AAV particles comprising RGD-containing peptides for delivering dystrophin payloads

Abstract

The present disclosure pertains to AAV particles comprising: AAV capsids comprising a RGD- containing peptide, and a dystrophin payload. Also disclosed herein are methods of using the disclosed AAV particles for treating and / or preventing a muscular dystrophy, e.g., Duchenne muscular dystrophy.

Description

Attorney Docket No.: 2011256-2608 (P1879PCT01)AAV PARTICLES COMPRISING RGD-CONTAINING PEPTIDES FOR DELIVERING DYSTROPHIN PAYLOADSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 685,506, filed on August 21, 2024, the entire contents of which is hereby incorporated by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on August 18, 2025, is named 2011256-2608(WO), and is 2,081,635 bytes in size.BACKGROUND

[0003] Targeted delivery of payloads (e.g., using recombinant adeno-associated viruses) to muscle cells or tissues for treating and / or preventing muscular dystrophies such as Duchenne muscular dystrophy remains a challenge.SUMMARY

[0004] The present disclosure identifies certain challenges with existing delivery of dystrophin payloads in the treatment of Duchenne muscular dystrophy (DMD). Safe and efficient delivery of a dystrophin payload to muscle cells and / or tissue, e.g., skeletal muscle cells and / or muscle tissue, remains a major challenge in the treatment of DMD. For example, the present disclosure identifies that a lack of targeting moieties that can specifically deliver dystrophin payloads to muscle cell or tissue with decreased (e.g., minimal) non-specific delivery to other cells or tissues is a key challenge. Another challenge identified by the present disclosure is the poor potency associated with currently available delivery vectors, e.g., recombinant adeno- associated virus (rAAV) vectors, for delivering dystrophin payloads. In some embodiments, improving the potency of delivery vectors could be beneficial in obtaining clinically relevant outcomes and allow for the use of lower doses of delivery vectors to obtain a meaningful clinical output, e.g., a reduction in disease severity and / or improvement in one or more symptoms.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0005] Among other things, the present disclosure provides technologies that can address certain limitations identified in existing technologies for delivery of dystrophin payloads. The technologies provided herein are particularly useful for specifically delivering a dystrophin payload to a muscle cell or muscle tissue. In some embodiments, by specifically delivering a dystrophin payload to a muscle cell or muscle tissue, technologies provided here can also increase the potency of a dystrophin payload and / or allow for use of reduced doses of a delivery vector to achieve clinically relevant outcomes.

[0006] The present disclosure encompasses rAAV particles comprising a variant AAV capsid comprising a muscle-targeting moiety disclosed herein. In some embodiments, a muscletargeting moiety in a variant AAV capsid is also referred to as a “peptide insertion.” In some embodiments, a muscle-targeting moiety, e.g., in a variant AAV capsid, provides muscle-cell tropism. In some embodiments, rAAV particles comprising a variant capsid having a peptide insertion disclosed herein binds to and / or recognizes a target on a muscle cell. Also disclosed herein are compositions comprising rAAV particles disclosed herein, and uses of the same.

[0007] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises the sequence of RGDYERI (SEQ ID NO: 1551); and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprisesAttorney Docket No.: 2011256-2608 (P1879PCT01) the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0008] Also provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises the sequence of RGDYREI (SEQ ID NO: 1825); and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0009] This disclosure further provides is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises the sequence of RGDYREV (SEQ ID NO: 1829); and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ IDAttorney Docket No.: 2011256-2608 (P1879PCT01)NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0010] This disclosure provides, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDYERI (SEQ ID NO: 1551); and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at leastAttorney Docket No.: 2011256-2608 (P1879PCT01)80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0011] Also provided herein is, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDYREI (SEQ ID NO: 1825); and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0012] This disclosure further provides, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDYREV (SEQ ID NO: 1829); and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding aAttorney Docket No.: 2011256-2608 (P1879PCT01) dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0013] Disclosed herein is a recombinant adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of (I) RGDX1X2RX3 (SEQ ID NO: 1), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (II) RGDX3QX1X2 (SEQ ID NO: 2), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (III) RGDHX1X2 X3 (SEQ ID NO: 3), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (IV) RGDPX1X2X3 (SEQ ID NO: 4), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (V) RGDX3X1X2V (SEQ ID NO: 5), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (VI) RGDX1QX2 X3 (SEQ ID NO: 6), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (VII) RGDX3X1SX2 (SEQ ID NO: 7), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; or (VIII) LRGDX1X2X3 (SEQ ID NO: 8), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (IX) RGDX1GLX2 (SEQ ID NO: 9), wherein Xi is Y, W, or F, and X2is any amino acid; or (X) RGDYX1X2X3 (SEQ ID NO: 10), wherein Xi is E or R, X2is E or R, and X3is V or I; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. InAttorney Docket No.: 2011256-2608 (P1879PCT01) some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0014] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein Xi and X2 are independently any amino acid and X3 is Y, W, or F ; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments X3 is W or F. In some embodiments X3 is Y. In some embodiments X3 is W. In some embodiments X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at leastAttorney Docket No.: 2011256-2608 (P1879PCT01)80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0015] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein Xi and X2 are independently any amino acid and X3 is Y, W, or F ; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., of an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence.. In some embodiments X3 is W or F. In some embodiments X3 is Y. In some embodiments X3 is W. In some embodiments X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0016] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In someAttorney Docket No.: 2011256-2608 (P1879PCT01) embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0017] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO:Attorney Docket No.: 2011256-2608 (P1879PCT01)3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0018] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO:3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0019] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consist of theAttorney Docket No.: 2011256-2608 (P1879PCT01) sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0020] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000,Attorney Docket No.: 2011256-2608 (P1879PCT01)SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0021] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consist of the sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In someAttorney Docket No.: 2011256-2608 (P1879PCT01) embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0022] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043 . In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0023] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoterAttorney Docket No.: 2011256-2608 (P1879PCT01) sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO:3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0024] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of RGDX1QX2 X3 (SEQ ID NO: 6), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO:3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or aAttorney Docket No.: 2011256-2608 (P1879PCT01) sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0025] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDX1QX2 X3 (SEQ ID NO: 6), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0026] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence ofAttorney Docket No.: 2011256-2608 (P1879PCT01)RGDX3X1SX2 (SEQ ID NO: 7), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0027] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity toAttorney Docket No.: 2011256-2608 (P1879PCT01)SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO:3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0028] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO:3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at leastAttorney Docket No.: 2011256-2608 (P1879PCT01)80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0029] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0030] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein Xi is Y, W, or F, and X2 is any amino acid; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W orAttorney Docket No.: 2011256-2608 (P1879PCT01)F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0031] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein Xi is Y, W, or F, and X2 is any amino acid; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, X3 is W or F. In some embodiments, X3 is Y. In some embodiments, X3 is W. In some embodiments, X3 is F. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at leastAttorney Docket No.: 2011256-2608 (P1879PCT01)80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0032] Provided herein is a rAAV particle comprising: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein Xi is E or R, X2is E or R, and X3is V or I; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0033] In some embodiments, a peptide insertion comprises the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide insertion comprises the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, a peptide insertion comprises the sequence of RGDYREV (SEQ ID NO: 1829).Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0034] In some embodiments, a rAAV particle comprises: (a) a variant AAV capsid protein, wherein a variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion consists of the sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein Xi is E or R, X2 is E or R, and X3 is V or I; and (ii) the peptide insertion site is in a variable region (VR) of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0035] In some embodiments, a peptide insertion consists of the sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide insertion consists of the sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, a peptide insertion consists of the sequence of RGDYREV (SEQ ID NO: 1829).

[0036] This disclosure further provides a rAAV particle comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises a sequence provided in Table 7; and (ii) the peptide insertion site is in a variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and (b) a heterologous nucleic acid comprising: (I) a nucleotide sequence encoding a dystrophin payload and (II) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence havingAttorney Docket No.: 2011256-2608 (P1879PCT01) at least 80% identity to SEQ ID NO: 3001 , SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0037] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion site is located between two adjacent amino acids in the variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0038] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion site is located between two non-adjacent amino acids in a variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0039] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0040] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a peptide insertion is in VR-VIII of a parental AAV capsid protein (e.g., an AAV9 capsid protein). In some embodiments, a parental AAV capsid protein is chosen from an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV1 1, AAV12, AAV13 or AAVrh74 capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP 1, VP2, or VP3 of an AAV9 capsid protein or the corresponding positions in a capsid protein (e.g., VP1, VP2 or VP3) of another parental AAV capsid protein, e.g., an AAV1,Attorney Docket No.: 2011256-2608 (P1879PCT01)AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV1 1, AAV12, AAV13 or AAVrh74 capsid protein.

[0041] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of an AAV9 capsid protein.

[0042] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion of the heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0043] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion of the heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0044] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, an insertion site is located between amino acids 588 and 589 of VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding position in the capsid protein (e.g., VP1, VP2 or VP3) of another parental AAV capsid protein.

[0045] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising a consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2034, and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0046] In some embodiments of a rAAV particle disclosed herein or a variant AAV capsid protein disclosed herein, a variant AAV capsid protein comprises: (1) a peptide insertion comprising any one sequence provided in any one of Table 7, and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein.

[0047] This disclosure further provides a rAAV particle comprising: (a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises aAttorney Docket No.: 2011256-2608 (P1879PCT01) sequence provided in Table 7; and (ii) the peptide insertion site is in a variable region of a parental AAV capsid protein (e.g., an AAV9 capsid protein), and a dystrophin payload disclosed herein. In some embodiments, a dystrophin payload comprises a nucleic acid molecule, e.g., an antisense oligonucleotide. In some embodiments, a dystrophin payload comprises a nucleic acid molecule, e.g., a U7 snRNA. In some embodiments, a dystrophin payload comprises a geneediting system e.g., a CRISPR / Cas system. In some embodiments, a dystrophin payload comprises a CRISPR-Cas protein.

[0048] In some embodiments, disclosed herein is a targeting moiety, e.g., a muscletargeting moiety, conjugated to a dystrophin payload. In some embodiments, a targeting moiety, e g., a muscle-targeting moiety, comprises a peptide sequence provided in any one of Table 7. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, is part of (e.g., incorporated into) a vector, e.g., a viral vector or a non-viral vector. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, encapsidates a dystrophin payload, e.g., as described herein. In some embodiments, a targeting moiety, e.g., a muscle-targeting moiety, is tethered to a payload, e.g., as described herein. In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 1825. In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 1551. In some embodiments, a peptide insertion in a targeting moiety disclosed herein comprises a sequence of SEQ ID NO: 1829.

[0049] Also disclosed herein is an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein as disclosed herein.

[0050] Further disclosed herein is an isolated cell transduced with an rAAV particle disclosed herein. In some embodiments, a cell is a muscle cell. In some embodiments, a muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or any combination thereof. In some embodiments, a muscle cell is derived from an induced pluripotent stem cells (iPSC).

[0051] This disclosure further provides a pharmaceutical composition comprising: (a) a rAAV particle disclosed herein; and (b) a pharmaceutically acceptable excipient.

[0052] Also provided herein is a method of delivering a payload to a muscle cell, comprising administering a pharmaceutical composition disclosed herein to a muscle cell.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0053] In some embodiments, a muscle cell is in vitro.

[0054] In some embodiments, a muscle cell is in vivo.

[0055] In some embodiments, a muscle cell is from a subject that has, or has been determined to have, a muscle disorder.

[0056] In some embodiments, a muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or any combination thereof. In some embodiments, a muscle cell is derived from an induced pluripotent stem cells (iPSC).

[0057] This disclosure provides a method of delivering a dystrophin payload to a muscle cell, comprising administering a rAAV particle or a pharmaceutical composition comprising a rAAV particle disclosed herein to a muscle cell.

[0058] In some embodiments, a muscle cell is in vitro.

[0059] In some embodiments, a muscle cell is in vivo.

[0060] In some embodiments, a muscle cell is from a subject that has, or has been determined to have, a muscle disorder.

[0061] In some embodiments, a muscle disorder is Duchenne Muscular Dystrophy.

[0062] Also provided herein is a method of treating a subject having a muscle disorder and / or ameliorating a symptom of a muscle disorder in a subject, the method comprising administering to the subject a rAAV particle or a pharmaceutical composition comprising a rAAV particle disclosed herein.

[0063] In some embodiments, a muscle disorder is Duchenne Muscular Dystrophy.

[0064] In some embodiments, administration of a rAAV particle or a pharmaceutical composition comprising a rAAV particle delivers a nucleotide sequence encoding a dystrophin payload to a muscle cell.

[0065] In some embodiments, a muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e g., a muscle satellite cell), or any combination thereof.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0066] In some embodiments, administration of a rAAV particle or a pharmaceutical composition comprising a rAAV particle increases dystrophin polypeptide level and / or activity in a muscle tissue in the subject as compared to a muscle tissue in a control subject, wherein the control subject: (i) is the same subject prior to administration of the pharmaceutical composition; (ii) is a different subject who has not been administered the pharmaceutical composition; (iii) is the same or different subject who has been administered the pharmaceutical composition at a lower dose; or (iv) is a different subject who has been administered a pharmaceutical composition comprising a control AAV particle comprising the corresponding parental AAV capsid protein at the same dose.

[0067] In some embodiments, increase in dystrophin polypeptide level and / or activity is at least 1.5-fold.

[0068] In some embodiments, increase in dystrophin polypeptide level and / or activity is about 1.5-fold to about 50-fold.

[0069] In some embodiments, administration of a rAAV particle or a pharmaceutical composition comprising a rAAV particle increases dystrophin RNA level in a muscle tissue in the subject as compared to a muscle tissue in a control subject, wherein the control subject: (i) is the same subject prior to administration of the pharmaceutical composition; (ii) is a different subject who has not been administered the pharmaceutical composition; (iii) is the same or different subject who has been administered the pharmaceutical composition at a lower dose; or (iv) is a different subject who has been administered a pharmaceutical composition comprising a control AAV particle comprising the corresponding parental AAV capsid protein at the same dose.

[0070] In some embodiments, increase in dystrophin RNA level is at least 1.5-fold.

[0071] In some embodiments, increase in dystrophin RNA level is about 1.5-fold to about 500-fold.

[0072] In some embodiments, administration of a rAAV particle or a pharmaceutical composition comprising a rAAV particle results in: (i) increased muscle force, e.g., increased specific force generating capacity; (ii) increased muscle strength; (iii) reduced muscle fatigue and / or injury, (iv) increased muscle contractility; (v) increased localization of neuronal nitricAttorney Docket No.: 2011256-2608 (P1879PCT01) oxide synthase; (vi) reduced fibrosis; or (vii) any combination of (i)-(vi). In some embodiments, (i)-(vii) is compared to a control subject, wherein the control subject: (i) is the same subject prior to administration of the pharmaceutical composition; (ii) is a different subject who has not been administered the pharmaceutical composition; (iii) is the same or different subject who has been administered the pharmaceutical composition at a lower dose; or (iv) is a different subject who has been administered a pharmaceutical composition comprising a control AAV particle comprising the corresponding parental AAV capsid protein at the same dose.

[0073] Further provided herein is a method of increasing muscle force and / or muscle contractility in a subject, comprising administering to a subject a rAAV particle or a pharmaceutical composition comprising a rAAV particle, wherein the subject has or has been diagnosed with having Duchenne Muscular Dystrophy.

[0074] In some embodiments, a rAAV particle or a pharmaceutical composition comprising a rAAV particle is delivered in combination with one or more additional agents.

[0075] In some embodiments, an additional agent is a therapeutic agent.

[0076] In some embodiments, an additional agent is an agent that promotes exon skipping in a DMD gene.

[0077] In some embodiments, an additional agent comprises a gene editing agent, a U7 snRNA, an antisense oligonucleotide (AON), a morpholino, or a 2’-0 methyl oligonucleotide.

[0078] In some embodiments, an additional agent comprises Drisapersen, Vitolarsen, Eteplirsen, Golodirsen, Casimersen, or Suvodirsen.

[0079] In some embodiments, a pharmaceutical composition is administered via a route of administration chosen from: intramuscular, intravenous, intraarterial, intracoronary, intraparenchymal, subpial, subcutaneous, intradermal, intrathecal, intraperitoneal, intranasal, intraocular, intra-cistema magna, or limb perfusion.

[0080] In some embodiments, a subject is a human.

[0081] In some embodiments of a rAAV particle, a variant AAV capsid, a composition, or a method disclosed herein, a payload is a polypeptide that is encoded by a nucleic acid sequence within the rAAV particle. In some embodiments, a payload is a dystrophin payload.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0082] In some embodiments, a dystrophin payload is a dystrophin polypeptide. In some embodiments, a dystrophin payload is a micro-dystrophin (e g., a micro-dystrophin payload).

[0083] In some embodiments, a dystrophin payload comprises : (i) an N-terminal domain, (ii) one or more hinge domains, (iii) one or more spectrin-like repeats (SR), (iv) a cysteine-rich domain; (v) a C-terminal domain; or (vi) any combination of (i)-(v).

[0084] In some embodiments, an N-terminal domain comprises an actin binding domain.

[0085] In some embodiments, one or more hinge domains comprise a hinge domain 1(Hl), a hinge domain 2 (H2), a hinge domain 3 (H3), a hinge domain 4 (H4), or any combination thereof.

[0086] In some embodiments, one or more spectrin-like repeats (SRs) comprise: (a) an actin binding domain, (b) a neuronal nitric oxide synthase (nNOS) binding domain, (c) a microtubule binding domain, (d) any combination of (a)-(c). In some embodiments, a dystrophin payload comprises at least two, at least three, at least four, or at least five spectrin-like repeats (SR). In some embodiments, a spectrin-like repeats (SR) comprise: a spectrin-like repeat 1 (SRI), a spectrin-like repeat 2 (SR2), a spectrin-like repeat 3 (SR3), a spectrin-like repeat 4 (SR4), a spectrin-like repeat 5 (SR5), a spectrin-like repeat 6 (SR6), a spectrin-like repeat 7 (SR7), a spectrin-like repeat 8 (SR8), a spectrin-like repeat 9 (SR9), a spectrin-like repeat 10 (SR10), a spectrin-like repeat 1 1 (SRI 1), a spectrin-like repeat 12 (SR12), a spectrin-like repeat 13 (SR13), a spectrin-like repeat 14 (SR14), a spectrin-like repeat 15 (SR15), a spectrin-like repeat 16 (SR16), a spectrin-like repeat 17 (SR17), a spectrin-like repeat 18 (SR18), a spectrinlike repeat 19 (SR19), a spectrin-like repeat 20 (SR20), a spectrin-like repeat 21 (SR21), a spectrin-like repeat 22 (SR22), a spectrin-like repeat 23 (SR23), a spectrin-like repeat 24 (SR24), or any combination thereof.

[0087] In some embodiments, one or more spectrin-like repeats (SRs) comprise a rod domain. In some embodiments, one or more spectrin-like repeats (SRs) are separated by one or more hinge domains.

[0088] In some embodiments, a C-terminal domain comprises a syntrophin binding domain or a fragment or variant thereof, a dystrobrevin binding domain or a fragment or variant thereof, or both. In some embodiments, a syntrophin binding domain comprises an al -syntrophinAttorney Docket No.: 2011256-2608 (P1879PCT01) binding site, a [H-syntrophin binding site or both. In some embodiments, a dystrobrevin binding domain comprises an a-dystrobrevin binding site.

[0089] In some embodiments, a dystrophin payload comprises: (i) an N terminal domain, (ii) hinge domains Hl, H2 and H4, (iii) spectrin-like repeats SRI, SR2, SR3, and SR24, and (iv) a cysteine-rich domain.

[0090] In some embodiments, a dystrophin payload comprises in 5’ to 3’ order: an N terminal domain, a Hl domain, a spectrin-like repeat SRI, a spectrin-like repeat SR2, a spectrinlike repeat SR3, a H2 domain, a spectrin-like repeat SR24, a H4 domain and a cysteine-rich domain.

[0091] In some embodiments, a nucleotide sequence encoding the dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 5.

[0092] In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 13.

[0093] In some embodiments, dystrophin payload comprises: (i) an N terminal domain, (ii) hinge domains Hl, H3 and H4, (iii) spectrin-like repeats SRI, SR2, SR22, SR23, and SR24, and (iv) a cysteine-rich domain.

[0094] In some embodiments, a dystrophin payload comprises in 5’ to 3’ order: an N terminal domain, a Hl domain, a spectrin-like repeat SRI, a spectrin-like repeat SR2, a H3 domain, a spectrin-like repeat SR22, a spectrin-like repeat SR23, a spectrin-like repeat SR24, a H4 domain and a cysteine-rich domain.

[0095] In some embodiments, a nucleotide sequence encoding the dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 4.

[0096] In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 12.

[0097] In some embodiments, a nucleic acid further comprises a promoter.

[0098] In some embodiments, a promoter is or comprises a muscle specific promoter.

[0099] In some embodiments, a muscle-specific promoter comprises a Spc5-12,MHCK7, CK8, or MCK promoter.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0100] In some embodiments, a promoter comprises a MHCK7 promoter. In some embodiments, a MHCK7 promoter comprises the sequence of SEQ ID NO: 10.

[0101] In some embodiments, a promoter comprises a MCK promoter. In some embodiments, a MCK promoter comprises the sequence of SEQ ID NO: 9.

[0102] In some embodiments, a promoter comprises a Spc5-12 promoter. In some embodiments, a Spc5-12 promoter comprises the sequence of SEQ ID NO: 11.

[0103] In some embodiments, a promoter comprises a CK8 promoter. In some embodiments, a CK8 promoter comprises the sequence of SEQ ID NO: 8.

[0104] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a payload is an RNA molecule. In some embodiments, an RNA molecule is an siRNA, a miRNA, a gRNA, antisense RNA, circular RNA, a snRNA, or an aptamer.

[0105] In some embodiments, a dystrophin payload is a nucleic acid molecule. In some embodiments, a dystrophin payload is a heterologous nucleic acid with a therapeutic purpose, e.g., an miRNA, siRNA, shRNA, mRNA, snRNA, or CRISPR / Cas guide RNA, or a precursor thereof, which when administered to a cell, tissue or subject alters, e.g., increases, one or more activities and / or functions of a dystrophin protein. In some embodiments, a dystrophin payload is a heterologous nucleic acid that promotes exon-skipping in an RNA transcribed from a DMD gene. In some embodiments, a dystrophin payload promotes skipping of exon 53, exon 51, exon 45, exon 44 and / or exon 2. In some embodiments, a dystrophin payload is or comprises an antisense oligonucleotide (AON). In some embodiments, a dystrophin payload is or comprises a gene-editing system, e.g., a CRISPR / Cas system. In some embodiments, a dystrophin payload comprises a CRISPR-Cas protein. In some embodiments, a dystrophin payload is or comprises a nucleic acid sequence comprising a U7 small nuclear RNA (snRNA) sequence and an antisense oligonucleotide that can bind to a portion of an RNA transcript encoded by a DMD gene.

[0106] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a payload is a DNA molecule, e.g., a donor DNA molecule that is integrated into a host genome via homologous recombination. In some embodiments, a DNA molecule comprises a nucleic acid sequence of up to about 5,100 nt inAttorney Docket No.: 2011256-2608 (P1879PCT01) length, e.g., up to about 5,000 nt, up to about 4,900, up to about 4,800, up to about 4,700, up to about 4,600, up to about 4,500, up to about 4,400, etc. In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a nucleotide sequence encoding a payload comprises a promoter.

[0107] In some embodiments of a rAAV particle, a variant AAV capsid, a composition, or a method disclosed herein a variant AAV capsid protein confers increased infectivity and / or transduction of a muscle cell compared to the infectivity and / or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.

[0108] In some embodiments of a rAAV particle, a variant AAV capsid, a composition, or a method disclosed herein, a variant AAV capsid protein confers at least 5-fold, at least 10- fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, or at least 50-fold increased infectivity and / or transduction of a muscle cell compared to the infectivity and / or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.

[0109] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein confers about 5-fold, about 10-fold, about 15-fold, about 20 fold, about 25-fold, about 30-fold, about 40-fold, or about 50-fold increased infectivity and / or transduction of a muscle cell compared the infectivity and / or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.

[0110] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein confers about 5-fold to about 50-fold, about 5-fold to about 40-fold, about 5-fold to about 30 fold, about 5-fold to about 25-fold, about 5-fold to about 20-fold, about 5-fold to about 15-fold, about 5-fold to about 10- fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, 20-fold to about 50-fold, 25- fold to about 50-fold, 30-fold to about 50-fold, or 40-fold to about 50-fold increased infectivity and / or transduction of a muscle cell compared the infectivity and / or transduction of the muscle cell by a control AAV particle comprising the corresponding parental AAV capsid protein.

[0111] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a muscle cell is chosen from: a cardiac muscle cell, aAttorney Docket No.: 2011256-2608 (P1879PCT01) smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or combinations thereof.

[0112] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a peptide insertion site is located at or between amino acids 581 and 593 of VP1, VP2 or VP3 of AAV9 or the corresponding position in the capsid protein (e.g., VP1, VP2 or VP3) of another parental AAV capsid protein.

[0113] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein.

[0114] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence flanking the peptide insertion site. In some embodiments, one or more modifications are within about 10 amino acids upstream or downstream of the location of a peptide insertion site. In some embodiments, one or more modifications are within about 5 amino acids upstream or downstream of the location of a peptide insertion site.

[0115] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications are located in a variable region of parental AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, one or more modifications are located in an AAV9 capsid protein variable region, e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX, or any combination thereof.

[0116] In some embodiments, one or more modifications are located in: VR-VIII of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein, VR-IV of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein, or VR-V of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein, or any combination thereof.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0117] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a VR-IV comprises amino acids 451-475 of VP1, VP2 or VP3 of an AAV9 capsid protein.

[0118] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, VR-V comprises amino acids 488-506 of VP1, VP2 or VP3 of an AAV9 capsid protein.

[0119] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications comprises an insertion, deletion, mutation, or a combination thereof.

[0120] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region.

[0121] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications reduces glycan binding. In some embodiments, a glycan is galactose.

[0122] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP 1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP 1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f). In some embodiments, a parental AAV capsid protein is an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 orAttorney Docket No.: 2011256-2608 (P1879PCT01)AAVrh74capsid protein. In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein.

[0123] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, a variant capsid has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity relative to a parental AAV capsid protein. In some embodiments, percent identity is determined by comparing the sequence of the variant capsid without the peptide insertion, with a parental AAV capsid protein (e.g., an AAV9 capsid protein).

[0124] In some embodiments, a variant capsid protein and a parental AAV capsid protein have 100% identity when: (a) the peptide insertion in the variant capsid protein is not taken into account in the sequence comparison; and (b) the variant capsid protein does not have one or more modifications other than the peptide insertion.

[0125] In some embodiments, a variant capsid protein and a parental AAV capsid protein have less than 100% identity when: (a) the peptide insertion in the variant capsid protein is not taken into account in the sequence comparison; and (b) the variant capsid protein comprises one or more modifications other than the peptide insertion.

[0126] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001.

[0127] In some embodiments, a parental AAV capsid protein is an AAV1 capsid protein of SEQ ID NO: 2002.

[0128] In some embodiments, a parental AAV capsid protein is an AAV2 capsid protein of SEQ ID NO: 2003.

[0129] In some embodiments, a parental AAV capsid protein is an AAV3B capsid protein of SEQ ID NO: 2050.

[0130] In some embodiments, a parental AAV capsid protein is an AAV4 capsid protein of SEQ ID NO: 2051.

[0131] In some embodiments, a parental AAV capsid protein is an AAV5 capsid protein of SEQ ID NO: 2004.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0132] In some embodiments, a parental AAV capsid protein is an AAV6 capsid protein of SEQ ID NO: 2005.

[0133] In some embodiments, a parental AAV capsid protein is an AAV7 capsid protein of SEQ ID NO: 2052.

[0134] In some embodiments, a parental AAV capsid protein is an AAV8 capsid protein of SEQ ID NO: 2006.

[0135] In some embodiments, a parental AAV capsid protein is an AAV10 capsid protein of SEQ ID NO: 2053.

[0136] In some embodiments, a parental AAV capsid protein is an AAV11 capsid protein of SEQ ID NO: 2054.

[0137] In some embodiments, a parental AAV capsid protein is an AAV12 capsid protein of SEQ ID NO: 2055.

[0138] In some embodiments, a parental AAV capsid protein is an AAV13 capsid protein of SEQ ID NO: 2056.

[0139] In some embodiments, a parental AAV capsid protein is an AAVrh74 capsid protein of SEQ ID NO: 2057.

[0140] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description.BRIEF DESCRIPTION OF THE DRAWING

[0141] The patent or application file contains at least one drawing executed in color.Copies of this patent or patent application publication with color drawings(s) will be provided by the Office upon request and payment of the necessary fee.

[0142] The Figures described below, which together make up the Drawing, are for illustration purposes only, not for limitation.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0143] FTG. 1 provides a schematic of two exemplary micro-dystrophin constructs (Dys3579 [SEQ ID NO: 3002] and Dys3978 [SEQ ID NO: 3000]) utilized in the assessment of novel capsid variants RGDYREI (SEQ ID NO: 1825) and RGDYERI (SEQ ID NO: 1551), with the specified peptides inserted between AAV9 VP1 positions 588 and 589 in all viral protein subunits. Each of the micro-dystrophins was packaged with either AAV particles comprising a wild type AAV9 capsid or AAV particles comprising one of the novel capsid variants.

[0144] FIGS. 2A-2D show micro-dystrophin expression in 3D myobundles and myobundle contractile properties generated from two dystrophin-deficient lines. DMD_dup2 (FIGS. 2A-2B) and DMD_del52 (FIGS. 2C-2D) contain mutations that result in the complete absence of full-length dystrophin from the myoblasts. 3D myobundles were transduced with AAV particles comprising RGDYERI (SEQ ID NO: 1551) capsid variant packaged with the Dys3978 (SEQ ID NO: 3000) cargo. As shown in FIGS. 2A and 2C, samples were harvested and western blot was used to determine micro-dystrophin expression at different doses. FIG. 2A shows micro-dystrophin expression, as measured by western blot, in DMD_dup2 myobundles. FIG. 2B shows the results of the tetanic fatigue protocol on DMD_dup2 myobundles. FIG. 2C shows micro-dystrophin expression, as measured by western blot, in DMD_del52 myobundles. FIG. 2D shows the results of the tetanic fatigue protocol on DMD_del52 myobundles. As shown in FIGS. 2B and 2D, myobundles were tested, prior to harvest, with a tetanic fatigue protocol to assess force loss. Higher percentages indicate a protective effect. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0145] FIGS. 3A-3B show the reduction in force drop upon delivery of two microdystrophin cargos Dys3579 (SEQ ID NO: 3002) and Dys3978 (SEQ ID NO: 3000) in 3D DMD myobundles. Micro-dystrophins were delivered using AAV particles comprising RGDYERI (SEQ ID NO: 1551) capsid variant. FIG. 3A shows the reduction in force drop in DMD-dup2 myobundles. FIG. 3B shows the reduction in force drop in DMD-del52 myobundles. In both dystrophin-deficient lines (DMD_dup2, DMD_del52) the expression of micro-dystrophins conveys a reduction in force loss following a tetanic fatigue protocol. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0146] FIG. 4 shows the expression of micro-dystrophin following treatment of dystrophin-deficient 3D myobundles. Micro-dystrophin can be observed in transverse sectionsAttorney Docket No.: 2011256-2608 (P1879PCT01) from 3D myobundles treated with AAV particles comprising capsid variant RGDYERI (SEQ ID NO: 1551) packaged with either Dys3978 (SEQ ID NO: 3000) or Dys3579 (SEQ ID NO: 3002). At higher magnification, membrane localization can be observed. No micro-dystrophin signal is observed in untreated 3D myobundles. (Scale bars: 20x = 200pm; 63x = 20pm).

[0147] FIGS. 5A-5B compare the expression of micro-dystrophin in 3D myobundles following transduction by AAV particles comprising the capsid variants (RGDYREV [SEQ ID NO: 1829], RGDYERI [SEQ ID NO: 1551], and RGDYREI [SEQ ID NO: 1825]) to that of AAV particles comprising a wild type AAV9 capsid. FIG. 5A shows the Western blot performed on IMM_DMD del52 transduced with the various AAV particles. Myobundle Con4 is a positive control and untreated is a negative control. FIG. 5B shows the quantification of results from the Western blot analysis. These results indicate AAV particles comprising novel capsid variants achieved significantly higher expression of micro-dystrophin compared to AAV particles comprising a wild type AAV9 capsid.

[0148] FIGS. 6A-6B show the force drop comparison of micro-dystrophin in 3D myobundles following transduction by AAV particles comprising the novel capsid variants (RGDYREV [SEQ ID NO: 1829], RGDYERI [SEQ ID NO: 1551], and RGDYREI [SEQ ID NO: 1825]) to that of AAV particles comprising a wild type AAV9 capsid. FIG. 6A shows myobundles transduced with AAV particles comprising capsid variants packaged with Dys3579 cargo (SEQ ID NO: 3002) achieved reduced force drop compared to the untreated group and AAV9 group at the equivalent MOI . FIG. 6B shows myobundles transduced with AAV particles comprising capsid variants packaged with Dys3579 cargo (SEQ ID NO: 3002) achieved improved resistance to fatiguability compared to the untreated group and AAV9 group at the equivalent MOI. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0149] FIGS. 7A-7B show force frequency curves generated from plantar flexion of mouse lower limbs 12 weeks after a single treatment of micro-dystrophins delivered with either AAV particles comprising a wild type AAV9 capsid (1E13 vg / kg and 1E14 vg / kg) or AAV particles comprising the novel capsid variant RGDYERI (SEQ ID NO: 1551) (1E13 vg / kg). FIG. 7A shows force frequency results after treatment with AAV particles packaged with Dys3579 (SEQ ID NO: 3002). FIG. 7B shows force frequency results after treatment with AAV particles packaged with Dys3978 (SEQ ID NO: 3000). The RGDYERI treatment group producesAttorney Docket No.: 2011256-2608 (P1879PCT01) comparable traces to the higher dose treatment group of AAV9. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0150] FIGS. 8A-8B show the change in serum creatine kinase (CK) levels of dystrophin-deficient Dmdmdxmice following treatment with micro-dystrophins delivered with either AAV particles comprising a wild type AAV9 capsid (1E13 vg / kg and 1E14 vg / kg) or AAV particles comprising the novel capsid variant RGDYERI (SEQ ID NO: 1551) (1E13 vg / kg). FIG. 8A shows baseline serum CK levels. FIG. 8B shows serum CK levels 4 weeks after treatment with AAV particles comprising the specified capsid variants, AAV particles comprising a wild type AAV9 capsid, or the mdx control. Serum CK is a marker of muscle damage and is indicative of a leaky sarcolemma.

[0151] FIGS. 9A-9B show the in vivo eccentric force loss in Dmdmdxm\ce treated with vehicle (control) or AAV particles having the specified capsids and packaged with exemplary micro-dystrophin cargos Dys3579 (SEQ ID NO: 3002) or Dys3978 (SEQ ID NO: 3000) (16 weeks post-treatment). Force is expressed as the percentage of the force measured after the first contraction. Mice treated with 1E13 vg / kg AAV particles comprising the novel RGDYERI (SEQ ID NO: 1551) capsid variant carrying either micro-dystrophin cargos (Dys3579 [SEQ ID NO: 3002] or Dys3978 [SEQ ID NO: 3000]) showed enhanced resistance to eccentric contraction- induced damage compared to untreated controls, similar to those treated with high dose (lE14vg / kg) AAV particles comprising a wild type AAV9 capsid. FIG. 9A shows results for AAV particles carrying Dys3579 (SEQ ID NO: 3002). FIG. 9B shows results for AAV particles carrying Dys3978 (SEQ ID NO: 3000). Statistical analysis is shown comparing AAV treated / N?c / "'",‘ mice groups to vehicle-treated mdx controls. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0152] FIGS. 10A-10B show mRNA and protein expression of micro-dystrophin Dys3579 and Dys3978 in the gastrocnemius muscle of L)md"'kmice 16 weeks post-treatment with AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002) or Dys3978 (SEQ ID NO: 3000). FIG. 10A shows results for AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002). FIG. 10B shows results for AAV particles having the specified capsids carrying Dys3978 (SEQ ID NO: 3000). For mRNA expression, micro-dystrophin expression was normalized to Gapdh before further normalization to theAttorney Docket No.: 2011256-2608 (P1879PCT01)AAV9 1E13 vg / kg dose group (animals administered AAV particles having a wildtypes AAV9 capsid). For protein expression, micro-dystrophin expression was normalized to alpha-actinin. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0153] FIGS. 11A-11B show protein expression of micro-dystrophin Dys3579 (SEQ ID NO: 3003) and Dys3978 (SEQ ID NO: 3001) quantified via western blot in the tibialis anterior muscle of Dmd”^ mice 16 weeks post-treatment with AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002) or Dys3978 (SEQ ID NO: 3000). Micro-dystrophin expression was normalized to alpha-actinin. FIG. 11A shows results for AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002). FIG. 11B shows results for AAV particles having the specified capsids carrying Dys3978 (SEQ ID NO: 3000). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0154] FIGS. 12A-12B show protein expression of micro-dystrophin Dys3579 (SEQ ID NO: 3003) and Dys3978 (SEQ ID NO: 3001) quantified via western blot in the diaphragm of DmdmdxVMCQ 16 weeks post-treatment with AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002) or Dys3978 (SEQ ID NO: 3000). Micro-dystrophin expression was normalized to alpha-actinin. FIG. 12A shows results for AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002). FIG. 12B shows results for AAV particles having the specified capsids carrying Dys3978 (SEQ ID NO: 3000). *p<0.05, **p<0.01, * * *p<0.001 , * * * *p<0.0001 .

[0155] FIGS. 13A-13B show protein expression of micro-dystrophin Dys3579 (SEQ ID NO: 3003) and Dys3978 (SEQ ID NO: 3001) quantified via western blot in the heart of Dmdndxmice 16 weeks post-treatment with AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002) or Dys3978 (SEQ ID NO: 3000). Micro-dystrophin expression was normalized to alpha-actinin. FIG. 13A shows results for AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002). FIG. 13B shows results for AAV particles having the specified capsids carrying Dys3978 (SEQ ID NO: 3000). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0156] FIGS. 14A-14B show the lack of micro-dystrophin protein expression (Dys3579 [SEQ ID NO: 3003]; Dys3978 [SEQ ID NO: 3001]) in the liver of L)md!,dxmice 16 weeks posttreatment with AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO:Attorney Docket No.: 2011256-2608 (P1879PCT01)3002) or Dys3978 (SEQ ID NO: 3000). FIG. 14A shows results for AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002). FIG. 14B shows results for AAV particles having the specified capsids carrying Dys3978 (SEQ ID NO: 3000).

[0157] FIGS. 15A-15B show LC / MS-MS quantification of micro-dystrophin proteins in the gastrocnemius muscle of l)mdr, / 'Jxmice 16 weeks post-treatment with AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002) or Dys3978 (SEQ ID NO: 3000). One-way ANOVA was performed on the treated I)md'“Jxmice, comparing expression in those samples to that of untreated mdx control. FIG. 15A shows results in atmol per micrograms protein. FIG. 15B shows results normalized to the wildtype control (C57BL / 6). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0158] FIGS. 16A-16B show the response to in vivo eccentric contractions in wild type (WT) controls and Dmd"^ mice treated with vehicle or AAV particles having the specified capsids and packaged with Dys3579 (SEQ ID NO: 3002) at 6 weeks post-treatment. Force is expressed as the percentage of the force measured after the first eccentric contraction. Mice treated with a lower dose of AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) or RGDYREV (SEQ ID NO: 1829) and packaged with Dys3579 (SEQ ID NO: 3002) showed increased resistance to eccentric force loss compared to vehicle-treated Dmd™1* mice, and similar to those treated with a high dose (lE14vg / kg) of AAV particles comprising a wild type AAV9 capsid. High-dose AAV particles comprising a wild type AAV9 capsid and lower- dose AAV particles comprising capsid variants RGDYERI or RGDYREV treated groups showed partial rescue of eccentric force loss, approaching that of wild-type mice. FIG. 16A shows results for AAV particles comprising capsid variant RGDYREV. FIG. 16B shows results for AAV particles comprising capsid variant RGDYERI. Statistical analysis is shown comparing AAV treated l)mdn ldxmice groups to vehicle-treated mdx controls. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0159] FIGS. 17A-17E show that lower doses (5E12vg / kg and 1E13 vg / kg) of AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) or RGDYREV (SEQ ID NO: 1829) mediated similar or enhanced micro-dystrophin mRNA expression compared to high dose (lE14vg / kg) of AAV particles comprising a wild type AAV9 capsid in skeletal muscle tissues (gastrocnemius, tibialis anterior, triceps and diaphragm). Lower micro-dystrophin mRNAAttorney Docket No.: 2011256-2608 (P1879PCT01) levels were observed in the heart of DmcT^ mice treated with AAV particles comprising capsid variants RGDYERI or RGDYREV at both doses compared to those treated with high-dose AAV particles comprising a wild type AAV9 capsid. FIG. 17A shows micro-dystrophin mRNA levels observed in the gastrocnemius. FIG. 17B shows micro-dystrophin mRNA levels observed in the tibialis anterior. FIG. 17C shows micro-dystrophin mRNA levels observed in the diaphragm. FIG. 17D shows micro-dystrophin mRNA levels observed in the triceps. FIG. 17E shows micro-dystrophin mRNA levels observed in the heart. Samples were normalized to Gapdh and compared to high dose (1E14 vg / kg) AAV9 expression. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0160] FIGS. 18A-18C shows protein expression of micro-dystrophin Dys3579 (SEQ ID NO: 3003) quantified via Jess automated western blot in the gastrocnemius of Dmd ^ mice 6 weeks post-treatment with AAV particles having the specified capsids carrying Dys3579 (SEQ ID NO: 3002) at the indicated doses. FIG. 18A shows results from plate 1 ( replicate 1). FIG. 18B shows results from plate 2 ( replicate 2). FIG. 18C shows a quantification of results from plate 1 and plate 2. Micro-dystrophin expression was normalized to alpha-actinin before being compared to the AAV9 1E14 vg / kg dose group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0161] FIGS. 19A-19C shows protein expression of micro-dystrophin Dys3579 (SEQ ID NO: 3003) quantified via Jess automated western blot in the heart of DmcTdxmice 6 weeks posttreatment with AAV particles having the specified capsids packaged with Dys3579 (SEQ ID NO: 3002) at the indicated doses. Micro-dystrophin expression was normalized to alpha-actinin before being compared to the AAV9 1E14 vg / kg dose group. FIG. 19A shows results from plate 1 (replicate 1). FIG. 19B shows results from plate 2 ( replicate 2). FIG. 19C shows a quantification of results from plate 1 and plate 2. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0162] FIGS. 20A-20C shows protein expression of micro-dystrophin Dys3579 (SEQ ID NO: 3003) quantified via Jess automated western blot in the diaphragm of DmcF”dxmice 6 weeks post-treatment with AAV particles having the specified capsids packaged with Dys3579 (SEQ ID NO: 3002) at the indicated doses. FIG. 20A shows results from plate 1 (replicate 1). FIG. 20B shows results from plate 2 (replicate 2). FIG. 20C shows a quantification of results fromAttorney Docket No.: 2011256-2608 (P1879PCT01) plate 1 and plate 2. Micro-dystrophin expression was normalized to alpha-actinin before being compared to the AAV9 1E14 vg / kg dose group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0163] FIGS. 21A-21B shows protein expression of micro-dystrophin Dys3579 (SEQ ID NO: 3003) quantified via Jess automated western blot in the tibialis anterior ofmice 6 weeks post -treatment with AAV particles having the specified capsids packaged with Dys3579 (SEQ ID NO: 3002) at the indicated doses. FIG. 21A shows results from the capillary-based protein analysis. FIG. 21B shows a quantification of micro-dystrophin Dys3579 expression in tibialis anterior muscle based on the data in FIG. 21A. Micro-dystrophin expression was normalized to alpha-actinin before being compared to the AAV9 1E14 vg / kg dose group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0164] FIGS. 22A-22H show the quantification of vector genomes per diploid genome (vg / dg) in / )fl?t / "'lAmice treated with AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) or RGDYREV (SEQ ID NO: 1829) at two doses (1E13 vg / kg and 5E12 vg / kg) or AAV particles comprising a wild type AAV9 capsid (1E14 vg / kg) packaged with a MHCK-7- microdystrophin (Dys3579 [SEQ ID NO: 3002]) cargo 6 weeks post-treatment in indicated tissues. Micro-dystrophin copy numbers were normalized to Rpp30 and quantified using a standard curve of both. FIG. 22A shows a comparison of vd / dg between all treatment groups in gastrocnemius. FIG. 22B shows a comparison of vd / dg between all treatment groups in heart. FIG. 22C shows a comparison of vd / dg between all treatment groups in liver. FIG. 22D shows results from the AAV9 (1E14 vg / kg) treatment group. FIG. 22E shows results from the RGDYERI (5E12 vg / kg) treatment group. FIG. 22F shows results from the RGDYERI (1E13 vg / kg) treatment group. FIG. 22G shows results from the RGDYREV (5E12 vg / kg) treatment group. FIG. 22H shows results from the RGDYREV (1E13 vg / kg) treatment group. Y-axis denotes vector genome copy number per diploid genome (vg / dg). **p<0.01, ****p<0.0001.

[0165] FIG. 23 shows JESS Western blot quantification of micro-dystrophin protein expression in 2D DMD_del52 myotubes seven days post-transduction with AAV particles delivering cargo constructs encoding micro-dystrophin (Dys3579 [SEQ ID NO: 3003]). Several cargo constructs encoding micro-dystrophin (Dys3579), including codon-optimized cargos, were delivered by AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551), RGDYREI (SEQ ID NO: 1825), or RGDYREV (SEQ ID NO: 1829). The codon-optimizedAttorney Docket No.: 2011256-2608 (P1879PCT01) constructs used were: Dysl 329 (SEQ ID NO: 3043), Dysl 33O (SEQ ID NO: 3038), Dysl 331 (SEQ ID NO: 3035), Dysl332 (SEQ ID NO: 3036), Dysl334 (SEQ ID NO: 3040), Dysl335 (SEQ ID NO: 3037), and Dysl336 (SEQ ID NO: 3041). Three additional constructs were used: Dys3579 (corresponding to SEQ ID NO: 3002), Dysl308 ([SEQ ID NO: 3042], all CpGs removed) and Dysl333 ([SEQ ID NO: 3039], corresponding to naturally occurring sequence encoding micro-dystrophin Dys3579 [SEQ ID NO: 3003]).

[0166] FIG. 24 demonstrates micro-dystrophin expression in DMD_del52 3D myobundles seven days post-transduction or control. Quantification was carried out using the JESS automated Western blot system. All micro-dystrophin cargo constructs (Dys3579 [SEQ ID NO: 3002] or codon -optimized Dysl331 [SEQ ID NO: 3035], Dysl332 [SEQ ID NO: 3036] and Dysl335 [SEQ ID NO: 3037]) were delivered by AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) (representative blot shown on the left) or RGDYREI (SEQ ID NO: 1825). For protein levels, micro-dystrophin expression was first normalized to alpha-actinin and then to Dys3579 delivered with the same AAV particles. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0167] FIG. 25 shows confocal immunofluorescence images of 2D iPSC cardiomyocytes from a DMD patient. Cells were stained using an anti -Dystrophin antibody (DYSB) and counterstained with DAPI to identify nuclei. Micro-dystrophin cargo constructs (Dys3579 [SEQ ID NO: 3002] or codon-optimized Dysl 332 [SEQ ID NO: 3036] and Dysl 335 [SEQ ID NO: 3037]) were delivered to iPSC cardiomyocytes by AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) or RGDYREI (SEQ ID NO: 1825).

[0168] FIGS. 26A-26B show micro-dystrophin protein expression in cardiomyocytes. Quantification was performed using the JESS automated Western blot system, with a representative blot shown on the left. For protein levels, micro-dystrophin expression was first normalized to alpha-actinin and then to Dys3579 delivered with the same AAV particles. Microdystrophin cargo constructs (Dys3579 [SEQ ID NO: 3002] or codon-optimized Dysl332 [SEQ ID NO: 3036] and Dysl335 [SEQ ID NO: 3037]) were delivered to cardiomyocytes by AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) or RGDYREI (SEQ ID NO: 1825). FIG. 26A shows micro-dystrophin protein expression in 2D DMD patient derived iPSC cardiomyocytes (CM) containing the del52 mutation. FIG. 26B shows micro-dystrophin proteinAttorney Docket No.: 2011256-2608 (P1879PCT01) expression in 2D DMD iPSC cardiomyocytes containing the de!52 mutation generated using CRISPR from a healthy isogenic control line. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0169] FIGS. 27A-27B show the effect of delivering micro-dystrophin cargo constructs on creatine kinase (CK) release. Micro-dystrophin cargo constructs were delivered by AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) or RGDYREI (SEQ ID NO: 1825). FIG. 27A shows results from DMD patient-derived iPSC cardiomyocytes. FIG. 27B shows results from DMD iPSC cardiomyocytes containing the del52 mutation generated using CRISPR from a healthy isogenic control line. CK release in both FIG. 27A and FIG. 27B was compared to untreated cardiomyocytes. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0170] FIG. 28 shows the effects of delivering micro-dystrophin cargo constructs (Dys3579 [SEQ ID NO: 3002], Dys3810 [SEQ ID NO: 3004], Dys3978 [SEQ ID NO: 3000], and Dys4017 [SEQ ID NO: 3006]) on resistance to eccentric contraction (ECC)-induced damage 15 weeks post-treatment. Micro-dystrophin cargos (top panel) were delivered with AAV9 particles at 1E14 vg / kg, AAVrh74 particles at 1E14 vg / kg, or AAV particles comprising capsid variant RGDYERI (SEQ ID NO: 1551) at 1E13 vg / kg.. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0171] FIGS. 29A-29B show micro-dystrophin protein expression. The data shows a comparison of micro-dystrophin protein levels when the same micro-dystrophin cargo constructs described in FIG. 28 were delivered by: (a) AAV particles comprising capsid variant RGDYERI (SEQ ID NO: 1551) at a 1E13 vg / kg dose, (b) AAV9 at a 1E14 vg / kg dose, or (c) AAVrh74 at a 1E14 vg / kg dose. LC / MS-MS quantification was performed on gastrocnemius (FIG. 29A) and heart (FIG. 29B) tissues.

[0172] FIGS. 30A-30F show force frequency response and resistance to eccentric contraction (ECC) damage in mdx mice following treatment with codon-optimized microdystrophin cargo constructs. Force frequency analysis was performed 6 weeks post-treatment. Codon-optimized micro-dystrophin cargo constructs Dysl332 (SEQ ID NO: 3036) and Dysl335 (SEQ ID NO: 3037) were delivered with AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) (FIG. 30B) or RGDYREI (FIG. 30C) at a 2E13 vg / kg dose. The force frequency in these groups was compared to the force frequency in mice receiving the microdystrophin cargo construct Dys3579 (SEQ ID NO: 3002) with: AAV particles comprising capsidAttorney Docket No.: 2011256-2608 (P1879PCT01) variants RGDYERI (SEQ ID NO: 1551 ) or RGDYREI (SEQ ID NO: 1825) at 2E13vg / kg, or with AAVrh74 particles at a 2E13 vg / kg or a 1E14 vg / kg dose. FIG. 30D shows results of the eccentric contraction (ECC) injury assay performed 7 weeks post-treatment. Codon-optimized micro-dystrophin cargo constructs Dysl332 (SEQ ID NO: 3036) and Dysl335 (SEQ ID NO: 3037) were delivered with AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) (FIG. 30E) and RGDYREI (FIG. 30F) at a 2E13 vg / kg dose. The results from these groups was compared to micro-dystrophin cargo construct Dys3579 (SEQ ID NO: 3002) delivered with: AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) and RGDYREI (SEQ ID NO: 1825) at 2E 13 vg / kg, or AAVrh74 particles at a dose of 2E13 vg / kg or at a dose oflE14 vg / kg. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0173] FIG. 31 shows results of statistical tests comparing eccentric contraction (ECC) damage-induced force in wildtype (wt) mice, or mdx mice that were untreated or treated with AAV particles comprising capsid variants RGDYERI (SEQ ID NO: 1551) or RGDYREI (SEQ ID NO: 1825), or AAVrh74 delivering the specified micro-dystrophin cargos. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

[0174] FIG. 32 shows the changes in serum creatine kinase (CK) levels in mdx mice following treatment with micro-dystrophin cargo constructs: Dys3579 (SEQ ID NO: 3002), or codon-optimized Dysl332 (SEQ ID NO: 3036) and Dysl335 (SEQ ID NO: 3037). The microdystrophin cargo constructs were delivered by: AAVrh74 particles at a dose of at 2E13 vg / kg or 1E14 vg / kg, AAV particles comprising capsid variant RGDYERI (SEQ ID NO: 1551) at a dose of 2E13 vg / kg, or AAV particles comprising capsid variant RGDYREI (SEQ ID NO: 1825) at a dose of 2E13 vg / kg. Tissues were collected and analyzed 8 weeks post-treatment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0175] FIGS. 33A-33C shows JESS western blot images of lysates from mdx mice treated with micro-dystrophin cargo constructs: Dys3579 (SEQ ID NO: 3002), or codon- optimized Dysl332 (SEQ ID NO: 3036) and Dysl335 (SEQ ID NO: 3037). The microdystrophin cargo constructs were delivered by: AAVrh74 particles at a dose of at 2E13 vg / kg or 1E14 vg / kg, AAV particles comprising capsid variant RGDYERI (SEQ ID NO: 1551) at a dose of 2E13 vg / kg, or AAV particles comprising capsid variant RGDYREI (SEQ ID NO: 1825) at a dose of 2E13 vg / kg. FIG. 33A shows western blot images of gastrocnemius muscle lysates. FIG.Attorney Docket No.: 2011256-2608 (P1879PCT01)33B shows JESS western blot images of heart muscle lysates. FIG. 33C shows JESS western blot images of diaphragm muscle lysates.

[0176] FIGS. 34A-34G demonstrate improved micro-dystrophin expression in tissue samples from mdx mice treated with micro-dystrophin cargo constructs: Dys3579 (SEQ ID NO: 3002), or codon-optimized Dysl332 (SEQ ID NO: 3036) and Dysl335 (SEQ ID NO: 3037). Micro-dystrophin cargo constructs were delivered by: AAVrh74 particles at a dose of 2E13 vg / kg or at a dose of 1E14 vg / kg, AAV particles comprising capsid variant RGDYERI (SEQ ID NO: 1551) at a dose of 2E13 vg / kg, or AAV particles comprising capsid variant RGDYREI (SEQ ID NO: 1825) at dose of 2E13 vg / kg. For protein levels, micro-dystrophin expression was first normalized to alpha-actinin and then to AAVrh74 delivering Dys3579 at dose of 1E14 vg / kg. Micro-dystrophin expression is shown for the following tissues: gastrocnemius (FIG.34A), heart (FIG. 34B), diaphragm (FIG. 34C), tibialis anterior (FIG. 34D), triceps (FIG. 34E), soleus (FIG. 34F), and quadricep (FIG. 34G). *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.

[0177] FIGS. 35A-35D show quantification of viral vector genomes per diploid genome (vg / dg) in heart (FIG. 35A), diaphragm (FIG. 35B), gastrocnemius (FIG. 35C), and liver (FIG. 35D) tissues. Samples are from mdx mice treated with micro-dystrophin cargo constructs: Dys3579 (SEQ ID NO: 3002), or codon-optimized Dysl332 (SEQ ID NO: 3036) and Dysl335 (SEQ ID NO: 3037). Micro-dystrophin cargo constructs were delivered by: AAVrh74 particles at a dose of 2E13 vg / kg or a dose of 1 El 4 vg / kg, AAV particles comprising capsid variant RGDYERI (SEQ ID NO: 1551) at a dose of 2E13 vg / kg, or AAV particles comprising capsid variant RGDYREI (SEQ ID NO: 1825) at a dose of 2E13 vg / kg. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001.DEFINITIONS

[0178] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” may be understood to permit standardAttorney Docket No.: 2011256-2608 (P1879PCT01) variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0179] 5’ and 3’ The terms “5”’ and “3”’ are relative terms to define a spatial relationship or directionality between two or more segment of a nucleic acid sequence. Thus, 3’ of a nucleic acid indicates a segment of the nucleic acid that is downstream of another segment, while 5’ indicates a segment of the nucleic acid that is upstream of another segment. For example, 3’ may indicate that a segment is in the 3’ half of the nucleic acid sequence or even at the 3’ end of the nucleic acid sequence. Similarly, 5’ may indicate that a segment is in the 5’ half of the nucleic acid sequence or even at the 5’ end of the nucleic acid sequence. Unless indicated otherwise, the directionality of a nucleic acid will be in the 5’ to 3’ direction of translation.

[0180] About or approximately. As used herein, the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0181] Adeno-associated virus (AA V): As used herein, the terms “Adeno-associated virus” and “AAV” refer to viral particles, in whole or in part, of the family Parvoviridae and the genus Dependoparvovirus . AAV is a small replication-defective, nonenveloped virus. AAV includes, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotypes 4, AAV serotypes 5, AAV serotypes 6, AAV serotypes 7, AAV serotypes 8, AAV serotypes 9, AAV serotypes 10, AAV serotypes 11, AAV serotypes 12, AAV serotype 13, AAVrh74, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, non-human primate AAV, e.g., from rhesus monkeys, and any variant of any of the foregoing. Wild-type AAV is replication deficient and requires coinfection of cells by a helper virus, e.g., adenovirus, herpes, or vaccinia virus, e.g., an Ad2 or Ad5 virus, or supplementation of helper viral genes, in order to replicate.

[0182] Ad2 helper: As used herein, the term “Ad2 helper” refers to the Adenovirus serotype 2 (Ad2) helper virus (e.g., wildtype or recombinantly engineered Ad2 helper virus) and various Ad2 helper genes and / or Ad2 helper polypeptides, including, but not limited, to El a, Elb, E2a, E40rf6, VA RNA, and any variant or fragment of any of the foregoing. In someAttorney Docket No.: 2011256-2608 (P1879PCT01) embodiments, an Ad2 helper vector (e.g., plasmid) encodes Ad2 helper polypeptides (e.g., one, two, three, or four of El (e.g., Ela and / or Elb), E2A, E4, or VA RNA) necessary to generate functional rAAV particles. In certain embodiments, the Ad2 helper vector is transfected into an El complementing cell line (e.g., HEK293). The nucleotide sequence of an Ad2 helper vector and Ad2 helper virus genes can be derived from the Adenovirus 2 genome (Genbank Accession No. J01917.1).

[0183] Ad5 helper. As used herein, the term “Ad5 helper” refers to the Adenovirus serotype 5 (Ad5) helper virus (e.g., wildtype or recombinantly engineered Ad5 helper virus) and various Ad5 helper genes and / or Ad5 helper polypeptides, including, but not limited, to Ela, Elb, E2a, E40rf6, and / or VA RNA. In some embodiments, an Ad5 helper vector (e.g., plasmid) comprises Ad5 helper genes (e.g., one, two, three, or four of El (e.g., Ela and / or Elb), E2A, E4, or VA RNA) necessary to generation functional rAAV particles. In certain embodiments, the Ad5 helper vector is transfected into an El complementing cell line (e.g., HEK293). The nucleotide sequence of an Ad5 helper vector and Ad5 helper genes can be derived from the Adenovirus 5 genome (Genbank Accession No. AY601635).

[0184] Administration: As used herein, the term “administration” refers to the administration of a composition comprising rAAV particles as described herein to a subject. Administration may be by any appropriate route. For example, in some embodiments, administration may be local or systemic administration (e.g., to a mammal, e.g., to a human, e.g., a patient). A composition of the disclosure may be administered by injection or infusion by any route. For example, a composition may be administered by retinal, subretinal, intravitreal, suprachoroidal, intraspinal, intra-ci sterna magna, or intrathecal injection or infusion. Additional exemplary routes of administration may include, but are not limited to, bronchial (e.g., bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), transdermal, vaginal, and vitreal.

[0185] Bioreactor: The term “bioreactor,” as used herein, refers to any vessel used for the growth of a cell culture (e.g., a mammalian cell culture). The bioreactor can be of any size and / or any shape so long as it is useful for culturing a cell culture (e.g., a mammalian cell culture).Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0186] Cap polypeptide: As used herein, the term “Cap polypeptide” refers to the structural proteins that form a functional AAV capsid, which can in turn package DNA and infect or transduce a target cell. In some embodiments, a Cap polypeptide comprises a variant AAV capsid as disclosed herein. In some embodiments, Cap polypeptides will comprise all of the AAV capsid subunits, but less than all of the capsid subunits may be present as long as a functional capsid is produced. In some embodiments, the nucleic acid sequence encoding Cap polypeptides will be present on a single vector (e.g., plasmid). In some embodiments, the Cap polypeptide comprises an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13 or AAVrh74 Cap polypeptide, or a variant of any of the foregoing. AAV capsid genes and proteins have been described in, e.g., Knipe et al., Fields Virology, Volume 1, (6th ed., Lippincott-Raven Publishers), which is hereby incorporated by reference in its entirety.

[0187] Cell Density. As used herein, the term “cell density” refers to that number of cells present in a given volume of medium or the number of cells present in a given surface area. For example, cell density may be represented as viable cells (vc) / cm2of culture medium or vc / mL.

[0188] Culture-. As used herein, the terms “culture” and “cell culture” refer to a cell population (e.g., a eukaryotic cell population) that is suspended in or covered by a medium under conditions suitable to survival and / or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms can also refer to the combination comprising the cell population and the medium.

[0189] Dystrophin. The term “dystrophin” as used herein refers to a dystrophin protein, or a fragment or variant thereof; or a nucleic acid encoding a dystrophin protein, or a fragment or a variant thereof. In some embodiments, a dystrophin is a dystrophin polypeptide. In some embodiments, a dystrophin is a nucleic acid sequence encoding a dystrophin polypeptide. Amino acid sequences for full-length dystrophin and / or for nucleic acids that encode it can be found in a public database such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of full-length human dystrophin having 3,685 amino acids can be found as GenBank Accession No.: AAA53189.1 or UniProt Accession No. Pl 1532 (SEQ ID NO: 3034). The gene encoding human Dystrophin is known as the DMD gene and is located on the X chromosome. The DMD gene has a 11 ,5kb coding sequence with 8 promoters and 79 exons. The DMD gene encodes several isoforms of the dystrophin protein. Those skilled in the art will appreciate thatAttorney Docket No.: 2011256-2608 (P1879PCT01) sequences for the DMD gene can be readily identified using known public databases and that certain variations such as codon-optimized variants of known DMD gene sequences can be readily obtained and are understood to also encode a dystrophin protein. Full-length human dystrophin protein has several domains including: an N-terminal domain, a rod domain comprising spectrin like repeats, a cysteine rich domain, and a C-terminal domain. Several truncations of dystrophin also naturally exist which can include one or more domains of a dystrophin protein. In some embodiments, a micro-dystrophin is a truncated dystrophin comprising one or more domains of a dystrophin protein.

[0190] Dystrophin payload: The term “dystrophin payload” as used herein refers to a payload which when administered to a subject having a muscular dystrophy (e.g., Duchenne muscular dystrophy) can ameliorate one or more symptoms of the disease and / or treat and / or prevent the disease. In some embodiments, a “dystrophin payload” is also referred to herein as a cargo. In some embodiments, a dystrophin payload has one or more activities and / or functions of a dystrophin protein, e.g., when expressed in a cell. In some embodiments, a dystrophin payload alters (e.g., increases), one or more activities and / or functions of a dystrophin protein, e.g., by altering one or more activities and / or functions of an RNA transcribing a dystrophin protein. In some embodiments, a dystrophin payload (e.g., cargo) is or comprises a dystrophin polypeptide, e.g., a fragment or variant of a dystrophin polypeptide. In some embodiments, a dystrophin payload is a heterologous nucleic acid (e.g., cargo or cargo construct) with a therapeutic purpose, e.g., an antisense oligonucleotide, an miRNA, a siRNA, a shRNA, a mRNA, a snRNA (e.g., a U7 snRNA or a complex comprising the same [e g., a U7 snRNP]), or a CRISPR / Cas guide RNA, or a precursor thereof, which when administered to a cell, tissue or subject alters, e.g., increases, one or more activities and / or functions of a dystrophin protein. In some embodiments, a dystrophin payload is a heterologous nucleic acid that promotes exon-skipping. In some embodiments, a dystrophin payload is or comprises an antisense oligonucleotide (AON). In some embodiments, a dystrophin payload is or comprises a gene-editing system, e.g., a CRISPR / Cas system. In some embodiments, a dystrophin payload is or comprises a nucleic acid sequence comprising a U7 small nuclear RNA (snRNA) sequence and an antisense oligonucleotide that can bind to a portion of an RNA transcript encoded by a DMD gene.

[0191] Encode: As used herein, “encode” or “encodes” means directs the expression of or processed into. For example, as used herein, a nucleic acid encodes a polypeptide sequence ifAttorney Docket No.: 2011256-2608 (P1879PCT01) it directs the expression of that polypeptide sequence. As another example, as used herein, a nucleic acid precursor (e.g., a pri-miRNA or pre-miRNA) encodes a further processed version of the nucleic acid (e.g., mature miRNA) if it is processed into the further processed version.

[0192] Fragment: As used herein, the terms “fragment” or “portion” refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide. The whole material or entity may in some embodiments be referred to as the “parent” of the whole.

[0193] Gene. As used herein, the term “gene” refers to a DNA sequence that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene includes non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequence. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or effect one or more aspects of gene expression (e.g., inducible expression, etc.).

[0194] Gene therapy . As used herein, the term “gene therapy” refers to insertion or deletion of specific genomic DNA sequences to treat or prevent a disorder or condition for which such therapy is sought. In some embodiments, the insertion or deletion of genomic DNA sequences occurs in specific cells (e.g., target cells). Target cells may be from a mammal and / or may be cells in a mammalian subject. Mammals include but are not limited to humans, dogs, cats, cows, sheep, pigs, llamas, etc. In some embodiments, heterologous DNA is transferred to target cells. The heterologous DNA may be introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby isAttorney Docket No.: 2011256-2608 (P1879PCT01) produced. Additionally or alternatively, the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product, or it may encode a product, such as a peptide or RNA that in some manner mediates or modulates, directly or indirectly, expression of a therapeutic product. Genetic therapy may also be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced. The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof. Genetic therapy may also involve delivery of an inhibitor or repressor or other modulator of gene expression. Such an inhibitor or repressor or other modulator can be a polypeptide, peptide, or nucleic acid (e.g., DNA or RNA). Gene therapy may include in vivo or ex vivo techniques. In some embodiments, viral and non-viral based gene transfer methods can be used to introduce a nucleic acid encoding a polypeptide of interest or to introduce a therapeutic nucleic acid into mammalian cells or target tissues. Non- viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as poloxamers or liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808- 813 (1992); Miller, Nature 357:455-460 (1992); Feuerbach et al., Kidney International 49: 1791- 1794 (1996); Umov et al., Nature Reviews Genetics 11, 636-646 (2010); and Collins et al., Proceedings Biological Sciences / The Royal Society, 282(1821 ):pii 20143003 (2015), each of which is hereby incorporated by reference in its entirety.

[0195] Host Cell. As used herein, the term “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life that are suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence).Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0196] Identity As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CAB IOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0197] Improve, increase, inhibit, or reduce: As used herein the terms “improve”, “increase,” “inhibit,” “reduce,” or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single sample, e.g., of a culture medium) under otherwise comparable conditions absent presenceAttorney Docket No.: 2011256-2608 (P1879PCT01) of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0198] Medium: As used herein, the terms “medium,” “culture medium,” and “growth medium” refer to a solution comprising nutrients to nourish cells (e.g., growing cells, e.g., eukaryotic cells). Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for survival and / or minimal growth. The solution can also comprise components that enhance survival and / or growth above the minimal rate, including hormones and growth factors. The solution can be formulated to a pH and concentration of one or more salts that are optimal for cellular survival and / or proliferation. For example, the medium can also be a “defined medium” or “chemically defined medium,” e.g., a serum -free medium that contains no proteins, hydrolysates, or components of unknown composition. Defined media are free of animal -derived components and all components have a known chemical structure. One of skill in the art understands a defined medium can comprise recombinant polypeptides, for example, but not limited to, hormones, cytokines, interleukins, and / or other signaling molecules.

[0199] Micro-dystrophin. The term “micro-dystrophin” as used herein refers to a fragment of a dystrophin protein (or a nucleic acid sequence encoding the same) comprising one or more domains of a dystrophin protein. Exemplary micro-dystrophins are disclosed in Duan 2018, the entire contents of which are hereby incorporated by reference. In some embodiments, a micro-dystrophin protein is encoded by a nucleic acid that is less than 4kb in size.

[0200] Muscle targeting moiety: The phrase “muscle targeting moiety” as used herein refers to a peptide containing an RGD-motif which is effective in targeting a muscle cell or muscle tissue. In some embodiments, a muscle-targeting moiety can target a muscle cell or tissue by: (i) contacting a muscle cell or muscle tissue (e.g., binding to one or more receptors expressed on a muscle cell or tissue); (ii) contacting a cell in contact with a muscle cell or tissue (e.g., binding to one or more receptors expressed on a cell in contact with a muscle cell or tissue); (iii) delivering a payload to a muscle cell or tissue; or (iv) any combination of (i)-(iii). In some embodiments, delivering a payload to a muscle cell or muscle tissue comprises transducing a muscle cell or muscle tissue. In some embodiments, delivering a payload to a muscle cell orAttorney Docket No.: 2011256-2608 (P1879PCT01) muscle tissue results in expression of (e.g., detectable expression of) a payload in a muscle cell or muscle tissue. In some embodiments, a muscle cell comprises a skeletal muscle cell, a cardiac muscle cell, a smooth muscle cell, or a muscle stem cell, e.g., a muscle satellite cell. In some embodiments, a muscle targeting moiety can be conjugated to a payload. In some embodiments, a muscle targeting moiety can be incorporated into a vector, e.g., a viral vector or a non-viral vector. In some embodiments, a muscle targeting moiety can be inserted in an AAV capsid to form a variant AAV capsid as disclosed herein.

[0201] Nucleic acid-. The term “nucleic acid” includes any nucleotides, analogs thereof, and polymers thereof. The term “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecules and, thus, include double- and singlestranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligodeoxyribonucleotides (DNA); RNA or DNA derived from N-gly cosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus-atom bridges (also referred to herein as “intemucleotide linkages”). The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moi eties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. In some embodiments, the prefix poly- refers to a nucleic acid containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units. In some embodiments, the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units. In accordance with the methods and compositions described herein, in some embodiments, an RNA comprises a short hairpin RNA (shRNA), small interfering RNA (siRNA), mRNA, snRNA, CRISPR / Cas guide RNA, microRNA (miRNA), and / or a precursor thereof.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0202] Pharmaceutical composition. As used herein, the term “pharmaceutical composition” refers to a composition comprising rAAV particles that is suitable for administration to a human or animal subject. In some embodiments, a pharmaceutical composition comprises an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen. In some embodiments, a therapeutic regimen comprises one or more doses administered according to a schedule that has been determined to achieve a desired therapeutic effect when administered to a subject or population in need thereof (e.g., by a statistically significant probability). A pharmaceutical composition may be specially formulated for administration in solid or liquid form. In some embodiments, a pharmaceutical composition is formulated for administration by parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection. In some embodiments, a pharmaceutical composition is formulated as a sterile solution or suspension, e.g., in a sustained- release formulation. Pharmaceutical compositions of the disclosure may be formulated for administration by injection or infusion (e.g., subcutaneous, intramuscular, intravenous or epidural injection or infusion). For example, compositions may be formulated for administration by retinal, subretinal, intravitreal, suprachoroidal, intraspinal, intra-cistema magna, or intrathecal injection or infusion. In some embodiments, a pharmaceutical composition is intended and suitable for administration to a human subject. In some embodiments, a pharmaceutical composition is substantially free of contaminants (e.g., sterile and substantially pyrogen-free). Formulations of the pharmaceutical compositions may include, but are not limited to, formulations for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; topical application, such as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0203] Polypeptide. The term “polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompassAttorney Docket No.: 2011256-2608 (P1879PCT01) polypeptides that represent functional fragments (e.g., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0204] Recombinant. As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc) that is transgenic for or otherwise has been manipulated to express a gene or genes, or gene components that encode and / or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., inAttorney Docket No.: 2011256-2608 (P1879PCT01) vivo or in vitro of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc).

[0205] Recombinant AAV (rAAV) particle: A “recombinant AAV particle”, or “rAAV particle,” as used herein, refers to an infectious, replication-defective viral particle comprising an AAV protein shell encapsulating a payload that is flanked on both sides by ITRs. An AAV particle is produced in a suitable host cell (e.g., a HEK293 cell). For example, the host cell is transfected with at least one vector encoding one or more helper polypeptides (e.g., Ad2 helper polypeptides), at least one Rep polypeptide, at least one Cap polypeptide, and at least one payload (e.g., for polypeptide expression or a therapeutic nucleic acid), such that the host cell is capable of producing the Rep and Cap polypeptides necessary for packing the rAAV particle. rAAV particles may be used for subsequent gene delivery.

[0206] Rep polypeptide: The term “Rep polypeptide”, as used herein, refers to the AAV non- structural proteins that mediate AAV replication for the production of AAV particles. The AAV replication genes and proteins have been described in, e.g., Knipe et al., FIELDS VIROLOGY, Volume 1, (6th ed., Lippincott-Raven Publishers), which is hereby incorporated by reference in its entirety.

[0207] RGD motif. The phrase “RGD motif’ as used herein refers to a peptide comprising the amino acids R, G, and D in consecutive order. Peptides comprising an RGD- motif are provided in Table 7.

[0208] Seeding: The term “seeding” as used herein refers to the process of providing a cell culture to a vessel (e.g., a bioreactor or culture flask). For example, the process of providing a cell culture may include propagation of the cells in another bioreactor or vessel before providing to the bioreactor or other vessel. The cells have been frozen and thawed immediately prior to providing them to the bioreactor or vessel. The term “seeding” refers to providing any number of cells, including a single cell.

[0209] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., Duchenne muscular dystrophy, or Becker muscular dystrophy. In some embodiments, a subject is susceptible to a disease, disorder, or conditionAttorney Docket No.: 2011256-2608 (P1879PCT01)(e.g., Duchenne muscular dystrophy, or Becker muscular dystrophy); in some embodiments, a susceptible subject is predisposed to and / or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder, or condition (e.g., Duchenne muscular dystrophy, or Becker muscular dystrophy). In some embodiments, a subject does not display a particular symptom (e.g,. clinical manifestation of disease) or characteristic of a disease, disorder, or condition (e.g., Duchenne muscular dystrophy, or Becker muscular dystrophy). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., Duchenne muscular dystrophy, or Becker muscular dystrophy). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0210] Titer: As used herein, the term “titer” refers to the quantity of virus in a given volume. Titer, for example, can be expressed as viral genome copies (vg) per given volume or plaque forming units (pfu) per given volume. In some embodiments, titer can be expressed as number of capsids per given volume.

[0211] Transfection: As used herein, the term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as eukaryotic cells (e.g., mammalian cells). For example, transfection can include vector-based transfection, viral-based transfection, electroporation, lipofection (e.g., with cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, and / or transfection based on cationic polymers (e.g., DEAE-dextran or polyethylenimine). In some embodiments, viral-based transfection is also referred to herein as transduction.

[0212] Treating: As used herein, the term “treating” refers to providing treatment, e.g., providing any type of medical or surgical management of a subject, e.g., a subject having Duchenne muscular dystrophy, or Becker muscular dystrophy. The treatment can be provided in order to reverse, alleviate, inhibit the progression of, prevent or reduce the likelihood of a disease, disorder, or condition (e.g., Duchenne muscular dystrophy, or Becker muscular dystrophy), or in order to reverse, alleviate, inhibit or prevent the progression of, prevent or reduce the likelihood of one or more symptoms or manifestations of a disease, disorder or condition (e.g., Duchenne muscular dystrophy, or Becker muscular dystrophy). “Prevent” refersAttorney Docket No.: 2011256-2608 (P1879PCT01) to causing a disease, disorder, condition, or symptom or manifestation of such not to occur for at least a period of time in at least some individuals. Treating can include administering an agent to the subject following the development of one or more symptoms or manifestations indicative of a condition, disease, or disorder (e.g., Duchenne muscular dystrophy, or Becker muscular dystrophy), e.g., in order to reverse, alleviate, reduce the severity of, and / or inhibit or prevent the progression of the condition and / or to reverse, alleviate, reduce the severity of, and / or inhibit or one or more symptoms or manifestations of the condition, e.g., Duchenne muscular dystrophy, or Becker muscular dystrophy. A composition comprising rAAV particles of the disclosure can be administered to a subject who has developed a disorder or is at increased risk of developing such a disorder relative to a member of the general population. A composition of the disclosure can be administered prophylactically or before development of any symptom or manifestation of the condition. Typically, in this case, the subject will be at risk of developing the condition.

[0213] Variant: As used herein in the context of molecules, e.g., nucleic acids, or proteins, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and / or contributing to a particular structural motif and / or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to another in linear or three- dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g.,Attorney Docket No.: 2011256-2608 (P1879PCT01) that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (z.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0214] Vector As used herein, the term “vector” refers to a molecule comprising a nucleic acid molecule, where the vector is capable of transporting the nucleic acid molecule into a cell. By way of non-limiting example, one type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.Another type of vector is a viral vector, wherein additional DNA segments may be packaged into a viral capsid and can be transferred into another cell and / or organism. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0215] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference in its entirety.

[0216] VP: As used herein, the term “VP” refers to an AAV VP1 capsid protein, an AAV VP2 capsid protein, an AAV VP3 capsid protein, or variants or fragments or combinations of any of the foregoing. The term “capsid protein” is used interchangeably herein with VP. The numbering used herein in describing exemplary locations of peptide insertions in VP1, VP2 or VP3 are used relative to AAV VP1 numbering. For example VP1, VP2 and VP3 of the AAV9 capsid protein correspond to amino acids 1 to 736 of VP1, amino acids 138 to 736 of VP1 and amino acids 203 to 736 of VP1, respectively. Thus, reference to a peptide insertion between positions 588 and 589 in an AAV capsid variant refers to positions 588 and 589 in VP1, VP2 or VP3 relative to VP1 numbering. Those with knowledge in the pertinent field would be able to readily ascertain the corresponding position in VP2 and VP3, e.g., by comparing the sequencesAttorney Docket No.: 2011256-2608 (P1879PCT01) of VP1, VP2 and VP3 of the parental AAV capsid proteins using methods known in the field such as sequence alignment. In some embodiments, a VP capsid protein is a VP1 capsid protein. In some embodiments, a VP capsid protein is a VP2 capsid protein. In some embodiments, a VP capsid protein is a VP3 capsid protein. In some embodiments, a VP protein comprises a peptide insertion disclosed herein.

[0217] Variant AAV capsid protein. As used herein, the term “variant AAV capsid protein” refers to a VP capsid protein (e.g., a VP1, VP2, or VP3) comprising a peptide insertion relative to a corresponding parental AAV capsid protein (e.g., a parental VP1, VP2, or VP3).DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0218] Duchenne muscular dystrophy (DMD) is one of the most severe childhood-onset muscular dystrophies caused by mutations in the DMD gene located on the X-chromosome. The DMD gene encodes the dystrophin protein which is part of the dystrophin-associated glycoprotein complex (DGC) which is critical to maintain the structural integrity and normal function of muscle (see ZXNQC, S. et al., (2024) “Dystrophin- and Utrophin-Based Therapeutic Approaches for Treatment of Duchenne Muscular Dystrophy: A Comparative Review" vol. 38, pp. 95-119, the entire contents of which is hereby incorporated by reference). DMD is one of the most common genetic conditions affecting approximately 1 in 3500 male births worldwide. The life expectancy of patients diagnosed with DMD is about 30 years. DMD is a recessively inherited muscle wasting disorder and often results from a mutation in the DMD gene that results in aberrant or absent expression of the dystrophin protein. DMD patients experience progressive wasting of skeletal muscles and cardiac dysfunction, leading to loss of ambulation and premature death, primarily due to cardiac or respiratory failure (see Gao et al. “Genome Editing Therapy for Duchenne Muscular Dystrophy,” Academic Press, (2018); Fang et al. Gene editing in Regenerative Medicine,” Third Edition, Academic Press, (2019), the entire contents each of which are hereby incorporated by reference).

[0219] Currently available treatments are generally only able to slow the pathology of DMD (see Emery, A E. H. and Muntoni, F. “Duchenne Muscular Dystrophy,” Third Edition, Oxford University Press, (2003), the entire contents of which are hereby incorporated by reference). Gene therapy approaches for DMD have been demonstrated in dystrophic animal models by either directly targeting a class of mutations, as with exon skipping, or replacing theAttorney Docket No.: 2011256-2608 (P1879PCT01) mutated gene with viral -vector mediated delivery (see Koo, T. and Wood, M. J. Human Gene Therapy 24, pp. 479-488, (2013); Benedetti, S., et al., The FEBS Journal vol. 280, pp. 4263- 4280, (2013); and Seto, J. T., et al., Current Gene Therapy vol. 12, pp. 139-151 (2012) the entire contents of each of which are hereby incorporated by reference in their entirety). Currently, there are three approved exon skipping therapies which received accelerated approval in the US, Exondys 51 (eteplirsen), Vyondys 53 (golodirsen), and Viltepso (viltolarsen). Notably, these exon skipping therapies provide only a low-level increase in dystrophin (< 1%) and limited functional benefits.

[0220] Recombinant adeno-associated virus (rAAV) vectors are a potential vehicle for gene therapy in DMD. The first gene therapy for all DMD patients over age of 4 for microdystrophin delivery with AAV particles having the AAVrh74 serotype received accelerated approval; however, this therapy did not achieve its primary endpoint (change in NSAA score compared to baseline). Pre-clinical studies designing and testing therapeutic constructs for treatment of DMD are typically confined by the approximately 4.9 kb size of a single stranded rAAV vector genome (see Dong, B., et al., Molecular Therapy vol. 18, pp. 87-92, (2010), and Wu, Z., et al., Molecular Therapy vol. 18, pp. 80-86, (2010), the entire contents of each of which are hereby incorporated by reference in their entirety). Packaging the entire -13.9 kb DNA sequence of the muscle-specific isoform of dystrophin into a single rAAV particle cannot be achieved; accordingly, miniaturized, synthetic versions of the muscle-specific isoform of dystrophin are often used. Although in vivo recombination of two and three rAAV vector genomes has been demonstrated to deliver a mini- or full-length dystrophin coding sequence (see, Odom, G. L., et al., Molecular Therapy vol. 19, pp. 36-45, (2011 ); Lostal, W., et al., Human Gene Therapy: vol. 25, pp. 552-562, (2014); and Koo, T., et al., Human Gene Therapy, vol. 25, pp. 98-108, (2014) ), the entire contents of each of which are hereby incorporated by reference in their entirety), the efficiency of delivering multiple vectors for reconstituting full- length dystrophin is suboptimal and can increase the overall dose of AAV particles needed for delivering the dystrophin payload. This in turn can result in unwanted dose-related toxicities.

[0221] While rAAVs have emerged as some of the most promising vectors for in vivo gene therapy and are currently under clinical evaluation for a number of disorders including DMD, naturally occurring AAV capsids sub-optimally target skeletal muscle, and require extremely high doses to achieve minimum effective transgene expression. This poses dauntingAttorney Docket No.: 2011256-2608 (P1879PCT01) manufacturing challenges as well as safety concerns. For example, low muscle tropism in existing AAV vectors necessitates high viral doses for systemic delivery, which has been associated with adverse events across clinical trials including patient deaths (see Lek, A., et al., New England Journal of Medicine: vol. 389, pp. 1203-1210, (2023); Duan, D., et al., Molecular Therapy: vol. 31, pp. 3123-3126, (2023), the entire contents of each of which are hereby incorporated by reference in their entireties). Among the adverse events in DMD patients treated with AAV particles having an AAV9 capsid or a AAVrh74 capsid delivering micro-dystrophin are acute liver injury. This adverse event is observed in a high percentage of cases (35-50% of patients), with a subset resulting in severe adverse events (see Asokan et al. “Redirecting AAV vectors to extrahepatic tissues,” Molecular Therapy, vol. 31, pp. 3371-3375, (2023) the entire contents of which are hereby incorporated by reference in its entirety). Additionally, a number of patients experienced myocarditis as an adverse event. Thus, there is a need to develop gene therapies to deliver therapeutic polypeptides and / or nucleic acid molecules encoding the same, e.g., cargos, using novel vectors with enhanced muscle targeting to improve gene delivery efficiency with lower doses (e.g., titer).

[0222] The present disclosure recognizes the challenges in current treatments of DMD and provides solutions with the AAV particles disclosed herein which can be used to deliver a dystrophin payload to muscle cells or tissue. The present disclosure is based, in part, on the discovery that AAV muscle tropism can be obtained by inserting a short peptide onto an AAV capsid to direct said AAV capsid to a muscle cell and / or muscle tissue. In some embodiments, rAAV particles comprising a variant capsid having a peptide insertion disclosed herein bind to and / or recognize a target on a muscle cell and / or muscle tissue. Without wishing to be bound by any particular theory, in some embodiments, rAAV particles comprising a variant capsid comprising a peptide insertion disclosed herein can enhance vector attachment, internalization, and / or payload expression in muscle cells and / or muscle tissue.

[0223] Accordingly, the present disclosure features compositions and methods for treating DMD with rAAV particles comprising a variant capsid having a peptide insertion disclosed herein and a dystrophin payload, e.g., a heterologous nucleic acid encoding a dystrophin payload. Also provided herein are methods for delivering micro-dystrophin proteins for treating a subject having a muscular dystrophy (e.g., DMD or BMD), sarcopenia, heart failure, and / or cachexia.Attorney Docket No.: 2011256-2608 (P1879PCT01)Dystrophin

[0224] Dystrophin protein is encoded by the DMD gene located on the X-chromosome. The DMD gene is also referred to as the dystrophin gene, and both terms are used interchangeably herein. The full-length DMD gene is 2.6 Mb, with an 11.5 kb coding sequence that translates into a 427 kilodalton (kDa) dystrophin protein. The DMD gene is the largest known human gene with 79 exons. Due to its size, mutation rate in DMD is high, with 30% of DMD patients exhibiting de novo mutations. The DMD gene encodes seven different tissuespecific transcripts (see, Szwec 2024) The full-length striated muscle isoform of dystrophin can play a role in transmitting contractile force through the sarcolemma and out to the extracellular matrix (see Kaplan, Katherine M, and Kathleen G Morgan. (2022) “The importance of dystrophin and the dystrophin associated proteins in vascular smooth muscle.” Frontiers in physiology vol. 13, 1059021).

[0225] Generally, the roles of the different dystrophin proteins are described in terms of the complexes they assemble. Most dystrophins can bind beta-dystroglycan and assemble dystrophin glycoprotein complexes (DGC) on the plasmalemma of various cell types, including muscle fibers, satellite cells, cardiomyocytes, and neurons (Szwec 2024). In DGCs, dystrophins can have mechanical as well as signaling functions. For example, dystrophins connect the internal cytoskeleton with the extracellular matrix (e.g., with muscle cells) to transmit forces created during contraction, and establish cell polarity (e g., of satellite cells). Dystrophins also regulate the maturation of neurotransmitter receptor complexes and their release at neuromuscular junctions (NMJs) and central synapses in the nervous system, as well as the binding of several regulatory proteins important in signal transduction inside the cell and between different cell types. In addition to maintaining the mechanical link between the intracellular cytoskeleton and the membrane bound DGC, dystrophin can also be a scaffold for signaling proteins (see Ozawa, E. in Myology (ed. Franzini-Armstrong C Engel A) 455-470 (McGraw-Hill, 2004); Winder, S. J. Journal of Muscle Research and Cell Motility 18, 617-629 (1997); and Campbell, K. P. and Kahl, S. D. Nature 338, 259-262, (1989), the entire contents of each of which are hereby incorporated by reference in their entireties).

[0226] Dystrophin reference protein sequence (SEQ ID NO: 3034):Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0227] MLWWEEVEDCYEREDVQKKTFTKWVNAQFSKFGKQHIENLFSDLQDGRRLLDLLEGLTGQKLPKEKGSTRVHALNNVNKALRVLQNNNVDLVNIGSTDIVDGNHKLTLGLIWNIILHWQVKNVMKNIMAGLQQTNSEKILLS WVRQSTRNYPQVNVINFTTSWSDGLALNALIHSHRPDLFDWNSVVCQQSATQRLEHAFNIARYQLGIEKLLDPEDVD TTYPDKKSILMYITSLFQVLPQQVSIEAIQEVEMLPRPPKVTKEEHFQLHHQMHYSQQITVSLAQGYERTSSPKPRF KSYAYTQAAYVTTSDPTRSPFPSQHLEAPEDKSFGSSLMESEVNLDRYQTALEEVLSWLLSAEDTLQAQGEISNDVE VVKDQFHTHEGYMMDLTAHQGRVGNILQLGSKLIGTGKLSEDEETEVQEQMNLLNSRWECLRVASMEKQSNLHRVLM DLQNQKLKELNDWLTKTEERTRKMEEEPLGPDLEDLKRQVQQHKVLQEDLEQEQVRVNSLTHMVVVVDESSGDHATA ALEEQLKVLGDRWANICRWTEDRWVLLQDILLKWQRLTEEQCLFSAWLSEKEDAVNKIHTTGFKDQNEMLSSLQKLA VLKADLEKKKQSMGKLYSLKQDLLSTLKNKSVTQKTEAWLDNFARCWDNLVQKLEKSTAQISQAVTTTQPSLTQTTV METVTTVTTREQILVKHAQEELPPPPPQKKRQITVDSEIRKRLDVDITELHSWITRSEAVLQSPEFAI FRKEGNFSD LKEKVNAIEREKAEKFRKLQDASRSAQALVEQMVNEGVNADSIKQASEQLNSRWIEFCQLLSERLNWLEYQNNIIAF YNQLQQLEQMTTTAENWLKIQPTTPSEPTAIKSQLKICKDEVNRLSGLQPQIERLKIQSIALKEKGQGPMFLDADFV AFTNHFKQVFSDVQAREKELQTIFDTLPPMRYQETMSAIRTWVQQSETKLSIPQLSVTDYEIMEQRLGELQALQSSL QEQQSGLYYLSTTVKEMSKKAPSEISRKYQSEFEEIEGRWKKLSSQLVEHCQKLEEQMNKLRKIQNHIQTLKKWMAE VDVFLKEEWPALGDSEILKKQLKQCRLLVSDIQTIQPSLNSVNEGGQKIKNEAEPEFASRLETELKELNTQWDHMCQ QVYARKEALKGGLEKTVSLQKDLSEMHEWMTQAEEEYLERDFEYKTPDELQKAVEEMKRAKEEAQQKEAKVKLLTES VNSVIAQAPPVAQEALKKELETLTTNYQWLCTRLNGKCKTLEEVWACWHELLSYLEKANKWLNEVEFKLKTTENI PG GAEEISEVLDSLENLMRHSEDNPNQIRILAQTLTDGGVMDELINEELETFNSRWRELHEEAVRRQKLLEQSIQSAQE TEKSLHLIQESLTFIDKQLAAYIADKVDAAQMPQEAQKIQSDLTSHEISLEEMKKHNQGKEAAQRVLSQIDVAQKKL QDVSMKFRLFQKPANFELRLQESKMILDEVKMHLPALETKSVEQEVVQSQLNHCVNLYKSLSEVKSEVEMVIKTGRQ IVQKKQTENPKELDERVTALKLHYNELGAKVTERKQQLEKCLKLSRKMRKEMNVLTEWLAATDMELTKRSAVEGMPS NLDSEVAWGKATQKEIEKQKVHLKSITEVGEALKTVLGKKETLVEDKLSLLNSNWIAVTSRAEEWLNLLLEYQKHMETFDQNVDHITKWIIQADTLLDESEKKKPQQKEDVLKRLKAELNDIRPKVDSTRDQAANLMANRGDHCRKLVEPQISE LNHRFAAISHRIKTGKASIPLKELEQFNSDIQKLLEPLEAEIQQGVNLKEEDFNKDMNEDNEGTVKELLQRGDNLQQ RITDERKREEIKIKQQLLQTKHNALKDLRSQRRKKALEISHQWYQYKRQADDLLKCLDDIEKKLASLPEPRDERKIK EIDRELQKKKEELNAVRRQAEGLSEDGAAMAVEPTQIQLSKRWREIESKFAQFRRLNFAQIHTVREETMMVMTEDMP LEISYVPSTYLTEITHVSQALLEVEQLLNAPDLCAKDFEDLFKQEESLKNIKDSLQQSSGRIDIIHSKKTAALQSAT PVERVKLQEALSQLDFQWEKVNKMYKDRQGRFDRSVEKWRRFHYDIKI FNQWLTEAEQFLRKTQI PENWEHAKYKWY LKELQDGIGQRQTVVRTLNATGEEIIQQSSKTDASILQEKLGSLNLRWQEVCKQLSDRKKRLEEQKNILSEFQRDLN EFVLWLEEADNIASIPLEPGKEQQLKEKLEQVKLLVEELPLRQGILKQLNETGGPVLVSAPISPEEQDKLENKLKQT NLQWIKVSRALPEKQGEIEAQIKDLGQLEKKLEDLEEQLNHLLLWLSPIRNQLEIYNQPNQEGPFDVQETEIAVQAK QPDVEEILSKGQHLYKEKPATQPVKRKLEDLSSEWKAVNRLLQELRAKQPDLAPGLTTIGASPTQTVTLVTQPVVTK ETAISKLEMPSSLMLEVPALADFNRAWTELTDWLSLLDQVIKSQRVMVGDLEDINEMIIKQKATMQDLEQRRPQLEE LITAAQNLKNKTSNQEARTI ITDRIERIQNQWDEVQEHLQNRRQQLNEMLKDSTQWLEAKEEAEQVLGQARAKLESW KEGPYTVDAIQKKITETKQLAKDLRQWQTNVDVANDLALKLLRDYSADDTRKVHMITENINASWRSIHKRVSEREAA LEETHRLLQQFPLDLEKFLAWLTEAETTANVLQDATRKERLLEDSKGVKELMKQWQDLQGEIEAHTDVYHNLDENSQ KILRSLEGSDDAVLLQRRLDNMNFKWSELRKKSLNIRSHLEASSDQWKRLHLSLQELLVWLQLKDDELSRQAPIGGD FPAVQKQNDVHRAFKRELKTKEPVIMSTLETVRIFLTEQPLEGLEKLYQEPRELPPEERAQNVTRLLRKQAEEVNTEAttorney Docket No.: 2011256-2608 (P1879PCT01)WEKLNLHSADWQRKIDETLERLQELQEATDELDLKLRQAEVIKGSWQPVGDLLIDSLQDHLEKVKALRGE IAPLKEN VSHVNDLARQLTTLGIQLS PYNLSTLEDLNTRWKLLQVAVEDRVRQLHEAHRDFGPASQHFLSTSVQGPWERAI S PN KVPYYINHETQTTCWDHPKMTELYQSLADLNNVRFSAYRTAMKLRRLQKALCLDLLSLSAACDALDQHNLKQNDQPM DILQI INCLTTIYDRLEQEHNNLVNVPLCVDMCLWLLNVYDTGRTGRIRVLSFKTGI I SLCKAHLEDKYRYLFKQV ASSTGFCDQRRLGLLLHDS IQI PRQLGEVASFGGSNIE PSVRSCFQFANNKPEIEAALFLDWMRLEPQSMVWLPVLH RVAAAETAKHQAKCNI CKECPI I GFRYRSLKHFNYDICQSCFFSGRVAKGHKMHYPMVEYCTPTTSGEDVRDFAKVL KNKFRTKRYFAKHPRMGYLPVQTVLEGDNMETPVTLINFWPVDSAPASS PQLSHDDTHSRIEHYASRLAEMENSNGS YLNDS I S PNES IDDEHLLIQHYCQSLNQDS PLSQPRSPAQI LI SLESEERGELERILADLEEENRNLQAEYDRLKQQ HEHKGLS PLPS PPEMMPTS PQS PRDAE LI AEAKLLRQHKGRLEARMQI LEDHNKQLE SQLHRLRQLLEQPQAEAKVN GTTVSS PSTSLQRSDSSQPMLLRVVGSQTSDSMGEEDLLS PPQDTSTGLEEVMEQLNNS FPSSRGRNTPGKPMREDT M

[0228] Dystrophin can be divided into four major domains, including an N-terminal domain, a central rod domain, a cysteine-rich (CR) domain, and a C-terminal (CT) domain, as described in Duan D. (2018), “Systemic AAV Micro-dystrophin Gene Therapy for Duchenne Muscular Dystrophy,” Molecular Therapy vol. 26, no. 10, pp. 2337-2356. The central rod domain can be further divided into 24 spectrin-like repeats (SRI to SR24) and 4 hinge domains (H1-H4). The N-terminal domain constitutes the first actin-binding domain (ABDI), the central rod domain has spectrin-like repeats (SR), and the cysteine-rich (CR) domain contains 14 cysteines. ABDI contains three actin binding sites responsible for binding F- and y-actin and links the sarcolemma to the subsarcolemmal network (Szwec 2024). Additionally, ABDI can interact with intermediate filament protein cytokeratins that allow dystrophin to associate with the contractile apparatus in muscle cells. The central rod domain is the largest and has an alpha helical structure, stemming from 24 tandem spectrin-like repeats (SR1-SR24) and four hinge domains that each comprise proline-rich regions (H1-H4). Hinge domain 1 (Hl) is positioned before the first spectrin-like repeat (SRI), H2 is positioned between SR3 and SR4, H3 is positioned between SRI 9 and SR20, and H4 is positioned after SR24 and is followed by the cysteine rich domain. The central rod domain can give dystrophin elasticity and flexibility for maintaining the integrity of the sarcolemma during muscle contractility (see Winder, S. J. Journal of Muscle Research and Cell Motility 18, 617-629 (1997)). The central rod domain is involved in, among other things, interaction of dystrophin with microtubules which are necessary for the proper organization of the microtubule network in skeletal muscle cells. The central rod domain also provides a flexible connection between the N- and C-terminus of dystrophin.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0229] Certain spectrin-like repeats in the central rod domain provide unique regions that can serve as additional binding sites for cytoskeleton, the sarcolemma, as well as members of the DGC. For example, SRI 6 and SRI 7 contain a binding domain for an integral constituent of the DGC, the neuronal nitric oxide synthase (nNOS). Nitric oxide produced by nNOS diffuses to the surrounding vasculature and counteracts sympathetic vasoconstriction, allowing sufficient blood perfusion in working muscle. In DMD, nNOS no longer localizes to the sarcolemma, compromising the ability of muscle to counteract functional ischemia and leading to focal ischemic damage (see Rybakova, I. N., et al., The Journal of Cell Biology 135, 661-672 (1996); Warner, L. E., et al., Human Molecular Genetics 11, 1095-1105 (2002); Metzinger, L., et al., Human Molecular Genetics 6, 1185-1191 (1997); Lai, Y., et al., The Journal of Clinical Investigation 119, 624-635, (2009); and Duan 2018). The cysteine-rich domain and the adjacent H4 region form the 13-dystroglycan binding domain (Dg BD) (see Blake, D. J., et al., Physiological Reviews 82, 291-329).

[0230] The C-terminal (CT) domain contains two alpha helices that resemble the spectrin repeats of the central rod domain. The structure of the CT domain provides binding sites for dystrobrevins and syntrophins, specifying their location on the sarcolemma. The size of the CT domain varies and is based on alternative splicing of exons 71-74 and 78. Although the CT domain may not be required for assembly of the DGC, its length may regulate the syntrophin and dystrobrevin isoform composition. The N-terminal domain of dystrophin can bind to F-actin filaments of the intracellular cytoskeleton (see Way, M., et al., FEBS Letters 301, 243-245 (1992); Hemmings, L., et al., The Journal of Cell Biology 116, 1369-1380 (1992); Fabbrizio, E., et al., Biochemistry 32, 10457- 10463 (1993); and Pavalko, F. M. and Otey, C. A. Proceedings of the Society for Experimental Biology and Medicine 205, 282-293 (1994)).Dystrophin payload

[0231] An rAAV particle disclosed herein comprises a variant AAV capsid protein disclosed herein and a dystrophin payload.

[0232] In some embodiments, a dystrophin payload is or comprises a dystrophin polypeptide, e.g., a fragment or variant of a dystrophin polypeptide. In some embodiments, anAttorney Docket No.: 2011256-2608 (P1879PCT01) rAAV particle comprises a variant AAV capsid protein disclosed herein and a heterologous nucleic acid sequence encoding a dystrophin payload.

[0233] In some embodiments, a dystrophin payload is a heterologous nucleic acid, e.g., an antisense oligonucleotide, an miRNA, a siRNA, a shRNA, a mRNA, a snRNA (e.g., a U7 snRNA or a complex comprising the same [e.g., a U7 snRNP]), or a CRISPR / Cas guide RNA, or a precursor thereof. In some embodiments, a dystrophin payload is a heterologous nucleic acid that promotes exon-skipping in an RNA transcribed from a DMD gene. In some embodiments, a dystrophin payload promotes skipping of exon 53, exon 51, exon 45, exon 44 and / or exon 2.

[0234] In some embodiments, a dystrophin payload is or comprises an antisense oligonucleotide (AON). AONs that can be used for treating and / or preventing DMD are known in the field, e.g., as described in Wilton-Clark and Yokota (2023), “Recent Trends in Antisense Therapies for Duchenne Muscular Dystrophy,” Phramaceutics vol. 15(3), the entire contents of which are hereby incorporated by reference. As will be appreciated by one with knowledge in the field, any of the AONs disclosed in Wilton-Clark and Yokota (2023) can be used in an AAV particle disclosed herein and can be useful for treating and / or preventing DMD, or ameliorating one or more symptoms of DMD in a subject. In some embodiments, an antisense oligonucleotide is Viltolarsen, Golodirsen, WVE-N531, Eteplirsen, SRP-5051, PGN-EDO51, Casimersen, Drisapersen, Suvodirsen, DS-5141B, NS-089 / NCNP-02, ENTR-601-44, or ATL1102.

[0235] In some embodiments, a dystrophin payload is or comprises a gene-editing system, e.g., a CRISPR / Cas system. In some embodiments, a dystrophin payload comprises a CRISPR-Cas protein. In some embodiments, a CRISPR-Cas protein is chosen from: a Type II, Type V or Type VI CRISPR-Cas protein (e.g., a Cas9 protein), a Casl2a protein, a Casl2b protein, a Cas 12c protein, a Casl2d protein, a Casl2e protein, a Casl2f protein, a Cast 2g protein, a Casl2h protein, a Casl2i protein, a Casl3a protein, a Casl3b protein or a variant or fragment thereof. In some embodiments, the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA / tracrRNA that interacts with the CRISPR-Cas protein. In some embodiments a CRISPR-Cas protein is fused with one or more domains, e.g., an activator domain and / or a repressor domain. In some embodiments a CRISPR-Cas protein is a nuclease. In some embodiment a CRISPR-Cas protein is a nickase and only cleaves one strand of a target nucleicAttorney Docket No.: 2011256-2608 (P1879PCT01) acid molecule. In some embodiment a CRISPR-Cas protein is inactivated and binds to but does not cleave a target nucleic acid molecule.

[0236] In some embodiments, a dystrophin payload is or comprises a nucleic acid sequence comprising a U7 small nuclear RNA (snRNA) sequence and an antisense oligonucleotide that can bind to a portion of an RNA transcript encoded by a DMD gene. Exemplary U7 snRNA that can be useful in the treatment and / or prevention of DMD are described in Gadgil and Raczynska (2021), “U7 snRNA: A Tool for Gene Therapy,” Journal of Gene Medicine, vol. 23(4), e3321 and Wein et al., (2022), “Systemic delivery of an AAV9 exonskipping vector significantly improves or prevents features of Duchenne muscular dystrophy in the Dup2 mouse,” Mol Ther Methods Clin Dev, vol. 26, pp. 239-293, the entire contents of each of which are hereby incorporated by reference. As will be appreciated by one with knowledge in the field, any of the U7 snRNA disclosed in Gadgil and Raczynska (2021) can be used in an AAV particle disclosed herein and can be useful for treating and / or preventing DMD, or ameliorating one or more symptoms of DMD in a subject.Micro-dystrophin payload

[0237] It has been discovered that fragments of dystrophin proteins comprising certain domains of the full-length dystrophin protein (e g., including one or more of an N-terminal domain, hinge domains, spectrin-like repeats, cysteine-rich domain, and / or C-terminal domain of dystrophin) can restore dystrophin function to levels sufficient to reduce certain symptoms of muscular dystrophy and / or DMD (see Szwec 2024, and Duan 2018). Fragments of dystrophin proteins that are encoded by a nucleic acid sequence that is less than about 4kb in size are also referred to as micro-dystrophins herein (Chamberlain J.S. et al., (2023) Human Gene Therapy, vol. 23(9-10), pp. 404-415, the entire contents of which is hereby incorporated by reference). Such micro-dystrophins are amenable for packaging in AAV particles which have a packaging size capacity, e.g., cargo size limitation, of about 3.7kb.

[0238] The design of micro-dystrophins started in the late 1990s / early 2000s based on truncated versions of naturally occurring dystrophin found in Becker muscular dystrophy patients which show a mild form of the disease (Duan 2018). Researchers in the field developed numerous versions of micro-dystrophins which could restore sarcolemmal integrity of dystrophicAttorney Docket No.: 2011256-2608 (P1879PCT01) muscles and improve function in DMD preclinical models, including mouse, rat, and dog DMD models. Therapeutic optimization was significantly improved through codon optimization of the sequences, as well as addition of certain domains (e.g., nNOS domains and C-terminal domain). Clinical efficacy in DMD patients upon micro-dystrophin delivery has been observed to some extent but has reached a ceiling effect since higher doses cannot be administered due to safety concerns arising from the use of natural AAV capsids. Currently, the first approved gene therapy for DMD uses a dose of 1.33E14 vg / kg and is capped at 9.31E15 vg total fixed dose for patients weighing 70 kg or greater (see Elevidys prescribing information).

[0239] Without wishing to be bound by any particular theory, in some embodiments, gene therapies that can achieve a therapeutic affect against DMD at a lower dose, e.g., a lower vg / kg dose, can ameliorate some of the negative effects of currently available rAAV gene therapy.

[0240] In some embodiments, a dystrophin payload is or comprises a dystrophin polypeptide, e.g., a fragment or variant of a dystrophin polypeptide. In some embodiments, an rAAV particle comprises a variant AAV capsid protein disclosed herein and a heterologous nucleic acid sequence encoding a dystrophin (e.g., a micro-dystrophin) payload.

[0241] In some embodiments, a dystrophin (e.g., a micro- dystrophin) payload comprises: (i) an N-terminal domain, (ii) one or more hinge domains, (iii) one or more spectrin-like repeats (SR), (iv) a cysteine-rich domain, (v) a C-terminal domain, or (vi) any combination of (i)-(v).

[0242] In some embodiments, a dystrophin(e.g., a micro-dystrophin) payload comprises a hinge domain 1 (Hl), a hinge domain 2 (H2), a hinge domain 3 (H3), a hinge domain 4 (H4), or any combination thereof.

[0243] In some embodiments, a dystrophin (e.g., a micro-dystrophin) payload comprise one or more spectrin-like repeats (SRs). In some embodiments, one or more spectrin-like repeats comprise: (a) an actin binding domain, (b) a neuronal nitric oxide synthase (nNOS) binding domain, (c) a microtubule binding domain, (d) any combination of (a)-(c). In some embodiments, a dystrophin payload comprises a spectrin-like repeat 1 (SRI), a spectrin-like repeat 2 (SR2), a spectrin-like repeat 3 (SR3), a spectrin-like repeat 4 (SR4), a spectrin-like repeat 5 (SR5), a spectrin-like repeat 6 (SR6), a spectrin-like repeat 7 (SR7), a spectrin-like repeat 8 (SR8), a spectrin-like repeat 9 (SR9), a spectrin-like repeat 10 (SR 10), a spectrin-likeAttorney Docket No.: 2011256-2608 (P1879PCT01) repeat 11 (SRI 1), a spectrin-like repeat 12 (SR12), a spectrin-like repeat 13 (SR13), a spectrinlike repeat 14 (SRI 4), a spectrin-like repeat 15 (SRI 5), a spectrin-like repeat 16 (SRI 6), a spectrin-like repeat 17 (SRI 7), a spectrin-like repeat 18 (SRI 8), a spectrin-like repeat 19 (SRI 9), a spectrin-like repeat 20 (SR20), a spectrin-like repeat 21 (SR21), a spectrin-like repeat 22 (SR22), a spectrin-like repeat 23 (SR23), a spectrin-like repeat 24 (SR24), or any combination thereof.

[0244] In some embodiments, a dystrophin (e.g., a micro- dystrophin) payload comprises at least two, at least three, at least four, or at least five spectrin-like repeats (SR). In some embodiments, a dystrophin payload comprises SRI, SR2, SR3, and SR24. In some embodiments, a dystrophin payload comprises SRI , SR2, SR22, SR23, and SR24.

[0245] In some embodiments, a dystrophin (e.g., a micro- dystrophin) payload comprises (i) an N terminal domain, (ii) hinge domains Hl, H2 and H4, (iii) spectrin-like repeats SRI, SR2, SR3, and SR24, and (iv) a cysteine-rich domain. In some embodiments, a dystrophin payload comprises in 5’ to 3’ order: an N terminal domain, a Hl domain, a spectrin-like repeat SRI, a spectrin-like repeat SR2, a spectrin-like repeat SR3, a H2 domain, a spectrin-like repeat SR24, a H4 domain and a cysteine-rich domain.

[0246] In some embodiments, a dystrophin (e.g., a micro- dystrophin) payload comprises (i) an N terminal domain, (ii) hinge domains Hl, H3 and H4, (iii) spectrin-like repeats SRI, SR2, SR22, SR23, and SR24, and (iv) a cysteine-rich domain. In some embodiments, a dystrophin payload comprises in 5’ to 3’ order: an N terminal domain, a Hl domain, a spectrin-like repeat SRI, a spectrin-like repeat SR2, a H3 domain, a spectrin-like repeat SR22, a spectrin-like repeat SR23, a spectrin-like repeat SR24, a H4 domain and a cysteine-rich domain.

[0247] In some embodiments, a dystrophin (e.g., a micro-dystrophin) payload comprises the amino acid sequence of SEQ ID NO: 3001 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3001.

[0248] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3000 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%,Attorney Docket No.: 2011256-2608 (P1879PCT01) at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3000.

[0249] In some embodiments, a dystrophin (e.g., a micro-dystrophin) payload comprises the amino acid sequence of SEQ ID NO: 3003 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3003.

[0250] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3035 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3035.

[0251] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3036 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3036.

[0252] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3037 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3037.

[0253] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3038 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3038.

[0254] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3039 or aAttorney Docket No.: 2011256-2608 (P1879PCT01) nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3039.

[0255] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3040 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3040.

[0256] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3041 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3041.

[0257] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3042 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3042.

[0258] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3043 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3043.

[0259] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3002 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3002.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0260] In some embodiments, a dystrophin (e.g., a micro-dystrophin) payload comprises the amino acid sequence of SEQ ID NO: 3005 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3005.

[0261] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3004 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3004.

[0262] In some embodiments, a dystrophin (e.g., a micro- dystrophin) payload comprises the amino acid sequence of SEQ ID NO: 3007 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3007.

[0263] In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises the nucleic acid sequence of SEQ ID NO: 3006 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3006.

[0264] Exemplary dystrophin (e.g., a micro-dystrophin) payload sequences are provided in Table 1. In some embodiments, a dystrophin (e.g., a micro-dystrophin) payload comprises an amino acid sequence provided in Table 1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to an amino acid sequence provided in Table 1. In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises a codon-optimized nucleic acid sequence. In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a microdystrophin) payload comprises a nucleic acid sequence provided in Table 1 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%,Attorney Docket No.: 2011256-2608 (P1879PCT01) at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a nucleic acid sequence provided in Table 1. In some embodiments, a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload comprises a codon-optimized sequence of a nucleotide sequence provided in Table 1.

[0265] Methods of codon-optimization are known in the field and could be readily ascertained by those with ordinary skill in the relevant art. Exemplary algorithms that can be used for codon-optimization are available from companies such as: IDT, GeneWiz (Azenta Life Sciences, Vector Builder, GenScript, GeneArt (Thermo Fisher), and Twist Bioscience.Table 1: Exemplary Dystrophin SequencesAttorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0266] As will be appreciated by those with knowledge in the field, additional exemplary micro-dystrophin proteins disclosed in Table 2 of Duan 2018 (hereby expressly incorporated by reference) can also be useful in rAAV particles disclosed herein.Exemplary muscle-specific promoters

[0267] Among other things, provided herein are muscle-specific promoters for use in a nucleic acid comprising nucleotide sequences encoding a dystrophin (e.g., a micro-dystrophin) payload.

[0268] In some embodiments, a nucleic acid comprising a nucleotide sequence encoding a dystrophin (e.g., a micro-dystrophin) payload further comprises a promoter. In some embodiments, a promoter is a muscle-specific promoter. In some embodiments, a promoter comprises a promoter sequence provided in Table 2 or a nucleic acid sequence having at leastAttorney Docket No.: 2011256-2608 (P1879PCT01)80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to a promoter sequence provided in Table 2.

[0269] In some embodiments, a muscle-specific promoter is a MHCK7 promoter. In some embodiments, a muscle-specific promoter comprises the nucleic acid sequence of SEQ ID NO: 3010 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3010.

[0270] In some embodiments, a muscle-specific promoter is a CK8 promoter. In some embodiments, a muscle-specific promoter comprises the nucleic acid sequence of SEQ ID NO: 3008 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3008.

[0271] In some embodiments, a muscle-specific promoter is a SPc5-12 promoter. In some embodiments, a muscle-specific promoter comprises the nucleic acid sequence of SEQ ID NO: 3011 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3011.

[0272] In some embodiments, a muscle-specific promoter is a desmin promoter.

[0273] In some embodiments, a muscle-specific promoter is a tMCK promoter.

[0274] In some embodiments, a muscle-specific promoter is a dMCK promoter.

[0275] In some embodiments, a muscle-specific promoter comprises the nucleic acid sequence of SEQ ID NO: 3009 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity to the sequence of SEQ ID NO: 3009.

[0276] In some embodiments, a muscle-specific promoter is a CK6 promoter.Table 2: Exemplary Promoter SequencesAttorney Docket No.: 2011256-2608 (P1879PCT01)Attorney Docket No.: 2011256-2608 (P1879PCT01)AAV9

[0277] Adeno-associated viruses (AAVs) are small, nonenveloped, single-stranded DNA (ssDNA) viruses that belong to the Parvoviridae family. At least twelve distinct AAV serotypes have been identified from human and nonhuman primate sources (see DiMattia MA et al., (2012) J. Virology 86( 12): 6947, the entire contents of which is hereby incorporated by reference).AAV9 is one of the human AAV serotypes that has enhanced transduction efficiency in cardiac and skeletal muscle, liver tissue, pancreatic tissue, and the eye compared to other serotypes (DiMattia 2012).

[0278] The AAV wild-type genome contains at least three genes, rep, cap and X (Buning and Srivastava, (2019) Molecular Therapy: Methods & Clinical Development ' vol. 12 pages 248- 265). The cap gene encodes for viral proteins VP1, VP2, and VP3, and assembly-activating protein (AAP). All three VP proteins are capsid monomers. Transcription of the cap gene results in two messenger RNA: a messenger RNA which encodes VP1 and a messenger RNA which encodes VP2 and VP3 (as described in Warrington KH et al., (2004) Journal of Virology volume 78(12) pages 6595-6609). VP1, VP2, and VP3 are present at ratios of 1 : 1 : 10, respectively. The VP3 region is observed in all capsid structures of AAV serotypes that have been studied (DiMattia 2012).

[0279] VPs comprise beta strands, alpha helical regions, and structurally variable regions (VRs) in the surface loops which connect the beta strands. Without wishing to be bound by any particular theory, it is believed that differences in sequence and / or conformations of VRs contribute to the variability in cellular tropism, differences in tissue transduction efficiently,Attorney Docket No.: 2011256-2608 (P1879PCT01) and / or antigenic reactivity among different AAV serotypes. In some embodiments, differences in VR sequence and / or structure among different AAV serotypes allow for differential recognition of cell surface glycans and / or tissue specific protein or lipid receptor interaction for internalization.

[0280] Wild type AAV9 (WT AAV9) has nine variable regions VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX (DiMattia 2012, see also Table 3 therein). AAV9 VR-1 encompasses amino acid positions 262-269. AAV VR-II encompasses amino acid positions 327-332 and has a role, e.g., in genome packaging. AAV9 VR-III encompasses amino acid positions 382-386. AAV9 VR-IV encompasses amino acid positions 452-460 and has a role, e g., in liver transduction and / or a delayed blood clearance phenotype. AAV9 VR-V encompasses amino acid positions 488-505 and has a role, e.g., in LamR receptor binding, liver and / or muscle-specific transduction, and / or a delayed blood clearance phenotype. AAV9 VR-VI encompasses amino acid positions 527-539 and has a role, e.g., in LamR receptor binding, and / or a delayed blood clearance phenotype. AAV9 VR-VII encompasses amino acid positions 545-558 and has a role, e.g., in liver transduction and / or delayed blood clearance phenotype. AAV9 VR- VIII encompasses amino acid positions 581-593 and has a role, e.g., in LamR receptor binding and / or transduction. AAV9 VR-IX encompasses amino acid positions 704-714 and has a role, e.g., in heart tropism, melanoma tropism and / or altered tropism.

[0281] In some embodiments, a rAAV particle disclosed herein is a recombinant AAV (rAAV) particle. In some embodiments, a rAAV particle comprises a variant AAV9 capsid protein comprising a peptide insertion disclosed herein. In some embodiments, a peptide insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of a parental AAV capsid protein.

[0282] In some embodiments, a parental AAV capsid protein comprises the sequence of a wildtype AAV capsid protein, or a sequence having at least 95% identity to the sequence of a wildtype AAV capsid protein, or a sequence having no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to the sequence of a wildtype AAV capsid protein. In some embodiments, aAttorney Docket No.: 2011256-2608 (P1879PCT01) parental AAV capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein.

[0283] In some embodiments, a parental AAV capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to the sequence of a wildtype AAV capsid protein and one or more mutations, e.g., as disclosed herein.

[0284] In some embodiments, one or more mutations comprises a mutation that alters a binding profde of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation.

[0285] In some embodiments, a parental AAV capsid protein is other than an AAV9 capsid protein and comprises one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) at a position of VP1, VP2 or VP3 corresponding to position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein.

[0286] In some embodiments, one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more mutations reduces glycan binding. In some embodiments, a glycan is galactose.

[0287] In some embodiments, one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a correspondingAttorney Docket No.: 2011256-2608 (P1879PCT01) position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP 1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f).

[0288] In some embodiments, one or more mutations comprises a Tryptophan (W) to Arginine (R) mutation at amino acid 503 (W503R mutation). In some embodiments, an AAV capsid protein disclosed herein comprises a Arginine (R) at amino acid 503.

[0289] In some embodiments, a parental AAV capsid protein is chosen from: an AAV9 capsid protein, an AAV1 capsid protein, an AAV2 capsid protein, an AAV3B capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, or an AAVrh74 capsid protein.

[0290] In some embodiments, a parental AAV capsid protein comprises: an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises: the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.

[0291] In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0292] In some embodiments, one or more mutations comprises a mutation that alters a binding profde of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. Exemplary mutations including liver de-targeting mutations are disclosed in Pulicherla N. et al., (2011) Molecular Therapy volume 19, pages 1070-1078, the entire contents of which are hereby incorporated by reference.

[0293] In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VPlof an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de- targeting mutation) comprises a mutation at position 503, e.g., a W5O3R mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e g., a liver de-targeting mutation) comprises a mutation at position 595, e.g., a W595C mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 457, e g., a N457H mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 574, e.g., a T574S mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 592, e.g., a Q592L mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 498, e.g., a N498Y or an N498I mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 602, e.g., a L602F mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 468, e.g., a P468T mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 500, e.g., a E500D mutation.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0294] In some embodiments, one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more mutations reduces glycan binding. In some embodiments, a glycan is galactose.

[0295] In some embodiments, one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP 1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f).

[0296] In some embodiments, a parental AAV capsid protein comprises an AAV1 capsid protein. In some embodiments, an AAV1 capsid protein sequence is provided in SEQ ID NO:2002.

[0297] In some embodiments, a parental AAV capsid protein comprises an AAV2 capsid protein. In some embodiments, an AAV2 capsid protein sequence is provided in SEQ ID NO:2003.

[0298] In some embodiments, a parental AAV capsid protein comprises an AAV3B capsid protein. In some embodiments, an AAV3B capsid protein sequence is provided in SEQ ID NO: 2050.

[0299] In some embodiments, a parental AAV capsid protein comprises an AAV4 capsid protein. In some embodiments, an AAV4 capsid protein sequence is provided in SEQ ID NO: 2051.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0300] In some embodiments, a parental AAV capsid protein comprises an AAV5 capsid protein. In some embodiments, an AAV5 capsid protein sequence is provided in SEQ ID NO:2004.

[0301] In some embodiments, a parental AAV capsid protein comprises an AAV6 capsid protein. In some embodiments, an AAV6 capsid protein sequence is provided in SEQ ID NO:2005.

[0302] In some embodiments, a parental AAV capsid protein comprises an AAV7 capsid protein. In some embodiments, an AAV7 capsid protein sequence is provided in SEQ ID NO: 2052.

[0303] In some embodiments, a parental AAV capsid protein comprises an AAV8 capsid protein. In some embodiments, an AAV8 capsid protein sequence is provided in SEQ ID NO:2006.

[0304] In some embodiments, a parental AAV capsid protein comprises an AAV10 capsid protein. In some embodiments, an AAV10 capsid protein sequence is provided in SEQ ID NO: 2053.

[0305] In some embodiments, a parental AAV capsid protein comprises an AAV11 capsid protein. In some embodiments, an AAV11 capsid protein sequence is provided in SEQ ID NO: 2054.

[0306] In some embodiments, a parental AAV capsid protein comprises an AAV12 capsid protein. In some embodiments, an AAV12 capsid protein sequence is provided in SEQ ID NO: 2055.

[0307] In some embodiments, a parental AAV capsid protein comprises an AAV13 capsid protein. In some embodiments, an AAV 13 capsid protein sequence is provided in SEQ ID NO: 2056.

[0308] In some embodiments, a parental AAV capsid protein comprises an AAVrh74 capsid protein. In some embodiments, an AAVrh74 capsid protein sequence is provided in SEQ ID NO: 2057.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0309] In some embodiments, a peptide insertion is in VR-I of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV 12, AAV 13 or AAVrh74.

[0310] In some embodiments, a peptide insertion is in VR-II of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV 12, AAV 13 or AAVrh74.

[0311] In some embodiments, a peptide insertion is in VR-III of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV 12, AAV 13 or AAVrh74.

[0312] In some embodiments, a peptide insertion is in VR-IV of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV 12, AAV13 or AAVrh74.

[0313] In some embodiments, a peptide insertion is in VR-V of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV 12, AAV 13 or AAVrh74.

[0314] In some embodiments, a peptide insertion is in VR-VI of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV 12, AAV 13 or AAVrh74.

[0315] In some embodiments, a peptide insertion is in VR-VII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV 12, AAV 13 or AAVrh74.

[0316] In some embodiments, a peptide insertion is in VR-VIII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV 12, AAV 13 or AAVrh74.

[0317] In some embodiments, a peptide insertion is in VR-IX of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV 12, AAV13 or AAVrh74.

[0318] In some embodiments, a parental AAV capsid protein is chosen from an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12,Attorney Docket No.: 2011256-2608 (P1879PCT01)AAV13 or AAVrh74capsid protein, and VR-VIIT comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding positions in the capsid proteins of another parental AAV capsid protein, e.g., an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74 capsid protein.

[0319] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein.

[0320] In some embodiments, a peptide insertion is in a VP (e.g., VP1, VP2, and / or VP3) of a parental AAV capsid protein.

[0321] In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of a parental AAV capsid protein (e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13 or AAVrh74).

[0322] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of an AAV9 capsid protein.

[0323] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP2 of an AAV9 capsid protein.

[0324] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP3 of an AAV9 capsid protein.

[0325] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 and VP3 of an AAV9 capsid protein.

[0326] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0327] In some embodiments, a peptide insertion site is located between two adjacent amino acids in VR-VIII of a parental AAV capsid protein.

[0328] In some embodiments, a peptide insertion site is located between two nonadj acent amino acids in VR-VIII of a parental AAV capsid protein.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0329] In some embodiments, insertion of a heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein.

[0330] In some embodiments, insertion of a heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein.

[0331] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid protein, e.g., as compared to a WT AAV9 capsid protein. In some embodiments, an AAV9 WT capsid protein sequence is provided in SEQ ID NO: 2001. In some embodiments, a variant AAV9 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV9 capsid protein.

[0332] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV1 capsid protein, e.g., as compared to a WT AAV1 capsid protein. In some embodiments, an AAV1 WT capsid protein is provided in SEQ ID NO: 2002. In some embodiments, a variant AAV1 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV1 capsid.

[0333] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV2 capsid protein, e.g., as compared to a WT AAV2 capsid protein. In some embodiments, an AAV2 WT capsid protein is provided in SEQ ID NO: 2003. In some embodiments, a variant AAV2 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV2 capsid protein.

[0334] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV3B capsid protein, e.g., as compared to a WT AAV3B capsid protein. In some embodiments, an AAV3B WT capsid protein is provided in SEQ ID NO: 2050. In some embodiments, a variant AAV3B capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV3B capsid protein.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0335] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV5 capsid protein, e.g., as compared to a WT AAV5 capsid protein. In some embodiments, an AAV5 WT capsid protein is provided in SEQ ID NO: 2004. In some embodiments, a variant AAV5 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV5 capsid protein.

[0336] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV6 capsid protein, e.g., as compared to a WT AAV6 capsid protein. In some embodiments, an AAV6 WT capsid protein is provided in SEQ ID NO: 2005. In some embodiments, a variant AAV6 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV6 capsid protein.

[0337] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV8 capsid protein, e.g., as compared to a WT AAV8 capsid protein. In some embodiments, an AAV8 WT capsid protein is provided in SEQ ID NO: 2006. In some embodiments, a variant AAV8 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV8 capsid protein.

[0338] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV4 capsid protein, e.g., as compared to a WT AAV4 capsid protein. In some embodiments, an AAV4 WT capsid protein is provided in SEQ ID NO: 2051. In some embodiments, a variant AAV4 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV4 capsid protein.

[0339] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV7 capsid protein, e.g., as compared to a WT AAV7 capsid protein. In some embodiments, an AAV7 WT capsid protein is provided in SEQ ID NO: 2052. In some embodiments, a variant AAV7 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV7 capsid protein.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0340] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV10 capsid protein, e.g., as compared to a WT AAV10 capsid protein. In some embodiments, an AAV10 WT capsid protein is provided in SEQ ID NO: 2053. In some embodiments, a variant AAV10 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV 10 capsid protein.

[0341] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV1 1 capsid protein, e.g., as compared to a WT AAV11 capsid protein. In some embodiments, an AAV11 WT capsid protein is provided in SEQ ID NO: 2054. In some embodiments, a variant AAV11 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV11 capsid protein.

[0342] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV12 capsid protein, e.g., as compared to a WT AAV12 capsid protein. In some embodiments, an AAV12 WT capsid protein is provided in SEQ ID NO: 2055. In some embodiments, a variant AAV12 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV12 capsid protein.

[0343] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV13 capsid protein, e g., as compared to a WT AAV13 capsid protein. In some embodiments, an AAV13 WT capsid protein is provided in SEQ ID NO: 2056. In some embodiments, a variant AAV13 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV13 capsid protein.

[0344] In some embodiments, a rAAV particle disclosed herein comprises a variant AAVrh74 capsid protein, e.g., as compared to a WT AAVrh74 capsid protein. In some embodiments, an AAVrh74 WT capsid protein is provided in SEQ ID NO: 2057. In some embodiments, a variant AAVrh74 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAVrh74 capsid protein.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0345] Additional modifications to an AAV9 capsid protein (not including peptide insertions disclosed herein) are possible including, for example, variants disclosed in International Patent Application WO 2003 / 052052 filed on November 12, 2002, the entire contents of which are hereby incorporated by reference. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein and one or more AAV9 capsid modifications disclosed in WO 2003 / 052052.

[0346] Several other reports disclose modifications to an AAV9 capsid protein, including: Pulicherla N. et al., (2011) Mol Ther. 19(6): pp. 1070-1078; Wang D. et al., (2018) Mol Ther Methods Clin Dev. (9): pp. 234-246; Adachi K. et al., (2014) Nat. Comm. (5): art.3075; or Bell CL. Et al ., (2012) J Virol. 86(13): pp. 7326-7333, the entire contents each of which are hereby incorporated by reference. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein and one or more AAV9 capsid modifications disclosed in any of the reports referenced herein.AAV particles comprising AAV9 capsid variants with peptide insertion

[0347] Among other things, disclosed herein, are rAAV particles comprising a AAV capsid protein variants having one or more peptide insertions, e.g., as disclosed herein, and a dystrophin payload.

[0348] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a peptide insertion disclosed herein. In some embodiments, a peptide insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of AAV9.

[0349] In some embodiments, a peptide insertion is in VR-I of an AAV9 capsid protein.

[0350] In some embodiments, a peptide insertion is in VR-II of an AAV9 capsid protein.

[0351] In some embodiments, a peptide insertion is in VR-III of an AAV9 capsid protein.

[0352] In some embodiments, a peptide insertion is in VR-IV of an AAV9 capsid protein.

[0353] In some embodiments, a peptide insertion is in VR-V of an AAV9 capsid protein.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0354] In some embodiments, a peptide insertion is in VR-VI of an AAV9 capsid protein.

[0355] In some embodiments, a peptide insertion is in VR-VII of an AAV9 capsid protein.

[0356] In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein.

[0357] In some embodiments, a peptide insertion is in VR-IX of an AAV9 capsid protein.

[0358] In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion site is located between amino acids 580-585, amino acids 585-590, amino acids 590-595, or amino acids 595-601 of VP1, VP2 or VP3 of AAV9. In some embodiments, a peptide insertion site is located between amino acids 580-581, between amino acids 581-582, between amino acids 582-583, between amino acids 583-584, between amino acids 584-585, between amino acids 585-586, between amino acids 586-587, between amino acids 587-588, between amino acids 588-589, between amino acids 589-590, between amino acids 590-591, between amino acids 591-592, between amino acids 592-593, between amino acids 593-594, between amino acids 594-595, between amino acids 595-596, between amino acids 596-597, between amino acids 597-598, between amino acids 598-599, between amino acids 599-600, or between amino acids 600-601 of VP1, VP2 or VP3 of an AAV9 capsid protein.

[0359] In some embodiments, a peptide insertion site is located between amino acids 588 and 589 of VP1, VP2 or VP3 of an AAV9 capsid protein.

[0360] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 of an AAV9 capsid protein. In some embodiments, aAttorney Docket No.: 2011256-2608 (P1879PCT01) peptide insertion is located between amino acids 588 and 589 of a VP3 of an AAV9 capsid protein.

[0361] In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP 1 and VP2 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP2 and VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1 and VP3 of an AAV9 capsid protein. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0362] In some embodiments, a peptide insertion site is located between two adjacent amino acids in VR-VIII of an AAV9 capsid protein.

[0363] In some embodiments, a peptide insertion site is located between two nonadj acent amino acids in VR-VIII of an AAV9 capsid protein.

[0364] In some embodiments, insertion of a heterologous peptide replaces a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein.

[0365] In some embodiments, insertion of a heterologous peptide does not replace a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein.

[0366] In some embodiments, a rAAV particle comprises: (1) a variant AAV9 capsid protein comprising a peptide insertion disclosed herein, and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein. In some embodiments, a peptide insertion comprises a sequence provided in Table 7.

[0367] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV capsid protein comprising a consensus sequence of any one of SEQ ID NOs: 1-10 or any one of SEQ ID NOs: 2026-2034, and (2) one or more sequences of a VP (e.g., VP1, VP2, or VP3) of an AAV9 capsid protein.

[0368] In some embodiments, a peptide insertion does not comprise an additional sequence N-terminal of a peptide sequence provided in Table 7.

[0369] In some embodiments, a peptide insertion does not comprise an additional sequence C-terminal of a peptide sequence provided in Table 7.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0370] In some embodiments, a peptide insertion does not comprise an additional sequence N-terminal and C-terminal of a peptide sequence provided in Table 7.

[0371] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDXIX2R[W / F] (SEQ ID NO: 2026), wherein Xi and X2 are independently any amino acid.

[0372] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W / F]QXiX2 (SEQ ID NO: 2027), wherein Xi and X2 are independently any amino acid.

[0373] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F, with the proviso that the peptide insertion does not comprise RGDYQAV (SEQ ID NO: 1764).

[0374] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDHXIX2[W / F] (SEQ ID NO: 2028), wherein Xi and X2 are independently any amino acid.

[0375] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDPXiX2[W / F] (SEQ ID NO: 2029), wherein Xi and X2 are independently any amino acid.

[0376] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion inAttorney Docket No.: 2011256-2608 (P1879PCT01) an AAV capsid protein disclosed herein comprises a sequence of RGD[W / F]XIX2V (SEQ ID NO: 2030), wherein Xi and X2 are independently any amino acid.

[0377] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1QX2X3 (SEQ ID NO: 6), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDXIQX2[W / F] (SEQ ID NO: 2031), wherein Xi and X2 are independently any amino acid.

[0378] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W / F]XISX2 (SEQ ID NO: 2032), wherein Xi and X2 are independently any amino acid.

[0379] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of LRGDXIX2[W / F] (SEQ ID NO: 2033), wherein Xi and X2 are independently any amino acid.

[0380] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein Xi is Y, W, or F, and X2 is any amino acid. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGD[W / F]GLX (SEQ ID NO: 2034) wherein X is any amino acid. In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYGLX (SEQ ID NO: 2035), wherein X is any amino acid.

[0381] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein Xi is E or R, X2 is E or R, and X3 is V or I.

[0382] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2063), wherein Xi is E, X2 is E or R, and X3 is V or I.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0383] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2068), wherein Xi is R, X2 is E or R, and X3 is V or I.

[0384] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2064), wherein Xi is E or R, X2 is E, and X3 is V or I.

[0385] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2067), wherein Xi is E or R, X2 is R, and X3 is V or I.

[0386] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2066), wherein Xi is E or R, X2 is E or R, and X3 is V.

[0387] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 2065), wherein Xi is E or R, X2 is E or R, and X is I.

[0388] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYERI (SEQ ID NO: 1551). In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein consists of the sequence of RGDYERI (SEQ ID NO: 1551).

[0389] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2047 as shown below:

[0390] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNL TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNAttorney Docket No.: 2011256-2608 (P1879PCT01)QQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYERIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPH TDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL.

[0391] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein Xi is E or R, X2 is E or R, and X3 is V or I with the proviso that the peptide insertion is not NATRGDYREI (SEQ ID NO: 2069).

[0392] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein Xi is E or R, X2 is E or R, and X3 is V or I with the proviso that the peptide insertion is not RRGDYREIPL (SEQ ID NO: 2059).

[0393] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYREI (SEQ ID NO: 1825). In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein consists of the sequence of RGDYREI (SEQ ID NO: 1825).

[0394] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2046 as shown below:

[0395] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLP GYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPI<RLNFI<LFNIQVI<EVTDNNGVKTIANNL TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKF S VAGP SNMAVQGRNYIPGP S YRQQRVSTT VTQNNNSEF AWPGAS S WALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYREIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHAttorney Docket No.: 2011256-2608 (P1879PCT01)TDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0396] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include a T residue immediately upstream (or 5’) of the peptide insertion.

[0397] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include an N residue at the third position immediately upstream (or 5’) of the peptide insertion.

[0398] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include a R residue immediately upstream (or 5’) of the peptide insertion.

[0399] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include a P residue immediately downstream (or 3’) of the peptide insertion.

[0400] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprising or having a sequence of RGDYREI (SEQ ID NO: 1825) does not include an L residue at the second position immediately downstream (or 3’) of the peptide insertion.

[0401] In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein comprises a sequence of RGDYREV (SEQ ID NO: 1829). In some embodiments, a peptide insertion in an AAV capsid protein disclosed herein consists of the sequence of RGDYREV (SEQ ID NO: 1829).

[0402] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the sequence of SEQ ID NO: 2058 as shown below:

[0403] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLAttorney Docket No.: 2011256-2608 (P1879PCT01)TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYREVAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0404] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the amino acid sequence of SEQ ID NO: 3032 as shown below.MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSDYQLPYVLGSAHEGCLPPFP AD VFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSV AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFARPGASSWALNGRNSLMNPGPA MASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVAT NHQSAQRGDYREIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPS PLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENS KRWNPEIQ YT SNYYK SNNVEF A VNTEGVYSEPRPIGTRYLTRNL

[0405] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising the amino acid sequence of SEQ ID NO: 3033 as shown below.

[0406] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERL KEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSG AQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLAttorney Docket No.: 2011256-2608 (P1879PCT01)TSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCL EYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQN QQTLKF S V AGP SNMAVQGRNYIPGP S YRQQRVSTT VTQNNNSEF ARPGAS S W ALNGRN SLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVAT ESYGQVATNHQSAQRGDYERIAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIE WELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

[0407] In some embodiments, an AAV capsid protein disclosed herein comprises a Tryptophan (W) to Arginine (R) mutation at amino acid 503 (e.g., W503R mutation). In some embodiments, an AAV capsid protein disclosed herein comprises a Arginine (R) at amino acid 503.

[0408] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid protein comprising a peptide insertion disclosed in Table 7 and one or more modifications to an amino acid sequence flanking the peptide insertion site. In some embodiments, one or more modifications are within about 10 amino acids, e.g., within about 5 amino acids, upstream or downstream of the location of the peptide insertion site.

[0409] In some embodiments, one or more modifications are located in a variable region IV (VR-IV) of a VP1, VP2, or VP3 of an AAV9 capsid protein, or a variable region V (VR-V) of VP1, VP2, or VP3 of an AAV9 capsid protein, or both.

[0410] In some embodiments, VR-IV of a VP1, VP2, or VP3 of an AAV9 capsid protein comprises amino acids 451-475 of a VP1, VP2, or VP3 of an AAV9 capsid protein.

[0411] In some embodiments, VR-V of a VP1, VP2, or VP3 of an AAV9 capsid protein comprises amino acids 488-506 of a VP1, VP2, or VP3 of an AAV9 capsid protein.

[0412] In some embodiments, one or more modifications comprises an insertion, deletion, mutation, or a combination thereof.

[0413] In some embodiments, a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more modifications reduces glycan binding. In some embodiments, a glycan is galactose.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0414] In some embodiments, one or more modifications is at or between amino acids: (a) 271 and 272 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f).Exemplary rAAV particles comprising capsid variants with peptide insertions in VR-VIII and a dystrophin payload

[0415] In some embodiments, a rAAV particle disclosed herein comprises: a capsid variant (A), and a heterologous nucleic acid comprising: (i) a nucleotide sequence encoding a dystrophin payload (B), and (ii) a promoter sequence (C), as provided in Table 4.

[0416] In Table 4, A is a capsid variant, B is a dystrophin payload, and C is a promoter. Three capsid variants are provided in Table 4: Al represents RGDYREI (SEQ ID NO: 1825), A2 represents RGDYERI (SEQ ID NO: 1551), A3 represents RGDYREV (SEQ ID NO: 1829). Four dystrophin payloads are provided in Table 4: Bl represents Dys3978 (SEQ ID NO: 3001), B2 represents Dys3579 (SEQ ID NO: 3003), B3 represents Dys3810 (SEQ ID NO: 3005), and B4 represents Dys4017 (SEQ ID NO: 3007). Four promoters are provided in Table 4: Cl represents CK8 promoter (SEQ ID NO: 3008), C2 represents MCK promoter (SEQ ID NO: 3009), C3 represents MHCK7 promoter (SEQ ID NO: 3010), and C4 represents Spc5-12 promoter (SEQ ID NO: 3011). For example, the combination A1+B1+C1 as shown in Table 4 represents an rAAV particle comprising: a variant AAV capsid protein with an (Al) RGDYREI peptide insertion, and a heterologous nucleic acid comprising (i) a nucleotide sequence encoding a (Bl) Dys3978 payload and (ii) a (Cl) CK8 promoter.Table 4: Exemplary capsid, dystrophin, and promoter combinations.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0417] In some embodiments, a rAAV particle disclosed herein comprises: a capsid variant (A), and a heterologous nucleic acid comprising: (i) a nucleotide sequence (D) encoding a dystrophin payload, and (ii) a promoter sequence (C), as provided in Table 5.

[0418] In Table 5, A is a capsid variant, D is a nucleotide sequence encoding a dystrophin payload, and C is a promoter. Three capsid variants are provided in Table 5: Al represents RGDYREI (SEQ ID NO: 1825), A2 represents RGDYERI (SEQ ID NO: 1551), A3 represents RGDYREV (SEQ ID NO: 1829). Four nucleotide sequences encoding microdystrophin payload Dys3579 (SEQ ID NO: 3003) are provided in Table 5: DI represents Dys3579 (SEQ ID NO: 3002), D2 represents Dysl331 (SEQ ID NO: 3035), D3 represents Dysl332 (SEQ ID NO: 3036), and D4 represents Dysl335 (SEQ ID NO: 3037). Four promoters are provided in Table 5: Cl represents CK8 promoter (SEQ ID NO: 3008), C2 represents MCK promoter (SEQ ID NO: 3009), C3 represents MHCK7 promoter (SEQ ID NO: 3010), and C4 represents Spc5-12 promoter (SEQ ID NO: 3011). For example, the combination A1+D1+C1 as shown in Table 5 represents an rAAV particle comprising: a variant AAV capsid protein with an (Al) RGDYREI peptide insertion, and a heterologous nucleic acid comprising (i) a (DI) Dys3579 nucleotide sequence (SEQ ID NO: 3002) encoding a dystrophin payload and (ii) a (Cl) CK8 promoter.Table 5: Exemplary capsid, dystrophin, and promoter combinations.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0419] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises any one of the peptide insertions disclosed herein; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein.

[0420] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a peptide insertion provided in Table 7; (2) the peptide insertion site is in a VR- VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, noAttorney Docket No.: 2011256-2608 (P1879PCT01) more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.Attorney Docket No.: 2011256-2608 (P1879PCT01)

[0421] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of RGDX1X2RX3 (SEQ ID NO: 1), wherein Xi and X2 are independently any amino acid and X3 is Y, W, or F; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or theAttorney Docket No.: 2011256-2608 (P1879PCT01) corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profde of a parental AAV capsid protein (e.g., a liver detargeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0422] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of RGDX3QX1X2 (SEQ ID NO: 2), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ IDAttorney Docket No.: 2011256-2608 (P1879PCT01)NO: 2001 ; or a sequence having at least 95% identity to SEQ ID NO: 2001 ; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profde of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profde of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profde of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profde of a parental AAV capsid protein (e.g., a liver detargeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophinAttorney Docket No.: 2011256-2608 (P1879PCT01) payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0423] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of RGDHX1X2X3 (SEQ ID NO: 3), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profde of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one orAttorney Docket No.: 2011256-2608 (P1879PCT01) more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver detargeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0424] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of RGDPX1X2X3 (SEQ ID NO: 4), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsidAttorney Docket No.: 2011256-2608 (P1879PCT01) protein. Tn some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver detargeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002,Attorney Docket No.: 2011256-2608 (P1879PCT01)SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0425] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of RGDX3X1X2V (SEQ ID NO: 5), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In someAttorney Docket No.: 2011256-2608 (P1879PCT01) embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver detargeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0426] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAVAttorney Docket No.: 2011256-2608 (P1879PCT01) particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of RGDX1QX2 X3 (SEQ ID NO: 6), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver detargeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleicAttorney Docket No.: 2011256-2608 (P1879PCT01) acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0427] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of RGDX3X1SX2 (SEQ ID NO: 7), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001 ; or a sequence having at least 95% identity to SEQ ID NO: 2001 ; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, atAttorney Docket No.: 2011256-2608 (P1879PCT01) least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver detargeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In someAttorney Docket No.: 2011256-2608 (P1879PCT01) embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0428] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of LRGDX1X2X3 (SEQ ID NO: 8), wherein Xi and X2 are independently any amino acid, and X3 is Y, W, or F; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parentalAttorney Docket No.: 2011256-2608 (P1879PCT01)AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver detargeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0429] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of RGDX1GLX2 (SEQ ID NO: 9), wherein Xi is Y, W, or F, and X2 is any amino acid; (2) the peptide insertion site is in a VR-VIII of a parental AAV capsid protein; and (3) the parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, a peptide insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a peptide insertion is located between amino acids 588 and 589 of VP1, VP2, orAttorney Docket No.: 2011256-2608 (P1879PCT01)VP3 of an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation. In some embodiments, a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001, SEQ ID NO: 3003, SEQ ID NO: 3005 or SEQ ID NO: 3007. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000, SEQ ID NO: 3002, SEQ ID NO: 3004, SEQ ID NO: 3006, SEQ ID NO: 3035, SEQ ID NO: 3036, SEQ ID NO: 3037, SEQ ID NO: 3038, SEQ ID NO: 3039, SEQ ID NO: 3040, SEQ ID NO: 3041, SEQ ID NO: 3042, or SEQ ID NO: 3043. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence ofAttorney Docket No.: 2011256-2608 (P1879PCT01)SEQ ID NO: 3035, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3036, or a sequence having at least 80% identity thereto. In some embodiments, a nucleotide sequence encoding a dystrophin payload comprises the sequence of SEQ ID NO: 3037, or a sequence having at least 80% identity thereto. In some embodiments, a promoter sequence comprises the sequence of any one of SEQ ID NOS: 3008, 3009, 3010, or 3011.

[0430] In some embodiments, a rAAV particle disclosed herein comprises (I) a variant AAV capsid protein and (II) a heterologous nucleic acid comprising (a) a nucleotide sequence encoding a dystrophin payload and (b) a promoter sequence. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a peptide insertion relative to a parental AAV capsid protein. In some embodiments, (1) the peptide insertion comprises a sequence of RGDYX1X2X3 (SEQ ID NO: 10), wherein Xi is E or R, X2 is E or R, X3 is V or I; (2...

Claims

1. Attorney Docket No.: 2011256-2608 (P1879PCT01)CLAIMS1. A recombinant adeno-associated virus (rAAV) particle comprising:(a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a peptide insertion relative to a corresponding parental AAV capsid protein, wherein: (i) the peptide insertion comprises the sequence of: (I) RGDYREI (SEQ ID NO: 1825); or (II) RGDYERI (SEQ ID NO: 1551); and (ii) the peptide insertion site is in a variable region (VR) of the parental AAV capsid protein, and(b) a heterologous nucleic acid comprising a nucleotide sequence encoding a dystrophin payload.

2. The rAAV particle of claim 1, wherein the insertion site is located between two adjacent amino acids in the variable region of the parental AAV capsid protein.

3. The rAAV particle of claim 2, wherein the peptide insertion replaces a contiguous stretch of amino acids of the parental AAV capsid protein.

4. The rAAV particle of any one of the preceding claims, wherein the peptide insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein.

5. The rAAV particle of any one of the preceding claims, wherein the peptide insertion is in VR-VIII of the parental AAV capsid protein.

6. The rAAV particle of claim 5, wherein the parental AAV capsid protein is an AAV9 capsid protein and VR-VIII of AAV9 comprises amino acids 580 to 601 of VP1, VP2 or VP3 of an AAV9 capsid protein.

7. The rAAV particle of claim 5 or 6, wherein the insertion site is located between amino acids 588 and 589 of VP1, VP2 and / or VP3 of an AAV9 capsid protein or the corresponding position in the VP1 of another parental AAV capsid protein.Attorney Docket No.: 2011256-2608 (P1879PCT01)8. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein comprises a peptide insertion comprising the sequence of SEQ ID NO: 1825.

9. The rAAV particle of any one of claims 1-7, wherein the variant AAV capsid protein comprises a peptide insertion comprising the sequence of SEQ ID NO: 1551.

10. The rAAV particle of any one of the preceding claims, wherein the dystrophin payload comprises:(i) an N-terminal domain,(ii) one or more hinge domains,(iii) one or more spectrin-like repeats (SR),(iv) a cysteine-rich domain;(v) a C-terminal domain; or(vi) any combination of (i)-(v).

11. The rAAV particle of claim 10, wherein:(a) the N-terminal domain comprises an actin binding domain;(b) the one or more hinge domains comprise a hinge domain 1 (Hl), a hinge domain 2 (H2), a hinge domain 3 (H3), a hinge domain 4 (H4), or any combination thereof; and / or(c) the one or more SRs comprise:(i) an actin binding domain,(ii) a neuronal nitric oxide synthase (nNOS) binding domain,(iii) a microtubule binding domain,(iv) any combination of (i)-(iii).

12. The rAAV particle of claim 10 or 11, wherein the dystrophin payload comprises at least two, at least three, at least four, or at least five SRs.

13. The rAAV particle of claim 12, wherein the SRs comprise: a spectrin-like repeat 1 (SRI), a spectrin-like repeat 2 (SR2), a spectrin-like repeat 3 (SR3), a spectrin-like repeat 4 (SR4), a spectrin-like repeat 5 (SR5), a spectrin-like repeat 6 (SR6), a spectrin-like repeat 7Attorney Docket No.: 2011256-2608 (P1879PCT01)(SR7), a spectrin-like repeat 8 (SR8), a spectrin-like repeat 9 (SR9), a spectrin-like repeat 10 (SR10), a spectrin-like repeat 11 (SR11), a spectrin-like repeat 12 (SR12), a spectrin-like repeat 13 (SR13), a spectrin-like repeat 14 (SR14), a spectrin-like repeat 15 (SR15), a spectrin-like repeat 16 (SR16), a spectrin-like repeat 17 (SR17), a spectrin-like repeat 18 (SR18), a spectrinlike repeat 19 (SR19), a spectrin-like repeat 20 (SR20), a spectrin-like repeat 21 (SR21), a spectrin-like repeat 22 (SR22), a spectrin-like repeat 23 (SR23), a spectrin-like repeat 24 (SR24), or any combination thereof.

14. The rAAV particle of any one of claims 10-13, wherein the one or more SRs comprise a rod domain and / or are separated by one or more hinge domains.

15. The rAAV particle of any one of claims 10-14, wherein the C-terminal domain comprises a syntrophin binding domain or a fragment or variant thereof, a dystrobrevin binding domain or a fragment or variant thereof, or both, optionally wherein:(a) the syntrophin binding domain comprises an al -syntrophin binding site, a 01- syntrophin binding site or both; and / or(b) the dystrobrevin binding domain comprises an a-dystrobrevin binding site.

16. The rAAV particle of any one of claims 10-15, wherein the dystrophin payload comprises:(i) an N terminal domain,(ii) hinge domains Hl, H2 and H4,(iii) spectrin-like repeats SRI, SR2, SR3, and SR24, and(iv) a cysteine-rich domain.

17. The rAAV particle of claim 16, wherein the dystrophin payload comprises in 5’ to 3’ order: an N terminal domain, a Hl domain, a spectrin-like repeat SRI, a spectrin-like repeat SR2, a spectrin-like repeat SR3, a H2 domain, a spectrin-like repeat SR24, a H4 domain and a cysteine-rich domain.Attorney Docket No.: 2011256-2608 (P1879PCT01)18. The rAAV particle of claim 16 or 17, wherein the nucleotide sequence encoding the dystrophin payload comprises a nucleotide sequence having at least 80% identity to SEQ ID NO: 3002, SEQ ID NO: 3035, SEQ ID NO: 3036 or SEQ ID NO: 3037.

19. The rAAV particle of any one of claims 16-18, wherein the dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3003.

20. The rAAV particle of any one of claims 10-15, wherein the dystrophin payload comprises:(i) an N terminal domain,(ii) hinge domains Hl, H3 and H4,(iii) spectrin-like repeats SRI, SR2, SR22, SR23, and SR24, and(iv) a cysteine-rich domain, optionally wherein the dystrophin payload comprises in 5’ to 3’ order: an N terminal domain, a Hl domain, a spectrin-like repeat SRI, a spectrin-like repeat SR2, a H3 domain, a spectrin-like repeat SR22, a spectrin-like repeat SR23, a spectrin-like repeat SR24, a H4 domain and a cysteine-rich domain.

21. The rAAV particle of claim 20, wherein the nucleotide sequence encoding the dystrophin payload comprises a sequence having at least 80% identity to SEQ ID NO: 3000.

22. The rAAV particle of 20 or 21, wherein the dystrophin payload comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3001.

23. The rAAV particle of any one of the preceding claims, wherein the dystrophin payload comprises a micro-dystrophin.

24. The rAAV particle of any one of the preceding claims, wherein the nucleic acid further comprises a promoter, optionally wherein the promoter is or comprises a muscle specific promoter.Attorney Docket No.: 2011256-2608 (P1879PCT01)25. The rAAV particle of claim 24, wherein the muscle-specific promoter comprises a Spc5- 12, MHCK7, CK8, or MCK promoter.

26. The rAAV particle of any one of the preceding claims, wherein the dystrophin payload is an RNA.

27. The rAAV particle of any one of claims 1-25, wherein the dystrophin payload is a polypeptide.

28. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence flanking the peptide insertion site, optionally wherein the one or more modifications:(a) are within about 5 to 10 amino acids upstream or downstream of the location of the peptide insertion site; and / or(b) reduces glycan binding.

29. The rAAV particle of claim 28, wherein the one or more modifications is at or between amino acids:(a) 271 and 272 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein;(b) 446 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein;(c) 470 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein;(d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein;(e) 489 and 545 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein;(f) 591 and 621 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein;Attorney Docket No.: 2011256-2608 (P1879PCT01)(g) 503 of an AAV9 capsid protein or the corresponding position in the capsid protein of another parental AAV capsid protein, optionally wherein the modification is a W503R modification; or(h) any combination or all of (a)-(g).

30. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein has at least 90% identity relative to a parental AAV capsid protein, optionally wherein percent identity is determined by comparing the sequence of the variant AAV capsid protein without the peptide insertion, with the parental AAV capsid protein.

31. The rAAV particle of any one of the preceding claims, wherein the parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001.

32. A pharmaceutical composition comprising:(a) a rAAV particle of any one of the preceding claims; and(b) a pharmaceutically acceptable excipient.

33. A method of delivering a dystrophin payload to a muscle cell, comprising administering the pharmaceutical composition of claim 32 to the muscle cell.

34. The method of claim 33, wherein the muscle cell is from a subject that has, or has been determined to have, a muscle disorder.

35. A method of treating a subject having a muscle disorder and / or ameliorating a symptom of a muscle disorder in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 32.

36. The method of claim 34 or 35, wherein the muscle disorder is Duchenne Muscular Dystrophy.Attorney Docket No.: 2011256-2608 (P1879PCT01)37. The method of any one of claims 33-36, wherein administration of the pharmaceutical composition delivers the nucleotide sequence encoding a dystrophin payload to a muscle cell.

38. The method of any one of claims 33-37, wherein administration of the pharmaceutical composition confers at least 5-fold increased infectivity and / or transduction of a muscle cell compared to the infectivity and / or transduction of the muscle cell when a pharmaceutical composition comprising a control AAV particle comprising the corresponding parental AAV capsid protein is administered.

39. The method of any one of claims 33-38, wherein the muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or any combination thereof.

40. The method of any one of claims 33-39, wherein administration of the pharmaceutical composition increases dystrophin polypeptide level and / or activity in a muscle tissue in the subject as compared to a muscle tissue in a control subject, wherein the control subject:(i) is the same subject prior to administration of the pharmaceutical composition;(ii) is a different subject who has not been administered the pharmaceutical composition;(iii) is the same or different subject who has been administered the pharmaceutical composition at a lower dose; or(iv) is a different subject who has been administered a pharmaceutical composition comprising a control AAV particle comprising the corresponding parental AAV capsid protein at the same dose, optionally wherein the increase in dystrophin polypeptide level and / or activity is about 1.5-fold to about 50-fold.

41. The method of any one of claims 33-40, wherein administration of the pharmaceutical composition increases dystrophin RNA level in a muscle tissue in the subject as compared to a muscle tissue in a control subject, wherein the control subject:(i) is the same subject prior to administration of the pharmaceutical composition;(ii) is a different subject who has not been administered the pharmaceutical composition;Attorney Docket No.: 2011256-2608 (P1879PCT01)(iii) is the same or different subject who has been administered the pharmaceutical composition at a lower dose; or(iv) is a different subject who has been administered a pharmaceutical composition comprising a control AAV particle comprising the corresponding parental AAV capsid protein at the same dose, optionally wherein the increase in dystrophin RNA level is about 1.5-fold to about 500- fold.

42. The method of any one of claims 33-41, wherein administration of the pharmaceutical composition results in:(i) increased muscle force, e.g., increased specific force generating capacity;(ii) increased muscle strength;(iii) reduced muscle fatigue, damage and / or injury;(iv) increased muscle contractility;(v) increased localization of neuronal nitric oxide synthase;(vi) reduced fibrosis;(vii) increased sacrolemmal integrity; or(viii) any combination of (i)-(vii).

43. The method of claim 42, wherein (i)-(vii) is compared to a control subject, wherein the control subject:(i) is the same subject prior to administration of the pharmaceutical composition;(ii) is a different subject who has not been administered the pharmaceutical composition;(iii) is the same or different subject who has been administered the pharmaceutical composition at a lower dose; or(iv) is a different subject who has been administered a pharmaceutical composition comprising a control AAV particle comprising the corresponding parental AAV capsid protein at the same dose.

44. A method of increasing muscle force and / or muscle contractility in a subject, comprising administering to the subject the pharmaceutical composition of claim 32,Attorney Docket No.: 2011256-2608 (P1879PCT01) wherein the subject has or has been diagnosed with having Duchenne Muscular Dystrophy.

45. The method of any one of claims 33-44, wherein the pharmaceutical composition is delivered in combination with one or more additional agents.

46. The method of claim 45, wherein the additional agent is an agent that promotes exon skipping in aDMD gene, optionally wherein the additional agent comprises a gene editing agent, a U7 snRNA, an antisense oligonucleotide (AON), a morpholino, or a 2’-0 methyl oligonucleotide.

47. The method of any one of claims 35-46, wherein the pharmaceutical composition is administered via a route of administration chosen from: intramuscular, intra-ci sterna magna, intravenous, intraarterial, intracoronary, intraparenchymal, subpial, subcutaneous, intradermal, intrathecal, intraperitoneal, or limb perfusion.

48. The method of any one of claims 34-47, wherein the subject is a human.

49. An isolated cell transduced with the rAAV particle of any one of claims 1-31.

50. The isolated cell of claim 49, wherein the cell is a muscle cell, optionally wherein:(a) the muscle cell is chosen from: a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell, a muscle stem cell (e.g., a muscle satellite cell), or any combination thereof; and / or(b) the muscle cell is derived from an induced pluripotent stem cells (iPSC).

51. A composition comprising the rAAV particle of any one of claims 1-31 for use in delivering a dystrophin payload to a muscle cell, the use comprising administering the composition comprising the rAAV particle to the muscle cell.Attorney Docket No.: 2011256-2608 (P1879PCT01)52. Use of a composition comprising the rAAV particle of any one of claims 1 -31 in the manufacture of a medicament for delivering a dystrophin payload to a muscle cell, the use comprising administering the composition comprising the rAAV particle to the muscle cell.

53. A composition comprising the rAAV particle of any one of claims 1-31 for use in treating a subject having a muscle disorder and / or ameliorating a symptom of a muscle disorder in a subject, the use comprising administering the composition comprising the rAAV particle to the subject.

54. Use of a composition comprising the rAAV particle of any one of claims 1-31 in the manufacture of a medicament for treating a subject having a muscle disorder and / or ameliorating a symptom of a muscle disorder in a subject, the use comprising administering the composition comprising the rAAV particle to the subject.

55. The composition for use of claim 53, or the use of claim 54, wherein the subject has, or has been determined to have, a muscle disorder.

56. The composition for use of claim 55, or the use of claim 55, wherein the muscle disorder is Duchenne Muscular Dystrophy.

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