Engineered AAV capsid polypeptides and methods of use

Abstract

Described herein are engineered and chimeric VP capsid polypeptides with enhanced tropism for target tissues (e.g., CNS or eye tissues) and enhanced functional transduction. The engineered VP capsid polypeptides are capable of assembling into recombinant AAV (rAAV) capsids for delivery of payload sequences to target tissues (e.g., CNS or eye tissues). Also described herein are methods of using rAAV capsids assembled from the engineered VP capsid polypeptides to deliver and express a payload, such as a guide RNA, in target tissues (e.g., CNS or eye tissues).

Description

ENGINEERED AAV CAPSID POLYPEPTIDES AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Nos 63 / 681,482, filed August 9, 2024, 63 / 695,270, filed September 16, 2024, 63 / 717,633, filed November 7, 2024, 63 / 738,197, filed December 23, 2024, 63 / 744,726, filed January 13, 2025, 63 / 758,992, filed February 14, 2025, 63 / 776,791, filed March 24, 2025, 63 / 781,907, filed April 1, 2025, 63 / 802,456, filed May 8, 2025, and 63 / 842,529, filed July 11, 2025. The disclosure of the prior applications is considered part of and is herein incorporated by reference in the disclosure of this application in its entirety. BACKGROUND

[0002] Recombinant adeno-associated viruses (rAAV) provide the leading platform for in vivo delivery of gene therapies. Current clinical trials employ a limited number of AAV capsids, primarily from naturally occurring human or primate serotypes such as AAV1, AAV2, AAV5, AAV6, AAV8, AAV9, AAVrh.10, AAV4rh.74, and AAVhu.67. These capsids often provide suboptimal targeting to tissues of interest, both due to poor infectivity of the tissue of interest and competing liver tropism. Increasing the dose to ensure infection of desired tissues can lead to dose-dependent liver toxicity. In addition, use of naturally- occurring capsids presents an immunological memory challenge – pre-immune patient populations are excluded from treatment and repeat dosing in a previously immune naïve patient is often not possible. Thus, there is a need for additional AAV capsids for use in gene therapy, in particular capsids that confer upon the rAAV high infectivity for specific tissues. INCORPORATION BY REFERENCE

[0003] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation byreference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. SUMMARY

[0004] In various aspects, the present disclosure provides an engineered viral protein (VP) capsid polypeptide, wherein the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 7 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1).

[0005] In various aspects, the present disclosure provides an engineered viral protein (VP) capsid polypeptide, wherein the engineered VP capsid polypeptide comprises at least 6, 7, 8, 9, or 10 contiguous amino acids of SEQ ID NO: 7 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1).

[0006] In some aspects, the engineered VP capsid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to an AAV5, AAV9, AAV2, AAV1, AAV3, AAV4, AAV6, AAV7, AAV8, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 capsid polypeptide, optionally, wherein the engineered VP capsid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to an AAV5 VP1 capsid, further optionally wherein the AAV5 VP1 capsid is SEQ ID NO: 1.

[0007] In some aspects, the engineered VP capsid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to a chimera polypeptide of comprising at least one serotype of any one of or two serotypes selected from AAV5, AAV9, AAV2, AAV1, AAV3, AAV4, AAV6, AAV7, AAV8, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, -2- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOAAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 capsid polypeptide, optionally, wherein the at least one serotype is AAV5 or AAV9 or the two serotypes selected are AAV5 and AAV9, further optionally wherein the AAV5 is SEQ ID NO: 1 and the AAV9 is SEQ ID NO: 4.

[0008] In some aspects, the chimera polypeptide comprises an AAV9 / AAV5 chimera polypeptide. In some aspects, the chimera polypeptide comprises an AAV9 / AAV5 chimera polypeptide. In some aspects, the chimera polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 85. In some aspects, the chimera polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 98.5% sequence identity to SEQ ID NO: 2. In some aspects, the chimera polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 100% sequence identity to SEQ ID NO: 35. In some aspects, the chimera polypeptide comprises a first region derived from a first AAV polypeptide and a second region derived from a second polypeptide, wherein the first region comprises a VP1 unique region of the first AAV polypeptide and the second region comprises the VP2 and VP3 region of the second AAV polypeptide. In some aspects, the chimera polypeptide comprises a first region comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 85 and a second region comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 98.5% sequence identity to SEQ ID NO: 2.

[0009] In some aspects, the chimera polypeptide increases functional transduction of the engineered VP capsid polypeptide. In some aspects, the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24. In some aspects, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 24. In some aspects, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 24. In some aspects, the engineered VP capsid polypeptide comprises SEQ ID NO: 24.

[0010] In some aspects, the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20. In some aspects, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 20. In some aspects, the engineered VP capsid polypeptide comprises at least -3- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO98% sequence identity to SEQ ID NO: 20. In some aspects, the engineered VP capsid polypeptide comprises SEQ ID NO: 20.

[0011] In some aspects, the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 100% sequence identity to SEQ ID NO: 16. In some aspects, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 16. In some aspects, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 16. In some aspects, the engineered VP capsid polypeptide comprises at least 98.5% sequence identity to SEQ ID NO: 16. In some aspects, the engineered VP capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 16. In some aspects, the engineered VP capsid polypeptide comprises SEQ ID NO: 16.

[0012] In various aspects, the present disclosure provides an engineered capsid comprising the engineered VP capsid polypeptide as described herein.

[0013] In various aspects, the present disclosure provides an engineered capsid comprising a VP1 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 100% sequence identity to SEQ ID NO: 16, a VP2 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20, a VP3 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24, or any combination thereof.

[0014] In various aspects, the present disclosure provides an engineered capsid comprising a VP1 sequence of SEQ ID NO: 16, a VP2 sequence of SEQ ID NO: 20, and a VP3 sequence of SEQ ID NO: 24.

[0015] In various aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising the engineered VP capsid polypeptide as described herein or the engineered capsid as described herein, and a payload.

[0016] In some aspects, the payload encodes a therapeutic polynucleotide or a therapeutic peptide. In some aspects, the payload encodes a heterologous polynucleotide or a heterologous peptide. In some aspects, the payload encodes an antibody or antibody fragment.

[0017] In some aspects, the payload encodes a transgene. In some aspects, the therapeutic polynucleotide encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof. In some aspects, the therapeutic polynucleotide encodes a guide RNA. In some aspects, the guide RNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR) enzyme when bound to a target. -4- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOIn some aspects, the guide RNA facilitates editing of a target. In some aspects, the guide RNA facilitates altered splicing of a target. In some aspects, the guide RNA facilitates exon skipping in a target. In some aspects, the target is a CNS target. In some aspects, the target is SNCA. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 75. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence with at least 90% sequence identity to SEQ ID NO: 75. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence with at least 98% sequence identity to SEQ ID NO: 75. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence comprising SEQ ID NO: 75.

[0018] In various aspects, the present disclosure provides an engineered viral protein (VP) chimeric capsid polypeptide, comprising a tropic variant region corresponding to positions 582-591 relative to SEQ ID NO: 35, wherein the engineered VP chimeric capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35.

[0019] In some aspects, the engineered VP chimeric capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 35. In some aspects, the engineered VP chimeric capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 35. In some aspects, the engineered VP chimeric capsid polypeptide comprises SEQ ID NO: 35.

[0020] In various aspects, the present disclosure provides an engineered viral protein (VP) chimeric capsid polypeptide, comprising a tropic variant region corresponding to positions 582-591 relative to SEQ ID NO: 77 or SEQ ID NO: 81, wherein the engineered VP chimeric capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 77 or SEQ ID NO: 81.

[0021] In some aspects, the engineered VP chimeric capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 77 or SEQ ID NO: 81. In some aspects, the engineered VP chimeric capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 77 or SEQ ID NO: 81. In some aspects, the engineered VP chimeric capsid polypeptide comprises SEQ ID NO: 77 or SEQ ID NO: 81.

[0022] In some aspects, wherein the functional transduction of an engineered VP chimeric capsid comprising the engineered VP chimeric capsid polypeptide is increased. In some aspects, the tropic variant region increases tissue tropism of the engineered VP chimeric capsid polypeptide for a target tissue. -5- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0023] In some aspects, the target tissue is a central nervous system (CNS) tissue. In some aspects, the tropic variant region comprises at least four contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some aspects, the tropic variant region comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some aspects, the tropic variant region comprises any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65.

[0024] In some aspects, X1in SEQ ID NO: 35 is L, T, or A. In some aspects, X1in SEQ ID NO: 35 is L. In some aspects, X2 in SEQ ID NO: 35 is W or A. In some aspects, X2 in SEQ ID NO: 35 is W. In some aspects, X3in SEQ ID NO: 35 is G or A. In some aspects, X3in SEQ ID NO: 35 is G. In some aspects, X4 in SEQ ID NO: 35 is W, G, or R. In some aspects, X4in SEQ ID NO: 35 is W. In some aspects, X5in SEQ ID NO: 35 is T, A, or Y. In some aspects, X5 in SEQ ID NO: 35 is T. In some aspects, X6 in SEQ ID NO: 35 is L, Y, or N. In some aspects, X6 in SEQ ID NO: 35 is L. In some aspects, X7 in SEQ ID NO: 35 is Q, K, S, or Y. In some aspects, X7 in SEQ ID NO: 35 is Q. In some aspects, X8 in SEQ ID NO: 35 is V, E, H, or V. In some aspects, X8 in SEQ ID NO: 35 is V. In some aspects, X9 in SEQ ID NO: 35 is Q, E, F, or G. In some aspects, X9in SEQ ID NO: 35 is Q. In some aspects, X10in SEQ ID NO: 35 is T or I. In some aspects, X10 in SEQ ID NO: 35 is T.35.

[0025] In some aspects, X1in SEQ ID NO: 77 or SEQ ID NO: 81 is L, T, or A. In some aspects, X1 in SEQ ID NO: 77 or SEQ ID NO: 81 is L. In some aspects, X2 in SEQ ID NO: 77 or SEQ ID NO: 81 is W or A. In some aspects, X2in SEQ ID NO: 77 or SEQ ID NO: 81 is W. In some aspects, X3 in SEQ ID NO: 77 or SEQ ID NO: 81 is G or A. In some aspects, X3in SEQ ID NO: 77 or SEQ ID NO: 81 is G. In some aspects, X4in SEQ ID NO: 77 or SEQ ID NO: 81 is W, G, or R. In some aspects, X4 in SEQ ID NO: 77 or SEQ ID NO: 81 is W. In some aspects, X5in SEQ ID NO: 77 or SEQ ID NO: 81 is T, A, or Y. In some aspects, X5 in SEQ ID NO: 77 or SEQ ID NO: 81 is T. In some aspects, X6 in SEQ ID NO: 77 or SEQ ID NO: 81 is L, Y, or N. In some aspects, X6in SEQ ID NO: 77 or SEQ ID NO: 81 is L. In some aspects, X7 in SEQ ID NO: 77 or SEQ ID NO: 81 is Q, K, S, or Y. In some aspects, X7 in SEQ ID NO: 77 or SEQ ID NO: 81 is Q. In some aspects, X8in SEQ ID NO: 77 or SEQ ID NO: 81 is V, E, H, or V. In some aspects, X8 in SEQ ID NO: 77 or SEQ ID NO: 81 is V. In some aspects, X9in SEQ ID NO: 77 or SEQ ID NO: 81 is Q, E, F, or G. In some aspects, -6- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0026] In some aspects, X9 in SEQ ID NO: 77 or SEQ ID NO: 81 is Q. In some aspects, X10 in SEQ ID NO: 77 or SEQ ID NO: 81 is T or I. In some aspects, X10in SEQ ID NO: 77 or SEQ ID NO: 81 is T.

[0027] In some aspects, the engineered viral protein (VP) chimeric capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 100% sequence identity to a sequence of any one of: i) SEQ ID NO: 16, ii) SEQ ID NO: 14, iii) SEQ ID NO: 15, iv) SEQ ID NO: 17, or v) SEQ ID NO: 124 – SEQ ID NO: 153. In some aspects, wherein the engineered viral protein (VP) chimeric capsid polypeptide comprises a sequence of any one of: i) SEQ ID NO: 16, ii) SEQ ID NO: 14, iii) SEQ ID NO: 15, iv) SEQ ID NO: 17, or v) SEQ ID NO: 124 – SEQ ID NO: 153.

[0028] In some aspects, the target tissue is a retinal tissue. In some aspects, the variant polypeptide sequence comprises at least four amino acids from any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some aspects, the variant polypeptide sequence comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some aspects, the variant polypeptide sequence comprises a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71.

[0029] In various aspects, the present disclosure provides an engineered capsid comprising the engineered VP chimeric capsid polypeptide as described herein.

[0030] In various aspects, the present disclosure provides an engineered capsid comprising a VP1 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35, SEQ ID NO: 77, or SEQ ID NO: 81.

[0031] In various aspects, the present disclosure provides an engineered capsid comprising a VP1 sequence of SEQ ID NO: 16, a VP2 sequence of SEQ ID NO: 20, and a VP3 sequence of SEQ ID NO: 24.

[0032] In various aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising the engineered VP chimeric capsid polypeptide as described herein or the engineered capsid as described herein, and a payload.

[0033] In some aspects, the payload encodes a therapeutic polynucleotide or a therapeutic peptide. In some aspects, the payload encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof. In some aspects, the guide RNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR) enzyme when bound to a target. In some aspects, the guide RNA facilitates editing of a target. In some aspects, the guide RNA facilitates knockdown of a target. In some aspects, the -7- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOguide RNA facilitates altered splicing of a target. In some aspects, the guide RNA facilitates exon skipping in a target. In some aspects, the target is a CNS target. In some aspects, the target is SNCA.

[0034] In some aspects, the engineered VP capsid polypeptide is encoded by a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 75, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 160 – SEQ ID NO: 189, or SEQ ID NO: 190 – SEQ ID NO: 195. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence with at least 90% sequence identity to SEQ ID NO: 75, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 160 – SEQ ID NO: 189, or SEQ ID NO: 190 – SEQ ID NO: 195. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence with at least 98% sequence identity to SEQ ID NO: 75, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 160 – SEQ ID NO: 189, or SEQ ID NO: 190 – SEQ ID NO: 195. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence comprising SEQ ID NO: 75, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 160 – SEQ ID NO: 189, or SEQ ID NO: 190 – SEQ ID NO: 195.

[0035] In some aspects, the engineered viral protein (VP) chimeric capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of any one of SEQ ID NO: 154 – SEQ ID NO: 159. In some aspects, the engineered viral protein (VP) chimeric capsid polypeptide comprises a sequence of any one of SEQ ID NO: 154 – SEQ ID NO: 159.

[0036] In various aspects, the present disclosure provides an engineered capsid comprising a VP1 sequence of any one of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17, a VP2 sequence of any one of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21, and a VP3 sequence of any one of SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25.

[0037] In various aspects, the present disclosure provides an engineered capsid comprising a VP1 sequence of any one of SEQ ID NO: 124 – SEQ ID NO: 153 or SEQ ID NO: 154 – SEQ ID NO: 159, a VP2 sequence of residues 138-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153 or SEQ ID NO: 154 – SEQ ID NO: 159, and a VP3 sequence of residues 194-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153 or SEQ ID NO: 154 – SEQ ID NO: 159.

[0038] In some aspects, the payload encodes an antibody or antibody fragment. In some aspects, the payload encodes a heterologous polynucleotide or a heterologous peptide. In some aspects, the payload encodes a transgene. In some aspects, the therapeutic polynucleotide encodes a guide RNA. -8- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0039] In various aspects, the present disclosure provides an engineered viral protein (VP) chimeric capsid polypeptide, comprising a tropic variant region corresponding to positions 582-591 relative to SEQ ID NO: 77, wherein the engineered VP chimeric capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 77.

[0040] In some aspects, the engineered VP chimeric capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 77. In some aspects, the engineered VP chimeric capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 77. In some aspects, the engineered VP chimeric capsid polypeptide comprises SEQ ID NO: 77.

[0041] In various aspects, the present disclosure provides an engineered viral protein (VP) chimeric capsid polypeptide, comprising a tropic variant region corresponding to positions 582-591 relative to SEQ ID NO: 81, wherein the engineered VP chimeric capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 81.

[0042] In some aspects, the engineered VP chimeric capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 81. In some aspects, the engineered VP chimeric capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 81. In some aspects, the engineered VP chimeric capsid polypeptide comprises SEQ ID NO: 81.

[0043] In various aspects, the present disclosure provides an engineered viral protein (VP) capsid polypeptide, wherein the engineered VP capsid polypeptide comprises a sequence of any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 36 – SEQ ID NO: 65, or SEQ ID NO: 66 – SEQ ID NO: 71 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1), and optionally wherein the engineered VP capsid polypeptide comprises a sequence of NM or QM at residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1).

[0044] In some aspects, the engineered VP capsid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to an AAV5, AAV9, AAV2, AAV1, AAV3, AAV4, AAV6, AAV7, AAV8, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, -9- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOAAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 capsid polypeptide, optionally, wherein the engineered VP capsid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to an AAV5 VP1 capsid, further optionally wherein the AAV5 VP1 capsid is SEQ ID NO: 1. In some aspects, the engineered VP capsid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to a chimera polypeptide of comprising at least one serotype of any one of or two serotypes selected from AAV5, AAV9, AAV2, AAV1, AAV3, AAV4, AAV6, AAV7, AAV8, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 capsid polypeptide, optionally, wherein the at least one serotype is AAV5 or AAV9 or the two serotypes selected are AAV5 and AAV9, further optionally wherein the AAV5 is SEQ ID NO: 1 and the AAV9 is SEQ ID NO: 4.

[0045] In some aspects, the chimera polypeptide comprises an AAV9 / AAV5 chimera polypeptide. In some aspects, the chimera polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 85. In some aspects, the chimera polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5% sequence identity to SEQ ID NO: 2. In some aspects, the chimera polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 100% sequence identity to SEQ ID NO: 35.

[0046] In some aspects, the chimera polypeptide comprises a first region derived from a first AAV polypeptide and a second region derived from a second polypeptide, wherein the first region comprises a VP1 unique region of the first AAV polypeptide and the second region comprises the VP2 and VP3 region of the second AAV polypeptide. In some aspects, the chimera polypeptide comprises a first region comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 85 and a second region comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 98.5% sequence identity to SEQ ID NO: 2. In some aspects, the chimera polypeptide increases functional transduction of the engineered VP capsid polypeptide. -10- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0047] In some aspects, the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25. In some aspects, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25. In some aspects, wherein the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25. In some aspects, the engineered VP capsid polypeptide comprises SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25.

[0048] In some aspects, the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21. In some aspects, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21. In some aspects, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21. In some aspects, the engineered VP capsid polypeptide comprises SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21.

[0049] In some aspects, the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 124 – SEQ ID NO: 153. In some aspects, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 124 – SEQ ID NO: 153. In some aspects, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 124 – SEQ ID NO: 153. In some aspects, the engineered VP capsid polypeptide comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, or SEQ ID NO: 124 – SEQ ID NO: 153.

[0050] In various aspects, the present disclosure provides an engineered capsid comprising the engineered VP capsid polypeptide as described herein. In various aspects, the present disclosure provides an engineered capsid comprising a VP1 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17, a VP2 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21, a VP3 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, -11- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO99%, or 100% sequence identity to SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25, or any combination thereof.

[0051] In various aspects, the present disclosure provides an engineered capsid comprising a VP1 sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17, a VP2 sequence of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21, and a VP3 sequence of SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25.

[0052] In various aspects, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising the engineered VP capsid polypeptide as described herein or the engineered capsid as described herein, and a payload.

[0053] In some aspects, the payload encodes a therapeutic polynucleotide or a therapeutic peptide. In some aspects, the therapeutic polynucleotide encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof.

[0054] In some aspects, the guide RNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR) enzyme when bound to a target. In some aspects, the guide RNA facilitates editing of a target. In some aspects, the guide RNA facilitates knockdown of a target. In some aspects, the guide RNA facilitates altered splicing of a target. In some aspects, the guide RNA facilitates exon skipping in a target. In some aspects, the target is a CNS target. In some aspects, the target is SNCA.

[0055] In some aspects, the engineered VP capsid polypeptide is encoded by a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 160 – SEQ ID NO: 189, or SEQ ID NO: 190 – SEQ ID NO: 195. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence with at least 90% sequence identity to SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 160 – SEQ ID NO: 189, or SEQ ID NO: 190 – SEQ ID NO: 195. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence with at least 98% sequence identity to SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 160 – SEQ ID NO: 189, or SEQ ID NO: 190 – SEQ ID NO: 195. In some aspects, the engineered VP capsid polypeptide is encoded by a sequence comprising SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 160 – SEQ ID NO: 189, or SEQ ID NO: 190 – SEQ ID NO: 195.

[0056] In various aspects, the present disclosure provides an engineered viral protein (VP) capsid polypeptide, wherein the engineered VP capsid polypeptide comprises at least 6, 7, 8, 9, or 10 contiguous amino acids of any one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, -12- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOSEQ ID NO: 36 – SEQ ID NO: 65, or SEQ ID NO: 66 – SEQ ID NO: 71 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1), and optionally wherein the engineered VP capsid polypeptide comprises a sequence of NM or QM at residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1).

[0057] In some aspects, the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to residues 194-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153. In some aspects, the engineered VP capsid polypeptide comprises at least 90% sequence identity to residues 194-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153. In some aspects, the engineered VP capsid polypeptide comprises at least 98% sequence identity to residues 194-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153. In some aspects, the engineered VP capsid polypeptide comprises residues 194-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153.

[0058] In some aspects, the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to residues 138-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153 or SEQ ID NO: 154 – SEQ ID NO: 159. In some aspects, the engineered VP capsid polypeptide comprises at least 90% sequence identity to residues 138-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153 or SEQ ID NO: 154 – SEQ ID NO: 159. In some aspects, the engineered VP capsid polypeptide comprises at least 98% sequence identity to residues 138-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153 or SEQ ID NO: 154 – SEQ ID NO: 159. In some aspects, the engineered VP capsid polypeptide comprises residues 138-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153 or SEQ ID NO: 154 – SEQ ID NO: 159.

[0059] In some aspects, the payload encodes an antibody or antibody fragment. In some aspects, the payload encodes a heterologous polynucleotide or a heterologous peptide. In some aspects, the payload encodes a transgene. In some aspects, the therapeutic polynucleotide encodes a guide RNA.

[0060] In various aspects, the present disclosure provides a pharmaceutical composition comprising the engineered VP capsid polypeptide as described herein, the engineered capsid as described herein, or the rAAV as described herein, and a pharmaceutically acceptable carrier or excipient.

[0061] In various aspects, the present disclosure provides a method of increasing transduction in a target brain tissue as compared to a non-target liver tissue in a subject, comprising: i) administering the rAAV as described herein or the pharmaceutical composition as described herein, ii) collecting the target brain tissue and the non-target liver tissue from the subject -13- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOafter administration, and iii) quantifying the transduction in the target brain tissue and the non-target liver tissue; wherein, the transduction is greater in the target brain tissue than the non-target liver tissue.

[0062] In some aspects, the transduction is at least two-fold, three-fold, four-fold, or five-fold greater in the target brain tissue as compared to the transduction in the non-target liver tissue. In some aspects, the transduction is at least two-fold, three-fold, four-fold, or five-fold greater in the target brain tissue as compared to a comparative capsid. In some aspects, the transduction is at least two-fold, three-fold, four-fold, or five-fold lesser in the non-target liver tissue as compared to a comparative capsid. In some aspects, the comparative capsid is a wild type AAV capsid or an engineered control capsid. In some aspects, the transduction is measured by RNA expression.

[0063] In various aspects, the present disclosure provides a method of increasing payload expression in a target brain tissue as compared to a non-target liver tissue in a subject, comprising: i) administering the rAAV as described herein or the pharmaceutical composition as described herein, ii) collecting the target brain tissue and the non-target liver tissue from the subject after administration, and iii) quantifying the payload expression in the target brain tissue and the non-target liver tissue; wherein, the payload expression is greater in the target brain tissue than the non-target liver tissue.

[0064] In some aspects, the payload expression is at least two-fold, three-fold, four-fold, or five-fold greater in the target brain tissue as compared to the transduction in the non-target liver tissue. In some aspects, the payload expression is at least two-fold, three-fold, four-fold, or five-fold greater in the target brain tissue as compared to a comparative capsid. In some aspects, the payload expression is at least two-fold, three-fold, four-fold, or five-fold lesser in the non-target liver tissue as compared to a comparative capsid. In some aspects, the comparative capsid is a wild type AAV capsid or an engineered control capsid. In some aspects, the payload expression is measured by RNA expression.

[0065] In various aspects, the present disclosure provides a method of increasing target RNA editing in a target brain tissue in a subject, comprising: i) administering the rAAV as described herein or the pharmaceutical composition as described herein, ii) collecting the target brain tissue and the non-target liver tissue from the subject after administration, and iii) quantifying the target RNA editing in the target brain tissue; wherein, the target RNA editing is at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% target RNA editing.

[0066] In some aspects, the target brain tissue is a substantia nigra tissue and the target RNA editing is greater than 80%. In some aspects, the target RNA editing is at least two-fold, at -14- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOleast three-fold, at least four-fold, or at least five-fold greater in the target brain tissue as compared to a comparative capsid. In some aspects, the target brain tissue is a substantia nigra tissue and the target RNA editing is at least 4-fold greater as compared to the target RNA editing of the comparative capsid. In some aspects, the comparative capsid is a wild type AAV capsid or an engineered control capsid.

[0067] In various aspects, the present disclosure provides a method of increasing AAV delivery to a target brain tissue as compared to a non-target liver tissue in a subject, comprising: i) administering the rAAV as described herein or the pharmaceutical composition as described herein, ii) collecting the target brain tissue and the non-target liver tissue from the subject after administration, iii) quantifying the number of viral genomes (vg) and diploid genomes (dg), and iv) calculating a ratio of vg / dg in the target brain tissue and the non-target liver tissue; wherein: the vg / dg ratio of the administered capsid is greater in the target brain tissue as compared to the vg / dg ratio of a comparative capsid, and the vg / dg ratio of the administered capsid is lesser in the non-target liver tissue as compared to the vg / dg ratio of a comparative capsid.

[0068] In some aspects, the vg / dg ratio of the administered capsid is at least two-fold, three- fold, four-fold, or five-fold greater in the target brain tissue as compared to the vg / dg ratio of a comparative capsid. In some aspects, the vg / dg ratio of the administered capsid is at least two-fold, three-fold, four-fold, or five-fold lesser in the target brain tissue as compared to the vg / dg ratio of a comparative capsid. In some aspects, the comparative capsid is a wild type AAV capsid or an engineered control capsid.

[0069] In various aspects, the present disclosure provides a method of transducing a neuron, the method comprising administering to a subject the rAAV as described herein or the pharmaceutical composition as described herein, and expressing the payload in the neuron.

[0070] In some aspects, the neuron is a human neuron. In some aspects, the human neuron is an iPSC-derived neuron. In some aspects, the neuron is in a tissue and wherein at least 50%, at least 60%, at least 70%, or at least 80% of the neurons in the tissue are transduced. In some aspects, the tissue is a brain tissue.

[0071] In various aspects, the present disclosure provides a method of transducing an astrocyte, the method comprising administering to a subject the rAAV as described herein or the pharmaceutical composition as described herein, and expressing the payload in the astrocyte. -15- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0072] In various aspects, the present disclosure provides a method of transducing a glial cell, the method comprising administering to a subject the rAAV as described herein or the pharmaceutical composition as described herein, and expressing the payload in the glial cell.

[0073] In some aspects, the glial cell is in a tissue and wherein at least 50%, at least 60%, at least 70%, or at least 80% of the glial cells in the tissue are transduced. In some aspects, the tissue is a brain tissue.

[0074] In various aspects, the present disclosure provides a method of delivering a payload to a target tissue, the method comprising administering to a subject the rAAV as described herein or the pharmaceutical composition as described herein.

[0075] In various aspects, the present disclosure provides a method of administering the rAAV as described herein or the pharmaceutical composition as described herein to a subject and delivering a payload to a target tissue.

[0076] In various aspects, the present disclosure provides a method of treating a condition in a subject, the method comprising administering to the subject the rAAV as described herein or the pharmaceutical composition as described herein and delivering a payload to a target tissue, thereby treating the condition.

[0077] In some aspects, the rAAV as described herein or the pharmaceutical composition as described herein is administered by systemic administration. In some aspects, the method further comprises infecting the target tissue with the rAAV. In some aspects, a ratio of payload expression in a brain tissue relative to a non-brain tissue upon administration of the rAAV is higher than upon administration of a wild type AAV. In some aspects, a ratio of target editing in a brain tissue relative to a non-brain tissue upon administration of the rAAV is higher than upon administration of a wild type AAV. In some aspects, upon delivery to the target tissue, the payload exhibits increased transduction compared to a payload delivered via an AAV5 comprising a wild type VP1 capsid polypeptide, a wild type VP2 capsid polypeptide, and a wild type VP3 capsid polypeptide.

[0078] In some aspects, the target tissue is a central nervous system tissue, a muscle tissue, an eye tissue, a retina tissue, or a retinal pigment epithelium tissue. In some aspects, the brain tissue is a deep brain tissue. In some aspects, the deep brain tissue is a substantia nigra tissue. In some aspects, the non-brain tissue is a liver tissue. In some aspects, the non-brain tissue is a peripheral nervous system tissue. In some aspects, the non-brain tissue is a dorsal root ganglion tissue. In some aspects, the non-brain tissue is a muscle tissue. -16- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0079] In some aspects, administration of the rAAV produces lower toxicity than administration of a wild type AAV. In some aspects, administration of the rAAV produces a lower immunogenicity response than administration of a wild type AAV. In some aspects,

[0080] In some aspects, the payload encodes a therapeutic peptide or a therapeutic polynucleotide. In some aspects, the payload encodes an antibody or fragment thereof. In some aspects, the payload encodes a therapeutic polynucleotide.

[0081] In some aspects, the method further comprises expressing the payload in a CNS tissue of the subject. In some aspects, the therapeutic polynucleotide encodes a peptide.

[0082] In some aspects, the therapeutic polynucleotide is a guide RNA. In some aspects, the guide RNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR) enzyme when bound to a target sequence. In some aspects, the method further comprises editing of the target sequence. In some aspects, the method further comprises facilitating gene or protein knockdown. In some aspects, the method further comprises facilitating altered splicing of the target sequence. In some aspects, the method further comprises facilitating exon skipping in the target sequence.

[0083] In some aspects, the target sequence is a CNS target sequence. In some aspects, the target sequence is implicated in the condition. In some aspects, the target sequence is an SNCA target sequence. In some aspects, the target sequence is present in deep brain tissue. In some aspects, the condition is Parkinson’s disease, Alzheimer’s disease, Amyotrophic Lateral Sclerosis, Huntington’s disease, Lewy body dementia, essential tremor, dystonia, or epilepsy.

[0084] The present disclosure is based on the identification of viral capsid polypeptides that confer CNS targeting to the payload encoded by the rAAV particles disclosed herein.

[0085] In one embodiment, the present disclosure provides an engineered viral protein (VP) capsid polypeptide.

[0086] In one embodiment, the present disclosure provides an engineered viral protein (VP) capsid polypeptide, wherein the engineered VP capsid polypeptide comprises a sequence of QM at residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) and a sequence of SEQ ID NO: 7 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 11 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 24. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 24. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 24. In one aspect, the engineered VP -17- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOcapsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 20. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 20. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 20. In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 16. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 16. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 16.

[0087] In one aspect, the engineered VP capsid polypeptide comprises a sequence of NM at residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) and a sequence of SEQ ID NO: 5 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 9 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 22. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 22. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 22. In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 18. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 18. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 18. In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 14. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 14. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 14. In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 13. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 13. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 13.

[0088] In one embodiment, the present disclosure provides an engineered viral protein (VP) capsid polypeptide, wherein the engineered VP capsid polypeptide comprises a sequence of QM at residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) and a sequence of SEQ ID NO: 6 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 10 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence -18- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOidentity to SEQ ID NO: 23. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 23. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 23. In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 19. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 19. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 19. In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 15. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 15. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 15.

[0089] In one embodiment, the present disclosure provides an engineered viral protein (VP) capsid polypeptide, wherein the engineered VP capsid polypeptide comprises a sequence of QM at residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) and a sequence of SEQ ID NO: 8 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 12 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 25. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 25. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 25. In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 21. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 21. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 21. In one aspect, the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 17. In one aspect, the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 17. In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 17. In one aspect, the engineered VP capsid polypeptide is a chimeric VP capsid polypeptide.

[0090] The present disclosure provides an engineered viral protein (VP) capsid polypeptide, comprising an amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 8. In one aspect, the engineered VP capsid polypeptide comprises the sequence of SEQ ID NO: 7, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 85 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of NM or QM at residues -19- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOcorresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 12 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the VP capsid polypeptide is a chimeric VP capsid polypeptide comprising a first region, wherein the first region is a region of an AAV5 VP capsid polypeptide. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising at least 90% sequence identity to residues 137-724 of SEQ ID NO: 27, SEQ ID NO: 13, SEQ ID NO: 26, or SEQ ID NO: 28. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising at least 98% sequence identity to residues 137-724 of SEQ ID NO: 27, SEQ ID NO: 13, SEQ ID NO: 26, or SEQ ID NO: 28. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising residues 137-724 of SEQ ID NO: 27, SEQ ID NO: 13, SEQ ID NO: 26, or SEQ ID NO: 28. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising at least 90% sequence identity to residues 137-724 of SEQ ID NO: 27. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising at least 98% sequence identity to residues 137-724 of SEQ ID NO: 27. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising residues 137-724 of SEQ ID NO: 27. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising at least 90% sequence identity to residues 138-725 of SEQ ID NO: 16, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising at least 98% sequence identity to residues 138-725 of SEQ ID NO: 16, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising residues 138-725 of SEQ ID NO: 16, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising at least 90% sequence identity to residues 138-725 of SEQ ID NO: 16. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising at least 98% sequence identity to residues 138-725 of SEQ ID NO: 16. In one aspect, the first region is a region of an AAV VP capsid polypeptide comprising residues 138-725 of SEQ ID NO: 16. In one aspect, the engineered VP capsid polypeptide comprises a second region, wherein the second region comprises a region of a AAV9 VP polypeptide. In one aspect, the AAV9 polypeptide comprises at least 90% sequence identity to SEQ ID NO: 4. In one aspect, the AAV9 polypeptide comprises at least 98% sequence identity to SEQ ID NO: 4. In one aspect, the AAV9 polypeptide comprises SEQ ID NO: 4. In one aspect, the second region comprises at least 90% sequence identity to -20- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOresidues 1-137 of SEQ ID NO:4. In one aspect, the second region comprises at least 98% sequence identity to residues 1-137 of SEQ ID NO:4. In one aspect, the second region comprises residues 1-137 of SEQ ID NO:4. In one aspect, the engineered VP capsid polypeptide comprises a second region, wherein the second region comprises a region of the AAV9 VP polypeptide. In one aspect, the chimeric VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 16, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 16, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 16, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 26, or SEQ ID NO: 28. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 26, or SEQ ID NO: 28. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 26, or SEQ ID NO: 28. In one aspect, the VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 20, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 20, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 20, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 21. In one aspect, the VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO: 24, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 24, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 24, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25.

[0091] The present disclosure provides a viral protein (VP) capsid polypeptide. In some embodiments, the VP capsid polypeptide is a chimeric VP capsid polypeptide. In some embodiments, the AAV5 VP capsid polypeptide is an AAV5 VP1 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the chimeric VP capsid polypeptide can include a first region and a second region. In some embodiments, the first region is a region of an AAV5 VP capsid polypeptide. In some embodiments, the AAV5 VP capsid polypeptide is an AAV5 VP1 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1, an AAV5 -21- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP2 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2, or an AAV5 VP3 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the region of the AAV5 VP capsid polypeptide includes one or more of residues 47-724 of the AAV5 VP1 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1; residues 110-724 of the AAV5 VP1 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1; and residues 137-724 of the AAV5 VP1 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the second region of an AAV VP capsid polypeptide, can be a region having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity compared to a wild type of an AAV9 VP polypeptide, an AAV1 VP polypeptide, an AAV2 VP polypeptide, an AAV3 VP polypeptide, an AAV4 VP polypeptide, an AAV6 VP polypeptide, an AAV7 VP polypeptide, an AAV8 VP polypeptide, an AAV 10 VP polypeptide, an AAV11 VP polypeptide, an AAV12 VP polypeptide, an AAV13 VP polypeptide, an AAV14 VP polypeptide, an AAV15 VP polypeptide, an AAV16 VP polypeptide, an AAV-DJ VP polypeptide, an AAV-DJ / 8 VP polypeptide, an AAV-DJ / 9 VP polypeptide, an AAV1 / 2 VP polypeptide, an AAV.rh8 VP polypeptide, an AAV.rhlO VP polypeptide, an AAV.rh20 VP polypeptide, an AAV.rh39 VP polypeptide, an AAV.Rh43 VP polypeptide, an AAV.Rh74 VP polypeptide, an AAV.v66 VP polypeptide, an AAV.OligoOOl VP polypeptide, an AAV.SCH9 VP polypeptide, an AAV.r3.45 VP polypeptide, an AAV.RHM4-1 VP polypeptide, an AAV.hu37 VP polypeptide, an AAV.Anc80 VP polypeptide, an AAV.Anc80L65 VP polypeptide, an AAV.7m8 VP polypeptide, an AAV.PhP.eB VP polypeptide, an AAV.PhP.Vl VP polypeptide, an AAV.PHP.B VP polypeptide, an AAV.PhB.Cl VP polypeptide, an AAV.PhB.C2 VP polypeptide, an AAV.PhB.C3 VP polypeptide, an AAV.PhB.C6 VP polypeptide, an AAV.cy5 VP polypeptide, an AAV2.5 VP polypeptide, an AAV2tYF VP polypeptide, an AAV3B VP polypeptide, an AAV.LK03 VP polypeptide, an AAV.HSC1 VP polypeptide, an AAV.HSC2 VP polypeptide, an AAV.HSC3 VP polypeptide, an AAV.HSC4 VP polypeptide, an AAV.HSC5 VP polypeptide, an AAV.HSC6 VP polypeptide, an AAV.HSC7 VP polypeptide, an AAV.HSC8 VP polypeptide, an AAV.HSC9 VP polypeptide, an AAV.HSC10 VP polypeptide, an AAV.HSC11 VP polypeptide, an AAV.HSC12 VP polypeptide, an AAV.HSC13 VP polypeptide, an AAV.HSC14 VP polypeptide, an AAV.HSC15 VP polypeptide, an -22- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOAAV.HSC16 VP polypeptide, an AAV.HSC17 VP polypeptide, or an AAVhu68 VP polypeptide; wherein the first region, which is the region of the AAV5 VP capsid polypeptide, and the second region of the AAV VP capsid polypeptide, are fused to form the chimeric VP capsid polypeptide. As a non-limiting example, the second region of the AAV VP capsid polypeptide is a region of the AAV9 VP polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity compared to a wild type of the AAV9 VP polypeptide. In some aspects, the AAV9 VP polypeptide is an AAV9 VP1 polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 4, or an AAV9 VP2 polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 31. In some aspects, the second region of the AAV VP capsid polypeptide includes residues 1-47 of the AAV9 VP1 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 4; residues 1-110 of the AAV9 VP1 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 4; or residues 1-137 of the AAV9 VP1 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the VP capsid polypeptide includes residues 47-724 of the VP capsid polypeptide derived from the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1, and residues 1-47 of the VP capsid polypeptide derived from the AAV9 VP1 capsid polypeptide of SEQ ID NO: 4. In some embodiments, the VP capsid polypeptide includes residues 110-724 of the VP capsid polypeptide from the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1, and residues 1-110 of the VP capsid polypeptide from the AAV9 VP1 capsid polypeptide of SEQ ID NO: 4. In some embodiments, the VP capsid polypeptide includes residues 137-724 of the VP capsid polypeptide from the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1, and residues 1-137 of the VP capsid polypeptide from the AAV9 VP1 capsid polypeptide of SEQ ID NO: 4. In some embodiments, the viral protein capsid polypeptide includes a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identity compared to any one of SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159. In some embodiments, the VP capsid polypeptide comprises a mutation that increases tissue tropism of the VP capsid polypeptide for a target tissue. In some embodiments, the target tissue is a central nervous system tissue, a muscle tissue, a retinal tissue, or a retinal pigment epithelium tissue. -23- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0092] In one embodiment, the present disclosure provides an engineered capsid comprising the engineered VP capsid polypeptide disclosed herein. In one embodiment, the present disclosure provides an engineered capsid comprising a VP1 sequence of SEQ ID NO: 14, a VP2 sequence of SEQ ID NO: 18 and a VP3 sequence of SEQ ID NO: 22. In one embodiment, the present disclosure provides an engineered capsid comprising a VP1 sequence of SEQ ID NO: 16, a VP2 sequence of SEQ ID NO: 20 and a VP3 sequence of SEQ ID NO: 24. In one aspect, the present disclosure provides an engineered capsid comprising a VP1 sequence of SEQ ID NO: 15, a VP2 sequence of SEQ ID NO: 19 and a VP3 sequence of SEQ ID NO: 23. In one embodiment, the present disclosure provides an engineered capsid comprising a VP1 sequence of SEQ ID NO: 17, a VP2 sequence of SEQ ID NO: 21 and a VP3 sequence of SEQ ID NO: 25.

[0093] The present disclosure also provides pharmaceutical compositions of the VP capsid polypeptides described herein. In some embodiments, the VP capsid polypeptide is assembled into a recombinant viral capsid.

[0094] In one embodiment, the present disclosure provides a recombinant adeno-associated virus (rAAV) comprising the engineered VP capsid polypeptide described herein and a payload. In one aspect, the payload encodes a therapeutic payload. In one aspect, the payload encodes a peptide. In one aspect, the payload encodes an antibody or fragment thereof. In one aspect, the payload encodes a gene. In one aspect, the payload encodes a polynucleotide. In one aspect, the polynucleotide is a guide RNA. In one aspect, the guide RNA facilitates editing of a target. In one aspect, the guide RNA facilitates knockdown of a target. In one aspect, the guide RNA facilitates altered splicing of a target. In one aspect, the guide RNA facilitates exon skipping in a target. In one aspect, the guide RNA facilitates exon skipping in a target. In one aspect, the guide RNA facilitates editing of a target and exon skipping. In one aspect, the target is a CNS target. In one aspect, the target is SNCA.

[0095] In one embodiment, the present disclosure provides a pharmaceutical composition comprising the rAAV disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0096] The present disclosure also provides pharmaceutical compositions of the VP capsid polypeptides described herein. In some embodiments, the VP capsid polypeptide is assembled into a recombinant viral capsid. In some embodiments, the pharmaceutical composition further includes a payload encapsidated by the recombinant viral capsid. In some embodiments, the payload encodes a therapeutic polynucleotide or a therapeutic peptide. In some embodiments, the payload encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide -24- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO(ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof. In some embodiments, the payload encodes a component of a CRISPR / Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, or a transcriptional repressor.

[0097] The present disclosure provides a recombinant adeno-associated virus (rAAV) which includes the VP capsid polypeptide described herein assembled into a recombinant viral capsid and a payload encapsidated by the recombinant viral capsid. In some embodiments, the pharmaceutical composition further includes a payload encapsidated by the recombinant viral capsid. In some embodiments, the payload encodes a therapeutic polynucleotide or a therapeutic peptide. In some embodiments, the payload encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof. In some embodiments, the payload encodes a component of a CRISPR / Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, or a transcriptional repressor.

[0098] The present disclosure also provides a method of increasing transduction of a payload in a target tissue by administering the rAAV described herein and infecting the target tissue with the rAAV.

[0099] In one embodiment, the present disclosure provides a method of delivering a payload to a CNS tissue, the method comprising administering to a subject the rAAV or the pharmaceutical composition disclosed herein. In one aspect, the administering is systemic administration. In one aspect, the payload encodes a peptide. In one aspect, the payload encodes a polynucleotide.

[0100] In one embodiment, the present disclosure provides a method of treating a condition in a subject, the method comprising administering to the subject the rAAV or the pharmaceutical composition described herein and expressing a polynucleotide encoded by the payload in a CNS tissue of the subject. In one aspect, the polynucleotide encodes a peptide. In one aspect, the polynucleotide is a guide RNA. In one aspect, the polynucleotide encodes an antibody or fragment thereof. In one aspect, the method includes further comprising editing a target in the subject. In one aspect, the method further includes facilitating knockdown of a target. In one aspect, the method further includes facilitating altered splicing of a target. In one aspect, the method includes further comprising exon skipping of a target in the subject. In one aspect, the method includes further comprising editing a target in the subject and exon skipping of the target in the subject. In one aspect, the target is a CNS -25- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOtarget. In one aspect, the target is implicated in the condition. In one aspect, the target is SNCA. In one aspect, the condition affects deep brain tissue. In one aspect, the condition is Parkinson’s disease, Alzheimer’s disease, Amyotrophic Lateral Sclerosis, Huntington’s disease, Lewy body dementia, essential tremor, dystonia, or epilepsy. In one embodiment, the present disclosure provides a method of transducing a neuron, the method comprising administering to a subject the rAAV or the pharmaceutical composition described herein and expressing a polynucleotide encoded by the payload in the neuron.

[0101] In one embodiment, the present disclosure provides a method of transducing an astrocyte, the method comprising administering to a subject the rAAV or the pharmaceutical composition described herein and expressing a polynucleotide encoded by the payload in the astrocyte. In one aspect, a ratio of payload expression in a brain tissue relative to a non-brain tissue upon administration of the rAAV is higher than upon administration of a wild type AAV. In one aspect, a ratio of target editing in a brain tissue relative to a non-brain tissue upon administration of the rAAV is higher than upon administration of a wild type AAV. In one aspect, the brain tissue is a deep brain tissue. In one aspect, the deep brain tissue is a substantia nigra tissue. In one aspect, the non-brain tissue is a liver tissue. In one aspect, the non-brain tissue is a peripheral nervous system tissue. In one aspect, the non-brain tissue is a dorsal root ganglion (DRG) tissue. In one aspect, the non-brain tissue is a muscle tissue. In one aspect, the administration of the rAAV produces lower toxicity than administration of a wild type AAV. In one aspect, the administration of the rAAV produces lower toxicity than administration of a wild type AAV as a result of decreased delivery, editing, or transduction of the AAV and payload in a liver tissue. In one aspect, the administration of the rAAV produces lower toxicity than administration of a wild type AAV as a result of decreased delivery, editing, or transduction of the AAV and payload in a dorsal root ganglion (DRG) tissue. In one aspect, the administration of the rAAV produces a lower immunogenicity response than administration of a wild type AAV.

[0102] The present disclosure also provides a method of delivering a payload to a target tissue by administering the rAAV described herein and infecting the target tissue with the rAAV.

[0103] The present disclosure also provides methods of transcribing a payload in a target tissue by administering the rAAV described herein and infecting the target tissue with the rAAV. Also provided herein are methods of treating a condition in a subject by administering the rAAV described herein and infecting a target tissue with the rAAV. In some embodiments, upon delivery to the target tissue, the payload exhibits increased transduction -26- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOcompared to a payload delivered via an AAV5 comprising a wild type VP1 capsid polypeptide, a wild type VP2 capsid polypeptide, and a wild type VP3 capsid polypeptide. In some embodiments, the target tissue is a central nervous system tissue, a muscle tissue, a retina tissue, or a retinal pigment epithelium tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings where:

[0105] FIG.1 shows a dot plot of percentage of on-target ADAR-mediated editing of an SNCA 3’ untranslated region (UTR) sequence in different brain regions of a non-human primate (NHP) upon systemic administration of an engineered AAV encapsidating a payload encoding an SNCA-targeting guide RNA (gRNA). Percent editing was compared across three different engineered AAV capsids: an engineered control capsid (“Engineered AAV Control,” top), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera,” middle), and an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 14 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 Chimera,” bottom). Individual points represent percent editing in different brain regions, including medulla, pons, midbrain, cerebellum, frontal lobe, temporal lobe, occipital lobe, motor cortex, Wernicke’s area, Broca’s area, thalamus, hypothalamus, hippocampus, substantia nigra, globus pallidus, putamen, and caudate. The point representing percent editing in the substantia nigra is labeled along with the percent editing in the substantia nigra. Data shown in FIG.1 is also included in FIG.4, FIG.5, and FIG.6.

[0106] FIG.2A shows a dot plot of payload DNA levels, quantified as viral genomes per delivered genomes (vg / dg) and indicative of payload delivery, in different brain regions upon systemic administration of an engineered AAV encapsidating a payload encoding an SNCA- targeting gRNA. Payload DNA level was compared across six different engineered AAV capsids: an engineered control capsid (“Engineered AAV Control”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 13 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 AAV5”), -27- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOan engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 14 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 15 containing a sequence of SEQ ID NO: 10 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 2 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”), and an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 17 containing a sequence of SEQ ID NO: 12 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 4 Chimera”). Individual points represent payload DNA levels in different brain regions, including medulla, pons, midbrain, cerebellum, frontal lobe, temporal lobe, occipital lobe, motor cortex, Wernicke’s area, Broca’s area, thalamus, hypothalamus, hippocampus, substantia nigra, globus pallidus, putamen, and caudate.

[0107] FIG.2B shows a dot plot of payload DNA levels, quantified as vg / dg and indicative of payload delivery, in different liver regions upon systemic administration of an engineered AAV encapsidating a payload encoding an SNCA-targeting gRNA. Payload DNA level was compared across six different engineered AAV capsids: an engineered control capsid (“Engineered AAV Control”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 13 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 AAV5”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 14 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 15 containing a sequence of SEQ ID NO: 10 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 2 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”), and an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 17 containing a sequence of SEQ ID NO: 12 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 4 Chimera”). Individual points represent payload DNA levels in different liver biopsy samples.

[0108] FIG.3A shows a dot plot of gRNA levels, quantified as gRNA / U1 snRNA (gRNA / U1) and indicative of payload expression, in different brain regions upon systemic administration of an engineered AAV encapsidating a payload encoding an SNCA-targeting gRNA. Guide RNA level was compared across six different engineered AAV capsids: an -28- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOengineered control capsid (“Engineered AAV Control”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 13 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 AAV5”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 14 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 15 containing a sequence of SEQ ID NO: 10 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 2 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”), and an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 17 containing a sequence of SEQ ID NO: 12 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 4 Chimera”). Individual points represent gRNA levels in different brain regions, including medulla, pons, midbrain, cerebellum, frontal lobe, temporal lobe, occipital lobe, motor cortex, Wernicke’s area, Broca’s area, thalamus, hypothalamus, hippocampus, substantia nigra, globus pallidus, putamen, and caudate.

[0109] FIG.3B shows a dot plot of gRNA levels, quantified as gRNA / U1 and indicative of payload expression, in different liver regions upon systemic administration of an engineered AAV encapsidating a payload encoding an SNCA-targeting gRNA. Guide RNA level was compared across six different engineered AAV capsids: an engineered control capsid (“Engineered AAV Control”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 13 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 AAV5”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 14 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 15 containing a sequence of SEQ ID NO: 10 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 2 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”), and an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 17 containing a sequence of SEQ ID NO: 12 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 4 Chimera”). Individual points represent gRNA levels in different liver biopsy samples. No points are shown for Variant 2 Chimera as they were below the limit of detection. -29- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0110] FIG.4 shows a dot plot of percentage of on-target ADAR-mediated editing of an SNCA 3’ UTR sequence in different brain regions of an NHP upon systemic administration of an engineered AAV encapsidating a payload encoding an SNCA-targeting gRNA. FIG.4 includes the data shown in FIG.1. Percent editing was compared across six different engineered AAV capsids: an engineered control capsid (“Engineered AAV Control”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 13 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 AAV5”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 14 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 15 containing a sequence of SEQ ID NO: 10 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 2 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”), and an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 17 containing a sequence of SEQ ID NO: 12 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 4 Chimera”). Individual points represent percent editing in different brain regions, including medulla, pons, midbrain, cerebellum, frontal lobe, temporal lobe, occipital lobe, motor cortex, Wernicke’s area, Broca’s area, thalamus, hypothalamus, hippocampus, substantia nigra, globus pallidus, putamen, and caudate.

[0111] FIG.5 shows a dot plot comparing the percent on-target editing in brain tissue following systemic intravenous (IV) administration of the Variant 1 Chimera (“Variant 1 Chimera (IV)”) capsid and the Variant 3 Chimera (“Variant 1 Chimera (IV)”) capsid, as shown and described in FIG.1 and FIG.4, to on-target editing following direct injection (intraperitoneal and intracerebroventricular) of an AAV9 capsid (“AAV9 (IP + ICV)”).

[0112] FIG.6 shows a dot plot of the percent on-target editing upon systemic administration of the Variant 1 Chimera (dark blue points), the Variant 3 chimera (light blue points), or the engineered AAV control (gray points), as shown and described in FIG.1 and FIG.4, separated by brain region.

[0113] FIG.7A shows a bar plot of percentage of CHO cells expressing a gRNA (% gRNA+) following treatment with AAV capsids encoding the gRNA at three different concentrations (Vg / cell): 5E3, 5E4, and 5E5 viral genomes per cell (vg / cell). At each concentration, cells were treated with (from left to right), wild type AAV5, an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 17 containing a sequence of SEQ -30- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOID NO: 12 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 4 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 13 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 AAV5”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 14 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”), or an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 15 containing a sequence of SEQ ID NO: 10 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 2 Chimera”).

[0114] FIG.7B shows a bar plot of gRNA signal intensity (gRNA gMFI) in CHO cells following treatment with AAV capsids encoding the gRNA at three different concentrations (Vg / cell): 5E3, 5E4, and 5E5 viral genomes per cell (vg / cell). At each concentration, cells were treated with (from left to right), wild type AAV5, an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 17 containing a sequence of SEQ ID NO: 12 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 4 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 13 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 AAV5”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 14 containing a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 Chimera”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”), or an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 15 containing a sequence of SEQ ID NO: 10 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 2 Chimera”).

[0115] FIG.8A shows a plot of percentage on-target editing of a target mRNA in human iPSC-derived neurons produced from two donors (“Donor 1” and “Donor 2”) following treatment with AAV capsids encoding a gRNA targeting the mRNA at three different concentrations (Vg / cell): 2E4, 1E5, and 5E5 viral genomes per cell (vg / cell). At each concentration, cells from Donor 1 and Donor 2 were treated with wild type AAV5 (lower cluster of points at each concentration), or an engineered AAV5 capsid (upper cluster of points at each concentration) comprising either a VP1 sequence of SEQ ID NO: 14 -31- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOcontaining a sequence of SEQ ID NO: 9 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 1 Chimera”) or a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”). Cells not treated with virus (“No Virus”) are shown as a negative control (bottom points at 2E4).

[0116] FIG.8B shows a plot of gRNA levels, quantified as gRNA / U1 snRNA (gRNA / U1) and indicative of payload expression, in ex vivo mouse neurons following treatment with AAV capsids encoding a gRNA targeting the mRNA at three different concentrations of viral genomes per cell (Vg / Cell). At each concentration, cells were treated with wild type AAV5 (“WT AAV5”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”), an AAV positive control (“AAV Positive Control”), an AAV control (“AAV Control”), an engineered control capsid (“Engineered AAV Control”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 29 containing a sequence of SEQ ID NO: 30 at amino acid positions corresponding to 568-590 of AAV5 (“AAV5 / 9 Chimera”), or an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 15 containing a sequence of SEQ ID NO: 10 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 2 Chimera”). Cells not treated with virus (“untransduced”) are shown as a negative control.

[0117] FIG.8C shows a plot Thy1.2 expression levels, quantified as Thyl.1 / House Keeping Gene (Thy1.1 / HKG) and indicative of payload expression in ex vivo mouse neurons following treatment with AAV capsids encoding a Thy1.2 mRNA at three different concentrations of viral genomes per cell (Vg / Cell). At each concentration, cells were treated with wild type AAV5 (“WT AAV5”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 16 containing a sequence of SEQ ID NO: 11 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 3 Chimera”), an AAV positive control (“AAV Positive Control”), an AAV control (“AAV Control”), an engineered control capsid (“Engineered AAV Control”), an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 29 containing a sequence of SEQ ID NO: 30 at amino acid positions corresponding to 568-590 of AAV5 (“AAV5 / 9 Chimera”), or an engineered AAV capsid comprising a VP1 sequence of SEQ ID NO: 15 containing a sequence of SEQ ID NO: 10 at amino acid positions corresponding to residues 568-590 of AAV5 (“Variant 2 Chimera”). Cells not treated with virus (“untransduced”) are shown as a negative control. -32- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0118] FIG.9 shows a dot plot of the biodistribution (vg / dg) of capsid genomes upon systemic administration of the Variant 1 Chimera (dark blue points), the Variant 3 chimera (light blue points), or the engineered AAV control (gray points) in different tissues including, from left to right: Medulla; Pons; Midbrain; Cerebellum; Frontal lobe; Temporal lobe; Occipital lobe; Motor cortex; Thalamus; Hypothalamus; Hippocampus; Substantia nigra; Globus pallidus; Putamen; Caudate; Spinal cord, lumbar; Spinal cord, cervical; Spinal cord, thoracic; Nerve, median; Nerve, radial; Nerve, sural; Nerve, tibial; Nerve, peroneal; Never, sciatic, left; Nerve, sciatic, right; Nerve roots, cervical, dorsal; Nerve roots, cervical, ventral; Nerve roots, thoracic, dorsal; Nerev roots, thoracic, ventral; Nerve roots, lumbar, dorsal; Nerve roots, lumbar, ventral; DRG, cervical; DRG, thoracic; DRG, lumbar; Skeletal muscle, quadricep, left; Skeletal muscle, quadricep, right; Lung, left caudal lobe; Heart, left atrium; Heart, left ventricle; Heart, right atrium; Heart, right ventricle; Bone marrow; Lymph node, mandibular; Adrenal glands; Pancreas; Stomach; Duodenum; Jejunum; Ileum; Cecum; Colon; Ovaries; Kidney, left; Ureter (slab 2); Ureter (slab 1); Urinary bladder; Spleen; Liver, anterior left lateral lobe; Liver, posterior left lateral lobe; Liver, anterior right lateral lobe; Liver, posterior lateral lobe; Liver, left median lobe; Liver, right median lobe.

[0119] FIG.10 shows a dot plot of expression of a thy1.2 mRNA marker encoded by the capsid genome relative to TBP (thy1.1 mRNA / TBP) upon systemic administration of the Variant 1 Chimera (dark blue points), the Variant 3 chimera (light blue points), or the engineered AAV control (gray points) in different tissues including, from left to right: Medulla; Pons; Midbrain; Cerebellum; Frontal lobe; Temporal lobe; Occipital lobe; Motor cortex; Thalamus; Hypothalamus; Hippocampus; Substantia nigra; Globus pallidus; Putamen; Caudate; Spinal cord, lumbar; Spinal cord, cervical; Spinal cord, thoracic; Nerve, median; Nerve, radial; Nerve, sural; Nerve, tibial; Nerve, peroneal; Never, sciatic, left; Nerve, sciatic, right; Nerve roots, cervical, dorsal; Nerve roots, cervical, ventral; Nerve roots, thoracic, dorsal; Nerev roots, thoracic, ventral; Nerve roots, lumbar, dorsal; Nerve roots, lumbar, ventral; DRG, cervical; DRG, thoracic; DRG, lumbar; Skeletal muscle, quadricep, left; Skeletal muscle, quadricep, right; Lung, left caudal lobe; Heart, left atrium; Heart, left ventricle; Heart, right atrium; Heart, right ventricle; Bone marrow; Lymph node, mandibular; Adrenal glands; Pancreas; Stomach; Duodenum; Jejunum; Ileum; Cecum; Colon; Ovaries; Kidney, left; Ureter (slab 2); Ureter (slab 1); Urinary bladder; Spleen; Liver, anterior left lateral lobe; Liver, right lateral lobe, anterior.

[0120] FIG.11A shows a bar graph of percentage of cells that were positive for gRNA expression (% Cells gRNA+), as measured by RNA fluorescence in situ hybridization (RNA- -33- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOFISH), upon systemic administration of the Variant 3 Chimera, the Variant 1 Chimera, or the engineered AAV control. Each point corresponds to a coronal section of a brain hemisphere.

[0121] FIG.11B shows a bar graph of percentage of cells that were positive for Thy1.2 mRNA expression (% Cells Thy+), as measured by RNA-FISH, upon systemic administration of the Variant 3 Chimera, the Variant 1 Chimera, or the engineered AAV control. Each point corresponds to a coronal section of a brain hemisphere.

[0122] FIG.12A shows a bar graph of percentage of astrocytes that were positive for gRNA expression (% Astrocytes gRNA+), as measured by RNA-FISH, upon systemic administration of the Variant 3 Chimera, the Variant 1 Chimera, or the engineered AAV control. Each point corresponds to a coronal section of a brain hemisphere.

[0123] FIG.12B shows a bar graph of percentage of astrocytes that were positive for Thy1.2 mRNA expression (% Astrocytes Thy+), as measured by RNA-FISH, upon systemic administration of the Variant 3 Chimera, the Variant 1 Chimera, or the engineered AAV control. Each point corresponds to a coronal section of a brain hemisphere.

[0124] FIG.13A shows a bar graph of percentage of neurons that were positive for gRNA expression (% Neurons gRNA+), as measured by RNA-FISH, upon systemic administration of the Variant 3 Chimera, the Variant 1 Chimera, or the engineered AAV control. Each point corresponds to a coronal section of a brain hemisphere.

[0125] FIG.13B shows a bar graph of percentage of neurons that were positive for Thy1.2 mRNA expression (% Neurons Thy+), as measured by RNA-FISH, upon systemic administration of the Variant 3 Chimera, the Variant 1 Chimera, or the engineered AAV control. Each point corresponds to a coronal section of a brain hemisphere.

[0126] FIG.14A shows a bar graph of percentage of neurons in different brain tissues that were positive for gRNA expression (% positive cells), as measured by RNA-FISH, upon systemic administration of the Variant 3 Chimera. Tissues in which gRNA positive neurons were quantified include substantia nigra, globus pallidus, hypothalamus, thalamus, caudate, putamen, hippocampus, temporal cortex, frontal cortex, motor cortex, occipital lobe, and cerebellum.

[0127] FIG.14B shows a bar graph of percentage of neurons in different brain tissues that were positive for Thy1.2 mRNA expression (% positive cells), as measured by RNA-FISH, upon systemic administration of the Variant 3 Chimera. Tissues in which mRNA positive neurons were quantified include substantia nigra, globus pallidus, hypothalamus, thalamus, caudate, putamen, hippocampus, temporal cortex, frontal cortex, motor cortex, occipital lobe, and cerebellum. -34- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0128] FIG.15A shows a bar graph of percentage of neurons in different brain tissues that were positive for gRNA expression (% positive cells), as measured by RNA-FISH, upon systemic administration of the Variant 1 Chimera. Tissues in which gRNA positive neurons were quantified include substantia nigra, globus pallidus, thalamus, caudate, hippocampus, temporal cortex, frontal cortex, motor cortex, occipital lobe, and cerebellum.

[0129] FIG.15B shows a bar graph of percentage of neurons in different brain tissues that were positive for Thy1.2 mRNA expression (% positive cells), as measured by RNA-FISH, upon systemic administration of the Variant 1 Chimera. Tissues in which mRNA positive neurons were quantified include substantia nigra, globus pallidus, thalamus, caudate, hippocampus, temporal cortex, frontal cortex, motor cortex, occipital lobe, and cerebellum.

[0130] FIG.15C is a dot plot of percentage of cells that were positive for AAV genome transduction (% positive cells) as measured by DNA-FISH, upon systemic administration of Variant 3 Chimera, Variant 1 Chimera, and an engineered AAV control (“Control”).

[0131] FIG.15D is a dot plot showing percentage of neurons that were positive for AAV genome transduction (% positive cells) as measured by DNA-FISH, upon systemic administration of Variant 3 Chimera, Variant 1 Chimera, and an engineered AAV control (“Control”).

[0132] FIG.15E is a scatter plot showing DNA payload (vg / dg) as a function of percentage of cells that were positive for AAV genome transduction (genome +) in various regions of the brain, upon systemic administration of Variant 3 Chimera or Variant 1 Chimera.

[0133] FIG.15F is a scatter plot showing DNA payload (vg / dg) as a function of percentage of cells that were positive for AAV genome transduction (genome +) in various regions of the brain, but excluding the cortex, upon system administration of Variant 3 Chimera or Variant 1 Chimera.

[0134] FIG.15G is a dot plot showing percentage of cells that were positive for AAV genome transduction (% cells AAV genome +) as measured by DNA-FISH either without denaturing DNA (stains only for ssDNA; “ssDNA FISH”) or with the denaturing step (stains total DNA; “normal DNA FISH”) in cortical sections from primates administered Variant 3 Chimera or Variant 1 Chimera.

[0135] FIG.16A shows a scatter plot of percent target editing (%editing) as a function of percentage of neurons that were positive for gRNA expression (%Neurons gRNA+) in brain tissue, upon systemic administration of the Variant 3 Chimera. -35- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0136] FIG.16B shows a scatter plot of percent target editing (%editing) as a function of percentage of neurons that were positive for gRNA expression (%Neurons gRNA+) in brain tissue, upon systemic administration of the Variant 1 Chimera.

[0137] FIG.16C shows a scatter plot of percent target editing (% targeted RNA editing) as a function of percentage of neurons that were positive for gRNA expression (%Neurons gRNA+) in brain tissue, upon systemic administration of the Variant 3 Chimera (light blue) and Variant 1 Chimera (dark blue).

[0138] FIG.16D shows a bar graph of percentage of neurons or glial cells in different brain tissues that were positive for gRNA expression (% positive), as measured by RNA-FISH, for neuron cell marker NeuN positive and gRNA positive (“NeuN gRNA+”), neuron cell marker NeuN positive and Thy1.2 mRNA positive (“NeuN+ Thy1.2+”), astrocyte cell marker GFAP positive and gRNA positive (“GFAP+ gRNA+”), astrocyte cell marker GFAP positive and Thy1.2 mRNA positive (“GFAP+ Thy1.2+”), upon systemic administration of the Variant 3 Chimera.

[0139] FIG.16E shows a bar graph of percentage of neurons (neuron cell marker NeuN positive), astrocytes (astrocyte cell marker GFAP positive), or all cells in different brain tissues that were positive for gRNA expression (% gRNA+), as measured by RNA-FISH, upon systemic administration of the Variant 3 Chimera.

[0140] FIG.16F shows a bar graph of percentage of neurons (neuron cell marker NeuN positive), astrocytes (astrocyte cell marker GFAP positive), or all cells in different brain tissues that were positive for Thy1.2 mRNA expression (% Thy+), as measured by RNA- FISH, upon systemic administration of the Variant 3 Chimera.

[0141] FIG.16G shows a bar graph of percentage of neurons (neuron cell marker NeuN positive), astrocytes (astrocyte cell marker GFAP positive), or all cells in different brain tissues that were positive for gRNA expression (% gRNA+), as measured by RNA-FISH, upon systemic administration of the Variant 1 Chimera.

[0142] FIG.16H shows a bar graph of percentage of neurons (neuron cell marker NeuN positive), astrocytes (astrocyte cell marker GFAP positive), or all cells in different brain tissues that were positive for Thy1.2 mRNA expression (% Thy1.1+), as measured by RNA- FISH, upon systemic administration of the Variant 1 Chimera.

[0143] FIG.17A shows a scatter plot of binding antibody (Bab) activity against Variant 3 Chimera as measured by absorbance at 450 nm (OD450) as a function of serum dilution from serum collected from serotyped macaques that were AAV5-positive, AAV9-positive, and AAV5 and AAV9-negative (“Double-negative”). -36- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0144] FIG.17B shows a scatter plot of BAb activity against Variant 1 Chimera as measured by absorbance at 450 nm (OD450) as a function of serum dilution from serum collected from serotyped macaques that were AAV5-positive, AAV9-positive, and AAV5 and AAV9- negative (“Double-negative”).

[0145] FIG.17C shows a scatter plot of BAb activity against the Engineered AAV Control as measured by absorbance at 450 nm (OD450) as a function of serum dilution from serum collected from serotyped macaques that were AAV5-positive, AAV9-positive, and AAV5 and AAV9-negative (“Double-negative”).

[0146] FIG.18A shows a scatter plot of BAb activity against Variant 1 Chimera, Variant 3 Chimera, and the Engineered AAV Control as measured by absorbance at 450 nm (OD450) as a function of serum dilution from serum collected from serotyped macaques that were AAV5 and AAV9-negative (“AAV5-AAV9-”).

[0147] FIG.18B shows a scatter plot of BAb activity against Variant 1 Chimera, Variant 3 Chimera, and the Engineered AAV Control as measured by absorbance at 450 nm (OD450) as a function of serum dilution from serum collected from serotyped macaques that were AAV5-positive (“AAV5+AAV9-”).

[0148] FIG.18C shows a scatter plot of BAb activity against Variant 1 Chimera, Variant 3 Chimera, and the Engineered AAV Control as measured by absorbance at 450 nm (OD450) as a function of serum dilution from serum collected from serotyped macaques that were AAV9-positive (“AAV5-AAV9+”).

[0149] FIG.18D shows a scatter plot of BAb activity against Variant 1 Chimera, Variant 3 Chimera, and the Engineered AAV Control as measured by absorbance at 450 nm (OD450) as a function of serum dilution from serum collected from serotyped macaques that were AAV5 and AAV9-positive (“AAV5+AAV9+”).

[0150] FIG.19A shows a scatter plot of BAb activity against wtAAV5 as measured by absorbance at 450 nm (OD450) as a function of serum dilution from serum collected from macaques that were treated with Variant 1 Chimera, Variant 3 Chimera, and the Engineered AAV Control.

[0151] FIG.19B shows a scatter plot of BAb activity against wtAAV9 as measured by absorbance at 450 nm (OD450) as a function of serum dilution from serum collected from macaques that were treated with Variant 1 Chimera, Variant 3 Chimera, and the Engineered AAV Control. -37- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0152] FIG.20 shows a dot plot of virus production (“average vg per hyperflask”) of the capsids of Variant 3 chimera, Variant 1 Chimera, and wild type AAVs including AAV5, AAV1, AAV2, AAV6, AAV8, and AAV9. Each point represents a single production run.

[0153] FIG.21A shows a scatter plot of percentage of gRNA positive cells (“% gRNA +”) at the tested multiplicities of infection (MOIs) (5e3, 5e4, and 5e5 vg / cell) for Variant 4 AAV5, Variant 4 Chimera, Variant 2 AAV5, Variant 2 Chimera, Variant 3 AAV5, Variant 3 Chimera, Variant 1 AAV5, and Variant 1 Chimera.

[0154] FIG.21B shows a scatter plot of gRNA expression (“gRNA gMFI (AF555)”) at the tested MOIs (5e3, 5e4, and 5e5 vg / cell) for Variant 4 AAV5, Variant 4 Chimera, Variant 2 AAV5, Variant 2 Chimera, Variant 3 AAV5, Variant 3 Chimera, Variant 1 AAV5, and Variant 1 Chimera.

[0155] FIG.21C shows a scatter plot of percentage of capsid positive cells (“% Capsid +”) at the tested MOIs (5e3, 5e4, and 5e5 vg / cell) for Variant 4 AAV5, Variant 4 Chimera, Variant 2 AAV5, Variant 2 Chimera, Variant 3 AAV5, Variant 3 Chimera, Variant 1 AAV5, and Variant 1 Chimera.

[0156] FIG.21D shows a scatter plot of capsid quantification (“Capsid gMFI (PacBlue)”) at the tested MOIs (5e3, 5e4, and 5e5 vg / cell) for Variant 4 AAV5, Variant 4 Chimera, Variant 2 AAV5, Variant 2 Chimera, Variant 3 AAV5, Variant 3 Chimera, Variant 1 AAV5, and Variant 1 Chimera.

[0157] FIG.22 shows the gRNA expression relative to U1 expression (“gRNA / U1”) for the WT AAV5 / 9 Chimera, Variant 1 Chimera, and Variant 3 Chimera in primary human hepatocytes.

[0158] FIG.23 shows (top) plots of payload DNA levels, quantified as viral genomes per delivered genomes (vg / dg) and indicative of payload delivery, in different brain regions (left plot) versus liver (right plot) for payloads delivered using the engineered control capsid (“Engineered AAV Control”), the Variant 1 AAV5 capsid, and the Variant 1 Chimera capsid and (bottom) plots of gRNA levels, quantified as gRNA / U1 snRNA (gRNA / U1) and indicative of payload expression, in different brain regions (left plot) versus liver (right plot) for payloads delivered using the engineered control capsid (“Engineered AAV Control”), the Variant 1 AAV5 capsid, and the Variant 1 Chimera capsid.

[0159] FIG.24 shows luciferase expression in H4 cells transduced with AAV5 wild type or chimeric VP capsid polypeptides. In FIG.24, “chimera 1” corresponds to cells transduced with SEQ ID NO: 33, “chimera 2” corresponds to cells transduced with SEQ ID NO: 34, and “chimera 3” corresponds to cells transduced with SEQ ID NO: 29. -38- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0160] FIG.25A and FIG.25B show transgene expression, measured by ddPCR for guide RNA quantification, in H4 cells for AAV5 capsid B (FIG.25A) and AAV5 capsid A (FIG. 25B). Capsid A contains a sequence of SEQ ID NO: 5 at amino acid positions corresponding to residues 581-590 of AAV5 in either a non-chimera VP1 sequence of SEQ ID NO: 13 (“WT-VP1”) or a chimeric VP1 sequence of SEQ ID NO: 14 (“Chimeric-VP1”). Capsid B was tested in either a non-chimera VP1 sequence (“WT-VP1”) or a chimeric VP1 sequence (“Chimeric-VP1”). The chimeric AAV5 capsid B with VP1 exhibited five times greater transgene expression than wild type AAV5 capsid B (FIG.25A). The chimeric AAV5 capsid A with VP1 also exhibited five times greater transgene expression than wild type AAV5 capsid A (FIG.25B). DETAILED DESCRIPTION OF THE INVENTION

[0161] Unless described otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention pertains.

[0162] Unless otherwise stated, whenever a range is recited, the range is inclusive of the recited endpoints. For example, the region from amino acid residue 568 to amino acid residue 590 of SEQ ID NO: 1 includes amino acid residues 568 and 590.

[0163] “Homology” or “identity” or “sequence identity” or “similarity” can refer to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which can be aligned for purposes of comparison. When a position in the compared sequence can be occupied by the same base or amino acid, then the molecules can be homologous at that position. A degree of homology between sequences can be a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the disclosure. Sequence homology can refer to a % identity of a sequence to a reference sequence. As a practical matter, whether any particular sequence can be at least 50%, 60%, 70%, 77.7%, 80%, 85%, 88.8%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to any sequence described herein (which can correspond with a particular nucleic acid or amino acid sequence described herein), such particular polypeptide sequence can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis.53711). When using Bestfit or any other -39- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOsequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence, the parameters can be set such that the percentage of identity can be calculated over the full length of the reference sequence and that gaps in sequence homology of up to 5% of the total reference sequence can be allowed. The term percent “identity” or percent “sequence identity” or percent “homology,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” or the percent “sequence identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. For purposes herein, determination of percent identity and sequence similarity is performed using the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ). For example, where an amino acid sequence consisting of 9 residues is compared with another 9 residue amino acid sequence, if 8 residues match then the sequences have 88.8% identity, if 7 residues match then the sequences have 77.7% identity.

[0164] In some cases, the identity between a reference sequence (query sequence, e.g., a sequence of the disclosure) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program. In some embodiments, parameters for a particular embodiment in which identity can be narrowly construed, used in a FASTDB amino acid alignment, can include: Scoring Scheme=PAM (Percent Accepted Mutations) 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject sequence, whichever can be -40- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOshorter. According to this embodiment, if the subject sequence can be shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction can be made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity can be corrected by calculating the number of residues of the query sequence that can be lateral to the N- and C-terminal of the subject sequence, which can be not matched / aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue can be matched / aligned can be determined by results of the FASTDB sequence alignment. This percentage can then be subtracted from the percent identity, calculated by the FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score can be used for the purposes of this embodiment. In some cases, only residues to the N- and C-termini of the subject sequence, which cannot be matched / aligned with the query sequence, can be considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C- terminal residues of the subject sequence can be considered for this manual correction. For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence, and therefore, the FASTDB alignment does not show a matching / alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched / total number of residues in the query sequence) so 10% can be subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percent identity can be 90%. In another example, a 90-residue subject sequence can be compared with a 100-residue query sequence. This time the deletions can be internal deletions, so there can be no residues at the N- or C-termini of the subject sequence which can be not matched / aligned with the query. In this case, the percent identity calculated by FASTDB cannot be manually corrected. Once again, only residue positions outside the N- and C- terminal ends of the subject sequence, as displayed in the FASTDB alignment, which can be not matched / aligned with the query sequence can be manually corrected for.

[0165] Peptide sequences for use in the present invention may include one or more conservative amino acid substitutions, such that the resulting sequence has a similar amino acid sequence and / or retains the same function. The skilled person is aware that various -41- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOamino acids have similar biochemical properties and thus are “conservative”. One or more such amino acids of a protein, polypeptide or peptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that protein, polypeptide or peptide. Thus, the amino acids glycine, alanine, valine, leucine, and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids which can often be substituted for one another include: phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulfur containing side chains). It should be appreciated that amino acid substitutions within the scope of the present invention can be made using naturally occurring or non-naturally occurring amino acids. For example, the methyl group on an alanine may be replaced with an ethyl group, and / or minor changes may be made to the peptide backbone. Whether or not natural or synthetic amino acids are used, it is preferred that only L- amino acids are present. Substitutions of this nature are often referred to as “conservative substitutions”.

[0166] For simplicity throughout this disclosure, viral capsid protein is generally referred to as “VP.” In some embodiments, viral capsid sequences disclosed herein pertain to one or more isoforms of the capsid protein (e.g. VP1, VP2, and / or VP3), as a mixture of these isoforms assemble to form virions.

[0167] As used herein, “wild type AAV5 VP1” or “wild type AAV5 VP1 capsid polypeptide” refers to a VP1 capsid polypeptide of SEQ ID NO: 1 (MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPG NGLDRGEPVNRADEVAREHDISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGG NLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDA EAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDST WMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRF HSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFT DDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCLEYFP SKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQF NKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQ -42- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQPVNR VAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKIPETG AHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEME WELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL). In some embodiments, the AAV5 VP1 capsid polypeptide has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1.

[0168] As used herein, “wild type AAV5 VP2” or “wild type AAV5 VP2 capsid polypeptide” refers to a VP2 capsid polypeptide of SEQ ID NO: 2 (TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTM SAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNNHQ YREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSL RVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQ VFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSF APSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRT QGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTM IFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGT YNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKN TPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQ FVDFAPDSTGEYRTTRPIGTRYLTRPL). In some embodiments, the AAV5 VP2 capsid polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2.

[0169] As used herein, “wild type AAV5 VP3” or “wild type AAV5 VP3 capsid polypeptide” refers to a VP3 capsid polypeptide of SEQ ID NO: 3 (MSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNN HQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPR SLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPP QVFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSS FAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGR TQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENT MIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPAT GTYNLQEIVPGSVWMERDVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLI KNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYND PQFVDFAPDSTGEYRTTRPIGTRYLTRPL). In some embodiments, the AAV5 VP3 capsid -43- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOpolypeptide has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 3.

[0170] As used herein, “wild type AAV5” or “wild type AAV5 capsid polypeptide” refers to a VP1 capsid polypeptide of SEQ ID NO: 1, a VP2 capsid polypeptide of SEQ ID NO: 2, a VP3 capsid polypeptide of SEQ ID NO: 3, or a combination thereof.

[0171] As used herein, “wild type AAV9 VP1” or “wild type AAV9 VP1 capsid polypeptide” refers to a VP1 capsid polypeptide of SEQ ID NO: 4 (MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWP GASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQ GPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTR YLTRNL). In some embodiments, the AAV9 VP1 capsid polypeptide has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 4.

[0172] As used herein, “AAV5 / 9 Chimera VP1” or “AAV5 / 9 VP1 capsid polypeptide” refers to a VP1 capsid polypeptide of SEQ ID NO: 29 (MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPTGKRIDDHFPKRKKARTEEDSKPST SSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWH CDSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFD FNRFHSHWSPRDWQRLINNYWGFRPRSLRVKIFNIQVKEVTVQDSTTTIANNLTSTV QVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDNTENPTERSSFFCL EYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTG GVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEG -44- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTTATYLEGNMLITSESETQ PVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERDVYLQGPIWAKI PETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTV EMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL).

[0173] In some embodiments, the AAV9 VP2 capsid polypeptide has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of TAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAP SGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALP TYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNW GFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGC LPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVP FHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRN YIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDR FFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQ AQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPP QILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTS NYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 31).

[0174] In some embodiments, the AAV9 VP3 capsid polypeptide has an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the amino acid sequence of MASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHL YKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKR LNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPA DVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSY AHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPS YRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLS GSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTG WVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIK NTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYK SNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 32).

[0175] As used herein, “wild type AAV9” or “wild type AAV9 capsid polypeptide” refers to a VP1 capsid polypeptide of SEQ ID NO: 4, a VP2 capsid polypeptide of SEQ ID NO: 31, a VP3 capsid polypeptide of SEQ ID NO: 32, or a combination thereof. -45- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0176] AAV engineered capsids and engineered capsid polypeptides described herein may comprise variations from a wild type AAV sequence or other AAV sequence in an engineered residue region. The variation positions within an engineered residue region (e.g., a chimeric region or a variant tropic region) in an AAV sequence (e.g., an AAV engineered capsid sequence or an engineered AAV capsid polypeptide sequence) may be described as the residue positioning corresponding to an AAV capsid sequence (e.g., an AAV5, an AAV9, an engineered AAV described herein, or any other AAV capsid known in the art). When a sequence is described as “corresponding to residues” of an AAV capsid sequence described herein, this is used to describe the positioning of the sequence as corresponding to the positions of residues in the AAV capsid sequence. For example, when a sequence is described as corresponding to residues 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1), this means that the sequence corresponds to the numerical positions of the 581-590 residues in wild type AAV5 VP1 (SEQ ID NO: 1) as determined by a sequence alignment rather than the amino acid identities of residues 581-590 residues in wild type AAV5 VP1 (SEQ ID NO: 1). For example, a sequence of SEQ ID NO: 7 may be described as corresponding to residues of 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) since upon alignment of a sequence comprising SEQ ID NO: 7 (e.g., SEQ ID NO: 16), the position of SEQ ID NO: 7 corresponds to the position of residues 581-590 in wild type AAV5 VP1 (SEQ ID NO: 1). In some embodiments, engineered residue region in an AAV sequence is described as corresponding to AAV5. In some embodiments, engineered residue region in an AAV sequence is described as corresponding to AAV9. In some embodiments, engineered residue region in an AAV sequence is described as corresponding to an AAV sequence as described herein.

[0177] Engineered residue regions as described herein and their positioning corresponding to AAV sequences described herein are provided in TABLE 1. TABLE 1 – Residue Positions Corresponding to AAV Sequences Described Herein Exemplary Region Exemplary Exemplary Region Positioning r-46- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOExemplary Region Exemplary Exemplary Region Positioning Positioning Region Corresponding to a Chimeric rEngineered AAV Viral Protein Capsid Polypeptides with CNS Tissue Tropism

[0178] Described herein are engineered AAV viral protein (VP) capsid polypeptides with enhanced tropism for CNS tissue. In some embodiments, an engineered AAV VP capsid polypeptide comprises one or more amino acid substitutions relative to an AAV VP capsid polypeptide (e.g., AAV5, AAV9, AAV2, AAV1, AAV3, AAV4, AAV6, AAV7, AAV8, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV- DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, -47- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOAAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 capsid polypeptide). In some embodiments, an engineered AAV VP capsid polypeptide comprises one or more amino acid substitutions in a tropic variant region relative to an AAV VP capsid polypeptide (e.g., AAV5, AAV9, AAV2, AAV1, AAV3, AAV4, AAV6, AAV7, AAV8, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 capsid polypeptide).

[0179] An engineered AAV VP capsid polypeptide comprising one or more amino acid substitutions or a tropic variant region relative to an AAV VP capsid polypeptide may describe the position of the amino acid substitution or the position of the tropic variant region as corresponding to a position in an AAV VP capsid polypeptide sequence (e.g., a wild type AAV5 VP1 capsid sequence of SEQ ID NO: 1 or an wild type AAV9 VP1 capsid sequence of SEQ ID NO: 4). To determine the position of the amino acid substitution or the position of the tropic variant region corresponding to a position in an AAV VP capsid polypeptide sequence (e.g., a wild type AAV5 VP1 capsid sequence of SEQ ID NO: 1 or a wild type AAV9 VP1 capsid sequence of SEQ ID NO: 4), an alignment between the engineered AAV VP capsid polypeptide sequence and a reference AAV VP capsid polypeptide sequence (e.g., a wild type AAV5 VP1 capsid sequence of SEQ ID NO: 1 or a wild type AAV9 VP1 capsid sequence of SEQ ID NO: 4) may be performed, wherein the position of the amino acid substitution or the position of the tropic variant region is determined as corresponding to the position of the aligned sequence or residue of the reference AAV VP capsid polypeptide sequence (e.g., a wild type AAV5 VP1 capsid sequence of SEQ ID NO: 1 or a wild type AAV9 VP1 capsid sequence of SEQ ID NO: 4). For example, a position of an amino acid substitution or a tropic variant region comprising one or more amino acid substitutions in an engineered AAV VP capsid may be determined as corresponding to a position in a wild type AAV5 VP1 sequence of SEQ ID NO: 1 by performing an alignment (e.g., a protein BLAST) between the engineered AAV VP capsid sequence and the wild type AAV5 VP1 sequence of SEQ ID NO: 1 and determining the position of the amino acid substitution or the tropic -48- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOvariant region as corresponding to the positions of the aligned amino acid residue or residues in SEQ ID NO: 1.

[0180] In some embodiments, an engineered AAV VP capsid polypeptide comprises a tropic variant region corresponding to residues 581-590 of a wild type AAV5 VP1 capsid polypeptide of SEQ ID NO: 1. In some embodiments, an engineered AAV VP capsid polypeptide comprises a tropic variant region corresponding to residues 592-601 corresponding to a wild type AAV9 VP1 capsid polypeptide of SEQ ID NO: 4.

[0181] In some embodiments, an engineered AAV VP capsid polypeptide comprises one or more amino acid substitutions relative to a wild type AAV5 VP capsid polypeptide (e.g., SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3,) within a region corresponding to residues 568-569 of AAV5 VP1. The amino acid sequence of AAV5 VP1 (also referred to as wild type AAV5 VP1) is as set forth in SEQ ID NO: 1. In some embodiments, an engineered AAV VP capsid polypeptide comprises one or more amino acid substitutions in a tropic variant corresponding to residues 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). Amino acid sequences corresponding to residues 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) (“568-689 Sequence”) and residues 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) (“581- 590 Sequence”) that may be included in an engineered AAV VP capsid polypeptide of the present disclosure are provided in TABLE 2. Also provided in TABLE 2 are engineered amino acid sequences corresponding to residues 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) (“568-590 Sequence”), comprising the amino acid sequences corresponding to residues 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) and residues 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) (e.g., a tropic variant region), that may be included in an engineered AAV VP capsid polypeptide. TABLE 2 – AAV VP Capsid Polypeptide Variant Sequences Variant 568-569 Sequence 581-590 Sequence 568-590 Sequence I I T I-49- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVariant 568-569 Sequence 581-590 Sequence 568-590 Sequence QM MDDICEFYAI QMATNNQSSTTAPMDDICEFYAI I I I I I I I I-50- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVariant 568-569 Sequence 581-590 Sequence 568-590 Sequence QM DAWQMLSGNI QMATNNQSSTTAPDAWQMLSGNI I I I GI I I

[0182] In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least four contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID -51- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WONO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581- 590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least four contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least five contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least five contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least six contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least six contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least seven contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least seven contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least eight contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581- 590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least eight contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of -52- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOwild type AAV5 VP1 (SEQ ID NO: 1), comprises at least nine contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least nine contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least ten contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least ten contiguous amino acids of SEQ ID NO: 7.

[0183] In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises four contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises four contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises five contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises five contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises six contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises six contiguous amino acids of SEQ ID -53- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WONO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises seven contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises seven contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises eight contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises eight contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises nine contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises nine contiguous amino acids of SEQ ID NO: 7. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises ten contiguous amino acids selected from a sequence of any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises ten contiguous amino acids of SEQ ID NO: 7.

[0184] In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least four contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to -54- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOthe 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least five contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least six contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581- 590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least seven contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least eight contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least nine contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least ten contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71.

[0185] In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises four contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581- 590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises five contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises six contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises seven contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic -55- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOvariant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises eight contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises nine contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises ten contiguous amino acids selected from a sequence of any one of SEQ ID NO: 66 – SEQ ID NO: 71.

[0186] In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, or v) SEQ ID NO: 36 – SEQ ID NO: 65.

[0187] In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 66 – SEQ ID NO: 71. In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), comprises any one of SEQ ID NO: 66 – SEQ ID NO: 71.

[0188] In some embodiments, the tropic variant region of an AAV capsid or AAV polypeptide as described herein, corresponding to the 581-590 residues of wild type AAV5 VP1 (SEQ ID NO: 1), may comprises SEQ ID NO: 87 (X1X2X3X4X5X6X7X8X9X10), wherein each of X1X2X3X4X5X6X7X8X9X10 is independently selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X1in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X2 in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X3 in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, -56- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOF, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X4 in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X5in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X6in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X7 in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X8in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X9in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X10 in SEQ ID NO: 87 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q.

[0189] In some embodiments, X1 in SEQ ID NO: 87 is selected from L, T, or A. In some embodiments, X1in SEQ ID NO: 87 is L.

[0190] In some embodiments, X2 in SEQ ID NO: 87 is selected from W or A. In some embodiments, X2in SEQ ID NO: 87 is W.

[0191] In some embodiments, X3 in SEQ ID NO: 87 is selected from G or A. In some embodiments, X3 in SEQ ID NO: 87 is G.

[0192] In some embodiments, X4 in SEQ ID NO: 87 is selected from W, G, or R. In some embodiments, X4 is W.

[0193] In some embodiments, X5in SEQ ID NO: 87 is selected from T, A, or Y. In some embodiments, X5 in SEQ ID NO: 87 is T.

[0194] In some embodiments, X6in SEQ ID NO: 87 is selected from L, Y, or N. In some embodiments, X6 in SEQ ID NO: 87 is selected from L.

[0195] In some embodiments, X7in SEQ ID NO: 87 is selected from Q, K, S, or Y. In some embodiments, X7 in SEQ ID NO: 87 is Q.

[0196] In some embodiments, X8in SEQ ID NO: 87 is selected from V, E, H, or V. In some embodiments, X8 in SEQ ID NO: 87 is V.

[0197] In some embodiments, X9in SEQ ID NO: 87 is selected from Q, E, F, or G. In some embodiments, X9 in SEQ ID NO: 87 is Q.

[0198] In some embodiments, X10in SEQ ID NO: 87 is selected from T or I. In some embodiments, X10 in SEQ ID NO: 87 is T.

[0199] A variant sequence provided in TABLE 2 (e.g., a sequence corresponding to residues 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1), a sequence corresponding to residues 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1), or a sequence corresponding to residues 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1)) may be included in an engineered VP1 -57- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOcapsid polypeptide. In some embodiments, the engineered VP1 capsid polypeptide is an engineered AAV5 VP1 capsid polypeptide. In some embodiments, these variant sequences are substituted at the recited residues into AAV5 VP1 (e.g., SEQ ID NO: 1), AAV5 VP2 (e.g., SEQ ID NO: 2), and / or AAV5 VP3 (e.g., SEQ ID NO: 3) In some embodiments, the engineered VP1 capsid polypeptide is a chimeric VP1 capsid polypeptide. The chimeric VP1 capsid polypeptide may comprise an N-terminal portion of an AAV9 VP1 capsid polypeptide and a C-terminal portion derived from an AAV5 VP1 capsid polypeptide. The chimeric VP1 capsid polypeptide may comprise an N-terminal portion of an AAV9 VP1 capsid polypeptide and a C-terminal portion from an engineered AAV5 VP1 capsid polypeptide as disclosed herein. For example, a chimeric VP1 capsid polypeptide may comprise an engineered VP1 capsid polypeptide in which amino acid corresponding to residues 1-136 of an AAV5 VP1 capsid polypeptide (e.g., residues 1-136 of SEQ ID NO: 1) are replaced with amino acid residues 1-137 of an AAV9 VP1 capsid polypeptide (e.g., residues 1-137 of SEQ ID NO: 4). For example, a chimeric VP1 capsid polypeptide may comprise an engineered VP1 capsid polypeptide in which amino acid corresponding to residues 1-136 of an AAV capsid or AAV polypeptide having a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1 that is a variant of the 581-590 region (e.g., a tropic variant region) of AAV5 VP1 are replaced with amino acid residues 1-137 of an AAV9 VP1 capsid polypeptide (e.g., residues 1-137 of SEQ ID NO: 4). The region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1 that is a variant of the 581-590 region (e.g., a tropic variant region) of AAV5 VP1 comprise any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, v) SEQ ID NO: 36 – SEQ ID NO: 65, or vi) SEQ ID NO: 66 – SEQ ID NO: 71. Examples of engineered VP1 capsid polypeptides comprising the AAV VP capsid polypeptide variant sequences of TABLE 2 are provided in TABLE 3. In TABLE 3, “*” indicates translation of the stop codon. TABLE 3 – Engineered VP1 Capsid Polypeptides VP1 Variant SEQ ID NO VP1 Sequence D Y A R T W-58- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence QVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNG E P G P E A P S F H N D Y V S K N S A A F I Q D Y A R T W G E P G P E S Y-59- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence STGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVD H N D Y V S K N S A A F I Q D Y A R T W G E P G P E P Q V H N D Y-60- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence STPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRV V S K N S A A H F D Y A R T W G E P G P E S Y D H N D Y V S K N S A A F I-61- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence TQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQ H N D Y V S K N S A A I Q H N D Y V S K N S A A F I Q H N D Y-62- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence STPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRV V S K N S A A H T F H N D Y V S K N S A A F I Q H N D Y V S K N S A A F I-63- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence TQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQ H N D Y V S K N S A A T F H N D Y V S K N S A A F I Q H N D Y-64- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence STPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRV V S K N S A A F I Q H N D Y V S K N S A A F H N D Y V S K N S A A F I-65- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence TQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQ H N D Y V S K N S A A F I Q H N D Y V S K N S A A H T F H N D Y-66- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence STPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRV V S K N S A A F I Q H N D Y V S K N S A A F I Q H N D Y V S K N S A A F-67- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence ITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDP H N D Y V S K N S A A F I Q H N D Y V S K N S A A F I Q H N D Y-68- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence STPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRV V S K N S A A F I Q H N D Y V S K N S A A F I Q H N D Y V S K N S A A F-69- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence ITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDP H N D Y V S K N S A A F H N D Y V S K N S A A F I Q H N D Y-70- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence STPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRV V S K N S A A I Q H N D Y V S K N S A A F I Q H N D Y V S K N S A A H T-71- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence QYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQF H N D Y V S K N S A A F I Q H N D Y V S K N S A A S D H N D Y-72- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence STPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRV V S K N S A A I Q H N D Y V S K N S A A F I Q H N D Y V S K N S A A H F-73- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence ITQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDP H N D Y V S K N S A A F I Q H N D Y V S K N S A A F I Q H N D Y-74- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence STPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPRSLRV V S K N S A A H T F H N D Y V S K N S A A F I Q H N D Y V S K N S A A H T-75- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 Variant SEQ ID NO VP1 Sequence QYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQF H N D Y V S K N S A A F I Qso e e o e s, a e g ee e caps poypep e co p ses a a o acid sequence of any one of SEQ ID NO: 13 – SEQ ID NO: 17, SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 16. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 13. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 14. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 15. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 17. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 26. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 27. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 28. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 17 , SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at -76- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 13 , SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159. In some embodiments, an engineered AAV5 VP1 comprises a sequence of SEQ ID NO: 13 or at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 13. In some embodiments, an engineered AAV5 VP1 comprises a sequence of SEQ ID NO: 14 or at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 14. In some embodiments, an engineered AAV5 VP1 comprises a sequence of SEQ ID NO: 15 or at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 15. In some embodiments, an engineered AAV5 VP1 comprises a sequence of SEQ ID NO: 16 or at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 16. In some embodiments, an engineered AAV5 VP1 comprises a sequence of SEQ ID NO: 17 or at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 17. In some embodiments, an engineered AAV5 VP1 comprises a sequence of SEQ ID NO: 26 or at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 26. In some embodiments, an engineered AAV5 VP1 comprises a sequence of SEQ ID NO: 27 or at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 27. In some embodiments, an engineered AAV5 VP1 comprises a sequence of SEQ ID NO: 28 or at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 28. In some embodiments, a chimeric VP1 comprises a sequence of any one of SEQ ID NO: 14 – SEQ ID NO: 17 , SEQ -77- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159 or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 14 – SEQ ID NO: 17 , SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159. In some embodiments, a chimeric VP1 comprises a sequence of any one of SEQ ID NO: 14 – SEQ ID NO: 17 at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 14 – SEQ ID NO: 17. In some embodiments, a chimeric VP1 comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 16. In some embodiments, a chimeric VP1 is SEQ ID NO: 16. In some embodiments, a chimeric VP1 comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 14. In some embodiments, a chimeric VP1 is SEQ ID NO: 14. In some embodiments, a chimeric VP1 comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 15. In some embodiments, a chimeric VP1 is SEQ ID NO: 15. In some embodiments, a chimeric VP1 comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 17. In some embodiments, a chimeric VP1 is SEQ ID NO: 17.

[0201] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild -78- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOtype AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 13, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an -79- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOengineered VP1 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98.5% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 13, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 13, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9.

[0202] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid -80- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOpolypeptide comprises at least 98% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98.5% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 14, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5.

[0203] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-590 of wild type AAV5 -81- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98.5% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 14, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 14, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9.

[0204] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 26, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 6. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 26, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 10.

[0205] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 15, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 6. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 15, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 10. -82- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0206] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98.5% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 27, -83- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOwherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7.

[0207] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11.. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98.5% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 27, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 27, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11.

[0208] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-569 of wild type -84- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOAAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 16, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7.

[0209] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an -85- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOengineered VP1 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 98.5% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 16, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP1 capsid polypeptide comprises SEQ ID NO: 16, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11.

[0210] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 28, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 8. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 28, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 12. -86- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0211] In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 17, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 8. In some embodiments, an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 17, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 12.

[0212] In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 13 – SEQ ID NO: 17 , SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 16. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 13. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 14. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 15. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 17. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 26. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 27. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 28. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a nucleotide sequence encoding any one of SEQ ID NO: 13 – SEQ ID NO: 17 , SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ -87- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOID NO: 154 – SEQ ID NO: 159. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide is a codon optimized nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 13 – SEQ ID NO: 17 , SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 16. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 13. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 14. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 15. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 17. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 26. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 27. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 28. In some embodiments, a nucleotide sequence encoding an engineered VP1 capsid polypeptide is a codon optimized nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a nucleotide sequence encoding any one of SEQ ID NO: 13 – SEQ ID NO: 17 , SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159.

[0213] A variant sequence provided in TABLE 2 (e.g., a sequence corresponding to residues 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1), a sequence corresponding to residues 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1), or a sequence corresponding to residues 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1)) may be included in an engineered VP2 capsid polypeptide. In some embodiments, the engineered VP2 capsid polypeptide is an engineered AAV5 VP2 capsid polypeptide. In some embodiments, an engineered VP2 capsid polypeptide may comprise residues 138-725 of an engineered VP1 capsid polypeptide (e.g., an engineered VP1 capsid polypeptide of any one of SEQ ID NO: 13 – SEQ ID NO: 17 , SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: -88- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO154 – SEQ ID NO: 159. For example, an engineered VP2 capsid polypeptide may comprise residues 138-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153 or SEQ ID NO: 154 – SEQ ID NO: 159. Examples of engineered VP2 capsid polypeptides comprising the AAV VP capsid polypeptide variant sequences of TABLE 2 are provided in TABLE 4. TABLE 4 – Engineered VP2 Capsid Polypeptides VP2 Variant SEQ ID NO VP2 Sequence Variant 1 VP2 SEQ ID NO: TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQ A S L V T V N Y P R R Q A S L V T V N Y P R R Q A S L V T-89- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP2 Variant SEQ ID NO VP2 Sequence VPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQF V N V T T Q A S L V T V N T T

[0000] n some embod men s, an eng neered caps d poypep de comprses an amno acid sequence of any one of SEQ ID NO: 18 – SEQ ID NO: 21. In some embodiments, an engineered VP2 capsid polypeptide comprises SEQ ID NO: 20. In some embodiments, an engineered VP2 capsid polypeptide comprises SEQ ID NO: 18. In some embodiments, an engineered VP2 capsid polypeptide comprises SEQ ID NO: 19. In some embodiments, an engineered VP2 capsid polypeptide comprises SEQ ID NO: 21. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 18 – SEQ ID NO: 21. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 20. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at -90- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 18. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 19. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 21.

[0215] In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ -91- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOID NO: 5. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP2 capsid polypeptide comprises SEQ ID NO: 18, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 18, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP2 capsid polypeptide comprises SEQ ID NO: 18, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9.

[0216] In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, -92- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOat least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 19, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 6. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 19, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 10.

[0217] In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ -93- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOID NO: 7. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP2 capsid polypeptide comprises SEQ ID NO: 20, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 20, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP2 capsid polypeptide comprises SEQ ID NO: 20, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11.

[0218] In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, -94- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOat least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 21, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 8. In some embodiments, an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 21, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 12.

[0219] In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide comprises a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 18 – SEQ ID NO: 21. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 18. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 19. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 20. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 21. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a nucleotide sequence encoding any one of SEQ ID NO: 18 – SEQ ID NO: 21. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide is a codon optimized nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 18 – SEQ ID NO: 21. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 18. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 19. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 20. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 21. In some embodiments, a nucleotide sequence encoding an engineered VP2 capsid polypeptide is a codon optimized nucleotide sequence having at least -95- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a nucleotide sequence encoding any one of SEQ ID NO: 18 – SEQ ID NO: 21.

[0220] A variant sequence provided in TABLE 2 (e.g., a sequence corresponding to residues 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1), a sequence corresponding to residues 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1), or a sequence corresponding to residues 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1)) may be included in an engineered VP3 capsid polypeptide. In some embodiments, the engineered VP3 capsid polypeptide is an engineered AAV5 VP3 capsid polypeptide. In some embodiments, an engineered VP3 capsid polypeptide may comprise residues 194-725 of an engineered VP1 capsid polypeptide (e.g., an engineered VP1 capsid polypeptide of any one of SEQ ID NO: 13 – SEQ ID NO: 17 , SEQ ID NO: 26 – SEQ ID NO: 28, SEQ ID NO: 124 – SEQ ID NO: 153, or SEQ ID NO: 154 – SEQ ID NO: 159. For example, an engineered VP3 capsid polypeptide may comprise residues 194-725 of any one of SEQ ID NO: 124 – SEQ ID NO: 153 or SEQ ID NO: 154 – SEQ ID NO: 159. Examples of engineered VP3 capsid polypeptides comprising the AAV VP capsid polypeptide variant sequences of TABLE 2 are provided in TABLE 5. TABLE 5 – Engineered VP3 Capsid Polypeptides VP3 Variant SEQ ID NO VP3 Sequence R S I F L S A A F I Q R S I F L-96- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP3 Variant SEQ ID NO VP3 Sequence ANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKN S A A F I Q R S I F L S A A H F R S I F L S A A F I Q

[0221] In some embodiments, an engineered VP3 capsid polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 22 – SEQ ID NO: 25. In some embodiments, an engineered VP3 capsid polypeptide comprises SEQ ID NO: 24. In some embodiments, an engineered VP3 capsid polypeptide comprises SEQ ID NO: 22. In some embodiments, an engineered VP3 capsid polypeptide comprises SEQ ID NO: 23. In some embodiments, an engineered VP3 capsid polypeptide comprises SEQ ID NO: 25. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at -97- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to any one of SEQ ID NO: 22 – SEQ ID NO: 25. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 24. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 22. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 23. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 25.

[0222] In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP3 capsid -98- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOpolypeptide comprises at least 98% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP3 capsid polypeptide comprises SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 5. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 99.5% sequence -99- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOidentity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9. In some embodiments, an engineered VP3 capsid polypeptide comprises SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 9.

[0223] In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 23, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 6. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 23, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 10.

[0224] In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP3 capsid -100- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOpolypeptide comprises at least 98% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 99.5% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP3 capsid polypeptide comprises SEQ ID NO: 24, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 7. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 95% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 96% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 97% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO: 24, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 99.5% sequence -101- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOidentity to SEQ ID NO: 24, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11. In some embodiments, an engineered VP3 capsid polypeptide comprises SEQ ID NO: 24, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 11.

[0225] In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 25, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 8. In some embodiments, an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 25, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) have a sequence of SEQ ID NO: 12.

[0226] In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide comprises a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 22 – SEQ ID NO: 25. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 22. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 23. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 24. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 25. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a nucleotide sequence encoding any one of SEQ ID NO: 22 – SEQ ID NO: 25. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide is a codon optimized nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NO: 22 – SEQ ID NO: 25. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide comprises a nucleotide sequence encoding SEQ ID NO: 22. In some -102- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOembodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 23. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 24. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide is a codon optimized nucleotide sequence encoding SEQ ID NO: 25. In some embodiments, a nucleotide sequence encoding an engineered VP3 capsid polypeptide is a codon optimized nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to a nucleotide sequence encoding any one of SEQ ID NO: 22 – SEQ ID NO: 25.

[0227] The present disclosure provides an engineered viral protein (VP) capsid polypeptide, comprising an amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 8. The present disclosure provides an engineered viral protein (VP) capsid polypeptide, comprising an amino acid sequence of SEQ ID NO: 36 - SEQ ID NO: 65. The present disclosure provides an engineered viral protein (VP) capsid polypeptide, comprising an amino acid sequence of SEQ ID NO: 66 - SEQ ID NO: 71. In one aspect, the engineered VP capsid polypeptide comprises SEQ ID NO: 7. In one aspect, the engineered VP capsid polypeptide comprises SEQ ID NO: 5. In one aspect, the engineered VP capsid polypeptide comprises SEQ ID NO: 6. In one aspect, the engineered VP capsid polypeptide comprises SEQ ID NO: 8. In one aspect, the engineered VP capsid polypeptide comprises the sequence of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 6, or SEQ ID NO: 8 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises the sequence of SEQ ID NO: 36 - SEQ ID NO: 65 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises the sequence of SEQ ID NO: 66 - SEQ ID NO: 71 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises the sequence of SEQ ID NO: 7 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises the sequence of SEQ ID NO: 5 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises the sequence of SEQ ID NO: 6, at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises the sequence of SEQ ID NO: 8, at residues -103- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOcorresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of NM or QM at residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of NM at residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of QM at residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 10, or SEQ ID NO: 12 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 88 - SEQ ID NO: 117 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 118 - SEQ ID NO: 123 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 11 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 9 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 10 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 12 at residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1). In one aspect, the VP capsid polypeptide is a chimeric VP capsid polypeptide comprising a first region, wherein the first region is a region of an AAV5 VP capsid polypeptide. In one aspect, the first region comprises residues corresponding to residues 137-724 of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In one aspect, the first region comprises residues corresponding to residues 138-725 of SEQ ID NO: 16, SEQ ID NO: 14; SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the engineered VP capsid polypeptide comprises a second region, wherein the second region comprises a region of the AAV9 VP polypeptide. In one aspect, the second region comprises residues corresponding to residues 1- 137 of SEQ ID NO: 4. In one aspect, the chimeric VP capsid polypeptide comprises residues corresponding to residues 1-137 of SEQ ID NO: 4 substituted for residues 1-136 of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. In one aspect, the chimeric VP capsid polypeptide comprises residues corresponding to residues 1-137 of SEQ ID NO: 4 substituted for residues 1-136 of SEQ ID NO: 1 resulting in SEQ ID NO: 29. -104- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOIn one aspect, the chimeric VP capsid polypeptide comprises residues corresponding to residues 1-137 of SEQ ID NO: 4 substituted for residues 1-136 of SEQ ID NO: 13 resulting in SEQ ID NO: 14. In one aspect, the chimeric VP capsid polypeptide comprises residues corresponding to residues 1-137 of SEQ ID NO: 4 substituted for residues 1-136 of SEQ ID NO: 26 resulting in SEQ ID NO: 15. In one aspect, the chimeric VP capsid polypeptide comprises residues corresponding to residues 1-137 of SEQ ID NO: 4 substituted for residues 1-136 of SEQ ID NO: 27 resulting in SEQ ID NO: 16. In one aspect, the chimeric VP capsid polypeptide comprises residues corresponding to residues 1-137 of SEQ ID NO: 4 or substituted for residues 1-136 of SEQ ID NO: 28 resulting in SEQ ID NO: 17. In one aspect, the chimeric VP capsid polypeptide comprises a first region comprising residues corresponding to residues 137-724 of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, and a second region comprising residues corresponding to residues 1-137 of SEQ ID NO: 4, wherein first region is 3’ to the second region. In one aspect, the chimeric VP capsid polypeptide comprises a first region comprising residues corresponding to residues 137-724 of SEQ ID NO: 1 SEQ ID NO: 13, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, and a second region comprising residues corresponding to residues 1-137 of SEQ ID NO: 4, wherein second region is 5’ to the first region. In one aspect, the chimeric VP1 capsid polypeptide comprises an engineered VP1 capsid polypeptide in which amino acid corresponding to residues 1-136 of an AAV5 VP1 capsid polypeptide (e.g., residues 1-136 of SEQ ID NO: 1) are replaced with amino acid residues 1- 137 of an AAV9 VP1 capsid polypeptide (e.g., residues 1-137 of SEQ ID NO: 4). In one aspect, the chimeric VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the chimeric VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 16. In one aspect, the chimeric VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 14. In one aspect, the chimeric VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 15. In one aspect, the chimeric VP capsid polypeptide comprises at least 90%, at least 91%, at -105- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 17. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 16. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 14. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 15. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 17. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 15, or SEQ ID NO: 17. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 16. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 14. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 15. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 17. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 26, or SEQ ID NO: 28. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 13. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 27. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 26. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 28. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 26, or SEQ ID NO: 28. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 13. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID SEQ ID NO: 27. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence -106- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOidentity to SEQ ID NO: 26. In one aspect, the chimeric VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 28. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 13, SEQ ID NO: 27, SEQ ID NO: 26, OR SEQ ID NO: 28. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 13. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 27. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 26. In one aspect, the chimeric VP capsid polypeptide comprises a sequence of SEQ ID NO: 28. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 19, or SEQ ID NO: 21. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 20. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 18. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 19. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 21. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 19, or SEQ ID NO: 21. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 20. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 18. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 19. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 21. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 19, or SEQ ID NO: 21. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 20. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 18. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 19. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 21. In one aspect, the VP capsid polypeptide comprises at least 90%, at least -107- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 23, or SEQ ID NO: 25. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 24. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 22. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 23. In one aspect, the VP capsid polypeptide comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 25. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 23, or SEQ ID NO: 25. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 24,. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 22. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 23. In one aspect, the VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO: 25. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 23, or SEQ ID NO: 25. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 24. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 22. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 23. In one aspect, the VP capsid polypeptide comprises a sequence of SEQ ID NO: 25. Viral Protein (VP) Capsid Polypeptides

[0228] Described herein are viral protein capsid polypeptides and engineered capsid polypeptides. In some embodiments, an engineered capsid may comprise one or more engineered capsid polypeptides assembled into a recombinant adeno-associated virus (rAAV) viral capsid.

[0229] In some embodiments, the capsid is a capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, -108- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOAAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 (for example, as described in WO2020 / 033842, incorporated herein by reference in its entirety). For example, the capsid may be an AAV5 capsid and / or AAV9. The hu68 capsid is described in WO 2018 / 160582, incorporated herein by reference in its entirety.

[0230] In some embodiments, the capsid is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68. For example, the capsid may be a derivative of AAV5 and / or AAV9. Chimeric VP Capsid Polypeptides

[0231] In some embodiments, the VP capsid polypeptides are chimeric VP capsid polypeptides. In some embodiments, the chimeric VP capsid polypeptides have regions or domains or individual amino acids that are derived from at least one serotype of AAV. In some embodiments, the chimeric VP capsid polypeptides have regions or domains or individual amino acids that are derived from at least two serotypes of AAV. In some embodiments, the chimeric VP capsid polypeptides can include a first region that is derived from a first AAV serotype, and a second region that is derived from a second AAV serotype. In some embodiments, the first region is in a portion of the VP capsid polypeptide that is present in VP1 but absent in VP2 and VP3. In some embodiments, the second region is in a portion of the VP capsid polypeptide that is present in VP1 but absent in VP2 and VP3. A portion present in VP1 but not in VP2 or VP3 may be referred to as a VP1 unique region. For -109- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOexample, a VP1 unique region may comprise amino acids corresponding to residues 1-136 of wild type AAV5 VP1 (SEQ ID NO: 1), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to residues 1-136 of wild type AAV5 VP1 (SEQ ID NO: 1). For example, a VP1 unique region may comprise amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). For example, a VP1 unique region may comprise amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4), or an amino acid sequence having more than 98.5% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). As another example, a VP1 unique region may comprise amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4), or an amino acid sequence having at least 99% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). A VP1 unique region may comprise amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1u of a first serotype may replace or be substituted by a VP1u of a second serotype. For example, the VP1u of wild type AAV5 VP1 (SEQ ID NO: 1) may be replaced or substituted by a VP1 unique region comprising amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). For example, the VP1 unique region comprising residues 1-136 of wild type AAV5 VP1 (SEQ ID NO: 1) may be replaced or substituted by a VP1 unique region comprising amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). For example, the VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide having a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1 that is a variant of the 581-590 region (e.g., a tropic variant region) of AAV5 VP1 may be replaced or substituted by a VP1 unique region comprising amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). For example, the VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises any one of: i) SEQ ID NO: 7, ii) SEQ ID NO: 5, iii) SEQ ID NO: 6, iv) SEQ ID NO: 8, v) SEQ ID NO: 36 – SEQ ID NO: 65, or vi) -110- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOSEQ ID NO: 66-71, may be replaced or substituted by a VP1 unique region comprising amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). As another example, the VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 7, may be replaced or substituted by a VP1 unique region comprising amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4), or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 7, may be replaced or substituted by a VP1 unique region comprising amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 7, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having at least 99% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 7, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having more than 98.5% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 5, may be replaced or substituted by a VP1 unique region comprising amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 5, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having at least 99% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to -111- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOthe 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 5, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having more than 98.5% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 6, may be replaced or substituted by a VP1 unique region comprising amino acids corresponding to residues 1- 137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 6, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having at least 99% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 6, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having more than 98.5% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 8, may be replaced or substituted by a VP1 unique region comprising amino acids corresponding to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 8, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having at least 99% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises SEQ ID NO: 8, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having more than 98.5% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises any one of SEQ ID NOs: 36-65, may be replaced or substituted by a VP1 unique region comprising residues 1-137 of AAV9 -112- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises any one of SEQ ID NOs: 36-65, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having at least 99% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises any one of SEQ ID NOs: 36-65, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having more than 98.5% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises any one of SEQ ID NOs: 66-71, may be replaced or substituted by a VP1 unique region comprising residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises any one of SEQ ID NOs: 66-71, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having at least 99% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). The VP1 unique region comprising residues 1-136 of an AAV capsid or AAV polypeptide comprising a region corresponding to the 581-590 region (e.g., a tropic variant region) of AAV5 VP1, wherein the region comprises any one of SEQ ID NOs: 66-71, may be replaced or substituted by a VP1 unique region comprising amino acid sequence having more than 98.5% sequence identity to residues 1-137 of AAV9 VP1 (SEQ ID NO: 4). In some embodiments, the first region is in a portion of the VP capsid polypeptide that is present in VP1 and VP2 but absent in VP3. In some embodiments, the second region is in a portion of the VP capsid polypeptide that is present in VP1 and VP2 but absent in VP3. A portion of a VP capsid polypeptide present in VP1 and VP2 but not in VP3 may be referred to as a VP1 / 2 unique region. For example, a VP1 / 2 unique region may comprise amino acids corresponding to an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to residues 137-192 of wild type AAV5 VP1 (SEQ ID NO: 1).

[0232] The AAV serotypes may be human AAV serotypes or non-human AAV serotypes, such as bovine, avian, and caprine AAV serotypes. In particular, non-primate mammalian AAV serotypes, such as AAV sequences from rodents (e.g., mice, rats, rabbits, and hamsters) -113- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOand carnivores (e.g., dogs, cats, and raccoons), may be used. By including individual amino acids or regions from multiple AAV serotypes in one capsid protein, capsid proteins that have multiple desired properties that are separately derived from the multiple AAV serotypes may be obtained.

[0233] In some embodiments, capsid protein is a chimera of capsid proteins from two or more serotypes such as but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 (for example, as described in WO2020 / 033842, incorporated herein by reference in its entirety). In certain embodiments, the capsid is an rh32.33 capsid, described in US Pat. No.8,999,678, incorporated herein by reference in its entirety.

[0234] In some embodiments, the chimeric VP capsid polypeptide can include a first region e.g., a region of an AAV5 VP capsid polypeptide. In some embodiments, the AAV5 capsid polypeptide can be a AAV5 VP1 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO:1, an AAV5 VP2 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2 or an AAV5 VP3 capsid polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 3. In one embodiment, the AAV5 capsid polypeptide is an AAV5 VP1 capsid polypeptide.

[0235] In some embodiments, the residues 47-724 of the chimeric VP capsid polypeptide are derived from the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1.

[0236] In some embodiments, the residues 110-724 of the chimeric VP capsid polypeptide are derived from the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1.

[0237] In some embodiments, the residues 137-724 of the chimeric VP capsid polypeptide are derived from the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1. -114- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0238] In some embodiments, the chimeric VP polypeptide comprises a first region of an AAV5 VP capsid polypeptide and a second region of a AAV9 VP polypeptide, an AAV1 VP polypeptide VP polypeptide, an AAV2 VP polypeptide VP polypeptide, an AAV3 VP polypeptide, an AAV4 VP polypeptide, an AAV6 VP polypeptide, an AAV7 VP polypeptide, an AAV8 VP polypeptide, an AAV 10 VP polypeptide, an AAV11 VP polypeptide, an AAV12 VP polypeptide, an AAV13 VP polypeptide, an AAV14 VP polypeptide, an AAV15 VP polypeptide, an AAV16 VP polypeptide, an AAV-DJ VP polypeptide, an AAV-DJ / 8 VP polypeptide, an AAV-DJ / 9 VP polypeptide, an AAV1 / 2 VP polypeptide, an AAV.rh8 VP polypeptide, an AAV.rhlO VP polypeptide, an AAV.rh20 VP polypeptide, an AAV.rh39 VP polypeptide, an AAV.Rh43 VP polypeptide, an AAV.Rh74 VP polypeptide, an AAV.v66 VP polypeptide, an AAV.OligoOOl VP polypeptide, an AAV.SCH9 VP polypeptide, an AAV.r3.45 VP polypeptide, an AAV.RHM4-1 VP polypeptide, an AAV.hu37 VP polypeptide, an AAV.Anc80 VP polypeptide, an AAV.Anc80L65 VP polypeptide, an AAV.7m8 VP polypeptide, an AAV.PhP.eB VP polypeptide, an AAV.PhP.Vl VP polypeptide, an AAV.PHP.B VP polypeptide, an AAV.PhB.Cl VP polypeptide, an AAV.PhB.C2 VP polypeptide, an AAV.PhB.C3 VP polypeptide, an AAV.PhB.C6 VP polypeptide, an AAV.cy5 VP polypeptide, an AAV2.5 VP polypeptide, an AAV2tYF VP polypeptide, an AAV3B VP polypeptide, an AAV.LK03 VP polypeptide, an AAV.HSC1 VP polypeptide, an AAV.HSC2 VP polypeptide, an AAV.HSC3 VP polypeptide, an AAV.HSC4 VP polypeptide, an AAV.HSC5 VP polypeptide, an AAV.HSC6 VP polypeptide, an AAV.HSC7 VP polypeptide, an AAV.HSC8 VP polypeptide, an AAV.HSC9 VP polypeptide, an AAV.HSC10 VP polypeptide, an AAV.HSC11 VP polypeptide, an AAV.HSC12 VP polypeptide, an AAV.HSC13 VP polypeptide, an AAV.HSC14 VP polypeptide, an AAV.HSC15 VP polypeptide, an AAV.HSC16 VP polypeptide, an AAV.HSC17 VP polypeptide, or an AAVhu68 VP polypeptide.

[0239] In some embodiments, the chimeric VP capsid polypeptide comprises a second region of which is a region of the AAV9 VP polypeptide. In some embodiments, the AAV9 VP polypeptide can be an AAV9 VP1 polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 4, an AAV9 VP2 polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 31 or an AAV9 VP3 polypeptide having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 32.

[0240] In some embodiments, the chimeric VP capsid polypeptide comprises residues 1-47 of the AAV9 VP1 capsid polypeptide of SEQ ID NO: 4. -115- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0241] In some embodiments, the chimeric VP capsid polypeptide comprises residues 1-110 of the AAV9 VP1 capsid polypeptide of SEQ ID NO: 4.

[0242] In some embodiments, the chimeric VP capsid polypeptide comprises residues 1-137 of the AAV9 VP1 capsid polypeptide of SEQ ID NO: 4.

[0243] As a non-limiting example, residues 47-724 of the chimeric VP capsid polypeptide are derived from the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1 and residues 1-47 of the VP capsid polypeptide are derived from the AAV9 VP1 capsid polypeptide of SEQ ID NO: 4.

[0244] As a non-limiting example, residues 110-724 of the chimeric VP capsid polypeptide are derived from the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1 and residues 1-110 of the VP capsid polypeptide are derived from the AAV9 VP1 capsid polypeptide of SEQ ID NO: 4.

[0245] As a non-limiting example, residues 137-724 of the chimeric VP capsid polypeptide are derived from the AAV5 VP1 capsid polypeptide of SEQ ID NO: 1 and residues 1-137 of the VP capsid polypeptide are derived from the AAV9 VP1 capsid polypeptide of SEQ ID NO: 4.

[0246] Exemplary chimeric VP capsid polypeptide sequences are provided in TABLE 6. In TABLE 6, “*” indicates translation of the stop codon and X indicates a residue selected from any amino acid residue. Also provided in TABLE 6 are exemplary capsid regions comprised by the chimeric VP capsid polypeptides described herein. TABLE 6 – Chimeric VP Capsid Polypeptide Sequences Amino Amino Chimeric VP Capsid protein sequence SEQ ID acid acid NO:-116- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGE YRTTRPIGTRYLTRPL* 1-47 47-724 MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQD SEQ ID-117- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNN QGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVLPSYNN HQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRD-118- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMA TNNQSSTTAPX1X2X3X4X5X6X7X8X9X10VPGSVWMERDVYLQG PIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITS-119- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO137-724 TAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQI SEQ ID PAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHC NO: 86 DSTWMGDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANon that is derived from an AAV serotype, and a second region that is derived from a non-AAV viral protein. In some embodiments, the chimeric VP capsid polypeptides can include a first region that is derived from an AAV serotype, and a second region that is derived from a non- viral protein. In some embodiments, chimeric VP capsid polypeptides can include more than two regions, with additional regions derived from an AAV serotype, a non-AAV viral protein, and / or a non-viral protein.

[0248] In some embodiments, a tropic variant region of a AAV capsid or AAV capsid polypeptide as described herein may comprise SEQ ID NO: 77 or SEQ ID NO: 80 (X1X2X3X4X5X6X7X8X9X10), wherein each of X1X2X3X4X5X6X7X8X9X10 is independently selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X1 in SEQ ID NO: 77 or SEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X2in SEQ ID NO: 77 or SEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X3in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X4 in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X5 in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X6in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X7in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X8in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X9 in SEQ ID NO: 77 OR -120- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOSEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X10in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q.

[0249] In some embodiments, X1in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from L, T, or A. In some embodiments, X1 in SEQ ID NO: 77 OR SEQ ID NO: 80 is L.

[0250] In some embodiments, X2in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from W or A. In some embodiments, X2 in SEQ ID NO: 77 OR SEQ ID NO: 80 is W.

[0251] In some embodiments, X3in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from G or A. In some embodiments, X3 in SEQ ID NO: 77 OR SEQ ID NO: 80 is G.

[0252] In some embodiments, X4in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from W, G, or R. In some embodiments, X4 in SEQ ID NO: 77 OR SEQ ID NO: 80 is W.

[0253] In some embodiments, X5in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from T, A, or Y. In some embodiments, X5 in SEQ ID NO: 77 OR SEQ ID NO: 80 is T.

[0254] In some embodiments, X6in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from L, Y, or N. In some embodiments, X6 in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from L.

[0255] In some embodiments, X7 in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from Q, K, S, or Y. In some embodiments, X7 in SEQ ID NO: 77 OR SEQ ID NO: 80 is Q.

[0256] In some embodiments, X8 in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from V, E, H, or V. In some embodiments, X8in SEQ ID NO: 77 OR SEQ ID NO: 80 is V.

[0257] In some embodiments, X9 in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from Q, E, F, or G. In some embodiments, X9in SEQ ID NO: 77 OR SEQ ID NO: 80 is Q.

[0258] In some embodiments, X10 in SEQ ID NO: 77 OR SEQ ID NO: 80 is selected from T or I. In some embodiments, X10in SEQ ID NO: 77 OR SEQ ID NO: 80 is T.

[0259] In some embodiments, a tropic variant region of a AAV capsid or AAV capsid polypeptide as described herein may comprise SEQ ID NO: 81 OR SEQ ID NO: 84 (X1X2X3X4X5X6X7X8X9X10), wherein each of X1X2X3X4X5X6X7X8X9X10 is independently selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X1 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X2in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X3in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X4 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X5 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected -121- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOfrom G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X6 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X7 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X8 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X9in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X10in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q.

[0260] In some embodiments, X1in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from L, T, or A. In some embodiments, X1 in SEQ ID NO: 81 OR SEQ ID NO: 84 is L.

[0261] In some embodiments, X2in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from W or A. In some embodiments, X2 in SEQ ID NO: 81 OR SEQ ID NO: 84 is W.

[0262] In some embodiments, X3in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from G or A. In some embodiments, X3 in SEQ ID NO: 81 OR SEQ ID NO: 84 is G.

[0263] In some embodiments, X4 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from W, G, or R. In some embodiments, X4 in SEQ ID NO: 81 OR SEQ ID NO: 84 is W.

[0264] In some embodiments, X5 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from T, A, or Y. In some embodiments, X5in SEQ ID NO: 81 OR SEQ ID NO: 84 is T.

[0265] In some embodiments, X6 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from L, Y, or N. In some embodiments, X6in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from L.

[0266] In some embodiments, X7 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from Q, K, S, or Y. In some embodiments, X7in SEQ ID NO: 81 OR SEQ ID NO: 84 is Q.

[0267] In some embodiments, X8 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from V, E, H, or V. In some embodiments, X8in SEQ ID NO: 81 OR SEQ ID NO: 84 is V.

[0268] In some embodiments, X9 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from Q, E, F, or G. In some embodiments, X9in SEQ ID NO: 81 OR SEQ ID NO: 84 is Q.

[0269] In some embodiments, X10 in SEQ ID NO: 81 OR SEQ ID NO: 84 is selected from T or I. In some embodiments, X10in SEQ ID NO: 81 OR SEQ ID NO: 84 is T.

[0270] In some embodiments, a tropic variant region of a AAV capsid or AAV capsid polypeptide as described herein may comprise SEQ ID NO: 35 OR SEQ ID NO: 86 (X1X2X3X4X5X6X7X8X9X10), wherein each of X1X2X3X4X5X6X7X8X9X10 is independently selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X1 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, -122- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOM, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X2 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X3 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X4in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X5in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X6 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X7 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X8 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X9in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q. In some embodiments, X10in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G, A, V, L, I, T, S, M, C, P, F, Y, W, H, K, R, D, E, N, or Q.

[0271] In some embodiments, X1 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from L, T, or A. In some embodiments, X1 in SEQ ID NO: 35 OR SEQ ID NO: 86 is L.

[0272] In some embodiments, X2 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from W or A. In some embodiments, X2in SEQ ID NO: 35 OR SEQ ID NO: 86 is W.

[0273] In some embodiments, X3 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from G or A. In some embodiments, X3in SEQ ID NO: 35 OR SEQ ID NO: 86 is G.

[0274] In some embodiments, X4 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from W, G, or R. In some embodiments, X4in SEQ ID NO: 35 OR SEQ ID NO: 86 is W.

[0275] In some embodiments, X5 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from T, A, or Y. In some embodiments, X5in SEQ ID NO: 35 OR SEQ ID NO: 86 is T.

[0276] In some embodiments, X6 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from L, Y, or N. In some embodiments, X6in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from L.

[0277] In some embodiments, X7 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from Q, K, S, or Y. In some embodiments, X7in SEQ ID NO: 35 OR SEQ ID NO: 86 is Q.

[0278] In some embodiments, X8 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from V, E, H, or V. In some embodiments, X8in SEQ ID NO: 35 OR SEQ ID NO: 86 is V.

[0279] In some embodiments, X9 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from Q, E, F, or G. In some embodiments, X9in SEQ ID NO: 35 OR SEQ ID NO: 86 is Q. -123- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

[0280] In some embodiments, X10 in SEQ ID NO: 35 OR SEQ ID NO: 86 is selected from T or I. In some embodiments, X10in SEQ ID NO: 35 OR SEQ ID NO: 86 is T.

[0281] It should be understood that the present disclosure includes polynucleotide sequences encoding for any sequence disclosed herein. For example, if an amino acid sequence is provided, the present disclosure also encompasses a polynucleotide sequence encoding for said amino acid sequence.

[0282] It should be understood that further embodiments include mutations in VP1, VP2, VP3, or any combination thereof that do not alter the desired properties (e.g., a particular tissue tropism) or affect viral assembly, as described herein.

[0283] In some embodiments, an rAAV virion is made of a capsid that may include the chimeric VP capsid polypeptides disclosed herein. Tissue Tropic Variants

[0284] In some embodiments, a chimeric VP capsid polypeptide may comprise a mutation (e.g., an insertion, a substitution, a deletion, or combinations thereof) that confers tropism for a tissue of interest. For example, a chimeric VP capsid polypeptide may comprise a mutation in a region corresponding to amino acid residues 581-589 of an AAV5 VP1 capsid polypeptide. For example, the chimeric VP capsid polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, or 9 substitutions in a region corresponding to amino acid residues 581-589 of an AAV5 VP1 capsid polypeptide. In some embodiments, the tissue may be a central nervous system (CNS) tissue, a muscle tissue, or an eye tissue. For example, the tissue may be a retinal tissue, a retinal pigment epithelium (RPE) tissue, a choroid tissue, or a combination thereof. In some embodiments, the tissue of interest may be a tissue associated with a disease or condition. Examples of mutations for increased CNS tissue tropism that may be incorporated into a chimeric VP capsid polypeptide of the present disclosure are described in WO / 2023 / 102079, incorporated herein by reference in its entirety. Examples of mutations for increased muscle tissue tropism that may be incorporated into a chimeric VP capsid polypeptide of the present disclosure are described in WO / 2023 / 102079, incorporated herein by reference in its entirety. Examples of mutations for increased retinal tissue tropism that may be incorporated into a chimeric VP capsid polypeptide of the present disclosure are described in WO / 2023 / 102078, incorporated herein by reference in its entirety. Examples of mutations for increased RPE tissue tropism that may be incorporated into a chimeric VP capsid polypeptide of the present disclosure are described in WO / 2023 / 102078, incorporated herein by reference in its entirety.

[0285] In some embodiments, the mutation may be in an AAV5 portion of a chimeric VP capsid polypeptide. In some embodiments, the mutation may be an AAV9 portion of a -124- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOchimeric VP capsid polypeptide. In some embodiments, a chimeric VP capsid polypeptide may comprise mutations in both an AAV5 portion and an AAV9 portion of the chimeric VP capsid polypeptide. Recombinant Adeno-Associated Viruses with CNS Tissue Tropism

[0286] Described herein are recombinant adeno-associated viruses (rAAVs) comprising the engineered AAV VP capsid polypeptides described herein. An rAAV may comprise engineered VP1, engineered VP2, and engineered VP3 capsid polypeptides.

[0287] In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide of SEQ ID NO: 13, an engineered VP2 capsid polypeptide of SEQ ID NO: 18, and an engineered VP3 capsid polypeptide of SEQ ID NO: 22. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 13, an engineered VP2 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 5. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 13, an engineered VP2 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 22, -125- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOwherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 9.

[0288] In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide of SEQ ID NO: 14, an engineered VP2 capsid polypeptide of SEQ ID NO: 18, and an engineered VP3 capsid polypeptide of SEQ ID NO: 22. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 5. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 95% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 95% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 95% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 5. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 96% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 96% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 96% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID -126- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WONO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 5. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 97% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 97% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 97% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 5. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 98% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 98% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 98% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 5. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 99% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 99% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 99% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 5. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 99.5% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 99.5% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 99.5% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of NM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 5. In some embodiments, an rAAV may comprise an engineered -127- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOVP1 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 9. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 95% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 95% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 95% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 9. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 96% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 96% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 96% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 9. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 97% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 97% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 97% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 9. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 98% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 98% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 98% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each -128- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOof the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 9. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 99% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 99% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 99% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 9. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 99.5% sequence identity to SEQ ID NO: 14, an engineered VP2 capsid polypeptide having at least 99.5% sequence identity to SEQ ID NO: 18, and an engineered VP3 capsid polypeptide having at least 99.5% sequence identity to SEQ ID NO: 22, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 9.

[0289] In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide of SEQ ID NO: 26, an engineered VP2 capsid polypeptide of SEQ ID NO: 19, and an engineered VP3 capsid polypeptide of SEQ ID NO: 23. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 26, an engineered VP2 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 19, and an engineered VP3 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 23, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 6. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% identity to SEQ ID NO: 26, an engineered VP2 capsid -129- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOpolypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 19, and an engineered VP3 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 23, wherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 10.

[0290] In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide of SEQ ID NO: 15, an engineered VP2 capsid polypeptide of SEQ ID NO: 19, and an engineered VP3 capsid polypeptide of SEQ ID NO: 23. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 15, an engineered VP2 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 19, and an engineered VP3 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 23, wherein residues corresponding to 568-569 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of QM and residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 6. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 15, an engineered VP2 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 19, and an engineered VP3 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 23, -130- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOwherein residues corresponding to 568-590 of wild type AAV5 VP1 (SEQ ID NO: 1) in each of the engineered VP1, the engineered VP2, and the engineered VP3 have a sequence of SEQ ID NO: 10.

[0291] In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide of SEQ ID NO: 27, an engineered VP2 capsid polypeptide of SEQ ID NO: 20, and an engineered VP3 capsid polypeptide of SEQ ID NO: 24. In some embodiments, an rAAV may comprise an engineered VP1 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO: 27, an engineered VP2 capsid polypeptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, or at least 99.5% sequence identity to SEQ ID...

Claims

1. CLAIMS WHAT IS CLAIMED IS:

1. An engineered viral protein (VP) capsid polypeptide, wherein the engineered VP capsid polypeptide comprises a sequence of SEQ ID NO: 7 at residues corresponding to 581- 590 of wild type AAV5 VP1 (SEQ ID NO: 1).

2. An engineered viral protein (VP) capsid polypeptide, wherein the engineered VP capsid polypeptide comprises at least 6, 7, 8, 9, or 10 contiguous amino acids of SEQ ID NO: 7 at residues corresponding to 581-590 of wild type AAV5 VP1 (SEQ ID NO: 1).

3. The engineered viral protein (VP) capsid polypeptide of claim 1 or claim 2, wherein the engineered VP capsid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to an AAV5, AAV9, AAV2, AAV1, AAV3, AAV4, AAV6, AAV7, AAV8, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV- DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 capsid polypeptide, optionally, wherein the engineered VP capsid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to an AAV5 VP1 capsid, further optionally wherein the AAV5 VP1 capsid is SEQ ID NO:

1.

4. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-3, wherein the engineered VP capsid comprises at least 80%, 85%, 90%, 95%, 96%, 97%, or 98% sequence identity to a chimera polypeptide of comprising at least one serotype of any one of or two serotypes selected from AAV5, AAV9, AAV2, AAV1, AAV3, AAV4, AAV6, AAV7, AAV8, AAV 10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-DJ, AAV-DJ / 8, AAV-DJ / 9, AAV1 / 2, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh43, AAV.Rh74, AAV.v66, AAV.Oligo001, AAV.SCH9, AAV.r3.45, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PhP.eB, AAV.PhP.V1, -245- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO AAV.PHP.B, AAV.PhB.C1, AAV.PhB.C2, AAV.PhB.C3, AAV.PhB.C6, AAV.cy5, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, AAV.HSC17, or AAVhu68 capsid polypeptide, optionally, wherein the at least one serotype is AAV5 or AAV9 or the two serotypes selected are AAV5 and AAV9, further optionally wherein the AAV5 is SEQ ID NO: 1 and the AAV9 is SEQ ID NO:

4.

5. The engineered viral protein (VP) capsid polypeptide of claim 4, wherein the chimera polypeptide comprises an AAV9 / AAV5 chimera polypeptide.

6. The engineered viral protein (VP) capsid polypeptide of claim 4 or claim 5, wherein the chimera polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

85.

7. The engineered viral protein (VP) capsid polypeptide of any one of claims 4-6, wherein the chimera polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 98.5% sequence identity to SEQ ID NO:

2.

8. The engineered viral protein (VP) capsid polypeptide of any one of claims 4-7, wherein the chimera polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 100% sequence identity to SEQ ID NO:

35.

9. The engineered viral protein (VP) capsid polypeptide of any one of claims 4-8, wherein the chimera polypeptide comprises a first region derived from a first AAV polypeptide and a second region derived from a second polypeptide, wherein the first region comprises a VP1 unique region of the first AAV polypeptide and the second region comprises the VP2 and VP3 region of the second AAV polypeptide.

10. The engineered viral protein (VP) capsid polypeptide of any one of claims 4-9, wherein the chimera polypeptide comprises a first region comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 85 and a second region comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 98.5% sequence identity to SEQ ID NO:

2. -246- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO11. The engineered viral protein (VP) capsid polypeptide of any one of claims 4-10, wherein the chimera polypeptide increases functional transduction of the engineered VP capsid polypeptide.

12. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-11, wherein the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

24.

13. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-12, wherein the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO:

24.

14. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-13, wherein the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO:

24.

15. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-14, wherein the engineered VP capsid polypeptide comprises SEQ ID NO:

24.

16. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-15, wherein the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

20.

17. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-16, wherein the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO:

20.

18. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-17, wherein the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO:

20.

19. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-18, wherein the engineered VP capsid polypeptide comprises SEQ ID NO:

20.

20. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-19, wherein the engineered VP capsid polypeptide comprises at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 100% sequence identity to SEQ ID NO:

16. -247- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO21. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-20, wherein the engineered VP capsid polypeptide comprises at least 90% sequence identity to SEQ ID NO:

16.

22. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-21, wherein the engineered VP capsid polypeptide comprises at least 98% sequence identity to SEQ ID NO:

16.

23. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-22, wherein the engineered VP capsid polypeptide comprises at least 98.5% sequence identity to SEQ ID NO:

16.

24. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-23, wherein the engineered VP capsid polypeptide comprises at least 99% sequence identity to SEQ ID NO:

16.

25. The engineered viral protein (VP) capsid polypeptide of any one of claims 1-24, wherein the engineered VP capsid polypeptide comprises SEQ ID NO:

16.

26. An engineered capsid comprising the engineered VP capsid polypeptide of any one of claims 1-25.

27. An engineered capsid comprising a VP1 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 100% sequence identity to SEQ ID NO: 16, a VP2 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 20, a VP3 sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24, or any combination thereof.

28. An engineered capsid comprising a VP1 sequence of SEQ ID NO: 16, a VP2 sequence of SEQ ID NO: 20, and a VP3 sequence of SEQ ID NO:

24.

29. A recombinant adeno-associated virus (rAAV) comprising the engineered VP capsid polypeptide of any one of claims 1-25 or the engineered capsid of any one of claims 26-28, and a payload.

30. The rAAV of claim 29, wherein the payload encodes a therapeutic polynucleotide or a therapeutic peptide. -248- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO31. The rAAV of claim 29, wherein the payload encodes a heterologous polynucleotide or a heterologous peptide.

32. The rAAV of claim 29, wherein the payload encodes an antibody or antibody fragment.

33. The rAAV of claim 29, wherein the payload encodes a transgene.

34. The rAAV of claim 30, wherein the therapeutic polynucleotide encodes a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, or an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof.

35. The rAAV of claim 30, wherein the therapeutic polynucleotide encodes a guide RNA.

36. The rAAV of claim 34 or claim 35, wherein the guide RNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR) enzyme when bound to a target.

37. The rAAV of any one of claims 34-36, wherein the guide RNA facilitates editing of a target.

38. The rAAV of any one of claims 34-37, wherein the guide RNA facilitates knockdown of a target.

39. The rAAV of any one of claims 34-38, wherein the guide RNA facilitates altered splicing of a target.

40. The rAAV of any one of claims 34-39, wherein the guide RNA facilitates exon skipping in a target.

41. The rAAV of any one of claims 36-40, wherein the target is a CNS target.

42. The rAAV of any one of claims 36-41, wherein the target is SNCA.

43. The rAAV of any one of claims 29-42, wherein the engineered VP capsid polypeptide is encoded by a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:

75. -249- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO44. The rAAV of any one of claims 29-43, wherein the engineered VP capsid polypeptide is encoded by a sequence with at least 90% sequence identity to SEQ ID NO:

75.

45. The rAAV of any one of claims 29-44, wherein the engineered VP capsid polypeptide is encoded by a sequence with at least 98% sequence identity to SEQ ID NO:

75.

46. The rAAV of any one of claims 29-45, wherein the engineered VP capsid polypeptide is encoded by a sequence comprising SEQ ID NO:

75.

47. A pharmaceutical composition comprising the engineered VP capsid polypeptide of any one of claims 1-25, the engineered capsid of any one of claims 26-28, or the rAAV of any one of claims 29-46, and a pharmaceutically acceptable carrier or excipient.

48. A method of increasing transduction in a target brain tissue as compared to a non- target liver tissue in a subject, comprising: i) administering the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47, ii) collecting the target brain tissue and the non-target liver tissue from the subject after administration, and iii) quantifying the transduction in the target brain tissue and the non-target liver tissue; wherein, the transduction is greater in the target brain tissue than the non-target liver tissue.

49. The method of claim 48, wherein the transduction is at least two-fold, three-fold, four- fold, or five-fold greater in the target brain tissue as compared to the transduction in the non- target liver tissue.

50. The method of claim 48 or claim 49, wherein the transduction is at least two-fold, three-fold, four-fold, or five-fold greater in the target brain tissue as compared to a comparative capsid.

51. The method of any one of claims 48-50, wherein the transduction is at least two-fold, three-fold, four-fold, or five-fold lesser in the non-target liver tissue as compared to a comparative capsid.

52. The method of claim 50 or claim 51, wherein the comparative capsid is a wild type AAV capsid or an engineered control capsid. -250- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO53. The methods of any one of claims 48-52, wherein the transduction is measured by RNA expression.

54. A method of increasing payload expression in a target brain tissue as compared to a non-target liver tissue in a subject, comprising: i) administering the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47, ii) collecting the target brain tissue and the non-target liver tissue from the subject after administration, and iii) quantifying the payload expression in the target brain tissue and the non-target liver tissue; wherein, the payload expression is greater in the target brain tissue than the non-target liver tissue.

55. The method of claim 54, wherein the payload expression is at least two-fold, three- fold, four-fold, or five-fold greater in the target brain tissue as compared to the transduction in the non-target liver tissue.

56. The method of claim 54 or claim 55, wherein the payload expression is at least two- fold, three-fold, four-fold, or five-fold greater in the target brain tissue as compared to a comparative capsid.

57. The method of any one of claims 54-56, wherein the payload expression is at least two-fold, three-fold, four-fold, or five-fold lesser in the non-target liver tissue as compared to a comparative capsid.

58. The method of claim 56 or claim 57, wherein the comparative capsid is a wild type AAV capsid or an engineered control capsid.

59. The methods of any one of claims 54-58, wherein the payload expression is measured by RNA expression.

60. A method of increasing target RNA editing in a target brain tissue in a subject, comprising: i) administering the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47, -251- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WOii) collecting the target brain tissue and the non-target liver tissue from the subject after administration, and iii) quantifying the target RNA editing in the target brain tissue; wherein, the target RNA editing is at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% target RNA editing.

61. The method of claim 60, wherein the target brain tissue is a substantia nigra tissue and the target RNA editing is greater than 80%.

62. The method of claim 60 or claim 61, wherein the target RNA editing is at least two- fold, at least three-fold, at least four-fold, or at least five-fold greater in the target brain tissue as compared to a comparative capsid.

63. The method of claim 62, wherein the target brain tissue is a substantia nigra tissue and the target RNA editing is at least 4-fold greater as compared to the target RNA editing of the comparative capsid.

64. The method of claim 62 or claim 63, wherein the comparative capsid is a wild type AAV capsid or an engineered control capsid.

65. A method of increasing AAV delivery to a target brain tissue as compared to a non- target liver tissue in a subject, comprising: i) administering the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47, ii) collecting the target brain tissue and the non-target liver tissue from the subject after administration, iii) quantifying the number of viral genomes (vg) and diploid genomes (dg), and iv) calculating a ratio of vg / dg in the target brain tissue and the non-target liver tissue; wherein: the vg / dg ratio of the administered capsid is greater in the target brain tissue as compared to the vg / dg ratio of a comparative capsid, and the vg / dg ratio of the administered capsid is lesser in the non-target liver tissue as compared to the vg / dg ratio of a comparative capsid. -252- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO66. The method of claim 65, wherein the vg / dg ratio of the administered capsid is at least two-fold, three-fold, four-fold, or five-fold greater in the target brain tissue as compared to the vg / dg ratio of a comparative capsid.

67. The method of claim 65 or claim 66, wherein the vg / dg ratio of the administered capsid is at least two-fold, three-fold, four-fold, or five-fold lesser in the target brain tissue as compared to the vg / dg ratio of a comparative capsid.

68. The method of any one of claims 65-67, wherein the comparative capsid is a wild type AAV capsid or an engineered control capsid.

69. A method of transducing a neuron, the method comprising administering to a subject the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47, and expressing the payload in the neuron.

70. The method of claim 69, wherein the neuron is a human neuron.

71. The method of claim 70, wherein the human neuron is an iPSC-derived neuron.

72. The method of any one of claims 69-71, wherein the neuron is in a tissue and wherein at least 50%, at least 60%, at least 70%, or at least 80% of the neurons in the tissue are transduced.

73. The method of claim 72, wherein the tissue is a brain tissue.

74. A method of transducing an astrocyte, the method comprising administering to a subject the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47, and expressing the payload in the astrocyte.

75. A method of transducing a glial cell, the method comprising administering to a subject the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47, and expressing the payload in the glial cell.

76. The method of claim 75, wherein the glial cell is in a tissue and wherein at least 50%, at least 60%, at least 70%, or at least 80% of the glial cells in the tissue are transduced.

77. The method of claim 76, wherein the tissue is a brain tissue. -253- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO78. A method of delivering a payload to a target tissue, the method comprising administering to a subject the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47.

79. A method of administering the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47 to a subject and delivering a payload to a target tissue.

80. A method of treating a condition in a subject, the method comprising administering to the subject the rAAV of any one of claims 29-46 or the pharmaceutical composition of claim 47 and delivering a payload to a target tissue, thereby treating the condition.

81. The method of any one of claims 78-80, wherein the rAAV of any one of claims 29- 46 or the pharmaceutical composition of claim 47 is administered by systemic administration.

82. The method of any one of claims 78-81, further comprising infecting the target tissue with the rAAV.

83. The method of any one of claims 78-82, wherein a ratio of payload expression in a brain tissue relative to a non-brain tissue upon administration of the rAAV is higher than upon administration of a wild type AAV.

84. The method of any one of claims 78-83, wherein a ratio of target editing in a brain tissue relative to a non-brain tissue upon administration of the rAAV is higher than upon administration of a wild type AAV.

85. The method of any one of claims 78-84, wherein, upon delivery to the target tissue, the payload exhibits increased transduction compared to a payload delivered via an AAV5 comprising a wild type VP1 capsid polypeptide, a wild type VP2 capsid polypeptide, and a wild type VP3 capsid polypeptide.

86. The method of any one of claims 78-85, wherein the target tissue is a central nervous system tissue, a muscle tissue, an eye tissue, a retina tissue, or a retinal pigment epithelium tissue.

87. The method of claim 86, wherein the brain tissue is a deep brain tissue.

88. The method of claim 87, wherein the deep brain tissue is a substantia nigra tissue. -254- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO89. The method of any one of claims 83-88, wherein the non-brain tissue is a liver tissue.

90. The method of any one of claims 83-89, wherein the non-brain tissue is a peripheral nervous system tissue.

91. The method of any one of claims 83-90, wherein the non-brain tissue is a dorsal root ganglion tissue.

92. The method of any one of claims 83-91, wherein the non-brain tissue is a muscle tissue.

93. The method of any one of claims 78-92, wherein administration of the rAAV produces lower toxicity than administration of a wild type AAV.

94. The method of any one of claims 78-93, wherein administration of the rAAV produces a lower immunogenicity response than administration of a wild type AAV.

95. The method of any one of claims 78-94, wherein the payload encodes a therapeutic peptide or a therapeutic polynucleotide.

96. The method of claim 78-95, wherein the payload encodes an antibody or fragment thereof.

97. The method of any one of claims 78-95, wherein the payload encodes a therapeutic polynucleotide.

98. The method of any one of claims 78-97¸ further comprising expressing the payload in a CNS tissue of the subject.

99. The method of any one of claims 95-98, wherein the therapeutic polynucleotide encodes a peptide.

100. The method of any one of claims 95-99, wherein the therapeutic polynucleotide is a guide RNA.

101. The method of claim 100, wherein the guide RNA is capable of recruiting an adenosine deaminase acting on RNA (ADAR) enzyme when bound to a target sequence.

102. The method of claim 101, further comprising editing of the target sequence. -255- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO103. The method of claim 101 or claim 102, further comprising facilitating gene or protein knockdown.

104. The method of any one of claims 101-103, further comprising facilitating altered splicing of the target sequence.

105. The method of any one of claims 101-104, comprising facilitating exon skipping in the target sequence.

106. The method of any one of claims 101-105, wherein the target sequence is a CNS target sequence.

107. The method of any one of claims 101-106, wherein the target sequence is implicated in the condition.

108. The method of any one of claims 101-107, wherein the target sequence is an SNCA target sequence.

109. The method of any one of claims 101-108, wherein the target sequence is present in deep brain tissue.

110. The method of any one of claims 80-109, wherein the condition is Parkinson’s disease, Alzheimer’s disease, Amyotrophic Lateral Sclerosis, Huntington’s disease, Lewy body dementia, essential tremor, dystonia, or epilepsy. -256- KTS Docket No.: 116779-1518674 (745WO1) Client Ref. No.: STX-128WO

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