Adeno-associated virus with engineered capsid

Abstract

The present disclosure provides engineered adeno-associated capsid proteins and recombinant virus vectors comprising an engineered capsid protein, and uses thereof. In some embodiments, the present dislosure provides engineered AAV9 capsid proteins. In particular, the disclosure provides AAV (e.g., AAV9) virions comprising an engineered AAV capsid protein that achieves increased transduction efficiency in heart, increased heart-to-liver ratio, and / or other desirable properties.

Description

ADENO-ASSOCIATED VIRUS WITH ENGINEERED CAPSIDCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 516,134 filed July 27, 2023, U.S. Provisional Patent Application No. 63 / 580,941 filed September 6, 2023, and U.S. Provisional Patent Application No. 63 / 654,556 filed May 31, 2024, the entire disclosures of each of which are incorporated herein be reference in their entireties.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The present application is being filed electronically via USPTO Patent Center and includes an electronically submitted Sequence Listing in XML format. The name of the XML file containing the Sequence Listing is TENA_049_03WO_SeqList_ST26.xml. The XML file, created on July 26, 2024 is 1,069,989 bytes in size and is being submitted electronically via USPTO Patent Center. The contents of the electronic Sequence Listing are incorporated by reference herein in their entirety.FIELD OF THE INVENTION

[0003] The present disclosure generally relates to adeno-associated capsid proteins and virus vectors. In particular, the disclosure relates to engineered capsid proteins, and recombinant adeno-associated virus virions having an engineered capsid protein, and uses thereof.BACKGROUND

[0004] Adeno-associated virus (AAV) holds promise for gene therapy and other biomedical applications. In particular, AAV can be used to deliver gene products to various tissues and cells, both in vitro and in vivo. The capsid proteins of AAV largely determine the immunogenicity and tropism of AAV vectors.

[0005] For cardiac tissues, AAV subtype 9 (AAV9) is a preferred AAV vector due to its ability to transduce the heart following systemic delivery. While AAV9 can achieve moderate transduction of the heart, the majority of vector trafficks to the liver. Moreover, in order to achieve therapeutic levels of transduction in the heart, relatively high systemic doses are required, potentially leading to systemic inflammation and in turn, toxicity.

[0006] There is a need for developing an Adeno-associated virus with engineered capsid protein that achieves improved cardiac tropism, and optionally improved selectivity of cardiac tissues over liver. The present disclosure provides variants of AAV capsids with modified VR-VIII site, for example, AAV9 capsid variants, that form rAAV virions capable of transducing cardiac tissues and / or cell types more efficiently and / or with more selectivity than rAAV virions comprising wild-type AAV9 capsid proteins, which can be used for safe and efficacious cardiac gene therapy.SUMMARY OF THE DISCLOSURE

[0007] In some embodiments, the present disclosure provides an engineered adeno- associated virus (rAAV) capsid protein, wherein the capsid protein comprises an amino acid sequence X1DVQX2X3PGFX4X5X6X7X8 in the VR-VIII site, wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs is any amino acid. In some embodiments, the capsid protein is a variant of an AAV5, AAV9, AAVrh.74, or AAVrh.10 capsid protein.

[0008] In some embodiments, the present disclosure provides an engineered adeno- associated virus (rAAV) capsid protein, wherein the capsid protein comprises: (i) at least 80% amino acid sequence identity to the AAV5 VP3 capsid protein of SEQ ID NO: 324, and comprises the amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 at amino acid positions 570 to 583, wherein the amino acid numbering is according to SEQ ID NO: 322; (ii) at least 80% amino acid sequence identity to the AAV9 VP3 capsid protein of SEQ ID NO: 487, and comprisesthe amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 at amino acid positions 581 to 594, wherein the amino acid numbering is according to SEQ ID NO: 1; (iii) at least 80% amino acid sequence identity to the AAVrh.74 VP3 capsid protein of SEQ ID NO: 338, and comprises the amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 at amino acid positions 583 to 596, wherein the amino acid numbering is according to SEQ ID NO: 336; or (iv) at least 80% amino acid sequence identity to the AAVrh.10 VP3 capsid protein of SEQ ID NO: 331, and comprises the amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 at amino acid positions 583 to 596 of AAVrh.10, wherein the amino acid numbering is according to SEQ ID NO: 329; wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs is any amino acid.

[0009] In some embodiments, Xi is Alanine (A). In some embodiments, X2 is Glutamine (Q). In some embodiments, X7 is Threonine (T). In some embodiments, X5 is Alanine (A) or Proline (P). In some embodiments, Xe is Glutamine (Q) or Glutamic Acid (E). In some embodiments, X4 is Q, G, R, N, H, M, P, or S. In some embodiments, Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments, X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, Xi is Alanine (A), X2 is Glutamine (Q), and X7 is Threonine (T). In some embodiments, X4 is Glutamine (Q), X5 is Alanine (A), and Xe is Glutamine (Q). In some embodiments, Xs is Glutamic Acid (E).

[0010] In some embodiments, the capsid protein comprises an amino acid sequence selected from X1DVQX2X3PGFX4AQX7X8 (SEQ ID NO: 1039), X1DVQX2X3PGFX4AEX7X8 (SEQ ID NO: 1040), X1DVQX2X3PGFX4PQX7X8 (SEQ ID NO: 1041), X1DVQX2X3PGFX4PEX7X8 (SEQ ID NO: 1042), ADVQQX3PGFX4AQTX8 (SEQ ID NO: 1033), ADVQQX3PGFX4AETX8 (SEQ ID NO: 1043), ADVQQX3PGFX4PQTX8 (SEQ ID NO:1044), and ADVQQX3PGFX4PETX8 (SEQ ID NO: 1032) in the VR-VIII site, and wherein each of Xi, X2, X3, X4, X7, and Xs , if present, is any amino acid. In some embodiments, X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; and X8is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0011] In some embodiments, the capsid protein comprises an amino acid sequence selected from ADVQQX3PGFX4X5X6X7X8 (SEQ ID NO: 315), ADVQQX3PGFQAQTX8 (SEQ ID NO: 312), ADVQQX3PGFX4X5X6TX8 (SEQ ID NO: 311), ADVQQX3PGFX4X5X6X7E (SEQ ID NO: 316), ADVQQX3PGFX4X5X6TE (SEQ ID NO: 313), and X1DVQX2X3PGFX4X5X6X7E (SEQ ID NO: 306) in the VR-VIII site, and wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs , if present, is any amino acid. In some embodiments, X3 is selected from Leucine (L) and Histidine (H). In some embodiments, X3 is L, or the capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E (SEQ ID NO: 1045) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 317) wherein each of Xi, X2, X4, X5, X6, and X7, if present, is any amino acid, or the capsid protein comprises amino acid sequence ADVQQLPGFQAQTE (SEQ ID NO: 320). In some embodiments, X3 is H, or the capsid protein comprises an amino acid sequence X1DVQX2HPGFX4X5X6X7E (SEQ ID NO: 1051) or ADVQQHPGFX4X5X6TE (SEQ ID NO: 318) wherein each of Xi, X2, X4, X5, X6, and X7, if present, is any amino acid, or the capsid protein comprises the amino acid sequence ADVQQHPGFQAQTE (SEQ ID NO: 321). In some embodiments, X3 is L and Xs is E, or the capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E (SEQ ID NO:1045) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 317) wherein each of Xi, X2, X4, X5, X6, andX7, if present, is any amino acid, or the capsid protein comprises the amino acid sequence selected from the group consisting of: ADVQQLPGFQAQTE (SEQ ID NO: 320), ADVQQLPGFGAQTE (SEQ ID NO: 906), ADVQQLPGFRPETE (SEQ ID NO: 907),ADVQQLPGFNAQTE (SEQ ID NO: 908), ADVQQLPGFQAETE (SEQ ID NO: 951),ADVQQLPGFGAETE (SEQ ID NO: 952), ADVQQLPGFGPQTE (SEQ ID NO: 953),ADVQQLPGFHAQTE (SEQ ID NO: 954), ADVQQLPGFRAQTE (SEQ ID NO: 955),ADVQQLPGFRAETE (SEQ ID NO: 956), ADVQQLPGFMAQTE (SEQ ID NO: 957),ADVQQLPGFPAQTE (SEQ ID NO: 958), ADVQQLPGFSAQTE (SEQ ID NO: 959), ADVQQLPGFSAETE (SEQ ID NO: 960), and ADVQQLPGFRPQTE (SEQ ID NO: 961).

[0012] In some embodiments, X4 is Q, or the capsid protein comprises an amino acid sequence X1DVQX2X3PGFQX5X6X7X8 (SEQ ID NO: 307), ADVQQXsPGFQXsXeTXs (SEQ ID NO: 1046), or ADVQQX3PGFQAQTX8(SEQ ID NO: 312) wherein each of Xi, X2, X3, X5, Xe, X7, and X8, if present, is any amino acid, or the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 320, 321, 904, 905, and 909-950.

[0013] In some embodiments, the present disclosure provides a recombinant adeno- associated virus (rAAV) capsid protein, wherein the capsid protein comprises a sequence sharing at least 80% amino acid sequence identity to AAV9 VP3 capsid protein of SEQ ID NO: 487, and comprises relative to the AAV9 VP1 capsid protein sequence of SEQ ID NO: 1 : (i) an amino acid sequence XIDVQX2X3PGFX4XSX6X7X8in the VR-VIII site or at positions 581 to 594, wherein each of Xi, X2, X3, X4, X5, Xe, X7, and X8is any amino acid (SEQ ID NO: 303), or (ii) amino acid substitutions T582D, N583V, H584Q, A587P, Q588G, and A589F. In some embodiments, Xi is Alanine (A), or the capsid protein comprises amino acid A at position 581. In some embodiments, X2 is Glutamine (Q), or the capsid protein comprises amino acid Q at position 585. In some embodiments, X7 is Threonine (T), or the capsid protein comprises amino acid T at position 593. In some embodiments, X5 is Alanine (A) or Proline (P), or the capsid protein comprises amino acid A or P at position 591. In some embodiments, Xe is Glutamine (Q) or Glutamic Acid (E), or the capsid protein comprises Q or E at position 592. In some embodiments, X4 is Q, G, R, N, H, M, P, or S, or the capsid protein comprises Q, G, R, N, H, M, P, or S at position 590. In some embodiments, X8is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N, or the capsid protein comprises E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N at position 594. In some embodiments, X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y, or the capsid protein comprises L, H, V, C, Q, G, I, M, F, P, T, or Y at position 586.

[0014] In some embodiments, Xi is Alanine (A), X2 is Glutamine (Q), and X7 is Threonine (T); or the capsid protein comprises amino acids A, Q and T at position 581, 585 and 593, respectively. In some embodiments, X4 is Glutamine (Q), X5 is Alanine (A), and Xe is Glutamine (Q); or the capsid protein comprises amino acids Q, A and Q and positions 590, 591 and 592, respectively. In some embodiments, X8is Glutamic Acid (E), or the capsid protein comprises amino acid substitution G594E.

[0015] In some embodiments, the capsid protein comprises in the VR-VIII site an amino acid sequence XIDVQX2X3PGFX4AQX7X8(SEQ ID NO: 1039), XIDVQX2X3PGFX4AEX7X8(SEQ ID NO: 1040), X1DVQX2X3PGFX4PQX7X8 (SEQ ID NO: 1041), X1DVQX2X3PGFX4PEX7X8 (SEQ ID NO: 1042), ADVQQX3PGFX4AQTX8 (SEQ ID NO: 1033), ADVQQX3PGFX4AETX8 (SEQ ID NO: 1043), ADVQQX3PGFX4PQTX8 (SEQ ID NO: 1044), or ADVQQX3PGFX4PETX8 (SEQ ID NO: 1032), wherein each of Xi, X2, X3, X4, X7, and Xs, if present, is any amino acid. In some embodiments, X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; and X8is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0016] In some embodiments, the capsid protein comprises in the VR-VIII site an amino acid sequence ADVQQX3PGFX4X5X6X7X8 (SEQ ID NO: 315), ADVQQX3PGFQAQTX8 (SEQ ID NO: 312), ADVQQX3PGFX4X5X6TX8 (SEQ ID NO: 311), ADVQQX3PGFX4X5X6X7E (SEQ ID NO: 316), ADVQQX3PGFX4X5X6TE (SEQ ID NO: 313), or X1DVQX2X3PGFX4X5X6X7E (SEQ ID NO: 306), wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs, if present, is any amino acid. In some embodiments, X3 is selected from Leucine (L) and Histidine (H), or wherein the capsid protein comprises amino acid substitution S586L or S586H. In some embodiments, X3 is L, or the capsid protein comprises amino acid substitution S586L, or the capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E (SEQ ID NO: 1045) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 317) wherein each of Xi, X2, X4, X5, Xe, and X7, if present, is any amino acid, or the capsid protein comprises amino acid sequence ADVQQLPGFQAQTE (SEQ ID NO: 320). In some embodiments, X3 is H, or the capsid protein comprises amino acid substitution S586H, or the capsid protein comprises an amino acid sequence ADVQX2HPGFX4X5X6X7E (SEQ ID NO: 1050) or ADVQQHPGFX4X5X6TE (SEQ ID NO: 318) wherein each of X2, X4, X5, X6, and X7, if present, is any amino acid, or the capsid protein comprises amino acid sequence ADVQQHPGFQAQTE (SEQ ID NO: 321). In some embodiments, X3 is L and Xs is E, or the capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E (SEQ ID NO: 1045) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 317) wherein each of Xi, X2, X4, X5, X6, and X7 is any amino acid or, or the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 320, 906-908, and 951-961. In some embodiments, X4 is Q, or the capsid protein comprises an amino acid sequence X1DVQX2X3PGFQX5X6X7X8, ADVQQXsPGFQXsXeTXs (SEQ ID NO: 1046), or ADVQQX3PGFQAQTX8 (SEQ ID NO: 312) wherein each of Xi, X2, X3, X5, Xe, X7, and Xs, if present, is any amino acid, or the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 320, 321, 904, 905, and 909-950.

[0017] In some embodiments, the capsid protein comprises relative to AAV9 VP1 capsid protein sequence, at position 452 an amino acid selected from the group consisting of: K and N. In some embodiments, the capsid protein comprises relative to AAV9 VP1 capsid protein sequence, at position 452 an amino acid substitution N452K.

[0018] In some embodiments, the present disclosure provides an engineered adeno- associated virus (rAAV) capsid protein, wherein the capsid protein comprises a variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10, wherein Xi is T or D; X2 is N or V; X3 is H, Q, or R; X4is Q, M, N, or S; X5is S, G, M, T, or L; X6is A, N, K, T, or P; X7 is Q, Y, R, L, T, or G; X8is A, V, or F; X9is Q, R, G, M, or N; X10 is A or P (SEQ ID NO: 1047). In some embodiments, the capsid protein is a variant of an AAV5, AAV9, AAVrh.74, or AAVrh.10 capsid protein.

[0019] In some embodiments, the capsid protein: (i) comprises a sequence sharing at least 80% amino acid sequence identity to AAV5 VP3 capsid protein, and comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 571 to 580 relative to AAV5 VP1 capsid protein sequence of SEQ ID NO: 322; (ii) comprises a sequence sharing at least 80% amino acid sequence identity to AAV9 VP3 capsid protein, and comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 582 to 591 relative to AAV9 VP1 capsid protein sequence of SEQ ID NO: 1; (iii) comprises a sequence sharing at least 80% amino acid sequence identity to AAVrh.74 VP3 capsid protein, and comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 584 to 593 relative to AAVrh.74 VP1 capsid protein sequence of SEQ ID NO: 336; or (iv) comprises a sequence sharing at least 80% amino acid sequence identity to AAVrh.10 VP3 capsid protein, and comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 584 to 593 relative to AAVrh.10 VP1 capsid protein sequence of SEQ ID NO: 329. In some embodiments, the capsid protein comprises the variant polypeptide sequence of any one of SEQ ID NOs: 1006 - 1027.

[0020] In some embodiments, the capsid protein is a variant of AAV9 capsid protein, and comprise the amino acid sequence of any one of SEQ ID NOs: 962-983 at amino acid positions 581 to 594 relative to the AAV9 VP1 capsid protein sequence of SEQ ID NO: 1.

[0021] In some embodiments, the capsid protein: (i) is cardiotrophic, (ii) mediates increased transduction efficiency in cardiac cells compared to the parental sequence, (iii) mediates decreased transduction efficiency in liver cells compared to the parental sequence,and / or (iv) mediates increased selectivity for the cardiac cells over liver cells compared to the parental sequence.

[0022] In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 343. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 344. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 846. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 847. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 848. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 849. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 850. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 851. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 852. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 853. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 854. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 855. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 856. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 857. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 858. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 859. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 860. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 861. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 862. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 863. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 864. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 865. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 866. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 867. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 868. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 869. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 870. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 871. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO:872. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 873. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 874. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 875. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 876. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 877. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 878. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 879. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 880. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 881. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 882. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 883. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 884. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 885. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 886. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 887. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 888. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 889. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 890. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 891. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 892. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 893. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 894. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 895. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 896. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 897. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 898. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 899. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 900. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 901. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO:902. In some embodiments, the present disclosure provides a capsid protein comprising SEQ ID NO: 903.

[0023] In some embodiments, the present disclosure provides capsid protein comprising any one of the sequences selected from the group consisting of: SEQ ID NOs: 846 to SEQ ID NO:903. In some embodiments, the capsid comprises the sequence selected from the group consisting of: SEQ ID NO:846, 847, 848, 849, 850, 891, 864, 867, 858, 878, 890, 870, 856, 886, 900, 901, and 898. In some embodiments, the capsid comprises the sequence selected from the group consisting of: SEQ ID NO:846, 847, 848, 849, and 850.

[0024] In some embodiments, the present disclosure provides a recombinant adeno- associated virus (rAAV) virion, comprising a capsid protein described herein and a vector genome comprising a polynucleotide cassette flanked by inverted terminal repeats (ITRs). In some embodiments, the rAAV virion transduces heart cells. In some embodiments, the rAAV virion transduces cardiomyocytes. In some embodiments, the rAAV virion traffics to at least one organ other than the liver. In some embodiments, the rAAV virion traffics to the heart.

[0025] In some embodiments, the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1. In some embodiments, the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher. In some embodiments, administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower. In some embodiments, the rAAV virion exhibits a higher transduction efficiency, optionally higher heart transduction efficiency, than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, assessed in a primate. In some embodiments, the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher. In some embodiments, administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

[0026] In some embodiments, the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322. In some embodiments, the rAAV virion exhibits a higher heart-to-liver transductionratio than an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher. In some embodiments, administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower. In some embodiments, the rAAV virion exhibits a higher transduction efficiency, optionally higher heart transduction efficiency, than an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, assessed in a primate. In some embodiments, the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher. In some embodiments, administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

[0027] In some embodiments, the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329. In some embodiments, the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher. In some embodiments, administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower. In some embodiments, the rAAV virion exhibits a higher transduction efficiency, optionally higher heart transduction efficiency, than an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, assessed in a primate. In some embodiments, the rAAV virion exhibits a higher heart- to-liver transduction ratio than an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher. In some embodiments, administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

[0028] In some embodiments, the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336. In some embodiments, the rAAV virion exhibits a higher heart-to-liver transductionratio than an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher. In some embodiments, administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower. In some embodiments, the rAAV virion exhibits a higher transduction efficiency, optionally higher heart transduction efficiency, than an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, assessed in a primate. In some embodiments, the rAAV virion exhibits a higher heart- to-liver transduction ratio than an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher. In some embodiments, administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

[0029] In some embodiments, the polynucleotide cassette comprises a polynucleotide sequence encoding MYBPC3, DWORF, PKP2, KCNH2, TRPM4, DSG2, TGFBR2, TGFBR1, EMD, KCNQ1, TAZ, COL3A1, JUP, CASQ2, MLRP44, DNAJC19, LMNA, TNNI3, DSP, DSG2, RAFI, S0S1, FBN1, LAMP2, FXN, RAFI, BAG3, KCNQ1, MYLK3, CRYAB, ALPK3, ACTN2, JPH2, PLN, ATP2A2, CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, S0S1, NPR1, ERBB4, VIP, MYH6, MYH7, Cas9, RBM20, MYOCD, ASCL1, GATA4, MEF2C, TBX5, miR-133, or MESP1. In some embodiments, the polynucleotide cassette comprises a polynucleotide sequence which encodes a protein selected from the group consisting of: MYBPC3, DWORF, PKP2, LMNA, LAMP2, BAG3, CRYAB, JPH2, PLN, TTNI3, MYOCD, ASCL1, DSP, JUP, DSP, MYH6, MYH7, RBM20, and Cas9.

[0030] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a rAAV virion described herein and a pharmaceutically acceptable carrier.

[0031] In some embodiments, the present disclosure provides a polynucleotide encoding a capsid protein described herein.

[0032] In some embodiments, the present disclosure provides a method of transducing a cardiac cell, comprising contacting the cardiac cell with a rAAV virion described herein, wherein the rAAV virion transduces the cardiac cell. In some embodiments, the cardiac cell is a cardiomyocyte. In some embodiments, the rAAV virion exhibits higher transductionefficiency in the cell than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, an AAV5 VP1 capsid protein according to SEQ ID NO: 322, an AAVrh.10 capsid protein according to SEQ ID NO: 329, or an AAVrh.74 capsid protein according to SEQ ID NO: 336.

[0033] In some embodiments, the present disclosure provides a method of delivering one or more gene products to a cardiac cell, comprising contacting the cardiac cell with a rAAV virion described herein. In some embodiments, the cardiac cell is a cardiomyocyte.

[0034] In some embodiments, the present disclosure provides a method of treating a cardiac pathology in a subject in need thereof, comprising administering a therapeutically effective amount of a rAAV virion described herein to the subject, or a pharmaceutical composition thereof, wherein the rAAV virion transduces cardiac tissue.

[0035] In some embodiments, the present disclosure provides a method of treating a heart disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a rAAV virion described herein to the subject or a pharmaceutical composition thereof.

[0036] In some embodiments, the present disclosure provides a kit comprising a pharmaceutical composition described herein and instructions for use.BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 shows a schematic of in silico design facilitated capsid engineering approach. Single and double substitutions and insertions were introduced to the VR-VIII region of AAV9 capsid. The resulting variants and some previously characterized variants were tested in mice together to measure their transduction efficiencies in heart and liver following systemic delivery. Based on this experimental dataset, multiple in silico methods were utilized to predict / generate over 9000 novel variants, which were manufactured and tested in mice.

[0038] FIG. 2A-FIG. 2B are graphs showing heart transduction (FIG. 2A) and liver viral load (FIG. 2B) of ZC621 and ZC623 capsids compared to AAV9 wild-type control in mice from a pooled experiment. All the values are relative to the performance of wild-type AAV9 control.

[0039] FIG. 3 shows a schematic of measuring heart, liver, and skeletal muscle transduction in mice 3-weeks after retro-orbital administration of 2E+13 vg / kg of wild-type AAV9, ZC621, or ZC623 capsid in C57BL / 6NCrl mice. Heart tissue transduction is measuredby RNA / DNA qPCR analysis and liver and skeletal muscle are measured by protein ELISA analysis.

[0040] FIG. 4A-FIG. 4B are graphs showing heart transduction measured by viral RNA expression (FIG. 4A) and viral DNA expression (FIG. 4B) of ZC621 and ZC623 capsids compared to AAV9 wild-type control in C57BL / 6NCrl mice administered as described in FIG. 3. All values are relative to the performance of the wild-type AAV9 control.

[0041] FIG. 5A-FIG. 5B are graphs showing liver (FIG. 5A) and skeletal muscle (tibialis anterior) (FIG. 5B) transduction measured by viral protein expression (ELISA) of ZC621 and ZC623 capsids compared to AAV9 wild-type control in C57BL / 6NCrl mice administered as described in FIG. 3. All values are relative to the performance of the wild-type AAV9 control.

[0042] FIG. 6 shows a schematic of measuring heart, liver, and skeletal muscle transduction in mice 3-weeks after retro-orbital administration of 2E+13 vg / kg of wild-type AAV9, ZC621, or ZC623 capsid in CD-I mice.

[0043] FIG. 7A-FIG. 7C are graphs showing heart (FIG. 7 A), liver (FIG. 7B), and skeletal (FIG. 7C) muscle transduction measured by viral protein expression (ELISA) of ZC621 and ZC623 capsids compared to AAV9 wild-type control in CD-I mice administered as described in FIG. 6. All values are relative to the performance of the wild-type AAV9 control.

[0044] FIG. 8 shows a schematic of measuring heart, liver, and skeletal muscle transduction in C57BL / 6NCrl mice 3-weeks after retro-orbital administration of 2E+13 vg / kg of wild-type AAV9 or ZC621 capsid, or 5E+12 vg / kg of ZC621 capsid.

[0045] FIG. 9A-FIG. 9B are graphs showing heart transduction measured by viral RNA expression (FIG. 9A) and viral DNA expression (FIG. 9B) of ZC621 capsid compared to AAV9 wild-type control in C57BL / 6NCrl mice administered as described in FIG. 8. All values are relative to the performance of the wild-type AAV9 control.

[0046] FIG. 10A-FIG. 10B are graphs showing liver (FIG. 10A) and skeletal muscle (tibialis anterior) (FIG. 10B) transduction measured by viral protein expression of ZC621 capsid compared to AAV9 wild-type control in C57BL / 6NCrl mice administered as described in FIG. 8. All values are relative to the performance of the wild-type AAV9 control.

[0047] FIG. 11 shows a schematic of measuring heart transduction in mice after administration of capsid variants generated by amino acid substitutions introduced to positions 586 and 594 of the ZC621 capsid or positions 590, 591 and 592 of the ZC621 capsid.

[0048] FIG. 12A-FIG. 12B are heatmaps demonstrating packaging efficiency (FIG. 12A) and heart transduction efficiency (FIG. 12B) for ZC621-based position 586 + 594 rediversification variants. All values are relative to the performance of the wild-type AAV9 control.

[0049] FIG. 13 is a graph showing heart transduction measured by viral RNA expression of ZC621 based positions 586 + 594 re-diversification variant capsids compared to AAV9 wildtype control. All values are relative to the performance of the wild-type AAV9 control.

[0050] FIG. 14A-FIG. 14B are a graph panel highlighting top ZC621-based positions 590 + 591 + 592 re-diversification variants in the 3-position sequence space (FIG. 14A). These variants packaged efficiently and showed improved heart transduction efficiency in mouse compared to wild-type AAV9 (FIG. 14B). All values are relative to the performance of the wild-type AAV9 control.

[0051] FIG. 15 shows graphs measuring transduction / viral load levels of capsids without an N452K mutation (ZC404, ZC470, ZC428, and ZC416) and with an N452K mutation (ZC373, ZC374, ZC375, and ZC376) in cynomolgus monkey heart and liver, mouse heart and liver, and human iPSCs

[0052] FIG. 16 is a schematic of the design of sequence variants of the AAV9 VR-VIII region. Sequence variants (also called “reconstitution variants”) were generated by reconstitution of various combinations of short sequence seeds, some of which contain substitutions, into full sequences at the AAV9 VR-VIII site (581 to 594, based on VP1 numbering).

[0053] FIG. 17 shows heart transduction efficiencies (relative to the weighted average of all capsids in the pooled study) of top AAV9 VR-VIII reconstitution variants, measured in C57BL / 6 and CD-I mouse strains.DETAILED DESCRIPTION

[0054] The disclosure provides engineered capsid proteins and recombinant adeno- associated virus (rAAV) virions comprising the same. The disclosure also provides methods of making and using engineered capsid proteins and rAAV virions.

[0055] In some aspects, provided are engineered capsid proteins, e.g., AAV capsid proteins, comprising a variant polypeptide relative to a corresponding wild-type or parental sequence. Capsid proteins are structural proteins that make up the assembled icosahedral packaging of an AAV virion and largely determine the immunogenicity and tropism of the virus.In some embodiments, the engineered capsid protein comprises one or more amino acid substitutions relative to a corresponding wild-type or parental sequence (also referred to as “substitution motifs”). In some embodiments, the variant polypeptide is in the VR-VIII site. In some embodiments, the reference wild-type or parental capsid protein is of any serotype known in the field or described herein, including, for example, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rh.10, rh.20, rh.74, and any chimeric or mosaic variant derived therefrom.

[0056] In some embodiments, provided herein is an engineered AAV9 capsid protein. In some embodiments, provided herein is an engineered AAV5 capsid protein. In some embodiments, provided herein is an engineered AAVrh.74 capsid protein. In some embodiments, provided herein is an engineered AAVrh.10 capsid protein. In some embodiments, provided herein is a chimeric AAV5 / AAV9 capsid protein.

[0057] In some embodiments, the present disclosure provides recombinant adeno- associated virus (rAAV) virions comprising: a) an engineered capsid protein as described herein; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products.

[0058] In some embodiments, provided herein is an rAAV virion comprising any engineered AAV9 capsid protein disclosed herein. In some embodiments, provided here in is an rAAV virion comprising any engineered AAV5 capsid protein disclosed herein. In some embodiments, provided herein is an rAAV virion comprising any engineered AAVrh.74 capsid protein disclosed herein. In some embodiments, provided herein is an rAAV virion comprising any engineered AAVrh.10 capsid protein disclosed herein. In some embodiments, provided herein is an rAAV virion comprising any chimeric capsid protein disclosed herein. In some embodiments, provided herein is an rAAV virion comprising any chimeric AAV5 / AAV9 capsid protein disclosed herein.

[0059] In some embodiments, an rAAV virion described herein transduces a cardiac cell or cardiac tissue. In some embodiments, an rAAV virion described herein trafficks to the heart. In some embodiments, an rAAV virion described herein transduces cardiac cells or tissue better than wild type parental AAV (e.g., wild type AAV9).

[0060] In some embodiments, an rAAV virion described herein exhibits decreased transduction of liver cells or liver tissue. In some embodiments, an rAAV virion described herein has increased transduction of heart and decreased transduction of liver, i.e., exhibits selectivity for the heart over liver or high heart-to-liver ratio (e.g., more than 2x, 3x, 4x, 5x orlOx). In some embodiments, an rAAV virion described herein transduces liver less than wild type parental AAV (e.g., wild type AAV9).

[0061] In some embodiments, the recombinant adeno-associated virus (rAAV) virion comprises a vector genome comprising an expression cassette flanked by inverted terminal repeats (ITRs). In some embodiments, the recombinant AAV virion transduces heart cells and / or cardiomyocytes, traffics to at least one organ other than the liver, traffics to the heart, and / or exhibits a higher transduction efficiency than an rAAV virion having a wild-type AAV9 VP1 capsid protein. In some embodiments, the rAAV virion comprises a polynucleotide cassette comprising a polynucleotide sequence, optionally encoding a protein.

[0062] In some embodiments, provided herein is a kit comprising a pharmaceutical composition or an rAAV described herein, and optionally instructions for use.

[0063] In some embodiments, provided herein is a pharmaceutical composition comprising any rAAV virion described herein and a pharmaceutically acceptable carrier or excipient.

[0064] In some embodiments, provided herein is a kit comprising a pharmaceutical composition or an rAAV described herein, and optionally instructions for use.

[0065] In some embodiments, provided herein is a polynucleotide encoding any engineered capsid protein described herein.

[0066] In some embodiments, provided herein is a method of transducing a cardiac cell, comprising contacting the cardiac cell with any rAAV virion described herein.

[0067] In some embodiments, provided herein is a method of delivering one or more gene products to a cardiac cell comprising contacting the cardiac cell with any rAAV virion described herein.

[0068] In some embodiments, provided herein is a method of treating cardiac pathology, or a heart disease or condition, in a subject in need thereof, comprising administering any rAAV virion described herein. In some embodiments, the subject is a human..Variant Polypeptides

[0069] In some embodiments, the engineered capsid proteins described herein comprise one or more variant polypeptides relative to a wild-type or parental capsid protein sequence. The variant polypeptides described herein comprise one or more substitutions or substitution motifs. The wild-type or parental capsid protein can be any wild-type, chimeric, or mosaic capsid protein as described herein or as known in the art, or a variant thereof.

[0070] In some embodiments, the non-naturally occurring amino acid motifs described herein comprise substitution of one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids in the wild-type or parental AAV capsid protein.

[0071] In some embodiments, the engineered AAV capsid protein provided herein comprises a variant polypeptide sequence at the VR-VIII site of the capsid. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the capsid protein comprises any one or more substitutions or substitution motifs described herein. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least six substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least eight substitutions at the VR-VIII site relative to the parental sequence at the VR-VIII site.

[0072] In some embodiments, the parental sequence is the AAV9 VP1 parental sequence (SEQ ID NO: 1). In some embodiments, the parental sequence is the AAV5 VP1 parental sequence (SEQ ID NO: 322). In some embodiments, the parental sequence is the AAVrh.10 VP1 parental sequence (SEQ ID NO: 329). In some embodiments, the parental sequence is the AAVrh.74 VP1 parental sequence (SEQ ID NO: 336). In some embodiments, the variant polypeptide sequence occurs only at the VR-VIII site.Substitution motifs

[0073] In some embodiments, the engineered capsid proteins described herein comprise a variant polypeptide comprising one or more amino acid substitutions relative to a wild-type or parental capsid protein described herein (referred to herein as a “substitution motif’). In some embodiments, the wild-type or parental capsid protein is an AAV9, AAV5, AAVrh.10, or AAVrh.74 capsid. In some embodiments, the substitution motif does not comprise amino acid insertions.

[0074] In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, or ten positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at two positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acidsubstitution at three positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at four positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at five positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at six positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at seven positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at eight positions relative to a wild-type AAV capsid protein sequence. In some embodiments, the engineered capsid protein comprises a substitution motif comprising an amino acid substitution at one, two, three, four, five, six, seven, or eight of the following positions relative to a wild-type AAV9 capsid protein sequence.VR-VIII Substitution Motifs

[0075] In some embodiments, the engineered capsid comprises a variant polypeptide in the VR-VII site. In some embodiments, the engineered capside comprises a variant polypeptide at positions 581-594 of the capsid protein, wherein the amino acid numbering is according to the AAV9 VP1 sequence of SEQ ID NO: 1. A person of skill in the art will recognize the equivalent positions in other AAV serotype capsid proteins, e.g., positions 570-583 of the AAV5 VP1 capsid sequence of SEQ ID NO: 322, positions 583-596 of the AAVrh.10 VP1 capsid sequence of SEQ ID NO: 329, and positions 583-596 of the AAVrh.74 VP1 capsid sequence of SEQ ID NO: 336. The variant polypeptide may comprise substitutions across the entire VR-VIII site (e.g., between positions 581-594) or may comprise substitutions in a portion of the VR-VIII site (e.g., between positions 582-591).

[0076] In some embodiments, the variant polypeptide comprises one or more substitutions (e.g., a substitution motif). In some embodiments, the non-naturally occurring amino acid motif comprises a substitution motif and does not comprise an insertion motif.

[0077] In some embodiments, provided herein is an engineered capsid protein comprising an amino acid sequence X1DVQX2X3PGFX4X5X6X7X8 in the VR-VIII site, wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 303). In some embodiments, X5 is A or P. In some embodiments, Xe is Q or E. In some embodiments, X5 is A or P, and Xe is Q or E.

[0078] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising:X1DVQX2X3PGFX4X5X6X7X8, wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 303). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; X7 is T; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments of the variant polypeptide sequence, X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; X7 is T; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N (SEQ ID NO: 1034). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L or H; X4 is Q; X5 is A; Xe is Q; X7 is T; and Xs is E.

[0079] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising:X1DVQX2X3PGFX4X5X6X7X8 wherein each of Xi, X2, X3, X4, X7, and Xs is any amino acid (SEQ ID NO: 303). In some embodiments, X5 is A or P. In some embodiments, Xe is Q or E. In some embodiments, at least one, at least two, at least three, at least four, at least five, or at least six other X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X7 is T; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0080] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising:X1DVQX2X3PGFX4X5X6X7X8 wherein X3 is Leucine (L) or Histidine (H) and Xi, X2, X4, X5, Xe, X7, and Xs are any amino acid (SEQ ID NO: 319). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X4 is Q, G, R, N, H, M, P, or S; X5 is A or P; Xe is Q or E; X7 is T; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments of the variant polypeptide sequence, X amino acids are selected from the group consisting of: Xi is A;X2is Q; X3is L or H; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; X7is T; and X8is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N (SEQ ID NO: 1067). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X4is Q; X5 is A; Xe is Q; X7 is T; and X8is E.

[0081] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising:X1DVQX2X3PGFX4X5X6X7E wherein each of Xi, X2, X3, X4, X5, Xe, and X7is any amino acid (SEQ ID NO: 306). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; and X7 is T. In some embodiments of the variant polypeptide sequence, X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; and X7 is T (SEQ ID NO: 1035). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L or H; X4is Q; X5 is A; Xe is Q; and X7 is T.

[0082] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising:ADVQX2X3PGFX4X5X6X7X8wherein each of X2, X3, X4, X5, Xe, X7, and X8is any amino acid (SEQ ID NO: 304). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; X7 is T; and X8is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments of the variant polypeptide sequence, X amino acids are selected from the group consisting of: X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; X7is T; and X8is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N (SEQ ID NO: 1036). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: X2 is Q; X3 is L or H; X4is Q; X5 is A; Xe is Q; X7 is T; and X8is E.

[0083] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising:X1DVQQX3PGFX4X5X6X7X8 wherein each of Xi, X3, X4, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 305). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; X7 is T; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments of the variant polypeptide sequence, X amino acids are selected from the group consisting of: Xi is A; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; X7is T; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N (SEQ ID NO: 1037). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X3 is L or H; X4 is Q; X5 is A; Xe is Q; X7 is T; and Xs is E.

[0084] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising:X1DVQX2X3PGFX4X5X6TX8 wherein each of Xi, X2, X3, X4, X5, Xe, and Xs is any amino acid (SEQ ID NO: 310). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments of the variant polypeptide sequence, X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N (SEQ ID NO: 1038). In some embodiments of the variant polypeptide sequence, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L or H; X4 is Q; X5 is A; Xe is Q; and Xs is E.

[0085] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: ADVQQX3PGFX4X5X6TX8, wherein each of X3, X4, X5, Xe, and Xs is any amino acid (SEQ ID NO: 311).

[0086] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: ADVQQX3PGFX4X5X6TE, wherein each of X3, X4, X5, and Xe is any amino acid (SEQ ID NO: 313).

[0087] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: X1DVQX2X3PGFQX5X6X7X8, wherein each of Xi, X2, X3, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 307).

[0088] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: X1DVQX2X3PGFX4AX6X7X8, wherein each of Xi, X2, X3, X4, Xe, X7, and Xs is any amino acid (SEQ ID NO: 308).

[0089] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: X1DVQX2X3PGFX4X5QX7X8, wherein each of Xi, X2, X3, X4, X5, X7, and Xs is any amino acid (SEQ ID NO: 309).

[0090] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: ADVQQX3PGFQAQTX8, wherein each of X3 and Xs is any amino acid (SEQ ID NO: 312).

[0091] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: ADVQQX3PGFX4PETX8, wherein each of X3, X4 and / or Xs is any amino acid (SEQ ID NO: 1032). In some embodiments, X3 is L, H, S, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4 is Q, G, R, N, H, M, P, or S. In some embodiments, Xs is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0092] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: AD VQQX3PGFX4AQTX8, wherein each of X3, X4 and / or Xs is any amino acid (SEQ ID NO: 1033). In some embodiments, X3 is L, H, S, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4 is Q, G, R, N, H, M, P, or S. In some embodiments, Xs is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0093] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: ADVQQX3PGFQAQTE, wherein each of X3 is any amino acid (SEQ ID NO: 314).

[0094] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: ADVQQX3PGFX4X5X6X7X8, wherein each of X3, X4, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 315).

[0095] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: ADVQQX3PGFX4X5X6X7E, wherein each of, X3, X4, X5, Xe, and X7 is any amino acid (SEQ ID NO: 316).

[0096] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: ADVQQLPGFX4X5X6TE, wherein each of X4, X5, and Xe is any amino acid (SEQ ID NO: 317).

[0097] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising: ADVQQHPGFX4X5X6TE, wherein each of X4, X5, and Xe is any amino acid (SEQ ID NO: 318).

[0098] In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises an amino acid sequence selected from X1DVQX2X3PGFX4AQX7X8 (SEQ ID NO: 1039), XDVQX2X3PGFX4AEX7X8 (SEQ ID NO: 1040), X1DVQX2X3PGFX4PQX7X8 (SEQ ID NO: 1041), X1DVQX2X3PGFX4PEX7X8 (SEQ ID NO: 1042), ADVQQX3PGFX4AQTX8 (SEQ ID NO: 1033), ADVQQX3PGFX4AETX8 (SEQ ID NO: 1043), ADVQQX3PGFX4PQTX8 (SEQ ID NO: 1044), or ADVQQX3PGFX4PETX8 (SEQ ID NO: 1032) wherein each of Xi, X2, X3, X4, X7, and X8, if present, is any amino acid. In some embodiments, X3 is selected from L, H, V, C, Q, G, I, M, F, P, T, or Y, X4 is selected from Q, G, R, N, H, M, P, or S, and Xs is selected from E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments, X3 is L and Xs is E.

[0099] In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E (SEQ ID NO: 1045) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 317), wherein each of Xi, X2, X4, X5, Xe, and X7, if present, is any amino acid. In some embodiments, X4 is Q. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises an amino acid sequence selected from the group consisting of: ADVQQLPGFQAQTE (SEQ ID NO: 320), ADVQQLPGFGAQTE (SEQ ID NO: 906), ADVQQLPGFRPETE (SEQ ID NO: 907), ADVQQLPGFNAQTE (SEQ ID NO: 908), ADVQQLPGFQAETE (SEQ ID NO: 951), ADVQQLPGFGAETE (SEQ ID NO: 952),ADVQQLPGFGPQTE (SEQ ID NO: 953), ADVQQLPGFHAQTE (SEQ ID NO: 954),ADVQQLPGFRAQTE (SEQ ID NO: 955), ADVQQLPGFRAETE (SEQ ID NO: 956),ADVQQLPGFMAQTE (SEQ ID NO: 957), ADVQQLPGFPAQTE (SEQ ID NO: 958),ADVQQLPGFSAQTE (SEQ ID NO: 959), ADVQQLPGFSAETE (SEQ ID NO: 960), and ADVQQLPGFRPQTE (SEQ ID NO: 961).

[0100] In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises amino acid sequence X1DVQX2X3PGFQX5X6X7X8 (SEQ ID NO: 307), ADVQQXsPGFQXsXeTXs (SEQ ID NO: 1046), or ADVQQX3PGFQAQTX8 (SEQ ID NO: 312), wherein each of Xi, X2, X3, X5, X6, X7, and X8, if present, is any amino acid. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises amino acid sequence selected from the group consisting of: ADVQQLPGFQAQTE (SEQ ID NO: 320), ADVQQHPGFQAQTE (SEQ ID NO: 321), ADVQQVPGFQAQTM (SEQ ID NO: 904), ADVQQVPGFQAQTQ (SEQ ID NO: 905), ADVQQCPGFQAQTE (SEQ ID NO: 909), ADVQQQPGFQAQTE (SEQ ID NO: 910), ADVQQGPGFQAQTD (SEQ ID NO: 911), ADVQQHPGFQAQTD (SEQ ID NO: 912), ADVQQHPGFQAQTE (SEQ ID NO: 913), ADVQQIPGFQAQTA (SEQ ID NO: 914), ADVQQIPGFQAQTC (SEQ ID NO: 915), ADVQQIPGFQAQTQ (SEQ ID NO: 916), ADVQQIPGFQAQTH (SEQ ID NO: 917), ADVQQIPGFQAQTM (SEQ ID NO: 918), ADVQQLPGFQAQTD (SEQ ID NO: 919), ADVQQLPGFQAQTC (SEQ ID NO: 920), ADVQQLPGFQAQTQ (SEQ ID NO: 921), ADVQQLPGFQAQTE (SEQ ID NO: 922), ADVQQLPGFQAQTM (SEQ ID NO: 923), ADVQQMPGFQAQTD (SEQ ID NO: 924), ADVQQMPGFQAQTE (SEQ ID NO: 925), ADVQQMPGFQAQTL (SEQ ID NO: 926), ADVQQMPGFQAQTF (SEQ ID NO: 927), ADVQQMPGFQAQTY (SEQ ID NO: 928), ADVQQFPGFQAQTA (SEQ ID NO: 929), ADVQQFPGFQAQTQ (SEQ ID NO: 930), ADVQQFPGFQAQTH (SEQ ID NO: 931), ADVQQFPGFQAQTT (SEQ ID NO: 932), ADVQQFPGFQAQTV (SEQ ID NO: 933), ADVQQPPGFQAQTE (SEQ ID NO: 934), ADVQQTPGFQAQTE (SEQ ID NO: 935), ADVQQYPGFQAQTA (SEQ ID NO: 936), ADVQQYPGFQAQTC (SEQ ID NO: 937), ADVQQYPGFQAQTQ (SEQ ID NO: 938), ADVQQYPGFQAQTH (SEQ ID NO: 939), ADVQQYPGFQAQTI (SEQ ID NO: 940), ADVQQYPGFQAQTM (SEQ ID NO: 941), ADVQQYPGFQAQTS (SEQ ID NO: 942), ADVQQYPGFQAQTT (SEQ ID NO: 943), ADVQQYPGFQAQTV (SEQ ID NO: 944), ADVQQVPGFQAQTA (SEQ ID NO: 945), ADVQQVPGFQAQTN (SEQ ID NO: 946), ADVQQVPGFQAQTD (SEQ ID NO: 947), ADVQQVPGFQAQTC (SEQ ID NO: 948), ADVQQVPGFQAQTH (SEQ ID NO: 949), and ADVQQVPGFQAQTL (SEQ ID NO: 950).

[0101] In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises a sequence selected from the DVQ, PGF, DVQQL (SEQ ID NO: 1028), DVQQH (SEQ ID NO: 1029), ADVQQLPGF (SEQ ID NO: 1030), and ADVQQHPGF (SEQ ID NO: 1031). In some embodiments, the variant polypeptide sequenceat the VR-VIII site of the engineered AAV capsid protein comprises amino acid sequence DVQ. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises amino acid sequence PGF. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises amino acid sequence DVQQL (SEQ ID NO: 1028). In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises amino acid sequence DVQQH (SEQ ID NO: 1029). In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises amino acid sequence ADVQQLPGF (SEQ ID NO: 1030). In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV capsid protein comprises amino acid sequence ADVQQHPGF (SEQ ID NO: 1031).

[0102] In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to ADVQQLPGFQAQTE (SEQ ID NO: 320). In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to ADVQQHPGFQAQTE (SEQ ID NO: 321).

[0103] In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises the sequence of ADVQQLPGFQAQTE (SEQ ID NO: 320), or a sequence having not more than one, two, three, four, five or six amino acid substitutions relative to SEQ ID NO: 320 (in some embodiments, the substitutions are in one or more of Xi, X2, X4, X5, Xe, X7, and / or Xs in the following sequence X1DVQX2LPGFX4X5X6X7X8 , wherein each of Xi, X2, X4, X5, Xe, X7, and Xs ia any amino acid (SEQ ID NO: 1048)). In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises the sequence of ADVQQHPGFQAQTE (SEQ ID NO: 321), or a sequence having not more than one, two, three, four, five or six amino acid substitutions relative to SEQ ID NO: 321 (in some embodiments, the substitutions are in one or more of Xi, X2, X4, X5, Xe, X7, and / or Xs in the following sequence X1DVQX2HPGFX4X5X6X7X8, wherein each of Xi, X2, X4, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 1049)).

[0104] In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises a sequence selected from ADVQQLPGFQAQTE (SEQ ID NO: 320) and ADVQQHPGFQAQTE (SEQ ID NO: 321). In some embodiments, the variant polypeptidesequence at the VR-VIII site comprises the sequence of ADVQQLPGFQAQTE (SEQ ID NO: 320). In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises the sequence of ADVQQHPGFQAQTE (SEQ ID NO: 321).

[0105] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site comprising X1X2X3X4X5X6X7X8X9X10, wherein Xi is T or D; X2 is N or V; X3 is H, Q, or R; X4is Q, M, N, or S; X5is S, G, M, T, or L; X6is A, N, K, T, or P; X7 is Q, Y, R, L, T, or G; X8is A, V, or F; X9is Q, R, G, M, or N; and X10 is A or P (SEQ ID NO: 1047). In some embodiments, the variant polypeptide sequence at the VR- VIII site comprises a sequence selected from SEQ ID NOs: 903-983. In some embodiments, the capsid protein is a variant of an AAV5, AAVrh.74, or AAVrh.10 capsid protein.

[0106] In some embodiments, the capsid protein comprises the variant polypeptide sequence selected from the group consisting of: DVQQGAYARA (SEQ ID NO: 1006), DVQQMNRVGA (SEQ ID NO: 1007), DVQMSAQARA (SEQ ID NO: 1008),DVQQTKLAGA (SEQ ID NO: 1009), DVQQGAYAQA (SEQ ID NO: 1010),DVQQSAQARA (SEQ ID NO: 1011), DVQQGAYARP (SEQ ID NO: 1012),DVQMSAQAMA (SEQ ID NO: 1013), DVQQMTTARA (SEQ ID NO: 1014),DVQMSAQAQA (SEQ ID NO: 1015), DVQQMTTAMA (SEQ ID NO: 1016),DVQQMNRVNA (SEQ ID NO: 1017), DVQNSAQARP (SEQ ID NO: 1018),DVQQGAYAMA (SEQ ID NO: 1019), TNRMGAYAQA (SEQ ID NO: 1020),TNRQLPGFQA (SEQ ID NO: 1021), DVQQSAQARP (SEQ ID NO: 1022), DNQQLPGFQA (SEQ ID NO: 1023), TNHSGAYAQA (SEQ ID NO: 1024), DVQMSAQARP (SEQ ID NO: 1025), TNHMGAYAQA (SEQ ID NO: 1026), and DVQQTKLANA (SEQ ID NO: 1027).

[0107] In some embodiments, the disclosure provides an engineered adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV5 VP3 reference sequence according to SEQ ID NO: 324, and wherein the capsid protein comprises at the VR-VIII site the sequence of X1DVQX2X3PGFX4X5X6X7X8 (SEQ ID NO: 303), wherein each of Xi, X2, X3, X4, X7, and X8is any amino acid or any amino acid as specified herein.

[0108] In some embodiments, the disclosure provides an engineeredadeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV5 VP3 reference sequence according to SEQ ID NO: 324, and wherein the capsid protein comprises at the VR-VIII site the sequence of ADVQQHPGFQAQTE (SEQ ID NO: 321). In some embodiments, the disclosure provides arecombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV5 VP3 reference sequence according to SEQ ID NO: 324, and wherein the capsid protein comprises at the VR- VIII site the sequence of AD VQQLPGFQ AQTE (SEQ ID NO: 320).

[0109] In some embodiments, the disclosure provides an engineeredadeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV5 VP3 reference sequence according to SEQ ID NO: 324, and wherein the capsid protein comprises at the VR-VIII site the sequence of X1X2X3X4X5X6X7X8X9X10, wherein Xi is T or D; X2 is N or V; X3 is H, Q, or R; X4is Q, M, N, or S; X5is S, G, M, T, or L; X6is A, N, K, T, or P; X7 is Q, Y, R, L, T, or G; X8is A, V, or F; X9 is Q, R, G, M, or N; and X10 is A or P (SEQ ID NO: 1047). In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises a sequence selected from SEQ ID NOs: 903-983.

[0110] In some embodiments, the disclosure provides an engineered adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises at the VR-VIII site the sequence of X1DVQX2X3PGFX4X5X6X7X8 (SEQ ID NO: 303), wherein each of Xi, X2, X3, X4, X5, X6, X7, and Xs is any amino acid or any amino acid as specified herein.

[0111] In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises at the VR-VIII site the sequence of ADVQQHPGFQAQTE (SEQ ID NO: 321). In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises at the VR- VIII site the sequence of AD VQQLPGFQ AQTE (SEQ ID NO: 320).

[0112] In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAVrh.10 VP3 reference sequence according to SEQ ID NO: 331, and wherein the capsid protein comprises at the VR-VIII site the sequence ofX1DVQX2X3PGFX4X5X6X7X8 (SEQ ID NO: 303), wherein Xi, X3, X4, X5, X6, X7, and / or X8is any amino acid or any amino acid as specified herein.

[0113] In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAVrh.10 VP3 reference sequence according to SEQ ID NO: 331, and wherein the capsid protein comprises at the VR-VIII site the sequence of ADVQQHPGFQAQTE (SEQ ID NO: 321). In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAVrh.10 VP3 reference sequence according to SEQ ID NO: 331, and wherein the capsid protein comprises at the VR- VIII site the sequence of AD VQQLPGFQ AQTE (SEQ ID NO: 320).

[0114] In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAVrh.74 VP3 reference sequence according to SEQ ID NO: 338, and wherein the capsid protein comprises at the VR-VIII site the sequence of X1DVQX2X3PGFX4X5X6X7X8 (SEQ ID NO: 303), wherein each of Xi, X2, X3, X4, X5, X6, X7, and / or Xsis any amino acid or any amino acid as specified herein.

[0115] In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAVrh.74 VP3 reference sequence according to SEQ ID NO: 338, and wherein the capsid protein comprises at the VR-VIII site the sequence of ADVQQHPGFQAQTE (SEQ ID NO: 321). In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAVrh.74 VP3 reference sequence according to SEQ ID NO: 338, and wherein the capsid protein comprises at the VR- VIII site the sequence of AD VQQLPGFQ AQTE (SEQ ID NO: 320).

[0116] In some embodiments, the disclosure provides an engineered adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV5 VP3 reference sequence according to SEQ ID NO: 324, and wherein the capsid protein comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 571 to 580 relative to AAV5 VP1 capsid protein sequence of SEQ ID NO: 329, wherein Xi is T or D; X2 is N or V; X3 is H, Q, or R; X4is Q, M, N, or S; X5is S, G, M, T, or L; X6is A, N, K, T, or P; X7 is Q, Y, R, L, T, or G; X8isA, V, or F; X9 is Q, R, G, M, orN; and X10 is A or P (SEQ ID NO: 1047). In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises a sequence selected from SEQ ID NOs: 903-983.

[0117] In some embodiments, the disclosure provides an engineeredadeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAVrh.74 VP3 reference sequence according to SEQ ID NO: 338, and wherein the capsid protein comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 584 to 593 relative to the AAVrh.74 VP1 capsid protein sequence of SEQ ID NO: 336, wherein Xi is T or D; X2 is N or V; X3 is H, Q, or R; X4is Q, M, N, or S; X5is S, G, M, T, or L; X6is A, N, K, T, or P; X7 is Q, Y, R, L, T, or G; X8is A, V, or F; X9is Q, R, G, M, or N; and X10 is A or P (SEQ ID NO: 1047). In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises a sequence selected from SEQ ID NOs: 903-983.

[0118] In some embodiments, the disclosure provides an engineered adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAVrh.10 VP3 reference sequence according to SEQ ID NO: 331, and wherein the capsid protein comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 584 to 593 relative to AAVrh.10 VP1 capsid protein sequence of SEQ ID NO: 329, wherein Xi is T or D; X2 is N or V; X3 is H, Q, or R; X4 is Q, M, N, or S; X5is S, G, M, T, or L; X6is A, N, K, T, or P; X7 is Q, Y, R, L, T, or G; X8is A, V, or F; X9 is Q, R, G, M, orN; and X10 is A or P (SEQ ID NO: 1047). In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises a sequence selected from SEQ ID NOs: 903-983.

[0119] In some embodiments, the disclosure provides an engineered adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises at the VR-VIII site the sequence of X1X2X3X4X5X6X7X8X9X10, wherein Xi is T or D; X2 is N or V; X3 is H, Q, or R; X4is Q, M, N, or S; X5is S, G, M, T, or L; X6is A, N, K, T, or P; X7 is Q, Y, R, L, T, or G; X8is A, V, or F; X9 is Q, R, G, M, or N; and X10 is A or P (SEQ ID NO: 1047). In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises a sequence selected from SEQ ID NOs: 903-983.Table 1: Exemplary VR-VIII modifications

[0121] Any of the modifications described herein may be used either alone or in combination with other modifications (e.g., in combination with other modifications described herein).

[0122] Any of the VR-VIII modifications or variant VR-VIII site sequences described herein can be combined with any one or more additional modifications in an AAV capsid protein (such as a modification outside of the VR-VIII sequence), including substitution of one or more amino acids, or insertion of one or more amino acids, at any site in the AAV capsid protein (e.g., VR-IV site, VR-V site and / or VR-VII site). In some embodiments, the additional modification in an AAV capsid protein is not in the VR-VIII site of the AAV capsid protein.

[0123] In some embodiments, the engineered AAV capsid protein comprises a variant polypeptide sequence that is either rationally designed, introduced by mutagenesis, or randomized through generating a library of sequences with random codon usage at one or more sites. The AAV capsid proteins of the disclosure include any variant polypeptide sequences identified as enriched by directed evolution followed by sequencing.

[0124] Various amino acid substitutions, insertions, and deletion are provided herein. Any of these modifications may be combined with any of the others, though where the modifications overlap one must be selected or an insertion maybe used to put two modifications adjacent to or near one another. The combination of modifications may be tested, using methods described herein (e.g., in vitro test in iPSC-CMs, in vivo testing in model organisms individually, and in vivo re-screening of pooled rAAV virions) or other known methods to identify combinations having desired packing efficiency, tropism, or other desired properties. Similarly, modifications may be made at exactly the position desired herein, or the same modification may be may at any position near to the described position. Structural modeling of the capsid protein may be used to select modification for testing.

[0125] In some embodiments, the engineered AAV capsid provided comprises any of the VR-VIII-modifications described herein. In some embodiments, the engineered AAV capsid provided herein comprises any of the VR-VIII-modifications described herein, and an additional modification in the VR-IV site, such as any one or more VR-IV modifications described herein or known in the art. In some embodiments, the engineered AAV capsid provided herein comprises any of the VR-VIII-modifications described herein, and an additional modification in the VR-V and / or VR-VII sites, such as any one or more VR-V and VR-VII modifications described herein or known in the art.

[0126] In some embodiments, the variant VP1, VP2 and / or VP3 AAV capsid protein comprises one of the amino acid sequences of Table 2 (e.g., at the VR-VIII site), or comprises one of the amino acid sequences of Table 2 with not more than 1, 2, 3, 4 or 5 amino acid substitutions (e.g., not more than 1, 2, or 3 amino acid substitutions):Table 2: Exemplary variant polypeptide sequences

[0127] In some embodiments, the engineered AAV capsid provided herein is any of the capsids described in any of the examples, tables or figures provided herein.AA V9 Variant Capsid Sequences

[0128] In one aspect, the present disclosure provides an engineered AAV9 capsid protein, wherein the AAV9 capsid protein comprises a variant polypeptide sequence with respect to the parental sequence at one or more sites of the parental sequence, such as any of the variant polypeptide sequences described herein. In some embodiments, the engineered AAV9 capsid protein of the present disclosure comprises a variant polypeptide sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen substitutions at the VR-VIII site relative to the AAV9 capsid protein parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least six substitutions at the VR-VIII site relative to the AAV9 capsid protein parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least eight substitutions at the VR-VIII site relative to the AAV9 capsid protein parental sequence at the VR-VIII site.

[0129] In another aspect, the present disclosure provides an engineered chimeric capsid protein, wherein the chimeric capsid protein comprises AAV9 parental sequence at the VR-VIII site. In some embodiments, the engineered chimeric capsid protein of the present disclosure comprises a variant polypeptide sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen substitutions at the VR-VIII site relative to the AAV9 capsid protein parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least six substitutions at the VR- VIII site relative to the AAV9 capsid protein parental sequence at the VR-VIII site. In some embodiments, the variant polypeptide sequence comprises at least eight substitutions at the VR- VIII site relative to the AAV9 capsid protein parental sequence at the VR-VIII site.

[0130] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:X1DVQX2X3PGFX4X5X6X7X8 wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 303). In some embodiments, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; X7 is T; and X8is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments, X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4 is Q, G, R, N, H, M, P, or S; X5is A or P; X6is Q or E; X7 is T; and X8is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments, at least one, at least two, at least three, or at least four X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L or H; X4 is Q; X5 is A; Xe is Q; X7 is T; and X8is E. In some embodiments, 3 is L or H, and at least one, at least two, at least three, or at least four other X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X4 is Q; X5 is A; Xe is Q; X7 is T; and X8is E.

[0131] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:X1DVQX2X3PGFX4X5X6X7X8wherein each of Xi, X2, X3, X4, X7, and X8is any amino acid (SEQ ID NO: 303). In some embodiments, X5 is A or P. In some embodiments, Xe is Q or E. In some embodiments, at least one, at least two, at least three, at least four, at least five, or at least six other X amino acids are selected from the group consisting of: Xi is A; X2 is Q; X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4is Q, G, R, N, H, M, P, or S; X7 is T; and X8is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

[0132] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:ADVQX2X3PGFX4X5X6X7X8wherein each of X2, X3, X4, X5, Xe, X7, and X8is any amino acid (SEQ ID NO: 304).

[0133] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:X1DVQQX3PGFX4X5X6X7X8 wherein each of Xi, X3, X4, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 305).

[0134] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:X1DVQX2X3PGFX4X5X6X7E wherein each of Xi, X2, X3, X4, X5, Xe, and X?is any amino acid (SEQ ID NO: 306).

[0135] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:X1DVQX2X3PGFQX5X6X7X8 wherein each of Xi, X2, X3, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 307).

[0136] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:X1DVQX2X3PGFX4AX6X7X8 wherein each of Xi, X2, X3, X4, Xe, X7, and Xs is any amino acid (SEQ ID NO: 308).

[0137] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:X1DVQX2X3PGFX4X5QX7X8 wherein each of Xi, X2, X3, X4, X5, X7, and Xs is any amino acid (SEQ ID NO: 309).

[0138] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:X1DVQX2X3PGFX4X5X6TX8 wherein each of Xi, X2, X3, X4, X5, Xe, and Xs is any amino acid (SEQ ID NO: 310).

[0139] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:ADVQQX3PGFX4X5X6TX8 wherein each of X3, X4, X5, Xe, and Xs is any amino acid (SEQ ID NO: 311).

[0140] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:ADVQQX3PGFX4PETX8 wherein each of X3, X4 and / or Xs is any amino acid (SEQ ID NO: 1032). In some embodiments, X3 is L, H, S, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4is Q, G, R, N, H, M, P, or S. In some embodiments, Xs is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, N.

[0141] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:ADVQQX3PGFX4AQTX8 wherein each of X3, X4 and / or Xs is any amino acid (SEQ ID NO: 1033). In some embodiments, X3 is L, H, S, V, C, Q, G, I, M, F, P, T, or Y. In some embodiments, X4is Q, G, R, N, H, M, P, or S. In some embodiments, Xs is E, G, M, Q, D, L, A, C, H, F, Y, T, V, I, S, N.

[0142] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:ADVQQX3PGFQAQTX8 wherein each of X3 and Xs is any amino acid (SEQ ID NO: 312).

[0143] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:ADVQQX3PGFX4X5X6TE wherein each of X3, X4, X5, and Xe is any amino acid (SEQ ID NO: 313).

[0144] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula:ADVQQX3PGFQAQTE wherein each of X3 is any amino acid (SEQ ID NO: 314).

[0145] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula: AD VQQX3PGFX4X5X6X7X8, wherein each of X3, X4, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 315).

[0146] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula: ADVQQX3PGFX4X5X6X7E, wherein each of, X3, X4, X5, Xe, and X7 is any amino acid (SEQ ID NO: 316).

[0147] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula: ADVQQLPGFX4X5X6TE, wherein each of X4, X5, and Xe is any amino acid (SEQ ID NO: 317).

[0148] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula: ADVQQHPGFX4X5X6TE, wherein each of X4, X5, and Xe is any amino acid (SEQ ID NO: 318).

[0149] In some embodiments, the amino acids at positions 581-594 (relative to reference sequence of SEQ ID NO: 1) of the AAV9 VR-VIII site (“ATNHQSAQAQAQTG” SEQ ID NO: 6) are substituted by a peptide of formula: X1DVQX2X3PGFX4X5X6X7X8, wherein X3 is Leucine (L) or Histidine (H) and Xi, X2, X4, X5, Xe, X7, and Xs are any amino acid (SEQ ID NO: 319).

[0150] In some embodiments, the engineered AAV9 capsid protein comprises one or more amino acid substitutions selected from the group consisting of A581R, A581N, A581D, A581C, A581Q, A581E, A581G, A581H, A581I, A581L, A581K, A581M, A581F, A581P, A581O, A581S, A581T, A581W, A581Y, A581V, T582D, N583V, H584Q, Q585T, Q585C, Q585V, Q585L, Q585N, Q585S, Q585P, Q585A, Q585M, Q585E, Q585Y, Q585G, Q585H, Q585I, Q585R, Q585D, Q585K, Q585F, Q585O, Q585, S586D, S586T, S586G, S586K, S586M, S586N, S586I, S586Q, S586L, S586P, S586F, S586R, S586A, S586C, S586E, S586H, S586O, S586W, S586Y, S586V, A587P, Q588G, A589F, Q590I, Q590S, Q590N, Q590G, Q590D, Q590R, Q590H, Q590T, Q590M, Q590F, Q590Y, Q590L, Q590A, Q590C, Q590E, Q590K, Q590P, Q590O, Q590W, Q590V, A591I, A591R, A591N, A591D, A591C, A591Q, A591E,A591G, A591H, A591 L, A591K, A591M, A591F, A591P, A591O, A591S, A591T, A591W, A591Y, A591V, Q592I, Q592R, Q592N, Q592D, Q592C, Q592A, Q592E, Q592G, Q592H, Q592 L, Q592K, Q592M, Q592F, Q592P, Q592O, Q592S, Q592T, Q592W, Q592Y, Q592V, T593I, T593R, T593N, T593D, T593C, T593A, T593E, T593G, T593H, T593L, T593K, T593M, T593F, T593P, T593O, T593S, T593Q, T593W, T593Y, T593V, G594I, G594R, G594N, G594D, G594C, G594A, G594E, G594Q, G594H, G594 L, G594K, G594M, G594F, G594P, G594O, G594S, G594T, G594W, G594Y, and G594V.

[0151] In some embodiments, the capsid protein comprises an amino acid substitution at position 582 consisting of T582D. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises T582D substitution (z.e., D at position 582) relative to reference sequence SEQ ID NO: 1.

[0152] In some embodiments, the capsid protein comprises an amino acid substitution at position 583 consisting of N583V. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises N583V substitution (z.e., V at position 583) relative to reference sequence SEQ ID NO: 1.

[0153] In some embodiments, the capsid protein comprises an amino acid substitution at position 584 consisting of H584Q. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises H584Q substitution (z.e., Q at position 584) relative to reference sequence SEQ ID NO: 1.

[0154] In some embodiments, the capsid protein comprises an amino acid substitution at position 586 selected from the group consisting of: S586L, S586H, S586V, S586C, S586Q, S586G, S586I, S586M, S586F, S586P, S586T, and S586Y.

[0155] In some embodiments, the capsid protein comprises an amino acid substitution at position 586 consisting of S586L. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises S586L substitution (z.e., L at position 586) relative to reference sequence SEQ ID NO: 1.

[0156] In some embodiments, the capsid protein comprises an amino acid substitution at position 586 consisting of S586H. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises S586H substitution (z.e., H at position 586) relative to reference sequence SEQ ID NO: 1.

[0157] In some embodiments, the capsid protein comprises an amino acid substitution at position 587 consisting of A587P. In some embodiments, the variant polypeptide sequence atthe VR-VIII site comprises A587P substitution (z.e., P at position 587) relative to reference sequence SEQ ID NO: 1.

[0158] In some embodiments, the capsid protein comprises an amino acid substitution at position 588 consisting of Q588G. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q588G substitution (z.e., G at position 588) relative to reference sequence SEQ ID NO: 1.

[0159] In some embodiments, the capsid protein comprises an amino acid substitution at position 589 consisting of A589F. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises A589F substitution (z.e., F at position 589) relative to reference sequence SEQ ID NO: 1.

[0160] In some embodiments, the capsid protein comprises an amino acid substitution at position 590 selected from the group consisting of: Q590G, Q590R, Q590N, Q590H, Q590M, Q590P, and Q590S.

[0161] In some embodiments, the capsid protein comprises an amino acid substitution at position 591 consisting of A591P. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises A591P substitution (z.e., P at position 591) relative to reference sequence SEQ ID NO: 1.

[0162] In some embodiments, the capsid protein comprises an amino acid substitution at position 592 consisting of Q592E. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q592E substitution (z.e., E at position 592) relative to reference sequence SEQ ID NO: 1.

[0163] In some embodiments, the capsid protein comprises an amino acid substitution at position 594 selected from the group consisting of: G594E, G594M, G594Q, G594D, G594L, G594A, G594C, G594H, G594F, G594Y, G594T, G594V, G594I, G594S, and G594N.

[0164] In some embodiments, the capsid protein comprises an amino acid substitution at position 594 consisting of G594E. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises G594E substitution (z.e., E at position 594) relative to reference sequence SEQ ID NO: 1.

[0165] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five or six) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, A587P, Q588G, and A589F, relative to reference sequence SEQ ID NO: 1.

[0166] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, six or seven) amino acid substitutions selected from the group consisting of S586L, T582D, N583V, H584Q, A587P, Q588G, and A589F, relative to reference sequence SEQ ID NO: 1.

[0167] In some embodiments, the capsid protein may comprise one or more (e.g., three, four, five, six or seven) amino acid substitutions selected from the group consisting of S586H, T582D, N583V, H584Q, A587P, Q588G, and A589F, relative to reference sequence SEQ ID NO: 1.

[0168] In some embodiments, the capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected from the group consisting of S586L, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 1.

[0169] In some embodiments, the capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected from the group consisting of S586H, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 1.

[0170] In some embodiments, the capsid protein may comprise one or more (e.g., three, four, five, six, seven, or eight) amino acid substitutions selected from the group consisting of T582D, N583 V, H584Q, A587P, Q588G, A589F, A591P, Q592E relative to reference sequence SEQ ID NO: 1.

[0171] In some embodiments, the capsid protein may comprise one or more (e.g., two, three, four, five, six, seven, eight, nine or 10) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586L, S586H, S586V, S586C, S586Q, S586G, S586I, S586M, S586F, S586P, S586T, S586Y, A587P, Q588G, A589F, Q590G, Q590R, Q590N, Q590H, Q590M, Q590P, Q590S, A591P, Q592E, G594E, G594M, G594Q, G594D, G594L, G594A, G594C, G594H, G594F, G594Y, G594T, G594V, G594I, G594S, G594N relative to reference sequence SEQ ID NO: 1.

[0172] In some embodiments, the capsid protein may comprise one, two, three, four, five, six, seven or all of the VR-VIII modifications of any of the following engineered AAV9 capsid protein described herein and in Table 3, relative to reference sequence SEQ ID NO: 1. In some embodiments, the capsid protein may comprise the VR-VIII sequence of any one of the capsid proteins listed below. In some embodiments, the capsid protein provided herein comprises thefull length sequence of any of the capsid proteins listed below (the full length sequences of which are provided herein).Table 3: Exemplary VR-VIII modifications

[0173] In some embodiments, the variant VP1, VP2 and / or VP3 capsid protein comprises the VR-VIII site positions 581-594 of any one of the below-noted capsids, relative to SEQ ID NO: 1, or the VR-VIII site positions 581-594 of any one of the below-noted capsids with not more than 1, 2, 3, 4 or 5 amino acid substitutions (e..g, not more than 1, 2, or 3 substitutions). The full sequences of such capsids are provided herein, each of which comprises the AAV9 capsid sequence with the below-noted VR-VIII modification in Table 4 and 5.

[0174] In some embodiments, the variant VP1, VP2 and / or VP3 capsid protein comprises one or more (or all) of the amino acids at the VR-VIII site positions 581-594, relative to SEQ ID NO: 1 (e.g., where position 586 comprises L or H, and position 594 comprises E or G) as shown in Table 4 below.TABLE 4: Summary of Substitution Motifs for VR-VIII site

[0175] In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises an amino acid sequence selected from X1DVQX2X3PGFX4AQX7X8 (SEQ ID NO: 1039), X1DVQX2X3PGFX4AEX7X8 (SEQ ID NO: 1040), X1DVQX2X3PGFX4PQX7X8 (SEQ ID NO: 1041), X1DVQX2X3PGFX4PEX7X8 (SEQ ID NO: 1042), ADVQQX3PGFX4AQTX8 (SEQ ID NO: 1033), ADVQQX3PGFX4AETX8 (SEQ ID NO: 1043), ADVQQX3PGFX4PQTX8 (SEQ ID NO: 1044), or ADVQQX3PGFX4PETX8 (SEQ ID NO: 1032) wherein each of Xi, X2, and / or X7 is independently any amino acid. In some embodiments, X3 is selected from L, H, V, C, Q, G, I, M, F, P, T, or Y, X4 is selected from Q, G, R, N, H, M, P, or S, and Xs is selected from E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N. In some embodiments, X3 is L and Xs is E. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E or ADVQQLPGFX4X5X6TE, wherein each of Xi, X2, X4, X5, Xe, and / or X7 is any amino acid (SEQ ID NO: 1045 or SEQ ID NO: 317). In some embodiments, X4 is Q. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 320, 906-908, and 951-961. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineeredAAV9 capsid protein comprises amino acid sequence X1DVQX2X3PGFQX5X6X7X8 (SEQ ID NO: 307), ADVQQX3PGFQX5X6TX8 (SEQ ID NO: 1046), or ADVQQX3PGFQAQTX8 (SEQ ID NO: 312), wherein each of Xi, X2, , X3, X5, Xe, X7 and / or Xs is any amino acid. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 320, 321, 904, 905, and 909-950.

[0176] In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises a sequence selected from the DVQ, PGF, DVQQL (SEQ ID NO: 1028), DVQQH (SEQ ID NO: 1029), ADVQQLPGF (SEQ ID NO: 1030), and ADVQQHPGF (SEQ ID NO: 1031). In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises amino acid sequence DVQ. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises amino acid sequence PGF. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises amino acid sequence DVQQL. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises amino acid sequence DVQQH. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises amino acid sequence ADVQQLPGF. In some embodiments, the variant polypeptide sequence at the VR-VIII site of the engineered AAV9 capsid protein comprises amino acid sequence ADVQQHPGF.

[0177] In some embodiments of the engineered AAV9 capsid protein, the variant polypeptide sequence at the VR-VIII site comprises a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to ADVQQLPGFQAQTE (SEQ ID NO: 320). In some embodiments of the engineered AAV9 capsid protein, the variant polypeptide sequence at the VR-VIII site comprises a sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to ADVQQHPGFQAQTE (SEQ ID NO: 321).

[0178] In some embodiments, the variant polypeptide sequence of an engineered AAV9 capsid protein comprises the sequence of ADVQQLPGFQAQTE (SEQ ID NO: 320), or a sequence having not more than one, two, three, four, five or six amino acid substitutions relative to SEQ ID NO: 320 (in some embodiments, the substitutions are in one or more of Xi, X2, X4, X5, Xe, X7, and / or Xs in the following sequence X1DVQX2LPGFX4X5X6X7X8, wherein each ofXi, X2, X4, X5, Xe, X7, and Xs ia any amino acid (SEQ ID NO: 1048)). In some embodiments, the variant polypeptide sequence of an engineered AAV9 capsid protein comprises the sequence of ADVQQHPGFQAQTE (SEQ ID NO: 321), or a sequence having not more than one, two, three, four, five or six amino acid substitutions relative to SEQ ID NO: 321 (in some embodiments, the substitutions are in one or more of Xi, X2, X4, X5, Xe, X7, and / or Xs in the following sequence X1DVQX2HPGF X4X5X6X7X8).

[0179] In some embodiments of the engineered AAV9 capsid protein, the variant polypeptide sequence at the VR-VIII site comprises a sequence selected from ADVQQLPGFQAQTE (SEQ ID NO: 320) and ADVQQHPGFQAQTE (SEQ ID NO: 321). In some embodiments of the engineered AAV9 capsid protein, the variant polypeptide sequence at the VR-VIII site comprises the sequence of ADVQQLPGFQAQTE (SEQ ID NO: 320). In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises the sequence of ADVQQHPGFQAQTE (SEQ ID NO: 321).

[0180] In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, 4 or 5 amino-acid substitutions relative to SEQ ID NO: 320 or SEQ ID NO: 321. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, 4 or 5 conservative aminoacid substitutions relative to SEQ ID NO: 320 or SEQ ID NO: 321. In some embodiments, the variant polypeptide sequence at the VR-VIII site is SEQ NO: 320 or SEQ ID NO: 321.

[0181] In some embodiments, the engineered AAV9 capsid protein comprises a variant polypeptide sequence comprising, or the variant polypeptide sequence at the VR-VIII site is X1X2X3X4X5X6X7X8X9X10, wherein a) Xi is T or D; b) X2is N or V; c) X3 is H, Q, or R; d) X4is Q, M, N, or S; e) X5 is S, G, M, T, or L; f) X6is A, N, K, T, or P; g) X7is Q, Y, R, L, T, or G; h) Xs is A, V, or F; i) X9 is Q, R, G, M, or N; and j) X10 is A or P (SEQ ID NO: 1047).

[0182] In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein that shares at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity to the AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 582 to 591 relative to AAV9 VP1 capsid protein sequence, wherein a) Xi is T or D; b) X2is N or V; c) X3 is H, Q, or R; d) X4is Q, M, N, or S; e) X5is S, G, M, T, or L; f) X6is A, N, K, T, or P; g) X7is Q, Y, R, L, T, or G; h) Xs is A, V, or F; i) X9 is Q, R, G, M, or N; and j) X10 is A or P (SEQ ID NO: 1047).

[0183] In some embodiments, the capsid protein comprises the variant polypeptide sequence selected from any one of SEQ ID NOs: 1006-1027.

[0184] In some embodiments, the capsid protein is a variant of AAV9 capsid protein, and comprises the amino acid sequence of any one of SEQ ID NOs: 962-983 at amino acid positions 581 to 594 relative to the AAV9 VP1 capsid protein sequence of SEQ ID NO: 1.

[0185] In some embodiments, the engineered AAV9 capsid protein comprises one or more amino acid substitutions selected from the group consisting of A581R, A581N, A581D, A581C, A581Q, A581E, A581G, A581H, A581I, A581L, A581K, A581M, A581F, A581P, A581O, A581S, A581T, A581W, A581Y, A581V, T582D, N583V, H584Q, H584R, Q585T, Q585C, Q585V, Q585L, Q585N, Q585S, Q585P, Q585A, Q585M, Q585E, Q585Y, Q585G, Q585H, Q585I, Q585R, Q585D, Q585K, Q585F, Q585O, Q585, S586D, S586T, S586G, S586K, S586M, S586N, S586I, S586Q, S586L, S586P, S586F, S586R, S586A, S586C, S586E, S586H, S586O, S586W, S586Y, S586V, A587P, A587N, A587K, A587T, Q588G, Q588Y, Q588R, Q588L, Q588T, A589V, A589F, Q590I, Q590S, Q590N, Q590G, Q590D, Q590R, Q590H, Q590T, Q590M, Q590F, Q590Y, Q590L, Q590A, Q590C, Q590E, Q590K, Q590P, Q590O, Q590W, Q590V, A591I, A591R, A591N, A591D, A591C, A591Q, A591E, A591G, A591H, A591 L, A591K, A591M, A591F, A591P, A591O, A591S, A591T, A591W, A591Y, A591V,Q592I, Q592R, Q592N, Q592D, Q592C, Q592A, Q592E, Q592G, Q592H, Q592 L, Q592K, Q592M, Q592F, Q592P, Q592O, Q592S, Q592T, Q592W, Q592Y, Q592V, T593I, T593R, T593N, T593D, T593C, T593A, T593E, T593G, T593H, T593L, T593K, T593M, T593F, T593P, T593O, T593S, T593Q, T593W, T593Y, T593V, G594I, G594R, G594N, G594D, G594C, G594A, G594E, G594Q, G594H, G594 L, G594K, G594M, G594F, G594P, G594O, G594S, G594T, G594W, G594Y, and G594V.

[0186] In some embodiments, the engineered AAV9 capsid protein comprises one or more amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, H584R, Q585M, Q585N, Q585S, S586G, S586M, S586T, S586L, A587N, A587K, A587T, A587P, Q588Y, Q588R, Q588L, Q588T, Q588G, A589V, A589F, Q590R, Q590G, Q590M, Q590N, and A591P.

[0187] In some embodiments, the capsid protein comprises an amino acid substitution at position 582 consisting of T582D. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises T582D substitution (i.e., D at position 582) relative to reference sequence SEQ ID NO: 1.

[0188] In some embodiments, the capsid protein comprises an amino acid substitution at position 583 consisting of N583V. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises N583V substitution (i.e., V at position 583) relative to reference sequence SEQ ID NO: 1.

[0189] In some embodiments, the capsid protein comprises an amino acid substitution at position 584 selected from H584Q and H584R. In some embodiments, the capsid protein comprises an amino acid substitution at position 584 consisting of H584Q. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises H584Q substitution (i.e., Q at position 584) relative to reference sequence SEQ ID NO: 1. In some embodiments, the capsid protein comprises an amino acid substitution at position 584 consisting of H584R. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises H584R substitution (i.e., R at position 584) relative to reference sequence SEQ ID NO: 1.

[0190] In some embodiments, the capsid protein comprises an amino acid substitution at position 585 selected from Q585M, Q585N, and Q585S. In some embodiments, the capsid protein comprises an amino acid substitution at position 585 consisting of Q585M. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q585Msubstitution (i.e., M at position 585) relative to reference sequence SEQ ID NO: 1. In some embodiments, the capsid protein comprises an amino acid substitution at position 585 consisting of Q585N. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q585N substitution (i.e., N at position 585) relative to reference sequence SEQ ID NO: 1. In some embodiments, the capsid protein comprises an amino acid substitution at position 585 consisting of Q585S. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q585S substitution (i.e., S at position 585) relative to reference sequence SEQ ID NO: 1.

[0191] In some embodiments, the capsid protein comprises an amino acid substitution at position 586 selected from the group consisting of: S586G, S586M, S586T, and S586L.

[0192] In some embodiments, the capsid protein comprises an amino acid substitution at position 586 consisting of S586G. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises S586G substitution (i.e., G at position 586) relative to reference sequence SEQ ID NO: 1.

[0193] In some embodiments, the capsid protein comprises an amino acid substitution at position 586 consisting of S586M. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises S586M substitution (i.e., M at position 586) relative to reference sequence SEQ ID NO: 1.

[0194] In some embodiments, the capsid protein comprises an amino acid substitution at position 586 consisting of S586T. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises S586T substitution (i.e., T at position 586) relative to reference sequence SEQ ID NO: 1.

[0195] In some embodiments, the capsid protein comprises an amino acid substitution at position 586 consisting of S586L. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises S586L substitution (i.e., L at position 586) relative to reference sequence SEQ ID NO: 1.

[0196] In some embodiments, the capsid protein comprises an amino acid substitution at position 587 selected from the group consisting of: A587N, A587K, A587T, and A587P.

[0197] In some embodiments, the capsid protein comprises an amino acid substitution at position 587 consisting of A587N. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises A587N substitution (i.e., N at position 587) relative to reference sequence SEQ ID NO: 1.

[0198] In some embodiments, the capsid protein comprises an amino acid substitution at position 587 consisting of A587K. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises A587K substitution (i.e., K at position 587) relative to reference sequence SEQ ID NO: 1.

[0199] In some embodiments, the capsid protein comprises an amino acid substitution at position 587 consisting of A587T. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises A587T substitution (i.e., T at position 587) relative to reference sequence SEQ ID NO: 1.

[0200] In some embodiments, the capsid protein comprises an amino acid substitution at position 587 consisting of A587P. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises A587P substitution (i.e., P at position 587) relative to reference sequence SEQ ID NO: 1.

[0201] In some embodiments, the capsid protein comprises an amino acid substitution at position 588 selected from the group consisting of: Q588Y, Q588R, Q588L, Q588T, and Q588G.

[0202] In some embodiments, the capsid protein comprises an amino acid substitution at position 588 consisting of Q588R. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q588R substitution (i.e., R at position 588) relative to reference sequence SEQ ID NO: 1.

[0203] In some embodiments, the capsid protein comprises an amino acid substitution at position 588 consisting of Q588L. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q588L substitution (i.e., L at position 588) relative to reference sequence SEQ ID NO: 1.

[0204] In some embodiments, the capsid protein comprises an amino acid substitution at position 588 consisting of Q588T. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q588T substitution (i.e., T at position 588) relative to reference sequence SEQ ID NO: 1.

[0205] In some embodiments, the capsid protein comprises an amino acid substitution at position 588 consisting of Q588G. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q588G substitution (i.e., G at position 588) relative to reference sequence SEQ ID NO: 1.

[0206] In some embodiments, the capsid protein comprises an amino acid substitution at position 589 selected from the group consisting of: A589V and A589F.

[0207] In some embodiments, the capsid protein comprises an amino acid substitution at position 589 consisting of A589V. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises A589V substitution (i.e., V at position 589) relative to reference sequence SEQ ID NO: 1.

[0208] In some embodiments, the capsid protein comprises an amino acid substitution at position 589 consisting of A589F. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises A589F substitution (i.e., F at position 589) relative to reference sequence SEQ ID NO: 1.

[0209] In some embodiments, the capsid protein comprises an amino acid substitution at position 590 selected from the group consisting of: Q590R, Q590G, Q590M, and Q590N.

[0210] In some embodiments, the capsid protein comprises an amino acid substitution at position 590 consisting of Q590R. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q590R substitution (i.e., R at position 590) relative to reference sequence SEQ ID NO: 1.

[0211] In some embodiments, the capsid protein comprises an amino acid substitution at position 590 consisting of Q590G. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q590G substitution (i.e., G at position 590) relative to reference sequence SEQ ID NO: 1.

[0212] In some embodiments, the capsid protein comprises an amino acid substitution at position 590 consisting of Q590M. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q590M substitution (i.e., M at position 590) relative to reference sequence SEQ ID NO: 1.

[0213] In some embodiments, the capsid protein comprises an amino acid substitution at position 590 consisting of Q590N. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises Q590N substitution (i.e., N at position 590) relative to reference sequence SEQ ID NO: 1.

[0214] In some embodiments, the capsid protein comprises an amino acid substitution at position 591 consisting of A591P. In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises A591P substitution (i.e., P at position 591) relative to reference sequence SEQ ID NO: 1.

[0215] In some embodiments, the capsid protein comprises one or more (e.g., three) amino acid substitutions selected from the group consisting of Q585S, S586G, and Q588Y, relative to reference sequence SEQ ID NO: 1.

[0216] In some embodiments, the capsid protein comprises one or more (e.g., three) amino acid substitutions selected from the group consisting of Q585M, S586G, and Q588Y, relative to reference sequence SEQ ID NO: 1.

[0217] In some embodiments, the capsid protein comprises one or more (e.g., three or four) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, and Q590R , relative to reference sequence SEQ ID NO: 1.

[0218] In some embodiments, the capsid protein comprises one or more (e.g., three or four) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, and Q585M , relative to reference sequence SEQ ID NO: 1.

[0219] In some embodiments, the capsid protein comprises one or more (e.g., three or four) amino acid substitutions selected from the group consisting of H584R, Q585M, S586G, and Q588Y, relative to reference sequence SEQ ID NO: 1.

[0220] In some embodiments, the capsid protein comprises one or more (e.g., three, four, or five) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, Q585M, and Q590R, relative to reference sequence SEQ ID NO: 1.

[0221] In some embodiments, the capsid protein comprises one or more (e.g., three, four, or five) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586G, and Q588Y, relative to reference sequence SEQ ID NO: 1.

[0222] In some embodiments, the capsid protein comprises one or more (e.g., three, four, or five) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, Q585M, and Q590M, relative to reference sequence SEQ ID NO: 1.

[0223] In some embodiments, the capsid protein comprises one or more (e.g., three, four, or five) amino acid substitutions selected from the group consisting of H584R, S586L, A587P, Q588G, and A589F, relative to reference sequence SEQ ID NO: 1.

[0224] In some embodiments, the capsid protein comprises one or more (e.g., three, four, or five) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, Q590R. and A591P, relative to reference sequence SEQ ID NO: 1.

[0225] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, or six) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586G, Q688Y, and Q590R, relative to reference sequence SEQ ID NO: 1.

[0226] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, or six) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, Q585N, Q590R, and A591P relative to reference sequence SEQ ID NO: 1.

[0227] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, or six) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586G, Q588Y, and Q590M relative to reference sequence SEQ ID NO: 1.

[0228] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, or six) amino acid substitutions selected from the group consisting of T582D, H584Q, S586L, A587P, Q588G, and A589F relative to reference sequence SEQ ID NO: 1.

[0229] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, or six) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, Q585M, Q590R, and A591P relative to reference sequence SEQ ID NO: 1.

[0230] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, six, or seven) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586T, A587K, Q588L, and Q590G, relative to reference sequence SEQ ID NO: 1.

[0231] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, six, or seven) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586G, Q588Y, Q590R, and A591P relative to reference sequence SEQ ID NO: 1.

[0232] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, six, or seven) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586M, A587T, Q588T, and Q590R, relative to reference sequence SEQ ID NO: 1.

[0233] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, six, or seven) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586M, A587T, Q588T, and Q590M, relative to reference sequence SEQ ID NO: 1.

[0234] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, six, or seven) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586T, A587K, Q588L, and Q590N, relative to reference sequence SEQ ID NO: 1.

[0235] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, six, seven, or eight) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586M, A587N, Q588R, A589V, and Q590N, relative to reference sequence SEQ ID NO: 1.

[0236] In some embodiments, the capsid protein comprises one or more (e.g., three, four, five, six, seven, or eight) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, S586M, A587N, Q588R, A589V, and Q590G, relative to reference sequence SEQ ID NO: 1.

[0237] In some embodiments, the capsid protein may comprise one or more (e.g., three, four, five, six or seven) amino acid substitutions selected from the group consisting of S586H, T582D, N583V, H584Q, A587P, Q588G, and A589F, relative to reference sequence SEQ ID NO: 1.

[0238] In some embodiments, the capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected from the group consisting of S586L, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 1.

[0239] In some embodiments, the capsid protein may comprise one or more (e.g., three, four, five, six, seven or eight) amino acid substitutions selected from the group consisting of S586H, T582D, N583V, H584Q, A587P, Q588G, A589F, and G594E, relative to reference sequence SEQ ID NO: 1.

[0240] In some embodiments, the capsid protein may comprise one or more (e.g., three, four, five, six, seven, or eight) amino acid substitutions selected from the group consisting of T582D, N583 V, H584Q, A587P, Q588G, A589F, A591P, Q592E relative to reference sequence SEQ ID NO: 1.

[0241] In some embodiments, the capsid protein may comprise one or more (e.g., two, three, four, five, six, seven, eight, or nine) amino acid substitutions selected from the group consisting of T582D, N583V, H584Q, H584R, Q585M, Q585N, Q585S, S586G, S586M, S586T, S586L, A587N, A587K, A587T, A587P, Q588Y, Q588R, Q588L, Q588T, Q588G,A589V, A589F, Q590R, Q590G, Q590M, Q590N, and A591P relative to reference sequence SEQ ID NO: 1.

[0242] In some embodiments, the variant VP1, VP2 and / or VP3 capsid protein comprises the VR-VIII site positions 581-594 of any one of the below-noted capsids, relative to SEQ ID NO: 1, or the VR-VIII site positions 581-594 of any one of the below-noted capsids with not more than 1, 2, 3, 4 or 5 amino acid substitutions (e..g, not more than 1, 2, or 3 substitutions). The full sequences of such capsids are provided herein, each of which comprises the AAV9 capsid sequence with the below-noted VR-VIII modification in Table 5 and 6.Table 5: Exemplary Capsid sequences

[0243] In some embodiments, the variant VP1, VP2 and / or VP3 capsid protein comprises one or more of the amino acids at the VR-VIII site positions 581-594, relative to SEQ ID NO: 1 (e.g., where position 586 comprises L or H, and position 594 comprises E or G) as shown in Table 6 below.TABLE 6: Summary of Substitution Motifs for VR-VIII site

[0244] In some embodiments, the capsid protein comprises, consists essentially of, or consists of a polypeptide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 343, or a functional fragment thereof (e.g., wherein any susbtitutions relative to SEQ ID NO: 343 are not at the VR-VIII site). In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 343.

[0245] In some embodiments, the capsid protein comprises, consists essentially of, or consists of a polypeptide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 344, or a functional fragment thereof (e.g., wherein any susbtitutions relative to SEQ ID NO: 344 are not at the VR-VIII site). In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein, wherein the capsid protein comprises the amino acid sequence of SEQ ID NO: 344.

[0246] In some embodiments, the capsid protein comprises, consists essentially of, or consists of a polypeptide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to one of SEQ ID NOs: 846-903 and 984-1005, or a functional fragment thereof (e.g., wherein any susbtitutions are not at the VR-VIII site). In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 846-903 and 984-1005.

[0247] In some embodiments, the capsid protein comprises, consists essentially of, or consists of a polypeptide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to one of SEQ ID NOs: 846-850 and 984-1005, or a functional fragment thereof (e.g., wherein any susbtitutions are not at the VR-VIII site). In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 846-850 and 984-1005.

[0248] In some embodiments, the capsid protein comprises, consists essentially of, or consists of a polypeptide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to one of SEQ ID NOs: 891, 864, 867, 858, 878, 890, 870, 856, 886, 900, 901, or 898, or a functional fragment thereof (e.g., wherein any susbtitutions are not at the VR- VIII site). In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) capsid protein, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 891, 864, 867, 858, 878, 890, 870, 856, 886, 900, 901, or 898.

[0249] In some embodiments, the capsid protein is, or consists essentially of, ZC621 as provided herein. In some embodiments, the capsid protein is, or consists essentially of, ZC623 as provided herein.

[0250] In some embodiments, the capsid potein comprises the same substitution motif (e.g., the same VR-VIII substitution motif) as ZC621 or ZC623 capsid.

[0251] Any of the AAV9 capsid protein modifications described herein may be used either alone or in combination with other modifications (e.g., in combination with other modifications described herein).

[0252] In some embodiments, the VR-VIII modifications described herein are the only modifications in an AAV9 capsid protein. In some embodiments, such modifications are the only modifications in the capsid protein of the present disclosure relative to reference SEQ ID NO: 1. In some embodiments, such modification is the only modification in the AAV9 capsid protein’s VR-III sites, VR-IV, VR-V and / or VR-VII sites.

[0253] Any of the VR-VIII modifications or variant VR-VIII site sequences described herein in the AAV9 capsid protein can be combined with any one or more additional modifications in AAV9 capsid protein (such as a modification outside of the VR-VIII sequence), including substitution of one or more amino acids, or insertion of one or more amino acids, at any site in the AAV9 capsid protein (e.g., VR-IV site, VR-V site and / or VR-VII site). In some embodiments, the additional modification in an AAV9 capsid protein is not in the VR- VIII site of the AAV9 capsid protein.

[0254] In some embodiments, the engineered AAV9 capsid provided herein comprises any of the VR-VIII-modifications described herein, and an additional modification in the VR-IV site, such as any one or more VR-IV modifications described herein or known in the art. In some embodiments, the engineered AAV9 capsid provided herein comprises any of the VR- VIII-modifications described herein, and an additional modification in the VR-V and / or VR- VII sites, such as any one or more VR-V and VR-VII modifications described herein or known in the art.

[0255] In some embodiments, a capsid protein described herein comprises an amino acid substitution or insertion in the VR-IV site (between residues 452 and 460 in SEQ ID NO: 1 or in the sequence of SEQ ID NO: 2 (NGSGQNQ)). In some embodiments, a capsid protein described herein comprises an amino acid substitution in the VR-IV site (between residues 452 and 460 in SEQ ID NO: 1 or in the sequence of SEQ ID NO: 2 (NGSGQNQ)). In some embodiments, the amino acid substitution or insertion in the VR-IV site is any amino acid substitution or insertion described herein. In some embodiments, a capsid protein described herein comprises an amino acid substitution at position 452 of SEQ ID NO: 1 or the first amino acid of SEQ ID NO:2 (NGSGQNQ) in the VR-IV site. In some embodiments, a capsid protein described herein comprises an amino acid substitution N452K in SEQ ID NO: 1 or comprises the sequence KGSGQNQ in the VR-IV site.

[0256] In some embodiments, a capsid protein described herein comprises an amino acid substitution or insertion in the VR-V site (between residues 497 and 502 in SEQ ID NO: 1 or in the sequence of SEQ ID NO: 3 (NNSEFA)). In some embodiments, a capsid protein described herein comprises an amino acid substitution in the VR-V site (between residues 497 and 502 in SEQ ID NO: 1 or in the sequence of SEQ ID NO: 3 (NNSEFA)). In some embodiments, the amino acid substitution or insertion in the VR-V site is any amino acid substitution or insertion described herein.

[0257] In some embodiments, a capsid protein described herein comprises an amino acid substitution or insertion in the VR-VII site (between residues 549 and 553 in SEQ ID NO: 1 or in the sequence of SEQ ID NO:4 (GRDNV)). In some embodiments, a capsid protein described herein comprises an amino acid substitution in the VR-VII site (between residues 549 and 553 in SEQ ID NO: 1 or in the sequence of SEQ ID NO: 4 (GRDNV)). In some embodiments, the amino acid substitution or insertion in the VR-VII site is any amino acid substitution or insertion described herein.

[0258] Solely for purposes of clarity and without limitation, it is noted that reference to amino acid position numbers at which modifications occur is relative to position of corresponding amino acids in SEQ ID NO: 1. In some embodiments, the capsid protein does not comprise the full-length sequence corresponding to SEQ ID NO: 1, but comprises a shorter variant of this sequence (e.g., comprises only a variant of SEQ ID NO:487, or a variant of SEQ ID NO:486). In such embodiments, the modifications described herein may not occur at the same numerical positions as in SEQ ID NO: 1 but occur at the same site or consensus sequence relative to reference sequence SEQ ID NO: 1. In some embodiments, the capsid protein is a variant of SEQ ID NO: 1, and the modifications described herein occur at the same numerical positions as in SEQ ID NO: 1.

[0259] In some embodiments, the engineered AAV9 capsid protein described herein shares at least 80% polypeptide sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises one or more of the modifications (such as VR-VIII modifications) described herein (where amino acid positions are relative to reference sequence SEQ ID NO: 1). In some embodiments, the engineered AAV9 capsid protein described herein shares at least 85% polypeptide sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises one or more of the modifications (such as VR-VIII modifications) described herein (where amino acid positions are relative to reference sequence SEQ ID NO: 1). In some embodiments, theengineered AAV9 capsid protein comprises a sequence sharing at least 90%, at least 95%, at least 96%, at least 97%, at least 99%, or 100% amino acid sequence identity to an AAV9 VP3 sequence according to SEQ ID NO: 487 and wherein the capsid protein comprises one or more of the modifications (such as VR-VIII modifications) described herein (where amino acid positions are relative to reference sequence SEQ ID NO: 1).

[0260] In some embodiments, the engineered AAV9 capsid protein comprises any of the variant polypeptide sequences described herein or any of the VR-VIII modificatons described herein, and further comprises one or more modifications described hereinbelow.Additional Modifications in VR-IV, VR-V and VR-VII Sites

[0261] In some embodiments, in addition to modification in the VR-VIII site described herein, engineered AAV capsid proteins described herein comprise one or more modifications in the VR-IV, VR-V and / or VR-VII sites. The respective locations of these sites in various AAV capsid proteins are described herein or known in the art.VR-IV site

[0262] In some embodiments, the entire VR-IV site (“NGSGQNQQT”, SEQ ID NO: 2) in an AAV9 capsid protein is substituted by a peptide of formula:-(X)„- wherein n is 7-11, and X represents any of the 20 standard amino acids (SEQ ID NO: 1053).

[0263] In some embodiments, the variant polypeptide sequence at the VR-IV site is:-X1-X2-X3-X4-X5-X6-X7-X8-X9- wherein Xi, X2, X3, X4, X5, Xe, X7, Xs, and / or X9 is any amino acid (SEQ ID NO: 478).

[0264] In some embodiments, the variant polypeptide sequence at the VR-IV site is:-X1-X2-X3-X4-X5-X6-X7-X8-X9- wherein Xi is G, S or V; X2 is Y, Q or I; X3 is H, W, V, or I; X4 is K or N; X5 is S, G or I; X6is G or R; X7 is A, P or V; X8is A or R; and / or X9is Q or D (SEQ ID NO: 477).

[0265] In some embodiments, the variant polypeptide sequence at the VR-IV site is:-X1-X2-X3-X4-X5-X6-X7-X8-X9- wherein Xi is K, G, S or V; X2 is Y, Q or I; X3 is H, W, V, or I; X4 is K or N; X5 is S, G or I; X6is G or R; X7 is A, P or V; X8is A or R; and / or X9is Q or D (SEQ ID NO: 729).

[0266] In some embodiments, the variant polypeptide sequence at the VR-IV site is:-X1-X2-X3-X4-X5-X6-X7-X8-X9-wherein Xi is K; X2, X3, X4, X5, Xe, X7, Xs, and / or Xg is any amino acid (SEQ ID NO: 730).

[0267] In some embodiments, the variant polypeptide sequence at the VR-IV site comprises or consists of the sequence KGSGQNQQT (SEQ ID NO:727).

[0268] In some embodiments, the capsid protein of the present disclosure comprises a variant polypeptide sequence with N452K substitution at the VR-IV site. In some embodiments, the capsid protein of the present disclosure comprises a variant polypeptide sequence with N452K substitution at the VR-IV site relative to reference SEQ ID NO: 1 or comprises the sequence of KGSGQNQQT (SEQ ID NO:727). In some embodiments, the capsid protein of the present disclosure (such as an AAV9 capsid protein) comprises amino acid substitution N452K at the VR-IV site in addition to any other substitution or insertion described herein or known in the art. In some embodiments, the capsid protein of the present disclosure comprises amino acid substitution N452K at the VR-IV site relative to reference SEQ ID NO: 1 or the sequence KGSGQNQQT (SEQ ID NO: 727) in addition to any other chimeric modification described herein or known in the art. In some embodiments, such substitution is combined with any substitution(s) in the VR-VIII site described herein. In some embodiments, the capsid protein of the present disclosure comprises amino acid substitution N452K at the VR-IV site in addition to any one, two, three or more substitutions or insertions at the VR-VIII site described herein. In some embodiments, the capsid protein of the present disclosure comprises amino acid substitution N452K, relative to reference sequence SEQ ID NO: 1, in addition to one, two, three or more substitutions or insertions at the VR-VIII site described herein. In some embodiments, the capsid protein, such as the capsid protein with N452K substitution at the VR-IV site relative to reference SEQ ID NO: 1, increases transduction efficiency (e.g., of any tissue, such as muscle, heart, skeletal muscle, brain, etc.). In some embodiments, the capsid protein of the present disclosure, such as the capsid protein with N452K substitution at the VR-IV site relative to reference SEQ ID NO: 1, increases transduction efficiency of the heart.

[0269] The capsid protein may comprise an amino acid sequence selected from KGSGQNQ (SEQ ID NO: 590), NASGQNQ (SEQ ID NO: 591), NGTGQNQ (SEQ ID NO: 592), NGSGLNQ (SEQ ID NO: 593), ANDNKLI (SEQ ID NO: 594), VNDNKVI (SEQ ID NO: 595), NGSGQNH (SEQ ID NO: 596), or ANDNKVI (SEQ ID NO: 597) at positions 452- 458 or at about positions 452-458 and wherein the capsid protein shares at least 80%, at least 90%, at least 95%, at least 98%, or 100% identity to SEQ ID NOs: 488-495.

[0270] In some embodiments, the capsid protein of the present disclosure comprises a variant polypeptide sequence at the VR-IV site. In some embodiments, the engineered AAV9 capsid protein comprises one or more amino acid substitutions selected from the group consisting of N452K, N452A, N452V, G453A, G453N, S454T, S454D, G455N, Q456L, Q456K, N457L, N457V, Q458I, and Q458H. In some embodiments, the capsid protein comprises the amino acid substitution N452K, N452A, or N452V. In some embodiments, the capsid protein comprises the amino acid substitution G453 A or G453N. In some embodiments, the capsid protein comprises the amino acid substitution S454T or S454D. In some embodiments, the capsid protein comprises the amino acid substitution G455N. In some embodiments, the capsid protein comprises the amino acid substitution Q456L or Q456K. In some embodiments, the capsid protein comprises the amino acid substitution N457L orN457V. In some embodiments, the capsid protein comprises the amino acid substitution Q458I or Q458H.

[0271] In some embodiments, the engineered AAV9 capsid protein comprises the amino acid substitution N452K. In some embodiments, the engineered AAV9 capsid protein comprises any of the variant polypeptide sequences described herein, and an amino acid substitution N452K. In some embodiments, the engineered AAV9 capsid protein comprises any of the VR-VIII modifications described herein, and an amino acid substitution N452K.

[0272] In some embodiments, an engineered AAV9 capsid protein comprises one or more amino acid substitutions selected from the group consisting of N452K, T582D, N583V, H584Q, A587P, Q588G, and A589F. In some embodiments, the capsid protein comprises one or more amino acid substitutions selected from the group consisting of N452K, S586L, T582D, N583V, H584Q, A587P, Q588G, and A589F. In some embodiments, the capsid protein comprises one or more amino acid substitutions selected from the group consisting of N452K, S586H, T582D, N583V, H584Q, A587P, Q588G, and A589F.

[0273] In some embodiments, the capsid protein of the present disclosure comprises wild type AAV9 amino acid (which is N) at position 452 of the VR-IV site relative to reference SEQ ID NO: 1.

[0274] In some embodiments, the engineered capsid protein of the present disclosure comprises N or K at position 452 of the VR-IV site relative to reference SEQ ID NO: 1.

[0275] In some embodiments, the variant polypeptide sequence at the VR-IV site comprises or consists of a sequence selected from GYHKSGAAQ (SEQ ID NO: 6),VIIKSGAAQ (SEQ ID NO: 7), GYHKIGAAQ (SEQ ID NO: 8), GYHKSGVAQ (SEQ ID NO: 9), VYHKSGAAQ (SEQ ID NO: 10), GYHKISAAQ (SEQ ID NO: 11), TTVPSSSRY (SEQ ID NO: 12), VIIRVVRLS (SEQ ID NO: 13), TVLGQNQQT (SEQ ID NO: 14), IYHKSGAAQ (SEQ ID NO: 15), TVLDKNQQT (SEQ ID NO: 16), YSGTDVRYK (SEQ ID NO: 17), VTASGKEHR (SEQ ID NO: 18), GYRKSGAAQ (SEQ ID NO: 19), NRTVSNGSE (SEQ ID NO: 20), TVLDRINKT (SEQ ID NO: 21), TGVGHLTSA (SEQ ID NO: 22), GYHKGGAAQ (SEQ ID NO: 23), VIAKSGAAQ (SEQ ID NO: 24), GYHKSGAAH (SEQ ID NO: 25), FIIKSGAAQ (SEQ ID NO: 26), GYHKVVRLS (SEQ ID NO: 27), GATRSAVES (SEQ ID NO: 28), TVSGQNQQT (SEQ ID NO: 29), LSHKSGAAQ (SEQ ID NO: 30), SSSGQNQQT (SEQ ID NO: 31), SGSGQNQQT (SEQ ID NO: 32), SQVNGRPRD (SEQ ID NO: 33), GYHKEWCGS (SEQ ID NO: 34), VVSSKSLNS (SEQ ID NO: 35), GYHKSGAAP (SEQ ID NO: 36), DASSREKVR (SEQ ID NO: 37), SYHKSGAAQ (SEQ ID NO: 38), TANGSQKYL (SEQ ID NO: 39), VIIRVGAAQ (SEQ ID NO: 40), SSTNKISTA (SEQ ID NO: 41), TVLDRIQQT (SEQ ID NO: 42), GYHKSGAVQ (SEQ ID NO: 43), TVLDQNQQT (SEQ ID NO: 44), VNMSSPIKT (SEQ ID NO: 45), AAYNSNSAF (SEQ ID NO: 46), GYHKSGAAR (SEQ ID NO: 47), VIIRVVRLQ (SEQ ID NO: 48), RFWTQNQQT (SEQ ID NO: 49), SSPRASSAL (SEQ ID NO: 50), IIIRVVRLS (SEQ ID NO: 51), KSSNLTAMP (SEQ ID NO: 52), NLNSDRHSA (SEQ ID NO: 53), LSLKSGAAQ (SEQ ID NO: 54), TVLDRNQQT (SEQ ID NO: 55), GSERVSNSG (SEQ ID NO: 56), VIAKIGAAQ (SEQ ID NO: 57), VYHKIGAAQ (SEQ ID NO: 58), LSYKSGAAQ (SEQ ID NO: 59), STVSQPVRT (SEQ ID NO: 60), GHHKSGAAQ (SEQ ID NO: 61), YAGIDPRYH (SEQ ID NO: 62), DRSRKSMCD (SEQ ID NO: 63), VIIRSGAAQ (SEQ ID NO: 64), GYHKSGGSA (SEQ ID NO: 65), VIIKIGAAQ (SEQ ID NO: 66), GYHKVVQLS (SEQ ID NO: 67), VIIKLVAAQ (SEQ ID NO: 68), KVSSHSVCD (SEQ ID NO: 69), GYHKRVRLS (SEQ ID NO: 70), GYHKSSAAQ (SEQ ID NO: 71), GYRKIGAAQ (SEQ ID NO: 72), GYHKSGAAC (SEQ ID NO: 73), GYRQSGAAQ (SEQ ID NO: 74), VIIKLIAAQ (SEQ ID NO: 75), VIIRVVRAQ (SEQ ID NO: 76), GYHKSGAAW (SEQ ID NO: 77), GYHKSGAVS (SEQ ID NO: 78), GYHKEWCSS (SEQ ID NO: 79), SSSSNRLAD (SEQ ID NO: 80), SNNSSSAKF (SEQ ID NO: 81), VKLSSTSSS (SEQ ID NO: 82), GYHKEWCAQ (SEQ ID NO: 83), AGSGQNQQT (SEQ ID NO: 84), NPHGTATYL (SEQ ID NO: 85), NGSGQNQHT (SEQ ID NO: 86), GYHKVGAAQ (SEQ ID NO: 87), VIIRVVRLK (SEQ ID NO: 88), NSIPSTSKW (SEQ ID NO: 89), VIIRVVQLQ (SEQ ID NO: 90), SQVNGRPQD (SEQ ID NO: 91), NGSGQDQQT (SEQ ID NO: 92), GLNSSDRRL (SEQ ID NO: 93), IYHKIGAAQ (SEQ ID NO: 94), YHKSGAAQL (SEQ ID NO: 95), YSGTDVQYK (SEQ ID NO: 96), LGSGQNQQT (SEQ ID NO: 97), PVSSGADRR(SEQ ID NO: 98), EHSTKLNAC (SEQ ID NO: 99), NGSDRINKR (SEQ ID NO: 100), VIIKGGAAQ (SEQ ID NO: 101), GYHRVVRLS (SEQ ID NO: 102), VIIRVVRLL (SEQ ID NO: 103), and VILKSGAAQ (SEQ ID NO: 104). In some of any of these embodiments, the first amino acid is substituted with K instead of any other amino acid at this position (or has N452K substitution relative to reference sequence SEQ ID NO: 1).

[0276] In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists of a polypeptide sequence at least about 60%, 70%, 80%, 90%, or 100% identical to one of SEQ ID NOs: 6-104.

[0277] In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists of a sequence at least about 60%, 70%, 77%, 80%, 88%, 90%, or 100% identical to KGSGQNQQT (SEQ ID NO:727). In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, or 4 amino-acid substitutions relative to KGSGQNQQT (SEQ ID NO:727). In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, or 4 conservative amino-acid substitutions relative KGSGQNQQT (SEQ ID NO:727). In some embodiments, the variant polypeptide sequence at the VR-IV site is KGSGQNQQT (SEQ ID NO:727).

[0278] In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists of a sequence at least about 60%, 70%, 77%, 80%, 88%, 90%, or 100% identical to GYHKSGAAQ (SEQ ID NO: 6). In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, or 4 amino-acid substitutions relative to GYHKSGAAQ (SEQ ID NO: 6). In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, or 4 conservative amino-acid substitutions relative GYHKSGAAQ (SEQ ID NO: 6). In some embodiments, the variant polypeptide sequence at the VR-IV site is GYHKSGAAQ (SEQ ID NO: 6). In some of any of these embodiments, the first amino acid is substituted with K (KYHKSGAAQ; SEQ ID NO: 757).

[0279] In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists of a sequence at least about 60%, 70%, 77%, 80%, 88%, 90%, or 100% identical to SQVNGRPRD (SEQ ID NO: 33). In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists ofa sequence consisting of at most 1, 2, 3, or 4 amino-acid substitutions relative to SQVNGRPRD (SEQ ID NO: 33). In some embodiments, the variant polypeptide sequence at the VR-IV site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, or 4 conservative amino-acid substitutions relative SQVNGRPRD (SEQ ID NO: 33). In some embodiments, the variant polypeptide sequence at the VR-IV site is SQVNGRPRD (SEQ ID NO: 33). In some of any of these embodiments, the first amino acid is substituted with K (KQVNGRPRD; SEQ ID NO: 758).

[0280] In some of these embodiments, the capsid protein comprises N or K at position 452 relative to reference sequence SEQ ID NO: 1 (in addition to the variant polypeptide sequence described herein).

[0281] In some of these embodiments, the capsid protein may further comprise N452K substitution relative to reference sequence SEQ ID NO: 1 (in addition to the variant polypeptide sequence described herein).

[0282] In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises, consists essentially of, or consists of a sequence at least about 60%, 70%, 80%, 83%, 90%, or 100% identical to SEQ ID NO:320, and further comprises N452K substitution (relative to reference sequence SEQ ID NO: 1) in the VR-IV site. In some embodiments, a capsid described herein comprises the variant polypeptide sequence at the VR-VIII site comprising the sequence SEQ ID NO:320, and does not comprise N452K substitution (relative to reference sequence SEQ ID NO: 1) in the VR-IV site. In some of these embodiments, the capsid protein comprises the sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to VP3 of SEQ ID NO:487 except for the specific substitutions at the VR-VIII site and, optionally, position 452 described herein.

[0283] In some embodiments, the variant polypeptide sequence at the VR-VIII site comprises, consists essentially of, or consists of a sequence at least about 60%, 70%, 80%, 83%, 90%, or 100% identical to SEQ ID NO: 321, and further comprises N452K substitution (relative to reference sequence SEQ ID NO: 1) in the VR-IV site. In some embodiments, a capsid described herein comprises the variant polypeptide sequence at the VR-VIII site comprising the sequence SEQ ID NO: 321, and does not comprise N452K substitution (relative to reference sequence SEQ ID NO: 1) in the VR-IV site. In some of these embodiments, the capsid protein comprises the sequence at least 85%, 90%, 95%, 98%, 99% or 100% identical to VP3 of SEQ ID NO:487 except for the specific substitutions at the VR-VIII site and, optionally, position 452 described herein.VR-V site

[0284] In some embodiments, the capsid protein of the present disclosure comprises a variant polypeptide sequence at the VR-V site. In some embodiments, the entire VR-V site (“NNSEFA”, SEQ ID NO: 3) is substituted by a peptide of formula:-(X)„- wherein n is 4-8, and X represents any of the 20 standard amino acids (SEQ ID NO: 1052).

[0285] In some embodiments, the variant polypeptide sequence at the VR-V site is:-X1-X2-X3-X4-X5-X6- wherein Xi, X2, X3, X4, X5, and / or Xe, is any amino acid (SEQ ID NO: 479)

[0286] In some embodiments, the variant polypeptide sequence at the VR-V site is:-X1-X2-X3-X4-X5-X6-Wherein Xi is S, L, H, N, or A; X2 is T, M, K, G, or N; X3 is S, T, M or I; X4is S, P, F, M, or N; X5is F, S, P or L; and X6is I, V, or T (SEQ ID NO: 474).

[0287] In some embodiments, the variant polypeptide sequence at the VR-V site comprises or consists of a sequence selected from LNSMLI (SEQ ID NO: 105), NGMSFT (SEQ ID NO: 106), HKTFSI (SEQ ID NO: 107), SMSNFV (SEQ ID NO: 108), ATIPPI (SEQ ID NO: 109), SSTHFD (SEQ ID NO: 110), NNQFSY (SEQ ID NO: 111), NMGHYS (SEQ ID NO: 112), SKQMFQ (SEQ ID NO: 113), WPSAGV (SEQ ID NO: 114), NGGYQC (SEQ ID NO: 115), STSPIV (SEQ ID NO: 116), SQSGLW (SEQ ID NO: 117), VNSQFS (SEQ ID NO: 118), SGIEFR (SEQ ID NO: 119), SASKFT (SEQ ID NO: 120), QLNWTS (SEQ ID NO: 121), SMGFPV (SEQ ID NO: 122), SSFMGL (SEQ ID NO: 123), GSNFHV (SEQ ID NO: 124), DMTLYA (SEQ ID NO: 125), MGCLFT (SEQ ID NO: 126), ALAFNS (SEQ ID NO: 127), SKFLFA (SEQ ID NO: 128), QDAGLL (SEQ ID NO: 129), QDASLL (SEQ ID NO: 130), RDDMFS (SEQ ID NO: 131), LSRCFQ (SEQ ID NO: 132), LSRDFQ (SEQ ID NO: 133), QGLTPV (SEQ ID NO: 134), QWDVFT (SEQ ID NO: 135), PRVSFA (SEQ ID NO: 136), QSYYNP (SEQ ID NO: 137), RASHLG (SEQ ID NO: 138), IILFVP (SEQ ID NO: 139), IISFSY (SEQ ID NO: 140), LDSMLI (SEQ ID NO: 141), NIGHYS (SEQ ID NO: 142), NRMSFT (SEQ ID NO: 143), NGMSFA (SEQ ID NO: 144), IILLLP (SEQ ID NO: 145), RMRSLL (SEQ ID NO: 146), RRRCRF (SEQ ID NO: 147), PKQMFQ (SEQ ID NO: 148), LMSNFV (SEQ ID NO: 149), GASHLG (SEQ ID NO: 150), CASISW (SEQ ID NO: 151), SMTTFR (SEQ ID NO: 152), AAIPPI (SEQ ID NO: 153), PGCESL (SEQ ID NO: 154), SMGFAC (SEQ ID NO: 155), FLPSLM (SEQ ID NO: 156), NGISFT (SEQ ID NO: 157),ESSRWA (SEQ ID NO: 158), QLYFVP (SEQ ID NO: 159), SSNFHV (SEQ ID NO: 160), LEFMLI (SEQ ID NO: 161), QFDSFD (SEQ ID NO: 162), SPVFAC (SEQ ID NO: 163), VRLIFD (SEQ ID NO: 164), NGMSFI (SEQ ID NO: 165), LLFPPI (SEQ ID NO: 166), GAGVTG (SEQ ID NO: 167), QWMSFT (SEQ ID NO: 168), SIGFPV (SEQ ID NO: 169), RMQSLL (SEQ ID NO: 170), TSALQV (SEQ ID NO: 171), SLTHFD (SEQ ID NO: 172), QELPFL (SEQ ID NO: 173), LYFLLP (SEQ ID NO: 174), LSFFFA (SEQ ID NO: 175), LSRIFQ (SEQ ID NO: 176), DEVILF (SEQ ID NO: 177), RAGVAG (SEQ ID NO: 178), NGMSLP (SEQ ID NO: 179), PFEDFQ (SEQ ID NO: 180), QYGSLF (SEQ ID NO: 181), NYTFVL (SEQ ID NO: 182), MSGYQC (SEQ ID NO: 183), NYAFVP (SEQ ID NO: 184), RAGVTG (SEQ ID NO: 185), WNSMLI (SEQ ID NO: 186), IRRFSI (SEQ ID NO: 187), NGMSFY (SEQ ID NO: 188), IIQFSY (SEQ ID NO: 189), NGCLFT (SEQ ID NO: 190), RDASLL (SEQ ID NO: 191), ADSMLI (SEQ ID NO: 192), VDSQFS (SEQ ID NO: 193), SIGNFV (SEQ ID NO: 194), NGMSLL (SEQ ID NO: 195), NYTFVP (SEQ ID NO: 196), IRRLVF (SEQ ID NO: 197), PMSNFV (SEQ ID NO: 198), LWVFPV (SEQ ID NO: 199), VRLHFD (SEQ ID NO: 200), SMSNLF (SEQ ID NO: 201), STSLIV (SEQ ID NO: 202), and HKTFGI (SEQ ID NO: 203).

[0288] In some embodiments, the variant polypeptide sequence at the VR-V site comprises, consists essentially of, or consists of a polypeptide sequence at least about 60%, 70%, 80%, 90%, 95%, or 100% identical to one of SEQ ID NOs: 105-203.

[0289] In some embodiments, the variant polypeptide sequence at the VR-V site comprises, consists essentially of, or consists of a sequence at least about 60%, 70%, 80%, 83%, 90%, or 100% identical to LNSMLI (SEQ ID NO: 105). In some embodiments, the variant polypeptide sequence at the VR-V site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, or 4 amino-acid substitutions relative to LNSMLI (SEQ ID NO: 105). In some embodiments, the variant polypeptide sequence at the VR-V site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, or 4 conservative amino-acid substitutions relative LNSMLI (SEQ ID NO: 105). In some embodiments, the variant polypeptide sequence at the VR-V site is LNSMLI (SEQ ID NO: 105).VR-VII site

[0290] In some embodiments, the capsid protein of the present disclosure comprises a variant polypeptide sequence at the VR-VII site. In some embodiments, the entire VR-VII site (“GRDNV”, SEQ ID NO: 4) is substituted by a peptide of formula:-(X)„- wherein n is 3-7, and X represents any of the 20 standard amino acids (SEQ ID NO: 480).

[0291] In some embodiments, the variant polypeptide sequence at the VR-VII site is:-X1-X2-X3-X4-X5- wherein Xi, X2, X3, X4, and / or X5, is any amino acid (SEQ ID NO: 1066)

[0292] In some embodiments, the variant polypeptide sequence at the VR-VII site is:-X1-X2-X3-X4-X5-Wherein Xi is V, L, Q, C, or R; X2 is S, H, G, C, or D; X3 is Y, S, L, G, or N; X4is S, L, H, Q, or N; and X5is V, I, or R (SEQ ID NO: 475).

[0293] In some embodiments, the variant polypeptide sequence at the VR-VII site comprises or consists of a sequence selected from RGNQV (SEQ ID NO: 204), VSLNR (SEQ ID NO: 205), CDYSV (SEQ ID NO: 206), QHGHI (SEQ ID NO: 207), LCSLV (SEQ ID NO: 208), PTIYV (SEQ ID NO: 209), DVIHI (SEQ ID NO: 210), AEFYA (SEQ ID NO: 211), NSVVC (SEQ ID NO: 212), VRSNC (SEQ ID NO: 213), LANNI (SEQ ID NO: 214), NLQFM (SEQ ID NO: 215), EFRDL (SEQ ID NO: 216), DFGSL (SEQ ID NO: 217), VTNYC (SEQ ID NO: 218), WNTNA (SEQ ID NO: 219), TESTC (SEQ ID NO: 220), SGAAV (SEQ ID NO: 221), GGCDI (SEQ ID NO: 222), SGSVV (SEQ ID NO: 223), SSNAC (SEQ ID NO: 224), YNTTV (SEQ ID NO: 225), SKCLA (SEQ ID NO: 226), SAYTV (SEQ ID NO: 227), VRDTV (SEQ ID NO: 228), WRSMV (SEQ ID NO: 229), AYHGV (SEQ ID NO: 230), GMNTI (SEQ ID NO: 231), AETSL (SEQ ID NO: 232), TLVYV (SEQ ID NO: 233), NHDWI (SEQ ID NO: 234), TVGIV (SEQ ID NO: 235), SLPTV (SEQ ID NO: 236), TGILC (SEQ ID NO: 237), TDTYI (SEQ ID NO: 238), LPVTY (SEQ ID NO: 239), GDVYI (SEQ ID NO: 240), LYGTV (SEQ ID NO: 241), GCEFI (SEQ ID NO: 242), SAGLL (SEQ ID NO: 243), IKSNI (SEQ ID NO: 244), VTTSL (SEQ ID NO: 245), AVTSV (SEQ ID NO: 246), RDIHI (SEQ ID NO: 247), SAISL (SEQ ID NO: 248), VASTC (SEQ ID NO: 249), IKGLL (SEQ ID NO: 250), GSYHT (SEQ ID NO: 251), RIGFV (SEQ ID NO: 252), NDIYI (SEQ ID NO: 253), AVSCV (SEQ ID NO: 254), QHNLL (SEQ ID NO: 255), VSSCV (SEQ ID NO: 256), LNLDV (SEQ ID NO: 257), LGATI (SEQ ID NO: 258), PVLCV (SEQ ID NO: 259), SARHI (SEQ ID NO: 260), RATLI (SEQ ID NO: 261), PYNHA (SEQ ID NO: 262), IGDSI (SEQ ID NO: 263), SPMLC (SEQ ID NO: 264), YDSTL (SEQ ID NO: 265), ALKHV (SEQ ID NO: 266), ADLLT (SEQ ID NO: 267), NNGHL (SEQ ID NO: 268), INSEV (SEQ ID NO: 269), SNKTT (SEQ ID NO: 270), GSTGL (SEQ ID NO: 271), DSDMI (SEQ ID NO: 272), TSNFI (SEQ ID NO: 273),RNFTT (SEQ ID NO: 274), SHKYS (SEQ ID NO: 275), VSDIV (SEQ ID NO: 276), RVVQA (SEQ ID NO: 277), AACAV (SEQ ID NO: 278), RGRQI (SEQ ID NO: 279), AVANI (SEQ ID NO: 280), AGYDL (SEQ ID NO: 281), LSEAA (SEQ ID NO: 282), MSNYL (SEQ ID NO: 283), NFSDN (SEQ ID NO: 284), SCCDV (SEQ ID NO: 285), LASSV (SEQ ID NO: 286), PDHAV (SEQ ID NO: 287), KFDII (SEQ ID NO: 288), NSSSA (SEQ ID NO: 289), HTMHV (SEQ ID NO: 290), TLSYC (SEQ ID NO: 291), ADTHR (SEQ ID NO: 292), SMYSV (SEQ ID NO: 293), SVNLV (SEQ ID NO: 294), MSGHL (SEQ ID NO: 295), KISDT (SEQ ID NO: 296), TGLLA (SEQ ID NO: 297), AWTTS (SEQ ID NO: 298), GGALI (SEQ ID NO: 299), SCIEV (SEQ ID NO: 300), PPVIC (SEQ ID NO: 301), and GTYNL (SEQ ID NO: 302).

[0294] In some embodiments, the variant polypeptide sequence at the VR-VII site comprises, consists essentially of, or consists of a polypeptide sequence at least about 60%, 70%, 80%, 90%, or 100% identical to one of SEQ ID NOs: 204-302.

[0295] In some embodiments, the variant polypeptide sequence at the VR-VII site comprises, consists essentially of, or consists of a sequence at least about 60%, 70%, 80%, 90%, or 100% identical to RGNQV (SEQ ID NO: 204). In some embodiments, the variant polypeptide sequence at the VR-VII site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, or 4 amino-acid substitutions relative to RGNQV (SEQ ID NO: 204). In some embodiments, the variant polypeptide sequence at the VR-VII site comprises, consists essentially of, or consists of a sequence consisting of at most 1, 2, 3, or 4 conservative amino-acid substitutions relative RGNQV (SEQ ID NO: 204). In some embodiments, the variant polypeptide sequence at the VR-VII site is RGNQV (SEQ ID NO: 204).Additional Mutations

[0296] Additional amino acid substitutions may be incorporated into the variant capsid proteins described herein, for example, to further improve transduction efficiency or tissue selectivity.

[0297] Exemplary non-limiting substitutions include, but are not limited to, S651A, T578A or T582A relative to the sequence of AAV5, in either an AAV5 or AAV9-based capsid.

[0298] In some embodiments, the capsid protein comprises a mutation selected from S651A, T578A, T582A, K251R, Y709F, Y693F, or S485A relative to the sequence of AAV5, in either an AAV5 or AAV9-based capsid. In some embodiments, the capsid protein comprisesa mutation selected from K251R, Y709F, Y693F, or S485A relative to the sequence of AAV5, in either an AAV5 or AAV9-based capsid.AAV9

[0299] In some embodiments, an engineered AAV9 capsid protein comprises a variant polypeptide sequence at the VR-VIII site described herein.

[0300] In some embodiments, the AAV9 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 1. In some embodiments, the AAV9 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 487. In some embodiments, the AAV9 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 486. The N-terminal residue of VP1, VP2, and VP3, as well as the VR sites (VR-IV, VR-V, VR-VII and VR-VIII), are indicated (in bold, and underlined) in the sequence of full-length VP1 (SEQ ID NO: 1) below. The wild type AAV9 VP1 has the amino acid sequence of SEQ ID NO: 1. The wild type AAV9 VP2 has the amino acid sequence of SEQ ID NO: 486. The wild type AAV9 VP3 has the amino acid sequence of SEQ ID NO: 487.

[0301] As labeled in the SEQ ID NO: 1 above, the VR-IV site is between residues 452 and 460 in the parental sequence (“NGSGQNQ”, SEQ ID NO: 2); the VR-V site is between residues 497 and 502 in the parental sequence (“NNSEFA”, SEQ ID NO: 3); the VR-VII site is between residues 549 and 553 in the parental sequence (“GRDNV”, SEQ ID NO: 4); the VR-VIII site is between residues 581 and 594 in the parental sequence (“ATNHQSAQAQAQTG”, SEQ ID NO: 5). In some embodiments, the AAV9 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 1, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAV9 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 1, excluding the VR-VIII site. In some embodiments, the AAV9 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 487, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAV9 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 487, excluding the VR-VIII site. In some embodiments, the AAV9 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 486, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAV9 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 486, excluding the VR-VIII site.AAV 5

[0302] In some embodiments, an engineered AAV5 capsid protein comprises a variant polypeptide sequence at the VR-VIII site described herein.

[0303] In some embodiments, the AAV5 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 322. In some embodiments, the AAV5 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 324. In some embodiments, the AAV5 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 323. The N-terminal residue of VP1, VP2, and VP3, as well as the VR-VIII site, are indicated (in bold, and underlined) in the sequence of full-length VP 1 (SEQ ID NO: 322) below. The wild type AAV5 VP1 has the aminoacid sequence of SEQ ID NO: 322. The wild type AAV5 VP2 has the amino acid sequence of SEQ ID NO: 323. The wild type AAV5 VP3 has the amino acid sequence of SEQ ID NO: 324.

[0304] As labeled in the SEQ ID NO: 322 above, the VR-IV site is between amino acids 437 and 461 in the parental sequence (“RFVSTNNTGGVQFNKNLAGRYANTY”, SEQ ID NO: 1054); the VR-V site is between amino acids 477 and 490 in the parental sequence (“LGSGVNRASVSAFA”, SEQ ID NO: 1055); the VR-VII site is between amino acids 533 and 546 in the parental sequence (“PANPGTTATYLEGN”, SEQ ID NO: 1056); the VR-VIII site is between amino acids 570 and 583 in the parental sequence (“ATNNQSSTTAPATG”, SEQ ID NO: 1057). In some embodiments, the AAV5 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 322, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAV5 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 322, excluding the VR-VIII site. In some embodiments, the AAV5 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 324, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAV5 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 324, excluding the VR-VIII site. In some embodiments, the AAV5 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,99.5%, or 100% identity to SEQ ID NO: 323, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAV5 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 323, excluding the VR-VIII site.AAVrh.10

[0305] In some embodiments, an engineered AAVrh.10 capsid protein comprises a variant polypeptide sequence at the VR-VIII site described herein.

[0306] In some embodiments, the AAVrh.10 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 329, as shown below. In some embodiments, the AAV rh.10 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 331. In some embodiments, the AAV rh.10 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 330. The N-terminal residue of VP1, VP2, and VP3, as well as the VR-VIII site, are indicated (in bold, and underlined) in the sequence of full-length VP 1 (SEQ ID NO: 329) below. The wild type AAV rh.10 VP 1 has the amino acid sequence of SEQ ID NO: 329. The wild type AAV rh.10 VP2 has the amino acid sequence of SEQ ID NO: 330. The wild type AAV rh.10 VP3 has the amino acid sequence of SEQ ID NO: 331.

[0307] As labeled in AAVrh.10 VP1 (SEQ ID NO: 329) above, the VR-IV site is between amino acids 449 and 464 in the parental sequence (“SRTQSTGGTAGTQQLL”, SEQ ID NO: 1058); the VR-V site is between amino acids 493 and 506 in the parental sequence (“TTLSQNNNSNFAWT”, SEQ ID NO: 1059); the VR-VII site is between amino acids 549 and 559 in the parental sequence (“GAGKDNVDYSS”, SEQ ID NO: 1060); the VR-VIII site is between amino acids 583 and 596 in the parental sequence (“ADNLQQQNAAPIVG”, SEQ ID NO: 1061). In some embodiments, the AAVrh.10 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 329, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAVrh.10 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 329, excluding the VR-VIII site. In some embodiments, the AAVrh.10 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 331, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAVrh.10 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 331, excluding the VR-VIII site. In some embodiments, the AAVrh.10 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 330, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAVrh.10 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 330, excluding the VR-VIII site.AA Vrh. 74

[0308] In some embodiments, an engineered AAVrh.74 capsid protein comprises a variant polypeptide sequence at the VR-VIII site described herein.

[0309] In some embodiments, the AAVrh.74 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 336, as shown below. In some embodiments, the AAV rh.74 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 338. In some embodiments, the AAV rh.74 capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQID NO: 337. The N-terminal residue of VP1, VP2, and VP3, as well as the VR-VIII site, are indicated (in bold, and underlined) in the sequence of full-length VP 1 (SEQ ID NO: 336) below. The wild type AAV rh.74 VP1 has the amino acid sequence of SEQ ID NO: 336. The wild type AAV rh.74 VP2 has the amino acid sequence of SEQ ID NO: 337. The wild type AAV rh.74 VP3 has the amino acid sequence of SEQ ID NO: 338.

[0310] As labeled in AAVrh.74 VP1 (SEQ ID NO: 336) above, the VR-IV site is between amino acids 449 and 464 in the parental sequence (“SRTQSTGGTAGTQQLL”, SEQ ID NO: 1062); the VR-V site is between amino acids 493 and 506 in the parental sequence (“TTLSQNNNSNFAWT”, SEQ ID NO: 1063); the VR-VII site is between amino acids 549 and 559 in the parental sequence (“GAGKDNVDYSS”, SEQ ID NO: 1064); the VR-VIII site is between amino acids 583 and 596 in the parental sequence (“ADNLQQQNAAPIVG”, SEQ ID NO: 1065). In some embodiments, the AAVrh.74 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 336, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAVrh.74 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 336, excluding the VR-VIII site. In some embodiments, the AAVrh.74 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,99.5%, or 100% identity to SEQ ID NO: 338, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAVrh.74 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 338, excluding the VR-VIII site. In some embodiments, the AAVrh.74 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 337, excluding the VR-IV site, VR-V site, VR-VII site and / or the VR-VIII site. In some embodiments, the AAVrh.74 capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 337, excluding the VR-VIII site.Chimeric AA V capsid proteins

[0311] In some embodiments, a chimeric AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site described herein.

[0312] In some embodiments, the AAV capsid protein is any chimeric capsid protein described herein or known in the art.Chimeric AA V5 / AA V9 Capsid

[0313] In some embodiments, the AAV capsid protein is a chimeric AAV5 / AAV9 capsid protein.

[0314] In some embodiments, the AAV capsid protein described herein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 463. (In SEQ ID NO:463, the amino acids residues labeled “X” are excluded from sequence identity calculation.) In some embodiments, the AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site of a parental sequence, wherein the parental sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 463. In some embodiments, the AAV capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to SEQ ID NO: 463, excluding the VR-VIII site. (In SEQ ID NO:463, the amino acids residues labeled “X” are excluded from sequence identity calculation.)

[0315] In some embodiments, the AAV5 / AAV9 chimeric capsid protein sequence is more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to the AAV9 capsid protein sequence (SEQ ID NO: 1). In some embodiments, the C-terminal 500 residues of the AAV5 / AAV9 chimeric capsid protein sequence is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to the C-terminal 500 residues of the AAV9 capsid protein sequence (SEQ ID NO: 1). In some embodiments, the residue at the position equivalent to Q688 of the AAV9 capsid protein sequence (SEQ ID NO: 1) is a lysine (K) in the chimeric capsid protein.

[0316] In some embodiments, the chimeric capsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptide segments that are derived from AAV5 capsid protein. In some embodiments, the chimeric capsid protein comprises at least 1, 2, 3, 4, 5 or more polypeptide segments that are derived from AAV9 capsid protein. In some embodiments, at least one polypeptide segment is derived from the AAV5 capsid protein and at least one polypeptide segment is derived from the AAV9 capsid protein.

[0317] In some embodiments, the first 250 residues at the N-terminus of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, the first 225 residues at the N-terminus of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, the first 200 residues at the N-terminus of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, the first 150 residues at the N-terminus of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, the first 100 residues at the N-terminus of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, the first 50 residues at the N-terminus of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, each of the one or more AAV5 capsid derived polypeptide segments has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the corresponding AAV5 capsid sequence.

[0318] In some embodiments, residues 50-250 of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, residues 50-200 of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, residues 50-150 of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, residues 100-250 of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, residues 100-200 of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, residues 150-250 of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptidesegments. In some embodiments, each of the one or more AAV5 capsid derived polypeptide segments has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the corresponding AAV5 capsid sequence.

[0319] In some embodiments, the last 100 residues at the C-terminus of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, the last 50 residues at the C-terminus of the chimeric capsid protein comprise one or more AAV5 capsid derived polypeptide segments. In some embodiments, each of the one or more AAV5 capsid derived polypeptide segments has at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the corresponding AAV5 capsid sequence. In some embodiments, the chimeric capsid protein comprises one or more AAV5 capsid derived polypeptide segments at or near the N-terminus of the chimeric capsid protein, as described above, and one or more AAV5 capsid derived polypeptide segments at or near the C-terminus of the chimeric capsid protein, as described in this paragraph.

[0320] In some embodiments, the chimeric capsid protein comprises, in N-terminal to C- terminal order, a first polypeptide segment having sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 411 or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 412; a second polypeptide segment having sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 413 or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 414; a third polypeptide segment having sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 415 or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 416; a fourth polypeptide segment having sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 417 or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 418; and a fifth polypeptide segment having sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 419 or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical to SEQ ID NO: 420. In some embodiments, at least one polypeptide segment is derived from the AAV5 capsid protein and at least one polypeptide segment is derived from the AAV9 capsid protein.

[0321] In some embodiments, the chimeric capsid protein comprises, consists essentially of, or consists of a polypeptide sequence at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 99.5%, or 100% identical to one of SEQ ID NOs: 421-444, or a functional fragment thereof.Table 7. Chimeric Capsid Protein SequencesExemplary embodiments of other AA V capsid proteins

[0322] In some embodiments, another AAV capsid protein known in the art or described herein comprises a variant polypeptide sequence at the VR-VIII site described herein.

[0323] In some embodiments, the capsid protein is any AAV capsid protein known in the art or described herein.

[0324] In some embodiments, the capsid protein is an AAV-SLB101 capsid protein or a variant thereof as known in the art or described in, e.g., WO 2021 / 072197, which is incorporated by reference herein in its entirety (See SEQ ID NO: 834). In some embodiments, the capsid protein is an AAVmod capsid protein or a variant thereof as known in the art or described in, e.g., WO 2022 / 173847 or in Olivieri et al. (2021) 24thAnnual Meeting of the American Society of Gene & Cell Therapy, both of which are incorporated by reference herein in their entirety. In some embodiments, the capsid protein is an AAVmutldecl(SEQ ID NO: 835), AAVdeco1(SEQ ID NO: 836), and / or AAV1111111(SEQ ID NO: 837) capsid protein or a variant thereof as known in the art or described in, e.g., WO 2022 / 173847. In some embodiments, the capsid protein is an AAVcc.47 capsid protein (SEQ ID NO: 838) or a variant thereof as known in the art or described in, e.g., Gonzalez et al. Nature Communications 13:5947 (2022), which is incorporated by reference herein in its entirety. In some embodiments, the capsid protein is an AAVHSC16 capsid protein (SEQ ID NO: 839) or a variant thereof as known in the art or described in, e.g., Smith et al. Molecular Therapy Methods & Clinical Development 26:224- 238 (2022), which is incorporated by reference herein in its entirety. In some embodiments, the capsid protein is a MyoAAV capsid protein or variant thereof as known in the art or described in, e.g., Tabebordbar et al. Cell 184(19):4919-4938. (2021), which is incorporated by reference herein in its entirety. In some embodiments, the capsid protein is an MyoAAV-4E (SEQ ID NO: 840), MyoAAV-3F (SEQ ID NO: 841), MyoAAV-4A (SEQ ID NO: 842), or MyoAAV- 4D capsid protein (SEQ ID NO: 843) or variant thereof as known in the art or described in, e.g., Tabebordbar et al. In some embodiments, the capsid protein is an 4D-C102 (SEQ ID NO: 844) or Cl 02 (SEQ ID NO: 845) capsid protein or a variant thereof as known in the art or described in, e.g., US2021 / 0380643.

[0325] In some embodiments, the AAV capsid protein comprises a variant polypeptide sequence at the VR-VIII site of a parental sequence, wherein the parental sequence comprises a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to any one of the above-listed sequences. In some embodiments, the AAV capsid protein comprises a sequence that shares at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity to any one of the above-listed sequences, excluding the VR-VIII site.Transduction Efficiency, Tropism, and nAb Evasion

[0326] Transduction efficiency can be determined using methods known in the art or those described in the Examples. In some embodiments, the rAAV virion with engineered capsidprotein exhibits increased transduction efficiency in cardiac cells compared to an AAV virion comprising the parental sequence. The rAAV virion referenced in this section is any rAAV virion with modified or engineered capsid protein described herein.

[0327] In some embodiments, the rAAV virion exhibits increased transduction efficiency in human cardiac fibroblast (hCF) cells compared to an AAV virion comprising the parental sequence. In some embodiments, the human cardiac fibroblasts are located in the left ventricle of the heart.

[0328] In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-,10-, 11-, 12-, 13-, 14, or 15-fold increased transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, the rAAV virion exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3-fold increased transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10,11-, 12-, 13-, 14, or 15-fold increased transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, the rAAV virion exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 100,000.

[0329] In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold increased transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 1,000. In some embodiments, the rAAV virion exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3-fold increased transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 1,000. In some embodiments, the rAAV virion exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased transduction efficiency in hCF cells at a multiplicity of infection (MOI) of 1,000.

[0330] In some embodiments, the rAAV virion exhibits increased transduction efficiency in induced pluripotent stem cell-derived cardiomyocyte (iPS-CM) cells compared to an AAV virion comprising the parental sequence. Accordingly, the fold improvement discussed in this section is as compared to an AAV virion comprising the parental sequence (e.g., AAV9).

[0331] In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold increased transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, the rAAV virion exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3-fold increased transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 100,000. In some embodiments, the rAAV virion exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 100,000.

[0332] In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold increased transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 75,000. In some embodiments, the rAAV virion exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3-fold increased transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 75,000. In some embodiments, the rAAV virion exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 75,000.

[0333] In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold increased transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 1,000. In some embodiments, the rAAV virion exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10- fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3 -fold increased transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 1,000. In some embodiments, the rAAV virion exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased transduction efficiency in iPS-CM cells at a multiplicity of infection (MOI) of 1,000.

[0334] In some embodiments, the rAAV virion comprising the engineered capsid protein of the present disclosure exhibits increased transduction efficiency in heart compared to an AAV virion comprising the parental sequence. In some embodiments, transduction efficiencyin heart is monitored by injecting C57BL / 6J mice with either AAV:CAG-GFP (e.g., AAV9:CAG-GFP) or CAG-GFP encapsulated by the engineered capsid protein of the present disclosure. In some embodiments, the injection dosage is 2.5E+11 vg / mouse. In some embodiments, the injection dosage is 2E+11 vg / mouse. In some embodiments, the injection dosage is 1E+11 vg / mouse. In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold increased transduction efficiency in heart. In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold increased transduction efficiency in heart relative to wild-type AAV9. In some embodiments, the rAAV virion exhibits about 2- to about 16-fold, about 2- to about 14- fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3 -fold increased transduction efficiency in heart. In some embodiments, the rAAV virion exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3 -fold increased transduction efficiency in heart relative to wild-type AAV9. In some embodiments, the rAAV virion exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased transduction efficiency in heart. In some embodiments, the rAAV virion exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased transduction efficiency in heart relative to wild-type AAV9.

[0335] In some embodiments, the rAAV virion comprising the engineered capsid protein of the present disclosure exhibits decreased transduction efficiency in liver cells compared to an AAV virion comprising the parental sequence. In some embodiments, liver transduction efficiency is monitored by injecting C57BL / 6J mice with either AAV9:CAG-GFP or CAG-GFP encapsulated by the engineered capsid protein of the present disclosure. In some embodiments, the injection dosage is 2.5E+11 vg / mouse. In some embodiments, the injection dosage is 2E+11 vg / mouse. In some embodiments, the injection dosage is 1E+11 vg / mouse. In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold decreased transduction efficiency in liver. In some embodiments, the injection dosage is 1E+11 vg / mouse. In some embodiments, the rAAV virion exhibits at least 2-, 3-, 4-, 5-, 6, 7- , 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold decreased transduction efficiency in liver relative to wild-type AAV9. In some embodiments, the rAAV virion exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3 -fold decreased transduction efficiency in liver. In some embodiments, the rAAV virion exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3-fold decreased transduction efficiency in liver relative to wild-type AAV9. In some embodiments, the rAAV virion exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, or about 80% to 100 decreased transduction efficiency in liver. In some embodiments, the rAAV virion exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, or about 80% to 100 decreased transduction efficiency in liver relative to wild-type AAV9.

[0336] Selectivity for a cell type and / or a tissue / organ type is increased when the ratio of the transduction efficiencies for one cell / tissue / organ type over another is increased for rAAV virions comprising the engineered capsid protein of the present disclosure compared to an AAV virion comprising the parental sequence. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits increased selectivity for iPS-CM cells over liver cells. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits increased selectivity for heart over liver when injected in vivo. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits increased selectivity for the left ventricle of the heart over liver when injected in vivo.

[0337] In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold increased selectivity of iPS-CM cells over liver cells and / or heart over liver. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3-fold increased selectivity of iPS- CM cells over liver cells and / or heart over liver. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased selectivity of iPS-CM cells over liver cells and / or heart over liver.

[0338] In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold increased selectivity of heart tissue over liver tissue. In some embodiments, the rAAV virion comprising theengineered capsid protein exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3-fold increased selectivity of heart tissue over liver tissue. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits at least or more than 30%, 40%, 50%, 80%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800% or 1000% increased selectivity of heart tissue over liver tissue. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased selectivity of heart tissue over liver tissue.

[0339] In some embodiments, the rAAV virion comprising the engineered capsid protein of the present disclosure exhibits improved ability to evade human nAb (neutralizing antibodies) compared to an AAV virion comprising the parental sequence. In some embodiments, the ability to evade human nAb is measured via an nAb inhibition assay. Nonlimiting examples of nAb inhibition assays are described in the Example section of the present disclosure. In some embodiments, nAb inhibition assays are performed by incubating AAV virions with pooled human nAb (e.g., IgG) before treating a target cell at a pre-determined MOI and measure the decrease of transduction efficiency compared to AAV virions not incubated with pooled human nAb. Less nAb inhibition indicates improved ability of the AAV virion to evade human nAb. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10, 11-, 12-, 13-, 14, or 15-fold improved ability to evade human nAb. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3 -fold improved ability to evade human nAb. In some embodiments, the rAAV virion comprising the engineered capsid protein exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% improved ability to evade human nAb.

[0340] In some embodiments, any rAAV further comprising N452K mutation as described herein exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold increased transduction efficiency in heart relative to wild-type AAV9 and / or relative to transduction of liver. In some embodiments, any rAAV comprising N452K mutation as described hereinexhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3-fold increased transduction efficiency in heart relative to wild-type AAV9 and / or relative to transduction of liver. In some embodiments, any rAAV virion comprising N452K mutation as described herein exhibits at least or more than 30%, 40%, 50%, 80%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800% or 1000% increased transduction efficiency in heart relative to wild-type AAV9 and / or relative to transduction of liver. In some embodiments, any rAAV comprising N452K mutation as described herein exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, about 80% to 100%, about 100% to 125%, about 125% to 150%, about 150% to 175%, or about 175% to 200% increased transduction efficiency in heart relative to wild-type AAV9 and / or relative to transduction of liver.

[0341] In some embodiments, any rAAV further comprising N452K mutation as described herein exhibits at least 2-, 3-, 4-, 5-, 6, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14, or 15-fold decreased transduction efficiency in liver relative to wild-type AAV9. In some embodiments, any rAAV comprising N452K mutation as described herein exhibits about 2- to about 16-fold, about 2- to about 14-fold, about 2- to about 12-fold, about 2- to about 10-fold, about 2- to about 8-fold, about 2- to about 6-fold, about 2- to about 4-fold, or about 2- to about 3 -fold decreased transduction efficiency in liver relative to wild-type AAV9. In some embodiments, any rAAV virion comprising N452K mutation as described herein exhibits at least or more than 30%, 40%, 50%, 80%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800% or 1000% decreased transduction efficiency in liver relative to wild-type AAV9. In some embodiments, any rAAV comprising N452K mutation as described herein exhibits about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 80%, or about 80% to 100 decreased transduction efficiency in liver relative to wild-type AAV9.Gene Products / T ransgenes

[0342] The transgenes and gene products described herein are non-limiting. Any transgene encoding any gene product may be used in the rAAV virions described herein.

[0343] In some embodiments, the rAAV virion of the present disclosure comprises a viral vector comprising a transgene.

[0344] A transgene can be a gene or nucleotide sequence that encodes a product, or a functional fragment thereof. A product can be, for example, a polypeptide or a non-coding nucleotide. By non-coding nucleotide, it is meant that the sequence transcribed from thetransgene or nucleotide sequence is not translated into a polypeptide. In some embodiments, the product encoded by the transgene or nucleotide operably linked to an enhancer described herein is a non-coding polynucleotide. A non-coding polynucleotide can be an RNA, such as for example a microRNA (miRNA or mIR), short hairpin RNA (shRNA), long non-coding RNA (InRNA), and / or a short interfering RNA (siRNA). In some embodiments, the transgene encodes a product natively expressed by a cardiac cell, e.g., a cardiomyocyte.

[0345] In some embodiments, the transgene encodes a polypeptide. In some embodiments, the transgene encodes a non-coding polynucleotide such as, for example, a microRNA (miRNA or mIR).

[0346] In some embodiments, the transgene comprises a nucleotide sequence encoding a human protein. In some embodiments, the transgene comprises a human nucleotide sequence (a human DNA sequence). In some embodiments, the transgene comprises a DNA sequence that has been codon-optimized. In some embodiments, the transgene comprises a nucleotide sequence encoding a wild-type protein, or a functionally active fragment thereof. In some embodiments, the transgene comprises a nucleotide sequence encoding a variant of a wild-type protein, such as a functionally active variant thereof.

[0347] In some embodiments, the transgene comprises a sequence encoding a product selected from vascular endothelial growth factor (VEGF), a VEGF isoform, VEGF-A, VEGF- B, VEGF-C, VEGF-D, VEGF-DdNdc, VEGF-AH6A, VEGF-A165, VEGF-A121, VEGF-2, placenta growth factor (PIGF), fibroblast growth factor 4 (FGF-4), human growth factor (HGF), human granulocyte colony-stimulating factor (hGCSF), and hypoxia inducible factor la (HIF- la).

[0348] In some embodiments, the transgene comprises a sequence encoding a product selected from SERCA2a, stromal cell-derived factor- 1 (SDF-1), adenylyl cyclase type 6, S100A1, miRNA-17-92, miR-302-367, anti-miR-29a, anti-miR-30a, antimiR-141, cyclin A2, cyclin-dependent kinase 2, Tbx20, miRNA-590, miRNA- 199, anti-sense oligonucleotide against Lp(a), interfering RNA against PCSK9, anti-sense oligonucleotide against apolipoprotein C-III, lipoprotein lipaseS447X, anti-sense oligonucleotide against apolipoprotein B, anti-sense oligonucleotide against c-myc, and E2F oligonucleotide decoy.

[0349] In some embodiments, the transgene encodes a gene product whose expression complements a defect in a gene responsible for a genetic disorder. In some embodiments, the disclosure provides, without limitation, polynucleotides encoding one or more of thefollowing — e.g. , for use, without limitation, in the disorder indicated in parentheses, or for other disorders caused by each: TAZ (Barth syndrome); FXN (Freidrich’s Ataxia); CASQ2 (CPVT); FBN1 (Marfan); RAFI and SOS Is (Noonan); SCN5A (Brugada); KCNQ1 and KCNH2s (Long QT Syndrome); DMPK (Myotonic Dystrophy 1); LMNA (Limb Girdle Dystrophy Type IB); JUP (Naxos); TGFBR2 (Loeys-Dietz); EMD (X-Linked EDMD); and ELN (SV Aortic Stenosis). In some embodiments, a polynucleotide encodes one or more of: cardiac troponin T (TNNT2); BAG family molecular chaperone regulator 3 (BAG3); myosin heavy chain (MYH7); tropomyosin 1 (TPM1); myosin binding protein C (MYBPC3); 5 ’-AMP-activated protein kinase subunit gamma-2 (PRKAG2); troponin I type 3 (TNNI3); titin (TTN); myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1); myocyte enhancer factor 2c (MEF2C); and cardiac LIM protein (CSRP3).

[0350] In some embodiments, the transgene comprises a nucleotide sequence encoding a protein selected from DWORF, junctophilin (e.g., JPH2), BAG family molecular chaperone regulator 3 (BAG3), phospholamban (PLN), alpha-crystallin B chain (CRY AB), LMNA (such as Lamin A and Lamin C isoforms), troponin I type 3 (TNNI3), lysosomal-associated membrane protein 2 (LAMP2, such as LAMP2a, LAMP2b and LAMP2c isoforms), desmoplakin (DSP, such as DPI and DPII isoforms), desmoglein 2 (DSG2), junction plakoglobin (JUP), and plakophilin-2 (PKP2). In some embodiments, the transgene comprises a nucleotide sequence encoding a matrix metallopeptidase 11 (MMP11) protein, a synaptopodin 2 like (SYNPO2L) protein (e.g., SYNPO2LA or SYNPO2LA), or an RNA binding motif protein 20 (RBM20). In some embodiments, the transgene comprises a nucleotide sequence encoding an inhibitory oligonucleotide targeting metastasis suppressor protein 1 (MTSS1).

[0351] In some embodiments, the transgene in the viral vector (such as that in the rAAV virion of the present disclosure) is selected from DWORF, JPH2, BAG3, CRY AB, LMNA (e.g., Lamin A isoform of LMNA, or Lamin C isoform of LMNA), TNNI3, PLN, LAMP2 (e.g, LAMP2a, LAMP2b, or LAMP2c), DSP (e.g., DPI isoform of DSP or DPII isoform of DSP), DSG2 and JUP.

[0352] In some embodiments, the transgene comprises a polynucleotide sequence encoding a MYBPC3 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human MYBPC3 polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 811. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding MYBPC3,e.g., human MYBPC3. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 811. In some embodiments, the MYBPC3 polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 815. In some embodiments, the MYBPC3 polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 815.

[0353] In some embodiments, the transgene comprises a polynucleotide sequence encoding a MYBPC3-delC3 variant polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 812. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding MYBPC3-delC3. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 812. In some embodiments, the MYBPC3-delC3 variant polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 816. In some embodiments, the MYBPC3-delC3 variant polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:816.

[0354] In some embodiments, the transgene comprises a polynucleotide sequence encoding a MYBPC3-delC4 variant polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 813. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding MYBPC3-delC4. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 813. In some embodiments, the MYBPC3-delC4 variant polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 817. In some embodiments, the MYBPC3-delC4 variant polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:817.

[0355] In some embodiments, the transgene comprises a polynucleotide sequence encoding a MYBPC3-delC4b variant polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 814. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding MYBPC3-delC4b. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 814. In some embodiments, the MYBPC3-delC4b variant polypeptide comprises, essentially consists of, orconsists of SEQ ID NO: 818. In some embodiments, the MYBPC3-delC4b variant polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 818.

[0356] In some embodiments, the transgene comprises a polynucleotide sequence encoding a DWORF polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human DWORF polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 827 or SEQ ID NO:828. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding DWORF, e.g., human DWORF. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 827 or SEQ ID NO:828. In some embodiments, the DWORF polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 826. In some embodiments, the DWORF polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 826.

[0357] In some embodiments, the transgene comprises a polynucleotide sequence encoding a junctophilin 2 (JPH2) polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a full-length JPH2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human JPH2 polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 782. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding JPH2, e.g., human JPH2. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 782. In some embodiments, the JPH2 polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 783. In some embodiments, the JPH2 polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 783.

[0358] In some embodiments, the transgene comprises a polynucleotide sequence encoding an N-terminal fragment of the JPH2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding an N-terminal fragment of the JPH2 polypeptide, which retains the JPH2 activity. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 809. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding N-terminal fragment of JPH2. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%,80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 809. In some embodiments, the N-terminal fragment of JPH2 polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 808. In some embodiments, the N-terminal fragment of JPH2 polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 808.

[0359] In some embodiments, the transgene comprises a polynucleotide sequence encoding a BAG3 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human BAG3 polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 785. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding BAG3, e.g., human BAG3. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 785. In some embodiments, the BAG3 polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 784. In some embodiments, the BAG3 polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 784.

[0360] In some embodiments, the transgene comprises a polynucleotide sequence encoding a C 151R mutant form of B AG3 polypeptide. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding a Cl 51R mutant form ofBAG3 polypeptide. In some embodiments, a C151R mutant form of BAG3 polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 829. In some embodiments, a C151R mutant form of BAG3 polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 829.

[0361] In some embodiments, the transgene comprises a polynucleotide sequence encoding a CRYAB polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human CRY AB polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 787. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding CRYAB, e.g., human CRY AB. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 787. In some embodiments, the CRYAB polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 786. In some embodiments, the CRY AB polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 786.

[0362] In some embodiments, the transgene comprises a polynucleotide sequence encoding a LMNA polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human LMNA polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding the LaminA isoform of LMNA. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 789. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding LaminA isoform of LMNA, e.g., human. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 789. In some embodiments, the LaminA isoform of LMNA polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 788. In some embodiments, the LMNA polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 788.

[0363] In some embodiments, the transgene comprises a polynucleotide sequence encoding the LaminC isoform of LMNA. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 791. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding LaminC isoform of LMNA, e.g., human. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 791. In some embodiments, the LaminC isoform of LMNA polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 790. In some embodiments, the LMNA polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 790.

[0364] In some embodiments, the transgene comprises a polynucleotide sequence encoding a TNNI3 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human TNNI3 polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 793. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding TNNI3, e.g., human TNNI3. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 793. In some embodiments, the TNNI3 polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 792. In some embodiments, the TNNI3 polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 792.

[0365] In some embodiments, the transgene comprises a polynucleotide sequence encoding a PLN polypeptide. In some embodiments, the transgene comprises a polynucleotidesequence encoding a human PLN polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 810. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding PLN, e.g., human PLN. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 810. In some embodiments, the PLN polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 830. In some embodiments, the PLN polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 830.

[0366] In some embodiments, the transgene comprises a polynucleotide sequence encoding a guide RNA targeting a mutant PLN gene (such as a deletious mutant of PLN, e.g., PLN-R14Del).

[0367] In some embodiments, the transgene comprises a polynucleotide sequence encoding a LAMP2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human LAMP2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding the LAMP2a isoform. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 795. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding LAMP2a, e.g., human LAMP2a. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 795. In some embodiments, the LAMP2a polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 794. In some embodiments, the LAMP2a polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 794.

[0368] In some embodiments, the transgene comprises a polynucleotide sequence encoding the LAMP2b isoform. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 797. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding LAMP2b, e.g., human LAMP2b. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 797. In some embodiments, the LAMP2b polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 796. In some embodiments, the LAMP2b polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 796.

[0369] In some embodiments, the transgene comprises a polynucleotide sequence encoding the LAMP2c isoform. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 799. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding LAMP2c, e.g., human LAMP2c. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 799. In some embodiments, the LAMP2c polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 798. In some embodiments, the LAMP2c polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 798.

[0370] In some embodiments, the transgene comprises a polynucleotide sequence encoding a DSP polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human DSP polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding the DPI isoform of DSP. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 801. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding DPI isoform of DSP, e.g., human. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 801. In some embodiments, the DPI isoform of DSP polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 800. In some embodiments, the DPI isoform of DSP polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 800.

[0371] In some embodiments, the transgene comprises a polynucleotide sequence encoding the DPII isoform of DSP. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 803. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding DPII isoform of DSP, e.g., human. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 803. In some embodiments, the DPII isoform of DSP polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 802. In some embodiments, the DPII isoform of DSP polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 802.

[0372] In some embodiments, the transgene comprises a polynucleotide sequence encoding a DSG2 polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human DSG2 polypeptide. In some embodiments, the transgenecomprises, essentially consists of, or consists of SEQ ID NO: 805. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding DSG2, e.g., human DSG2. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 805. In some embodiments, the DSG2 polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 804. In some embodiments, the DSG2 polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 804.

[0373] In some embodiments, the transgene comprises a polynucleotide sequence encoding a JUP polypeptide. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human JUP polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 807. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding JUP, e.g., human JUP. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 807. In some embodiments, the JUP polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 806. In some embodiments, the JUP polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 806.

[0374] In some embodiments, the transgene comprises a polynucleotide sequence encoding MMP11. In some embodiments, the transgene comprises a polynucleotide sequence encoding a human MMP11 polypeptide. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 819. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding MMP11, e.g., human MMP11. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 819. In some embodiments, the MMP11 polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 822. In some embodiments, the MMP11 polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 822.

[0375] In some embodiments, the transgene comprises a polynucleotide sequence encoding SYNPO2L (e.g., SYNPO2LA or SYNPO2LA). In some embodiments, the transgene comprises a polynucleotide sequence encoding a human SYNPO2L (e.g., SYNPO2LA or SYNPO2LA). In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding SYNPO2LA, e.g., human. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 820. In some embodiments, the transgenecomprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 820. In some embodiments, the SYNPO2LA polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 823. In some embodiments, the SYNPO2LA polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 823. In some embodiments, a polynucleotide sequence is a codon-optimized sequence encoding SYNPO2LB, e.g., human. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 821. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 821. In some embodiments, the SYNPO2LB polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 824. In some embodiments, the SYNPO2LB polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 824.

[0376] In some embodiments, the transgene comprises a polynucleotide sequence encoding an inhibitory oligonucleotide (e.g., siRNA) targeting MTSS1. In some embodiments, the transgene comprises a polynucleotide sequence encoding an inhibitory oligonucleotide (e.g., siRNA) targeting SEQ ID NO: 831.

[0377] In some embodiments, the transgene comprises a polynucleotide sequence encoding saCas9. In some embodiments, the transgene comprises, essentially consists of, or consists of SEQ ID NO: 832. In some embodiments, a polynucleotide sequence is a codon- optimized sequence encoding saCas9. In some embodiments, the transgene comprises a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 832. In some embodiments, the saCas9 polypeptide comprises, essentially consists of, or consists of SEQ ID NO: 833. In some embodiments, the saCas9 polypeptide has least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 833.

[0378] In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from MYBPC3, KCNH2, TRPM4, DSG2, ATP2A2, CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, S0S1, NPR1, ERBB4, VIP, MYH6, MYH7, or a mutant, variant, or fragment thereof. In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from TGFBR2, TGFBR1, EMD, KCNQ1, TAZ, COL3A1, JUP, CASQ2, MLRP44, DNAJC19, LMNA, TNNI3, DSP, DSG2, RAFI, S0S1, FBN1,LAMP2, FXN, RAFI, BAG3, KCNQ1, MYLK3, CRYAB, ALPK3 and ACTN2. In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from MYBPC3, DWORF, JPH2, BAG3, CRYAB, Lamin A isoform of LMNA, Lamin C isoform of LMNA, TNNI3, PLN, LAMP2a, LAMP2b, LAMP2c, DPI isoform of DSP, DPII isoform of DSP, DSG2, MYH6, MYH7, RBM20, and JUP.

[0379] In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from ASCL1, MYOCD, MEF2C, and TBX5. In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from ASCL1, MYOCD, MEF2C, AND TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, ZFPM2, and MIR- 133.

[0380] In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from MYBPC3, DWORF, KCNH2, TRPM4, DSG2, and ATP2A2.

[0381] In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from TGFBR2, TGFBR1, EMD, KCNQ1, TAZ, COL3A1, JUP, CASQ2, MLRP44, DNAJC19, LMNA, TNNI3, DSP, DSG2, RAFI, S0S1, FBN1, LAMP2, FXN, RAFI, BAG3, KCNQ1, MYLK3, CRYAB, ALPK3 and ACTN2.

[0382] In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, S0S1, NPR1, ERBB4, VIP, MYH6, MYH7, and Cas9. In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes saCas9.

[0383] In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from MYOCD, ASCL1, GATA4, MEF2C, TBX5, miR-133, and MESPL

[0384] In some embodiments, the rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence that encodes one or more gene products selected from MMP11, SYNPO2L (e.g., SYNPO2LA or SYNPO2LA), and an inhibitory oligonucleotide targeting MTSS1.

[0385] In some embodiments, the transgene in the rAAV virion of the present disclosure encodes any of the above-identified gene products.

[0386] In some embodiments, the capsids described herein improve heart transduction efficiency, liver viral load, and / or heart-to-liver transduction ratio of the rAAV virions carrying any of the transgenes described herein (and encoding, and resulting in the expression of, any of the gene products described herein).Additional description of capsids, transgenes and virions

[0387] Efforts to identify capsid variants with properties useful for gene therapy have included shuffling the DNA of AAV2 and AAV5 cap genes as described in U.S. Pat. No. 9,233,131; as well as directed evolution as described in Int’l Pat. Appl. Nos. WO2012 / 145601A2 and WO2018 / 222503A1. The disclosures of these documents are incorporated here for all purposes, and particularly for the methods of making and using AAV virions and for the polynucleotide sequences and gene products therein disclosed, as well as for the combinations of transcription factors useful in treating cardiac diseases or disorders.

[0388] The AAV capsid is encoded by the cap gene of AAV, which is also termed the right open-reading frame (ORF) (in contrast to the left ORF, rep). The structures of representative AAV capsids are described in various publications including Xie et al. (2002) Proc. Natl. Acad. Sci USA 99: 10405-1040 (AAV2); Govindasamy et al. (2006) J. Virol. 80: 11556-11570 (AAV4); Nam et a. (2007) J. Virol. 81 : 12260-12271 (AAV8) and Govindasamy et al. (2013) J. Virol. 87: 11187-11199 (AAV5).

[0389] The AAV capsid contain 60 copies (in total) of three viral proteins (VPs), VP1, VP2, and VP3, in a predicted ratio of 1 : 1 : 10, arranged with T=1 icosahedral symmetry. The three VPs are translated from the same mRNA, with VP1 containing a unique N-terminal domain in addition to the entire VP2 sequence at its C-terminal region. VP2 contains an extra N-terminal sequence in addition to VP3 at its C terminus. In most crystal structures, only the C-terminal polypeptide sequence common to all the capsid proteins (~530 amino acids) is observed. The N-terminal unique region of VP1, the VP1-VP2 overlapping region, and the first14 to 16 N-terminal residues of VP3 are thought to be primarily disordered. Cryo-electron microscopy and image reconstruction data suggest that in intact AAV capsids, the N-terminal regions of the VP1 and VP2 proteins are located inside the capsid and are inaccessible for receptor and antibody binding. Thus, receptor attachment and transduction phenotypes are, generally, determined by the amino acid sequences within the common C-terminal domain of VP1, VP2 and VP3.

[0390] In some embodiments, each of the VP1, VP2 and / or VP3 capsid protein comprises the VR-VIII substitution(s) or substitution motif described herein, and optionally any of the additional modifications described herein. In some embodiments, each of the VP1, VP2 and VP3 capsid proteins comprise the VR-VIII substitution(s) or substitution motif described herein, and optionally any of the additional modifications described herein. In some embodiments, an rAAV described herein comprises VP1, VP2 and VP3 capsid proteins, wherein each of the VP1, VP2 and VP3 capsid proteins comprise the VR-VIII substitution(s) or substitution motif described herein, and optionally any of the additional modifications described herein. In some embodiments, an rAAV described herein comprises a mixture of wild-type and engineered VP1, VP2 and VP3 capsid proteins, wherein the engineered VP1, VP2 and / or VP3 capsid protein comprises the VR-VIII substitution(s) or substitution motif described herein, and optionally any of the additional modifications described herein.

[0391] For the GH loop / loop IV of AAV capsid, see, e.g., van Vliet etal. (2006) Mol. Ther.14:809; Padron et al. (2005) Virol. 79:5047; and Shen et al. (2007) Afo / . Ther. 15: 1955. In some embodiments, a “parental” AAV capsid protein is a wild-type AAV9 capsid protein. In some embodiments, a “parental” AAV capsid protein is a wild-type AAV5 capsid protein. In some embodiments, a “parental” AAV capsid protein is a wild-type AAVrh.10 capsid protein. In some embodiments, a “parental” AAV capsid protein is a wild-type AAVrh.74 capsid protein. In some embodiments, a “parental” AAV capsid protein is a chimeric AAV capsid protein. Amino acid sequences of various AAV capsid proteins are known in the art. See, e.g., GenBank Accession No. NP_049542 for AAV1; GenBank Accession No. NP_044927 for AAV4; GenBank Accession No. AAD13756 for AAV5; GenBank Accession No. AAB95450 for AAV6; GenBank Accession No. YP_077178 for AAV7; GenBank Accession No. YP_077180 for AAV 8; GenBank Accession No. AAS99264 for AAV9 and GenBank Accession No. AAT46337 for AAV10. See, e.g., Santiago-Ortiz etal. (2015) Gene Ther. 22:934 for a predicted ancestral AAV capsid.

[0392] Adeno-associated virus (AAV) is a replication-deficient parvovirus, the singlestranded DNA genome of which is about 4.7 kb in length including two 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the AAV5 genome is provided in GenBank Accession No. AF085716. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology , 158: 97-129 (1992). Production of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). Illustrative AAV vectors are provided in US 7,105,345; US 15 / 782,980; US 7,259,151; US 6,962,815; US 7,718,424; US 6,984,517; US 7,718,424; US 6,156,303; US 8,524,446; US 7,790,449; US 7,906,111; US 9,737,618; US App 15 / 433,322; US 7,198,951, each of which is incorporated by reference in its entirety for all purposes.

[0393] The rAAV virions of the disclosure comprise a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene product. The gene product(s) may be either a polypeptide or an RNA, or both. When the gene product is a polypeptide, the nucleotide sequence encodes a messenger RNA, optionally with one or more introns, which is translated into the gene product polypeptide. The nucleotide sequence may encode one, two, three, or more gene products (though the number is limited by the packaging capacity of the rAAV virion, typically about 5.2 kb). The gene products may be operatively linked to one promoter (for a single transcriptional unit) or more than one. Multiple gene products may also be produced using internal ribosome entry signal (IRES) or a self-cleaving peptide (e.g., a 2 A peptide).

[0394] In some embodiments, the gene product is a polypeptide. In some embodiments, the polypeptide gene product is a polypeptide that induces reprogramming of a cardiac fibroblast, to generate an induced cardiomyocyte-like cell (iCM). In some embodiments, the polypeptide gene product is a polypeptide that enhances the function of a cardiac cell. In some embodiments, the polypeptide gene product is a polypeptide that provides a function that is missing or defective in the cardiac cell. In some embodiments, the polypeptide gene product is a genome-editing endonuclease.

[0395] In some embodiments, the gene product comprises a fusion protein that is fused to a heterologous polypeptide. In some embodiments, the gene product comprises a genome editing nuclease fused to an amino acid sequence that provides for subcellular localization, i.e..the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.).

[0396] In general, a viral vector is produced by introducing a viral DNA or RNA construct into a “producer cell” or “packaging cell” line. Packaging cell lines include but are not limited to any easily-transfectable cell line. Packaging cell lines can be based on HEK291, 293T cells, NIH3T3, COS, HeLa or Sf9 cell lines. Examples of packaging cell lines include but are not limited to: Sf9 (ATCC® CRL-1711™). Exemplary packing cell lines and methods for generating rAAV virions are provided by Int’l Pat. Pub. Nos. WO2017075627, WO20 15 / 031686, WO2013 / 063379, WO2011 / 020710, W02009 / 104964, W02008 / 024998, W02003 / 042361, and WO1995 / 013392; U.S. Pat. Nos. US9441206B2, US8679837, and US7091029B2.

[0397] In some embodiments, the gene product is a functional cardiac protein. In some embodiments, the gene product is a genome-editing endonuclease (optionally with a guide RNA, single-guide RNA, and / or repair template) that replaces or repairs a non-functional cardiac protein into a functional cardiac protein. Functional cardiac proteins include, but are not limited to cardiac troponin T; a cardiac sarcomeric protein; P-myosin heavy chain; myosin ventricular essential light chain 1; myosin ventricular regulatory light chain 2; cardiac a-actin; a-tropomyosin; cardiac troponin I; cardiac myosin binding protein C; four-and-a-half LIM protein 1; titin; 5 ’-AMP-activated protein kinase subunit gamma-2; troponin I type 3, myosin light chain 2, actin alpha cardiac muscle 1; cardiac LIM protein; caveolin 3 (CAV3); galactosidase alpha (GLA); lysosomal-associated membrane protein 2 (LAMP2); mitochondrial transfer RNA glycine (MTTG); mitochondrial transfer RNA isoleucine (MTTI); mitochondrial transfer RNA lysine (MTTK); mitochondrial transfer RNA glutamine (MTTQ); myosin light chain 3 (MYL3); troponin C (TNNC1); transthyretin (TTR); sarcoendoplasmic reticulum calcium-ATPase 2a (SERCA2a); stromal-derived factor- 1 (SDF-1); adenylate cyclase-6 (AC6); beta-ARKct (P-adrenergic receptor kinase C terminus); fibroblast growth factor (FGF); platelet-derived growth factor (PDGF); vascular endothelial growth factor (VEGF); hepatocyte growth factor; hypoxia inducible growth factor; thymosin beta 4 (TMSB4X); nitric oxide synthase-3 (NOS3); unocartin 3 (UCN3); melusin; apoplipoprotein-E (ApoE); superoxide dismutase (SOD); and S100A1 (a small calcium binding protein; see, e.g., Ritterhoff and Most (2012) Gene Ther. 19:613; Kraus el al. (2009) Afo / . Cell. Cardiol. 47:445).

[0398] In some embodiments, the gene product is a gene product whose expression complements a defect in a gene responsible for a genetic disorder. The disclosure providesrAAV virions comprising a polynucleotide encoding one or more of the following — e.g., for use, without limitation, in the disorder indicated in parentheses, or for other disorders caused by each: TAZ (Barth syndrome); FXN (Freidrich’s Ataxia); CASQ2 (CPVT); FBN1 (Marfan); RAFI and SOSls (Noonan); SCN5A (Brugada); KCNQ1 and KCNH2s (Long QT Syndrome); DMPK (Myotonic Dystrophy 1); LMNA (Limb Girdle Dystrophy Type IB); JUP (Naxos); TGFBR2 (Loeys-Dietz); EMD (X-Linked EDMD); and ELN (SV Aortic Stenosis). In some embodiments, the rAAV virion comprises a polynucleotide encoding one or more of cardiac troponin T (TNNT2); BAG family molecular chaperone regulator 3 (BAG3); myosin heavy chain (MYH7); tropomyosin 1 (TPM1); myosin binding protein C (MYBPC3); 5’-AMP- activated protein kinase subunit gamma-2 (PRKAG2); troponin I type 3 (TNNI3); titin (TTN); myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1); myocyte enhancer factor 2c (MEF2C); and cardiac LIM protein (CSRP3).

[0399] In some embodiments, the gene products of the disclosure are polypeptide reprogramming factors. Reprogramming factors are desirable as means to convert one cell type into another. Non-cardiomyocytes cells can be differentiated into cardiomyocytes cells in vitro or in vivo using any method available to one of skill in the art. For example, see methods described in leda et al. (2010) Cell 142:375-386; Christoforou et al. (2013) pLoS ONE 8:e63577; Addis et al. (2013) J. Mol. Cell Cardiol. 60:97-106; Jayawardena et al. (2012) Circ. Res. 110: 1465-1473; Nam Y et al. (2003) PNAS USA 110:5588-5593; Wada R et al. (2013) PNAS USA 110: 12667-12672; and Fu J et al. (2013) Stem Cell Reports 1:235-247.

[0400] In cardiac context, the reprogramming factors may be capable of converting a cardiac fibroblast to a cardiac myocyte either directly or through an intermediate cell type. In particular, direct reprogramming is possible, or reprogramming by first converting the fibroblast to a pluripotent or totipotent stem cell. Such a pluripotent stem cell is termed an induced pluripotent stem (iPS) cell. An iPS cell that is subsequently converted to a cardiac myocyte (CM) cell is termed an iPS-CM cell. In the examples, iPS-CM derived in vitro from cardiac fibroblasts are used in vivo to select capsid proteins of interest. The disclosure also envisions using the capsid proteins disclosure to in turn generate iPS-CM cells in vitro but, particular, in vivo, as part of a therapeutic gene therapy regimen. Induced cardiomyocyte-like (iCM) cells refer to cells directly reprogrammed into cardiomyocytes.

[0401] Induced cardiomyocytes express one or more cardiomyocyte-specific markers, where cardiomyocyte-specific markers include, but are not limited to, cardiac troponin I, cardiactroponin-C, tropomyosin, caveolin-3, myosin heavy chain, myosin light chain-2a, myosin light chain-2v, ryanodine receptor, sarcomeric a-actinin, Nkx2.5, connexin 43, and atrial natriuretic factor. Induced cardiomyocytes can also exhibit sarcomeric structures. Induced cardiomyocytes exhibit increased expression of cardiomyocyte-specific genes ACTC1 (cardiac a-actin), ACTN2 (actinin a2), MYH6 (a-myosin heavy chain), RYR2 (ryanodine receptor 2), MYL2 (myosin regulatory light chain 2, ventricular isoform), MYL7 (myosin regulatory light chain, atrial isoform), TNNT2 (troponin T type 2, cardiac), and NPPA (natriuretic peptide precursor type A), PLN (phospholamban). Expression of fibroblasts markers such as Colla2 (collagen la2) is downregulated in induced cardiomyocytes, compared to fibroblasts from which the iCM is derived.

[0402] Reprogramming methods involving polypeptide reprogramming factors (in some cases supplemented by small-molecule reprogramming factors supplied in conjunction with the rAAV) include those described in US2018 / 0112282A1, W02018 / 005546, WO2017 / 173137, US2016 / 0186141, US2016 / 0251624, US2014 / 0301991, and US2013 / 0216503 Al, which are incorporated in their entirety, particularly for the reprogramming methods and factors disclosed.

[0403] In some embodiments, cardiac cells are reprogrammed into induced cardiomyocyte-like (iCM) cells using one or more reprogramming factors that modulate the expression of one or more polynucleotides or proteins of interest, such as Achaete-scute homolog 1 (ASCL1), Myocardin (MYOCD), myocyte-specific enhancer factor 2C (MEF2C), and / or T-box transcription factor 5 (TBX5). In some embodiments, the one or more reprogramming factors are provided as a polynucleotide (e.g., an RNA, an mRNA, or a DNA polynucleotide) that encode one or more polynucleotides or proteins of interest. In some embodiments, the one or more reprogramming factors are provided as a protein.

[0404] In some embodiments, the reprogramming factors are microRNAs or microRNA antagonists, siRNAs, or small molecules that are capable of increasing the expression of one or more polynucleotides or proteins of interest. In some embodiments, expression of a polynucleotides or proteins of interest is increased by expression of a microRNA or a microRNA antagonist. For example, endogenous expression of an Oct polypeptide can be increased by introduction of microRNA-302 (miR-302), or by increased expression of miR- 302. See, e.g., Hu et al., Stem Cells 31(2): 259-68 (2013), which is incorporated herein by reference in its entirety. Hence, miRNA-302 can be an inducer of endogenous Oct polypeptide expression. The miRNA-302 can be introduced alone or with a nucleic acid that encodes theOct polypeptide. In some embodiments, a suitable nucleic acid gene product is a microRNA.Suitable microRNAs include, e.g., mir-1, mir-133, mir-208, mir-143, mir-145, and mir-499.

[0405] In some embodiments, the methods of the disclosure comprise administering an rAAV virion of the disclosure before, during, or after administration of the small-molecule reprogramming factor. In some embodiments, the small-molecule reprogramming factor is a small molecule selected from the group consisting of SB431542, LDN- 193189, dexamethasone, LY364947, D4476, myricetin, IWR1, XAV939, docosahexaenoic acid (DHA), S-Nitroso-TV- acetylpenicillamine (SNAP), Hh-Agl.5, alprostadil, cromakalim, MNITMT, A769662, retinoic acid p-hydoxyanlide, decamethonium dibromide, nifedipine, piroxicam, bacitracin, aztreonam, harmalol hydrochloride, amide-C2 (A7), Ph-C12 (CIO), mCF3-C-7 (J5), G856-7272 (A473), 5475707, or any combination thereof.

[0406] In some embodiments, the gene products comprise reprogramming factors that modulate the expression of one or more proteins of interest selected from ASCL1, MYOCD, MEF2C, and TBX5. In some embodiments, the gene products comprise one or more reprogramming factors selected from ASCL1, MYOCD, MEF2C, AND TBX5, CCNB1, CCND1, CDK1, CDK4, AURKB, OCT4, BAF60C, ESRRG, GATA4, GATA6, HAND2, IRX4, ISLL, MESP1, MESP2, NKX2.5, SRF, TBX20, ZFPM2, and miR-133.

[0407] In some embodiments, the gene products comprise GATA4, MEF2C, and TBX5 (z.e., GMT). In some embodiments, the gene products comprise MYOCD, MEF2C, and TBX5 (z.e., MyMT). In some embodiments, the gene products comprise MYOCD, ASCL1, MEF2C, and TBX5 (z.e., My AMT). In some embodiments, the gene products comprise MYOCD and ASCL1 (z.e., MyA). In some embodiments, the gene products comprise GATA4, MEF2C, TBX5, and MYOCD (z.e., 4F). In other embodiments, the gene products comprise GATA4, MEF2C, TBX5, ESSRG, MYOCD, ZFPM2, and MESP1 (z.e., 7F). In some embodiments, the gene products comprise one or more of ASCL1, MEF2C, GATA4, TBX5, MYOCD, ESRRG, AND MESPL.

[0408] In some embodiments, the rAAV virions generate cardiac myocytes in vitro or in vivo. Cardiomyocytes or cardiac myocytes are the muscle cells that make up the cardiac muscle. Each myocardial cell contains myofibrils, which are long chains of sarcomeres, the contractile units of muscle cells. Cardiomyocytes show striations similar to those on skeletal muscle cells, but unlike multinucleated skeletal cells, they contain only one nucleus. Cardiomyocytes have a high mitochondrial density, which allows them to produce ATP quickly, making them highly resistant to fatigue. Mature cardiomyocytes can express one or more of the following cardiacmarkers: a-Actinin, MLC2v, MY20, cMHC, NKX2-5, GATA4, cTNT, cTNI, MEF2C, MLC2a, or any combination thereof. In some embodiments, the mature cardiomyocytes express NKX2- 5, MEF2C or a combination thereof. In some embodiments, cardiac progenitor cells express early stage cardiac progenitor markers such as GATA4, ISL1 or a combination thereof.

[0409] In some embodiments, the gene product is a polynucleotide. In some embodiments, as described below, the gene product is a guide RNA capable of binding to an RNA-guided endonuclease. In some embodiments, the gene product is an inhibitory nucleic acid capable of reducing the level of an mRNA and / or a polypeptide gene product, e.g., in a cardiac cell. For example, in some embodiments, the polynucleotide gene product is an interfering RNA capable of selectively inactivating a transcript encoded by an allele that causes a cardiac disease or disorder. As an example, the allele is a myosin heavy chain 7, cardiac muscle, beta (MYH7) allele that comprises a hypertrophic cardiomyopathy-causing mutation. Other examples include, e.g., interfering RNAs that selectively inactivate a transcript encoded by an allele that causes hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM) or Left Ventricular Non-Compaction (LVNC), where the allele is a MYL3 (myosin light chain 3, alkali, ventricular, skeletal slow), MYH7, TNNI3 (troponin I type 3 (cardiac)), TNNT2 (troponin T type 2 (cardiac)), TPM1 (tropomyosin 1 (alpha)) or ACTC1 allele comprising an HCM-causing, a DCM-causing or a LVNC-causing mutation. See, e.g., U.S. Pat. Pub. No. 2016 / 0237430 for examples of cardiac disease-causing mutations.

[0410] In some embodiments, the gene product is a polypeptide-encoding RNA. In some embodiments, the gene product is an interfering RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide. In some embodiments, the gene product is a therapeutic polypeptide, e.g, a polypeptide that provides clinical benefit. In some embodiments, the gene product is a site-specific nuclease that provide for site-specific knock-down of gene function. In some embodiments, the gene product is an RNA-guided endonuclease that provides for modification of a target nucleic acid. In some embodiments, the gene products are: i) an RNA-guided endonuclease that provides for modification of a target nucleic acid; and ii) a guide RNA that comprises a first segment that binds to a target sequence in a target nucleic acid and a second segment that binds to the RNA-guided endonuclease. In some embodiments, the gene products are: i) an RNA-guided endonuclease that provides for modification of a target nucleic acid; ii) a first guide RNA that comprises a first segment that binds to a first target sequence in a target nucleic acid and a second segment that binds to the RNA- guided endonuclease; and iii) a first guide RNA that comprises a first segment that bindsto a second target sequence in the target nucleic acid and a second segment that binds to the RNA-guided endonuclease.

[0411] A nucleotide sequence encoding a heterologous gene product in an rAAV virion of the present disclosure can be operably linked to a promoter. For example, a nucleotide sequence encoding a heterologous gene product in an rAAV virion of the present disclosure can be operably linked to a constitutive promoter, a regulatable promoter, or a cardiac cell-specific promoter. Suitable constitutive promoters include a human elongation factor 1 a subunit (eFla) promoter, a P-actin promoter, an a-actin promoter, a P-glucuronidase promoter, CAG promoter, super core promoter, and a ubiquitin promoter. In some embodiments, a nucleotide sequence encoding a heterologous gene product in an rAAV virion of the present disclosure is operably linked to a cardiac-specific transcriptional regulator element (TRE), where cardiac-specific TREs include promoters and enhancers. Suitable cardiac-specific TREs include, but are not limited to, TREs derived from the following genes: myosin light chain-2 (MLC-2), a- myosin heavy chain (a-MHC), desmin, AE3, cardiac troponin C (cTnC), and cardiac actin. Franz et al. (1997) Cardiovasc. Res. 3 5:560-56 6; Robbins et al. (1995) Ann. NY. Acad. Sci. 752:492-505; Linn etal. (1995) Circ. Res. 76:584-591; Parmacek eta / . (1994)Afo / . Cell. Biol. 14: 1870-1885; Hunter et al. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) Proc. Natl. Acad. Sci. USA 89:4047-4051. See also, Pacak et al. (2008) Genet Vaccines Ther. 6: 13. In some embodiments, the promoter is an a-MHC promoter, an MLC-2 promoter, or cTnT promoter.

[0412] The polynucleotide encoding a gene product is operably linked to a promoter and / or enhancer to facilitate expression of the gene product. Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the rAAV virion (e.g., Bitter et al. (1987) Methods in Enzymology, 153:516-544).

[0413] Separate promoters and / or enhancers can be employed for each of the polynucleotides. In some embodiments, the same promoter and / or enhance is used for two or more polynucleotides in a single open reading frame. Vectors employing this configuration of genetic elements are termed “polycistronic.” An illustrative example of a polycistronic vector comprises an enhancer and a promoter operatively linked to a single open-reading frame comprising two or more polynucleotides linked by 2A region(s), whereby expression of the open-reading frame result in multiple polypeptides being generated co-translationally. The 2A region is believed to mediate generation of multiple polypeptide sequences through codonskipping; however, the present disclosure relates also to polycistronic vectors that employ post- translational cleavage to generate two or more proteins of interest from the same polynucleotide. Illustrative 2A sequences, vectors, and associated methods are provided in US20040265955A1, which is incorporated herein by reference.

[0414] Non-limiting examples of suitable eukaryotic promoters (promoters functional in a eukaryotic cell) include CMV, CMV immediate early, HSV thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. In some embodiments, promoters that are capable of conferring cardiac specific expression will be used. Non-limiting examples of suitable cardiac specific promoters include desmin (Des), alphamyosin heavy chain (a-MHC), myosin light chain 2 (MLC-2), cardiac troponin T (cTnT) and cardiac troponin C (cTnC). Non-limiting examples of suitable neuron specific promoters include synapsin I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase and platelet-derived growth factor beta chain promoters and hybrid promoters by fusing cytomegalovirus enhancer (E) to those neuron-specific promoters.

[0415] Examples of suitable promoters for driving expression reprogramming factors include, but are not limited to, retroviral long terminal repeat (LTR) elements; constitutive promoters such as CMV, HSV1-TK, SV40, EF-la, P-actin, phosphoglycerol kinase (PGK); inducible promoters, such as those containing Tet- operator elements; cardiac specific promoters, such as desmin (DES), alpha-myosin heavy chain (a-MHC), myosin light chain 2 (MLC-2), cardiac troponin T (cTnT) and cardiac troponin C (cTnC); neural specific promoters, such as nestin, neuronal nuclei (NeuN), microtubule-associate protein 2 (MAP2), beta III tubulin, neuron specific enolase (NSE), oligodendrocyte lineage (Oligl / 2), and glial fibrillary acidic protein (GFAP); and pancreatic specific promoters, such as Pax4, Nkx2.2, Ngn3, insulin, glucagon, and somatostatin.

[0416] In some embodiments, a polynucleotide is operably linked to a cell type-specific transcriptional regulator element (TRE), where TREs include promoters and enhancers. Suitable TREs include, but are not limited to, TREs derived from the following genes: myosin light chain-2, a-myosin heavy chain, AE3, cardiac troponin C, and cardiac actin. Franz et al. (1997) Cardiovasc. Res. 35:560-566; Robbins et al. (1995) Ann. N. Y. Acad. Sci. 752:492-505; Linn et al. (1995) Circ. Res. 76:584-591; Parmacek et al. (1994) Cell. Biol. 14: 1870-1885; Hunter etal. (1993) Hypertension 22:608-617; and Sartorelli et al. (1992) PNAS USA 89:4047- 4051.I l l

[0417] The promoter can be one naturally associated with a gene or nucleic acid segment. Similarly, for RNAs (e.g., microRNAs), the promoter can be one naturally associated with a microRNA gene (e.g., an miRNA-302 gene). Such a naturally associated promoter can be referred to as the “natural promoter” and may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Similarly, an enhancer may be one naturally associated with a nucleic acid sequence. However, the enhancer can be located either downstream or upstream of that sequence.

[0418] Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and / or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Pat. No. 4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by reference).

[0419] The promoters employed may be constitutive, inducible, developmentally-specific, tissue-specific, and / or useful under the appropriate conditions to direct high level expression of the nucleic acid segment. For example, the promoter can be a constitutive promoter such as, a CMV promoter, a CMV cytomegalovirus immediate early promoter, a CAG promoter, an EFla promoter, a HSV1-TK promoter, an SV40 promoter, a P-actin promoter, a PGK promoter, or a combination thereof. Examples of eukaryotic promoters that can be used include, but are not limited to, constitutive promoters, e.g., viral promoters such as CMV, SV40 and RSV promoters, as well as regulatable promoters, e.g., an inducible or repressible promoter such as the tet promoter, the hsp70 promoter and a synthetic promoter regulated by CRE. In certain embodiments, cell type-specific promoters are used to drive expression of reprogramming factors in specific cell types. Examples of suitable cell type-specific promoters useful for the methods described herein include, but are not limited to, the synthetic macrophage-specific promoter described in He et al (2006), Human Gene Therapy 17:949-959; the granulocyte andmacrophage-specific lysozyme M promoter (see, e.g., Faust et al (2000), Blood 96(2): 719-726); and the myeloid-specific CDl lb promoter (see, e.g., Dziennis et al (1995), Blood 85(2):319- 329). Other examples of promoters that can be employed include a human EFla elongation factor promoter, a CMV cytomegalovirus immediate early promoter, a CAG chicken albumin promoter, a viral promoter associated with any of the viral vectors described herein, or a promoter that is homologous to any of the promoters described herein (e.g., from another species). Examples of prokaryotic promoters that can be used include, but are not limited to, SP6, T7, T5, tac, bla, trp, gal, lac, or maltose promoters.

[0420] In some embodiments, an internal ribosome entry sites (IRES) element can be used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5 '-methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, Nature 334(6180):320-325 (1988)). IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, Nature 334(6180):320-325 (1988)), as well an IRES from a mammalian message (Macejak & Sarnow, Nature 353:90-94 (1991)). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

[0421] In some embodiments, a nucleotide sequence is operably linked to a polyadenylation sequence. Suitable polyadenylation sequences include bovine growth hormone poly A signal (bGHpolyA) and short poly A signal. Optionally the rAAV vectors of the disclosure comprise the Woodchuck Post-transcriptional Regulatory Element (WPRE). In some embodiments, the polynucleotide encoding gene products are join by sequences include so- called self-cleaving peptide, e.g. P2A peptides.

[0422] In some embodiments, the gene product comprises a site-specific endonuclease that provides for site-specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a cardiac disease or disorder. For example, where a dominant allele encodes a defective copy of a gene that, when wild-type, is a cardiac structural protein and / or provides for normal cardiac function, a site-specific endonuclease can be targeted to the defective allele and knock out the defective allele. In some embodiments, a site-specific endonuclease is an RNA-guided endonuclease.

[0423] In addition to knocking out a defective allele, a site-specific nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. For example, a subject rAAV virion can be used to deliver both a site-specific endonuclease that knocks out a defective allele a functional copy of the defective allele (or fragment thereof), resulting in repair of the defective allele, thereby providing for production of a functional cardiac protein (e.g., functional troponin, etc.). In some embodiments, a subject rAAV virion comprises a heterologous nucleotide sequence that encodes a site-specific endonuclease and a heterologous nucleotide sequence that encodes a functional copy of a defective allele, where the functional copy encodes a functional cardiac protein. Functional cardiac proteins include, e.g., troponin, a chloride ion channel, and the like.

[0424] Site-specific endonucleases that are suitable for use include, e.g., zinc finger nucleases (ZFNs); meganucleases; and transcription activator-like effector nucleases (TALENs), where such site-specific endonucleases are non-naturally occurring and are modified to target a specific gene. Such site-specific nucleases can be engineered to cut specific locations within a genome, and non-homologous end joining can then repair the break while inserting or deleting several nucleotides. Such site-specific endonucleases (also referred to as “INDELs”) then throw the protein out of frame and effectively knock out the gene. See, e.g, U.S. Pat. Pub. No. 2011 / 0301073. Suitable site-specific endonucleases include engineered meganuclease re-engineered homing endonucleases. Suitable endonucleases include an I-Tevl nuclease. Suitable meganucleases include I-Scel (see, e.g, Bellaiche etal. (1999) Genetics 152: 1037); and I-Crel (see, e.g, Heath et al. (1997) Nature Sructural Biology 4:468). Site-specific endonucleases that are suitable for use include CRISPRi systems and the Cas9-based SAM system.

[0425] In some embodiments, the gene product is an RNA-guided endonuclease. In some embodiments, the gene product comprises an RNA comprising a nucleotide sequence encoding an RNA-guided endonuclease. In some embodiments, the gene product is a guide RNA, e.g., a single -guide RNA. In some embodiments, the gene products are: 1) a guide RNA; and 2) an RNA-guided endonuclease. The guide RNA can comprise: a) a protein-binding region that binds to the RNA-guided endonuclease; and b) a region that binds to a target nucleic acid. An RNA-guided endonuclease is also referred to herein as a “genome editing nuclease.”

[0426] Examples of suitable genome editing nucleases are CRISPR / Cas endonucleases (e.g., class 2 CRISPR / Cas endonucleases such as a type II, type V, or type VI CRISPR / Cas endonucleases). A suitable genome editing nuclease is a CRISPR / Cas endonuclease (e.g., aclass 2 CRISPR / Cas endonuclease such as a type II, type V, or type VI CRISPR / Cas endonuclease). In some embodiments, the gene product comprises a class 2 CRISPR / Cas endonuclease. In some embodiments, the gene product comprises a class 2 type II CRISPR / Cas endonuclease (e.g., a Cas9 proteinsuch as saCas9). In some embodiments, the gene product comprises a class 2 type V CRISPR / Cas endonuclease (e.g., a Cpfl protein, a C2cl protein, or a C2c3 protein). In some embodiments, the gene product comprises a class 2 type VI CRISPR / Cas endonuclease (e.g., a C2c2 protein; also referred to as a “Casl3a” protein). In some embodiments, the gene product comprises a CasX protein. In some embodiments, the gene product comprises a CasY protein.Nucleic acids, Vectors, Cells and Generation of rAA V virions

[0427] In some embodiments, the disclosure provides a nucleic acid encoding any engineered AAV capsid protein described herein.

[0428] The polynucleotide encoding the capsid protein can comprise a sequence comprising either the native codons of the wild-type cap gene, or alternative codons selected to encode the same protein. The codon usage of the insertion can be varied. It is within the skill of those in the art to select appropriate nucleotide sequences and to derive alternative nucleotide sequences to encode any capsid protein of the disclosure. Reverse translation of the protein sequence can be performed using the codon usage table of the host organism, i.e. Eukaryotic codon usage for humans.

[0429] In some embodiments, the disclosure provides a polynucleotide encoding an AAV9 derived capsid protein comprising a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 343 or SEQ ID NO:344. In some embodiments, the disclosure provides a polynucleotide encoding an AAV9 derived capsid protein comprising a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 343 or SEQ ID NO:344, and the VR- III of SEQ ID NO: 343 or SEQ ID NO: 344.

[0430] In some embodiments, the disclosure provides a polynucleotide encoding an AAV9 derived capsid protein comprising a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NO: 846-903 and 984-1005. In some embodiments, the disclosure provides a polynucleotide encoding an AAV9 derived capsid protein comprising the VR-III of any one of SEQ ID NO:846-903 and 984-1005. In some embodiments, the disclosure provides a polynucleotide encoding an AAV9 derived capsid protein comprising a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 846, 847, 848, 849, 850, 891, 864, 867, 858, 878, 890, 870, 856, 886, 900, 901, and 898.

[0431] In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 / AAV9 chimeric capsid protein comprising a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 421-444. In some embodiments, the disclosure provides a polynucleotide encoding an AAV5 / AAV9 chimeric capsid protein comprising a sequence at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 421-444, and the VR-III of SEQ ID NO: 343 or SEQ ID NO: 344.

[0432] In some embodiments, the disclosure provides a vector or a plasmid comprising a nucleic acid encoding any AAV capsid protein described herein. In some embodiments, the vector or plasmid further comprises a promoter operably linked to the nucleic acid encoding the AAV capsid proteins. In some embodiments, the promoter is any promoter active in a cell to be used for expressing the capsid protein (e.g., a producer or host cell). In some embodiments, the promoter is P40 promoter. In some embodiments, the promoter is a polyhedrin promoter.

[0433] In some embodiments, the vector or plasmid comprising a nucleic acid encoding any AAV capsid protein described herein further comprises a nucleic acid encoding a replication (Rep) protein. In some embodiments, the Rep protein is a Rep protein from the same serotype of AAV as the inverted terminal repeats (ITRs) used to flank the transgene (to be packaged into virions using any of the AAV capsid proteins described herein). In some embodiments, the Rep protein is an AAV2 Rep protein. In some embodiments, the Rep protein is an AAV8 Rep protein. In some embodiments, the vector or plasmid comprising a nucleic acid encoding any AAV capsid protein described herein does not further comprise a nucleic acid encoding a Rep protein.

[0434] In some embodiments, the disclosure provides a cell comprising a nucleic acid encoding any AAV capsid protein described herein. In some embodiments, the disclosure provides a cell comprising a vector or a plasmid comprising a nucleic acid encoding any AAV capsid protein described herein. In some embodiments, the cell further comprises a vector or plasmid comprsing a nucleic acid encoding a Rep protein, wherein the Rep protein may be expressed by the same or different vector or plasmid as the AAV capsid protein described herein.

[0435] In some embodiments, the disclosure provides a host cell comprising a nucleic acid encoding any AAV capsid protein described herein. In some embodiments, the disclosure provides a host cell comprising a vector or a plasmid comprising a nucleic acid encoding any AAV capsid protein described herein.

[0436] In some embodiments, a host cell comprising a nucleic acid encoding any AAV capsid protein described herein is for producing an rAAV virion described herein (such as an rAAV virion comprising a modified AAV capsid protein as described herein). In some embodiments, the nucleic acid encoding any AAV capsid protein is transiently transfected into a cell. In some embodiments, the nucleic acid encoding any AAV capsid protein is stably inserted into the cell genome.

[0437] In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is selected from the group consisting of: are HEK293, HEK293T, HeLa, Vero, MDCK, MRC-5, PER.C6, BHK21 and CHO. In some embodiments, the host cell is HEK293 cell.

[0438] In some embodiments, the host cell is an insect cell. In some embodiments, the host cell is Sf9 insect cell. In some embodiments where the insect cells are used as host cells, the vectors or plasmids described herein are first introduced into a recombinant baculovirus and then carried into insect cells by baculovirus infection.

[0439] In some embodiments, the host cells are further transfected with one or more vectors or plasmids comprising helper functions and / or viral structural proteins necessary for replication and / or encapsidation of the vector(s) carrying the transgene.

[0440] In some embodiments, the host cells are further transfected with a viral vector carrying a transgene (such as any transgene described herein). In some embodiments, the transgene is flanked by inverted terminal repeats (ITRs). In some embodiments, the ITRs are of the same serotype as the Rep protein expressed in the host cells. In some embodiments, the ITRs are AAV2 ITRs. In some embodiments, the ITRs are AAV8 ITRs. Any combinations of Rep proteins and ITRs known in the art can be used in the cells and methods described herein.

[0441] In some embodiments, a host cell (e.g., a mammalian or an insect cell) further comprises a helper plasmid expression Adenovirus helper genes.

[0442] In some embodiments, a host cell comprises one or more packaging factors stably integrated into cell genome. In some embodiments, the host cell comprises a nucleic acid encoding any of the AAV capsid proteins described herein stably integrated into its genome. In some embodiments, the host cell comprises a nucleic acid encoding a Rep protein stably integrated into its genome. In some embodiments, the host cell comprises an Adenovirus helper gene stably integrated into its genome. In some embodiments, the host cell comprises a nucleicacid encoding an AAV capsid protein described herein, a nucleic acid encoding a Rep protein, and an Adenovirus helper gene(s) stably integrated into its genome.

[0443] The methods of production of rAAV virions are known in the art. In some embodiments, an rAAV virion can be generated using the host cells as described herein.

[0444] In some embodiments, the method of producing an rAAV virion in cell comprises: i. introducing (e.g., by transient transfection or stable integration techniques) a nucleic acid encoding any of the AAV capsid proteins described herein, a nucleic acid encoding a Rep protein (such as any AAV Rep protein known in the art or described herein), an Adenovirus helper gene(s) (such as any Adenovirus helper genes known in the art), and / or a transgene cassette comprising a transgene flanked by ITRs (e.g., wherein the transgene expresses a therapeutic protein) into the cell (e.g., via DNA transfection, viral infection, and / or stable integration), wherein each of the introduced nucleic acids or genes is operably linked to a promoter active in the cell; ii culturing the cell (e.g. , using a suspension cell culture or an adherent cell culture) under conditions suitable for production of an rAAV virion (e.g, suitable for packaging protein expression and / or suitable for viral packaging), and iii. collecting the produced rAAV virion (e..g, from media supernatant and / or from cell lysate following cell lysis), and iv. optionally further purifying the rAAV virion, e..g., by density gradient ultracentrifugation and / or chromatography-based methods.

[0445] In some embodiments, the vectors, promoters, packaging factors, packaging systems, host cells, and / or methods of rAAV virion production are any of those known in the art.Methods

[0446] In some embodiments, the disclosure provides methods of identifying AAV capsid proteins that confer on rAAV virions increased transduction efficiency in target cells. The methods comprise providing a population of rAAV virions whose rAAV genomes comprise a library of cap polynucleotides encoding variant AAV capsid proteins; optionally contacting the population with non-target cells for a time sufficient to permit attachment of undesired rAAV virions to the non-target cells; contacting the population with target cells for a time sufficient to permit transduction of the cap polynucleotide into the target cells by the rAAV virions; and sequencing the cap polynucleotides from the target cells, thereby identifying AAV capsidproteins that confer increased transduction efficiency in the target cells. In some embodiments, the method further comprises depleting the population of rAAV virions by contacting the population with non-target cells for time sufficient to permit attachment of the rAAV virions to the non-target cells.

[0447] The present disclosure provides methods for generating cardiomyocytes and / or cardiomyocyte-like cells in vitro using an rAAV virion. Selected starting cells are transduced with an rAAV and optionally exposed to small-molecule reprogramming factors (before, during, or after transduction) for a time and under conditions sufficient to convert the starting cells across lineage and / or differentiation boundaries to form cardiac progenitor cells and / or cardiomyocytes. In some embodiments, the starting cells are fibroblast cells. In some embodiments, the starting cells express one or more markers indicative of a differentiated phenotype. The time for conversion of starting cells into cardiac progenitor and cardiomyocyte cells can vary. For example, the starting cells can be incubated after treatment with one or more polynucleotides or proteins of interest until cardiac or cardiomyocyte cell markers are expressed. Such cardiac or cardiomyocyte cell markers can include any of the following markers: a-GATA4, TNNT2, MYH6, RYR2, NKX2-5, MEF2C, ANP, Actinin, MLC2v, MY20, cMHC, ISL1, cTNT, cTNI, and MLC2a, or any combination thereof. In some embodiments, the induced cardiomycocyte cells are negative for one or more neuronal cells markers. Such neuronal cell markers can include any of the following markers: DCX, TUBB3, MAP2, and ENO2.

[0448] Incubation can proceed until cardiac progenitor markers are expressed by the starting cells. Such cardiac progenitor markers include GATA4, TNNT2, MYH6, RYR2, or a combination thereof. The cardiac progenitor markers such as GATA4, TNNT2, MYH6, RYR2, or a combination thereof can be expressed by about 8 days, or by about 9 days, or by about 10 days, or by about 11 days, or by about 12 days, or by about 14 days, or by about 15 days, or by about 16 days, or by about 17 days, or by about 18 days, or by about 19 days, or by about 20 days after starting incubation of cells in the compositions described herein. Further incubation of the cells can be performed until expression of late stage cardiac progenitor markers such as NKX2-5, MEF2C or a combination thereof occurs.

[0449] Reprogramming efficiency may be measured as a function of cardiomyocyte markers. Such pluripotency markers include, but are not limited to, the expression of cardiomyocyte marker proteins and mRNA, cardiomyocyte morphology and electrophysiological phenotype. Non-limiting examples of cardiomyocyte markers include, a-sarcoglycan, atrial natriuretic peptide (ANP), bone morphogenetic protein 4 (BMP4), connexin 37, connexin 40, crypto, desmin, GATA4, GATA6, MEF2C, MYH6, myosin heavy chain, NKX2.5, TBX5, and Troponin T.

[0450] The expression of various markers specific to cardiomyocytes may be detected by conventional biochemical or immunochemical methods (e.g., enzyme- linked immunosorbent assay, immunohistochemical assay, and the like). Alternatively, expression of a nucleic acid encoding a cardiomyocyte- specific marker can be assessed. Expression of cardiomyocyte- specific marker-encoding nucleic acids in a cell can be confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) or hybridization analysis, molecular biological methods which have been commonly used in the past for amplifying, detecting and analyzing mRNA coding for any marker proteins. Nucleic acid sequences coding for markers specific to cardiomyocytes are known and are available through public databases such as GenBank. Thus, marker-specific sequences needed for use as primers or probes are easily determined.

[0451] Cardiomyocytes exhibit some cardiac-specific electrophysiological properties. One electrical characteristic is an action potential, which is a short-lasting event in which the difference of potential between the interior and the exterior of each cardiac cell rises and falls following a consistent trajectory. Another electrophysiological characteristic of cardiomyocytes is the cyclic variations in the cytosolic-free Ca2+concentration, named as Ca2+transients, which are employed in the regulation of the contraction and relaxation of cardiomyocytes. These characteristics can be detected and evaluated to assess whether a population of cells has been reprogrammed into cardiomyocytes.

[0452] The present disclosure provides a method of delivering a gene product to a cardiac cell, e.g., a cardiac fibroblast. The methods generally involve infecting a cardiac cell (e.g., a cardiac fibroblast) with an rAAV virion, where the gene product(s) encoded by the heterologous nucleic acid present in the rAAV virion is / are produced in the cardiac cell (e.g., cardiac fibroblast). Delivery of gene product(s) to a cardiac cell (e.g., cardiac fibroblast) can provide for treatment of a cardiac disease or disorder. Delivery of gene product(s) to a cardiac cell (e.g., cardiac fibroblast) can provide for generation of an induced cardiomyocyte-like (iCM) cell from the cardiac fibroblast. Delivery of gene product(s) to a cardiac cell (e.g., cardiac fibroblast) can provide for editing of the genome of the cardiac cell (e.g., cardiac fibroblast).

[0453] In some embodiments, infecting or transducing a cardiac cell (e.g., cardiac fibroblast) is carried out in vitro. In some embodiments, infecting or transducing a cardiac cell (e.g., cardiac fibroblast) is carried out in vitro, and the infected / transduced cardiac cell (e.g.,cardiac fibroblast) is introduced into (e.g., transfused into or implanted into) an individual in need thereof, e.g., directly into cardiac tissue of an individual in need thereof. For in vitro transduction, an effective amount of rAAV virions to be delivered to cells is from about 105to about 1013of the rAAV virions. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.

[0454] In some embodiments, infecting a cardiac cell (e.g., cardiac fibroblast) is carried out in vivo. For example, in some embodiments, an effective amount of an rAAV virion of the present disclosure is administered directly into cardiac tissue of an individual in need thereof. An “effective amount” will fall in a relatively broad range that can be determined through experimentation and / or clinical trials. For example, for in vivo injection, i.e., injection directly into cardiac tissue, a therapeutically effective dose will be on the order of from about 106to about 1015of the rAAV virions, e.g., from about 105to 1012rAAV virions, of the present disclosure. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via vascular delivery through the coronary artery. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery through the superior vena cava. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0455] For example, from about 104to about 105, from about 105to about 106, from about 106to about 107, from about 106to about 107, from about 107to about 108, from about 108to about 109, from about 109to about IO10from about IO10to about 10n,to about 1011, from about 1011to about 1012, from about 1012to about 1013, from about 1013to about 1014, from about 1014to about 1015genome copies, or more than 1015genome copies, of an rAAV virion of the present disclosure are administered to an individual, e.g., are administered directly into cardiac tissue in the individual, or are administered via another route. The number of rAAV virions administered to an individual can be expressed in viral genomes (vg) per kilogram (kg) body weight of the individual. In some embodiments, and effective amount of an rAAV virion of the present disclosure is from about 102vg / kg to 104vg / kg, from about 104vg / kg to about 106vg / kg, from about 106vg / kg to about 108vg / kg, from about 108vg / kg to about 1010vg / kg, from about IO10vg / kg to about 1012vg / kg, from about 1012vg / kg to about 1014vg / kg, from about 1014vg / kg to about 1016vg / kg, from about 1016vg / kg to about 1018vg / kg, or more than 1018vg / kg. In some embodiments, the rAAV viron is administered at, at least at, or at no more than,102vg / kg, 103vg / kg, 104vg / kg, IO5vg / kg, 106vg / kg, 108vg / kg, 109vg / kg, IO10vg / kg, IO11vg / kg, 1012vg / kg, 1013vg / kg, 2xl013vg / kg, 3xlO13vg / kg, 4xl013vg / kg, 5xlO13vg / kg, 6xl013vg / kg, 7xl013vg / kg, 8xl013vg / kg, 9xl013vg / kg, 1014vg / kg, 2xl014vg / kg, 3xl014vg / kg, 4xl014vg / kg, 5xl014vg / kg, 6xl014vg / kg, 7xl014vg / kg, 8xl014vg / kg, 9xl014vg / kg, IO15vg / kg, 1016vg / kg, 1017vg / kg, or 1018vg / kg (or at any range of amounts in between these values). In some embodiments, the rAAV virion is administered at 2xl013vg / kg. In some embodiments, the rAAV virion is administered at 1.43xl013vg / kg. In some embodiments, the rAAV virion is administered at 1.2xl014vg / kg.

[0456] In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered locally to the heart. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered by intracardiac catheterization. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via vascular delivery through the coronary artery. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery, e.g., intravenously. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery through the superior vena cava. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0457] In some embodiments, a single administration may be used to achieve the desired level of gene expression. In some embodiments, more than one administration (e.g., two, three, four or more administrations) may be employed to achieve the desired level of gene expression. In some embodiments, the more than one administration is administered at various intervals, e.g., daily, weekly, twice monthly, monthly, every 3 months, every 6 months, yearly, etc. In some embodiments, multiple administrations are administered over a period of time of from 1 month to 2 months, from 2 months to 4 months, from 4 months to 8 months, from 8 months to 12 months, from 1 year to 2 years, from 2 years to 5 years, or more than 5 years.

[0458] The present disclosure provides a method of reprogramming a cardiac fibroblast to generate an induced cardiomyocyte-like cell (iCM). The method generally involves infecting a cardiac fibroblast with an rAAV virion of the present disclosure, wherein the rAAV virion comprises a heterologous nucleic acid comprising a nucleotide sequence encoding one or more reprogramming factors.

[0459] The expression of various markers specific to cardiomyocytes is detected by conventional biochemical or immunochemical methods (e.g., enzyme-linked immunosorbent assay; immunohistochemical assay; and the like). Alternatively, expression of nucleic acid encoding a cardiomyocyte-specific marker can be assessed. Expression of cardiomyocyte- specific marker-encoding nucleic acids in a cell can be confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) or hybridization analysis, molecular biological methods which have been commonly used in the past for amplifying, detecting and analyzing mRNA coding for any marker proteins. Nucleic acid sequences coding for markers specific to cardiomyocytes are known and are available through public data bases such as GenBank; thus, marker-specific sequences needed for use as primers or probes is easily determined.

[0460] Induced cardiomyocytes can also exhibit spontaneous contraction. Whether an induced cardiomyocyte exhibits spontaneous contraction can be determined using standard electrophysiological methods (e.g., patch clamp).

[0461] In some embodiments, induced cardiomyocytes can exhibit spontaneous Ca2+oscillations. Ca2+oscillations can be detected using standard methods, e.g., using any of a variety of calcium-sensitive dyes, intracellular Ca2+ion-detecting dyes include, but are not limited to, fura-2, bis-fura 2, indo-1, Quin-2, Quin-2 AM, Benzothiaza-1, Benzothiaza-2, indo- 5F, Fura-FF, BTC, Mag-Fura-2, Mag-Fura-5, Mag-Indo-1, fluo-3, rhod-2, rhod-3, fura-4F, fura- 5F, fura-6F, fluo-4, fluo-5F, fluo-5N, Oregon Green 488 BAPTA, Calcium Green, Calcein, Fura-C18, Calcium Green-C18, Calcium Orange, Calcium Crimson, Calcium Green-5N, Magnesium Green, Oregon Green 488 BAPTA-1, Oregon Green 488 BAPTA-2, X-rhod-1, Fura Red, Rhod-5F, Rhod-5N, X-Rhod-5N, Mag-Rhod-2, Mag-X- Rhod-1, Fluo-5N, Fluo-5F, Fluo-4FF, Mag-Fluo-4, Aequorin, dextran conjugates or any other derivatives of any of these dyes, and others (see, e.g., the catalog or Internet site for Molecular Probes, Eugene, see, also, Nuccitelli, ed., Methods in Cell Biology, Volume 40: A Practical Guide to the Study of Calcium in Living Cells, Academic Press (1994); Lambert, ed., Calcium Signaling Protocols (Methods in Molecular Biology Volume 114), Humana Press (1999); W. T. Mason, ed., Fluorescent and Luminescent Probes for Biological Activity. A Practical Guide to Technology for Quantitative Real-Time Analysis, Second Ed, Academic Press (1999); Calcium Signaling Protocols (Methods in Molecular Biology), 2005, D.G. Lamber, ed., Humana Press.).

[0462] In some embodiments, an iCM is generated in vitro,' and the iCM is introduced into an individual, e.g., the iCM is implanted into a cardiac tissue of an individual in need thereof. A method of the present disclosure can comprise infecting a population of cardiac fibroblastsin vitro, to generate a population of iCMs; and the population of iCMs is implanted into a cardiac tissue of an individual in need thereof.

[0463] In some embodiments, an iCM is generated in vivo. For example, in some embodiments, an rAAV virion of the present disclosure that comprises a heterologous nucleic acid comprising a nucleotide sequence encoding one or more reprogramming factors is administered to an individual. In some embodiments, the rAAV virion is administered directly into cardiac tissue of an individual in need thereof. In some embodiments, from about 106to about 105, from about 105to about 109, from about 109to about IO10, from about IO10to about 1011, from about 1011to about 1012, from about 1012to about 1013, from about 1013to about 1014, from about 1014to about 1015genome copies, or more than 1015genome copies, of an rAAV virion of the present disclosure that comprises a heterologous nucleic acid comprising a nucleotide sequence encoding one or more reprogramming factors are administered to an individual, e.g., are administered directly into cardiac tissue in the individual or via another route of administration. The number of rAAV virions administered to an individual can be expressed in viral genomes (vg) per kilogram (kg) body weight of the individual. In some embodiments, and effective amount of an rAAV virion of the present disclosure is from about 102vg / kg to 104vg / kg, from about 104vg / kg to about 106vg / kg, from about 106vg / kg to about 108vg / kg, from about 108vg / kg to about IO10vg / kg, from about IO10vg / kg to about 1012vg / kg, from about 1012vg / kg to about 1014vg / kg, from about 1014vg / kg to about 1014vg / kg, from about 1014vg / kg to about 1016vg / kg, or more than 1016vg / kg. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via vascular delivery through the coronary artery. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery through the superior vena cava. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0464] The present disclosure provides a method of modifying (“editing”) the genome of a cardiac cell. The present disclosure provides a method of modifying (“editing”) the genome of a cardiac fibroblast. The present disclosure provides a method of modifying (“editing”) the genome of a cardiomyocyte. The methods generally involve infecting a cardiac cell (e.g., a cardiac fibroblast or a cardiomyocyte) with an rAAV virion of the present disclosure, wherein the rAAV virion comprises a heterologous nucleic acid comprising a nucleotide sequenceencoding a genome-editing endonuclease. In some embodiments, the method comprises infecting a cardiac fibroblast or a cardiomyocyte with an rAAV virion of the present disclosure, wherein the rAAV virion comprises a heterologous nucleic acid comprising a nucleotide sequence encoding an RNA-guided genome -editing endonuclease. In some embodiments, the method comprises infecting a cardiac fibroblast or a cardiomyocyte with an rAAV virion of the present disclosure, wherein the rAAV virion comprises a heterologous nucleic acid comprising a nucleotide sequence encoding: i) an RNA-guided genome-editing endonuclease; and ii) one or more guide RNAs. In some embodiments, the method comprises infecting a cardiac fibroblast or a cardiomyocyte with an rAAV virion of the present disclosure, wherein the rAAV virion comprises a heterologous nucleic acid comprising a nucleotide sequence encoding: i) an RNA- guided genome-editing endonuclease; ii) a guide RNAs; and iii) a donor template DNA. Suitable RNA-guided genome-editing endonucleases are described above.

[0465] In some embodiments, infecting a cardiac cell (e.g., cardiac fibroblast; a cardiomyocyte) is carried out in vitro. In some embodiments, infecting a cardiac cell (e.g., cardiac fibroblast; a cardiomyocyte) is carried out in vitro; and the infected cardiac cell (e.g., cardiac fibroblast) is introduced into (e.g., implanted into) an individual in need thereof, e.g., directly into cardiac tissue of an individual in need thereof. For in vitro transduction, an effective amount of rAAV virions to be delivered to cells will be on the order of from about 10sto about 1013of the rAAV virions. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.

[0466] In some embodiments, infecting a cardiac cell (e.g., cardiac fibroblast; a cardiomyocyte) is carried out in vivo. For example, in some embodiments, an effective amount of an rAAV virion of the present disclosure is administered directly into cardiac tissue of an individual in need thereof. An “effective amount” will fall in a relatively broad range that can be determined through experimentation and / or clinical trials. For example, for in vivo injection, z.e., injection directly into cardiac tissue, a therapeutically effective dose will be on the order of from about 106to about 1015of the rAAV virions, e.g., from about 1011to 1012rAAV virions, of the present disclosure. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via vascular delivery through the coronary artery. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemicdelivery through the superior vena cava. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0467] For example, from about 106to about 107, from about 107to about 108, from about 108to about 109, from about 109to about IO10, from about IO10to about 1011, from about 1011to about 1012, from about 1012to about 1013, from about 1013to about 1014, from about 1014to about 1015genome copies, or more than 1015genome copies, of an rAAV virion of the present disclosure are administered to an individual, e.g., are administered directly into cardiac tissue in the individual. The number of rAAV virions administered to an individual can be expressed in viral genomes (vg) per kilogram (kg) body weight of the individual. In some embodiments, and effective amount of an rAAV virion of the present disclosure is from about 102vg / kg to 104vg / kg, from about 104vg / kg to about 106vg / kg, from about 106vg / kg to about 108vg / kg, from about 108vg / kg to about IO10vg / kg, from about IO10vg / kg to about 1012vg / kg, from about 1012vg / kg to about 1014vg / kg, from about 1014vg / kg to about 1016vg / kg, from about 1016vg / kg to about 1018vg / kg, or more than 1018vg / kg. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via intramyocardial injection through the epicardium. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via vascular delivery through the coronary artery. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery through the superior vena cava. In some embodiments, an effective amount of an rAAV virion of the present disclosure is administered via systemic delivery through a peripheral vein.

[0468] In some embodiments, the genome editing comprises homology-directed repair (HDR). In some embodiments, the HDR corrects a defect in an endogenous target nucleic acid in the cardiac fibroblast or the cardiomyocyte, wherein the defect is associated with, or leads to, a defect in structure and / or function of the cardiac fibroblast or the cardiomyocyte, or a component of the cardiac fibroblast or the cardiomyocyte.

[0469] In some embodiments, the genome editing comprises non-homologous end joining (NHEJ). In some embodiments, the NHEJ deletes a defect in an endogenous target nucleic acid in the cardiac fibroblast or the cardiomyocyte, wherein the defect is associated with, or leads to, a defect in structure and / or function of the cardiac fibroblast or the cardiomyocyte, or a component of the cardiac fibroblast or the cardiomyocyte.

[0470] A method of the present disclosure for editing the genome of a cardiac cell can be used to correct any of a variety of genetic defects that give rise to a cardiac disease or disorder.Mutations of interest include mutations in one or more of the following genes: cardiac troponin T (TNNT2); myosin heavy chain (MYH7); tropomyosin 1 (TPM1); myosin binding protein C (MYBPC3); 5 ’-AMP-activated protein kinase subunit gamma-2 (PRKAG2); troponin I type 3 (TNNI3); titin (TTN); myosin, light chain 2 (MYL2); actin, alpha cardiac muscle 1 (ACTC1); potassium voltage-gated channel, KQT- like subfamily, member 1 (KCNQ1); myocyte enhancer factor 2c (MEF2C); and cardiac LIM protein (CSRP3). Specific mutations of interest include, without limitation, MYH7 R663H mutation; TNNT2 R173W; and KCNQ1 G269S missense mutation. Mutations of interest include mutations in one or more of the following genes: MYH6, ACTN2, SERCA2, GATA4, TBX5, MYOCD, NKX2-5, N0TCH1, MEF2C, HAND2, and HAND 1. In some embodiments, the mutations of interest include mutations in the following genes: MEF2C, TBX5, and MYOCD. Cardiac diseases and disorders that can be treated with a method of the present disclosure include coronary heart disease, cardiomyopathy, endocarditis, congenital cardiovascular defects, and congestive heart failure. Cardiac diseases and disorders that can be treated with a method of the present disclosure include hypertrophic cardiomyopathy; a valvular heart disease; myocardial infarction; congestive heart failure; long QT syndrome; atrial arrhythmia; ventricular arrhythmia; diastolic heart failure; systolic heart failure; cardiac valve disease; cardiac valve calcification; left ventricular non-compaction; ventricular septal defect; and ischemia.

[0471] In some embodiments, the disclosure provides a method of transducing a cardiac cell. In some embodiments, the disclosure provides a method of transducing a cardiac cell, comprising contacting the cardiac cell with an rAAV virion described herein, wherein the rAAV virion transduces the cardiac cell. In some embodiments, the cardiac cell is a cardiomyocyte.

[0472] In some embodiments, the disclosure provides a method of transducing a cardiac cell, comprising contacting the cardiac cell with an rAAV virion, wherein the rAAV virion comprises a capsid protein, wherein the capsid protein is any capsid protein described herein.

[0473] In some embodiments, the disclosure provides a method of transducing a cardiac cell, comprising contacting the cardiac cell with an rAAV virion, wherein the rAAV virion comprises a capsid protein, wherein the capsid protein shares at least 80%, 85% or 90% polypeptide sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1 :

[0474] In some embodiments, the disclosure provides a method of transducing a cardiac cell, comprising contacting the cardiac cell with an rAAV virion, wherein the rAAV virion comprises a capsid protein, wherein the capsid protein shares at least 80%, 85% or 90%polypeptide sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1 : amino acid substitutions T582D, N583V, H584Q, A587P, Q588G, and A589F.

[0475] In some embodiments, the disclosure provides a method of transducing a cardiac cell, comprising contacting the cardiac cell with an rAAV virion, wherein the rAAV virion comprises a capsid protein, wherein the capsid protein shares at least 80%, 85% or 90% polypeptide sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1 : amino acid substitutions S586L, T582D, N583V, H584Q, A587P, Q588G, and A589F.

[0476] In some embodiments, the disclosure provides a method of transducing a cardiac cell, comprising contacting the cardiac cell with an rAAV virion, wherein the rAAV virion comprises a capsid protein, wherein the capsid protein shares at least 80%, 85% or 90% polypeptide sequence identity to an AAV9 VP3 reference sequence according to SEQ ID NO: 487, and wherein the capsid protein comprises, relative to reference sequence SEQ ID NO: 1 : amino acid substitutions S586H, T582D, N583V, H584Q, A587P, Q588G, and A589F.

[0477] some embodiments, the disclosure provides a method of transducing a cardiac cell, comprising contacting the cardiac cell with an rAAV virion, wherein the rAAV virion comprises a capsid protein, wherein the capsid protein s...

Claims

WHAT IS CLAIMED IS:

1. An engineered adeno-associated virus (rAAV) capsid protein, wherein the capsid protein comprises an amino acid sequence X1DVQX2X3PGFX4X5X6X7X8 in the VR-VIII site, wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs is any amino acid.

2. The capsid protein of claim 1, wherein the capsid protein is a variant of an AAV5, AAV9, AAVrh.74, or AAVrh.10 capsid protein.

3. An engineered adeno-associated virus (rAAV) capsid protein, wherein the capsid protein comprises:(i) at least 80% amino acid sequence identity to the AAV5 VP3 capsid protein of SEQ ID NO: 324, and comprises the amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 at amino acid positions 570 to 583, wherein the amino acid numbering is according to SEQ ID NO: 322;(ii) at least 80% amino acid sequence identity to the AAV9 VP3 capsid protein of SEQ ID NO: 487, and comprisesthe amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 at amino acid positions 581 to 594, wherein the amino acid numbering is according to SEQ ID NO: 1;(iii) at least 80% amino acid sequence identity to the AAVrh.74 VP3 capsid protein of SEQ ID NO: 338, and comprises the amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 at amino acid positions 583 to 596, wherein the amino acid numbering is according to SEQ ID NO: 336; or(iv) at least 80% amino acid sequence identity to the AAVrh.10 VP3 capsid protein of SEQ ID NO: 331, and comprises the amino acid sequence of X1DVQX2X3PGFX4X5X6X7X8 at amino acid positions 583 to 596 of AAVrh.10, wherein the amino acid numbering is according to SEQ ID NO: 329; wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs is any amino acid.

4. The capsid protein of any one of claims 1-3, wherein Xi is Alanine (A).

5. The capsid protein of any one of claims 1-4, wherein X2 is Glutamine (Q).

6. The capsid protein of any one of claims 1-5, wherein X7 is Threonine (T).

7. The capsid protein of any one of claims 1-6, wherein X5 is Alanine (A) or Proline (P).

8. The capsid protein of any one of claims 1-7, wherein Xe is Glutamine (Q) or Glutamic Acid (E).

9. The capsid protein of any one of claims 1-8, wherein X4 is Q, G, R, N, H, M, P, or S.

10. The capsid protein of any one of claims 1-9, wherein Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

11. The capsid protein of any one of claims 1-10, wherein X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y.

12. The capsid protein of any one of claims 1-11, wherein Xi is Alanine (A), X2 is Glutamine (Q), and X7 is Threonine (T).

13. The capsid protein of claim 12, wherein X4 is Glutamine (Q), X5 is Alanine (A), and Xe is Glutamine (Q).

14. The capsid protein of any one of claims 11-13, wherein Xs is Glutamic Acid (E).

15. The capsid protein of any one of claims 1-3, wherein the capsid protein comprises i an amino acid sequence selected from X1DVQX2X3PGFX4AQX7X8 (SEQ ID NO: 1039), X1DVQX2X3PGFX4AEX7X8 (SEQ ID NO: 1040), X1DVQX2X3PGFX4PQX7X8 (SEQ ID NO: 1041), X1DVQX2X3PGFX4PEX7X8 (SEQ ID NO: 1042), ADVQQX3PGFX4AQTX8 (SEQ ID NO: 1033), ADVQQX3PGFX4AETX8 (SEQ ID NO: 1043), ADVQQX3PGFX4PQTX8 (SEQ ID NO: 1044), and ADVQQX3PGFX4PETX8 (SEQ ID NO: 1032) in the VR-VIII site, and wherein each of Xi, X2, X3, X4, X7, and Xs , if present, is any amino acid.

16. The capsid protein of claim 15, wherein X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4 is Q, G, R, N, H, M, P, or S; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

17. The capsid protein of any one of claims 1-3, wherein the capsid protein comprises an amino acid sequence selected from ADVQQX3PGFX4X5X6X7X8 (SEQ ID NO: 315), ADVQQX3PGFQAQTX8 (SEQ ID NO: 312), ADVQQX3PGFX4X5X6TX8 (SEQ ID NO: 311), ADVQQX3PGFX4X5X6X7E (SEQ ID NO: 316), ADVQQX3PGFX4X5X6TE (SEQ ID NO:313), and X1DVQX2X3PGFX4X5X6X7E (SEQ ID NO: 306) in the VR-VIII site, and wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs , if present, is any amino acid.

18. The capsid protein of any one of claims 1-17, wherein X3 is selected from Leucine (L) and Histidine (H).

19. The capsid protein of claim 18, wherein X3 is L, or the capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E (SEQ ID NO: 1045) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 317) wherein each of Xi, X2, X4, X5, X6, and X7, if present, is any amino acid, or the capsid protein comprises amino acid sequence ADVQQLPGFQAQTE (SEQ ID NO: 320).

20. The capsid protein of claim 18, wherein X3 is H, or the capsid protein comprises an amino acid sequence X1DVQX2HPGFX4X5X6X7E (SEQ ID NO: 1051) or ADVQQHPGFX4X5X6TE (SEQ ID NO: 318) wherein each of Xi, X2, X4, X5, X6, and X7, if present, is any amino acid, or the capsid protein comprises the amino acid sequence ADVQQHPGFQAQTE (SEQ ID NO: 321).

21. The capsid protein of claim 16, wherein X3 is L and Xs is E, or the capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E (SEQ ID NO: 1045) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 317) wherein each of Xi, X2, X4, X5, X6, and X7, if present, is any amino acid, or the capsid protein comprises the amino acid sequence selected from the group consisting of: ADVQQLPGFQAQTE (SEQ ID NO: 320), ADVQQLPGFGAQTE (SEQ ID NO: 906), ADVQQLPGFRPETE (SEQ ID NO: 907),ADVQQLPGFNAQTE (SEQ ID NO: 908), ADVQQLPGFQAETE (SEQ ID NO: 951),ADVQQLPGFGAETE (SEQ ID NO: 952), ADVQQLPGFGPQTE (SEQ ID NO: 953),ADVQQLPGFHAQTE (SEQ ID NO: 954), ADVQQLPGFRAQTE (SEQ ID NO: 955),ADVQQLPGFRAETE (SEQ ID NO: 956), ADVQQLPGFMAQTE (SEQ ID NO: 957),ADVQQLPGFPAQTE (SEQ ID NO: 958), ADVQQLPGFSAQTE (SEQ ID NO: 959),ADVQQLPGFSAETE (SEQ ID NO: 960), and ADVQQLPGFRPQTE (SEQ ID NO: 961).

22. The capsid protein of claim 16, wherein X4 is Q, or the capsid protein comprises an amino acid sequence X1DVQX2X3PGFQX5X6X7X8 (SEQ ID NO: 307), ADVQQX3PGFQX5X6TX8 (SEQ ID NO: 1046), or ADVQQX3PGFQAQTX8 (SEQ ID NO:312) wherein each of Xi, X2, X3, X5, Xe, X7, and Xs, if present, is any amino acid, or the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 320, 321, 904, 905, and 909-950.

23. A recombinant adeno-associated virus (rAAV) capsid protein, wherein the capsid protein comprises a sequence sharing at least 80% amino acid sequence identity to AAV9 VP3 capsid protein of SEQ ID NO: 487, and comprises relative to the AAV9 VP1 capsid protein sequence of SEQ ID NO: 1 : (i) an amino acid sequence X1DVQX2X3PGFX4X5X6X7X8 in the VR-VIII site or at positions 581 to 594, wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs is any amino acid (SEQ ID NO: 303), or (ii) amino acid substitutions T582D, N583V, H584Q, A587P, Q588G, and A589F.

24. The capsid protein of claim 23, wherein Xi is Alanine (A), or the capsid protein comprises amino acid A at position 581.

25. The capsid protein of claim 23 or 24, wherein X2 is Glutamine (Q), or the capsid protein comprises amino acid Q at position 585.

26. The capsid protein of any one of claims 23-25, wherein X7 is Threonine (T), or the capsid protein comprises amino acid T at position 593.

27. The capsid protein of any one of claims 23-26, wherein X5 is Alanine (A) or Proline (P), or the capsid protein comprises amino acid A or P at position 591.

28. The capsid protein of any one of claims 23-27, wherein Xe is Glutamine (Q) or Glutamic Acid (E), or the capsid protein comprises Q or E at position 592.

29. The capsid protein of any one of claims 23-28, wherein X4 is Q, G, R, N, H, M, P, or S, or the capsid protein comprises Q, G, R, N, H, M, P, or S at position 590.

30. The capsid protein of any one of claims 23-29, wherein Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N, or the capsid protein comprises E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N at position 594.

31. The capsid protein of any one of claims 23-30, wherein X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y, or the capsid protein comprises L, H, V, C, Q, G, I, M, F, P, T, or Y at position 586.

32. The capsid protein of any one of claims 23-31, wherein Xi is Alanine (A), X2 is Glutamine (Q), and X7 is Threonine (T); or the capsid protein comprises amino acids A, Q and T at position 581, 585 and 593, respectively.

33. The capsid protein of claim 32, wherein X4 is Glutamine (Q), X5 is Alanine (A), and Xe is Glutamine (Q); or the capsid protein comprises amino acids Q, A and Q and positions 590, 591 and 592, respectively.

34. The capsid protein of claim 32 or 33, wherein Xs is Glutamic Acid (E), or the capsid protein comprises amino acid substitution G594E.

35. The capsid protein of claim 23, wherein the capsid protein comprises in the VR-VIII site an amino acid sequence X1DVQX2X3PGFX4AQX7X8 (SEQ ID NO: 1039), X1DVQX2X3PGFX4AEX7X8 (SEQ ID NO: 1040), X1DVQX2X3PGFX4PQX7X8 (SEQ ID NO: 1041), X1DVQX2X3PGFX4PEX7X8 (SEQ ID NO: 1042), ADVQQX3PGFX4AQTX8 (SEQ ID NO: 1033), ADVQQX3PGFX4AETX8 (SEQ ID NO: 1043), ADVQQX3PGFX4PQTX8 (SEQ ID NO: 1044), or ADVQQX3PGFX4PETX8 (SEQ ID NO: 1032), wherein each of Xi, X2, X3, X4, X7, and Xs, if present, is any amino acid.

36. The capsid protein of claim 35, wherein X3 is L, H, V, C, Q, G, I, M, F, P, T, or Y; X4 is Q, G, R, N, H, M, P, or S; and Xs is E, M, Q, D, L, A, C, H, F, Y, T, V, I, S, or N.

37. The capsid protein of claim 23, wherein the capsid protein comprises in the VR-VIII site an amino acid sequence ADVQQX3PGFX4X5X6X7X8 (SEQ ID NO: 315), ADVQQX3PGFQAQTX8 (SEQ ID NO: 312), ADVQQX3PGFX4X5X6TX8 (SEQ ID NO: 311), ADVQQX3PGFX4X5X6X7E (SEQ ID NO: 316), ADVQQX3PGFX4X5X6TE (SEQ ID NO: 313), or X1DVQX2X3PGFX4X5X6X7E (SEQ ID NO: 306), wherein each of Xi, X2, X3, X4, X5, Xe, X7, and Xs, if present, is any amino acid.

38. The capsid protein of any one of claims 23-37, wherein X3 is selected from Leucine (L) and Histidine (H), or wherein the capsid protein comprises amino acid substitution S586L or S586H.

39. The capsid protein of claim 38, wherein X3 is L, or the capsid protein comprises amino acid substitution S586L, or the capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E (SEQ ID NO: 1045) or ADVQQLPGFX4X5X6TE (SEQ ID NO:317) wherein each of Xi, X2, X4, X5, Xe, and X7, if present, is any amino acid, or the capsid protein comprises amino acid sequence ADVQQLPGFQAQTE (SEQ ID NO: 320).

40. The capsid protein of claim 38, wherein X3 is H, or the capsid protein comprises amino acid substitution S586H, or the capsid protein comprises an amino acid sequence ADVQX2HPGFX4X5X6X7E (SEQ ID NO: 1050) or ADVQQHPGFX4X5X6TE (SEQ ID NO:318) wherein each of X2, X4, X5, Xe, and X7, if present, is any amino acid, or the capsid protein comprises amino acid sequence ADVQQHPGFQAQTE (SEQ ID NO: 321).

41. The capsid protein of claim 36, wherein X3 is L and Xs is E, or the capsid protein comprises an amino acid sequence X1DVQX2LPGFX4X5X6X7E (SEQ ID NO: 1045) or ADVQQLPGFX4X5X6TE (SEQ ID NO: 317) wherein each of Xi, X2, X4, X5, X6, and X7 is any amino acid or, or the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 320, 906-908, and 951-961.

42. The capsid protein of claim 36, wherein X4 is Q, or the capsid protein comprises an amino acid sequence X1DVQX2X3PGFQX5X6X7X8, ADVQQXsPGFQXsXeTXs (SEQ ID NO: 1046), or ADVQQX3PGFQAQTX8 (SEQ ID NO: 312) wherein each of Xi, X2, X3, X5, X6, X7, and Xs, if present, is any amino acid, or the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 320, 321, 904, 905, and 909-950.

43. The capsid protein of any one of claims 23-42, wherein the capsid protein comprises relative to AAV9 VP1 capsid protein sequence, at position 452 an amino acid selected from the group consisting of: K and N.

44. The capsid protein of any one of claims 23-43, wherein the capsid protein comprises relative to AAV9 VP1 capsid protein sequence, at position 452 an amino acid substitution N452K.

45. An engineered adeno-associated virus (rAAV) capsid protein, wherein the capsid protein comprises a variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10, wherein a) Xi is T or D. b) X2is N or V. c) X3 is H, Q, or R. d) X4is Q, M, N, or S. e) X5is S, G, M, T, or L. f) X6is A, N, K, T, or P. g) X7is Q, Y, R, L, T, or G. h) Xs is A, V, or F. i) X9is Q, R, G, M, or N. j) X10 is A or P (SEQ ID NO: 1047).

46. The capsid protein of claim 45, wherein the capsid protein is a variant of an AAV5, AAV9, AAVrh.74, or AAVrh.10 capsid protein.

47. The capsid protein of any one of claims 45-46, wherein the capsid protein:(i) comprises a sequence sharing at least 80% amino acid sequence identity to AAV5 VP3 capsid protein, and comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 571 to 580 relative to AAV5 VP1 capsid protein sequence of SEQ ID NO: 322;(ii) comprises a sequence sharing at least 80% amino acid sequence identity to AAV9 VP3 capsid protein, and comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 582 to 591 relative to AAV9 VP1 capsid protein sequence of SEQ ID NO: 1;(iii) comprises a sequence sharing at least 80% amino acid sequence identity to AAVrh.74 VP3 capsid protein, and comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 584 to 593 relative to AAVrh.74 VP1 capsid protein sequence of SEQ ID NO: 336 or(iv) comprises a sequence sharing at least 80% amino acid sequence identity to AAVrh.10 VP3 capsid protein, and comprises the variant polypeptide sequence X1X2X3X4X5X6X7X8X9X10 at amino acid positions 584 to 593 relative to AAVrh.10 VP1 capsid protein sequence of SEQ ID NO: 329.

48. The capsid protein of any one of claims 45-47, wherein the capsid protein comprises the variant polypeptide sequence of any one of SEQ ID NOs: 1006 - 1027.

49. The capsid protein of any one of claims 45-48, wherein the capsid protein is a variant of AAV9 capsid protein, and comprise the amino acid sequence of any one of SEQ ID NOs: 962- 983 at amino acid positions 581 to 594 relative to the AAV9 VP1 capsid protein sequence of SEQ ID NO: 1.

50. The capsid protein of any one of claims 1-49, wherein the capsid protein: (i) is cardiotrophic, (ii) mediates increased transduction efficiency in cardiac cells compared to the parental sequence, (iii) mediates decreased transduction efficiency in liver cells compared to the parental sequence, and / or (iv) mediates increased selectivity for the cardiac cells over liver cells compared to the parental sequence.

51. A capsid protein comprising SEQ ID NO: 343.

52. A capsid protein comprising SEQ ID NO: 344.

53. A capsid protein comprising any one of the sequences selected from the group consisting of: SEQ ID NOs: 846 to SEQ ID NO:903; optionally wherein the capsid comprises the sequence selected from the group consisting of: SEQ ID NO:846, 847, 848, 849, 850, 891, 864, 867, 858, 878, 890, 870, 856, 886, 900, 901, and 898; optionally wherein the capsid comprises the sequence selected from the group consisting of: SEQ ID NO:846, 847, 848, 849, and 850.

54. A recombinant adeno-associated virus (rAAV) virion, comprising the capsid protein according to any one of claims 1-53 and a vector genome comprising a polynucleotide cassette flanked by inverted terminal repeats (ITRs).

55. The rAAV virion of claim 54, wherein the rAAV virion transduces heart cells.

56. The rAAV virion of claim 54 or claim 55, wherein the rAAV virion transduces cardiomyocytes.

57. The rAAV virion of any one of claims 54-56, wherein the rAAV virion traffics to at least one organ other than the liver.

58. The rAAV virion of any one of claims 54-57, wherein the rAAV virion traffics to the heart.

59. The rAAV virion of any one of claims 54-58, wherein the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1.

60. The rAAV virion of any one of claims 54-59, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

61. The rAAV virion of any one of claims 54-60, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

62. The rAAV virion of any one of claims 54-61, wherein the rAAV virion exhibits a higher transduction efficiency, optionally higher heart transduction efficiency, than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, assessed in a primate.

63. The rAAV virion of any one of claims 54-62, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

64. The rAAV virion of any one of claims 54-63, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having anAAV9 VP1 capsid protein according to SEQ ID NO: 1, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

65. The rAAV virion of any one of claims 54-64, wherein the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322.

66. The rAAV virion of any one of claims 54-58 and 65, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

67. The rAAV virion of any one of claims 54-58 and 65-66, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

68. The rAAV virion of any one of claims 54-58 and 65-67, wherein the rAAV virion exhibits a higher transduction efficiency, optionally higher heart transduction efficiency, than an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, assessed in a primate.

69. The rAAV virion of any one of claims 54-58 and 65-68, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

70. The rAAV virion of any one of claims 54-58 and 65-69, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAV5 VP1 capsid protein according to SEQ ID NO: 322, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

71. The rAAV virion of any one of claims 54-58, wherein the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329.

72. The rAAV virion of any one of claims 54-58 and 71, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

73. The rAAV virion of any one of claims 54-58 and 71-72, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

74. The rAAV virion of any one of claims 54-58 and 71-73, wherein the rAAV virion exhibits a higher transduction efficiency, optionally higher heart transduction efficiency, than an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, assessed in a primate.

75. The rAAV virion of any one of claims 54-58 and 71-74, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

76. The rAAV virion of any one of claims 54-58 and 71-75, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAVrh.10 VP1 capsid protein according to SEQ ID NO: 329, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

77. The rAAV virion of any one of claims 54-58, wherein the rAAV virion exhibits a higher heart transduction efficiency than an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336.

78. The rAAV virion of any one of claims 54-58 and 77, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

79. The rAAV virion of any one of claims 54-58 and 77-78, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

80. The rAAV virion of any one of claims 54-58 and 77-79, wherein the rAAV virion exhibits a higher transduction efficiency, optionally higher heart transduction efficiency, than an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, assessed in a primate.

81. The rAAV virion of any one of claims 54-58 and 77-80, wherein the rAAV virion exhibits a higher heart-to-liver transduction ratio than an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times higher.

82. The rAAV virion of any one of claims 54-58 and 77-81, wherein administration of the rAAV virion to a subject leads to a lower liver viral load than administration of an rAAV virion having an AAVrh.74 VP1 capsid protein according to SEQ ID NO: 336, assessed in a primate, optionally at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 times lower.

83. The rAAV virion of any one of claims 54-82, wherein the polynucleotide cassette comprises a polynucleotide sequence encoding MYBPC3, DWORF, PKP2, KCNH2, TRPM4, DSG2, TGFBR2, TGFBR1, EMD, KCNQ1, TAZ, COL3A1, JUP, CASQ2, MLRP44, DNAJC19, LMNA, TNNI3, DSP, DSG2, RAFI, S0S1, FBN1, LAMP2, FXN, RAFI, BAG3, KCNQ1, MYLK3, CRYAB, ALPK3, ACTN2, JPH2, PLN, ATP2A2, CACNA1C, DMD, DMPK, EPG5, EVC, EVC2, FBN1, NF1, SCN5A, S0S1, NPR1, ERBB4, VIP, MYH6, MYH7, Cas9, RBM20, MYOCD, ASCL1, GATA4, MEF2C, TBX5, miR-133, or MESPl.

84. The rAAV virion of any one of claims 54-82, wherein the polynucleotide cassette comprises a polynucleotide sequence which encodes a protein selected from the group consisting of: MYBPC3, DWORF, PKP2, LMNA, LAMP2, BAG3, CRYAB, JPH2, PLN, TTNI3, MYOCD, ASCL1, DSP, JUP, DSP, MYH6, MYH7, RBM20, and Cas9.

85. A pharmaceutical composition comprising the rAAV virion according to any one of claims 54-84 and a pharmaceutically acceptable carrier.

86. A polynucleotide encoding the capsid protein of any one of claims 1-53.

87. A method of transducing a cardiac cell, comprising contacting the cardiac cell with the rAAV virion according to any one of claims 54-84, wherein the rAAV virion transduces the cardiac cell.

88. The method of claim 87, wherein the cardiac cell is a cardiomyocyte.

89. The method of claim 87 or claim 88, wherein the rAAV virion exhibits higher transduction efficiency in the cell than an rAAV virion having an AAV9 VP1 capsid protein according to SEQ ID NO: 1, an AAV5 VP1 capsid protein according to SEQ ID NO: 322, an AAVrh.10 capsid protein according to SEQ ID NO: 329, or an AAVrh.74 capsid protein according to SEQ ID NO: 336.

90. A method of delivering one or more gene products to a cardiac cell, comprising contacting the cardiac cell with the rAAV virion according to any one of claims 54-84.

91. The method of claim 90, wherein the cardiac cell is a cardiomyocyte.

92. A method of treating a cardiac pathology in a subject in need thereof, comprising administering a therapeutically effective amount of the rAAV virion according to any one of claims 54-84 to the subject, or the pharmaceutical composition of claim 85, wherein the rAAV virion transduces cardiac tissue.

93. A method of treating a heart disease or condition in a subj ect in need thereof, comprising administering a therapeutically effective amount of the rAAV virion according to any one of claims 54-84 to the subject or the pharmaceutical composition of claim 85.

94. A kit comprising the pharmaceutical composition according to claim 85 and instructions for use.

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