Adeno-associated virus variants targeting the heart and de-targeting liver

Abstract

The application relates to recombinant adeno-associated virus particles comprising variant AAV capsid proteins (e.g., variant capsid proteins of serotype AAVrh74, AAVrh10, AAV8, or AAV1). Recombinant adeno-associated virus particles of the disclosure can be used to deliver exogenous nucleic acids (e.g., nucleic acids comprising genes encoding therapeutic RNAs or therapeutic proteins) to target cells, such as heart cells. In some aspects of the disclosure, the variant capsid proteins that comprise amino acid variations that alter the binding activity of recombinant adeno-associated virus particles to target cells, such as by increasing binding to heart cells and / or decreasing binding to liver cells. In some embodiments, the recombinant adeno-associated virus particles are utilized in a method of treating subject. In some embodiments, such methods are utilized in delivering exogenous nucleic acids to target cells more specifically and / or at lower doses compared to other recombinant adeno-associated particle delivery systems.

Description

[0001] ADENO-ASSOCIATED VIRUS VARIANTS TARGETING THE HEART AND DETARGETING LIVER

[0002] RELATED APPLICATIONS

[0003] The application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application number 63 / 691,973 filed September 6, 2024, and U.S. Provisional Application number 63 / 766,044 filed March 3, 2025, each of which are incorporated by reference in entirety.

[0004] GOVERNMENT SUPPORT

[0005] This invention was made with government support under Grant No. W81XWH-19-1- 0515, awarded by the US Defense Health Agency, Medical Research and Development Branch. The government has certain rights in the invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007] The contents of the electronic sequence listing (U119670111WOOO-SEQ-GJM,. xml; Size: 5,490,183 bytes; and Date of Creation: September 5, 2025) are herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Adeno-associated virus (AAV) has emerged as a leading vector for gene therapy, owing to its favorable properties of safety, efficiency, stability, and persistence. In current applications, therapeutic gene sequences are substituted for viral sequences between ITRs, enabling delivery to target cells by infection. Despite these advantages, AAV-based therapies often require administration of high doses of purified vector, which substantially increases production costs and presents a significant barrier to clinical scalability.

[0010] Existing efforts to address these limitations have focused primarily on improving upstream or downstream manufacturing processes. However, such process optimizations do not address the intrinsic biological constraints of AAV. These limitations can also be addressed through engineering improved AAV systems and / or components. For example, recombinant AAV particles can be engineered to include modified capsids for improving delivery of exogenous nucleic acids to a target cell. The structural features of AAV capsid proteins govern essential properties, including tissue tropism, immunogenicity, and packaging capacity. Thus, continued advancement of AAV-based gene therapy would be facilitated by the development of improved capsid proteins that enhance function, such as targeting tropisms and / or delivery efficiencies.

[0011] #14348316v1 SUMMARY

[0012] In certain aspects of the disclosure, the invention provides variant adeno-associated virus (AAV) capsid proteins that include one or more amino acid substitutions, insertions, or deletions that modulate the binding profile of recombinant AAV particles to target cells. Such engineered capsid proteins can, for example, enhance binding affinity and transduction efficiency for desired cell types, such as cardiomyocytes, while concurrently reducing binding to non-target tissues, such as hepatocytes. By selectively redirecting viral tropism, these variants address limitations of naturally occurring AAV serotypes, which often exhibit broad biodistribution and undesirable liver accumulation.

[0013] In some embodiments, recombinant AAV particles comprising the disclosed variant capsid proteins are employed in methods of treatment for subjects. These methods may involve delivery of exogenous nucleic acids — including therapeutic genes, gene editing systems, or regulatory RNA molecules — to target tissues with greater specificity. In certain embodiments, the modified binding characteristics of the variant capsids enable effective therapeutic outcomes at lower vector doses compared to conventional AAV delivery systems, thereby reducing systemic exposure and potential toxicity. Accordingly, the disclosed capsid variants offer improved safety, efficacy, and translational potential for gene therapy applications, in particular, to cardiac tissues.

[0014] Cardiac adeno-associated virus (AAV) gene therapy can be utilized to treat intractable heart disorders. A focus of AAV therapy is inherited cardiomyopathy affecting over 1 per 500 persons. However, challenges persist with high-dose administration, liver toxicity, and neutralizing antibodies (NAb) for existing heart AAV variants.

[0015] Aspects of the application relate to a variant adeno-associated virus (AAV) capsid protein, not of an AAV1 serotype, wherein a portion of the VP1 sequence has been replaced with a portion of the VP1 sequence of AAV1 (for example, as shown in FIG. 1).

[0016] Aspects of the application relate to a variant adeno-associated virus (AAV) capsid protein (e.g., a variant AAV capsid of serotype AAVrh74) comprising one or more amino acids which are located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229 and are substituted relative to a corresponding amino acid located in a wild-type AAV capsid protein counterpart. For example, in some embodiments, a variant AAV capsid protein comprises one or more of the amino acid substitutions shown in SEQ ID NO: 2, relative to the wildtype sequence of the AAV capsid protein. In some embodiments, the variant AAV capsid protein comprises one or more variable regions (VR) located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229. In some embodiments, the one or more VRs comprises a VR-I, a

[0017] #14348316v1 VR-II, a VR-III, a VR-IV, a VR-V, a VR-VI, a VR-VII, and / or a VR-VIII. In some embodiments, the variant AAV capsid protein is variant of AAV viral protein 1 (VP1). In some embodiments, the variant AAV capsid protein is a variant AAVrh74 VP1, AAVrhlO, a variant AAV8 VP1, or a variant AAV1 VP1 capsid protein. In some embodiments, the variant AAV capsid protein is a variant of AAVrh74, AAVrhlO, AAV8, or AAV1 viral protein 1 (VP1). In some embodiments, the variant AAV capsid protein is a variant AAVrh74 capsid protein comprising any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229.

[0018] Aspects of the application also relate to a recombinant AAV (rAAV) particle comprising: a heterologous nucleic acid flanked by a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR; and a variant AAV capsid protein. In some embodiments, the variant AAV capsid protein is a variant AAVrh74 capsid protein. In some embodiments, the variant AAV capsid protein is a variant AAVrhlO capsid protein. In some embodiments, the variant AAV capsid protein is a variant AAV8 capsid protein. In some embodiments, the variant AAV capsid protein is an AAV 1 capsid protein.

[0019] In some embodiments, the heterologous nucleic acid comprises a gene. In some embodiments, the gene encodes a peptide or protein. In some embodiments, the heterologous nucleic acid comprises a regulatory sequence.

[0020] In some aspects, the application relates to a nucleic acid comprising a sequence encoding a variant AAV capsid protein described herein (e.g., a variant AAVrh74 capsid protein).

[0021] In some aspects, the application relates to a nucleic acid library comprising a plurality of nucleic acids, wherein each nucleic acid of the plurality comprises a sequence encoding any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229. Further aspects of the application relate to a cell or cell population thereof comprising a variant AAV capsid protein, a nucleic acid, or a nucleic acid library described herein. In some embodiments, the cell or cell population comprises: a helper nucleic acid; a nucleic acid comprising an AAV rep gene; and / or a nucleic acid comprising a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR that flank a heterologous nucleic acid. In some embodiments, the cell is a mammalian cell. The application also relates, at least in some aspects, to a composition comprising a variant AAV capsid protein, an rAAV particle, a nucleic acid, a nucleic acid library, or a cell or cell population thereof described herein.

[0022] In some aspects, the application relates to methods. In some embodiments, a method comprising contacting a cell with a variant AAV capsid protein, an rAAV particle, a nucleic acid, a nucleic acid library, or a composition described herein. In some embodiments, a method comprises administering a variant AAV capsid protein or an rAAV particle to a subject. In some embodiments, the subject has or is suspected of having a disease, a disorder, or a

[0023] #14348316v1 condition. In some embodiments, the subject has or is suspected of having a cardiac disease, a cardiac disorder, or a cardiac condition. In some embodiments, the subject has or is suspected of having cardiac myopathy. In some embodiments, the subject is a mammalian subject.

[0024] Further aspects of the application relate to a kit comprising a variant AAV capsid protein, an rAAV particle, a nucleic acid, a nucleic acid library, a cell or cell population thereof, or a composition.

[0025] Aspects of the application relate to a variant adeno-associated virus (AAV) capsid protein (e.g., a variant AAV capsid of serotype AAVrh74) comprising an amino acid sequence that has at least 80%, at least 90%, at least 95%, or at least 99% identity to any one of the amino acid sequences set forth in Table 13a, Table 14a, Table 15a, Table 16a, and Table 17. In some embodiments, the variant AAV capsid protein comprises the amino acid sequence of any one of the amino acid sequences set forth in Table 13a, Table 14a, Table 15a, Table 16a, and Table 17.

[0026] Aspects of the application relate to a variant adeno-associated virus (AAV) capsid protein (e.g., a variant AAV capsid of serotype AAVrh74) comprising an amino acid sequence, wherein a portion of the amino acid sequence has at least 80%, at least 90%, at least 95%, or at least 99% identity to any one of the amino acid sequences set forth in Table 13a, Table 14a, Table 15a, Table 16a, and Table 17. In some embodiments, the variant AAV capsid protein comprises an amino acid sequence wherein a portion of the amino acid sequence comprises any one of the amino acid sequences set forth in Table 13a, Table 14a, Table 15a, Table 16a, and Table 17.

[0027] In some embodiments, the variant AAV capsid protein comprises a variable region 1 (VR-1), or a portion thereof, of an AAV1 capsid protein. In some embodiments, the amino acids corresponding to the sequence NGTSGGSTNDNT (SEQ ID NO: 4252) in AAVrh74 are replaced with the amino acid sequence SASTGASNDNH (SEQ ID NO: 4253). In some embodiments, the amino acid sequence of VPI is substituted as shown in FIG. 1.

[0028] Aspects of the application relate to a recombinant AAV (rAAV) particle comprising (a) a heterologous nucleic acid flanked by a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR; and(b) a variant AAVrh74 capsid protein of the present disclosure. In some embodiments, the heterologous nucleic acid comprises a gene. In some embodiments, the gene encodes a peptide or protein. In some embodiments, the heterologous nucleic acid comprises a regulatory sequence.

[0029] Aspects of the application relate to a nucleic acid comprising a sequence encoding a variant AAV capsid protein of the present disclosure.

[0030] Aspects of the application relate to a cell or cell population thereof comprising a variant AAV capsid protein of the present disclosure or a nucleic acid of the present disclosure. In

[0031] #14348316v1 some embodiments, cell or cell population comprises: (a) a helper nucleic acid; (b) a nucleic acid comprising an AAV rep gene; and / or (c) a nucleic acid comprising a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR that flank a heterologous nucleic acid. In some embodiments, the cell is a mammalian cell.

[0032] Aspects of the application relate to a composition comprising a variant AAV capsid protein of the present disclosure, an rAAV particle of the present disclosure, a nucleic acid of the present disclosure, or a cell or cell population thereof of the present disclosure.

[0033] Aspects of the application relate to a method comprising contacting a cell with a variant AAV capsid protein of the present disclosure, an rAAV particle of the present disclosure, a nucleic acid of the present disclosure, or a composition of the present disclosure. In some embodiments, the method comprises administering a variant AAV capsid protein of the present disclosure or an rAAV particle of the present disclosure to a subject. In some embodiments, the subject has or is suspected of having a disease, a disorder, or a condition. In some embodiments, the subject has or is suspected of having a cardiac disease, a cardiac disorder, or a cardiac condition. In some embodiments, the subject has or is suspected of having cardiac myopathy. In some embodiments, the subject is a mammalian subject.

[0034] Further aspects of the application relate to a kit comprising a variant AAV capsid protein of the present disclosure, an rAAV particle of the present disclosure, a nucleic acid of the present disclosure, a cell or cell population thereof of the present disclosure, or a composition of the present disclosure.

[0035] BRIEF DESCRIPTION OF DRAWINGS

[0036] FIG. 1 shows a scheme of VRI swapping for disruption of liver affinity domain. Briefly, a portion of AAVrh74 VP1 is replaced with the corresponding portion of AAV1 VP1. The resulting sequence is one amino acid shorter than the native AAVrh74 VP1. From top to bottom, SEQ ID NOs: 4333 and 4334)

[0037] FIGs. 2A-2B show vector maps of acceptor backbones with swapped VRI. FIG. 2A shows a vector comprising a TNNT promoter. FIG. 2B shows a vector comprising a MHCK7 promoter.

[0038] FIG. 3 shows loglO enrichments in non-human primate (NHP) heart vs liver for VRI- AAV 1 library design.

[0039] FIG. 4 shows loglO enrichments in NHP heart vs liver for VRI-rh74 library design.

[0040] FIG. 5 shows loglO enrichments in Pig heart vs liver for VRI-AAV1 library design.

[0041] FIG. 6 shows loglO enrichments in Pig heart vs liver for VRI-rh74 library design.

[0042] FIG. 7 shows loglO enrichments in Mouse heart vs liver for VRI-AAV1 library design.

[0043] #14348316v1 FIG. 8 shows LoglO enrichments in Mouse heart vs liver for VRI-rh74 library design.

[0044] FIGs. 9A-9B show sequence alignments of capsid variants of interest with the relevant parental capsid sequence. FIG. 9A shows an alignment relative to AAVrh74_VRI-AAVl. From top to bottom SEQ ID NOs: 1, 4230, 4247, 4246, 4237, 4231, 4232, 4245, 4248, 4249, 4233, 4234, 4236, 4235, 4244)FIG. 9B shows an alignment relative to AAVrh74_VRI-rh74. From top to bottom SEQ ID NOs: 1, 4238, 4243, 4239, 4242, 4240, 4241)

[0045] FIG. 10 shows an alignment of the wild-type VP1 amino acid sequences from serotypes AAVHSC16 (SEQ ID NO: 4322), AAV1 (SEQ ID NO: 4323), AAV2 (SEQ ID NO: 4324), AAV4 (SEQ ID NO: 4325), AAV5 (SEQ ID NO: 4326), AAV7 (SEQ ID NO: 4327), AAV8 (SEQ ID NO: 4328), AAVrh.10 (SEQ ID NO: 4329), and AAV44.9 (SEQ ID NO: 4330).

[0046] DETAILED DESCRIPTION

[0047] Cardiac adeno-associated virus (AAV) gene therapy can be utilized to treat intractable heart disorders. A focus of AAV therapy is inherited cardiomyopathy affecting over 1 per 500 persons. However, challenges persist with high-dose administration, liver toxicity, and neutralizing antibodies (NAb) for existing heart AAV variants. For example, AAV9 is associated with liver toxicity and a moderate level of NAb in the human population while AAVrh74 shows a low level of NAb and moderate cardiac transduction.

[0048] Aspects of the application relate to variants of an AAV capsid protein (alternatively referred to herein as variant AAV capsid proteins) that comprise one or more mutations relative to a wildtype AAV capsid protein counterpart. In some embodiments, a variant AAV capsid protein is a variant of a capsid protein of serotype AAVrh74 (which may be referred to herein as a variant AAVrh74 capsid protein). In some embodiments, a variant AAV capsid protein is a variant of a capsid protein of serotype AAVrhlO (which may be referred to herein as a variant AAVrhlO capsid protein). In some embodiments, a variant AAV capsid protein is a variant of a capsid protein of serotype AAV8 (which may be referred to herein as a variant AAV8 capsid protein). In some embodiments, a variant AAV capsid protein is a variant of a capsid protein of serotype AAV 1 (which may be referred to herein as a variant AAV 1 capsid protein). In some embodiments, a variant AAV capsid protein described herein comprises one or more mutations relative to a wild-type AAV capsid protein counterpart, wherein the one or more mutations result in AAVs (e.g., recombinant AAVs (rAAVs)) having altered tissue tropism relative to AAVs comprising the wild-type AAV capsid protein counterpart. In some embodiments, variant AAVrh74, variant AAVrhlO, variant AAV8, or variant AAV1 capsid proteins comprise one or more liver de-targeting mutations. In some embodiments, mutations in a variant AAV capsid

[0049] #14348316v1 protein (e.g., a variant AAVrh74 capsid protein, a variant AAVrhlO capsid protein, a variant AAV8 capsid protein, or a variant AAV1 capsid protein) enhance heart transduction efficiency and / or reduce liver off-target transduction relative to a wild- type AAV capsid protein counterpart (e.g., a wild-type AAVrh74, AAVrhlO, AAV8, or AAV1 capsid protein counterpart). In some embodiments, a variant AAVrh74, AAVrhlO, AAV8, or AAV1 capsid protein comprises one or more amino acids which are located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229 and substituted relative to a corresponding amino acid position located in a wild-type AAVrh74 capsid protein counterpart (see, e.g., SEQ ID NO: 4269). In some embodiments, the one or more amino acids comprise an amino acid residue which is located at a corresponding amino acid position in an AAV capsid protein of a serotype that is not AAVrh74 (e.g., AAV capsid protein of serotype AAV-rhlO, AAV8, or AAV1).

[0050] In some embodiments, a variant AAVrh74, AAVrhlO, AAV8, or AAV1 capsid protein comprises a variable region (VR) (e.g., a VR I, a VR-II, a VR-III, a VR-IV, a VR-V, a VR-VI, a VR-VII, or a VR-VIII) located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229. In some embodiments, a variant AAVrh74, AAVrhlO, AAV8, or AAV1 capsid protein comprises a plurality of VRs (e.g., two or more of a VR I, a VR-II, a VR-III, a VR-IV, a VR-V, a VR-VI, a VR-VII, and / or a VR-VIII) located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229. In some embodiments, a variant AAVrh74, AAVrhlO, or AAV8 capsid protein comprises a VR-I, or a portion thereof, of an AAV1 capsid protein (see, e.g., FIG. 1). In some embodiments, a variant AAV capsid protein is a variant of a VP1 protein. In some embodiments, a variant AAVrh74 capsid protein is variant of a VP1 protein of serotype AAVrh74. In some embodiments, a variant AAVrhlO capsid protein is a variant of a VP1 protein of a serotype AAVrhlO. In some embodiments, a variant AAV8 capsid protein is a variant of a VP1 protein of a serotype AAV8. In some embodiments, a variant AAV1 capsid protein is a variant of a VP1 protein of a serotype AAV1. In some embodiments, a variant AAVrh74 capsid protein comprises any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229. An example of a wild-type AAVrh74 VP1 amino acid sequence is shown below, wherein the positions in double underline-only are comprised in VR-I, the positions in dashed underline-only are comprised in VR-II, the positions in bold-only are comprised in VR-III, the positions in italics-only are comprised in VR-IV, the positions underline-only are comprised in VR-V, the positions in both italics and bold are comprised in VR-VI, the positions in bold and underline are comprised in VR-VII, the positions in both italics and underline are comprised in VR-VIII, and the positions in bold, italics, and underline are comprised in VR-IX.

[0051] AAVrh74 VP1 amino acid sequence:

[0052] #14348316v1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESP VKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAA GGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKOISNGTSGGSTNDNT YFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEyTQNEGTKTIANNLTSTIQV FTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSY NFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTgSrGGrAGrggLLFSeAGPAWMSAQAKNWLPGPCYR OORVSTTLSONNNSNFAWTGATKYHLNGRDSLVNPGVAMATH DD££RFFPSSGVLMFGKOGAGKDN VDYSSVMETSEEEIKTTNPVATEOYGVVADNEQOGAAAPZVGAVASGGAEPGMVWQNRDVYEOGPIWAKI PHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKR WNPEIQYTSNY YAS7WVI>FAVNTEGTYSEPRPIGTRYLTRNL* (SEQ ID NO: 4269)

[0053] Aspects of the application relate to variants of an AAV capsid protein (alternatively referred to herein as variant AAV capsid proteins) that comprise one or more mutations relative to an AAVrh74_VRl-AAVl VP1 capsid protein counterpart. In some embodiments, a variant AAV capsid protein is a variant of a capsid protein of serotype AAVrh74 (which may be referred to herein as a variant AAVrh74 capsid protein). In some embodiments, a variant AAV capsid protein is a variant of a capsid protein of serotype AAVrhlO (which may be referred to herein as a variant AAVrhlO capsid protein). In some embodiments, a variant AAV capsid protein is a variant of a capsid protein of serotype AAV8 (which may be referred to herein as a variant AAV8 capsid protein). In some embodiments, a variant AAV capsid protein is a variant of a capsid protein of serotype AAV 1 (which may be referred to herein as a variant AAV 1 capsid protein). In some embodiments, variant AAVrh74, variant AAVrhlO, variant AAV8, or variant AAV 1 capsid proteins comprise one or more liver de-targeting mutations. In some embodiments, mutations in a variant AAVrh74 capsid protein, a variant AAVrhlO capsid protein, a variant AAV8 capsid protein, or an AAV1 capsid protein enhance heart transduction efficiency and / or reduce liver off-target transduction relative to a wild-type AAVrh74, AAVrhlO, AAV8, or AAV1 capsid protein counterpart. In some embodiments, a variant AAVrh74, AAVrhlO, AAV8, or AAV1 capsid protein comprises one or more amino acids which are located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229 and substituted relative to a corresponding amino acid position located in a wild-type AAVrh74 capsid protein counterpart (see, e.g., SEQ ID NO: 4254). In some embodiments, the one or more amino acids comprise an amino acid residue which is located at a corresponding amino acid position in an AAV capsid protein of a serotype that is not AAVrh74 (e.g., AAV capsid protein of serotype AAV-rhlO, AAV8, or AAV1).

[0054] In some embodiments, a variant AAVrh74, AAVrhlO, AAV8, or AAV1 capsid protein comprises a variable region (VR) (e.g., a VR I, a VR-II, a VR-III, a VR-IV, a VR-V, a VR-VI, a VR-VII, or a VR-VIII) located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229. In some embodiments, a variant AAVrh74, AAVrhlO, AAV8, or AAV1 capsid protein comprises a plurality of VRs (e.g., two or more of a VR I, a VR-II, a VR-III, a VR-IV, a VR-V,

[0055] #14348316v1 a VR-VI, a VR-VII, and / or a VR-VIII) located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229. In some embodiments, a variant AAVrh74, AAVrhlO, or AAV8 capsid protein comprises a VR-I, or a portion thereof, of an AAV1 capsid protein (see, e.g., FIG. 1). In some embodiments, a variant AAV capsid protein is a variant of a VP1 protein. In some embodiments, a variant AAVrh74 capsid protein is variant of a VP1 protein of serotype AAVrh74. In some embodiments, a variant AAVrhlO capsid protein is a variant of a VP1 protein of a serotype AAVrhlO. In some embodiments, a variant AAV8 capsid protein is a variant of a VP1 protein of a serotype AAV8. In some embodiments, a variant AAV1 capsid protein is a variant of a VP1 protein of a serotype AAV1. In some embodiments, a variant AAVrh74 capsid protein comprises any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229. An example of a AAVrh74_VRl-AAVl VP1 amino acid sequence is shown below.

[0056] AAVrh74 VR1-AAV1 VP1 amino acid sequence: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESP VKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAA GGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYF GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFT DSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNF EDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQ QRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVD YSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIP HTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRW NPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL*(SEQ ID NO: 4254)

[0057] In some embodiments, a variant AAV capsid protein (e.g., a variant AAV VP1, such as a variant AAVrh74 VP1, a variant AAVrhlO VP1, a variant AAV8 VP1, or a variant AAV1 VP1 protein) comprises one or more amino acid mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15- 20, 20-25, 25-30, or more than 30 mutations) relative to an AAV capsid protein counterpart (e.g., wherein the one or more amino acid mutations are relative to a VR-I, VR-II, VR-III, VR- IV, VR-V, VR-VI, VR-VII, VR-VIII, and / or a VR-IX of the AAV capsid protein counterpart). In some embodiments, the AAV capsid protein counterpart is a wild-type AAV capsid protein (e.g., a wild-type AAV capsid protein of an AAV serotype described herein, such as a wild-type AAVrh74 capsid protein). In some embodiments, the variant AAV capsid protein and the AAV capsid protein counterpart are of the same serotype (e.g., wherein the variant AAV capsid protein is a variant AAVrh74 capsid protein and the AAV capsid protein counterpart comprises the amino acid sequence set forth in SEQ ID NO: 4269). In some embodiments, the one or more mutations comprise one or more amino acids that are located at corresponding positions in an AAV capsid protein of a different AAV serotype (e.g., wherein the one or more mutations comprise amino acids in a VR of an AAV capsid protein of a different AAV serotype, such as

[0058] #14348316v1 wherein the variant AAV capsid protein is a variant AAVrh74 capsid protein and the one or more mutations comprise one or more amino acids in a VR of an AAV capsid protein). In some embodiments, a variant AAV capsid protein comprises one or more mutations relative to an AAV capsid protein counterpart that comprises the amino acid sequence set forth in SEQ ID NO: 4254. Tables 1-12, 13a, 14a, 15a, 16a, and 17 show examples of variant AAVrh74 capsid proteins and amino acid mutations in variant AAVrh74 capsid proteins. In each of the AAV capsid protein amino acid sequences in Tables 1-12, 13a, 14a, 15a, 16a, and 17, amino acid position 1 corresponds to amino acid position 224 in SEQ ID NO: 4269 or amino acid position 224 in SEQ ID NO: 4254. See FIGs. 9A-9B, wherein amino acid position 1 of the AAVrh74_VRI-AAVl VP1 amino acid sequence shown in FIG. 9A corresponds to amino acid position 224 in SEQ ID NO: 4254 and wherein amino acid position 1 of the AAVrh74_VRI- rh74 VP1 amino acid sequence shown in FIG. 9B corresponds to amino acid position 224 in SEQ ID NO: 4269. In Tables 1-12, 13a, 14a, and 15a, dots / periods / points indicate the same amino acid as the corresponding amino acid in an AAV capsid protein counterpart. In Tables 1- 12, 13a, 14a, and 15a, one-letter amino acid abbreviations indicate amino acids that are different from the corresponding amino acid in the AAV capsid protein counterpart.

[0059] In some embodiments, an amino acid sequence in a variant AAV capsid protein (e.g., any one of the variant AAV capsid protein amino acid sequences in Table 1-12, 13a, 14a, 15a, 16a, or 17) can be compared to a reference sequence in an AAV capsid protein counterpart. Examples of reference sequences are provided in Tables 1-12, 13a, 14a, and 15a, and may referred to as “parent” or “parental” capsid proteins. Examples of reference sequences also include amino acid sequences of full-length AAV capsid proteins. See, e.g., the wild-type AAVrh74 VP1 amino acid sequence set forth in SEQ ID NO: 4269 and the AAVrh74_VRI- AAV1 VP1 amino acid sequence set forth in SEQ ID NO: 4254; see also the AAV capsid protein amino acid sequences found at the following GenBank accession numbers: NC_002077.1 (AAV1) , NC_001401.2 (AAV2) , NP_043941.1 (AAV3) , NC_001829.1 (AAV4) , NC.006152.1 (AAV5) , AF028704.1 (AAV6) , NC_006260.1 (AAV7) , NC_006261.1 (AAV8) , AY530579.1 (AAV9) , MQ286650.1 (AAV44.9) , AY243015.1 (AAVrhlO) , and LP899424.1 (AAVrh74); see also the AAV capsid protein sequences in FIG. 10.

[0060] Alignment algorithms that are known in the art (e.g., in BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), COBALT, OPAL, Multlin, Clustal Omega, Clustal W2.0, or Clustal X2.0 software) can be used to identify amino acid differences (e.g., substitutions, deletions, and / or insertions) in variant AAV capsid proteins (e.g., the amino acid sequences of variant AAV capsid proteins in Tables 1-12, 13a, 14a, 15a, 16a, and 17) relative to an AAV capsid

[0061] #14348316v1 protein counterpart and to identify percent sequence identity between a variant AAV capsid protein and an AAV capsid protein counterpart. To determine the percent identity of two amino acid sequences (e.g., an amino acid sequence of a variant AAV capsid protein and an amino acid sequence of an AAV capsid protein counterpart), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The amino acids at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. In some embodiments, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., % homology=# of identical positions / total # of positionsxlOO), optionally penalizing the score for the number of gaps introduced and / or length of gaps introduced. Methods for comparing amino acid sequences in AAV capsid proteins is also described in International Patent Application: PCT / US2024 / 029789, which published as International Publication No. WO 2024 / 238852 and which is incorporated by reference herein for its teachings related to AAV production, AAV engineering, and AAV capsid protein mutagenesis.

[0062] For example, in some embodiments, a variant AAV capsid protein comprises a full- length AAV VP1 amino acid sequence, which can be generated by aligning a counterpart AAV capsid protein (e.g., the amino acid sequence of SEQ ID NO: 4269, the amino acid sequence of SEQ ID NO: 4254, or other AAV capsid protein, such as an AAV capsid protein of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV44.9, or AAVrhlO) with any one of the variant AAV capsid protein amino acid sequences in Tables 1-12, 13a, 14a, 15a, 16a, and 17. In some embodiments, following alignment of the two AAV capsid protein amino acid sequences, the full-length variant AAV capsid protein is generated by substituting one or more of the amino acid positions in the counterpart AAV capsid protein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 20-25, 25-30, or more than 30 amino acid positions) with the amino acid substitution at the corresponding position in any one of the variant AAV capsid protein amino acid sequence that is in any one of Tables 1-12, 13a, 14a, 15a, 16a, and 17 and that was used in the alignment. In some embodiments, the one or more amino acid substitutions in the full-length variant AAV capsid protein are located at positions that correspond to a VR (e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII, and / or a VR-IX) in the counterpart AAV capsid protein. In some embodiments, the one or more amino acid substitutions in the full-length variant AAV capsid protein correspond to one or more of the positions within amino acids 224-629 in AAVrh74 VP1 (see, e.g., the bold and underlined region in the AAVrh74 VP1 amino acid sequence below). In some embodiments, the one or

[0063] #14348316v1 more amino acid substitutions in the full-length variant AAV capsid protein correspond to one or more of the positions within amino acids 224-628 in AAVrh74_VRl-AAVl VP1 (see, e.g., the bold and underlined region in the AAVrh74_VRl-AAVl VP1 amino acid sequence below).

[0064] Accordingly, in some embodiments, a full-length variant AAV capsid protein is generated by substituting one or more amino acid residues at positions 224-629 in AAVrh74 VP1 (SEQ ID NO: 4269), substituting one or more amino acid residues at positions 224-628 in AAVrh74_VRl-AAVl VP1 (SEQ ID NO: 4254), or substituting one or more amino acid residues at corresponding positions in an AAV capsid protein of another serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV44.9, or AAVrhlO) with one or more of the amino acid substitutions in any one of the variant AAV capsid proteins in any one of Tables 1-12, 13a, 14a, 15a, 16a, and 17. In some embodiments, the full-length variant AAV capsid protein is generated by substituting amino acids 224-629 in AAVrh74 VP1 (SEQ ID NO: 4269) (see, e.g., the bold and underlined region in the AAVrh74 VP1 amino acid sequence below), substituting amino acids 224-628 in AAVrh74_VRl-AAVl VP1 (SEQ ID NO: 4254) (see, e.g., the bold and underlined region in the AAVrh74_VRl-AAVl VP1 amino acid sequence below), or substituting amino acid residues at the corresponding positions in an AAV capsid protein of another serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV44.9, or AAVrhlO) with any one of the variant AAV capsid protein amino acid sequences in Tables 1-12, 13a, 14a, 15a, 16a, or 17. In some embodiments, a full- length variant AAV capsid protein is generated by substituting amino acids 224-629 in AAVrh74 VP1 (see, e.g., the bold and underlined region in the AAVrh74 VP1 amino acid sequence below) or by substituting amino acids 224-628 in AAVrh74_VRl-AAVl VP1 (see, e.g., the bold and underlined region in the AAVrh74_VRl-AAVl VP1 amino acid sequence below).

[0065] AAVrh74 VP1 amino acid sequence:

[0066] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNA ADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESP

[0067] VKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAA GGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTND

[0068] NTYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNWGFRPKRLNFKLFNIOVKEVTQNEGTKTIANN LTSTIQVFTDSEYOLPYVLGSAHOGCLPPFPADVFMIPQYGYLTLNNGSOAVGRSSFYCLEYFPSOML RTGNNFEFSYNFEDVPFHSSYAHSOSLDRLMNPLIDOYLYYLSRTOSTGGTAGTOQLLFSOAGPNNM SAQAKNWLPGPCYROQRVSTTLSONNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFP SSGVLMFGKOGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLOOONAAPIVGAVNSOGAL

[0069] PGMVWONRDVYLOGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPOILIKNTPVPADPPTTFNQAKLASFIT QYSTGQVSVEIEWEEQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYETRNE* (SEQ ID NO: 4269)

[0070] AAVrh74 VR1-AAV1 VP1 amino acid sequence:

[0071] MAADGYEPDWEEDNESEGIREWWDEKPGAPKPKANQQKQDNGRGEVEPGYKYEGPFNGEDKGEPVNA ADAAAEEHDKAYDQQEQAGDNPYERYNHADAEFQEREQEDTSFGGNEGRAVFQAKKRVEEPEGEVESP

[0072] #14348316v1 VKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAA

[0073] GGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDN HYFGYSTPWGYFDFNRFHCHFSPRDWORLINNNWGFRPKRLNFKLFNIOVKEVTQNEGTKTIANNL TSTIQVFTDSEYOLPYVLGSAHOGCLPPFPADVFMIPQYGYLTLNNGSOAVGRSSFYCLEYFPSOMLR TGNNFEFSYNFEDVPFHSSYAHSOSLDRLMNPLIDOYLYYLSRTOSTGGTAGTOQLLFSOAGPNNMS

[0074] AQAKNWLPGPCYROQRVSTTLSONNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPS SGVLMFGKOGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLOOONAAPIVGAVNSOGALP GMVWONRDVYLOGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPOILIKNTPVPADPPTTFNOAKLASFITQ YSTGQVSVEIEWEEQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYETRNE*(SEQ ID NO: 4254)

[0075] Tables 1-12. Examples of Variant AAVrh74 Capsid Proteins

[0076] #14348316v1

[0077] #14348316v1

[0078]

[0079] #14348316v1

[0080] #14348316v1

[0081]

[0082] #14348316v1

[0083]

[0084] #14348316v1

[0085]

[0086] #14348316v1

[0087]

[0088] #14348316v1

[0089]

[0090] #14348316v1

[0091] #14348316v1

[0092] #14348316v1

[0093]

[0094] #14348316v1

[0095]

[0096] #14348316v1

[0097] #14348316v1

[0098] #14348316v1

[0099]

[0100] #14348316v1

[0101] #14348316v1

[0102]

[0103] #14348316v1

[0104]

[0105] #14348316v1

[0106] #14348316v1

[0107]

[0108] #14348316v1

[0109] #14348316v1

[0110]

[0111] #14348316v1

[0112] #14348316v1

[0113]

[0114] #14348316v1

[0115]

[0116] #14348316v1

[0117]

[0118] #14348316v1

[0119]

[0120] #14348316v1

[0121]

[0122] #14348316v1

[0123]

[0124] #14348316v1

[0125]

[0126] #14348316v1

[0127] #14348316v1

[0128]

[0129] #14348316v1

[0130] #14348316v1

[0131]

[0132] #14348316v1

[0133]

[0134] #14348316v1

[0135] #14348316v1

[0136] #14348316v1

[0137]

[0138] #14348316v1

[0139] #14348316v1

[0140]

[0141] #14348316v1

[0142]

[0143] #14348316v1

[0144]

[0145] #14348316v1

[0146]

[0147] #14348316v1

[0148]

[0149] #14348316v1

[0150]

[0151] #14348316v1

[0152]

[0153] #14348316v1

[0154]

[0155] #14348316v1

[0156]

[0157] #14348316v1

[0158] #14348316v1

[0159]

[0160] #14348316v1

[0161] #14348316v1

[0162]

[0163] #14348316v1

[0164]

[0165] #14348316v1

[0166]

[0167] #14348316v1

[0168]

[0169] #14348316v1

[0170]

[0171] #14348316v1

[0172]

[0173] #14348316v1

[0174]

[0175] #14348316v1

[0176] #14348316v1

[0177]

[0178] #14348316v1

[0179] #14348316v1

[0180]

[0181] #14348316v1

[0182] #14348316v1

[0183]

[0184] #14348316v1

[0185]

[0186] #14348316v1

[0187] #14348316v1

[0188]

[0189] #14348316v1

[0190]

[0191] #14348316v1

[0192]

[0193] #14348316v1

[0194]

[0195] #14348316v1

[0196]

[0197] #14348316v1

[0198]

[0199] #14348316v1

[0200]

[0201] #14348316v1

[0202]

[0203] #14348316v1

[0204]

[0205] #14348316v1

[0206]

[0207] #14348316v1

[0208]

[0209] #14348316v1

[0210]

[0211] #14348316v1

[0212] #14348316v1

[0213] #14348316v1

[0214] #14348316v1

[0215]

[0216] #14348316v1

[0217]

[0218] #14348316v1

[0219]

[0220] #14348316v1

[0221]

[0222] #14348316v1

[0223]

[0224] #14348316v1

[0225]

[0226] #14348316v1

[0227]

[0228] #14348316v1

[0229]

[0230] #14348316v1

[0231] #14348316v1

[0232] #14348316v1

[0233]

[0234] #14348316v1

[0235]

[0236] #14348316v1

[0237] #14348316v1

[0238]

[0239] #14348316v1

[0240] #14348316v1 - Ill -

[0241] #14348316v1

[0242] #14348316v1

[0243]

[0244] #14348316v1

[0245] #14348316v1

[0246]

[0247] #14348316v1

[0248] #14348316v1

[0249] #14348316v1

[0250] #14348316v1

[0251]

[0252] #14348316v1

[0253] #14348316v1

[0254]

[0255] #14348316v1

[0256]

[0257] #14348316v1

[0258] #14348316v1

[0259]

[0260] #14348316v1

[0261]

[0262] #14348316v1

[0263]

[0264] #14348316v1

[0265] #14348316v1

[0266] #14348316v1

[0267]

[0268] #14348316v1

[0269] #14348316v1

[0270]

[0271] #14348316v1

[0272] #14348316v1

[0273]

[0274] #14348316v1

[0275]

[0276] #14348316v1

[0277] #14348316v1

[0278] #14348316v1

[0279] #14348316v1

[0280] #14348316v1

[0281] #14348316v1

[0282]

[0283] #14348316v1

[0284] #14348316v1

[0285]

[0286] #14348316v1

[0287]

[0288] #14348316v1

[0289] #14348316v1

[0290]

[0291] #14348316v1

[0292] #14348316v1

[0293]

[0294] #14348316v1

[0295]

[0296] #14348316v1

[0297]

[0298] #14348316v1

[0299]

[0300] #14348316v1

[0301] #14348316v1

[0302]

[0303] #14348316v1

[0304] #14348316v1

[0305]

[0306] #14348316v1

[0307] #14348316v1

[0308]

[0309] #14348316v1

[0310]

[0311] #14348316v1

[0312]

[0313] #14348316v1

[0314] #14348316v1

[0315]

[0316] #14348316v1

[0317] #14348316v1

[0318]

[0319] #14348316v1

[0320] #14348316v1

[0321]

[0322] #14348316v1

[0323]

[0324] #14348316v1

[0325]

[0326] #14348316v1

[0327] #14348316v1

[0328] #14348316v1

[0329]

[0330] #14348316v1

[0331]

[0332] #14348316v1

[0333]

[0334] #14348316v1

[0335]

[0336] #14348316v1

[0337]

[0338] #14348316v1

[0339]

[0340] #14348316v1

[0341]

[0342] #14348316v1

[0343]

[0344] #14348316v1

[0345] #14348316v1

[0346] #14348316v1

[0347] #14348316v1

[0348]

[0349] #14348316v1

[0350]

[0351] #14348316v1

[0352]

[0353] #14348316v1

[0354]

[0355] #14348316v1

[0356] #14348316v1

[0357] #14348316v1

[0358] #14348316v1

[0359] #14348316v1

[0360] #14348316v1

[0361] #14348316v1

[0362] #14348316v1

[0363] #14348316v1

[0364] #14348316v1

[0365] #14348316v1

[0366] #14348316v1

[0367] #14348316v1

[0368] #14348316v1

[0369] #14348316v1

[0370] #14348316v1

[0371] #14348316v1

[0372] #14348316v1

[0373]

[0374] #14348316v1

[0375] #14348316v1

[0376] #14348316v1

[0377] Aspects of the application relate to variant AAV capsid proteins comprising one or more amino acids substitutions relative to a corresponding wildtype AAV capsid protein. For example, aspects of the application relate to variant AAVrh74 capsid proteins comprising one or more amino acid substitutions relative to a wildtype AAVrh74 capsid protein.

[0378] In some embodiments, the variant AAVrh74, AAVrhlO, or AAV8 capsid protein comprises a VRI region comprising a portion of the AAV1-VRI as shown in FIG. 1. In some embodiments, the variant capsid protein comprising a portion of the AAV 1 -VRI has one fewer amino acids relative to the wildtype AAVrh74, AAVrhlO, or AAV8 capsid protein, as shown in FIG. 1. In some embodiments, the variant capsid protein further comprises one or more amino acid substitutions at a position selected from E310; R311; L319; M320; G325; V331; D332; Y333; S334; V357; D360; L362; Q364; Q365; 1370; V371; and Q377. In some embodiments, the variant capsid comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen amino acid substitutions at positions selected from E310 (which corresponds to amino acid position 533 in SEQ ID NO: 4254); R311 (which corresponds to amino acid position 534 in SEQ ID NO: 4254); L319 (which corresponds to amino acid position 542 in SEQ ID NO: 4254); M320 (which corresponds to amino acid position 543 in SEQ ID NO: 4254); G325 (which corresponds to amino acid position 548 in SEQ ID NO: 4254); V331 (which corresponds to amino acid position 554 in SEQ ID NO: 4245); D332 (which corresponds to amino acid position 555 in SEQ ID NO: XX); Y333 (which corresponds to amino acid position 556 in SEQ ID NO: 4254); S334 (which corresponds to amino acid position 557 in SEQ ID NO: 4254); V357 (which corresponds to amino acid position 580 in SEQ ID NO: 4254); D360 (which corresponds to amino acid position 583 in SEQ ID NO: 4254); L362 (which corresponds to amino acid position 585 in SEQ ID NO: 4254); Q364 (which corresponds to amino acid position 587 in SEQ ID NO: 4254); Q365 (which corresponds to amino acid position 588 in SEQ ID NO: 4254); 1370 (which corresponds to amino acid position 593 in SEQ ID NO: 4254); V371 (which corresponds to amino acid position 594 in SEQ ID NO: 4254); and Q377 (which corresponds to amino acid position 600 in SEQ ID NO: 4254). In some

[0379] #14348316v1 - 2 1 - embodiments, the variant capsid comprises amino acid substitutions at positions E310 (which corresponds to amino acid position 533 in SEQ ID NO: 4254); R311 (which corresponds to amino acid position 534 in SEQ ID NO: 4254); L319 (which corresponds to amino acid position 542 in SEQ ID NO: 4254); M320 (which corresponds to amino acid position 543 in SEQ ID NO: 4254); G325 (which corresponds to amino acid position 548 in SEQ ID NO: 4254); V331 (which corresponds to amino acid position 554 in SEQ ID NO: 4254); D332 (which corresponds to amino acid position 555 in SEQ ID NO: 4254); Y333 (which corresponds to amino acid position 556 in SEQ ID NO: 4254); S334 (which corresponds to amino acid position 557 in SEQ ID NO: 4254); V357 (which corresponds to amino acid position 580 in SEQ ID NO: 4254); D360 (which corresponds to amino acid position 583 in SEQ ID NO: 4254); L362 (which corresponds to amino acid position 585 in SEQ ID NO: 4254); Q364 (which corresponds to amino acid position 587 in SEQ ID NO: 4254); Q365 (which corresponds to amino acid position 588 in SEQ ID NO: 4254); 1370 (which corresponds to amino acid position 593 in SEQ ID NO: 4254); V371 (which corresponds to amino acid position 594 in SEQ ID NO: 4254); and Q377 (which corresponds to amino acid position 600 in SEQ ID NO: 4254).

[0380] In some embodiments, the variant capsid comprises a substitution at E310 and R311, E310 and L319, E310 and M320, E310 and G325, E310 and V331, E310 and D332, E310 and Y333, E310 and S334, E310 and V357, E310 and D360, E310 and L362, E310 and Q364, E310 and Q365, E310 and 1370, E310 and V371, E310 and Q377, R311 and L319, R311 and M320, R311 and G325, R311 and V331, R311 and D332, R311 and Y333, R311 and S334, R311 and V357, R311 and D360, R311 and L362, R311 and Q364, R311 and Q365, R311 and 1370, R311 and V371, R311 and Q377, V357 and D360, V357 and 1370, D360 and 1370, or a combination thereof, wherein: E310 corresponds to amino acid position 533 in SEQ ID NO: 4254; R311 corresponds to amino acid position 534 in SEQ ID NO: 4254; L319 corresponds to amino acid position 542 in SEQ ID NO: 4254; M320 corresponds to amino acid position 543 in SEQ ID NO: 4254; G325 corresponds to amino acid position 548 in SEQ ID NO: 4254; V331 corresponds to amino acid position 554 in SEQ ID NO: 4254; D332 corresponds to amino acid position 555 in SEQ ID NO: 4254; Y333 corresponds to amino acid position 556 in SEQ ID NO: 4254; S334 corresponds to amino acid position 557 in SEQ ID NO: 4254; V357 corresponds to amino acid position 580 in SEQ ID NO: 4254; D360 corresponds to amino acid position 583 in SEQ ID NO: 4254; L362 corresponds to amino acid position 585 in SEQ ID NO: 4254; Q364 corresponds to amino acid position 587 in SEQ ID NO: 4254; Q365 corresponds to amino acid position 588 in SEQ ID NO: 4254; 1370 corresponds to amino acid position 593 in SEQ ID NO: 4254; V371 corresponds to amino acid position 594 in SEQ ID NO: 4254; and Q377 corresponds to amino acid position 600 in SEQ ID NO: 4254.

[0381] #14348316v1 In some embodiments, the variant capsid protein comprises one or more amino acid substitution, wherein the one or more amino acid substitution comprises E310D; R311K; L319M; M320I; G325N; G325S; V331T; V331A; D332A; Y333L; S334: V357T; V357I; D360V; L362F; Q364S; Q365S; I370A; I370Q; V371I; V371T; and / or Q377M, wherein: E310 corresponds to amino acid position 533 in SEQ ID NO: 4254; R311 corresponds to amino acid position 534 in SEQ ID NO: 4254; L319 corresponds to amino acid position 542 in SEQ ID NO: 4254; M320 corresponds to amino acid position 543 in SEQ ID NO: 4254; G325 corresponds to amino acid position 548 in SEQ ID NO: 4254; V331 corresponds to amino acid position 554 in SEQ ID NO: 4254; D332 corresponds to amino acid position 555 in SEQ ID NO: 4254; Y333 corresponds to amino acid position 556 in SEQ ID NO: 4254; S334 corresponds to amino acid position 557 in SEQ ID NO: 4254; V357 corresponds to amino acid position 580 in SEQ ID NO: 4254; D360 corresponds to amino acid position 583 in SEQ ID NO: 4254; L362 corresponds to amino acid position 585 in SEQ ID NO: 4254; Q364 corresponds to amino acid position 587 in SEQ ID NO: 4254; Q365 corresponds to amino acid position 588 in SEQ ID NO: 4254; 1370 corresponds to amino acid position 593 in SEQ ID NO: 4254; V371 corresponds to amino acid position 594 in SEQ ID NO: 4254; and Q377 corresponds to amino acid position 600 in SEQ ID NO: 4254. In some embodiments, the variant capsid comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, or at least sixteen amino acid substitutions comprising E310D; R311K; L319M; M320I; G325N; G325S; V331T; V331A; D332A; Y333L; S334; V357T; V357I; D360V; L362F; Q364S; Q365S; I370A; I370Q; V371I; V371T; and / or Q377M. In some embodiments, the variant capsid comprises amino acid substitutions comprising E310D; R311K; L319M; M320I; G325N / S; V331T / A; D332A; Y333L; S334; V357T / I; D360V; L362F; Q364S; Q365S; I370A / Q; V371VT; and / or Q377M, wherein: E310 corresponds to amino acid position 533 in SEQ ID NO: 4254; R311 corresponds to amino acid position 534 in SEQ ID NO: 4254; L319 corresponds to amino acid position 542 in SEQ ID NO: 4254; M320 corresponds to amino acid position 543 in SEQ ID NO: 4254; G325 corresponds to amino acid position 548 in SEQ ID NO: 4254; V331 corresponds to amino acid position 554 in SEQ ID NO: 4254; D332 corresponds to amino acid position 555 in SEQ ID NO: 4254; Y333 corresponds to amino acid position 556 in SEQ ID NO: 4254; S334 corresponds to amino acid position 557 in SEQ ID NO: 4254; V357 corresponds to amino acid position 580 in SEQ ID NO: 4254; D360 corresponds to amino acid position 583 in SEQ ID NO: 4254; L362 corresponds to amino acid position 585 in SEQ ID NO: 4254; Q364 corresponds to amino acid position 587 in SEQ ID NO: 4254; Q365 corresponds to amino acid position 588 in SEQ ID NO: 4254; 1370 corresponds to amino acid

[0382] #14348316v1 position 593 in SEQ ID NO: 4254; V371 corresponds to amino acid position 594 in SEQ ID NO: 4254; and Q377 corresponds to amino acid position 600 in SEQ ID NO: 4254.

[0383] In some embodiments, the variant capsid protein comprises an amino acid sequence having at least 70%, 80%, 90%. 95%, 99%, or 100% identity to any one or more of SEQ ID NOs: 4230-4237 and 4244-4249. In some embodiments, the variant capsid protein comprises an amino acid sequence, wherein a portion of the variant capsid protein has at least 70%, 80%, 90%. 95%, 99%, or 100% identity to any one or more of SEQ ID NOs: 4230-4237 and 4244- 4249.

[0384] In some embodiments, the variant AAVrh74 capsid protein comprises a VRI region from AAVrh74. In some embodiments, as described above, a wildtype AAVrh74-VRI has one additional amino acid residue relative to an AAVrh74-VRI substituted with a portion of an AAV1-VRI. Accordingly, residues E310 and R311 in an AAVrh74-VRI substituted with a portion of AAV1-VRI correspond to residues E311 and R312 in a wildtype AAVrh74-VRI. In some embodiments, the variant capsid comprises one or more amino acid substitutions at a position selected from E311 (which corresponds to amino acid position 534 in SEQ ID NO: 4269); R312 (which corresponds to amino acid position 535 in SEQ ID NO: 4269); S316 (which corresponds to amino acid position 539 in SEQ ID NO: 4269); L320 (which corresponds to amino acid position 543 in SEQ ID NO: 4269); G326 (which corresponds to amino acid position 549 in SEQ ID NO: 4269); D333 (which corresponds to amino acid position 556 in SEQ ID NO: 4269); Y334 (which corresponds to amino acid position 557 in SEQ ID NO: 4269); S335 (which corresponds to amino acid position 558 in SEQ ID NO: 4269); S336 (which corresponds to amino acid position 559 in SEQ ID NO: 4269); D361 (which corresponds to amino acid position 584 in SEQ ID NO: 4269); Q365 (which corresponds to amino acid position 588 in SEQ ID NO: 4269); Q366 (which corresponds to amino acid position 589 in SEQ ID NO: 4269); 1371 (which corresponds to amino acid position 594 in SEQ ID NO: 4269); and N376 (which corresponds to amino acid position 599 in SEQ ID NO: 4269), calculated relative to the wildtype AAVrh74 sequence. In some embodiments, the variant capsid comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen amino acid substitutions at positions selected from E311 (which corresponds to amino acid position 534 in SEQ ID NO: 4269); R312 (which corresponds to amino acid position 535 in SEQ ID NO: 4269); S316 (which corresponds to amino acid position 539 in SEQ ID NO: 4269); L320 (which corresponds to amino acid position 543 in SEQ ID NO: 4269); G326 (which corresponds to amino acid position 549 in SEQ ID NO: 4269); D333 (which corresponds to amino acid position 556 in SEQ ID NO: 4269); Y334 (which corresponds to amino acid position 557 in SEQ ID NO: 4269); S335 (which corresponds

[0385] #14348316v1 to amino acid position 558 in SEQ ID NO: 4269); S336 (which corresponds to amino acid position 559 in SEQ ID NO: 4269); D361 (which corresponds to amino acid position 584 in SEQ ID NO: 4269); Q365 (which corresponds to amino acid position 588 in SEQ ID NO: 4269); Q366 (which corresponds to amino acid position 589 in SEQ ID NO: 4269); 1371 (which corresponds to amino acid position 594 in SEQ ID NO: 4269); and N376 (which corresponds to amino acid position 599 in SEQ ID NO: 4269).

[0386] In some embodiments, the variant capsid comprises a substitution at E311 and R312, E311 and S316, E311 and L320, E311 and G326, E311 and D333, E311 and Y33,4 E311 and S335, E311 and S336, E311 and D361, E311 and Q365, E311 and Q366, E311 and 1371, E311 and N376, R312 and L316, R312 and L320, R312 and G326, R312 and D333, R312 and Y334, R312 and S335, R312 and S336, R312 and D361, R312 and Q365, R312 and Q366, R312 and 1371, R312 and N376; 1371 and N376, or a combination thereof, wherein: E311 (which corresponds to amino acid position 534 in SEQ ID NO: 4269); R312 (which corresponds to amino acid position 535 in SEQ ID NO: 4269); S316 (which corresponds to amino acid position 539 in SEQ ID NO: 4269); L320 (which corresponds to amino acid position 543 in SEQ ID NO: 4269); G326 (which corresponds to amino acid position 549 in SEQ ID NO: 4269); D333 (which corresponds to amino acid position 556 in SEQ ID NO: 4269); Y334 (which corresponds to amino acid position 557 in SEQ ID NO: 4269); S335 (which corresponds to amino acid position 558 in SEQ ID NO: 4269); S336 (which corresponds to amino acid position 559 in SEQ ID NO: 4269); D361 (which corresponds to amino acid position 584 in SEQ ID NO: 4269); Q365 (which corresponds to amino acid position 588 in SEQ ID NO: 4269); Q366 (which corresponds to amino acid position 589 in SEQ ID NO: 4269); 1371 (which corresponds to amino acid position 594 in SEQ ID NO: 4269); and N376 (which corresponds to amino acid position 599 in SEQ ID NO: 4269).

[0387] In some embodiments, the variant capsid comprises one or more amino acid substitution, wherein the one or more amino acid substitution comprises E311D; R312K; S316M; L320M; G326S; G326N; D333A; Y334L; S335D; S336N; S336D; D361V; Q365S; Q366S; I371A; 137 IQ; and / or N376H, wherein: E311 (which corresponds to amino acid position 534 in SEQ ID NO: 4269); R312 (which corresponds to amino acid position 535 in SEQ ID NO: 4269); S316 (which corresponds to amino acid position 539 in SEQ ID NO: 4269); L320 (which corresponds to amino acid position 543 in SEQ ID NO: 4269); G326 (which corresponds to amino acid position 549 in SEQ ID NO: 4269); D333 (which corresponds to amino acid position 556 in SEQ ID NO: 4269); Y334 (which corresponds to amino acid position 557 in SEQ ID NO: 4269); S335 (which corresponds to amino acid position 558 in SEQ ID NO: 4269); S336 (which corresponds to amino acid position 559 in SEQ ID NO: 4269); D361 (which corresponds to

[0388] #14348316v1 amino acid position 584 in SEQ ID NO: 4269); Q365 (which corresponds to amino acid position 588 in SEQ ID NO: 4269); Q366 (which corresponds to amino acid position 589 in SEQ ID NO: 4269); 1371 (which corresponds to amino acid position 594 in SEQ ID NO: 4269); and N376 (which corresponds to amino acid position 599 in SEQ ID NO: 4269). In some embodiments, the variant capsid comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, or at least thirteen amino acid substitutions comprising E311D; R312K; S316M; L320M; G326S; G326N; D333A; Y334L; S335D; S336N; S336D; D361V; Q365S; Q366S; I371A; I371Q; and / or N376H, wherein: E311 (which corresponds to amino acid position 534 in SEQ ID NO: 4269); R312 (which corresponds to amino acid position 535 in SEQ ID NO: 4269); S316 (which corresponds to amino acid position 539 in SEQ ID NO: 4269); L320 (which corresponds to amino acid position 543 in SEQ ID NO: 4269); G326 (which corresponds to amino acid position 549 in SEQ ID NO: 4269); D333 (which corresponds to amino acid position 556 in SEQ ID NO: 4269); Y334 (which corresponds to amino acid position 557 in SEQ ID NO: 4269); S335 (which corresponds to amino acid position 558 in SEQ ID NO: 4269); S336 (which corresponds to amino acid position 559 in SEQ ID NO: 4269); D361 (which corresponds to amino acid position 584 in SEQ ID NO: 4269); Q365 (which corresponds to amino acid position 588 in SEQ ID NO: 4269); Q366 (which corresponds to amino acid position 589 in SEQ ID NO: 4269); 1371 (which corresponds to amino acid position 594 in SEQ ID NO: 4269); and N376 (which corresponds to amino acid position 599 in SEQ ID NO: 4269). In some embodiments, the variant capsid comprises amino acid substitutions comprising E311D; R312K; S316M; L320M; G326S / N; D333A; Y334L; S335D; S336N / D; D361V; Q365S; Q366S; I371A / Q; and / or N376H, wherein: E311 (which corresponds to amino acid position 534 in SEQ ID NO: 4269); R312 (which corresponds to amino acid position 535 in SEQ ID NO: 4269); S316 (which corresponds to amino acid position 539 in SEQ ID NO: 4269); L320 (which corresponds to amino acid position 543 in SEQ ID NO: 4269); G326 (which corresponds to amino acid position 549 in SEQ ID NO: 4269); D333 (which corresponds to amino acid position 556 in SEQ ID NO: 4269); Y334 (which corresponds to amino acid position 557 in SEQ ID NO: 4269); S335 (which corresponds to amino acid position 558 in SEQ ID NO: 4269); S336 (which corresponds to amino acid position 559 in SEQ ID NO: 4269); D361 (which corresponds to amino acid position 584 in SEQ ID NO: 4269); Q365 (which corresponds to amino acid position 588 in SEQ ID NO: 4269); Q366 (which corresponds to amino acid position 589 in SEQ ID NO: 4269); 1371 (which corresponds to amino acid position 594 in SEQ ID NO: 4269); and N376 (which corresponds to amino acid position 599 in SEQ ID NO: 4269). In some embodiments, the variant capsid protein comprises an amino acid sequence having at least 70%, 80%, 90%. 95%, 99%, or 100% identity

[0389] #14348316v1 to any one or more of SEQ ID NOs: 4238-4243. In some embodiments, the variant capsid protein comprises an amino acid sequence, wherein a portion of the variant capsid protein has at least 70%, 80%, 90%. 95%, 99%, or 100% identity to any one or more of SEQ ID NOs: 4238- 4243.

[0390] In some embodiments, the variant capsid protein comprises an amino acid sequence having at least 70%, 80%, 90%. 95%, 99%, or 100% identity to any one or more of the sequences set forth in Table 17. In some embodiments, the variant capsid protein comprises an amino acid sequence, wherein a portion of the variant capsid protein has at least 70%, 80%, 90%. 95%, 99%, or 100% identity to any one or more of the sequences set forth in Table 17.

[0391] In some embodiments, the variant capsid protein comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% identity to any one of the sequences set forth in Table 13a, Table 14a, Table 15a, Table 16a, and Table 17. In some embodiments, the variant capsid protein comprises an amino acid sequence, wherein a portion of the variant capsid protein has at least 70%, 80%, 90%, 95%, 99%, or 100% identity to any one of the sequences set forth in Table 13 a, Table 14a, Table 15 a, Table 16 a, and Table 17.

[0392] Aspects of the application also relate to a recombinant AAV (rAAV) particle comprising a variant AAV capsid protein and a heterologous nucleic acid flanked by a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR. In some embodiments, the AAV ITRs are of the same serotype as the variant capsid protein (e.g., both the AAV ITRs and the variant AAV capsid protein are of serotype AAVrh74). In other embodiments, the AAV ITRs are of a different serotype than the variant capsid protein (e.g., the variant capsid protein is of serotype AAVrh74 and the AAV ITRs are of serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVrh.10.

[0393] In some embodiments, a heterologous nucleic acid in an rAAV particle comprises a nucleotide sequence encoding an RNA (e.g., a coding RNA or a non-coding RNA, such as wherein the heterologous nucleic acid comprises a gene). Examples of RNAs that can be encoded by a gene in a heterologous nucleic acid include a small-hairpin RNA (shRNA), a short-interfering RNA (siRNA), a prokaryotic-interfering RNA (pro-siRNA), a micro-RNA (miRNA), a long non-coding RNA (IncRNA), a Piwi-interacting RNA (piRNA), an exonskipping RNA, an enzymatic RNA, a guide RNA (gRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), an RNA aptamer, and a messenger RNA (mRNA). In some embodiments, an inhibitory RNA (e.g., a shRNA, an siRNA, a miRNA, a IncRNA, an exon-skipping RNA, an RNA aptamer, or an enzymatic RNA) encoded by a gene in a heterologous nucleic acid can be expressed in a target cell (e.g., a cardiac cell) to regulate an RNA and / or protein, wherein increased levels of the RNA

[0394] #14348316v1 or protein is associated with a disease, disorder, or condition (e.g., a cardiac disease, a cardiac disorder, or a cardiac condition, such as cardiomyopathy, hypertrophic dilated cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmia, catecholaminergic polymorphic ventricular tachycardia (CPVT), arrhythmogenic right ventricular cardiomyopathy (ARVC), Duchenne Muscular Dystrophy (DMD), or dysferlinopathy). In some embodiments, an RNA encoded by a gene in a heterologous nucleic acid is an mRNA encoding a peptide or a protein. Examples of peptides and proteins that can be encoded by a sequence in a heterologous nucleic acid include a cell-, organelle-, or tissue-targeting peptide or protein, an antibody, an antigen-binding fragment, an antigen, an enzyme or an enzymatic domain, a proteinaceous enzyme substrate, a glycoprotein, a lipoprotein, a secreted protein, an extracellular matrix protein or a fragment thereof, a viral coat protein, a transmembrane receptor or a fragment thereof, a toxin or a fragment thereof, a hormone, receptors, a peptibody, a growth factor, a clotting factor, a cytokine, a chemokine, an activating or inhibitory peptide, a thrombolytic, a bone morphogenetic protein, an Fc-fusion protein, an anticoagulant, a signaling protein, a cell surface protein, a nucleic acid-binding protein, and a reporter. In some embodiments, an antibody is a monoclonal antibody, a polyclonal antibody, a nanobody, or a single-chain antibody (e.g., an scFv). In some embodiments, a protein comprising an antigen-binding fragment is a chimeric antigen receptor, such as one comprising an antibody. In some embodiments, an enzyme is a protease, a signaling protein, a polymerase, a transcriptional regulator, a nuclease, an RNA- guided nuclease, a metabolic enzyme, a kinase, a phosphatase, a lipid-transferase, a glycosylase, a DNA ligase, a ubiquitin ligase, a methyltransferase, an acetyltransferase, a SUMO transferase, a foldase, a reductase, a lyase, a dehydrogenase, a phosphorylase, a decarboxylase, a dephosphorylase, a kinase, a synthase, or a hydrolase. In some embodiments, a nucleic acidbinding protein is a transcription regulator (e.g., a transcription factor), a splicing regulator, a translation regulator (e.g., a ribosome-binding protein, a signaling protein that post- translationally modifies RNA subunits, etc.), a nuclease (e.g., an RNA-guided nuclease, a zinc finger nuclease, or a transcription activator- like effector nuclease (TALEN). In some embodiments, a signaling protein is an enzyme (e.g., a kinase), a ligand (e.g., a ligand of a receptor), a secreted protein, a morphogen, a cell differentiation regulator. In some embodiments, a cell surface protein is a membrane protein, such as a receptor, a channel, a lineage- specific antigen, an extracellular matrix protein, or protein comprising a sequence that binds to an antigen-binding fragment, an antibody, or a chimeric antigen receptor. In some embodiments, a peptide or protein encoded by a gene in a heterologous nucleic acid can be expressed in a target cell (e.g., a cardiac cell) to treat a disease, disorder, or condition (e.g., a cardiac disease, a cardiac disorder, or a cardiac condition, such as cardiomyopathy, hypertrophic

[0395] #14348316v1 dilated cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmia, dilated cardiomyopathy (CPVT), arrhythmogenic right ventricular cardiomyopathy (ARVC), or Duchenne Muscular Dystrophy (DMD)). In some embodiments, a heterologous nucleic acid comprises a RYR2 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates a RYR2 gene. In some embodiments, a heterologous nucleic acid comprises a CASQ2 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates a CASQ2 gene. In some embodiments, a heterologous nucleic acid comprises a MYBPC3 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates a MYBPC3 gene. In some embodiments, a heterologous nucleic acid comprises a TNNT2 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates a TNNT2 gene. In some embodiments, a heterologous nucleic acid comprises a TMEM43 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates a TMEM43 gene. In some embodiments, a heterologous nucleic acid comprises a dystrophin gene or a nucleic acid encoding an RNA, peptide, or protein that regulates a dystrophin gene. In some embodiments, a heterologous nucleic acid comprises a Dysferlin gene or a nucleic acid encoding RNA, peptide, or protein that regulates a Dysferlin gene.

[0396] In some embodiments, a heterologous nucleic acid (e.g. a nucleotide sequence encoding a therapeutic protein, e.g. an antibody) comprises a regulatory sequence. In some embodiments, a heterologous nucleic acid comprises a regulatory sequence that is operably linked to a gene. In some embodiments, a heterologous nucleic acid comprises a sequence that regulates transcription (e.g., any one or more of a promoter, an enhancer, a transcription factor binding sequence, a transcriptional start sequence, transcription termination sequence, etc.), a sequence that regulates translation (e.g., any one or more of a 5’ UTR, a translation initiation regulatory sequence, a Kozack sequence, a Shine-Dalgamo sequence, a start codon, a ribosome binding site, a 3’ UTR, a translation termination sequence, a stop codon, etc.), a sequence that regulates splicing (e.g., binding sites for small nuclear ribonucleoproteins, splicing acceptor sites, splicing donor sites, etc.), and / or a sequence that regulates post-translational modifications (e.g., a split- intein for protein trans-splicing, a protease cleavage site, a phosphorylation site, etc.). In some embodiments, a promoter comprised in a heterologous nucleic acid is one that is active in a cell (e.g., a cardiac cell), or a cell of the same cell type or a similar cell type, that binds to a variant AAVrh74 capsid protein. In some embodiments, a promoter is a constitutive promoter. In some embodiments, a promoter is an inducible promoter. In some embodiments, a promoter is a tissue-specific promoter (e.g., a cardiac -specific promoter or a cardiac muscle-specific promoter). In some embodiments, a promoter is a TNNT promoter. In some embodiments, a promoter is a MHCK7 promoter.

[0397] #14348316v1 Aspects of the application also relate to nucleic acids that encode a variant AAV capsid protein (e.g., a variant AAVrh74 capsid protein). In some embodiments, a nucleic acid encoding a variant AAV capsid protein is a vector, such as a plasmid. In some embodiments, a nucleic acid encoding a variant AAV capsid protein comprises a sequence encoding any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229. In some aspects, the application relates to a nucleic acid library comprising a plurality of nucleic acids, wherein each nucleic acid of the plurality comprises a sequence encoding a variant AAV capsid protein. For example, in some embodiments, a nucleic acid library comprises a plurality of nucleic acids, wherein one or more of the amino acid sequences set forth in SEQ ID NOs: 1-4229 are encoded by nucleic acid in the library.

[0398] Methods that can be utilized to generate nucleic acids described herein include those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

[0399] Other aspects of the application relate to a cell or cell population thereof comprising a variant AAV capsid protein, a nucleic acid (e.g., a nucleic acid encoding a variant AAV capsid protein), and / or a nucleic acid library (e.g., a nucleic acid library encoding variant AAV capsid proteins) described herein. In some embodiments, cells are mammalian cells. In some embodiments, cells are non-human primate cells. In some embodiments, cells are pig cells. In some embodiments, cells are mouse cells. In some embodiments, cells are human cells. In some embodiments, cells comprise a helper nucleic acid (e.g., a nucleic acid comprising an El gene, an E2 gene, an E4 gene, and / or VA gene, such as a nucleic acid comprising a helper virus nucleic acid including, but not limited to, an adenovirus nucleic acid), a nucleic acid comprising an AAV rep gene (e.g., a nucleic acid comprising an AAV rep gene and a gene encoding a variant AAV capsid protein), and / or a nucleic acid comprising 5’ and 3’ AAV ITRs that flank a heterologous nucleic acid (e.g. a nucleotide sequence encoding a therapeutic protein). In some embodiments, cells are used for production of an rAAV particle (e.g., wherein the cells are used in a method of producing an rAAV particle, such as a double transfection method or a triple transfection method (see, e.g., disclosures related to compositions and methods for rAAV production in: K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745). In addition, AAV serotypes, AAV derivatives, and AAV pseudotypes, as well as methods for producing them, have been previously described (see, e.g., Mol. Ther. 2012 Apr;20(4):699- 708. doi: 10.1038 / mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan Al, Schaffer DV, Samulski RJ; Duan et al, J. Virol., 75:7662-7671, 2001; Halbert et al, J. Virol., 74:1524-1532, 2000; Zolotukhin et al, Methods, 28:158-167, 2002; and Auricchio et al., Hum. Molec. Genet., 10:3075-3081, 2001; see, e.g., US Patent Publication No.:

[0400] #14348316v1 US 2005 / 0100890 Al; International Publication No.: WO 01 / 83692 A2; US Patent Publication No.: US 2003 / 0103939 Al; U.S. Pat. No. 6,001,650; and Miller (1996). Proc. Natl. Acad. Sci., 93: 11407-11413)).

[0401] Further aspects of the application relate to compositions comprising a variant AAV capsid protein, an rAAV particle, a nucleic acid, a nucleic acid library, and / or a cell or cell population described herein. Additionally, in some aspects, the application relates to a kit comprising a variant AAV capsid protein, an rAAV particle, a nucleic acid, a nucleic acid library, a cell or cell population, and / or a composition described herein. In some embodiments, a composition is a pharmaceutical composition comprising a variant AAV capsid protein or an rAAV particle comprising a variant AAV capsid protein. In some embodiments, the amount of an rAAV particle in a composition comprises 1 x 105vector genomes (vg) to 1 x 1018vg. In some embodiments, a composition comprises one or more pharmaceutically acceptable buffers, excipients, and / or additives.

[0402] Further aspects of application relate to methods. In some embodiments, a method comprises contacting a cell (e.g., a mammalian cell, such as a human cell which includes, but is not limited to, a heart cell, or a cell population thereof which is ex vivo or in vivo) with a variant AAV capsid protein, an rAAV particle, a nucleic acid, a nucleic acid library, or a composition described herein. In some embodiments, a method comprises contacting a cell with a nucleic acid and producing an rAAV particle described herein. In some embodiments, a method comprises administering a variant AAV capsid protein or an rAAV particle described herein to a subject (e.g., by administering to the subject a composition, such as a pharmaceutical composition, described herein). In some embodiments, administration is performed subcutaneously, intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intracranially, intrathecally, orally, intraperitoneally, or by oral or nasal inhalation. In some embodiments, administration is performed by direct injection into a tissue (e.g., cardiac tissue) or organ (e.g., the heart). In some embodiments, a method of administration to a subject is performed by utilizing only one administration route. In some embodiments, multiple administration routes are utilized in a method of administration to a subject (e.g., wherein the multiple administrations are performed serially, or simultaneously). Treatment methods involving rAAV particles which are known in the art can also be utilized in a method of administration to a subject (see, e.g., methods of rAAV particle administration described in: Adeno- Associated Virus Vectors in Clinical Trials. Barrie J. Carter. Human Gene Therapy. May 2005, 16(5): 541-550. doi: 10.1089 / hum.2005.16.541. Published in Volume: 16 Issue 5: May 25, 2005; Neuropharmacology. 2013 Jun;69: 82-8. doi: 10.1016 / j.neuropharm.2012.03.004. Epub 2012 Mar 17.; Adeno-associated virus (AAV) gene

[0403] #14348316v1 therapy for neurological disease. Weinberg MSI, Samulski RJ, McCown TJ.Gene therapy for lysosomal storage disorders. Yew NS, Cheng SH. Pediatr Endocrinol Rev. 2013 Nov; 11 Suppl 1:99-109; Directed evolution of novel adeno-associated viruses for therapeutic gene delivery. Bartel MA, Weinstein JR, Schaffer DV.Gene Ther. 2012 Jun;19(6):694-700. doi: 10.1038 / gt.2012.20. Epub 2012 Mar 8; Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Mingozzi F, High KA. Nat Rev Genet. 2011 May;12(5):341-55. doi: 10.1038 / nrg2988). In some embodiments, a subject is a mammal. In some embodiments, a subject is a non-human primate (e.g., a cynomolgus monkey or a rhesus monkey). In some embodiments, a subject is a rodent (e.g., a rat or a mouse). In some embodiments, a subject is a human. In some embodiments, a subject has, is suspected of having, is at risk of having, or is expected to develop a disease, disorder, or condition. In some embodiments, a disease, a disorder, or a condition comprises a genetic disease. In some embodiments, a disease, a disorder, or a condition comprises inflammation of one or more tissues (e.g., cardiac tissues). In some embodiments, a disease, a disorder, or a condition comprises a cardiac disease, a cardiac disorder, or a cardiac condition, such as a cardiomyopathy. In some embodiments, a disease, a disorder, or a condition is cardiomyopathy, HCM, DCM, arrhythmia, CPVT, ARVC, DMD, dysferlinopathy. In some embodiments, administration of an rAAV particle described herein is used to treat a disease, disorder, or condition. In some embodiments, an rAAV particle (e.g., an rAAV particle comprising a RYR2 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates an RYR2 gene) is administered to a subject to treat CPVT / arrhythmia. In some embodiments, an rAAV particle (e.g., an rAAV particle comprising a CASQ2 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates an CASQ2 gene) is administered to a subject to treat CPVT / arrhythmia. In some embodiments, an rAAV particle (e.g., an rAAV particle comprising a MYBPC3 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates an MYBPC3 gene) is administered to a subject to treat hypertrophic cardiomyopathy (HCM). In some embodiments, an rAAV particle (e.g. an rAAV particle comprising a TNNT2 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates an TNNT2 gene) is administered to a subject to treat dilated cardiomyopathy (DCM). In some embodiments, an rAAV particle (e.g., an rAAV particle comprising a TMEM43 gene or a nucleic acid encoding an RNA, peptide, or protein that regulates an TMEM43 gene) is administered to a subject to treat ARVC. In some embodiments, an rAAV particle (e.g., an rAAV particle comprising a dystrophin gene or a nucleic acid encoding an RNA, peptide, or protein that regulates an dystrophin gene) is administered to a subject to treat DMD. In some embodiments, an rAAV particle (e.g., an rAAV particle

[0404] #14348316v1 comprising a Dysferlin gene or a nucleic acid encoding an RNA, peptide, or protein that regulates an Dysferlin gene) is administered to a subject to treat dysferlinopathy.

[0405] EXAMPLES

[0406] Example 1: Utilizing Cross-Species Directed Evolution of Adeno-associated Virus Vectors to Derive Variants Targeting the Heart and De-targeting Liver

[0407] Identification of novel efficient AAV capsids for human organ transduction can involve conducting directed evolution in primary human cells in a 3D scaffold or using large closely related animal models. Although primary human cells can be used as selection targets, the inability to emulate multiple natural organ filters (liver, kidney), to reproduce not only the 3D scaffold but also organ confinement and biological liquid circulation (blood, intercellular fluid, etc.), imposes restrictions on such models. The closest evolutionary large animal model, the non-human primate (NHP) system, has played a role in AAV directed evolution in recent years. Additionally, the porcine model provides insight into AAV distribution since pigs resemble human body weight, and organ sizes, and are even used as donors for xenotransplantation of some heart structures or skin. On the other hand, pigs are as distant from humans evolutionarily as mice, and using these models for AAV selection alone is insufficient for determining humanspecific capsid variants.

[0408] A cross-species directed evolution approach (NHP, pig, and mouse) heavily fine-tuned on the closest to human primate models was developed. To enhance heart transduction efficiency and reduce liver off-target transduction, a novel library was developed based on AAV-rhlO, AAV-rh74, AAV8, and AAV1 serotypes with the extra incorporation of liver detargeting mutations. The design of the novel AAV library was based on a virtual family shuffling approach spanning about 50 mutational sites in variable regions from IV to VIII (see, e.g., AAV library production methods described in International Patent Application: PCT / US2024 / 029789, which published as International Publication No. WO 2024 / 238852). The high complexity of the novel AAV library (>lE+8) allows the generation of capsid mutants for successful transduction of a wide range of tissues / cells.

[0409] To improve liver de-targeting of rh74-based capsids, several amino acid substitutions were introduced into variable region I (VR-I) by swapping the native domain with the partial domain VRI domain of AAV1 (FIG. 1).

[0410] Additionally, to increase the selectivity of directed evolution, AAV cap expression were confined to cardiac tissues by implementing cardiac-specific promoters (TNNT and MHCK7) for cap mRNA expression (FIGs. 2A-2B). For the first round of directed evolution, 4 different

[0411] #14348316v1 plasmid constructions (as acceptor backbones) were used to construct 4 independent AAV libraries: TNNT-VR-I-rh74-Eib, MHCK7-VR-I-rh74-Lib, TNNT-VR-I-AAVl-Eib, and MHCK-VR-I-AAVl-Eib, where Lib is the set of mutations in VR-IV, VR-V, VR-VI, VR-VII, and VR-VIII domains described in International Patent Application: PCT / US2024 / 029789, which published as International Publication No. WO 2024 / 238852.

[0412] All four libraries were independently assembled in HEK293 adherent cells and purified using the iodixanol protocol. After purification, the endotoxin level was determined (<1 EU / mL) and libraries were mixed at equimolar concentrations for NHP injection. To shape the trajectory of directed evolution in the closest to human direction, the NHP model was used for the first round of selection (Cynomolgus macaques). Four weeks later, the NHP organs were harvested, and heart tissue was used for RNA isolation. Further, RNA was converted to cDNA and amplified using Q5 polymerase. Using vector- specific primers, qPCR assessment of the library distribution was performed on NHP RNA. Substantial prevalence of TNNT or MHCK7 transcripts was not found (almost equal amounts), and the TNNT promoter was used for the second round of selection (in both vectors: with native VRI-rh74 and swapped VRLAAV1 rh74 - two independent AAV libraries). After the first round of selection, about 500,000 - 1,000,000 capsid mRNA copies were isolated, which theoretically restrict the complexity of the Round 2 library to IM variants. Since each round of directed evolution leads to a 10-100 times drop in complexity, it was assumed that only one extra round was necessary to narrow down valuable capsids for the validation step.

[0413] To diversify the directed evolution process and shape it toward human body size and emulate some human organ specificity, selection models for Round 2 were extended to NHP, Pig, and Mouse (mice with humanized liver) models. For the second round of selection, TNNT- VR-I-rh74-Eib2 and TNNT-VR-LAAV1-Eib2 libraries constructed by insertion of VR-IV to VR-VIII domains from amplified cDNA after Round 1 were used. The libraries were mixed in equimolar proportion and injected intravenously. Four weeks later, heart and liver tissues were collected, and total DNA / RNA was isolated. For the determination of variant distribution (capsid DNA level in liver and heart tissues) and transduction profiles (capsid RNA in heart tissue), long-read Sequel NGS approach with HiFi mode and the longest movie time was used. Bioinformatics analysis was performed using custom- written Python scripts (de-barcoding, translation, and statistics extraction) on Einux OS. For each capsid variant, the sequence, CN (copy number), and clonal concentration (CN(var) / sum of all CN (vars)) in corresponding entities was determined. It should be noted that prevalence in CN for some variants was observed, especially in RNA / DNA. Thus, variant compositions for AAV libraries (TNNT-VR- Lrh74-Eib2 and TNNT-VR-LAAV1-Eib2) before the second round of injections and in tissues 4

[0414] #14348316v1 weeks later (Heart-RNA, Heart-DNA, and Liver-DNA) were determined. This allowed determination of absolute clonal enrichment as the ratio between clone concentration in tissue (DNA or RNA level) and its concentration in the injected library. As the main selection criteria, clonal enrichment in Heart-RNA and Liver-DNA was used. Particularly, variants with positive heart enrichment on the RNA level (HeartRNA_Enr) and negative enrichment (depletion) in the liver on the DNA level (LiverDNA_Enr) were selected. To increase the statistical credibility of data analysis, variants that had less than 10 copies (CN<10) in the datasets from Heart RNA and less than 3 copies (CN<3) in the injected Library Round 2 were filtered out.

[0415] The ultimate goal of the work was to determine variants with superior heart transduction relative to parental capsids (rh74), therefore parental capsid statistics were used as an internal reference. In this case, the parental capsid has relative enrichment in heart and liver equal to 1 or in Log 10 coordinates - 0, and variant relative (to parent) enrichment is determined as the ratio of its absolute enrichment to parental capsid enrichment. To visualize HeartRNA_Enr and LiverDNA_Enr relative enrichments, XY scatter plot Log 10 representations were used. Lor each animal, an XY scatter plot was built and variants from quadrants 1 and 4 were selected for subsequent validation (QI and Q4) (EIGs. 3-8). In quadrant 1, clones with a maximal ratio of heart RNA to Liver DNA (showing much more efficient heart transduction relative to liver transduction) were selected. The whole list of Q4 selected variants (with positive heart and negative liver enrichments) are represented in Tables 1-3 (AAVrh74_VRI-AAVl parent) and in Tables 4-6 (AAVrh74 parent). Additionally, entities that were represented in Heart RNA and in the injected Round 2 library but were not represented in Liver DNA were extracted. This approach was utilized to yield variants completely avoid the liver (or transduce the liver at an undetectable level) and represent significant interest as low liver toxic variants. CN filtering in the same manner as described earlier (CN>10, and CN>3) was performed, and variants with positive enrichments in heart were selected: Tables 7-9 (AAVrh74_VRI-AAVl parent) and Tables 10-12 (AAVrh74_VRI-AAVl parent).

[0416] In summary, a set of capsids with significant heart transduction (enrichment) and liver de-targeting (depletion) in NHP, Pig, and Mouse models was identified. Lor subsequent validation analysis, a total of 100 capsid variants with the best heart enrichment and liver depletion were selected.

[0417] The 20 top-performing capsids in each animal model, and the corresponding levels of transduction measured in each model, are shown in the tables below.

[0418] Each of the sequences in Tables 13, 14a, and 15a below start from amino acid position 224. The amino acid sequence of the parental capsid protein is shown as the first sequence in each Table. In the rest of sequences, dots / periods / points / indicate the same amino acid as the

[0419] #14348316v1 corresponding amino acid in the parent capsid protein and one-letter amino acid abbreviations indicate amino acids that are different from the corresponding amino acid in the parent capsid protein. Table 13a: Top Performing capsids in NHP model. In certain embodiments, any one of the amino acid sequences set forth in Table 13a, or a full-length capsid protein comprising any one of said sequences, is utilized in the packaging of recombinant adeno-associated virus (rAAV) particles. In some aspects, the sequences of Tables 1- 12, or nucleic acids encoding such sequences, are provided on one or more packaging plasmids for use in the production of rAAV particles comprising the variant capsid proteins. In further embodiments, the rAAV particles may additionally comprise one or more heterologous nucleic acid molecules, for example, a nucleic acid encoding a therapeutic protein. Such rAAV particles may be employed for targeted transduction of a cell or tissue of interest, including, for example, muscle cells or cardiac cells, and may be used in methods of treating a disease or disorder, such as a muscle disorder or a cardiac disorder.

[0420] #14348316v1

[0421] #14348316v1

[0422] #14348316v1

[0423] Table 13b: Enrichment of Top 20 variants in non-human primate (NHP) heart and depleted in liver.

[0424] #14348316v1

[0425] Table 14a: Top Performing capsids in Pig Model. In certain embodiments, any one of the amino acid sequences set forth in Table 14a, or a full-length capsid protein comprising any one of said sequences, is utilized in the packaging of recombinant adeno-associated virus (rAAV) particles. In some aspects, the sequences of Tables 1- 12, or nucleic acids encoding such sequences, are provided on one or more packaging plasmids for use in the production of rAAV particles comprising the variant capsid proteins. In further embodiments, the rAAV particles may additionally comprise one or more heterologous nucleic acid molecules, for example, a nucleic acid encoding a therapeutic protein. Such rAAV particles may be employed for targeted transduction of a cell or tissue of interest, including, for example, muscle cells or cardiac cells, and may be used in methods of treating a disease or disorder, such as a muscle disorder or a cardiac disorder.

[0426] #14348316v1

[0427] #14348316v1

[0428] #14348316v1

[0429] Table 14b. Enrichment of Top 20 variants in swine (pig) heart and depleted in liver.

[0430] Table 15a: Top Performing capsids in Mouse Model. In certain embodiments, any one of the amino acid sequences set forth in Table 15a, or a full-length capsid protein comprising any one of said sequences, is utilized in the packaging of recombinant adeno-associated virus (rAAV) particles. In some aspects, the sequences of Tables 1- 12, or nucleic acids encoding such sequences, are provided on one or more packaging plasmids for use in the production of rAAV particles comprising the variant capsid proteins. In further embodiments, the rAAV particles may additionally comprise one or more heterologous nucleic acid molecules, for example, a nucleic acid encoding a

[0431] #14348316v1 therapeutic protein. Such rAAV particles may be employed for targeted transduction of a cell or tissue of interest, including, for example, muscle cells or cardiac cells, and may be used in methods of treating a disease or disorder, such as a muscle disorder or a cardiac disorder.

[0432] #14348316v1

[0433] #14348316v1

[0434] Table 15b: Enrichment of Top 20 variants in mouse heart and depleted in liver.

[0435] #14348316v1

[0436] The overall top 20 performing capsids are shown in Table 16a below. The parental capsid for capsids UFCC1-UFCC8 and UFCC15-UFCC20 was AAVrh74_VRI-AAVl. The parental capsid for capsids UFCC9-UFCC14 was AAVrh74_VRI-rh74. FIGs. 9A-9B show alignments of the amino acid sequence of capsid proteins variants of interest against the amino acid sequence of their respective parental capsid protein.

[0437] Each of the sequences in Table 16 below start from amino acid position 224.

[0438] Table 16a. Examples of Variant Capsid Amino Acid Sequences. In certain embodiments, any one of the amino acid sequences set forth in Table 16a, or a full-length capsid protein comprising any one of said sequences, is utilized in the packaging of recombinant adeno-associated virus (rAAV) particles. In some aspects, the sequences of Tables 1-12, or nucleic acids encoding such sequences, are provided on one or more packaging plasmids for use in the production of rAAV particles comprising the variant capsid proteins. In further embodiments, the rAAV particles may additionally comprise one or more heterologous nucleic acid molecules, for example, a nucleic acid encoding a therapeutic protein. Such rAAV particles may be employed for targeted transduction of a cell or tissue of interest, including, for example, muscle cells or cardiac cells, and may be used in methods of treating a disease or disorder, such as a muscle disorder or a cardiac disorder.

[0439] #14348316v1

[0440] #14348316v1

[0441] Table 16b.

[0442] #14348316v1

[0443] Example 2: Additional Capsid Variants

[0444] An additional set of capsids of interest was generated based on DNA alignment and clusterization of generated NGS data. These capsid variants are shown in Table 17 below. Each of the sequences in Table 17 below start from amino acid position 224.

[0445] Table 17. Additional Capsid Variants. In certain embodiments, any one of the amino acid sequences set forth in Table 17, or a full-length capsid protein comprising any one of said sequences, is utilized in the packaging of recombinant adeno-associated virus (rAAV) particles. In some aspects, the sequences of Tables 1-12, or nucleic acids encoding such sequences, are provided on one or more packaging plasmids for use in the production of rAAV particles comprising the variant capsid proteins. In further embodiments, the rAAV particles may additionally comprise one or more heterologous nucleic acid molecules, for example, a nucleic acid encoding a therapeutic protein. Such rAAV particles may be employed for targeted transduction of a cell or tissue of interest, including, for example, muscle cells or cardiac cells, and may be used in methods of treating a disease or disorder, such as a muscle disorder or a cardiac disorder.

[0446] #14348316v1

[0447] #14348316v1

[0448] #14348316v1 INCORPORATION BY REFERENCE

[0449] The present application refers to various issued patent, published patent applications, scientific journal articles, and other publications, all of which are incorporated herein by reference. The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the Detailed Description, the Figures, the Examples, and the Claims.

[0450] EQUIVALENTS AND SCOPE

[0451] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present application are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present application.

[0452] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0453] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0454] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0455] #14348316v1 The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0456] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0457] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A,

[0458] #14348316v1 and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0459] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0460] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

[0461] #14348316v1

Claims

1. CLAIMSWhat is claimed is:

1. A variant adeno-associated virus (AAV) capsid protein of serotype AAVrh74 comprising one or more amino acids which are located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229 and are substituted relative to a corresponding amino acid located in a wild-type AAVrh74 capsid protein counterpart.

2. The variant AAVrh74 capsid protein of claim 1, wherein the variant AAVrh74 capsid protein comprises one or more variable regions (VR) located in any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229.

3. The variant AAVrh74 capsid protein of claim 2, wherein the one or more VRs comprises a VR-I, a VR-II, a VR-III, a VR-IV, a VR-V, a VR-VI, a VR-VII, and / or a VR-VIII.

4. The variant AAVrh74 capsid protein of any one of claims 1-3, wherein the variant AAVrh74 capsid protein is variant of AAVrh74 viral protein 1 (VP1).

5. The variant AAVrh74 capsid protein of any one of claims 1-4, wherein the variant AAVrh74 capsid protein comprises any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229.

6. A recombinant AAV (rAAV) particle comprising:(a) a heterologous nucleic acid flanked by a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR; and(b) the variant AAVrh74 capsid protein of any one of claims 1-5.

7. The rAAV particle of claim 6, wherein the heterologous nucleic acid comprises a gene.

8. The rAAV particle of claim 7, wherein the gene encodes a peptide or protein.

9. The rAAV particle of any one of claims 6-8, wherein the heterologous nucleic acid comprises a regulatory sequence.

10. A nucleic acid comprising a sequence encoding the variant AAVrh74 capsid protein of any one of claims 1-5.#14348316v111. A nucleic acid library comprising a plurality of nucleic acids, wherein each nucleic acid of the plurality comprises a sequence encoding any one of the amino acid sequences set forth in SEQ ID NOs: 1-4229.

12. A cell or cell population thereof comprising the variant AAVrh74 capsid protein of any one of claims 1-5, the nucleic acid of claim 10, or the nucleic acid library of claim 11.

13. The cell or cell population thereof of claim 12, wherein the cell or cell population comprises:(a) a helper nucleic acid;(b) a nucleic acid comprising an AAV rep gene; and / or(c) a nucleic acid comprising a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR that flank a heterologous nucleic acid.

14. The cell or cell population thereof of claim 12 or 13, wherein the cell is a mammalian cell.

15. A composition comprising the variant AAVrh74 capsid protein of any one of claims 1-5, the rAAV particle of any one of claims 6-9, the nucleic acid of claim 10, the nucleic acid library of claim 11, or the cell or cell population thereof of any one of claims 12-14.

16. A method comprising contacting a cell with the variant AAVrh74 capsid protein of any one of claims 1-5, the rAAV particle of any one of claims 6-9, the nucleic acid of claim 10, the nucleic acid library of claim 11, or the composition of claim 15.

17. A method comprising administering the variant AAVrh74 capsid protein of any one of claims 1-5 or the rAAV particle of any one of claims 6-9 to a subject.

18. The method of claim 17, wherein the subject has or is suspected of having a disease, a disorder, or a condition.

19. The method of claim 17 or 18, wherein the subject has or is suspected of having a cardiac disease, a cardiac disorder, or a cardiac condition.#14348316v120. The method of any one of claims 17-19, wherein the subject has or is suspected of having cardiac myopathy.

21. The method of any one of claims 17-20, wherein the subject is a mammalian subject.

22. A kit comprising the variant AAVrh74 capsid protein of any one of claims 1-5, the rAAV particle of any one of claims 6-9, the nucleic acid of claim 10, the nucleic acid library of claim 11, the cell or cell population thereof of any one of claims 12-14, or the composition of claim 15.

23. A variant adeno-associated virus (AAV) capsid protein comprising an amino acid sequence wherein a portion of the amino acid sequence has at least 90% identity to any one of the amino acid sequences set forth in Table 13a, Table 14a, Table 15a, Table 16a, and Table 17.

24. The variant AAV capsid protein of claim 23, wherein the variant AAV capsid protein comprises an amino acid sequence wherein a portion of the amino acid sequence comprises any one of the amino acid sequences set forth in Table 13a, Table 14a, Table 15a, Table 16a, and Table 17.

25. The variant AAV capsid protein of claim 23 or 24, wherein the variant AAV capsid protein comprises a VR-I, or a portion thereof, of an AAV 1 capsid protein.

26. A recombinant AAV (rAAV) particle comprising:(a) a heterologous nucleic acid flanked by a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR; and(b) the variant AAV capsid protein of any one of claims 23-25.

27. The rAAV particle of claim 26, wherein the heterologous nucleic acid comprises a gene.

28. The rAAV particle of claim 27, wherein the gene encodes a peptide or protein.

29. The rAAV particle of any one of claims 26-28, wherein the heterologous nucleic acid comprises a regulatory sequence.#14348316v130. A nucleic acid comprising a sequence encoding the variant AAV capsid protein of any one of claims 23-25.

31. A cell or cell population thereof comprising the variant AAV capsid protein of any one of claims 23-25 or the nucleic acid of claim 30.

32. The cell or cell population thereof of claim 31, wherein the cell or cell population comprises:(a) a helper nucleic acid;(b) a nucleic acid comprising an AAV rep gene; and / or(c) a nucleic acid comprising a 5' AAV inverted terminal repeat (ITR) and a 3' AAV ITR that flank a heterologous nucleic acid.

33. The cell or cell population thereof of claim 31 or 32, wherein the cell is a mammalian cell.

34. A composition comprising the variant AAV capsid protein of any one of claims 23-25, the rAAV particle of any one of claims 26-29, the nucleic acid of claim 30, or the cell or cell population thereof of any one of claims 31-33.

35. A method comprising contacting a cell with the variant AAV capsid protein of any one of claims 23-25, the rAAV particle of any one of claims 26-29, the nucleic acid of claim 30, or the composition of claim 34.

36. A method comprising administering the variant AAV capsid protein of any one of claims 23-25 or the rAAV particle of any one of claims 26-29 to a subject.

37. The method of claim 36, wherein the subject has or is suspected of having a disease, a disorder, or a condition.

38. The method of claim 36 or 37, wherein the subject has or is suspected of having a cardiac disease, a cardiac disorder, or a cardiac condition.

39. The method of any one of claims 36-38, wherein the subject has or is suspected of having cardiac myopathy.#14348316v140. The method of any one of claims 36-39, wherein the subject is a mammalian subject.

41. A kit comprising the variant AAV capsid protein of any one of claims 23-25, the rAAV particle of any one of claims 26-29, the nucleic acid of claim 30, the cell or cell population thereof of any one of claims 31-33, or the composition of claim 34.

42. A variant adeno-associated virus (AAV) capsid protein of serotype AAV comprising a portion of an AAV1-VRI and an amino acid substitution at least one of E310D; R311K; L319M; M320I; G325N; G325S; V331T; V331A; D332A; Y333L; S334: V357T; V357I; D360V;L362F; Q364S; Q365S; I370A; I370Q; V371I; V371T; or Q377M.

43. A variant adeno-associated virus (AAV) capsid protein comprising an amino acid substitution at least one of E311; R312; S316; L320; G326; D333; Y334; S335; S336; D361; Q365; Q366; 1371; and N376.

44. A variant adeno-associated virus (AAV) capsid protein not of serotype AAV1, wherein a portion of the wildtype VRI is replaced with a portion of an AAV1-VRI, optionally as shown in FIG. 1.#14348316v1

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