AAV capsid engineering for cardiac and / or musculoskeletal gene therapy

Abstract

Provided herein are engineered viral capsid proteins and their use in methods of treating subjects using viral gene therapy, and in particular when a specific tissue tropism is desired. For example, in several embodiments, modified AAV capsids are engineered to have reduced tropism to a first tissue type, and enhanced tropism for a second and / or third tissue type that is different from the first tissue type. In several embodiments, the second and / or third tissue type is cardiac and / or skeletal muscle.

Description

AAV CAPSID ENGINEERING FOR CARDIAC AND / OR MUSCULOSKELETAL GENE THERAPYRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 612971, entitled “AAV CAPSID ENGINEERING FOR CARDIAC AND / OR MUSCULOSKELETAL GENE THERAPY” and filed on December 20, 2023, which is hereby incorporated by reference herein in its entirety.INCORPORATION BY REFERENCE OF MATERIAL IN SEQUENCE LISTING

[0002] This application incorporates the material provided in the accompanying XML file entitled SequenceListingAAVAN096WO.xml, created December 17, 2024, which is 83.5 KB in size.FIELD

[0003] The present disclosure relates in some aspects to engineered adeno- associated virus (AAV) capsid proteins and methods of use thereof.BACKGROUND

[0004] Adeno-associated virus (AAV) is a single-stranded DNA virus belonging to the Parvoviridae family. The absence of pathogenicity, low immunogenicity, episomal localization and stable transgene expression make AAV-derived vectors promising tools for human gene therapy applications. However, significant limitations to the clinical use of AAV are its promiscuity and its susceptibility to neutralization by human antibodies. Thus, there remains a need in the field for variant AAVs having the desired tropism and decreased susceptibility to neutralization by human antibodies. Provided are compositions, methods and uses that meet such needs.SUMMARY

[0005] Some embodiments provided herein are described by way of the following provided embodiments and also provided as possible combinations or overlapping embodiments.

[0006] In some embodiments, a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding non-engineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more nonmuscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein is provided. In some embodiments, the first engineered feature comprises insertion of at least 5 amino acids. In some embodiments, the first engineered feature comprises insertion of at least 7 amino acids. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle. In some embodiments, the first engineered feature enhances transduction or tropism to skeletal muscle. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle and skeletal muscle. In some embodiments, the first engineered feature comprises a peptide encoded by SEQ ID NO: 3. In some embodiments, the first engineered feature comprises a peptide having the sequence of SEQ ID NO: 4. In some embodiments, the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 5. In some embodiments, the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 6. In some embodiments, the second engineered feature reduces transduction or tropism to a soft tissue. In some embodiments, the second engineered feature reduces transduction or tropism to liver tissue. In some embodiments, the second engineered feature comprises a glycine to alanine mutation. In some embodiments, the second engineered feature comprises a glycine to alanine mutation at position 268 of SEQ ID NO: 2.

[0007] In some embodiments, a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding non-engineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variantcapsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more nonmuscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein is provided. In some embodiments, the second engineered feature reduces transduction or tropism to a soft tissue. In some embodiments, wherein the second engineered feature reduces transduction or tropism to liver tissue. In some embodiments, the second engineered feature comprises a glycine to alanine mutation. In some embodiments, the second engineered feature comprises a glycine to alanine mutation at position 268 of SEQ ID NO: 2. In some embodiments, the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 7. In some embodiments, the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 8. In some embodiments, the first engineered feature comprises insertion of at least 5 amino acids. In some embodiments, the first engineered feature comprises insertion of at least 7 amino acids. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle. In some embodiments, the first engineered feature enhances transduction or tropism to skeletal muscle. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle and skeletal muscle. In some embodiments, the first engineered feature comprises a peptide encoded by SEQ ID NO: 3. In some embodiments, the first engineered feature comprises a peptide having the sequence of SEQ ID NO: 4. In some embodiments, the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 9. In some embodiments, the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 10. In some embodiments, the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 10.

[0008] In some embodiments, a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding non-engineered rh74 VP1 polypeptide, the non-engineered rh74VPl having a sequence of SEQ ID NO: 2; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide resulting in maintained or enhanced targeting or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue, as compared to the non-engineered rh74VPl capsid; wherein the secondengineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing targeting or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more non-muscle tissue, as compared to the nonengineered rh74VPl capsid is provided. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle. In some embodiments, the first engineered feature enhances transduction or tropism to skeletal muscle. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle and skeletal muscle. In some embodiments, the first engineered feature comprises a peptide encoded by SEQ ID NO: 3. In some embodiments, the first engineered feature comprises a peptide having the sequence of SEQ ID NO: 4. In some embodiments, the second engineered feature reduces transduction or tropism to a soft tissue. In some embodiments, the second engineered feature reduces transduction or tropism to liver tissue. In some embodiments, the second engineered feature comprises a glycine to alanine mutation. In some embodiments, the second engineered feature comprises a glycine to alanine mutation at position 268 of SEQ ID NO: 2.

[0009] In some embodiments, a variant adeno-associated virus (AAV) capsid polypeptide, wherein said variant AAV capsid polypeptide exhibits maintained or increased transduction or tropism in human muscle tissue or cells as compared to a nonvariant parent capsid polypeptide, wherein said variant AAV capsid polypeptide exhibits reduced transduction or tropism in human liver tissue or cells as compared to a nonvariant parent capsid polypeptide, and wherein said variant AAV capsid polypeptide is encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 9 is provided. In some embodiments, the variant AAV capsid polypeptide is encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 9. In some embodiments, the variant AAV capsid polypeptide is encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 9. In some embodiments, the variant AAV capsid polypeptide has at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, the variant AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 10. In some embodiments, the variant AAV capsid polypeptide exhibits maintained or increased transduction as compared to a non-variant parent capsid polypeptide. In some embodiments, the muscle tissue or cells comprise cardiac muscle tissue or cells and / or skeletal muscle tissue or cells. In some embodiments, the variant AAV capsid polypeptide further exhibits an enhanced neutralization profile as compared to a non-variant parent capsid polypeptide. In some embodiments, the variant AAV capsid polypeptide exhibitsincreased transduction of human muscle tissue or cells in vivo as compared to a nonvariant parent capsid polypeptide. In some embodiments, the variant AAV capsid polypeptide exhibits increased transduction of human muscle tissue or cells in vitro as compared to a non-variant parent capsid polypeptide. In some embodiments, the variant AAV capsid polypeptide exhibits increased transduction of a human muscle tissue explant ex vivo as compared to anon-variant parent capsid polypeptide.

[0010] In some embodiments, the variant AAV capsid polypeptide is part of a functional AAV capsid, wherein the functional AAV capsid packages a nucleic acid sequence selected from the group consisting of a non-coding RNA, a protein coding sequence, an expression cassette, a multi-expression cassette, a sequence for homologous recombination, a genomic gene targeting cassette, and a therapeutic expression cassette In some embodiments, the therapeutic expression cassette encodes a therapeutic protein. In some embodiments, the therapeutic protein is selected from the group consisting of frataxin, GSD1A, PRKAG2, BAG3, Dysferlin, MYBPC-3, Danon / Lamp2, TMEM43, PKP2, LMNA, TNNT2. RBM20. MYH7, RYR2, CAMKII, PRKAG2, CALQ2, DM1, GRN, GBA. MSH3. UNCI 3 A. Stathmin-2, SMEI, and combinations thereof. In some embodiments, the expression cassette further encodes a consensus Kozak sequence of AGCCCCAAC. In some embodiments, the expression cassette further encodes a promoter element. In some embodiments, the promoter element is selected from a tissuespecific promoter, a cardiac-restricted promoter, desmin, creatine kinase, myogenin, alpha myosin heavy chain, and natriuretic peptide, a muscle cell specific promoter, albumin, alpha- 1 -antitrypsin, hepatitis B virus core protein promoters, a liver cell specific promoter, cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT), a constitutive promoter, inducible promoter, synthetic promoter, Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A, cytomegalovirus (CMV), housekeeping gene promoters, (3-actin, chicken (3-actin promoter (CBA), 6-myosin heavy chain gene, myosin light chain 2v (MLC-2v) gene, myosin light chain 2a gene, CARP gene, cardiac a-actin gene, cardiac m2 muscarinic acetylcholine gene, atrial natriuretic factor gene (ANF), cardiac sarcoplasmic reticulum Ca-ATPase gene, skeletal a-actin gene, an artificial cardiac promoter derived from MLC-2v gene, cytochrome P450 genes, heat shock protein genes, metallothionein genes, hormone-inducible genes, estrogen gene promoter. tetVP16. and / or any combination thereof.

[0011] In some embodiments, the first engineered feature enhances transduction or tropism to skeletal muscle by up to about 25-fold. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle by up to about 10-fold. In some embodiments, the second engineered feature reduces transduction or tropism to liver tissue by up to about 100%.

[0012] In some embodiments, a method of treating a cardiac or musculoskeletal disease comprising administering to a subject in need thereof, a therapeutically effective amount of an rh74 genome encapsidated within a capsid comprising a variant AAV capsid is provided.

[0013] In some embodiments, use of the variant AAV capsid in the manufacture of a medicament for the treatment of a cardiac or musculoskeletal disease is provided.

[0014] In some embodiments, use of the variant AAV capsid for the treatment of a cardiac or musculoskeletal disease is provided.

[0015] In some embodiments, a method of treating one or more diseases comprising administering to a subject in need thereof, a therapeutically effective amount of an rh74 genome encapsidated within a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding non-engineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more nonmuscle tissue, wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein is provided. In some embodiments, the one or more diseases comprises a cardiac disease. In some embodiments, the cardiac disease comprises cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, Marfan Syndrome, Loys-Dietz syndrome, Shprintzen-Goldberg syndrome, familial thoracic aortic aneurysm and dissection, familial hypercholesterolemia, heritable pulmonary arterial hypertension, and / or any combination thereof. In some embodiments, the one or more diseases comprises a musculoskeletal disease.

[0016] In some embodiments, a method of treating cardiac disease comprising administering to a subject in need thereof, a therapeutically effective amount of an rh74 genome encapsidated within a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding non-engineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more non-muscle tissue, wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using anon-engineered VP1 capsid protein is provided. In some embodiments, the cardiac disease comprises cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, Marfan Syndrome, Loys-Dietz syndrome, Shprintzen-Goldberg syndrome, familial thoracic aortic aneurysm and dissection, familial hypercholesterolemia, heritable pulmonary arterial hypertension, and / or any combination thereof.

[0017] In some embodiments, a method of treating musculoskeletal disease comprising administering to a subject in need thereof, a therapeutically effective amount of an rh74 genome encapsidated within a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding non-engineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more nonmuscle tissue, wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein is provided. In some embodiments, the first engineered feature comprises insertion of at least 5 amino acids. In some embodiments, the first engineered feature comprises insertion of at least 7 amino acids. In some embodiments, the first engineered feature enhances transduction ortropism to cardiac muscle. In some embodiments, the first engineered feature enhances transduction or tropism to skeletal muscle. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle and skeletal muscle. In some embodiments, the first engineered feature comprises a peptide encoded by SEQ ID NO: 3. In some embodiments, the first engineered feature comprises a peptide having the sequence of SEQ ID NO: 4 In some embodiments, the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 5. In some embodiments, the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 6. In some embodiments, the second engineered feature reduces transduction or tropism to a soft tissue. In some embodiments, the second engineered feature reduces transduction or tropism to liver tissue. In some embodiments, the second engineered feature comprises a glycine to alanine mutation. In some embodiments, the second engineered feature comprises a glycine to alanine mutation at position 268 of SEQ ID NO: 2. In some embodiments, the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 7. In some embodiments, the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 8. In some embodiments, the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 9. In some embodiments, the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 10 In some embodiments, the variant AAV capsid polypeptide is part of a functional AAV capsid, wherein the functional AAV capsid packages a nucleic acid sequence selected from the group consisting of a non-coding RNA, a protein coding sequence, an expression cassette, a multi-expression cassette, a sequence for homologous recombination, a genomic gene targeting cassette, and a therapeutic expression cassette. In some embodiments, the therapeutic expression cassette encodes a therapeutic protein. In some embodiments, the therapeutic protein is selected from the group consisting of frataxin, GSD1A, PRKAG2, BAG3, Dysferlin, MYBPC-3, Danon / Lamp2, TMEM43, PKP2, LMNA, TNNT2, RBM20, MYH7, RYR2, CAMKII, PRKAG2, CALQ2, DM1, GRN, GBA, MSH3, UNCI 3 A. Stathmin-2, SMEI. and combinations thereof In some embodiments, the expression cassette further encodes a consensus Kozak sequence of AGCCCCAAC. In some embodiments, the expression cassette further encodes a promoter element In some embodiments, the promoter element is selected from a tissue-specific promoter, a cardiac-restricted promoter, desmin, creatine kinase, myogenin. alpha myosin heavy chain, and natriuretic peptide, a muscle cell specific promoter, albumin, alpha- 1 -antitrypsin, hepatitis B virus core protein promoters,a liver cell specific promoter, cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT), a constitutive promoter, inducible promoter, synthetic promoter, Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad El A, cytomegalovirus (CMV), housekeeping gene promoters, P-actin. chicken -actin promoter (CBA), 6-myosin heavy chain gene, myosin light chain 2v (MLC-2v) gene, myosin light chain 2a gene, CARP gene, cardiac a-actin gene, cardiac m2 muscarinic acetylcholine gene, atrial natriuretic factor gene (ANF), cardiac sarcoplasmic reticulum Ca-ATPase gene, skeletal a-actin gene, an artificial cardiac promoter derived from MLC-2v gene, cytochrome P450 genes, heat shock protein genes, metallothionein genes, hormone-inducible genes, estrogen gene promoter, tetVP16, and / or any combination thereof. In some embodiments, the first engineered feature enhances transduction or tropism to skeletal muscle by up to about 25-fold. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle by up to about 10-fold. In some embodiments, the second engineered feature reduces transduction or tropism to liver tissue by up to about 100%. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle by up to about 10-fold and the second engineered feature reduces transduction or tropism to liver tissue by up to about 100%. In some embodiments, the first engineered feature enhances transduction or tropism to cardiac muscle by up to about 10-fold and the second engineered feature reduces transduction or tropism to soft tissue by up to about 100 In some embodiments, the first engineered feature enhances transduction or tropism to skeletal muscle by up to about 25-fold and the second engineered feature reduces transduction or tropism to liver tissue by up to about 100%. In some embodiments, the first engineered feature enhances transduction or tropism to skeletal muscle by up to about 25-fold and the second engineered feature reduces transduction or tropism to soft tissue by up to about 100%.BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is a non-limiting illustrative representation of altered transduction or tropism of a modified adeno-associated virus (AAV) to muscle tissue vs liver tissue as compared to an un-modified AAV.

[0019] FIG. 2 A is a rh74 VP1 capsid DNA sequence with some embodiments of insertion and mutation sites highlighted.

[0020] FIG. 2B is a rh74 VP1 capsid amino acid sequence with some embodiments of insertion and mutation sites highlighted.

[0021] FIG. 2C is a representative map of an expression construct encoding a rh74 VPl capsid polypeptide.

[0022] FIG. 3 A is a rh74-A VP1 capsid DNA sequence depicting some embodiments of an engineered insertion.

[0023] FIG. 3B is a rh74-A VP1 capsid amino acid sequence with some embodiments of an engineered insertion.

[0024] FIG. 3C is a representative map of an expression construct encoding a rh74-A VPl capsid polypeptide.

[0025] FIG. 4A is a rh74-001 VP1 capsid DNA sequence depicting some embodiments of an engineered point mutation.

[0026] FIG. 4B is a rh74-001 VP1 capsid amino acid sequence with some embodiments of an engineered point mutation.

[0027] FIG. 4C is a representative map of an expression construct encoding a rh74-001 VP1 capsid polypeptide.

[0028] FIG. 5A is a rh74-003 VP1 capsid DNA sequence depicting some embodiments of an engineered insertion and point mutation.

[0029] FIG. 5B is a rh74-003 VP1 capsid amino acid sequence with some embodiments of an engineered insertion and point mutation.

[0030] FIG. 5C is a representative map of an expression construct encoding a rh74-003 VP1 capsid polypeptide.

[0031] FIG. 6A is a non-limiting representation of in vivo luciferase bioluminescent activity in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0032] FIG. 6B is a bar graph depicting some embodiments of quantification of in vivo luciferase bioluminescent activity' in mice treated yvith AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0033] FIG. 7 is a non-limiting representation of showing liver transduction or tropism in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0034] FIG. 8A is a bar graph showing some embodiments of luciferase biodistribution to heart tissue as compared to Rpp30 in mice treated with AAV particlescomprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0035] FIG. 8B is a bar graph showing some embodiments of fold change in luciferase biodistribution to heart tissue in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0036] FIG. 9A is a bar graph showing some embodiments of luciferase biodistribution to liver tissue as compared to Rpp30 in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0037] FIG. 9B is a bar graph showing some embodiments of fold change in luciferase biodistribution to liver tissue in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0038] FIG. 10A is a bar graph showing some embodiments of luciferase biodistribution to muscle tissue as compared to Rpp30 in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0039] FIG. 10B is a bar graph showing some embodiments of fold change in luciferase biodistribution to muscle tissue in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0040] FIG. 11 is a set of bar graphs depicting some embodiments of liver detargeting in rh74-001 and rh74-003 VP1 capsid polypeptides as compared to rh74.

[0041] FIG. 12A is a bar graph showing some embodiments of luciferase expression in heart tissue in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0042] FIG. 12B is a bar graph showing some embodiments of fold change in luciferase expression in heart tissue in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0043] FIG. 13A is a bar graph showing some embodiments of luciferase expression in liver tissue in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0044] FIG. 13B is a bar graph showing some embodiments of fold change in luciferase expression in liver tissue in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0045] FIG. 14A is a bar graph showing some embodiments of luciferase expression in muscle tissue in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0046] FIG. 14B is a bar graph showing some embodiments of fold change in luciferase expression in muscle tissue in mice treated with AAV particles comprising a luciferase expression vector packaged within variant and non-variant rh74 VP1 capsid polypeptides.

[0047] FIG. 15 is a set of bar graphs depicting some embodiments of liver detargeting in rh74-001 and rh74-003 VP1 capsid polypeptides as compared to rh74.

[0048] FIG. 16A is a bar graph depicting some embodiments of a comparison of AAV9 and variant AAV9 receptor binding.

[0049] FIG. 16B is a bar graph depicting some embodiments of a comparison of AAV9 variant receptor binding.

[0050] FIG. 16C is a bar graph depicting embodiments of transduction of AAV9 variants in human DMD skeletal muscle cells.

[0051] FIG. 17A shows bar graphs of embodiments of biodistribution in mouse skeletal muscle (quadriceps), heart, and liver for AAV9 variants. FIG. 17B depicts embodiments of immunofluorescent images of biodistribution of AAV9 and AAV9 variants in quadriceps.

[0052] FIG. 18A is a bar graph depicting some embodiments of a comparison of microdystrophin expression in C2C12 cells treated with AAV9 variants.

[0053] FIG. 18B is a bar graph depicting some embodiments of a comparison of luciferase activity in C2C12 cells treated with AAV9 variants.

[0054] FIGs. 18C and 18D are bar graphs depicting embodiments of microdystrophin expression in mouse skeletal muscle (quadriceps; FIG. 18C) and heart (FIG. 18D) of AAV9 variants.

[0055] FIG. 19A is a bar graph depicting some embodiments of a comparison of AAV9 variant in vitro transduction. FIG. 19B is a bar graph depicting embodiments of AAV9 variant in vitro transduction of mouse skeletal muscle cells as measured by microdystrophin expression.

[0056] FIG. 20A is a bar graph that depicts embodiments of in vitro transduction of an AAV9 variant in mouse and human skeletal muscle cells and human hepatocellular carcinoma cells. FIG. 20B is a bar graphs that depicts embodiments of in vivo liver cell de-targeting of an AAV9 variant. FIG. 20C is a bar graph that depicts embodiments of AAV9 variant in vivo skeletal muscle cell transduction as measured by microdystrophin expression.DETAILED DESCRIPTION

[0057] Provided herein are genetically engineered viral capsid proteins (such as an AAV capsid protein) that exhibit an altered tissue tropism as compared to a nonengineered capsid protein, and methods and uses thereof. Also provided are articles of kits containing the provided genetically engineered viral capsid proteins and / or compositions comprising the same.

[0058] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0059] The term “AAV” is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or derivatives thereof. The term covers all subty pes and both naturally occurring and recombinant forms, unless otherwise indicated. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”), which refers to AAV comprising a polynucleotide sequence not of AAV origin (e.g., a trans gene). The term “AAV” includes AAV seroty pe 1 (AAV-1), AAV serotype 2 (AAV -2), AAV serotype 3 (AAV-3), AAV serotype 4 (AAV-4), AAV serotype 5 (AAV-5), AAV serotype 6 (AAV-6), AAV serotype 7 (AAV-7), AAV serotype 8 (AAV-8), AAV serotype 9 (AAV-9), serotype rhlO AAV, serotype rh74 AAV, or a pseudotyped rAAV (e.g., AAV2 / 9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9).

[0060] The term "AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least AAV capsid protein and an encapsidated polynucleotide.

[0061] The term “adeno-associated virus (AAV) capsid” refers to the three- dimensional proteinaceous shell encapsidating or enclosing the viral genetic material. The AAV capsid is a non-enveloped, icosahedral 60-mer of three repeating monomers: VP1, VP2. and VP3. The AAV capsid determines the properties of viral particles, including tissue tropism and antigenic properties.

[0062] The term “tropism” refers to preferential entry of the AAV virus or viral particle into certain cell or tissue type(s) and / or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) of one or more nucleic acid sequences (e.g., one or more transgene sequences) carried by the AAV virus or viral particle. Variations in capsid proteins VP1, VP2, and / or VP3, or a combination thereof may alter AAV virus tropism.

[0063] The terms “variant adeno-associated virus (AAV) capsid” and “engineered adeno-associated virus (AAV) capsid” refer to an AAV capsid polypeptide sequence differing by at least one amino acid from a parent, or non-engineered, polypeptide sequence. Variant or engineered AAV capsid may also refer to an AAV capsid comprising one or more engineered features.

[0064] The term “engineered feature” refers to alterations in the AAV capsid polypeptide at specific loci such that the one or more viral properties, for example, transduction or tropism of the AAV virus or viral particle, is altered.

[0065] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including therapeutic proteins and other peptides, e.g., linkers, tags, capsid proteins, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some respects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0066] Amino acids generally can be grouped according to the following common side- chain properties: (1) hydrophobic: Norleucine, Met. Ala, Vai. Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0067] Amino acid substitutions may also refer to one or more changes in a polypeptide sequence. The changes may include replacement of one amino acid in a polypeptide with another amino acid, insertion of one or amino acids, and / or deletion of one or more amino acids, or any combination thereof. Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.

[0068] A “nucleic acid” sequence refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence. The term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetyl cytosine, 8- hydroxy-N6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxyl- methyl) uracil, 5 -fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine. N6-isopentenyladenine. 1-methyladenme, 1- methylpseudouracil. 1 -methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyl adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxy- aminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil. 2-methylthio- N6-isopentenyladenine, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5 -methyluracil, N- uracil-5-oxyacetic acid methylester, uracil-5- oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0069] The term “polynucleotide,” refers to a polymeric form of nucleotides of any length, including DNA, RNA, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0070] The term “isolated” when referring to a nucleotide sequence, means that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. Thus, an “isolated nucleic acid molecule which encodes a particular polypeptide” refers to a nucleic acid molecule which is substantially free of other nucleic acid molecules that do not encode the subject polypeptide; however, the molecule may include some additional bases or moieties which do not materially affect the basic characteristics of the composition.

[0071] The terms “percent (%) amino acid sequence identity” and “percent identity” and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) refer to the percentage of amino acid residues in a candidate sequence (e.g. , the engineered AAV capsid) that are identical with the amino acid residues in the non-engineered capsid reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity7, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art. for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0072] For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated “upstream,” “downstream,” “3’,” or “5”’ relative to another sequence, it is to be understood that it is the position of the sequences in the “sense” or “coding” strand of a DNA molecule that is being referred to as is conventional in the art.

[0073] The term “recombinant,” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature and / or a combination of polynucleotides and viral proteins that is not found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

[0074] The term “gene,"’ refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the genes with which they are associated. Methods of isolating larger fragment sequences are known to those of skill in the art.

[0075] The term “transgene,'’ as used herein, refers to a nucleic acid sequence to be positioned within a viral vector and encoding a polypeptide, protein or other product of interest. In some embodiments, one rAAV vector, or engineered rAAV vector may comprise a sequence encoding one or more transgenes (which can optionally be the same gene, or different genes). For example, one rAAV vector may comprise the coding sequence for 1. 2, 3, 4. 5, 6, 7, 8. 9, or 10 transgenes.

[0076] The terms '‘gene transfer” or “gene delivery” refer to methods or systems for inserting DNA, such as a transgene, into host cells, such as those of a subject afflicted with a cardiomyopathy. In several embodiments, gene transfer yields transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes). In additional embodiments, gene transfer results in integration of transferred genetic material into the genomic DNA of host cells.

[0077] The terms “regulatory element"’ or “regulatory sequence”, or variations thereof, refer to a nucleotide sequence that participates in functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Regulatory' elements can be enhancing or inhibitory in nature, depending on the embodiment. Non-limiting examples of regulatory elements include transcriptional regulatory sequences such as promoter sequences, poly adenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (“IRES”), enhancers, and the like. These elements collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell, though not all of these sequences need always be present. It shall be appreciated that the structural components of a rAAV vector as provided for herein may be listed in individual paragraphs solely for clarity and may be used together in combination. For example, any regulatory element or other component can be used in combination with any transgene (or transgenes) provided for herein.

[0078] A “promoter” is a polynucleotide that interacts with an RNA polymerase and initiates transcription of a coding region (e.g.. a transgene) usually located downstream (in the 3' direction) from the promoter.

[0079] A “tissue-specific promoter”, as used herein, refers to promoters that can only function in a specific type of tissue, e.g., the heart. Thus, a “tissue- specific promoter” is not able to drive the expression of the transgenes in other types of tissues.

[0080] The term “operably linked” refers to an arrangement of elements wherein the components are configured to perform a function. For example, regulatory sequences operably linked to a coding sequence result in the expression of the coding sequence. Depending on the embodiment, a regulatory sequence need not be contiguous with the coding sequence. Thus, for example, one or more untranslated, yet transcribed, sequences can be present between a promoter sequence and a coding sequence, with those two sequences still being considered “operably linked.”

[0081] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0082] An “expression vector” is a vector comprising a region of nucleic acid (e.g., a transgene) which encodes a gene product (e.g., a polypeptide or protein) of interest. As disclosed herein, vectors are used for achieving expression, e.g., stable expression, of a protein in an intended target cell. An expression vector may also comprise control elements operatively linked to the transgene to facilitate expression of the encoded protein in the target cell. A combination of one or more regulatory elements and a gene or genes to which they are operably linked for expression may be referred to herein as an “expression cassette.”

[0083] A Kozak sequence is a functional sequence motif that is positioned near or at the translational initiation site of eukaryotic mRNAs. Kozak sequences mediate ribosome assembly and translation initiation and help regulate that a protein is correctly translated in the correct reading frame.

[0084] A “subject” refers to mammal that is the object of treatment using a method or composition as provided for herein. “Mammal” includes, without limitation, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates, such asmonkeys, chimpanzees, and apes, and humans. In some embodiments, the subject is human.

[0085] The terms ‘‘treating,’’ ‘‘treatment,” “therapeutic,” or “therapy” do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and / or therapy. To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0086] The term “effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect, such as reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0087] As used herein, a “composition” refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0088] As used herein, the term “kit” may be used to describe variations of the portable, self-contained enclosure that includes at least one set of components to conduct one or more of the diagnostic or therapeutic methods described herein.

[0089] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0090] As used herein, the singular forms “a,” “an.” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects, embodiments, and variations described herein include “comprising,” “consisting,” and / or “consisting essentially of aspects, embodiments and variations.

[0091] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between theupper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0092] The term '‘about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.Variant adeno-associated virus (AAV) Capsids

[0093] Some embodiments herein relate to genetically engineered viral capsid proteins (such as an AAV capsid protein) that exhibit an altered tissue tropism as compared to a non-engineered capsid protein.

[0094] FIG. 1 is a non-limiting illustrative representation of altered transduction or tropism of a modified adeno-associated virus (AAV) to muscle tissue vs liver tissue as compared to an un-modified AAV.

[0095] FIG. 2A is a rh74 VP1 capsid DNA sequence with some embodiments of insertion and mutation sites highlighted.

[0096] FIG. 2B is a rh74 VP1 capsid amino acid sequence with some embodiments of insertion and mutation sites highlighted.

[0097] FIG. 2C is a representative map of an expression construct encoding a rh74 VPl capsid polypeptide.

[0098] Some embodiments herein are directed to a variant adeno-associated virus (AAV) capsid polypeptide. In some embodiments, the variant AAV capsid polypeptide comprises a polypeptide sequence of a VP1 capsid protein. In some embodiments, the polypeptide comprises a polypeptide sequence of a VP 1 capsid protein of AAV serotype rh74. In some embodiments, the variant AAV capsid polypeptide comprises one or more engineered features. In some embodiments, the variant AAV capsid comprises at least two engineered features as compared to a corresponding non-engineered VP1 polypeptide. In some embodiments, the corresponding non-engineered VP1 polypeptide is anon-engineered AAV serotype rh74 VP1 polypeptide. In some embodiments, the first engineered feature comprises an insertion of one or more peptides within the polypeptide sequence of one or more AAV capsid polypeptides. In some embodiments, the insertion is within the VP1 polypeptide. In some embodiments, the inserted peptide enhances transduction or tropism of a recombinant AAV vector packaged using the variant capsid to muscle tissue. In some embodiments, the muscle tissue is cardiac muscle tissue. In some embodiments, the muscle tissue is skeletal muscle tissue. In some embodiments, the second engineered feature comprises one or more point mutations within the polypeptide sequence of one or more AAV capsid polypeptides. In some embodiments, the second engineered feature comprises one or more point mutations within the VP1 polypeptide. In some embodiments, the one or more point mutations reduce transduction or tropism of a recombinant AAV vector packaged using the variant capsid to one or more non-muscle tissue as compared to an AAV vector packed using a non-engineered VP1 capsid protein. In some embodiments, the one or more point mutations reduce transduction or tropism of a recombinant AAV vector packaged using the variant capsid to soft tissue. In some embodiments, the one or more point mutations reduce transduction or tropism of a recombinant AAV vector packaged using the variant capsid to liver tissue. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serot pe rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0099] In some embodiments, the variant AAV capsid polypeptide is an AAV serotype 1 (AAV-1), AAV serotype 2 (AAV -2), AAV serotype 3 (AAV-3). AAV serotype 4 (AAV-4), AAV serotype 5 (AAV-5), AAV serotype 6 (AAV-6), AAV serotype 7 (AAV- 7), AAV serotype 8 (AAV-8), AAV seroty pe 9 (AAV-9), serotype rhlO AAV, serotype rh74 AAV, and / or a pseudoty ped rAAV (e.g., AAV2 / 9, referring an AAV vector with the genome of AAV2 (e.g., the ITRs of AAV2) and the capsid of AAV9) capsid polypeptide. In some embodiments, the variant AAV capsid polypeptide is an AAV serotype rh74 capsid polypeptide. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0100] In some embodiments, the variant AAV capsid polypeptide comprises one or more engineered features as compared to a corresponding non-engineered AAV capsid polypeptide. In some embodiments, the variant AAV capsid polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 engineered features, or a number of engineered features that is within a range defined by any two of the preceding values. For example, in some embodiments, the variant AAV capsid polypeptide comprises between 1-10, 1-7. 1-5, 1-3, 1-2, 2-10, 2-7, 2-5. 2-3, 3-10, 3-7, 3-5. 5-10. 5-7, or 7-10 engineered features as compared to a corresponding non-engineered AAV capsid polypeptide. In some embodiments, the AAV capsid polypeptide is a VP1 capsid polypeptide.

[0101] In some embodiments, the variant AAV capsid polypeptide comprises one or more engineered features in the VP1 capsid protein of AAV serotype rh74 as compared to a corresponding non-engineered AAV rh74 VP1 polypeptide. In some embodiments, the variant AAV rh74 VP1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 engineered features, or a number of engineered features that is within a range defined by any two of the preceding values. For example, in some embodiments, the variant AAV rh74 VP1 polypeptide comprises between 1-10. 1-7, 1-5. 1-3, 1-2. 2-10. 2-7, 2-5. 2-3, 3- 10, 3-7, 3-5, 5-10, 5-7, or 7-10 engineered features as compared to a corresponding nonengineered AAV rh74 VP1 polypeptide.

[0102] In some embodiments, the variant AAV capsid polypeptide comprises one or more engineered features in the VP1 capsid protein of AAV9 as compared to a corresponding non-engineered AAV9 VP I polypeptide. In some embodiments, the variant AAV9 VP1 polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 engineered features, or a number of engineered features that is within a range defined by any two of the preceding values. For example, in some embodiments, the variant AAV9 VP1 polypeptide comprises between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 engineered features as compared to a corresponding non-engineered AAV9 VP1 polypeptide.

[0103] In some embodiments, the one or more engineered features comprise an insertion, deletion, substitution of one or more amino acids in an AAV capsid polypeptide, and / or any combination thereof. In some embodiments, the one or more engineered features comprise an insertion of one or more amino acids in an AAV capsid polypeptide. In some embodiments, the insertion is of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values. For example, in some embodiments, the insertion is of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7. 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids. In some embodiments, the insertion is within the VP1 polypeptide. In some embodiments, the insertion is an insertion within the VP1 polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values. For example, in some embodiments, the insertion within the VP1 polypeptide is of between 1- 10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5. 5-10. 5-7, or 7-10 amino acids. In some embodiments, the variant AAV capsid polypeptide is an AAV serotype rh74 capsid polypeptide. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0104] In some embodiments, the one or more engineered features enhances transduction or tropism to one or more tissues, cells, or systems. In some embodiments, the one or more engineered features enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, soft tissue, pancreas, kidney, and / or any combination thereof. In some embodiments, an insertion, deletion, substitution of one or more amino acids in an AAV capsid polypeptide, and / or any combination thereof, enhances transduction or tropism to the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, heart, muscle, and / or any combination thereof. In some embodiments, an insertion of one or more amino acids in an AAV capsid polypeptide enhances transduction or tropism to the heart. In some embodiments, an insertion of one or more amino acids in an AAV capsid polypeptide enhances transduction or tropism to the skeletal muscle. As discussed in more detail below, in several embodiments, wherein transduction and / or tropism is enhanced in the cardiac and / or skeletal muscle, an additional engineered feature(s) reduces transduction and / or tropism to soft tissue (e.g., the liver). In some embodiments, an insertion of one or more amino acids within the VP1 polypeptide, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof. In some embodiments, an insertion within the VP1 polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof. For example, in some embodiments, aninsertion within the VP1 polypeptide of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7. or 7-10 amino acids, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof. In some embodiments, when an insertion within the VP1 polypeptide of between 1-10, 1-7, 1-5, 1- 3, 1-2, 2-10. 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, enhances transduction or tropism to cardiac muscle or skeletal muscle, one or more additional engineered features reduces tropism or transduction to liver tissue and / or other soft tissue. In some embodiments, the variant AAV capsid polypeptide is an AAV serotype rh74 capsid polypeptide. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0105] In some embodiments, the one or more engineered features enhances transduction or tropism to one or more tissues, cells, or systems, by about 1%, 2%, 3%, 4%, 5%. 6%, 7%. 8%, 9%. 10%. 15%, 20%, 25%, 30%. 40%. 50%, 60%, 70%, 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. For example, in some embodiments, the one or more engineered features enhances transduction or tropism to one or more tissues, cells, or systems, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%. 25%-50%. 50%-1000%, 50%-750%. 50%-500%, 50%-250%, 50%-100%. 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. In some embodiments, the variant AAV capsid polypeptide is an AAV serotype rh74 capsid polypeptide. In some embodiments, the recombinant AAV vector is not a recombinantAAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0106] In some embodiments, the one or more engineered features enhances transduction or tropism to one or more tissues, cells, or systems. In some embodiments, the one or more engineered features enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. For example, in some embodiments, the one or more engineered features enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about l%-1000%, l%-750%, l%-500%, l%-250%, 1%- 100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%- 750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%, 50%-1000%. 50%-750%, 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%- 1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. In some embodiments, when one or more engineered features enhances transduction or tropism to cardiac muscle or skeletal muscle by between about 1%- 1000%. l%-750%, l%-500%. l%-250%. l%-100%. l%-75%, l%-50%. l%-25%. 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features, one or more additional engineered features reduce tropism or transduction to liver tissue or soft tissue. In some embodiments, the variant AAV capsid polypeptide is an AAV serotype rh74 capsid polypeptide. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV9. In some embodiments, the recombinant AAV vector is not a recombinant AAV9.

[0107] In some embodiments, the one or more engineered features is an insertion, deletion, substitution of one or more amino acids in an AAV capsid polypeptide. In some embodiments, the one or more engineered features is an insertion, deletion, substitution of one or more amino acids in an AAV VP1 capsid polypeptide. In some embodiments, the one or more engineered features is an insertion, deletion, substitution of one or more amino acids in an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, the one or more engineered features is an insertion, deletion, substitution of one or more amino acids in an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, an insertion, deletion, substitution of one or more amino acids in an AAV capsid polypeptide, and / or any combination thereof, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%. 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. For example, in some embodiments, an insertion, deletion, or substitution of one or more amino acids in an AAV capsid polypeptide, and / or any combination thereof, enhances transduction or tropism to cardiac muscle, skeletal muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%,5%-100%, 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%,10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%. 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding AAV capsid polypeptide not containing the insertion, deletion, or substitution of one or more amino acids.

[0108] In some embodiments, the one or more engineered features is an insertion, deletion, substitution of one or more amino acids in an AAV capsid polypeptide. In some embodiments, the one or more engineered features is an insertion, deletion, substitution of one or more amino acids in an AAV VP1 capsid polypeptide. In some embodiments, the one or more engineered features is an insertion, deletion, substitution of one or more amino acids in an AAV9 VP1 capsid polypeptide. In some embodiments, the one or more engineered features is an insertion, deletion, substitution of one or more amino acids in an AAV9 VP1 capsid polypeptide. In some embodiments, an insertion, deletion, substitution of one or more amino acids in an AAV capsid polypeptide, and / or any combination thereof, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. For example, in some embodiments, an insertion, deletion, or substitution of one or more amino acids in an AAV capsid polypeptide, and / or any combination thereof, enhances transduction or tropism to cardiac muscle, skeletal muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, I%-25%, l%-10%, I%-7%, I%-5%, l%-3%, 3%-I000%, 3%-750%, 3%-500%, 3%-250%, 3%-I00%, 3%-75%, 3%-50%, 3%-25%, 3%-I0%, 3%- 7%, 3%-5%, 5%-I000%, 5%-750%. 5%-500%, 5%-250%, 5%-I00%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, I0%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV capsid polypeptide not containing the insertion, deletion, or substitution of one or more amino acids.

[0109] In some embodiments, the one or more engineered features is an insertion of one or more amino acids in an AAV capsid polypeptide. In some embodiments, an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV capsid polypeptide enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%. 350%, 400%, 450%, 500%, 600%. 700%. 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the insertion. For example, in some embodiments, an insertion of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10. 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids within an AAV capsid polypeptide enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, , and / or any combination thereof, by between about l%-1000%, l%-750%, l%-500%, l%-250%, 1%- 100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%- 750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%. 25%-250%, 25%-100%, 25%-75%, 25%-50%. 50%-1000%, 50%-750%, 50%-500%. 50%-250%. 50%-100%. 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV capsid polypeptide not containing insertion. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0110] In some embodiments, the one or more engineered features is an insertion of one or more amino acids in an AAV VP1 polypeptide. In some embodiments, an insertion within the VP1 polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding value, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1%. 2%, 3%, 4%, 5%, 6%, 7%, 8%. 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the insertion within the VP1 polypeptide. For example, in some embodiments, an insertion within the VP1 polypeptide of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, enhances transduction or tropism to the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, heart, muscle, and / or any combination thereof, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%- 100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV capsid polypeptide not containing the insertion within the VP1 polypeptide. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serot pe 9.[OHl] In some embodiments, the one or more engineered features is an insertion of one or more amino acids within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, an insertion within the rh74 VP1 capsid polypeptideof at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding value, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, 100%. 150%, 200%, 250%, 300%, 350%. 400%. 450%, 500%, 600%. 700%, 750%, 800%, 900%. or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding rh74 VP1 capsid polypeptide not containing the insertion. For example, in some embodiments, an insertion within the rh74 VP1 capsid polypeptide of between 1-10. 1-7, 1-5. 1-3, 1-2, 2-10, 2-7, 2-5, 2-3. 3-10, 3-7, 3-5. 5-10, 5-7, or 7-10 amino acids, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%. l%-25%, l%-10%, l%-7%. l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%. 3%-25%. 3%-10%, 3%- 7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%, 10%-50%. 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%. 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding rh74 VP1 capsid polypeptide not containing the insertion. In some embodiments, when an insertion within the rh74 VP1 capsid polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids enhance tropism or transduction to cardiac muscle or skeletal muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0112] In some embodiments, the one or more engineered features is an insertion of one or more amino acids within an AAV9 VP1 capsid polypeptide. In some embodiments, an insertion within the AAV9 VP1 capsid polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding value, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%. 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%. 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the insertion. For example, in some embodiments, an insertion within the AAV9 VP1 capsid polypeptide of between 1-10. 1-7, 1-5, 1-3. 1-2, 2-10, 2-7. 2-5, 2-3, 3-10, 3-7. 3-5, 5-10, 5-7. or 7-10 amino acids, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about l%-1000%, l%-750%, l%-500%, l%-250%, 1%- 100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%- 750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%, 10%-50%. 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%. 50%-1000%. 50%-750%. 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the insertion. In some embodiments, when an insertion within the AAV9 VP1 capsid polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids enhance tropism or transduction to cardiac muscle or skeletal muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0113] In some embodiments, the one or more engineered features is an insertion of at least 5 amino acids within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, an insertion within the rh74 VP1 polypeptide of at least 5 amino acids enhances transduction or tropism to cardiac muscle by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%. 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%. 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%. 100%, 150%, 200%, 250%. 300%. 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding rh74 VP1 polypeptide not containing the insertion. For example, in some embodiments, an insertion within the rh74 VP1 polypeptide of at least 5 amino acids enhances transduction or tropism to cardiac muscle by between about l%-1000%, 1%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%. 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%. 100%-1000%. 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding rh74 VP1 polypeptide not containing the insertion. In some embodiments, when an insertion within the rh74 VP1 capsid polypeptide of at least 5 amino acids enhances tropism or transduction to cardiac muscle or skeletal muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0114] In some embodiments, the one or more engineered features is an insertion of at least 5 amino acids within an AAV9 VP1 capsid polypeptide. In some embodiments, an insertion within the AAV9 VP1 polypeptide of at least 5 amino acids enhances transduction or tropism to cardiac muscle by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%. 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 polypeptide not containing the insertion. For example, in some embodiments, an insertion within the AAV9 VP1 polypeptide of at least 5 amino acids enhances transduction or tropism to cardiac muscle by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%-1000%. 25%-750%. 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding AAV9 VP1 polypeptide not containing the insertion. In some embodiments, when an insertion within the AAV9 VP1 capsidpolypeptide of at least 5 amino acids enhances tropism or transduction to cardiac muscle or skeletal muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0115] In some embodiments, the one or more engineered features is an insertion of at least 7 amino acids within an AAV serotype rh74VPl capsid polypeptide. In some embodiments, an insertion within the rh74 VP1 polypeptide of at least 7 amino acids enhances transduction or tropism to cardiac muscle by about 1%, 2%, 3%. 4%, 5%. 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding rh74 VP1 polypeptide not containing the insertion. For example, in some embodiments, an insertion within the rh74 VP1 polypeptide of at least 7 amino acids enhances transduction or tropism to cardiac muscle by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%. 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%. 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding rh74 VP1 polypeptide not containing the insertion. In some embodiments, when an insertion within the rh74 VP1 capsid polypeptide of at least 7 amino acids enhances tropism or transduction to cardiac muscle or skeletal muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0116] In some embodiments, the one or more engineered features is an insertion of at least 7 amino acids within an AAV9 capsid polypeptide. In some embodiments, an insertion within the AAV9 VP1 polypeptide of at least 7 amino acids enhances transduction or tropism to cardiac muscle by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%. 200%, 250%, 300%. 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentagethat is in a range defined by any two of the preceding values, as compared to a corresponding AAV9VP1 polypeptide not containing the insertion. For example, in some embodiments, an insertion within the AAV9 VP1 polypeptide of at least 7 amino acids enhances transduction or tropism to cardiac muscle by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%, 3%-75%, 3%- 50%. 3%-25%. 3%-10%, 3%-7%. 3%-5%, 5%-1000%. 5%-750%. 5%-500%. 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%, 100%-1000%. 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding AAV9 VP1 polypeptide not containing the insertion. In some embodiments, when an insertion within the AAV9 VP1 capsid polypeptide of at least 7 amino acids enhances tropism or transduction to cardiac muscle or skeletal muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0117] In some embodiments, the one or more engineered features enhances transduction or tropism to one or more tissues, cells, or systems., by about 1, 2, 3, 4, 5, 6, 7, 8, 9. 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200. 250, 300, 400. 500, 600. 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. For example, in some embodiments, the one or more engineered features enhances transduction or tropism to one or more tissues, cells, or systems, by between about 1-1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10- 250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500. 25-250, 25-100, 25-75, 25- 50. 50-1000. 50-750, 50-500. 50-250, 50-100, 100-1000. 100-750, 100-500, 100-250. 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. In some embodiments, when one engineered feature enhances tropism or transduction to cardiac muscle and / or skeletal muscle, one or more additional engineered features reduces transduction or tropism to soft tissue (e.g., liver tissue). In someembodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0118] In some embodiments, the one or more engineered features enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. For example, in some embodiments, the one or more engineered features enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about 1-1000, 1-750, 1-500. 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3-250. 3-100, 3-75, 3-50, 3-25, 3-10. 3-7. 3-5, 5-1000. 5-750, 5-500. 5-250. 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. In some embodiments, when one or more engineered features enhances tropism or transduction to cardiac muscle or skeletal muscle, one and / or more additional engineered features reduces transduction or tropism to soft tissue (e.g., liver tissue). In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0119] In some embodiments, the one or more engineered features is an insertion, deletion, substitution of one or more amino acids in an AAV capsid polypeptide. In some embodiments, an insertion, deletion, substitution of one or more amino acids in an AAV capsid polypeptide, and / or any combination thereof, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervoussystem (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1, 2. 3, 4, 5. 6, 7, 8, 9, 10, 20, 25, 30, 40, 50. 60. 70. 75. 80. 90. 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more engineered features. For example, in some embodiments, an insertion, deletion, or substitution of one or more amino acids in an AAV capsid polypeptide, and / or any combination thereof, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about 1-1000, 1-750, 1-500, 1-250, 1-100. 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750. 3-500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10. 3-7, 3-5. 5-1000, 5-750, 5- 500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10- 75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50- 750, 50-500, 50-250. 50-100, 100-1000, 100-750. 100-500. 100-250. 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV capsid polypeptide not containing the insertion, deletion, or substitution of one or more amino acids. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0120] In some embodiments, the one or more engineered features is an insertion, deletion, or substitution, of one or more amino acids in an AAV VP1 capsid polypeptide. In some embodiments, the one or more engineered features is an insertion, deletion, or substitution, of one or more amino acids in an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by' any two of the preceding values, within an AAV capsid polypeptide enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the insertion. For example, in some embodiments, an insertion of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10amino acids within an AAV capsid polypeptide enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about 1- 1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3- 500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50. 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000. 25-750, 25-500, 25-250, 25-100. 25-75, 25-50, 50-1000, 50-750. 50-500, 50- 250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500- 1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV capsid polypeptide not containing insertion. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0121] FIG. 3A is a rh74-A VP1 capsid DNA sequence depicting some embodiments of an engineered insertion.

[0122] FIG. 3B is a rh74-A VP1 capsid amino acid sequence with some embodiments of an engineered insertion.

[0123] FIG. 3C is a representative map of an expression construct encoding a rh74-A VPl capsid polypeptide.

[0124] In some embodiments, the one or more engineered features is an insertion of one or more amino acids in an AAV VP1 polypeptide. In some embodiments, an insertion within the VP1 polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. amino acids, or of a number of amino acids that is in a range defined by any two of the preceding value, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100. 200, 250, 300, 400, 500. 600, 700, 750, 800, 900. or 1000- fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the insertion within the VP1 polypeptide. For example, in some embodiments, an insertion within the VP1 polypeptide of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver,pancreas, kidney, and / or any combination thereof, by between about 1-1000, 1-750, 1- 500, 1-250, 1-100. 1-75. 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000. 3-750, 3-500, 3-250, 3- 100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5- 25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25- 750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000. 250-750. 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV capsid polypeptide not containing the insertion within the VP1 polypeptide. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0125] In some embodiments, the one or more engineered features is an insertion of one or more amino acids within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, an insertion within the rh74 VP1 capsid polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding value, enhances transduction or tropism to cardiac muscle, skeletal muscle, heart muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding rh74 VP1 capsid polypeptide not containing the insertion. For example, in some embodiments, an insertion within the rh74 VP1 capsid polypeptide of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2- 5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about 1- 1000. 1-750, 1-500, 1-250, 1-100. 1-75. 1-50. 1-25. 1-10. 1-7, 1-5. 1-3, 3-1000, 3-750, 3- 500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50- 250, 50-100. 100-1000, 100-750, 100-500, 100-250. 250-1000, 250-750, 250-500. 500- 1000, 500-750, or 750-1000-fold, as compared to a corresponding rh74 VP1 capsidpolypeptide not containing the insertion. In some embodiments, when an insertion within the rh74 VP1 capsid polypeptide of between 1-10, 1-7. 1-5, 1-3. 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10, amino acids enhance tropism or transduction to cardiac muscle and / or skeletal muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0126] In some embodiments, the one or more engineered features is an insertion of one or more amino acids within an AAV9 VP1 capsid polypeptide. In some embodiments, an insertion within the AAV9 VP1 capsid polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding value, enhances transduction or tropism to cardiac muscle, skeletal muscle, heart muscle, the central nervous system (CNS). retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the insertion. For example, in some embodiments, an insertion within the AAV9 VP1 capsid polypeptide of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3- 10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, enhances transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about 1- 1000, 1 -750, 1 -500, 1-250, 1-100, 1 -75, 1 -50, 1-25, 1 -10, 1 -7, 1-5, 1 -3, 3-1000, 3-750, 3- 500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750. 50-500, 50- 250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500- 1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the insertion. In some embodiments, when an insertion within the AAV9 VP1 capsid polypeptide of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7. 2-5, 2-3, 3-10. 3-7, 3-5, 5-10, 5-7. or 7-10, amino acids enhance tropism or transduction to cardiac muscle and / or skeletal muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0127] In some embodiments, the one or more engineered features is an insertion within an AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide. In some embodiments, an insertion within the rh74 VP1 polypeptide of at least 1, 2, 3, 4, 5, 6, 7, 8,9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding value, enhances transduction or tropism to cardiac muscle by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding rh74 and / or AAV9 VP1 polypeptide not containing the insertion. For example, in some embodiments, an insertion within the rh74 and / or AAV9 VP1 polypeptide of between 1-10. 1-7, 1-5. 1-3, 1-2, 2-10.2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, enhances transduction or tropism to cardiac muscle by between about, 1-1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1- 50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10,3-7, 3-5, 5-1000, 5-750. 5-500, 5-250, 5-100, 5-75, 5-50, 5-25. 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding rh74 and / or AAV9 VP1 polypeptide not containing the insertion. In some embodiments, when an insertion within the rh74 VP1 capsid polypeptide of between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10, amino acids enhance tropism or transduction to cardiac muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0128] In some embodiments, the one or more engineered features is an insertion of at least 5 amino acids within an AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide. In some embodiments, an insertion within the rh74 and / or AAV9 VP1 polypeptide of at least 5 amino acids enhances transduction or tropism to cardiac muscle by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding rh74 and / or AAV9 VP1 polypeptide not containing the insertion. For example, in some embodiments, an insertion within the rh74 and / or AAV9 VP1 polypeptide of at least 5 amino acids enhances transduction or tropism to cardiac muscle by between about 1- 1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3- 500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50. 5-25, 5-10, 10-1000, 10-750. 10-500, 10-250, 10-100, 10-75, 10-50. 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500- 1000. 500-750, or 750-1000-fold, as compared to a corresponding rh74 and / or AAV9 VP1 polypeptide not containing the insertion. In some embodiments, when an insertion within the rh74 and / or AAV9 VP1 capsid polypeptide of at least 5 amino acids enhances tropism or transduction to cardiac muscle, one or more additional engineered features reduces transduction or tropism to soft tissue or liver tissue.

[0129] In some embodiments, the one or more engineered features is an insertion of at least 7 amino acids within an AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide. In some embodiments, an insertion within the rh74 and / or AAV9 VP1 polypeptide of at least 7 amino acids enhances transduction or tropism to cardiac muscle by about 1. 2, 3, 4, 5. 6, 7, 8. 9, 10, 20, 25. 30. 40. 50. 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding rh74 and / or AAV9 VP1 polypeptide not containing the insertion. For example, in some embodiments, an insertion within the rh74 and / or AAV9 VP1 polypeptide of at least 7 amino acids enhances transduction or tropism to cardiac muscle by between about 1- 1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3- 500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50. 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750. 50-500, 50- 250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500- 1000, 500-750, or 750-1000-fold, as compared to a corresponding rh74 and / or AAV9 VP1 polypeptide not containing the insertion. In some embodiments, when an insertion within the rh74 and / or AAV9 VP1 capsid polypeptide of at least 7 amino acids enhances tropism or transduction to cardiac muscle, one or more additional engineered features reduces transduction or tropism to soft tissue and / or liver tissue.

[0130] In some embodiments, the one or more engineered features is an insertion of at least 8 amino acids within an AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide. In some embodiments, an insertion within the rh74 and / or AAV9 VP1 polypeptide of at least 8 amino acids enhances transduction or tropism to cardiac muscle by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding rh74 and / or AAV9 VP1 polypeptide not containing the insertion. For example, in someembodiments, an insertion within the rh74 and / or AAV9 VP1 polypeptide of at least 8 amino acids enhances transduction or tropism to cardiac muscle by between about 1- 1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3- 500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750. 50-500, 50- 250, 50-100. 100-1000, 100-750, 100-500. 100-250. 250-1000. 250-750. 250-500. 500- 1000, 500-750, or 750-1000-fold, as compared to a corresponding rh74 and / or AAV9 VP1 polypeptide not containing the insertion. In some embodiments, when an insertion within the rh74 and / or AAV9 VP1 capsid polypeptide of at least 8 amino acids enhances tropism or transduction to cardiac muscle, one or more additional engineered features reduces transduction or tropism to soft tissue and / or liver tissue.

[0131] FIG. 4A is a rh74-001 VP1 capsid DNA sequence depicting some embodiments of an engineered point mutation.

[0132] FIG. 4B is a rh74-001 VP1 capsid amino acid sequence with some embodiments of an engineered point mutation.

[0133] FIG. 4C is a representative map of an expression construct encoding a rh74-001 VP1 capsid polypeptide.

[0134] In some embodiments, the one or more engineered features is a point mutation in one or more amino acids of an AAV capsid polypeptide. In some embodiments, at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, are mutated. For example, in some embodiments, at least 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7. or 7-10, amino acids are mutated. In some embodiments, the mutation is within the VP1 polypeptide. In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or a number of amino acids in a range defined by any two of the preceding values, are mutated within the VP1 polypeptide. For example, in some embodiments, between 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10, amino acids are mutated within the VPI polypeptide. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0135] In some embodiments, mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to one or more tissues, cells, or systems, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to one or more tissues, cells, or systems, by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%. 50%-750%. 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%. as compared to a corresponding AAV capsid polypeptide not containing the one or more mutated amino acids. In some embodiments, the one or more amino acid mutations are a glycine to alanine mutation. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0136] In some embodiments, mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to the liver, the central nervous system (CNS), retina, lung, diaphragm, pancreas, kidney, heart, muscle, and / or any combination thereof, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%. 30%. 40%. 50%, 60%, 70%, 75%, 80%. 90%. 91%. 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV capsid polypeptidereduces transduction or tropism to the liver, soft tissue, the central nervous system (CNS), retina, lung, diaphragm, pancreas, kidney, heart, muscle, and / or any combination thereof, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%. 10%-1000%, 10%-750%. 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%. or 750%-1000%, as compared to a corresponding AAV capsid polypeptide not containing the one or more mutated amino acids. In some embodiments, the one or more amino acid mutations are a glycine to alanine mutation. In some embodiments, when mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to the liver and / or soft tissue, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of seroty pe rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0137] In some embodiments, mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to the liver by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%. 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%. 99%, 100%. 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to the liver by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%. 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV capsid polypeptide not containing the one or more mutated amino acids. In some embodiments, the one or more amino acid mutations are a glycine to alanine mutation. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0138] In some embodiments, mutation of one or more amino acids within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to the liver by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%. 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%. 450%. 500%. 600%, 700%, 750%, 800%, 900%, or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to the liver by between about 1%- 1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, 1 %-25%, 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%. 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%. or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide not containing the one or more mutated amino acids. In some embodiments, the one or more amino acid mutations are a glycine to alanine mutation. In some embodiments, when mutation of one or more amino acids within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to the liver, one or moreadditional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0139] In some embodiments, mutation of one or more amino acids within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to the liver by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%. 350%. 400%, 450%, 500%, 600%. 700%. 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to the liver by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, 1%- 75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%. 5%-50%, 5%-25%, 5%-10%. 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%. 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%-500%. 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the one or more mutated amino acids. In some embodiments, the one or more amino acid mutations are a glycine to alanine mutation. In some embodiments, when mutation of one or more amino acids within an AAV AAV9 VP1 capsid polypeptide reduces transduction or tropism to the liver, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0140] In some embodiments, a gly cine to alanine mutation within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to the liver by about 1%, 2%. 3%, 4%. 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%. 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide not containing the glycine to alanine mutation. For example, in someembodiments, a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to the liver by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, 1%- 7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%- 250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%. 10%-1000%, 10%-750%, 10%- 500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%. 25%-1000%, 25%- 750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%- 750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%. or 750%- 1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide not containing the glycine to alanine mutation. In some embodiments, when a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to the liver, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0141] In some embodiments, a glycine to alanine mutation within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to the liver by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%. 150%, 200%, 250%, 300%, 350%. 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%. or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the glycine to alanine mutation. For example, in some embodiments, a glycine to alanine mutation within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to the liver by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%. 10%-1000%, 10%-750%. 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%. or 750%-1000%, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the glycine to alanine mutation. In someembodiments, when a glycine to alanine mutation within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to the liver, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0142] In some embodiments, mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to one or more tissues, cells, or systems, muscles by about 1, 2. 3, 4, 5. 6, 7. 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80. 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to one or more tissues, cells, or systems, by between about 1-1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1- 7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750. 10-500, 10-250, 10- 100, 10-75, 10-50. 10-25. 25-1000. 25-750, 25-500. 25-250, 25-100. 25-75. 25-50, 50- 1000, 50-750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV capsid poly peptide not containing the one or more mutated amino acids. In some embodiments, the one or more amino acid mutations are a glycine to alanine mutation. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0143] In some embodiments, mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to the liver, soft tissue, the central nervous system (CNS), retina, lung, diaphragm. , pancreas, kidney, heart, muscle, and / or any combination thereof, muscle by about 1, 2. 3, 4, 5. 6, 7. 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000- fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to the liver, softtissue, the central nervous system (CNS), retina, lung, diaphragm, pancreas, kidney, heart, muscle, soft tissue, and / or any combination thereof, by between about 1-1000, 1-750, 1- 500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3-250, 3- 100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5- 25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25- 750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000. 250-750. 250-500. 500-1000. 500-750. or 750-1000-fold, as compared to a corresponding AAV capsid polypeptide not containing the one or more mutated amino acids. In some embodiments, the one or more amino acid mutations are a glycine to alanine mutation. In some embodiments, when mutation of one or more amino acids within an AAV capsid polypeptide reduces transduction or tropism to the liver and / or soft tissue, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0144] In some embodiments, mutation of one or more amino acids within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to soft tissue by about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to soft tissue by between about 1-1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5- 500, 5-250. 5-100. 5-75. 5-50. 5-25. 5-10, 10-1000, 10-750, 10-500. 10-250, 10-100, 10- 75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50- 750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide not containing the one or more mutated amino acids. In some embodiments, when mutation of one or more amino acids within an AAVserotype rh74 VP1 capsid polypeptide reduces transduction or tropism to soft tissue, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0145] In some embodiments, mutation of one or more amino acids within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to soft tissue by about 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 20, 25. 30. 40. 50, 60, 70, 75, 80, 90, 100. 200, 250, 300. 400, 500, 600. 700, 750, 800. 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to soft tissue by between about 1-1000, 1- 750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3- 250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5- 50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250. 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100. 25-75, 25-50, 50-1000, 50-750, 50-500. 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000. 250-750. 250-500. 500-1000. 500-750. or 750-1000-fold, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the one or more mutated amino acids. In some embodiments, when mutation of one or more amino acids within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to soft tissue, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0146] In some embodiments, mutation of one or more amino acids within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to liver tissue by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide not containing the one or more mutations. For example, in some embodiments, mutation of one or more amino acids within an AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide reduces transduction or tropism to liver tissue by between about 1-1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1- 50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100. 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500. 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500,100-250, 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide not containing the one or more mutated amino acids. In some embodiments, when mutation of one or more amino acids within an AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide reduces transduction or tropism to the liver, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0147] In some embodiments, a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to soft tissue by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500, 600. 700. 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide not containing the glycine to alanine mutation. For example, in some embodiments, a glycine to alanine mutation within an AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide reduces transduction or tropism to soft tissue by between about 1-1000, 1-750, 1-500. 1-250, 1-100, 1-75, 1-50, 1- 25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3- 5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25. 25-1000, 25-750, 25-500. 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750. 50-500, 50-250, 50-100. 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide not containing the glycine to alanine mutation. In some embodiments, when a glycine to alanine mutation within an AAV serotype rh74 and / or AAV9 VP1 capsid polypeptide reduces transduction or tropism to soft tissue, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0148] In some embodiments, a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to liver tissue by about 1, 2, 3. 4, 5, 6. 7, 8, 9. 10. 20. 25, 30, 40, 50, 60, 70, 75, 80, 90, 100. 200, 250, 300. 400, 500, 600, 700, 750, 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide not containing the glycine to alanine mutation. For example, in some embodiments, a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to liver tissue by betweenabout 1-1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 1-7, 1-5, 1-3, 3-1000, 3-750, 3-500, 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7. 3-5, 5-1000, 5-750, 5-500. 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide not containing the glycine to alanine mutation. In some embodiments, when a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid polypeptide reduces transduction or tropism to the liver, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0149] In some embodiments, a glycine to alanine mutation within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to liver tissue by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 40, 50, 60, 70, 75, 80, 90, 100, 200, 250, 300, 400, 500. 600, 700, 750. 800, 900, or 1000-fold, or by an amount that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the glycine to alanine mutation. For example, in some embodiments, a glycine to alanine mutation within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to liver tissue by between about 1-1000, 1-750, 1-500. 1-250, 1-100, 1-75, 1-50, 1-25. 1-10. 1-7, 1-5, 1-3. 3-1000, 3-750, 3-500. 3-250, 3-100, 3-75, 3-50, 3-25, 3-10, 3-7, 3-5, 5-1000, 5-750, 5-500, 5-250, 5-100, 5-75, 5-50, 5-25, 5-10, 10-1 00, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50-250, 50-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500-1000, 500-750, or 750-1000-fold, as compared to a corresponding AAV9 VP1 capsid polypeptide not containing the glycine to alanine mutation. In some embodiments, when a glycine to alanine mutation within an AAV9 VP1 capsid polypeptide reduces transduction or tropism to the liver, one or more additional engineered features enhances transduction or tropism to cardiac muscle and / or skeletal muscle.

[0150] FIG. 5A is a rh74-003 VP1 capsid DNA sequence depicting some embodiments of an engineered insertion and point mutation.

[0151] FIG. 5B is a rh74-003 VP1 capsid amino acid sequence with some embodiments of an engineered insertion and point mutation.

[0152] FIG. 5C is a representative map of an expression construct encoding a rh74-003 VP1 capsid polypeptide.

[0153] In some embodiments, the variant AAV capsid comprises 2 engineered features. In some embodiments, the first engineered feature is an insertion of one or more amino acids within an AAV capsid polypeptide and the second engineered feature is a mutation of one or more amino acids within an AAV capsid polypeptide. In some embodiments, the first engineered feature enhances or maintains transduction or tropism to one or more tissues, cells, or systems, muscles, and the second engineered feature reduces or maintains transduction or tropism to one or more tissues, cells, or systems, muscles. For example, in some embodiments, the first engineered feature enhances or maintains transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, and the second engineered feature reduces or maintains transduction or tropism to the liver, soft tissue, the central nervous system (CNS), retina, lung, diaphragm, pancreas, kidney, heart, muscle, and / or any combination thereof. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0154] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within AAV capsid polypeptide, and the second engineered feature comprises mutation of at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, amino acids, or a number of amino acids in a range defined by any two of the preceding values. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10, amino acids in an AAV capsid polypeptide, and the second engineered feature comprises mutation of between about 1-10, 1-7. 1-5, 1-3. 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids within the same AAV capsid polypeptide. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAVvector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0155] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, resulting in enhanced transduction or tropism to one or more tissues by about 1%. 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3. 4, 5, 6. 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, resulting in reduced transduction or tropism to one or more tissues by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%. 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%. 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10. 1-7, 1-5, 1-3. 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7. 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in enhanced transduction or tropism to one or more tissues by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%- 100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%. 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7- 10 amino acids, resulting in reduced transduction or tropism to one or more tissues by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%,3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000% 5%-750%, 5%-500%. 5%-250%. 5%-100% 5%-75% 5%-50%, 5%-25% 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%- 500%, 500%-1000%. 500%-750%, or 750%-1000%. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0156] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, resulting in enhanced transduction or tropism to cardiac muscle, skeletal muscle, muscle, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%. 100%, 150%, 200%. 250%, 300%, 350%. 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, resulting in reduced transduction or tropism to the liver, soft tissue, the central nervous system (CNS), retina, lung, diaphragm, pancreas, kidney, heart, muscle, and / or any combination thereof, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%. 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10, 1-7, 1-5, 1-3. 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7. 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in enhanced transduction or tropism to cardiac muscle, skeletal muscle, muscle,the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, and / or any combination thereof, by between about l%-1000%, l%-750%, l%-500%. l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%- 7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%. 10%-50%. 10%-25%, 25%-1000%. 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7. 1-5, 1-3. 1-2, 2-10, 2- 7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in reduced transduction or tropism to the liver, soft tissue, the central nervous system (CNS), retina, lung, diaphragm, liver, pancreas, kidney, heart, muscle, and / or any combination thereof, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%. l%-75%, 1%- 50%. l%-25%, l%-10%. l%-7%, l%-5%, l%-3%. 3%-1000%. 3%-750%. 3%-500%. 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%. 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype rh74. In some embodiments, the recombinant AAV vector is a recombinant AAV vector of serotype 9. In some embodiments, the recombinant AAV vector is not a recombinant AAV vector of serotype 9.

[0157] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%. 3%, 4%. 5%, 6%. 7%, 8%. 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%. or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%. 6%, 7%. 8%. 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. l%-50%. 1%- 25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%- 750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%- 1000%, 10%-750%. 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%- 25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%- 50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3- 7, 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in reduced transduction or tropism to soft tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, 1%- 75%, l%-50%. l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%. 3%-50%, 3%-25%. 3%-10%, 3%-7%. 3%-5%. 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%. 50%-1000%, 50%-750%, 50%-500%. 50%-250%. 50%-100%,100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%. 500%-1000%, 500%-750%, or 750%-1000%.

[0158] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, w ithin an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%. 3%, 4%. 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%. 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%. 94%. 95%. 96%. 97%. 98%. 99%. 100%. 150%, 200%. 250%. 300%, 350%. 400%. 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any tw o of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10, 1 -7, 1 -5, 1-3, 1 -2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, l%-750%, l%-500%. l%-250%, l%-100%, l%-75%. l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%. 25%-250%, 25%-100%, 25%-75%, 25%-50%. 50%-1000%, 50%-750%, 50%-500%. 50%-250%. 50%-100%. 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in reduced transduction or tropism to soft tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, 1%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%- 750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0159] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%. 250%, 300%, 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%. or 1000%. or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP 1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, 1%- 25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%- 750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%- 1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%- 50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3- 7, 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%. l%-750%. l%-500%. l%-250%. l%-100%. 1%- 75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%. 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0160] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, w ithin an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%. 3%, 4%. 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%. 94%. 95%. 96%. 97%. 98%. 99%. 100%. 150%, 200%. 250%. 300%, 350%. 400%. 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 ammo acids withinan AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, l%-750%, l%-500%. l%-250%. l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%. 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7, 1-5, 1-3. 1-2, 2-10, 2-7. 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%. l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%- 7%, 3%-5%. 5%-1000%, 5%-750%. 5%-500%. 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0161] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%. 250%, 300%, 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%. or 1000%. or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%,80%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. 100%, 150%, 200%, 250%, 300%, 350%. 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10. 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5. 5-10, 5-7. or 7-10 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, 1%- 25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%, 3%-5%, 5%-1000%. 5%- 750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%- 1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%- 25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%- 50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%. 100%-500%. 100%-250%, 250%-1000%. 250%-750%. 250%-500%. 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3- 7, 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in reduced transduction or tropism to soft tissue by between about l%-1000%. l%-750%. l%-500%. l%-250%. l%-100%. 1%- 75%, 1 %-50%, 1 %-25%, 1 %-10%, l%-7%, l %-5%, l%-3%, 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%. 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0162] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, w ithin an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%. 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%,150%, 200%, 250%, 300%, 350%, 400%, 450%. 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%. l%-250%, l%-100%, l%-75%. l%-50%, l%-25%, l%-10%, l%-7%, l%-5%. l%-3%, 3%-1000%. 3%-750%. 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%. 10%-1000%, 10%-750%, 10%-500%. 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7, 1-5, 1-3. 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in reduced transduction or tropism to soft tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, 1%- 100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%- 750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%. 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0163] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2. 3, 4, 5, 6, 7. 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%. 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%. 99%. 100%, 150%, 200%. 250%, 300%, 350%, 400%. 450%, 500%, 600%. 700%. 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3, 4. 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. 100%, 150%, 200%, 250%, 300%, 350%. 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10. 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5. 5-10, 5-7. or 7-10 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, 1%- 25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%, 3%-5%, 5%-1000%. 5%- 750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%- 1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%- 25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%- 50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%. 100%-500%. 100%-250%, 250%-1000%. 250%-750%. 250%-500%. 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3- 7, 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%. l%-750%. l%-500%. l%-250%. l%-100%. 1%- 75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%. 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%. 500%-1000%, 500%-750%. or 750%-1000%.

[0164] In some embodiments, the first engineered feature comprises an insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%. 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises mutation of at least 1, 2, 3. 4, 5, 6, 7, 8, 9, or 10, amino acids, or of a number of amino acids that is in a range defined by any two of the preceding values, within an AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%. 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%. l%-250%, l%-100%, l%-75%. l%-50%, l%-25%, l%-10%, l%-7%, l%-5%. l%-3%, 3%-1000%. 3%-750%. 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%. 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%,100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%. 500%-1000%. 500%-750%, or 750%-1000%, and the second engineered feature comprises mutation of between about 1-10, 1-7, 1-5, 1-3, 1-2, 2-10, 2-7, 2-5, 2-3, 3-10, 3-7, 3-5, 5-10, 5-7, or 7-10 amino acids, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%. l%-25%, l%-10%, l%-7%. l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%- 7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%. 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0165] In some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%. 3%, 4%. 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%. 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%. 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%. 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%. or 750%- 1000%.

[0166] In some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%. 40%. 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%. 500%. 600%, 700%, 750%, 800%, 900%, or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%,l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%. 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%. 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%. 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%, l%-750%, l%-500%, l%-250%. l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%. 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0167] In some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, 100%, 150%, 200%. 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%. 3%, 4%. 5%, 6%. 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%. 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the firstengineered feature comprises an insertion of at least 5 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%, 5%-500%. 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%. 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%. 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%-500%. 500%-1000%, 500%-750%, or 750%-1000%. and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%. l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%, l%-7%, l%-5%. l%-3%, 3%-1000%, 3%-750%. 3%-500%. 3%-250%, 3%-100%, 3%-75%. 3%-50%, 3%-25%. 3%-10%, 3%-7%, 3%-5%, 5%-1000%. 5%-750%. 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%. 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%- 1000%, 250%-750%, 250%-500%, 500%-1 00%, 500%-750%, or 750%-1000%.

[0168] In some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%. 70%. 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%. 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%,900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. l%-50%, l%-25%, l%-10%, l%-7%, l%-5%. l%-3%, 3%-1000%. 3%-750%. 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%. 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%.

[0169] In some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%. 40%. 50%, 60%, 70%, 75%, 80%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, byabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%. 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%. 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%. 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%. 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%. 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%, l%-750%, l%-500%, l%-250%. l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%, l %-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0170] In some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%. 92%, 93%, 94%, 95%, 96%. 97%. 98%, 99%, 100%, 150%, 200%. 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by apercentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%. 500%. 600%, 700%, 750%, 800%, 900%, or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%. 5%-500%. 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%- 25%. 5%-10%. 10%-1000%. 10%-750%, 10%-500%. 10%-250%, 10%-100%. 10%-75%. 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%-500%. 500%-1000%. 500%-750%, or 750%-1000%. and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%. l%-750%, l%-500%. l%-250%. l%-100%. l%-75%, l%-50%, l%-25%. 1%- 10%, l%-7%, l%-5%. l%-3%, 3%-1000%, 3%-750%. 3%-500%. 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%. 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%. 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-l 000%.

[0171] In some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%. 92%, 93%, 94%, 95%, 96%. 97%. 98%, 99%, 100%, 150%, 200%. 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%. 3%, 4%. 5%, 6%. 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%. 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, l%-750%, l%-500%. l%-250%, l%-100%, l%-75%. l%-50%, l%-25%, l%-10%. l%-7%, l%-5%. l%-3%, 3%-1000%. 3%-750%. 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%. 10%-1000%, 10%-750%, 10%-500%. 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%. 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%. 5%-1000%, 5%-750%. 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0172] In some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%. 70%. 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%. 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%. 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%. 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%. l%-750%, l%-500%. l%-250%. l%-100%. l%-75%, l%-50%. l%-25%. 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%.

[0173] In some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%. 40%. 50%, 60%, 70%, 75%, 80%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%. 500%. 600%. 700%, 750%, 800%, 900%, or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about 1 %-l 000%, l%-750%, l %-500%, l %-250%, 1 %-l 00%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%. 5%-500%. 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%. 500%-1000%. 500%-750%, or 750%-1000%. and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%, l%-750%, l%-500%, l%-250%. l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%. l%-7%, l%-5%. l%-3%, 3%-1000%, 3%-750%. 3%-500%. 3%-250%. 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0174] In some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%. 92%, 93%, 94%, 95%, 96%. 97%. 98%, 99%, 100%, 150%, 200%. 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%. 3%, 4%. 5%, 6%. 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%. 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%. l%-250%. l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%. 10%-1000%, 10%-750%, 10%-500%. 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%. 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%-1000%. l%-750%, l%-500%, l%-250%. l%-100%. l%-75%, l%-50%, l%-25%. 1%- 10%, l%-7%, l%-5%. l%-3%, 3%-1000%, 3%-750%. 3%-500%. 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%. 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%. 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-l 000%.

[0175] In some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%. 70%. 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%. 99%. 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%. 3%-50%, 3%-25%. 3%-10%, 3%-7%. 3%- 5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%. 50%-1000%, 50%-750%, 50%-500%. 50%-250%. 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%,250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%. 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%. 5%-1000%, 5%-750%. 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0176] In some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%. 3%, 4%, 5%, 6%. 7%, 8%, 9%, 10%, 15%, 20%, 25%. 30%. 40%. 50%, 60%, 70%, 75%, 80%. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV serotype rh74 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%. 500%. 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%. 3%- 5%, 5%-1000%. 5%-750%. 5%-500%. 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%- 25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%,10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%. 50%-250%. 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about 1%- 1000%. l%-750%, l%-500%. l%-250%. l%-100%. l%-75%, l%-50%. l%-25%. 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0177] In some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%. 200%, 250%, 300%. 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV9 VP1 polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%. 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%. 500%-1000%, 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%. 3%-25%, 3%-10%. 3%-7%. 3%-5%. 5%- 1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%. 100%-500%, 100%-250%. 250%-1000%. 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0178] In some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, 100%. 150%, 200%, 250%, 300%, 350%. 400%. 450%, 500%, 600%. 700%, 750%, 800%, 900%. or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, 1%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%. 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%. 100%-1000%. 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%. 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%. 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%. 10%-25%. 25%-1000%, 25%-750%. 25%-500%. 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0179] In some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%. 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%. 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%. 6%, 7%, 8%. 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises aninsertion of at least 5 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%-1000%. 25%-750%. 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%. 3%-7%. 3%-5%, 5%- 1000%. 5%-750%, 5%-500%. 5%-250%. 5%-100%. 5%-75%, 5%-50%. 5%-25%. 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%- 500%, 500%-1 00%, 500%-750%, or 750%-1000%.

[0180] In some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%. 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%. or 1000%, or by a percentage that is in a range defined by any two of the preceding values, ascompared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 5 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%, 3%-75%, 3%- 50%. 3%-25%. 3%-10%, 3%-7%. 3%-5%, 5%-1000%. 5%-750%. 5%-500%. 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%, 100%-1000%. 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. 1%- 50%. l%-25%, l%-10%. l%-7%, l%-5%, l%-3%. 3%-1000%. 3%-750%. 3%-500%. 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%. 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0181] In some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%. 350%, 400%, 450%, 500%, 600%. 700%. 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%. 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%,250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%. 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%. l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to hver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%. 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0182] In some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%,3%, 4%, 5%, 6%, 7%, 8%. 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%. 99%, 100%. 150%. 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 7 ammo acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%, 3%-75%, 3%- 50%. 3%-25%. 3%-10%, 3%-7%. 3%-5%, 5%-1000%. 5%-750%. 5%-500%. 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%. 250%-500%. 500%-1000%. 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. 1%- 50%. l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%. 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0183] In some embodiments, the first engineered feature comprises an insertion of at least 7 ammo acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%. 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineeredfeature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%. 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%. 50%-750%. 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l %-100%, 1 %-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0184] In some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%,400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, 100%. 150%, 200%, 250%, 300%, 350%. 400%. 450%, 500%, 600%. 700%, 750%, 800%, 900%. or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 7 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%, 3%-75%, 3%- 50%, 3%-25%. 3%-10%, 3%-7%. 3%-5%, 5%-1000%. 5%-750%. 5%-500%, 5%-250%, 5%-100%, 5%-75%. 5%-50%. 5%-25%, 5%-10%, 10%-1000%. 10%-750%, 10%-500%. 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%. 500%-1000%. 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%. 25%-50%. 50%-1000%, 50%-750%. 50%-500%, 50%-250%, 50%-100%. 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0185] In some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%. 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%. 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%. 6%, 7%, 8%. 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by betw een about l%-1000%. 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%. 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%. 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%. 10%-1000%, 10%-750%. 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0186] In some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%. 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%. 99%, 100%. 150%. 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 8 ammo acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%, 3%-75%, 3%- 50%. 3%-25%. 3%-10%, 3%-7%. 3%-5%, 5%-1000%. 5%-750%. 5%-500%. 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. 1%- 50%. l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%. 3%-500%. 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%. 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0187] In some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%. 200%, 250%, 300%. 350%. 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue, by between about l%-1000%. 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%, l %-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%. 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0188] In some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue by about 1%, 2%. 3%, 4%. 5%, 6%. 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, and the second engineered feature comprises a glycine to alanine mutation within the AAV9 VP1 capsid polypeptide, resulting in reduced transduction or tropism to soft tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, 100%. 150%, 200%, 250%, 300%, 350%. 400%. 450%, 500%, 600%. 700%, 750%, 800%, 900%. or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features. For example, in some embodiments, the first engineered feature comprises an insertion of at least 8 amino acids within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about 1 %-l 000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%, 3%-75%, 3%- 50%, 3%-25%. 3%-10%, 3%-7%. 3%-5%, 5%-1000%. 5%-750%. 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%. 500%-1000%. 500%-750%, or 750%- 1000%, and the second engineered feature comprises a glycine to alanine mutation within an AAV9 VP1 capsid, resulting in reduced transduction or tropism to liver tissue by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. 1%- 50%. l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%. 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%. 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%.

[0189] In some embodiments, the variant AAV capsids described herein are encoded by any suitable nucleic acid sequence. In some embodiments, a suitable nucleic acid sequence is any sequence that translates to any amino acid sequence disclosed herein.

[0190] In some embodiments, the one or more engineered features comprises insertion of a peptide encoded by SEQ ID NO: 3 within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, the one or more engineered features comprises insertion of a peptide having the sequence of SEQ ID NO: 4. within an AAV seroty pe rh74 VP1 capsid polypeptide. In some embodiments, insertion of a peptide encoded by SEQ ID NO: 3 and having the sequence of SEQ ID NO: 4 within an AAV serotype rh74 VP1 capsid polypeptide enhances transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%. 100%, 150%, 200%. 250%, 300%, 350%. 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP 1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the first engineered feature comprises an insertion of a peptide having the sequence of SEQ ID NO: 4. within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by betw een about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%. 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%. 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2.

[0191] In some embodiments, the one or more engineered features comprises insertion of a peptide encoded by SEQ ID NO: 15 within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, the one or more engineered features comprises insertion of a peptide having the sequence of SEQ ID NO: 16. within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, insertion of a peptide encoded by SEQ ID NO: 15 and having the sequence of SEQ ID NO: 16 within an AAV serotype rh74 VP1 capsid polypeptide enhances transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%. or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP 1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the first engineered feature comprises an insertion of a peptide having the sequence of SEQ ID NO: 16. within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about 1 %-l 000%, 1 %- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%, 3%-75%, 3%- 50%, 3%-25%. 3%-10%, 3%-7%. 3%-5%, 5%-1000%. 5%-750%. 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%. 500%-1000%. 500%-750%, or 750%- 1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2.

[0192] In some embodiments, the one or more engineered features comprises insertion of a peptide encoded by SEQ ID NO: 19 within an AAV serotype rh74 VP1capsid polypeptide. In some embodiments, the one or more engineered features comprises insertion of a peptide having the sequence of SEQ ID NO: 20. within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, insertion of a peptide encoded by SEQ ID NO: 19 and having the sequence of SEQ ID NO: 20 within an AAV serotype rh74 VP1 capsid polypeptide enhances transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%. 3%, 4%. 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%. 40%. 50%. 60%, 70%, 75%, 80%, 90%. 91%. 92%. 93%, 94%, 95%, 96%, 97%. 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV seroty pe rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP 1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the first engineered feature comprises an insertion of a peptide having the sequence of SEQ ID NO: 20 within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%. 1%- 750%, l%-500%, I%-250%, l%-100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%. l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2.

[0193] In some embodiments, the one or more engineered features comprises insertion of a peptide encoded by SEQ ID NO: 23 within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, the one or more engineered features comprises insertion of a peptide having the sequence of SEQ ID NO: 24. within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, insertion of a peptide encoded by SEQ ID NO: 23 and having the sequence of SEQ ID NO: 24 within an AAV serotype rh74 VP1 capsid polypeptide enhances transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%. 100%, 150%, 200%. 250%, 300%, 350%, 400%, 450%, 500%, 600%. 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP 1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the first engineered feature compnses an insertion of a peptide having the sequence of SEQ ID NO: 23 within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%. 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%. 100%-1000%. 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2.

[0194] In some embodiments, the one or more engineered features comprises insertion of a peptide encoded by SEQ ID NO: 3 within an AAV9 VP1 capsid polypeptide. In some embodiments, the one or more engineered features comprises insertion of a peptide having the sequence of SEQ ID NO: 4. within an AAV9 VP1 capsid polypeptide. In some embodiments, insertion of a peptide encoded by SEQ ID NO: 3 and having the sequence of SEQ ID NO: 4 within an AAV9 VP1 capsid polypeptide enhances transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%. 50%, 60%, 70%, 75%, 80%, 90%. 91%. 92%, 93%, 94%, 95%, 96%. 97%. 98%, 99%, 100%, 150%. 200%. 250%. 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP 1 capsid polypeptide having the sequence of SEQ ID NO: 11. For example, in some embodiments, the first engineered feature comprises an insertion of apeptide having the sequence of SEQ ID NO: 4. within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about 1%- 1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%. or 750%-1000%. as compared to a corresponding AAV9VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP1 capsid polypeptide having the sequence of SEQ ID NO: 11.

[0195] In some embodiments, the one or more engineered features comprises insertion of a peptide encoded by SEQ ID NO: 15 within an AAV VP1 capsid polypeptide. In some embodiments, the one or more engineered features comprises insertion of a peptide having the sequence of SEQ ID NO: 16. within an AAV VP1 capsid polypeptide. In some embodiments, insertion of a peptide encoded by SEQ ID NO: 15 and having the sequence of SEQ ID NO: 16 within an AAV VP1 capsid polypeptide enhances transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP 1 capsid polypeptide having the sequence of SEQ ID NO: 11. For example, in some embodiments, the first engineered feature comprises an insertion of a peptide having the sequence of SEQ ID NO: 15. within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%. 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV9VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP1 capsid polypeptide having the sequence of SEQ ID NO: 11.

[0196] In some embodiments, the one or more engineered features comprises insertion of a peptide encoded by SEQ ID NO: 19 within an AAV VP1 capsid polypeptide. In some embodiments, the one or more engineered features comprises insertion of a peptide having the sequence of SEQ ID NO: 20. within an AAV VP1 capsid polypeptide. In some embodiments, insertion of a peptide encoded by SEQ ID NO: 19 and having the sequence of SEQ ID NO: 20 within an AAV VP1 capsid polypeptide enhances transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99%, 100%. 150%, 200%, 250%, 300%, 350%. 400%. 450%, 500%, 600%. 700%, 750%, 800%, 900%. or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP 1 capsid polypeptide having the sequence of SEQ ID NO: 11. For example, in some embodiments, the first engineered feature comprises an insertion of a peptide having the sequence of SEQ ID NO: 19. within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%. 25%-50%. 50%-1000%, 50%-750%. 50%-500%, 50%-250%, 50%-100%. 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV9VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP1 capsid polypeptide having the sequence of SEQ ID NO: 11.

[0197] In some embodiments, the one or more engineered features comprises insertion of a peptide encoded by SEQ ID NO: 23 within an AAV VP1 capsid polypeptide. In some embodiments, the one or more engineered features comprises insertion of a peptide having the sequence of SEQ ID NO: 24 within an AAV VP1 capsid polypeptide. In some embodiments, insertion of a peptide encoded by SEQ ID NO: 23 and having the sequence of SEQ ID NO: 24 within an AAV VP1 capsid polypeptide enhances transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%. 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP 1 capsid polypeptide having the sequence of SEQ ID NO: 11. For example, in some embodiments, the first engineered feature comprises an insertion of a peptide having the sequence of SEQ ID NO: 23. within an AAV9 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, 1%- 50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%. 3%-7%, 3%-5%, 5%- 1000%, 5%-750%, 5%-500%, 5%-250%. 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%- 10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%- 50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%- 75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%- 500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV9VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP1 capsid polypeptide having the sequence of SEQ ID NO: 11.

[0198] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises a peptide encoded by SEQ ID NO: 5. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 6. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 5 and having the sequence of SEQ ID NO: 6 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%. 4%, 5%. 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%. 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%. 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 6. has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, 1%- 75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%. 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, I0%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%. 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2.

[0199] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises a peptide encoded by SEQ ID NO: 27. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 28. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 27 and having the sequence of SEQ ID NO: 28 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%. 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP I capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 28. has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about l%-1000%. l%-750%. l%-500%. l%-250%. l%-100%. 1%- 75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-I000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%. 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%. 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2.

[0200] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises a peptide encoded by SEQ ID NO: 29 In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 30. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 29 and having the sequence of SEQ ID NO: 30 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%. 6%, 7%, 8%, 9%, 10%, 15%, 20%. 25%. 30%. 40%, 50%, 60%, 70%, 75%. 80%. 90%. 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 30 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about l%-1000%. l%-750%. l%-500%. l%-250%. l%-100%. 1%- 75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%. 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%. 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2.

[0201] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises a peptide encoded by SEQ ID NO: 31. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 32. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 31 and having the sequence of SEQ ID NO: 32 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%. 6%, 7%, 8%, 9%, 10%, 15%, 20%. 25%. 30%. 40%, 50%, 60%, 70%, 75%. 80%. 90%. 91%, 92%, 93%, 94%, 95%. 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 32 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about l%-1000%. l%-750%. l%-500%. l%-250%. l%-100%. 1%- 75%. l%-50%. l%-25%, l%-10%, l%-7%. l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2.

[0202] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises a peptide encoded by SEQ ID NO: 33. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 34. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 33 and having the sequence of SEQ ID NO: 34 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%, 99%, 100%, 150%. 200%, 250%, 300%, 350%, 400%, 450%. 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range definedby any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 34 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%. 1%- 75%. l%-50%, l%-25%, l%-10%, l%-7%. l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%. 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2.

[0203] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises a peptide encoded by SEQ ID NO: 35. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 36. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 35 and having the sequence of SEQ ID NO: 36 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%. 40%, 50%, 60%, 70%, 75%, 80%, 90%. 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, 100%, 150%. 200%, 250%, 300%, 350%, 400%, 450%. 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 36 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, 1%- 75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%. 3%-50%, 3%-25%, 3%-10%, 3%-7%. 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%,5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%. 25%-250%. 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2.

[0204] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises a peptide encoded by SEQ ID NO: 37. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 38. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 37 and having the sequence of SEQ ID NO: 38 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%. 98%, 99%, 100%, 150%. 200%, 250%, 300%, 350%, 400%, 450%. 500%, 600%, 700%, 750%. 800%, 900%, or 1000%. or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 38 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, 1%- 75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%. 3%-50%, 3%-25%, 3%-10%, 3%-7%. 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%. 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%. 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2.

[0205] In some embodiments, the variant AAV9 VP1 capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 12. In several embodiments, thevariant AAV9 capsid is encoded by a polynucleotide having at least 85% sequence identity to SEQ ID NO: 13. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 14. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide of SEQ ID NO: 14. In some embodiments, the variant capsid polypeptide having the sequence of SEQ ID NO: 12 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%. 3%, 4%. 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%. 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP1 capsid polypeptide having the sequence of SEQ ID NO: 11. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 12. has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about 1%- 1000%. l%-750%, l%-500%. l%-250%. l%-100%. l%-75%, l%-50%. l%-25%. 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%. or 750%-1000%, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 11.

[0206] In some embodiments, the variant AAV9 VP1 capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 17. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide having at least 85% sequence identity to SEQ ID NO: 13. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 18. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide of SEQ ID NO: 18. In some embodiments, the variant capsid polypeptide having the sequence of SEQ ID NO: 17 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue byabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%. 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%. 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP1 capsid polypeptide having the sequence of SEQ ID NO: 11. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 17. has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about 1%- 1000%, l%-750%, l%-500%, l%-250%. l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%. l%-7%, l%-5%. l%-3%, 3%-1000%, 3%-750%. 3%-500%. 3%-250%. 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%. 50%-500%. 50%-250%, 50%-100%, 100%-1000%, 100%-750%. 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 11.

[0207] In some embodiments, the variant AAV9 VP1 capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 21. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide having at least 85% sequence identity to SEQ ID NO: 13. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 22. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide of SEQ ID NO: 22. In some embodiments, the variant capsid polypeptide having the sequence of SEQ ID NO: 21 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%. 3%, 4%. 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%. 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP1 capsid polypeptide having thesequence of SEQ ID NO: 11. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 21 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about 1%- 1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, 1%- 10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%. 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%. 5%-1000%, 5%-750%. 5%- 500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%. 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%. 100%-250%, 250%-1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 11.

[0208] In some embodiments, the variant AAV9 VP1 capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 25. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide having at least 85% sequence identity7to SEQ ID NO: 13. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide having at least 90% sequence identity to SEQ ID NO: 26. In several embodiments, the variant AAV9 capsid is encoded by a polynucleotide of SEQ ID NO: 26. In some embodiments, the variant capsid polypeptide having the sequence of SEQ ID NO: 25 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%. 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%. 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV9 VP1 capsid polypeptide having the sequence of SEQ ID NO: 11. For example, in some embodiments, the variant capsid polypeptide comprising a peptide having the sequence of SEQ ID NO: 25 has enhanced transduction or tropism to cardiac tissue and / or muscle tissue, by between about 1%- 1000%, l%-750%, l%-500%, l%-250%. l%-100%. l%-75%, l%-50%, l%-25%, 1%- 10%. l%-7%, l%-5%. l%-3%, 3%-1000%, 3%-750%. 3%-500%. 3%-250%. 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%- 750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%- 1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%- 1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV9 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 11.

[0209] In some embodiments, the one or more engineered features comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, the one or more engineered features comprises a glycine to alanine mutation at position 268 of SEQ ID NO: 2. In some embodiments, a glycine to alanine mutation at position 268 of SEQ ID NO: 2 reduces transduction or tropism to soft tissue, or liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%. 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%, 94%, 95%, 96%, 97%, 98%. 99%. 100%. 150%, 200%. 250%. 300%, 350%, 400%. 450%, 500%, 600%. 700%. 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, a glycine to alanine mutation at position 268 of SEQ ID NO: 2 reduces transduction or tropism to soft tissue, or liver tissue, by between about 1%-1000%, l %-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%. 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%- 75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%- 1000%. 250%-750%, 250%-500%, 500%-1000%, 500%-750%. or 750%-1000%. as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2.

[0210] In some embodiments, the one or more engineered features comprises a glycine to alanine mutation within an AAV9 VP1 capsid polypeptide. In some embodiments, the one or more engineered features comprises a glycine to alaninemutation at position 267 of SEQ ID NO: 11. In some embodiments, a glycine to alanine mutation at position 267 of SEQ ID NO: 11 reduces transduction or tropism to soft tissue, or liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV9 VP1 capsid polypeptide having the sequence of SEQ ID NO: 11. For example, in some embodiments, a glycine to alanine mutation at position 267 of SEQ ID NO: 11 reduces transduction or tropism to soft tissue, or liver tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%. l%-50%. l%-25%, l%-10%, l%-7%. l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%- 7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%- 75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%- 100%, 25%-75%, 25%-50%. 50%-1000%. 50%-750%. 50%-500%, 50%-250%, 50%- 100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%, 250%- 750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV9 VP1 capsid poly peptide having the sequence of SEQ ID NO: 11.

[0211] In some embodiments, the one or more engineered features comprises a glycine to alanine mutation within an AAV serotype rh74 VP1 capsid polypeptide. In some embodiments, the variant capsid polypeptide having a glycine to alanine mutation at position corresponding to position 268 of SEQ ID NO: 2 is encoded by SEQ ID NO: 7. In some embodiments, the variant capsid polypeptide having a glycine to alanine mutation at position corresponding to position 268 of SEQ ID NO: 2 is a variant capsid polypeptide having SEQ ID NO: 8. In some embodiments, a variant AAV serotype rh74 capsid polypeptide comprising a peptide encoded by SEQ ID NO: 7 and having the sequence of SEQ ID NO: 8 has reduced transduction or tropism to soft tissue, or liver tissue, by about 1%, 2%, 3%. 4%, 5%. 6%, 7%. 8%, 9%. 10%. 15%. 20%, 25%, 30%, 40%, 50%, 60%. 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, in some embodiments, a variant AAV serotyperh74 capsid polypeptide comprising a peptide encoded by SEQ ID NO: 7 and having the sequence of SEQ ID NO: 8 has reduced transduction or tropism to soft tissue, or liver tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, 1%- 75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%- 500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%. 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-I00%, 10%-75%. 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%-1000%. 250%-750%, 250%-500%. 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2.

[0212] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises at least two engineered features. In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide having at least 2 engineered features comprises a peptide encoded by SEQ ID NO: 9. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 10. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 9 and having the sequence of SEQ ID NO: 10 has a first engineered feature comprising an insertion of a peptide having the sequence of SEQ ID NO: 4 into an AAV rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%. 75%. 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%. 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered feature, for example, an AAV capsid having the sequence of SEQ ID NO: 2. For example, in some embodiments, the first engineered feature in the variant capsid polypeptide encoded by SEQ ID NO: 9 and having the sequence of SEQ ID NO: 10 comprises an insertion of a peptide having the sequence of SEQ ID NO: 4. within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%. 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%,l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%. 3%-5%, 5%-1000%. 5%-750%. 5%-500%, 5%-250%,5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%,10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%. 500%-1000%. 500%-750%, or 750%- 1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 9 and having the sequence of SEQ ID NO: 10 has a second engineered feature comprising a glycine to alanine mutation at a position corresponding to position 268 of SEQ ID NO: 2. In some embodiments, a glycine to alanine mutation in the variant capsid polypeptide encoded by SEQ ID NO: 9 and having the sequence of SEQ ID NO: 10 at a position corresponding to position 268 of SEQ ID NO: 2 reduces transduction or tropism to soft tissue, or liver tissue, by about 1%, 2%. 3%, 4%. 5%, 6%. 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, some embodiments, a glycine to alanine mutation at a position corresponding to position 268 of SEQ ID NO: 2 in the variant capsid polypeptide encoded by SEQ ID NO: 9 and having the sequence of SEQ ID NO: 10 reduces transduction or tropism to soft tissue, or liver tissue, by between about 1%- 1000%, 1 %-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%. 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%- 75%. 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%. 10%-250%. 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%- 1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%. or 750%-1000%. ascompared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2.

[0213] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises at least two engineered features. In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide having at least 2 engineered features comprises a peptide encoded by SEQ ID NO: 33. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 34. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 33 and having the sequence of SEQ ID NO: 34 has a first engineered feature comprising an insertion of a peptide having the sequence of SEQ ID NO: 15 into an AAV rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%. 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered feature, for example, an AAV capsid having the sequence of SEQ ID NO: 2. For example, in some embodiments, the first engineered feature in the variant capsid polypeptide encoded by SEQ ID NO: 33 and having the sequence of SEQ ID NO: 34 comprises an insertion of a peptide having the sequence of SEQ ID NO: 15 within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by between about l%-1000%, 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%. 3%-1000%, 3%-750%, 3%-500%, 3%-250%. 3%-100%. 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%. 50%-100%. 100%- 1000%. 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 33 and having the sequence of SEQ ID NO: 34 has a second-I l l-engineered feature comprising a glycine to alanine mutation at a position corresponding to position 268 of SEQ ID NO: 2. In some embodiments, a glycine to alanine mutation in the variant capsid polypeptide encoded by SEQ ID NO: 33 and having the sequence of SEQ ID NO: 34 at a position corresponding to position 268 of SEQ ID NO: 2 reduces transduction or tropism to soft tissue, or liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%. 200%, 250%, 300%. 350%. 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, some embodiments, a glycine to alanine mutation at a position corresponding to position 268 of SEQ ID NO: 2 in the variant capsid polypeptide encoded by SEQ ID NO: 33 and having the sequence of SEQ ID NO: 34 reduces transduction or tropism to soft tissue, or liver tissue, by between about l%-1000%, l%-750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%. l%-3%, 3%-I000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%. 3%-50%, 3%-25%. 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%- 75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-I00%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%. 50%-750%. 50%-500%, 50%-250%, 50%-100%, 100%- 1000%, 100%-750%, 100%-500%, 100%-250%, 250%- 1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2.

[0214] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises at least two engineered features. In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide having at least 2 engineered features comprises a peptide encoded by SEQ ID NO: 35. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 36. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 35 and having the sequence of SEQ ID NO: 36 has a first engineered feature comprising an insertion of a peptide having the sequence of SEQ ID NO: 19 into an AAV rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered feature, for example, an AAV capsid having the sequence of SEQ ID NO: 2. For example, in some embodiments, the first engineered feature in the variant capsid polypeptide encoded by SEQ ID NO: 35 and having the sequence of SEQ ID NO: 36 comprises an insertion of a peptide having the sequence of SEQ ID NO: 19 within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by betw een about l%-1000%. 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%- 50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%. 25%-75%, 25%-50%, 50%-1000%. 50%-750%. 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%- 1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 35 and having the sequence of SEQ ID NO: 36 has a second engineered feature comprising a glycine to alanine mutation at a position corresponding to position 268 of SEQ ID NO: 2. In some embodiments, a glycine to alanine mutation in the variant capsid polypeptide encoded by SEQ ID NO: 35 and having the sequence of SEQ ID NO: 36 at a position corresponding to position 268 of SEQ ID NO: 2 reduces transduction or tropism to soft tissue, or liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, 100%, 150%. 200%, 250%, 300%. 350%. 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, some embodiments, a glycine to alanine mutation at a position corresponding to position 268 of SEQ ID NO: 2 in the variant capsid polypeptide encodedby SEQ ID NO: 35 and having the sequence of SEQ ID NO: 36 reduces transduction or tropism to soft tissue, or liver tissue, by between about l%-1000%. l%-750%. l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%- 75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%. 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%-1000%. 25%-750%. 25%-500%. 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%-250%, 250%- 1000%, 250%-750%, 250%-500%, 500%-1000%, 500%-750%, or 750%-1000%, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2.

[0215] In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide comprises at least two engineered features. In some embodiments, the variant AAV serotype rh74 VP1 capsid polypeptide having at least 2 engineered features comprises a peptide encoded by SEQ ID NO: 37. In some embodiments, the variant capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 38. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 37 and having the sequence of SEQ ID NO: 38 has a first engineered feature comprising an insertion of a peptide having the sequence of SEQ ID NO: 23 into an AAV rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to cardiac tissue and / or muscle tissue by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%. 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered feature, for example, an AAV capsid having the sequence of SEQ ID NO: 2. For example, in some embodiments, the first engineered feature in the variant capsid polypeptide encoded by SEQ ID NO: 37 and having the sequence of SEQ ID NO: 38 comprises an insertion of a peptide having the sequence of SEQ ID NO: 23 within an AAV serotype rh74 VP1 capsid polypeptide, resulting in enhanced transduction or tropism to muscle tissue, by betw een about l%-1000%. 1%- 750%, l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%. l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%. 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%-75%, 5%-50%. 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%, 10%-25%, 25%-1000%, 25%-750%, 25%-500%, 25%-250%, 25%-100%, 25%-75%, 25%-50%, 50%-1000%, 50%-750%, 50%-500%, 50%-250%, 50%-100%, 100%-1000%, 100%-750%, 100%-500%, 100%- 250%, 250%-1000%, 250%-750%, 250%-500%. 500%-1000%, 500%-750%, or 750%- 1000%. as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide without the engineered features, for example, an AAV capsid polypeptide having the sequence of SEQ ID NO: 2. In some embodiments, the variant capsid polypeptide encoded by SEQ ID NO: 37 and having the sequence of SEQ ID NO: 38 has a second engineered feature comprising a glycine to alanine mutation at a position corresponding to position 268 of SEQ ID NO: 2. In some embodiments, a glycine to alanine mutation in the variant capsid polypeptide encoded by SEQ ID NO: 37 and having the sequence of SEQ ID NO: 38 at a position corresponding to position 268 of SEQ ID NO: 2 reduces transduction or tropism to soft tissue, or liver tissue, by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. 10%. 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 750%, 800%, 900%, or 1000%, or by a percentage that is in a range defined by any two of the preceding values, as compared to a corresponding AAV serotype rh74 VP1 capsid polypeptide having the sequence of SEQ ID NO: 2. For example, some embodiments, a glycine to alanine mutation at a position corresponding to position 268 of SEQ ID NO: 2 in the variant capsid polypeptide encoded by SEQ ID NO: 37 and having the sequence of SEQ ID NO: 38 reduces transduction or tropism to soft tissue, or liver tissue, by between about l%-1000%. l%-750%. l%-500%, l%-250%, l%-100%, l%-75%, l%-50%, l%-25%, l%-10%, l%-7%, l%-5%, l%-3%, 3%-1000%, 3%-750%, 3%-500%, 3%-250%, 3%-100%, 3%-75%, 3%-50%, 3%-25%, 3%-10%, 3%-7%, 3%-5%, 5%-1000%, 5%-750%, 5%-500%, 5%-250%, 5%-100%, 5%- 75%, 5%-50%, 5%-25%, 5%-10%, 10%-1000%, 10%-750%, 10%-500%, 10%-250%, 10%-100%, 10%-75%, 10%-50%. 10%-25%, 25%-1000%. 25%-750%. 25%-500%....

Claims

WHAT IS CLAIMED:

1. A variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding nonengineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more non-muscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein.

2. A variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV9 having at least two engineered features as compared to a corresponding non-engineered AAV9 VP1 polypeptide: wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to one or more nonmuscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an AAV9 vector packed using a non-engineered VP1 capsid protein.

3. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the first engineered feature comprises insertion of at least 5 amino acids.

4. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the first engineered feature comprises insertion of at least 7 amino acids.

5. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the first engineered feature comprises insertion of at least 8 amino acids.

6. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the first engineered feature enhances transduction or tropism to cardiac muscle.

7. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the first engineered feature enhances transduction or tropism to skeletal muscle.

8. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the first engineered feature enhances transduction or tropism to cardiac muscle and skeletal muscle.

9. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the first engineered feature comprises a peptide encoded by SEQ ID NO: 3, 16, 20, or 24.

10. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the first engineered feature comprises a peptide having the sequence of SEQ ID NO: 4, 15, 19, or 23.

11. The variant AAV capsid polypeptide of Claim 1, wherein the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO:

5.

28.

30. or 32.

12. The variant AAV capsid polypeptide of Claim 1, wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 6, 29, 31, or 33.

13. The variant AAV capsid polypeptide of Claim 2, wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 12, 17, 21 , or 25.

14. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the second engineered feature reduces transduction or tropism to a soft tissue.

15. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the second engineered feature reduces transduction or tropism to liver tissue.

16. The variant AAV capsid polypeptide of Claim 1 or 2, wherein the second engineered feature comprises a glycine to alanine mutation.

17. The variant AAV capsid polypeptide of Claim 1, wherein the second engineered feature comprises a glycine to alanine mutation at position 268 of SEQ ID NO: 2.

18. The variant AAV capsid polypeptide of Claim 2, wherein the second engineered feature comprises a glycine to alanine mutation at position 267 of SEQ ID NO: 11.

19. A variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding nonengineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more non-muscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using anon-engineered VP1 capsid protein.

20. A variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV9 having at least two engineered features as compared to a corresponding non-engineered AAV9 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to one or more nonmuscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an AAV9 vector packed using a non-engineered VP1 capsid protein.

21. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the second engineered feature reduces transduction or tropism to a soft tissue.

22. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the second engineered feature reduces transduction or tropism to liver tissue.

23. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the second engineered feature comprises a glycine to alanine mutation.

24. The variant AAV capsid polypeptide of Claim 19, wherein the second engineered feature comprises a glycine to alanine mutation at position 268 of SEQ ID NO: 2.

25. The variant AAV capsid polypeptide of Claim 20, wherein the second engineered feature comprises a glycine to alanine mutation at position 267 of SEQ ID NO: 11.

26. The variant AAV capsid polypeptide of Claim 19, wherein the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 7.

27. The variant AAV capsid polypeptide of Claim 20, wherein the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 14.

28. The variant AAV capsid polypeptide of Claim 19, wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 8.

29. The variant AAV capsid polypeptide of Claim 20, wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 12.

30. The variant AAV capsid polypeptide of Claim 19, wherein the first engineered feature compnses insertion of at least 5 ammo acids.

31. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the first engineered feature comprises insertion of at least 7 amino acids.

32. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the first engineered feature comprises insertion of at least 8 amino acids.

33. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the first engineered feature enhances transduction or tropism to cardiac muscle.

34. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the first engineered feature enhances transduction or tropism to skeletal muscle.

35. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the first engineered feature enhances transduction or tropism to cardiac muscle and skeletal muscle.

36. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the first engineered feature comprises a peptide encoded by SEQ ID NO:

3. 16, 20, or 24.

37. The variant AAV capsid polypeptide of Claim 19 or 20, wherein the first engineered feature comprises a peptide having the sequence of SEQ ID NO: 4, 15, 19, or 23.

38. The variant AAV capsid polypeptide of Claim 1, 19, or 20, wherein the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 9, 34, 36, or 38.

39. The variant AAV capsid polypeptide of Claim 1, 19, or 20, wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 10, 33, 35, or 37.

40. The variant AAV capsid polypeptide of Claim 1, 19, or 20. wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 10.

41. A variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding nonengineered rh74 VP1 polypeptide, the non-engineered rh74VPl having a sequence of SEQ ID NO: 2; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide resulting in maintained or enhanced targeting or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue, as compared to the non-engineered rh74VPl capsid; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing targeting or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more non-muscle tissue, as compared to the non-engineered rh74VPl capsid.

42. A variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV9 having at least two engineered features as compared to a corresponding non-engineered AAV9 VP1 polypeptide, the non-engineered AAV9 VP1 having a sequence of SEQ ID NO: 11; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide resulting in maintained or enhanced targeting or tropism of a recombinant AAV9 vector packaged using the variant capsid to muscle tissue, as compared to the non-engineered AAV9 VP1 capsid;wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing targeting or tropism of a recombinant AAV9 vector packaged using the variant capsid to one or more nonmuscle tissue, as compared to the non-engineered AAV9 VP1 capsid.

43. The variant AAV capsid polypeptide of Claim 41 or 42, wherein the first engineered feature enhances transduction or tropism to cardiac muscle.

44. The variant AAV capsid polypeptide of Claim 41 or 42, wherein the first engineered feature enhances transduction or tropism to skeletal muscle.

45. The variant AAV capsid polypeptide of Claim 41 or 42, wherein the first engineered feature enhances transduction or tropism to cardiac muscle and skeletal muscle.

46. The variant AAV capsid polypeptide of Claim 41 or 42, wherein the first engineered feature comprises a peptide encoded by SEQ ID NO: 3, 16, 20, or 24.

47. The variant AAV capsid polypeptide of Claim 41 or 42, wherein the first engineered feature comprises a peptide having the sequence of SEQ ID NO: 4, 15, 19, or 23.

48. The variant AAV capsid polypeptide of Claim 41 or 42, wherein the second engineered feature reduces transduction or tropism to a soft tissue.

49. The variant AAV capsid polypeptide of Claim 41 or 42, wherein the second engineered feature reduces transduction or tropism to liver tissue.

50. The variant AAV capsid polypeptide of Claim 41 or 42, wherein the second engineered feature comprises a glycine to alanine mutation.

51. The variant AAV capsid polypeptide of Claim 41, wherein the second engineered feature comprises a glycine to alanine mutation at position 268 of SEQ ID NO: 2.

52. The variant AAV capsid polypeptide of Claim 42, wherein the second engineered feature comprises a glycine to alanine mutation at position 267 of SEQ ID NO: 11.

53. A variant adeno-associated virus (AAV) capsid polypeptide, wherein said variant AAV capsid polypeptide exhibits maintained or increased transduction or tropism in human muscle tissue or cells as compared to a non-variant parent capsid polypeptide, wherein said variant AAV capsid polypeptide exhibits reduced transduction or tropism in human liver tissue or cells as compared to a non-variant parent capsid polypeptide, andwherein said variant AAV capsid polypeptide is encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 9.

54. The variant AAV capsid polypeptide of Claim 53, wherein the variant AAV capsid polypeptide is encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 9.

55. The variant AAV capsid polypeptide of Claim 53 or 54, wherein the variant AAV capsid polypeptide is encoded by a sequence having at least 98% sequence identity to SEQ ID NO: 9, 34, 36, or 38.

56. The variant AAV capsid polypeptide of Claim 53, wherein the variant AAV capsid polypeptide has at least 95% sequence identity to SEQ ID NO: 10, 33, 35, or 37.

57. The variant AAV capsid polypeptide of Claim 53, wherein the variant AAV capsid polypeptide has at least 98% sequence identity to SEQ ID NO: 10.

58. A variant adeno-associated virus (AAV) capsid polypeptide, wherein said variant AAV capsid polypeptide exhibits maintained or increased transduction or tropism in human muscle tissue or cells as compared to a non-variant parent capsid polypeptide, wherein said variant AAV capsid polypeptide exhibits reduced transduction or tropism in human liver tissue or cells as compared to a non-variant parent capsid polypeptide, and wherein said variant AAV capsid polypeptide is encoded by a sequence having at least 90% sequence identity to SEQ ID NO: 14.

59. The variant AAV capsid polypeptide of Claim 58, wherein the variant AAV capsid polypeptide is encoded by a sequence having at least 95% sequence identity to SEQ ID NO: 14.

60. The variant AAV capsid polypeptide of Claim 58 or 59, wherein the variant AAV capsid polypeptide is encoded by a sequence having at least 98% sequence identity' to SEQ ID NO: 14.

61. The variant AAV capsid polypeptide of Claim 53 or 58, wherein the variant AAV capsid polypeptide exhibits maintained or increased transduction as compared to a non-variant parent capsid polypeptide.

62. The variant AAV capsid polypeptide of Claim 53 or 58, wherein the muscle tissue or cells comprise cardiac muscle tissue or cells and / or skeletal muscle tissue or cells.

63. The variant AAV capsid polypeptide of Claim 53 or 58, wherein the variant AAV capsid polypeptide further exhibits an enhanced neutralization profile as compared to a non-variant parent capsid polypeptide.

64. The variant AAV capsid polypeptide of Claim 53 or 58, wherein the variant AAV capsid polypeptide exhibits increased transduction of human muscle tissue or cells in vivo as compared to a non-variant parent capsid polypeptide.

65. The variant AAV capsid polypeptide of Claim 53 or 58, wherein the variant AAV capsid polypeptide exhibits increased transduction of human muscle tissue or cells in vitro as compared to a non-variant parent capsid polypeptide.

66. The variant AAV capsid polypeptide of Claim 53 or 58, wherein the variant AAV capsid polypeptide exhibits increased transduction of a human muscle tissue explant ex vivo as compared to anon-variant parent capsid polypeptide.

67. The variant AAV capsid polypeptide of Claim 1, 2, 19, 20, 41, 42, 53 or 58, wherein the variant AAV capsid polypeptide is part of a functional AAV capsid, wherein the functional AAV capsid packages a nucleic acid sequence selected from the group consisting of a non-coding RNA, a protein coding sequence, an expression cassette, a multi-expression cassette, a sequence for homologous recombination, a genomic gene targeting cassette, and a therapeutic expression cassette.

68. The variant AAV capsid polypeptide of Claim 67, wherein the therapeutic expression cassette encodes a therapeutic protein.

69. The variant AAV capsid polypeptide of Claim 68, wherein the therapeutic protein is selected from the group consisting of frataxin, GSD1A, PRKAG2, BAG3, Dysferlin, MYBPC-3. Danon / Lamp2, TMEM43, PKP2, LMNA, TNNT2, RBM20, MYH7, RYR2, CAMKII, PRKAG2, CALQ2, DM1. GRN, GBA, MSH3, UNCI 3 A, Stathmin-2, SMEI, and combinations thereof.

70. The variant AAV capsid polypeptide of Claim 68 or 69, wherein the expression cassette further encodes a consensus Kozak sequence of AGCCCCAAC.

71. The variant AAV capsid polypeptide of any one of Claims 67-70, wherein the expression cassette further encodes a promoter element.

72. The variant AAV capsid polypeptide of Claim 71, wherein the promoter element is selected from a tissue-specific promoter, a cardiac-restricted promoter, desmin, creatine kinase, myogenin, alpha myosin heavy chain, and natriuretic peptide, a muscle cell specific promoter, albumin, alpha- 1 -antitrypsin, hepatitis B virus core protein promoters, a liver cell specific promoter, cardiac troponin C, cardiac troponin I, andcardiac troponin T (cTnT), a constitutive promoter, inducible promoter, synthetic promoter, Herpes Simplex virus (HSV), thymidine kinase (TK). Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A, cytomegalovirus (CMV), housekeeping gene promoters, P-actin, chicken -actin promoter (CBA), 6-myosin heavy chain gene, myosin light chain 2v (MLC-2v) gene, myosin light chain 2a gene, CARP gene, cardiac a-actin gene, cardiac m2 muscarinic acetylcholine gene, atrial natriuretic factor gene (ANF). cardiac sarcoplasmic reticulum Ca-ATPase gene, skeletal a-actin gene, an artificial cardiac promoter derived from MLC- 2v gene, cytochrome P450 genes, heat shock protein genes, metallothionein genes, hormone-inducible genes, estrogen gene promoter, tetVP16, and / or any combination thereof.

73. The variant AAV capsid polypeptide of Claim 1, 2, 19, 20, 41, 42, 53 or 58, wherein the first engineered feature enhances transduction or tropism to skeletal muscle by up to about 25-fold.

74. The variant AAV capsid polypeptide of Claim 1, 2, 19, 20, 41, 42, 53 or 58. wherein the first engineered feature enhances transduction or tropism to cardiac muscle by up to about 10-fold.

75. The variant AAV capsid polypeptide of Claim 1, 2, 19, 20, 41, 42, 53 or 58, wherein the second engineered feature reduces transduction or tropism to liver tissue by up to about 100%.

76. A method of treating a cardiac or musculoskeletal disease comprising administering to a subject in need thereof, a therapeutically effective amount of an rh74 genome encapsidated within a capsid comprising the variant AAV capsid of Claim 1, 2, 19, 20, 41, 42, 53 or 58.

77. Use of the variant AAV capsid of Claim 1, 2, 19, 20, 41, 42, 53 or 58 in the manufacture of a medicament for the treatment of a cardiac or musculoskeletal disease.

78. Use of the variant AAV capsid of Claim 1, 2, 19, 20.

41.

42. 53 or 58 for the treatment of a cardiac or musculoskeletal disease.

79. A method of treating one or more diseases comprising administering to a subject in need thereof, a therapeutically effective amount of an rh74 genome encapsidated within a variant adeno-associated virus (AAV) capsid polypeptide comprising:a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding nonengineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more non-muscle tissue, wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein.

80. A method of treating one or more diseases comprising administering to a subject in need thereof, a therapeutically effective amount of an rh74 genome encapsidated within a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV9 having at least two engineered features as compared to a corresponding non-engineered AAV9 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to one or more nonmuscle tissue, wherein the enhanced or reduced transduction or tropism is in comparison to an AAV9 vector packed using a non-engineered VP1 capsid protein.

81. The method of Claim 79 or 80, wherein the one or more diseases comprises a cardiac disease.

82. The method of Claim 81, wherein the cardiac disease comprises cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, Marfan Syndrome, Loys-Dietz syndrome, Shprintzen-Goldberg syndrome, familial thoracic aortic aneurysm and dissection, familial hypercholesterolemia, heritable pulmonary arterial hypertension, and / or any combination thereof.

83. The method of Claim 79 or 80, wherein the one or more diseases comprises a musculoskeletal disease.

84. A method of treating cardiac disease comprising administering to a subject in need thereof, a therapeutically effective amount of an rh74 genome encapsidated within a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding nonengineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more non-muscle tissue, wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein.

85. A method of treating cardiac disease comprising administering to a subject in need thereof, a therapeutically effective amount of an AAV9 genome encapsidated within a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV9 having at least two engineered features as compared to a corresponding non-engineered AAV9 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancingtransduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to one or more nonmuscle tissue, wherein the enhanced or reduced transduction or tropism is in comparison to an AAV9 vector packed using a non-engineered VP1 capsid protein.

86. The method of Claim 84 or 85, wherein the cardiac disease comprises cardiomyopathy, dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, Marfan Syndrome, Loys-Dietz syndrome, Shprintzen-Goldberg syndrome, familial thoracic aortic aneurysm and dissection, familial hypercholesterolemia, heritable pulmonary arterial hypertension, and / or any combination thereof.

87. A method of treating musculoskeletal disease comprising administering to a subject in need thereof, a therapeutically effective amount of an rh74 genome encapsidated within a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV serotype rh74 having at least two engineered features as compared to a corresponding nonengineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant rh74 AAV vector packaged using the variant capsid to one or more non-muscle tissue, wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein.

88. A method of treating musculoskeletal disease comprising administering to a subject in need thereof, a therapeutically effective amount of an AAV9 genome encapsidated within a variant adeno-associated virus (AAV) capsid polypeptide comprising: a polypeptide sequence of a VP1 capsid protein of AAV9 having at least two engineered features as compared to a corresponding non-engineered rh74 VP1 polypeptide; wherein the first engineered feature comprises an insertion of a peptide within the VP1 polypeptide, the inserted peptide enhancing transduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to muscle tissue; wherein the second engineered feature comprises a point mutation within the VP1 polypeptide, the point mutation reducing transduction or tropism of a recombinant AAV9 vector packaged using the variant capsid to one or more nonmuscle tissue, wherein the enhanced or reduced transduction or tropism is in comparison to an AAV9 vector packed using a non-engineered VP1 capsid protein.

89. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the first engineered feature comprises insertion of at least 5 amino acids.

90. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the first engineered feature comprises insertion of at least 7 amino acids.

91. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the first engineered feature enhances transduction or tropism to cardiac muscle.

92. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the first engineered feature enhances transduction or tropism to skeletal muscle.

93. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the first engineered feature enhances transduction or tropism to cardiac muscle and skeletal muscle.

94. The method of Claim 79.

80.

84. 85, 87, or 88. wherein the first engineered feature comprises a peptide encoded by SEQ ID NO: 3, 16, 20, or 24.

95. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the first engineered feature comprises a peptide having the sequence of SEQ ID NO: 4, 15, 19, or 23.

96. The method of Claim 79, 80, 84, 85, 87, or 88. wherein the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 5, 28, 30, or 32.

97. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 6, 27, 29, or 31.

98. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the second engineered feature reduces transduction or tropism to a soft tissue.

99. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the second engineered feature reduces transduction or tropism to liver tissue.

100. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the second engineered feature comprises a glycine to alanine mutation.

101. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the second engineered feature comprises a glycine to alanine mutation at position 268 of SEQ IDNO: 2.

102. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the second engineered feature comprises a glycine to alanine mutation at position 267 of SEQ IDNO: 11.

103. The method of Claim 79, 80, 84, 85, 87, or 88. wherein the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 7.

104. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 8.

105. The method of Claim 79, 80, 84, 85, 87, or 88. wherein the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 9.

106. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 10.

107. The method of Claim 79, 80, 84, 85, 87, or 88. wherein the variant AAV capsid polypeptide comprises a peptide encoded by SEQ ID NO: 14.

108. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the variant AAV capsid polypeptide comprises a peptide having the sequence of SEQ ID NO: 12.

109. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the variant AAV capsid polypeptide is part of a functional AAV capsid, wherein the functional AAV capsid packages a nucleic acid sequence selected from the group consisting of a noncoding RNA, a protein coding sequence, an expression cassette, a multi-expression cassette, a sequence for homologous recombination, a genomic gene targeting cassette, and a therapeutic expression cassette.

110. The method of Claim 109, wherein the therapeutic expression cassette encodes a therapeutic protein.

111. The method of Claim 1 10, wherein the therapeutic protein is selected from the group consisting of frataxin, GSD1A, PRKAG2, BAG3, Dysferlin, MYBPC-3, Danon / Lamp2, TMEM43, PKP2, LMNA, TNNT2, RBM20, MYH7, RYR2, CAMKII, PRKAG2, CALQ2, DM1, GRN, GBA, MSH3, UNCI 3 A. Stathmin-2, SMEI. and combinations thereof.

112. The method of Claim 109, wherein the expression cassette further encodes a consensus Kozak sequence of AGCCCCAAC.

113. The method of Claim 109, wherein the expression cassette further encodes a promoter element.

114. The method of Claim 113, wherein the promoter element is selected from a tissue-specific promoter, a cardiac-restricted promoter, desmin, creatine kinase, myogenin, alpha myosin heavy' chain, and natriuretic peptide, a muscle cell specific promoter, albumin, alpha- 1 -antitrypsin, hepatitis B virus core protein promoters, a liver cell specific promoter, cardiac troponin C, cardiac troponin I. and cardiac troponin T (cTnT), a constitutive promoter, inducible promoter, synthetic promoter. Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A, cytomegalovirus (CMV), housekeeping gene promoters. P-actin, chicken -actin promoter (CBA). 6-myosin heavy chain gene, myosin light chain 2v (MLC-2v) gene, myosin light chain 2a gene, CARP gene, cardiac a-actin gene, cardiac m2 muscarinic acetylcholine gene, atrial natriuretic factor gene (ANF), cardiac sarcoplasmic reticulum Ca-ATPase gene, skeletal a-actin gene, an artificial cardiac promoter derived from MLC-2v gene, cytochrome P450 genes, heat shock protein genes, metallothionein genes, hormone-inducible genes, estrogen gene promoter, tetVP16, and / or any combination thereof.

115. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the first engineered feature enhances transduction or tropism to skeletal muscle by up to about 25-fold.

116. The method of Claim 79.

80.

84. 85, 87, or 88. wherein the first engineered feature enhances transduction or tropism to cardiac muscle by up to about 10-fold.

117. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the second engineered feature reduces transduction or tropism to liver tissue by up to about 100%.

118. The method of Claim 79. 80, 84, 85, 87, or 88. wherein the first engineered feature enhances transduction or tropism to cardiac muscle by up to about 10-fold and thesecond engineered feature reduces transduction or tropism to liver tissue by up to about 100%.

119. The method of Claim 79, 80, 84, 85, 87, or 88, wherein the first engineered feature enhances transduction or tropism to cardiac muscle by up to about 10-fold and the second engineered feature reduces transduction or tropism to soft tissue by up to about 100%.

120. The method of Claim 79.

80.

84. 85, 87, or 88. wherein the first engineered feature enhances transduction or tropism to skeletal muscle by up to about 25-fold and the second engineered feature reduces transduction or tropism to liver tissue by up to about 100%.

121. The method of Claim 79. 80, 84, 85, 87, or 88. wherein the first engineered feature enhances transduction or tropism to skeletal muscle by up to about 25-fold and the second engineered feature reduces transduction or tropism to soft tissue by up to about 100%.

122. A variant adeno-associated virus (AAV) capsid polypeptide comprising: an inserted peptide within a VP1 capsid protein of AAV serotype rh74. the inserted peptide imparting enhanced transduction or tropism of muscle tissue; and a point mutation within the VP1 polypeptide, the point mutation resulting in reduced transduction or tropism one or more non-muscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein.

123. A variant adeno-associated virus (AAV) capsid polypeptide comprising: an inserted peptide of between 5-8 amino acids within a VP1 capsid protein of AAV serotype rh74, the inserted peptide imparting enhanced transduction or tropism of muscle tissue; and a point mutation within the VP1 polypeptide, the point mutation resulting in reduced transduction or tropism one or more non-muscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein.

124. A variant adeno-associated virus (AAV) capsid polypeptide comprising: an inserted peptide within a VP1 capsid protein of AAV serotype rh74, the inserted peptide imparting enhanced transduction or tropism of muscle tissue; anda glycine to alanine mutation within the VP1 polypeptide, the glycine to alanine mutation resulting in reduced transduction or tropism one or more nonmuscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using anon-engineered VP1 capsid protein.

125. A variant adeno-associated virus (AAV) capsid polypeptide comprising: an inserted peptide comprising RGDLGLS, the inserted peptide placed within a VP1 capsid protein of AAV serotype rh74, the inserted peptide imparting enhanced transduction or tropism of muscle tissue; and a glycine to alanine mutation within the VP1 polypeptide, the point mutation resulting in reduced transduction or tropism one or more non-muscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an rh74 AAV vector packed using a non-engineered VP1 capsid protein.

126. A variant adeno-associated virus (AAV) capsid polypeptide comprising: an inserted peptide within an AAV9 VP1 capsid protein, the inserted peptide imparting enhanced transduction or tropism of muscle tissue; and a point mutation within the VP1 polypeptide, the point mutation resulting in reduced transduction or tropism one or more non-muscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an AAV9 vector packed using a non-engineered VP1 capsid protein.

127. A variant adeno-associated virus (AAV) capsid polypeptide comprising: an inserted peptide of between 5-8 amino acids within an AAV9 VP1 capsid protein, the inserted peptide imparting enhanced transduction or tropism of muscle tissue; and a point mutation within the VP1 polypeptide, the point mutation resulting in reduced transduction or tropism one or more non-muscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an AAV9 vector packed using a non-engineered VP1 capsid protein.

128. A variant adeno-associated virus (AAV) capsid polypeptide comprising: an inserted peptide within an AAV9 VP1 capsid protein, the inserted peptide imparting enhanced transduction or tropism of muscle tissue; anda glycine to alanine mutation within the VP1 polypeptide, the glycine to alanine mutation resulting in reduced transduction or tropism one or more nonmuscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an AAV9 vector packed using a non-engineered VP1 capsid protein.

129. A variant adeno-associated virus (AAV) capsid polypeptide comprising: an inserted peptide comprising RGDLGLS, the inserted peptide placed within an AAV9 VP1 capsid protein, the inserted peptide imparting enhanced transduction or tropism of muscle tissue; and a glycine to alanine mutation within the VP 1 polypeptide, the point mutation resulting in reduced transduction or tropism one or more non-muscle tissue, and wherein the enhanced or reduced transduction or tropism is in comparison to an AAV9 vector packed using a non-engineered VP1 capsid protein.

130. The variant adeno-associated virus (AAV) capsid polypeptide of any one of claims 122-129, wherein the variant AAV capsid polypeptide is part of a functional AAV capsid, wherein the functional AAV capsid packages a nucleic acid sequence selected from the group consisting of a non-coding RNA, a protein coding sequence, an expression cassette, a multi-expression cassette, a sequence for homologous recombination, a genomic gene targeting cassette, and a therapeutic expression cassette.

131. The variant AAV capsid polypeptide of Claim 130, wherein the therapeutic expression cassette encodes a therapeutic protein.

132. The variant AAV capsid polypeptide of Claim 131, wherein the therapeutic protein is selected from the group consisting of frataxin. GSD1A. PRKAG2, BAG3, Dysferlin, MYBPC-3, Danon / Lamp2, TMEM43, PKP2, LMNA, TNNT2, RBM20, MYH7, RYR2, CAMKII, PRKAG2, CALQ2, DM1, GRN, GBA, MSH3, UNCI 3 A, Stathmin-2, SMEI, and combinations thereof.

133. The variant AAV capsid polypepride of Claim 131, wherein the expression cassette further encodes a consensus Kozak sequence of AGCCCCAAC.

134. The variant AAV capsid polypeptide of Claim 130, wherein the expression cassette further encodes a promoter element.

135. The variant AAV capsid polypeptide of Claim 134, wherein the promoter element is selected from a tissue-specific promoter, a cardiac-restricted promoter, desmin, creatine kinase, myogenin, alpha myosin heavy chain, and natriuretic peptide, a musclecell specific promoter, albumin, alpha- 1 -antitrypsin, hepatitis B virus core protein promoters, a liver cell specific promoter, cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT), a constitutive promoter, inducible promoter, synthetic promoter, Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad El A, cytomegalovirus (CMV), housekeeping gene promoters, P-actin, chicken -actin promoter (CBA), 6-myosin heavy chain gene, myosin light chain 2v (MLC-2v) gene, myosin light chain 2a gene, CARP gene, cardiac a-actin gene, cardiac m2 muscarinic acety lcholine gene, atrial natriuretic factor gene (ANF), cardiac sarcoplasmic reticulum Ca-ATPase gene, skeletal a-actin gene, an artificial cardiac promoter derived from MLC- 2v gene, cytochrome P450 genes, heat shock protein genes, metallothionein genes, hormone-inducible genes, estrogen gene promoter, tetVP16, and / or any combination thereof.

136. The variant AAV capsid polypeptide of any one of Claims 122-129, wherein the first engineered feature enhances transduction or tropism to skeletal muscle by up to about 25-fold.

137. The variant AAV capsid polypeptide of any one of Claims 122-129, wherein the first engineered feature enhances transduction or tropism to cardiac muscle by up to about 10-fold.

138. The variant AAV capsid polypeptide of any one of Claims 122-129, wherein the second engineered feature reduces transduction or tropism to liver tissue by up to about 100%.

139. Use of the variant AAV capsid of any one of Claims 122-129, in the manufacture of a medicament for the treatment of a cardiac or musculoskeletal disease.

140. Use of the variant AAV capsid of any one of Claims 122-129, for the treatment of a cardiac or musculoskeletal disease.

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