L212P-D214P-K226E); P at residue 120, 1 at residue 156, G at residue 187, and P at residue 212 (V120P-V156I-V187G-L212P); I at residue 156, G at residue 187, P at residue 212, and E at residue 226 (V156I-V187G-L212P-K226E); P at residue 120, 1 at residue 156, P at residue 212, and E at residue 226 (V120P-V156I-L212P- K226E); Q at residue 20, G at residue 187, P at residue 212, and P at residue 214 (T20Q- V187G-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and E at residue 226 (V120P-V187G-L212P-K226E); Q at residue 20, 1 at residue 156, P at residue 212, and E at residue 226 (T20Q-V156I-L212P-K226E); G at residue 187, P at residue 212, P at residue 214, and E at residue 226 (V187G-L212P-D214P-K226E); Q at residue 20, P at residue 120, G at residue 187, and E at residue 226 (T20Q-V120P-V187G-K226E); Q at residue 20, P at residue 120, I at residue 156, and P at residue 212 (T20Q-V120P-V156I- L212P); Q at residue 20, P at residue 120, P at residue 214, and E at residue 226 (T20Q- V120P-D214P-K226E); Q at residue 20, 1 at residue 156, G at residue 187, and P at residue 212 (T20Q-V156I-V187G-L212P); I at residue 156, Gat residue 187, P at residue 212, and P at residue 214 (V156I-V187G-L212P-D214P); P at residue 120, I at residue 156, G at residue 187, and E at residue 226 (V120P-V156I-V187G-K226E); P at residue 120, I at residue 156, P at residue 212, and P at residue 214 (V120P-V156I-L212P-D214P); P at residue 120, G at residue 187, P at residue 212, and P at residue 214 (V120P-V187G- L212P-D214P); Q at residue 20, G at residue 187, P at residue 214, and E at residue 226 (T20Q-V187G-D214P-K226E); Q at residue 20, I at residue 156, P at residue 212, and P at residue 214 (T20Q-V156I-L212P-D214P); I at residue 156, P at residue 212, P at residue 214, and E at residue 226 (V156I-L212P-D214P-K226E); Q at residue 20, P at residue 120, G at residue 187, and P at residue 214 (T20Q-V120P-V187G-D214P); Q at residue 20, P at residue 120, I at residue 156, and G at residue 187 (T20Q-V120P-V156I-V187G); I at residue 156, G at residue 187, P at residue 214, and E at residue 226 (V156I-V187G- D214P-K226E); Q at residue 20, P at residue 120, I at residue 156, and E at residue 226 (T20Q-V120P-V156I-K226E); P at residue 120, 1 at residue 156, P at residue 214, and E at residue 226 (V120P-V156I-D214P-K226E); Q at residue 20, I at residue 156, G at residue 187, and E at residue 226 (T20Q-V156I-V187G-K226E); Q at residue 20, P atSUBSTITUTE SHEET (RULE 26)residue 120, P at residue 212, and E at residue 226 (T20Q-V120P-L212P-K226E); P at residue 120, G at residue 187, P at residue 214, and E at residue 226 (V120P-V187G- D214P-K226E); P at residue 120, P at residue 212, P at residue 214, and E at residue 226 (V120P-L212P-D214P-K226E); Q at residue 20, G at residue 187, P at residue 212, and E at residue 226 (T20Q-V187G-L212P-K226E); P at residue 120, I at residue 156, G at residue 187, and P at residue 214 (V120P-V156I-V187G-D214P); Q at residue 20, I at residue 156, P at residue 214, and E at residue 226 (T20Q-V156I-D214P-K226E); Q at residue 20, P at residue 120, I at residue 156, and P at residue 214 (T20Q-V120P-V156I- D214P); or A at residue 17, A at residue 20, P at residue 212, and E at residue 226 (Q17A- T20A-L212P-K226E).

128. The polypeptide of claim 127, wherein the four amino acid substitutions are A at residue 17, A at residue 20, P at residue 212, and E at residue 226 (Q17A-T20A-L212P-K226E).

129. The polypeptide of any one of claims 127-128, wherein the polypeptide comprises an amino acid sequence at of SEQ ID NO: 108-143.

130. The polypeptide of claim 129, wherein the polypeptide is encoded by a polynucleotide comprising a nucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 248- 282.

131. The polypeptide of any one of claims 91-130, wherein the polypeptide further comprises one or more additional portions of a dystrophin protein.

132. The polypeptide of claim 131, wherein the polypeptide further comprises:(i) at least one dystrophin protein spectrin-like repeat located downstream of the ABD 1 domain; and(ii) at least one cysteine-rich domain located downstream of the dystrophin protein spectrin-like repeat.

133. The polypeptide of claim 132, wherein the dystrophin protein spectrin-like repeat is a dystrophin protein spectrin-like repeat 1, a dystrophin protein spectrin-like repeat 2, a dystrophin protein spectrin-like repeat 3, a dystrophin protein spectrin-like repeat 4, a dystrophin protein spectrin-like repeat 5, a dystrophin protein spectrin-like repeat 6, aSUBSTITUTE SHEET (RULE 26)dystrophin protein spectrin-like repeat 7, a dystrophin protein spectrin-like repeat 8, a dystrophin protein spectrin-like repeat 9, a dystrophin protein spectrin-like repeat 10, a dystrophin protein spectrin-like repeat 11, a dystrophin protein spectrin-like repeat 12, a dystrophin protein spectrin-like repeat 13, a dystrophin protein spectrin-like repeat 14, a dystrophin protein spectrin-like repeat 15, a dystrophin protein spectrin-like repeat 16, a dystrophin protein spectrin-like repeat 17, a dystrophin protein spectrin-like repeat 18, a dystrophin protein spectrin-like repeat 19, a dystrophin protein spectrin-like repeat 20, a dystrophin protein spectrin-like repeat 21, a dystrophin protein spectrin-like repeat 22, a dystrophin protein spectrin-like repeat 23, or a dystrophin protein spectrin-like repeat 24.

134. The polypeptide of claim 133, wherein:(i) the spectrin-like repeat 1 comprises amino acids 339-447 of SEQ ID NO: 4;(ii) the spectrin-like repeat 2 comprises amino acids 448-556 of SEQ ID NO: 4;(iii) the spectrin-like repeat 3 comprises amino acids 559-667 of SEQ ID NO: 4;(iv) the spectrin-like repeat 4 comprises amino acids 719-828 of SEQ ID NO: 4;(v) the spectrin-like repeat 5 comprises amino acids 830-934 of SEQ ID NO: 4;(vi) the spectrin-like repeat 6 comprises amino acids 943-1045 of SEQ ID NO: 4;(vii) the spectrin-like repeat 7 comprises amino acids 1048-1154 of SEQ ID NO: 4;(viii) the spectrin-like repeat 8 comprises amino acids 1157-1263 of SEQ ID NO: 4;(ix) the spectrin-like repeat 9 comprises amino acids 1266-1367 of SEQ ID NO: 4;(x) the spectrin-like repeat 104 comprises amino acids 1368-1463 of SEQ ID NO: 4;(xi) the spectrin-like repeat 11 comprises amino acids 1468-1568 of SEQ ID NO: 4;(xii) the spectrin-like repeat 12 comprises amino acids 1571-1676 of SEQ ID NO: 4;(xiii) the spectrin-like repeat 13 comprises amino acids 1679-1778 of SEQ ID NO: 4;(xiv) the spectrin-like repeat 14 comprises amino acids 1779-1874 of SEQ ID NO: 4;(xv) the spectrin-like repeat 15 comprises amino acids 1877-1979 of SEQ ID NO: 4;(xvi) the spectrin-like repeat 16 comprises amino acids 1992-2101 of SEQ ID NO: 4;(xvii) the spectrin-like repeat 17 comprises amino acids 2104-2208 of SEQ ID NO: 4;(xviii) the spectrin-like repeat 18 comprises amino acids 2211-2318 of SEQ ID NO: 4;(xix) the spectrin-like repeat 19 comprises amino acids 2319-2423 of SEQ ID NO: 4;(xx) the spectrin-like repeat 20 comprises amino acids 2475-2577 of SEQ ID NO: 4;(xxi) the spectrin-like repeat 21 comprises amino acids 2580-2686 of SEQ ID NO: 4;(xxii) the spectrin-like repeat 22 comprises amino acids 2689-2802 of SEQ ID NO: 4;SUBSTITUTE SHEET (RULE 26)(xxiii) thespectrin-likerepeat23comprisesaminoacids2808-2930ofSEQIDNO:4; (xxiv) thespectrin-likerepeat24comprisesaminoacids2935-3040ofSEQIDNO:

4.

135. Thepolypeptideofanyoneofclaims132-134,whereinthecysteine-richdomaincomprises aminoacids3113-3360ofSEQIDNO:

4.

136. Thepolypeptideofanyoneofclaims132-135,whereinthepolypeptidefurthercomprises atleastonedystrophinproteinhingeregionlocateddownstreamoftheABDIandupstream ofthecysteine-richdomain.

137. Thepolypeptideofclaim 136,whereinthedystrophinproteinhingeregionisadystrophin proteinhingeregion 1, a dystrophin protein hinge region 2, a dystrophin protein hinge region3,oradystrophinproteinhingeregion4.

138. Thepolypeptideofclaim 137,wherein: (i) thehingeregion1 comprisesaminoacids247-338ofSEQIDNO:4; (ii) thehingeregion2comprisesaminoacids668-718ofSEQIDNO:4; (iii) thehingeregion3comprisesaminoacids2424-2474ofSEQIDNO:4; (iv) thehingeregion4comprisesaminoacids3041-3112ofSEQIDNO:

4.

139. Thepolypeptideofanyoneofclaims132-138,whereinthepolypeptidefurthercomprises adystrophinproteincarboxy-terminaldomain(CTD)locateddownstreamofthecysteine- richdomain.

140. The polynucleotide of claim 139, wherein the dystrophin protein carboxyl-terminus comprisesrminoacids3361-3685ofSEQIDNO:

4.

141. Thepolypeptideofanyoneofclaims137-138,whereinthepolypeptidecomprises: (i) theGoA-DysABD1; (ii) dystrophinproteinspectrin-likerepeats1,2,3,and24; (iii) dystrophinproteinhingeregions1,2,and4;and (iv) adystrophinproteincysteine-richdomain.

142. Thepolypeptideofclaim 141,whereinthepolypeptidefromamino-terminustocarboxyl- terminusconsistsof: (i) theGoA-DysABD1; SUBSTITUTE SHEET(RULE 26)(ii) a dystrophin protein hinge region 1;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 2;(v) a dystrophin protein spectrin-like repeat 3;(vi) a dystrophin protein hinge region 2;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4; and(ix) a dystrophin protein cysteine-rich domain.

143. The polypeptide of any one of claims 137-138, wherein the polypeptide comprises:(i) The GoA-DysABD1;(ii) dystrophin protein spectrin-like repeats 1, 2, 22, 23, and 24;(iii) dystrophin protein hinge regions 1, 3, and 4; and(iv) a dystrophin protein cysteine-rich domain.

144. The polypeptide of claim 143, wherein the polypeptide from amino-terminus to carboxylterminus consists of:(i) the Go A-Dy s ABD 1 ;(ii) a dystrophin protein hinge region 1;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 22;(v) a dystrophin protein spectrin-like repeat 23;(vi) a dystrophin protein spectrin-like repeat 24;(vii) a dystrophin protein hinge region 4; and(viii) a dystrophin protein cysteine-rich domain.

145. The polypeptide of any one of claims 137-138, wherein the polypeptide comprises:(i) the Go A-Dy s ABD 1 ;(ii) dystrophin protein spectrin-like repeats 1, 16, 17, 23, and 24;(iii) dystrophin protein hinge regions 1, and 4; and(iv) a dystrophin protein cysteine-rich domain.

146. The polypeptide of claim 145, wherein the polypeptide from amino-terminus to carboxylterminus consists of:SUBSTITUTE SHEET (RULE 26)(i) the Go A-Dy s ABD 1 ;(ii) a dystrophin protein hinge region 1;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 16;(v) a dystrophin protein spectrin-like repeat 17;(vi) a dystrophin protein spectrin-like repeat 23;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4; and(ix) a dystrophin protein cysteine-rich domain.

147. The polypeptide of any one of claims 139-140, wherein the polypeptide comprises:(i) the Go A-Dy s ABD 1 ;(ii) dystrophin protein spectrin-like repeats 1, 2, 3, and 24;(iii) dystrophin protein hinge regions 1, 3, and 4;(iv) a dystrophin protein cysteine-rich domain; and(v) a dystrophin protein carboxyl-terminus.

148. The polypeptide of claim 147, wherein the polypeptide from amino-terminus to carboxyl- terminus consists of:(i) the Go A-Dy s ABD 1 ;(ii) a dystrophin protein hinge region 1;(iii) a dystrophin protein spectrin-like repeat 1;(iv) a dystrophin protein spectrin-like repeat 2;(v) a dystrophin protein spectrin-like repeat 3;(vi) a dystrophin protein hinge region 3;(vii) a dystrophin protein spectrin-like repeat 24;(viii) a dystrophin protein hinge region 4;(ix) a dystrophin protein cysteine-rich domain; and(x) a dystrophin protein carboxyl-terminus.

149. The polypeptide of any one of claims 91-148, wherein the polypeptide is capable of linking a subsarcolemmal cytoskeleton with the extracellular matrix.SUBSTITUTE SHEET (RULE 26)150. The polypeptide of any one of claims 91-149, wherein the polypeptide is capable of recruiting a dystrophin-associated protein complex.

151. A dystrophin protein actin-binding domain consisting of the polypeptide of any one of claims 91-150.

152. An engineered dystrophin protein comprising the polypeptide of any one of claims 91-150 or the GoA-DysABD1 of claim 151.

153. A pharmaceutical formulation comprising the polynucleotide of any one of claims 1-60, the expression cassette of any one of claims 61-74, the vector of any one of claims 75-83, the rAAV particle of any one of claims 84-87, the polypeptide of any one of claims 88 or 91-150, the GoA-DysABD1 of any one of claims 89 or 151, the engineered dystrophin protein of any one of claims 90 or 152, or any combination thereof and at least one pharmaceutically acceptable excipient or adjuvant.

154. A method of increasing the amount of functional dystrophin protein in a cell comprising contacting the cell with the polynucleotide of any one of claims 1-60, the expression cassette of any one of claims 61-74, the vector of any one of claims 75-83, the rAAV particle of any one of claims 84-87, the polypeptide of any one of claims 88 or 91-150, the GoA-DysABD1 of any one of claims 89 or 151, the engineered dystrophin protein of any one of claims 90 or 152, the pharmaceutical formulation of claim 153, or any combination thereof.

155. The method of claim 154, wherein the cell is a muscle cell.

156. The method of any one of claims 154-155, wherein the cell is a human cell.

157. The method of any one of claims 154-156, wherein the cell is comprised in a subject.

158. The method of claim 157, wherein the subjects suffers from a muscular dystrophy.

159. The method of claim 158, wherein the muscular dystrophy is a dystrophin-deficient muscular dystrophy.

160. The method of any one of claims 158-159, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.SUBSTITUTE SHEET (RULE 26)161. The method of claims 154-160, wherein the amount of functional dystrophin protein in the cell is increased of at least 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to a same cell not administered the polynucleotide of any one of claims 1-60, the expression cassette of any one of claims 61- 74, the vector of any one of claims 75-83, the rAAV particle of any one of claims 84-87, the polypeptide of any one of claims 88 or 91-150, the GoA-DysABD1 of any one of claims 89 or 151, the engineered dystrophin protein of any one of claims 90 or 152, the pharmaceutical formulation of claim 153, or any combination thereof.

162. A method of treating a muscular dystrophy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the polynucleotide of any one of claims 1-60, the expression cassette of any one of claims 61-74, the vector of any one of claims 75-83, the rAAV particle of any one of claims 84-87, the polypeptide of any one of claims 88 or 91-150, the GoA-DysABD1 of any one of claims 89 or 151, the engineered dystrophin protein of any one of claims 90 or 152, the pharmaceutical formulation of claim 153, or any combination thereof.

163. The method of claim 162, wherein the muscular dystrophy is a dystrophin-deficient muscular dystrophy.

164. The method of any one of claims 162-163, wherein the muscular dystrophy is Duchenne muscular dystrophy or Becker muscular dystrophy.

165. The method of any one of claims 162-164, wherein a degeneration of a muscle of the subject is reduced of at least 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold.

166. The method of claim 165, wherein the muscle is a smooth muscle, a skeletal muscle, a cardiac muscle, or any combination thereof.

167. The method of any one of claims 162-166, wherein a symptom of the muscular dystrophy in the subject is reduced.

168. The method of any one of claims 162-167, wherein the muscular dystrophy in the subject is ameliorated.SUBSTITUTE SHEET (RULE 26)169. The method of any one of claims 162-168, wherein the muscular dystrophy in the subject is treated.

170. The method of any one of claims 162-169, wherein the subject is a human.SUBSTITUTE SHEET (RULE 26)