Engineered adeno-associated virus capsids

Abstract

Provided herein are engineered adeno-associated virus (AAV) capsids with altered (e.g., increased) affinity for the AAV receptor (AAVR); methods for using the same; and methods for determining the affinity of an AAV capsid for a query protein using virus-like particles (VLPs) having capsids composed of the VP3 subunit of the AAV capsid.

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. Said XML copy, created on November 28, 2023, is titled 50474-282WO2_Sequence_Listing_11_28_23 and is 6,015 bytes in size.

[0002] FIELD OF THE INVENTION Provided herein are engineered adeno-associated virus (AAV) capsids with increased affinity for the AAV receptor (AAVR); methods of using the same; and methods of determining the affinity of AAV capsids for query proteins using virus-like particles (VLPs) having capsids composed of the VP3 subunit of the AAV capsid. [Background technology]

[0003] Adeno-associated viruses (AAVs) are nonpathogenic single-stranded DNA (ssDNA) viruses that can be used as vectors for gene therapy, for example. AAVs have a nonenveloped icosahedral capsid composed of the major capsid protein VP1, the minor capsid protein VP2, and the minor capsid protein VP3, which are encoded by overlapping genes.

[0004] An important factor in the use of AAVs is the mitigation of their immunogenicity in subjects, which has been shown to correlate with viral genome dose.

[0005] The AAV receptor (AAVR) is important for cell transduction by most AAV serotypes. Improving the affinity of the AAV capsid for AAV can increase transduction efficiency, thereby reducing dose requirements and alleviating immunogenicity.

[0006] Therefore, the development of AAV capsids with altered (e.g., increased) affinity for AAVR represents an important unmet need. Summary of the Invention

[0007] In one aspect, the disclosure features a method for improving the transduction efficiency of an adeno-associated virus (AAV), the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 including an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an A472H amino acid substitution mutation; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0008] In another aspect, the disclosure features a method of improving the transduction efficiency of an adeno-associated virus (AAV), the method including contacting a target cell with a modified AAV having a capsid including: (a) a major capsid protein VP1 including an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 including an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an A472H amino acid substitution mutation, where the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0009] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0010] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0011] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0012] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0013] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and E500R amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and E500R amino acid substitution mutations.

[0014] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0015] In another aspect, the disclosure features a method for improving the transduction efficiency of AAV, the method including contacting a target cell with a modified AAV having a capsid including: (a) a major capsid protein VP1 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, A472H, and E500R amino acid substitution mutations, where the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0016] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 including A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0017] In some embodiments, the methods include contacting the target cell with a modified AAV, thereby improving transduction efficiency.

[0018] In some embodiments, the transduction efficiency is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0019] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 including an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an A472H amino acid substitution mutation, wherein the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV has an increased transduction rate into the target cell compared to an AAV9 having a capsid that does not include any of the indicated amino acid substitution mutations.

[0020] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0021] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0022] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0023] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0024] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and E500R amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and E500R amino acid substitution mutations.

[0025] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, A472H, and E500R amino acid substitution mutations, wherein the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV has an increased transduction rate of the target cell compared to an AAV9 having a capsid that does not include any of the indicated amino acid substitution mutations.

[0026] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 including A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A472H and E500P amino acid substitution mutations, wherein the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV has an increased transduction rate into the target cell compared to an AAV9 having a capsid that does not include any of the indicated amino acid substitution mutations.

[0027] In some embodiments, the modified AAV has increased affinity for the AAV receptor (AAVR) compared to an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0028] In some embodiments, the modified AAV has an increased rate of delivery to the nucleus of a target cell compared to an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0029] In some embodiments, the modified AAV transduces target cells at a lower dose than an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0030] In another aspect, the disclosure provides a method for improving affinity of AAV for an AAV receptor (AAVR), comprising: (i) (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 having A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) a modified AAV having a capsid comprising (a) a major capsid protein VP1 having A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 having A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 having A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0031] In another aspect, the disclosure provides a method for increasing the rate of delivery of AAV to the nucleus of a target cell, comprising: (i) (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H amino acid substitution mutations. or (iii) providing a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0032] In another aspect, the disclosure provides a method for transducing a target cell with a low dose of AAV, comprising: (i) (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation. or (iii) providing a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0033] In some embodiments, the method comprises contacting a target cell with a modified AAV.

[0034] In some embodiments, the target cell is an endothelial cell, a cancer cell, a central nervous system (CNS) cell, an eye cell, a retinal cell, a muscle cell, a stem cell, a heart cell, a lung cell, a skin cell, a kidney cell, or a liver cell. In some embodiments, the endothelial cell is a brain endothelial cell.

[0035] In some embodiments, the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or comprise one or more amino acid insertions or deletions.

[0036] In some embodiments, the serotype of the modified AAV is AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV 12. In some embodiments, the serotype of the modified AAV is AAV9 or a variant thereof.

[0037] In another aspect, the disclosure provides a use of a modified AAV in the manufacture of a medicament for transducing a target cell, wherein the modified AAV comprises: (i) (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0038] In another aspect, the disclosure provides a use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, wherein the modified AAV comprises: (i) (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H amino acid substitution mutations. or (iii) a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0039] In another aspect, the disclosure provides a modified AAV for use in transducing a target cell, comprising: (i) (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising an A273N, Q387K, A472H, and E500R amino acid substitution mutations. or (iii) a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0040] In another aspect, the disclosure provides a modified AAV for use in delivering cargo to a target cell, comprising: (i) (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations. or (iii) a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein a cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0041] In another aspect, the disclosure features an AAV having a capsid that includes: (a) a major capsid protein VP1 that includes an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 that includes an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 that includes an A472H amino acid substitution mutation; and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0042] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0043] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0044] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0045] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0046] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and E500R amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and E500R amino acid substitution mutations.

[0047] In another aspect, the disclosure features an AAV having a capsid including: (a) a major capsid protein VP1 that includes A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 that includes A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes A273N, Q387K, A472H, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0048] In another aspect, the disclosure features an AAV having a capsid that includes (a) a major capsid protein VP1 that includes A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 that includes A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0049] In some embodiments, the AAV serotype is AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV 12. In some embodiments, the modified AAV serotype is AAV9 or a variant thereof.

[0050] In another aspect, the disclosure features a method for improving the transduction efficiency of an adeno-associated virus (AAV), the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 including an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an E500R amino acid substitution mutation; where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0051] In another aspect, the disclosure features a method of improving the transduction efficiency of an adeno-associated virus (AAV), the method including contacting a target cell with a modified AAV having a capsid including: (a) a major capsid protein VP1 including an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 including an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an E500R amino acid substitution mutation, where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0052] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0053] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0054] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0055] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and A472H amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and A472H amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and A472H amino acid substitution mutations.

[0056] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, and E500R amino acid substitution mutations; where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0057] In another aspect, the disclosure features a method for improving AAV transduction efficiency, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, and E500R amino acid substitution mutations, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency.

[0058] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0059] In some embodiments, the methods include contacting the target cell with a modified AAV, thereby improving transduction efficiency.

[0060] In some embodiments, the transduction efficiency is improved compared to the transduction efficiency of an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0061] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 including an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an E500R amino acid substitution mutation, wherein the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV has an increased transduction rate into the target cell compared to an AAV9 having a capsid that does not include any of the indicated amino acid substitution mutations.

[0062] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0063] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0064] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0065] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and A472H amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and A472H amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and A472H amino acid substitution mutations.

[0066] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, and E500R amino acid substitution mutations, wherein the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV has an increased transduction rate into the target cell compared to an AAV9 having a capsid that does not include any of the indicated amino acid substitution mutations.

[0067] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0068] In some embodiments, the modified AAV has increased affinity for the AAV receptor (AAVR) compared to AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0069] In some embodiments, the modified AAV has an increased rate of delivery to the nucleus of a target cell compared to AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0070] In some embodiments, the modified AAV transduces target cells at a lower dose than AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0071] In another aspect, the disclosure provides a method for improving affinity of AAV for an AAV receptor (AAVR), comprising: (i) (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; or (ii) (a) a major capsid protein V comprising an A273N, Q387K, and E500R amino acid substitution mutation; (b) a minor capsid protein VP2 having A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 having A273N, Q387K, and E500R amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0072] In another aspect, the disclosure provides a method for increasing the rate of delivery of AAV to the nucleus of a target cell, comprising: (i) (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; or (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; The method includes providing a modified AAV having a capsid comprising (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0073] In another aspect, the present disclosure provides a method for transducing a target cell with a low dose of AAV, comprising: (i) (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; or (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) providing a modified AAV having a capsid comprising a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0074] In some embodiments, the methods include contacting a target cell with a modified AAV.

[0075] In some embodiments, the target cell is an endothelial cell, a cancer cell, a central nervous system (CNS) cell, an eye cell, a retinal cell, a muscle cell, a stem cell, a heart cell, a lung cell, a skin cell, a kidney cell, or a liver cell. In some embodiments, the endothelial cell is a brain endothelial cell.

[0076] In some embodiments, the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or comprise one or more amino acid insertions or deletions.

[0077] In another aspect, the disclosure provides a use of a modified AAV in the manufacture of a medicament for transducing a target cell, wherein the modified AAV comprises: (i) (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; or (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a capsid comprising a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0078] In another aspect, the disclosure provides a use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, wherein the modified AAV comprises: (i) (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; or (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation. and / or (c) a minor capsid protein VP2 comprising a minor capsid protein VP2 containing the A273N, Q387K, and E500R amino acid substitution mutations; and / or (d) a minor capsid protein VP3 containing the A273N, Q387K, and E500R amino acid substitution mutations; the cargo is encapsulated by an AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0079] In another aspect, the disclosure provides a modified AAV for use in transducing a target cell, comprising: (i) (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; or (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations. and / or (c) a capsid comprising a minor capsid protein VP2 comprising an amino acid substitution mutation; and / or (d) a minor capsid protein VP3 comprising an amino acid substitution mutation, A273N, Q387K, and E500R; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0080] In another aspect, the disclosure provides a modified AAV for use in delivering cargo to a target cell, comprising: (i) (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; or (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations. and / or (c) a capsid comprising a minor capsid protein VP2; and / or (c) a minor capsid protein VP3 comprising the A273N, Q387K, and E500R amino acid substitution mutations; the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0081] In another aspect, the disclosure features an AAV having a capsid that includes: (a) a major capsid protein VP1 that includes an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 that includes an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 that includes an E500R amino acid substitution mutation; and wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0082] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0083] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0084] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0085] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and A472H amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and A472H amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and A472H amino acid substitution mutations.

[0086] In another aspect, the disclosure features an AAV having a capsid that includes (a) a major capsid protein VP1 that includes A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 that includes A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes A273N, Q387K, and E500R amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0087] In another embodiment, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0088] In another aspect, the disclosure features a method for improving the transduction efficiency of an adeno-associated virus (AAV), the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations; where the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0089] In some embodiments, the methods include contacting the target cell with a modified AAV, thereby improving transduction efficiency.

[0090] In another aspect, the disclosure features a method for improving the transduction efficiency of an adeno-associated virus (AAV), the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations; where the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0091] In some embodiments, the transduction efficiency is improved compared to the transduction efficiency of an AAV2 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0092] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations, wherein the cargo is encapsulated by the AAV; the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2); and the AAV has an increased transduction rate into the target cell compared to an AAV2 having a capsid that does not include any of the indicated amino acid substitution mutations.

[0093] In some embodiments, the modified AAV has increased affinity for the AAV receptor (AAVR) compared to AAV2 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0094] In some embodiments, the modified AAV has an increased rate of delivery to the nucleus of a target cell compared to AAV2 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0095] In some embodiments, the modified AAV transduces target cells at a lower dose than AAV2 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0096] In another aspect, the disclosure features a method for improving affinity of AAV for an AAV receptor (AAVR), the method having a capsid including: (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations; where the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0097] In another aspect, the disclosure features a method for increasing the rate of delivery of AAV to the nucleus of a target cell, the method having a capsid that includes (a) a major capsid protein VP1 that includes T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 that includes T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes T592D and A593E amino acid substitution mutations; and the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0098] In another aspect, the disclosure features a method for transducing target cells with a low dose of AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0099] In some embodiments, the methods include contacting a target cell with a modified AAV.

[0100] In some embodiments, the target cell is an endothelial cell, a cancer cell, a CNS cell, an eye cell, a retinal cell, a muscle cell, a stem cell, a cardiac cell, a lung cell, a skin cell, a kidney cell, or a liver cell. In some embodiments, the endothelial cell is a brain endothelial cell.

[0101] In some embodiments, the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or comprise one or more amino acid insertions or deletions.

[0102] In another aspect, the disclosure features the use of a modified AAV in the manufacture of a medicament for transducing a target cell, wherein the modified AAV has a capsid that includes (a) a major capsid protein VP1 that includes T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 that includes T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes T592D and A593E amino acid substitution mutations; and the AAV is AAV2 or a variant thereof, wherein the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2), and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not include any of the indicated amino acid substitution mutations.

[0103] In another aspect, the disclosure features the use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations, wherein the cargo is encapsulated by the AAV; the AAV is AAV2 or a variant thereof, wherein the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0104] In another aspect, the disclosure features a modified AAV for use in transducing a target cell, having a capsid that includes (a) a major capsid protein VP1 that includes T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 that includes T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2), and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not include any of the indicated amino acid substitution mutations.

[0105] In another aspect, the disclosure features a modified AAV for use in delivering cargo to a target cell, having a capsid that includes (a) a major capsid protein VP1 that includes T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 that includes T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2), and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not include any of the indicated amino acid substitution mutations.

[0106] In another aspect, the disclosure features an AAV having a capsid including: (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0107] In another aspect, the disclosure features a method for improving the transduction efficiency of an adeno-associated virus (AAV), the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, and P504I amino acid substitution mutations; where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0108] In another aspect, the disclosure features a method for improving the transduction efficiency of an adeno-associated virus (AAV), the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, and P504I amino acid substitution mutations; where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0109] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0110] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0111] In another aspect, the disclosure features a method for improving the transduction efficiency of AAV, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0112] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0113] In another aspect, the disclosure features a method for improving the transduction efficiency of AAV, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0114] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0115] In some embodiments, the methods include contacting the target cells with a modified AAV, thereby improving transduction efficiency.

[0116] In some embodiments, the transduction efficiency is improved compared to the transduction efficiency of an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0117] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, and P504I amino acid substitution mutations; wherein the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the AAV has an increased transduction rate into the target cell compared to an AAV9 having a capsid that does not include any of the indicated amino acid substitution mutations.

[0118] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0119] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0120] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; wherein the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the AAV has an increased transduction rate into the target cell compared to an AAV9 having a capsid that does not include any of the indicated amino acid substitution mutations.

[0121] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the AAV has an increased transduction rate into the target cell compared to an AAV9 having a capsid that does not include any of the indicated amino acid substitution mutations.

[0122] In some embodiments, the modified AAV has increased affinity for the AAV receptor (AAVR) compared to AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0123] In some embodiments, the modified AAV has an increased rate of delivery to the nucleus of a target cell compared to AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0124] In some embodiments, the modified AAV transduces target cells at a lower dose than AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

[0125] In another aspect, the disclosure provides a method for improving affinity of AAV for an AAV receptor (AAVR), comprising: (i) (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; or (iii) providing a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0126] In another aspect, the disclosure provides a method for increasing the rate of delivery of AAV to the nucleus of a target cell, comprising: (i) (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; or (iii) providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0127] In another aspect, the disclosure provides a method for transducing target cells with a low dose of AAV, comprising: (i) (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations. or (iii) providing a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0128] In some embodiments, the methods include contacting a target cell with a modified AAV.

[0129] In some embodiments, the target cell is an endothelial cell, a cancer cell, a central nervous system (CNS) cell, an eye cell, a retinal cell, a muscle cell, a stem cell, a heart cell, a lung cell, a skin cell, a kidney cell, or a liver cell. In some embodiments, the endothelial cell is a brain endothelial cell.

[0130] In some embodiments, the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or comprise one or more amino acid insertions or deletions.

[0131] In another aspect, the disclosure provides a method for the manufacture of a medicament for transducing a target cell, the method comprising the steps of: (i) (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) a minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations. or (iii) providing a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid not comprising any of the indicated amino acid substitution mutations.

[0132] In another aspect, the disclosure provides a method for the manufacture of a medicament for delivering cargo to a target cell, the method comprising the steps of: (i) (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) a minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations. or (iii) a capsid comprising (a) a major capsid protein VP1 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the cargo is encapsulated by an AAV; and the AAV is AAV9 or a variant thereof, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0133] In another aspect, the disclosure provides a modified AAV for use in transducing a target cell, comprising: (i) (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations. (ii) (a) a major capsid protein VP1 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations. or (iii) a capsid comprising (a) a major capsid protein VP1 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0134] In another aspect, the disclosure provides a modified AAV for use in delivering cargo to a target cell, comprising: (i) (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations. (ii) (a) a major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations. or (iii) a capsid comprising (a) a major capsid protein VP1 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the cargo is encapsulated by the AAV; and the AAV is AAV9 or a variant thereof, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0135] In another aspect, the disclosure features an AAV having a capsid that includes: (a) a major capsid protein VP1 that includes I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 that includes I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes I451Y, S454I, and P504I amino acid substitution mutations; and wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0136] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0137] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0138] In another aspect, the disclosure features an AAV having a capsid that includes: (a) a major capsid protein VP1 that includes I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 that includes I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes I451Y, S454I, E500P, and P504I amino acid substitution mutations; and wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0139] In another aspect, the disclosure features an AAV having a capsid that includes: (a) a major capsid protein VP1 that includes I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 that includes I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 that includes I451Y, S454I, E500R, and P504I amino acid substitution mutations; and wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1). In another aspect, the disclosure features a method for determining the affinity of an AAV capsid for a query protein, the method including: (a) providing a virus-like particle (VLP) having a capsid consisting essentially of the VP3 subunit of the AAV capsid; (b) contacting the VLP of step (a) with a query protein under conditions that allow binding of the query protein to the VLP; and (c) quantitating the strength of the interaction between the query protein and the VLP, thereby determining the affinity of the AAV capsid for the query protein.

[0140] In some embodiments, the VLP is immobilized on a solid surface. In some embodiments, the solid surface comprises an antibody or antigen-binding fragment thereof having affinity for the VLP. In some embodiments, the antibody is a single-domain antibody. In some embodiments, the antigen-binding fragment is a Fab. In some embodiments, the antibody or antigen-binding fragment thereof binds to the VP3 subunit of the AAV capsid.

[0141] In some embodiments, the VLPs are produced in a cell line and immobilizing the VLPs on the solid surface comprises contacting the solid surface with a culture medium comprising the VLPs. In some embodiments, the culture medium comprises a lysate of the cell line in which the VLPs are produced.

[0142] In some embodiments, the quantifying is performed using biolayer interferometry (BLI) and / or surface plasmon resonance (SPR).

[0143] In some embodiments, the VP3 subunit of the AAV capsid comprises one or more amino acid substitution mutations relative to the sequence of the wild-type VP3 subunit of the AAV capsid.

[0144] In some embodiments, the AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12. [Brief explanation of the drawings]

[0145] [Figure 1] Schematic showing the workflow for screening AAV virus-like particle (VLP) mutants containing variants of the minor capsid protein VP3 for affinity to a target molecule. BLI: Biolayer Interferometry. SPR: Surface Plasmon Resonance. [Figure 2A] 1 is a chart showing amino acid substitution mutations made at residues in the AAV2 or AAV9 capsid / AAVR interface. [Figure 2B]

[0023] Figure 1 is a pair of protein structure diagrams showing the interaction between residue 385 of the AAV2 capsid and residue 437 of the AAV receptor (AAVR; SEQ ID NO: 3). The left panel shows an AAV2 capsid protein with the native residue Q at position 385 (Q385), which forms a hydrogen bond (H-bond) with D437 of AAVR. The right panel shows an AAV2 capsid protein with a K at position 385 (Q385K substitution mutation), which can form a salt bridge with D437 of AAVR. [Figure 2C]A pair of protein structure diagrams showing the interaction between residue 271 of the AAV2 capsid and residue 437 of AAVR. The left panel shows an AAV2 capsid protein with the native residue H (H271) at position 271. The right panel shows an AAV2 capsid protein with an N at position 271 (H271N substitution mutation), which can form a new backbone H bond with D437 of AAVR. [Figure 3A] FIG. 1 is a schematic showing the workflow for assessing the affinity of VLPs for AAVR using BLI. [Figure 3B] 10 is a sensorgram showing the results of a BLI experiment in which AAV2 VLPs containing mutants of the minor capsid protein VP3 were evaluated for affinity to AAVR. [Figure 3C] 1 is a set of sensorgrams showing the affinity of wild-type (WT) AAV2 VLPs and AAV2 VLPs containing T592D, A593E, and T592D / A593E amino acid substitution mutations (numbered relative to AAV2 VP1) for AAV2 VLPs as assessed using SPR. [Figure 4A] 10 is a sensorgram showing the results of a BLI experiment in which AAV9 VLPs containing mutants of the minor capsid protein VP3 were evaluated for affinity to AAVR. [Figure 4B] 1 is a set of sensorgrams showing the affinity of WT AAV9 VLP and AAV9 VLP containing A273N, Q387K, and E500R amino acid substitution mutations (numbered relative to AAV9 VP1) for AAVR as assessed using SPR. [Figure 5] Schematic diagram showing the location of the A273 residue in the major capsid protein VP1 and the minor capsid proteins VP2 and VP3; the location of the related P98 residue in the accessory protein assembly activating protein (AAP); the effect of introducing the A273N mutation on the P98 codon (by replacing the codon GCC with either AAT or AAC); and an alignment of AAP protein sequences from the indicated AAV serotypes. [Figure 6]1 is a chart showing capture levels from conditioned medium of AAV9 VLPs produced with co-expressed AAP mutants on an anti-AAV9 BLI biosensor. [Figure 7A] FIG. 1 is a bar graph showing the yield titer (vector genomes (vg) / mL) of wild-type AAV9 (AAV9) and AAV9 containing the Q387K amino acid substitution mutation (AAV9-Q387K) in 40 mL of EXPI293™ cell culture. [Figure 7B] 1 is a pair of bar graphs showing the transduction efficiency (percent of cells) of wild-type AAV9 and AAV9 containing the Q387K amino acid substitution mutation in MDA-MB-231 cells. Left panel: AAV derived from cell pellet; right panel: AAV derived from culture medium. MOI: multiplicity of infection. [Figure 7C] Figure 7C is a bar graph showing the transduction efficiency (percent of cells) of wild-type AAV9 and AAV9 containing the Q387K amino acid substitution mutation in GNE293T (293 / tsA1609neo) cells. [Figure 7D] 1 is a bar graph showing the transduction efficiency of wild-type AAV9 and AAV9 containing the Q387K amino acid substitution mutation in wild-type HEK293 cells; a cell line without AAVR or GPR108 (DSO-25); and a cell line without AAVR (KIAA0319L-1). [Figure 8] 1 is a set of sensorgrams showing the affinity of WT AAV9 VLP and AAV9 VLPs containing Q387K; A273N and Q387K; A273N and E500R; Q387K and E500R; and A273N, Q387K and E500R amino acid substitution mutations for AAVR as assessed using SPR. [Figure 9] 1 is a set of sensorgrams showing the affinity of WT AAV9 VLP and AAV9 VLP containing A273N, Q387K, E500R, and A472H amino acid substitution mutations (numbered relative to AAV9 VP1) for AAVR as assessed using SPR. [Figure 10A]1 is a bar graph showing the transduction efficiency of control AAV9 and AAV9 containing E500R ("ER"), A273N ("AN"), and / or Q387K ("QK") amino acid substitution mutations and a GFP marker in HEK293T cells as measured by the percentage of cells containing the GFP marker. Multiplicity of infection (MOI): 1 x 10. [Figure 10B] 1 is a bar graph showing the transduction efficiency of control AAV9 and AAV9 containing E500R ("ER"), A273N ("AN"), and / or Q387K ("QK") amino acid substitution mutations and the GFP marker in HEK293T cells as measured by the percentage of cells containing the GFP marker. MOI: 1 x 10. [Figure 10C] 1 is a bar graph showing the transduction efficiency of control AAV9 and AAV9 containing E500R ("ER"), A273N ("AN"), and / or Q387K ("QK") amino acid substitution mutations and a GFP marker in HEK293 cells as measured by geometric mean fluorescence intensity (Geo MFI) of the FITC marker. MOI: 1 x 10. [Figure 10D] 1 is a bar graph showing the transduction efficiency of control AAV9 and AAV9 containing E500R ("ER"), A273N ("AN"), and / or Q387K ("QK") amino acid substitution mutations and a GFP marker in HEK293 cells as measured by Geo MFI of the FITC marker. MOI: 1 x 10. [Figure 10E] 1 is a set of photomicrographs showing the transduction efficiency of control AAV9 and AAV9 containing E500R ("ER"), A273N ("AN"), and / or Q387K ("QK") amino acid substitution mutations in HEK293T cells as indicated by the percentage of cells containing the GFP marker. MOI: 1 x 10. [Figure 11A]1 is a bar graph showing the transduction efficiency of control AAV9 and AAV9 containing E500R ("ER"), A273N ("AN"), and / or Q387K ("QK") amino acid substitution mutations and a GFP marker in SH-SY5y (neuroblastoma) cells as measured by the percentage of cells containing the GFP marker. MOI: 1 x 10. [Figure 11B] 1 is a bar graph showing the transduction efficiency of control AAV9 and AAV9 containing E500R ("ER"), A273N ("AN"), and / or Q387K ("QK") amino acid substitution mutations and the GFP marker in SH-SY5y cells as measured by the percentage of cells containing the GFP marker. MOI: 1 x 10. [Figure 11C] 1 is a bar graph showing the transduction efficiency of control AAV9 and AAV9 containing E500R ("ER"), A273N ("AN"), and / or Q387K ("QK") amino acid substitution mutations and a GFP marker in SH-SY5y cells as measured by Geo MFI of the FITC marker. Multiplicity of infection (MOI): 1 x 10. [Figure 11D] 1 is a bar graph showing the transduction efficiency of control AAV9 and AAV9 containing E500R ("ER"), A273N ("AN"), and / or Q387K ("QK") amino acid substitution mutations and a GFP marker in SH-SY5y cells as measured by Geo MFI of the FITC marker. Multiplicity of infection (MOI): 1 x 10. [Figure 12] Figure 1 shows the affinity of VLPs containing the indicated amino acid substitution mutations for AAVR as measured using CARTERRA® LSA (shown as the ratio of the dissociation constant (KD) of wild-type VLP to the KD of the mutant (KD fold change)). X indicates mutants that were not tested. Blanks indicate mutants with a KD fold change of less than 0.05. Fold changes between 0.05 and 0.1 are rounded to 0.1. [Figure 13]A pair of ribbon diagrams showing AAV9 in complex with AAVR (Protein Data Bank (PDB) 7WJX), showing the locations of six AAVR residues associated with improved affinity when mutated. [Figure 14] 1 is a set of sensorgrams showing the affinity of unpurified WT VLPs (from cell culture media) and purified WT VLPs for target proteins as assessed using SPR. DETAILED DESCRIPTION OF THE INVENTION

[0146] I. Definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0147] As used herein, the term "about" refers to a normal error range for the respective value, which would be readily understood by one of ordinary skill in the art. Reference to "about" with respect to a value or parameter herein includes (and describes) aspects directed to the value or parameter itself.

[0148] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to an "isolated peptide" means one or more isolated peptides.

[0149] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0150] An "effective amount" refers to an amount of an agent (e.g., a therapeutic agent) effective to provide a therapeutic / prophylactic benefit (e.g., as described herein) that is not outweighed by any unwanted / undesirable side effects.

[0151] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient(s) is effective and which does not contain additional components that are unacceptably toxic to the subject to which the formulation is administered. Such formulations are sterile. In one embodiment, the formulation is for intravenous (IV) administration. In another embodiment, the formulation is for subcutaneous (SC) administration.

[0152] A "native sequence" protein, as used herein, refers to a protein comprising the amino acid sequence of a protein found in nature, including variants of a naturally occurring protein. As used herein, the term includes proteins isolated from their natural source or recombinantly produced.

[0153] The term "protein," as used herein, refers to any naturally occurring protein from any source, including viruses (e.g., adeno-associated virus (AAV)), mammals such as primates (e.g., humans), and rodents (e.g., mice and rats). The term encompasses any form of a protein that results from processing, e.g., processing within a cell, that is a "full-length," unprocessed protein. The term also encompasses naturally occurring variants of a protein, e.g., splice variants or allelic variants, such as amino acid substitution or deletion variants. The term also includes isolated regions or domains of a protein, e.g., the extracellular domain (ECD).

[0154] An "isolated" protein or peptide is one that is separated from a component of its natural environment. In some embodiments, the protein or peptide is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC).

[0155] An "isolated" nucleic acid refers to a nucleic acid molecule that is separated from components of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained within a cell that ordinarily contains the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0156] The terms "AAV9 major capsid protein VP1" and "AAV9 VP1," as used herein, refer to any native VP1 from adeno-associated virus serotype 9 (AAV9), unless otherwise indicated. The terms encompass full-length AAV9 VP1 and isolated regions or domains of AAV9 VP1 that contain at least a portion of the unique N-terminus of AAV9 VP1 (i.e., the AAV9 VP1 N-terminal sequence not present in wild-type VP2 or VP3). The terms also encompass naturally occurring variants of AAV9 VP1, such as splice variants or allelic variants. An exemplary amino acid sequence of AAV9 VP1 is provided as SEQ ID NO: 1 and GenBank accession number AAS99264.1. Minor sequence variants of AAV9 VP1, particularly conservative amino acid substitutions, that do not affect the function and / or activity of AAV9 VP1 are also contemplated by the present invention.

[0157] The terms "AAV2 major capsid protein VP1" and "AAV2 VP1," as used herein, refer to any native VP1 from adeno-associated virus serotype 2 (AAV2), unless otherwise indicated. The terms encompass full-length AAV2 VP1 and isolated regions or domains of AAV2 VP1 that contain at least a portion of the unique N-terminus of AAV2 VP1 (i.e., an AAV2 VP1 N-terminal sequence not present in wild-type VP2 or VP3). The terms also encompass naturally occurring variants of AAV2 VP1, such as splice variants or allelic variants. An exemplary amino acid sequence of AAV2 VP1 is provided as SEQ ID NO: 2 and in NCBI reference sequence YP_680426.1. Minor sequence variants of AAV2 VP1, particularly conservative amino acid substitutions, that do not affect the function and / or activity of AAV2 VP1 are also contemplated by the present invention.

[0158] The term "AAV receptor" or "AAVR," as used herein, unless otherwise indicated, refers to any naturally occurring AAVR from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses full-length AAVR and isolated regions or domains thereof. The term also encompasses naturally occurring variants of AAVR, such as splice variants or allelic variants. The amino acid sequence of an exemplary AAVR is provided as SEQ ID NO: 3 and as UniProt ID: Q8IZA0. Minor sequence variants of AAVR, particularly conservative amino acid substitutions, that do not affect the function and / or activity of the AAVR are also contemplated by the present invention.

[0159] The term "virus-like particle" or "VLP," as used herein, refers to an AAV-like particle having a capsid that includes (e.g., consists essentially of) only one or two of the AAV major capsid protein VP1, the AAV minor capsid protein VP2, and the AAV minor capsid protein VP3, e.g., includes only VP1; includes only VP2; includes only VP3; includes only VP1 and VP2; includes only VP1 and VP3; or includes only VP2 and VP3.

[0160] "Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D Affinity can be measured by common methods known in the art, including those described herein.

[0161] As used herein, "complex" or "complex-type" refers to an association of two or more molecules that interact with each other through bonds and / or forces (e.g., van der Waals, hydrophobic, hydrophilic forces) that are not peptide bonds. In one embodiment, the complex is a heteromultimer. The term "protein complex" or "polypeptide complex," as used herein, should be understood to include complexes having non-protein entities conjugated to proteins in the protein complex (e.g., including, but not limited to, chemical molecules such as toxins or detection agents).

[0162] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and cells, including the progeny of such cells. Host cells include "transfected cells," "transformed cells," and "transformants," which include the primary transformed cell and its progeny, regardless of the number of passages. The progeny may not have exactly the same nucleic acid content as the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. In some embodiments, the host cell is stably transformed with the exogenous nucleic acid. In other embodiments, the host cell is transiently transformed with the exogenous nucleic acid.

[0163] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors that integrate into the genome of a host cell into which they are introduced, as well as vectors that act as self-replicating nucleic acid structures. Certain vectors are capable of directing the expression of a nucleic acid to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0164] "Percent amino acid sequence identity (%)" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after sequence alignment and, if necessary, introducing gaps to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the full length of the sequences being compared. However, in this specification, percent amino acid sequence identity values ​​are calculated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, together with user documentation, has been submitted to the U.S. Copyright Office (Washington, DC, 20559) and is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, California) or can be compiled from source code. The ALIGN-2 program must be compiled to be used on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and should not be altered.

[0165] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or to a given amino acid sequence B (which can alternatively be said as "given amino acid sequence A has a certain % amino acid sequence identity to, with, or to a given amino acid sequence B, or is composed of a given amino acid sequence A") is calculated as follows: X / Y×100

[0166] where X is the number of amino acid residues scored as perfect matches by the sequence alignment program ALIGN-2 in a program alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless expressly stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0167] II. Engineered AAV Capsids A. Engineered AAV capsid containing the A472H mutation In some aspects, the disclosure features an adeno-associated virus (AAV) or virus-like particle (VLP) having a capsid comprising: (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0168] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV or VLP is AAV1, AAV2, or AAV9. In some embodiments, the AAV or VLP is AAV9 or a variant thereof. Additional serotypes that can be used in the present invention are provided, for example, in section II(F) below. One of skill in the art can identify corresponding residues in the VP1, VP2, and / or VP3 proteins of two or more AAV serotypes (e.g., one can determine which residues of a given serotype correspond to the listed residues of AAV9), for example, by generating a sequence alignment.

[0169] In some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0170] A472H in combination with A273N, Q387K, and / or E500R mutations In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise A273N and A472H amino acid substitution mutations.

[0171] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise Q387K and A472H amino acid substitution mutations.

[0172] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise E500R and A472H amino acid substitution mutations.

[0173] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and E500R amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and E500R amino acid substitution mutations, i.e., VP1, VP2, and / or VP3 comprise A273N, Q387K, A472H, and E500R amino acid substitution mutations.

[0174] In some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0175] In some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0176] In some embodiments, the modified AAV or VLP has increased affinity for the AAV receptor (AAVR) compared to a control AAV or VLP having a capsid that does not contain any of the indicated amino acid substitution mutations (e.g., an AAV or VLP of the same serotype that does not contain any of the indicated amino acid substitution mutations). In some embodiments, the modified AAV or VLP contains two of the four A273N, Q387K, A472H, and E500R amino acid substitution mutations, the modified AAV or VLP has increased affinity for the AAVR compared to a control AAV or VLP having a capsid that contains only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP contains three of the four A273N, Q387K, A472H, and E500R amino acid substitution mutations, the modified AAV or VLP has increased affinity for the AAVR compared to a control AAV or VLP having a capsid that contains only one or two of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP contains all four of the A273N, Q387K, A472H, and E500R amino acid substitution mutations, and the modified AAV or VLP has increased affinity for AAVR compared to a control AAV or VLP having a capsid containing only one, two, or three of the indicated amino acid substitution mutations.

[0177] In some embodiments, the modified AAV or VLP has increased affinity for AAVR compared to its control AAV or VLP derivative, e.g., an AAV or VLP derivative having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP. In some embodiments, the modified AAV or VLP is AAV9 or a variant thereof.

[0178] A472H in combination with T592D and / or A593E mutations In some embodiments, (a) major capsid protein VP1 further comprises a T592D amino acid substitution mutation; (b) minor capsid protein VP2 further comprises a T592D amino acid substitution mutation; and / or (c) minor capsid protein VP3 further comprises a T592D amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise A472H and T592D amino acid substitution mutations, wherein the A472H amino acid substitution mutation is numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1) and the T592D amino acid substitution mutation is numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0179] In some embodiments, (a) major capsid protein VP1 further comprises an A593E amino acid substitution mutation; (b) minor capsid protein VP2 further comprises an A593E amino acid substitution mutation; and / or (c) minor capsid protein VP3 further comprises an A593E amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise A472H and A593E amino acid substitution mutations, wherein the A472H amino acid substitution mutation is numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1) and the A593E amino acid substitution mutation is numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0180] In some embodiments, (a) major capsid protein VP1 further comprises T592D and A593E amino acid substitution mutations; (b) minor capsid protein VP2 further comprises T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 further comprises T592D and A593E amino acid substitution mutations, i.e., VP1, VP2, and / or VP3 comprise A472H, T592D, and A593E amino acid substitution mutations, where the A472H amino acid substitution mutation is numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1) and the T592D and A593E amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0181] In some embodiments, the modified AAV or VLP has increased affinity for the AAV receptor (AAVR) compared to a control AAV or VLP having a capsid that does not contain any of the indicated amino acid substitution mutations (e.g., an AAV or VLP of the same serotype that does not contain any of the indicated amino acid substitution mutations).

[0182] In some embodiments, the modified AAV or VLP has increased affinity for AAVR compared to its control AAV or VLP derivative, e.g., an AAV or VLP derivative having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP.

[0183] A472H in combination with the E500P mutation In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise A472H and E500P amino acid substitution mutations, wherein the A472H and E500P amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0184] In some aspects, the disclosure features an AAV having a capsid including: (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0185] In some embodiments, the modified AAV or VLP has increased affinity for the AAV receptor (AAVR) compared to a control AAV or VLP having a capsid that does not contain any of the indicated amino acid substitution mutations (e.g., an AAV or VLP of the same serotype that does not contain any of the indicated amino acid substitution mutations).

[0186] In some embodiments, the modified AAV or VLP has increased affinity for AAVR compared to its control AAV or VLP derivative, e.g., an AAV or VLP derivative having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP.

[0187] In some embodiments, the modified AAV or VLP is AAV2 or a variant thereof.

[0188] In some embodiments, the yield and / or titer in an expression system (e.g., EXPI293 brand cells) of any of the modified AAV or VLPs provided herein, e.g., AAV or VLPs comprising one or more of the A273N, Q387K, A472H, E500R, T592D, and A593E amino acid substitution mutations, is not substantially reduced compared to the yield and / or titer of wild-type AAV or VLP in the expression system (e.g., is similar to, equal to, or greater than the yield and / or titer of wild-type AAV or VLP in the expression system).

[0189] How to improve affinity for AAVR In some aspects, the disclosure features a method for improving the affinity of AAV for an AAV receptor (AAVR), the method including providing any of the modified AAVs described above (e.g., contacting a cell with any of the modified AAVs described above, wherein the contacting increases affinity for the AAVR).

[0190] For example, in some aspects, the disclosure features a method for improving affinity of AAV for an AAV receptor (AAVR), comprising providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0191] In some aspects, the disclosure features a method for improving affinity of AAV for an AAV receptor (AAVR), comprising providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0192] In some aspects, the disclosure features a method for improving affinity of AAV for an AAV receptor (AAVR), comprising providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0193] For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP.

[0194] Alternative Amino Acid Substitution at Position A472 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may include an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation instead of the A472H amino acid substitution mutation.

[0195] For example, in some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0196] An AAV or VLP comprising an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation may further comprise one or more additional amino acid substitutions provided herein, for example, a major capsid protein VP1, a minor capsid protein VP2, and / or a minor capsid protein VP3 comprising one, two, or all three of an A273N amino acid substitution mutation, a Q387K amino acid substitution mutation, and an E500R amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0197] Additionally or alternatively, an AAV or VLP comprising an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation may further comprise a major capsid protein VP1, a minor capsid protein VP2, and / or a minor capsid protein VP3 comprising one or both of a T592D amino acid substitution mutation and an A593E amino acid substitution mutation, wherein the A472H amino acid substitution mutation is numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and the T592D and A593E amino acid substitution mutations are numbered relative to the AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0198] Additionally or alternatively, an AAV or VLP comprising an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation may further comprise a major capsid protein VP1, a minor capsid protein VP2, and / or a minor capsid protein VP3 comprising an E500P amino acid substitution mutation, where the E500P amino acid substitution mutation is numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0199] B. Engineered AAV capsids containing the E500R mutation In some aspects, the disclosure features an adeno-associated virus (AAV) or virus-like particle (VLP) having a capsid that includes: (a) a major capsid protein VP1 that includes an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 that includes an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 that includes an E500R amino acid substitution mutation; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0200] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV or VLP is AAV1, AAV2, or AAV9. Additional serotypes that may be used in the present invention are provided, for example, in section II(F) below. One of skill in the art can identify corresponding residues in the VP1, VP2, and / or VP3 proteins of two or more AAV serotypes (e.g., one can determine which residues of a given serotype correspond to the listed residues of AAV9), for example, by generating a sequence alignment.

[0201] In some aspects, the disclosure features an AAV or VLP having a capsid that includes (a) a major capsid protein VP1 that includes an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 that includes an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 that includes an E500R amino acid substitution mutation; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0202] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise A273N and E500R amino acid substitution mutations.

[0203] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise Q387K and E500R amino acid substitution mutations.

[0204] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise A472H and E500R amino acid substitution mutations.

[0205] In some embodiments, (a) the major capsid protein VP1 further comprises A273N and Q387K amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N and Q387K amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N and Q387K amino acid substitution mutations, i.e., VP1, VP2, and / or VP3 comprise A273N, Q387K, and E500R amino acid substitution mutations.

[0206] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and A472H amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and A472H amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and A472H amino acid substitution mutations, i.e., VP1, VP2, and / or VP3 comprise A273N, Q387K, A472H, and E500R amino acid substitution mutations.

[0207] In some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0208] In some aspects, the disclosure features an AAV or VLP having a capsid including: (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0209] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation. In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise A472H and E500R amino acid substitution mutations.

[0210] In some embodiments, the modified AAV or VLP has increased affinity for the AAV receptor (AAVR) compared to a control AAV or VLP having a capsid that does not contain any of the indicated amino acid substitution mutations (e.g., an AAV or VLP of the same serotype that does not contain any of the indicated amino acid substitution mutations). In some embodiments, the modified AAV or VLP contains two of the three A273N, Q387K, and E500R amino acid substitution mutations, the modified AAV or VLP has increased affinity for the AAVR compared to a control AAV or VLP having a capsid that contains only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP contains all three of the A273N, Q387K, and E500R amino acid substitution mutations, the modified AAV or VLP has increased affinity for the AAVR compared to a control AAV or VLP having a capsid that contains only one or only two of the indicated amino acid substitution mutations.

[0211] In some embodiments, the modified AAV or VLP is AAV9 or a variant thereof and has increased affinity for AAVR compared to a control AAV or VLP derivative thereof, e.g., an AAV9 or VLP derivative thereof having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP.

[0212] How to improve affinity for AAVR In some aspects, the disclosure features a method for improving the affinity of AAV for an AAV receptor (AAVR), the method including providing any of the modified AAVs described above (e.g., contacting a cell with any of the modified AAVs described above, wherein the contacting increases affinity for the AAVR).

[0213] For example, in some aspects, the disclosure features a method for improving affinity of AAV for an AAV receptor (AAVR), comprising providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0214] In some aspects, the disclosure features a method for improving affinity of AAV for an AAV receptor (AAVR), comprising providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0215] For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP.

[0216] Alternative amino acid substitution at position E500 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may comprise an E500P amino acid substitution mutation in place of the E500R amino acid substitution mutation.

[0217] For example, in some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0218] An AAV or VLP comprising the E500P amino acid substitution mutation may further comprise one or more additional amino acid substitutions provided herein, for example, a major capsid protein VP1, a minor capsid protein VP2, and / or a minor capsid protein VP3 comprising one, two, or all three of the A273N amino acid substitution mutation, the Q387K amino acid substitution mutation, and the A472H amino acid substitution mutation, where the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0219] C. Engineered AAV capsids containing the T592D and A593E mutations In some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising a T592D and / or A593E amino acid substitution mutation; (b) a minor capsid protein VP2 comprising a T592D and / or A593E amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising a T592D and / or A593E amino acid substitution mutation; wherein the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0220] In some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0221] In some embodiments, the AAV or VLP is of a serotype that interacts with AAVR (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV or VLP is AAV1, AAV2, or AAV9. Additional serotypes that can be used in the present invention are provided, for example, in Section II(F), below.

[0222] In some aspects, the disclosure features a method including providing a modified AAV or VLP having a capsid including: (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0223] In some embodiments, the modified AAV or VLP has increased affinity for the AAV receptor (AAVR) compared to a control AAV or VLP (e.g., an AAV or VLP of the same serotype that does not contain either of the indicated amino acid substitution mutations or only one of the two indicated amino acid substitution mutations) having a capsid that does not contain either of the indicated amino acid substitution mutations or only one of the two indicated amino acid substitution mutations.

[0224] In some embodiments, the modified AAV or VLP is AAV2 or a variant thereof and has increased affinity for AAVR compared to its control AAV2 or VLP derivative, e.g., an AAV2 or VLP derivative having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains only one of the indicated amino acid substitution mutations. For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP.

[0225] How to improve affinity for AAVR In some aspects, the disclosure features a method for improving the affinity of AAV for an AAV receptor (AAVR), the method including providing any of the modified AAVs described above (e.g., contacting a cell with any of the modified AAVs described above, wherein the contacting increases affinity for the AAVR).

[0226] For example, in some aspects, the disclosure features a method for improving the affinity of AAV for an AAV receptor (AAVR), comprising providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0227] For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP.

[0228] D. Engineered AAV capsids containing I451Y, S454I, and P504I mutations In some aspects, the disclosure provides an adeno-associated virus (AAV) or virus-like particle (VLP) comprising: (a) a major capsid protein VP1 comprising one, two, or all three of an I451Y, I451T, or I451M amino acid substitution mutation, a S454I or S454F amino acid substitution mutation, and a P504I, P504T, or P504V amino acid substitution mutation; (b) a major capsid protein VP1 comprising one, two, or all three of an I451Y, I451T, or I451M amino acid substitution mutation, a S454I or S454F amino acid substitution mutation, and a P504I, P504T, or P504V amino acid substitution mutation; and / or (c) a minor capsid protein VP3 (SEQ ID NO: 1) that contains one, two, or all three of an I451Y, I451T, or I451M amino acid substitution mutation, a S454I or S454F amino acid substitution mutation, and a P504I, P504T, or P504V amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0229] In some aspects, the disclosure features an adeno-associated virus (AAV) or virus-like particle (VLP) having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0230] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV or VLP is AAV1, AAV2, or AAV9. Additional serotypes that may be used in the present invention are provided, for example, in section II(F) below. One of skill in the art can identify corresponding residues in the VP1, VP2, and / or VP3 proteins of two or more AAV serotypes (e.g., one can determine which residues of a given serotype correspond to the listed residues of AAV9), for example, by generating a sequence alignment.

[0231] In some aspects, the disclosure features an AAV or VLP having a capsid including: (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0232] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise I451Y, S454I, P504I, and E500P amino acid substitution mutations.

[0233] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation, i.e., VP1, VP2, and / or VP3 comprise I451Y, S454I, P504I, and E500R amino acid substitution mutations.

[0234] In some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0235] In some aspects, the disclosure features an AAV or VLP having a capsid including: (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0236] In some aspects, the disclosure features an AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0237] In some aspects, the disclosure features a method including providing a modified AAV or VLP having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0238] In some embodiments, the modified AAV or VLP has increased affinity for the AAV receptor (AAVR) compared to a control AAV or VLP having a capsid that does not contain any of the indicated amino acid substitution mutations (e.g., an AAV or VLP of the same serotype that does not contain any of the indicated amino acid substitution mutations). In some embodiments, the modified AAV or VLP contains three of the four I451Y, S454I, E500P, and P504I amino acid substitution mutations, or three of the four I451Y, S454I, E500R, and P504I amino acid substitution mutations, the modified AAV or VLP has increased affinity for the AAVR compared to a control AAV or VLP having a capsid that contains only one or only two of the indicated amino acid substitution mutations. In some embodiments, wherein the modified AAV or VLP comprises all four of the I451Y, S454I, E500P, and P504I amino acid substitution mutations, or all four of the four I451Y, S454I, E500R, and P504I amino acid substitution mutations, the modified AAV or VLP has increased affinity for AAVR compared to a control AAV or VLP having a capsid comprising only one, or only two, or only three of the indicated amino acid substitution mutations.

[0239] In some embodiments, the modified AAV or VLP is AAV9 or a variant thereof and has increased affinity for AAVR compared to a control AAV or VLP derivative thereof, e.g., an AAV9 or VLP derivative thereof having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP.

[0240] How to improve affinity for AAVR In some aspects, the disclosure features a method for improving the affinity of AAV for an AAV receptor (AAVR), the method including providing any of the modified AAVs described above (e.g., contacting a cell with any of the modified AAVs described above, wherein the contacting increases affinity for the AAVR).

[0241] For example, in some aspects, the disclosure features a method for improving affinity of AAV for an AAV receptor (AAVR), comprising providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0242] In some aspects, the disclosure features a method for improving affinity of AAV for an AAV receptor (AAVR), comprising providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0243] In some aspects, the disclosure features a method for improving affinity of AAV for an AAV receptor (AAVR), comprising providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), and wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

[0244] For example, in some embodiments, the affinity of the modified AAV or VLP for AAVR is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improved compared to the affinity of the control AAV or VLP.

[0245] Alternative amino acid substitution at position I451 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may comprise an I451Y or I451M amino acid substitution mutation in place of the I451T amino acid substitution mutation.

[0246] Alternative amino acid substitution at position S454 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may comprise a S454F amino acid substitution mutation in place of the S454I amino acid substitution mutation.

[0247] Alternative amino acid substitution at position P504 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may include a P504T or P504V amino acid substitution mutation in place of the P504I amino acid substitution mutation.

[0248] E. Further Capsid Mutations In some aspects, the disclosure provides an adeno-associated virus (AAV) or virus-like particle (VLP) comprising an I451T, I451Y, or I451M amino acid substitution mutation; an S454I or an S454F amino acid substitution mutation; an A472F, A472H, A472K, A472N, A472W, or A472Y amino acid substitution mutation; a P504I, P504T, or P504V amino acid substitution mutation; an E500P or E500R amino acid substitution mutation; an A273N amino acid substitution mutation; a Q387K amino acid substitution mutation; The present invention features an adeno-associated virus (AAV) or virus-like particle (VLP) having a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, each of which comprises one or more of the following amino acid substitution mutations: a S263A or S263Y amino acid substitution mutation, and a W503H amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some embodiments, the AAV or VLP has improved transduction affinity, increased affinity for AAVR, increased delivery rate to the nucleus of target cells, and / or transduces target cells at a lower dose than a control AAV or VLP, e.g., an AAV or VLP that does not comprise any of the indicated amino acid substitution mutations.

[0249] In some embodiments, the AAV or VLP comprises 2, 3, 4, 5, 6, 7, 8 or 9 of the listed amino acid substitution mutations. For example, in some embodiments, the disclosure features an AAV or VLP having a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, wherein the capsid comprises an amino acid substitution mutation of I451T and A472H, I451Y and A472H, S454I and A472H, A472H and P504I, A472H and P504T, A472H and E500R, A273N and A472H, A472H and E500P, Q387K and A472H, A273N and Q387K, A273N and E500R, or Q387K and E500R. In some aspects, the disclosure features an AAV or VLP having a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, wherein the capsid comprises Q387K, A472H, and E500P; A273N, A472H, and E500P; I451Y, S454I, and E500P; or A273N, Q387K, and E500R amino acid substitution mutations. In some aspects, the disclosure features an AAV or VLP having a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, wherein the capsid comprises I451Y, S454I, E500P, and P504I; I451Y, S454I, E500P, and P504T; or A273N, Q387K, A472H, and E500P amino acid substitution mutations.

[0250] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV or VLP is AAV1, AAV2, or AAV9. In some embodiments, the AAV or VLP is AAV9 or a variant thereof. Additional serotypes that can be used in the present invention are provided, for example, in section II(F) below. One of skill in the art can identify corresponding residues in the VP1, VP2, and / or VP3 proteins of two or more AAV serotypes (e.g., one can determine which residues of a given serotype correspond to the listed residues of AAV9), for example, by generating a sequence alignment.

[0251] F. Exemplary AAV Serotypes As used herein, the serotype of an AAV or virus-like particle (VLP) refers to the serotype of the capsid protein of the AAV or VLP.

[0252] The AAV or VLP of the disclosure can be, for example, a naturally occurring AAV serotype (e.g., AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12), an engineered AAV serotype, or any mutant, derivative, or pseudotype thereof. For example, AAVs include self-complementary AAVs (scAAVs), chimeric AAVs, hybrid AAVs, AAVrh74, AAVanc80L65, AAVrh.10, AAVrh.74, AAV2 / 1, AAV2 / 5, AAV2 / 6, AAV2 / 8, AAV2 / 9, AAV2-AAV3 hybrids, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, and AAV6(Y445F / Y73 IF), AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShHIO, AAV2(Y->F), AAV8(Y733F), AAV2.15, AAV2.4, AAVM41, or AAVr3.45. AAV serotypes that can be used in the present invention are described, for example, in Asokan et al., Mol Ther., 20(4):699-708, 2012.

[0253] G. AAV and VLPs In some embodiments, the present disclosure provides adeno-associated viruses (AAVs) having a capsid comprising all three of the major capsid protein VP1, the minor capsid protein VP2, and the minor capsid protein VP3. In some embodiments, all three of VP1, VP2, and VP3 comprise specific amino acid substitution mutations (e.g., all three of VP1, VP2, and VP3 comprise A273N, Q387K, A472H, and / or E500R amino acid substitution mutations, or all three of VP1, VP2, and VP3 comprise T592D and A593E amino acid substitution mutations). In other embodiments, only a subset of VP1, VP2, and VP3 comprise the indicated amino acid substitution mutations (e.g., VP1 only; VP2 only; VP3 only; VP1 and VP2 only; VP1 and VP3 only; or VP2 and VP3 only).

[0254] Provided herein are AAVs having a capsid comprising: (a) a major capsid protein VP1 comprising one or more amino acid substitution mutations ("mutant VP1"); (b) a minor capsid protein VP2 comprising one or more amino acid substitution mutations ("mutant VP2"); and / or (c) a minor capsid protein VP3 comprising one or more amino acid substitution mutations ("mutant VP3") (e.g., the amino acid substitution mutations described in Sections IIA-IIE, above). Thus, the AAVs provided herein may comprise (i) mutant VP1, wild-type VP2, and wild-type VP3; (ii) wild-type VP1, mutant VP2, and wild-type VP3; (iii) wild-type VP1, wild-type VP2, and mutant VP3; (iv) mutant VP1, mutant VP2, and wild-type VP3; (v) mutant VP1, wild-type VP2, and mutant VP3; or (vi) wild-type VP1, mutant VP2, and mutant VP3 (i.e., may be chimeric), or may comprise mutant VP1, mutant VP2, and mutant VP3.

[0255] In some embodiments, where one or more amino acid substitution mutations increase the affinity of an AAV subunit (VP1, VP2, or VP3) for AAVR, a chimeric AAV (e.g., an AAV containing amino acid substitution mutations in only one or two subunits) has lower affinity for AAVR than an AAV containing amino acid substitution mutations in all three subunits. Therefore, those skilled in the art will understand that the affinity of an AAV for its target can be optimized by providing a chimeric AAV. Of note, VP1 and VP2 are present in lower proportions in the AAV capsid than VP3; therefore, in some embodiments, provided herein are chimeric AAVs containing amino acid substitution mutations in only VP1, only VP2, or only VP1 and VP2. In other embodiments, provided herein are chimeric AAVs containing amino acid substitution mutations in only VP3.

[0256] AAVs containing capsids with differences between the VP1, VP2, and / or VP3 sequences can be generated using molecular biology techniques known in the art. For example, VP1, VP2, and / or VP3 can be expressed on separate plasmids or separate open reading frames (ORFs) on the same plasmid.

[0257] In other aspects, the disclosure provides virus-like particles (VLPs) having capsids that include only one or two of VP1, VP2, and VP3, e.g., only VP1; only VP2; only VP3; only VP1 and VP2; only VP1 and VP3; or only VP2 and VP3. In aspects that include two of VP1, VP2, and VP3, one or both of the proteins can include specific amino acid substitution mutations.

[0258] H. Further mutations In some embodiments, the major capsid protein VP1, minor capsid protein VP2, and / or minor capsid protein VP3 of any of the above embodiments comprises one or more additional amino acid substitution mutations and / or comprises one or more amino acid insertions or deletions.

[0259] In some embodiments, one or more additional amino acid substitution mutations, amino acid insertions, or amino acid deletions are substitutions, insertions, or deletions that affect the binding of AAV capsid to one or more attachment receptors.Exemplary mutations that affect (e.g., attenuate) binding to attachment receptors are provided in, for example, Cabanes-Creus et al., Molecular Therapy: Methods and Clinical Development, 17:1139-1154, 2020; Shen et al., Journal of Virology, 86(19):10408-10417, 2012; and Asokan et al., Journal of Virology, 80(18):8961-8969, 2006.

[0260] In some embodiments, the modified AAV or VLP has improved transduction into liver cells (e.g., human liver cells). For example, in some embodiments, one or more of the mutations provided herein are combined with R585A, R585S, R588A, R588T, T503A, or N596D amino acid substitution mutations (numbered relative to AAV2 major capsid protein VP1) (Cabanes-Creus et al., Molecular Therapy: Methods and Clinical Development, 17:1139-1154, 2020). In some embodiments, the modified AAV has reduced affinity for heparin.

[0261] In some embodiments, the modified AAV or VLP has reduced affinity for glycans. For example, in some embodiments, one or more of the mutations provided herein are combined with a W503R amino acid substitution mutation (numbered relative to the AAV9 major capsid protein VP1) (Shen et al., Journal of Virology, 86(19):10408-10417, 2012).

[0262] In some embodiments, the modified AAV or VLP has reduced affinity for integrin α5β1. For example, in some embodiments, one or more of the mutations provided herein are combined with an R513A amino acid substitution mutation (numbered relative to the AAV2 major capsid protein VP1) (Asokan et al., Journal of Virology, 80(18):8961-8969, 2006).

[0263] In some embodiments, the modified AAV or VLP has reduced sialic acid binding. For example, in some embodiments, one or more of the mutations provided herein are combined with an L587T amino acid substitution mutation and / or an M569V amino acid substitution mutation (see Afione et al., Journal of Virology, 89(3), 2015, which is incorporated herein by reference in its entirety).

[0264] In some embodiments, the modified AAV or VLP comprises an N272A amino acid substitution mutation (e.g., as described in Bell et al., Journal of Virology, 86(13), 2012, which is incorporated by reference in its entirety).

[0265] I. Target disease In some embodiments of any of the AAVs, VLPs, and methods provided herein, the AAVs or VLPs are for use in treating a disease or condition in a subject, for example, a human subject.Exemplary diseases and conditions that can be treated using the AAVs and VLPs provided herein include ophthalmological, neurological, or neurodegenerative diseases or conditions and proliferative diseases or conditions (e.g., cancer).For example, in some embodiments, the AAVs or VLPs provided herein are used to treat Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), diseases or conditions associated with the ataxia 2 (ATXN2) gene (e.g., ALS), age-related macular degeneration (AMD) (e.g., dry AMD or wet AMD), cystic fibrosis (CF), diseases or conditions associated with the cystic fibrosis transmembrane conductance regulator (CFTR) gene (e.g., CF), neuronal ceroid lipoprotein (NLP) and the like. In another embodiment, the AAV or VLP is used in the treatment of fuscinosis 2 (CLN2) disease, frontotemporal dementia (FTD), GRN-associated frontotemporal lobar degeneration (FTD-GRN), Friedreich's ataxia, generalized anxiety disorder (GAD), panic disorder (PD), Huntington's disease (HTT), Parkinson's disease (PD), a disease or condition associated with the glucocerebrosidase (GBD) gene (e.g., PD), Rett syndrome, spinal muscular atrophy (SMA), or a disease or condition associated with the tau gene. In another embodiment, the AAV or VLP is used in an in vitro system (e.g., for delivery to an in vitro cell).

[0266] III. Methods of Using Engineered AAV Capsids A. Methods for improving transduction efficiency using engineered AAV capsids Capsid containing the A472H mutation In one aspect, the disclosure features a method of improving the transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), the method including providing a modified AAV or VLP having a capsid including: (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV or VLP, thereby improving the transduction efficiency.

[0267] In another aspect, the disclosure features a method of improving the transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), comprising contacting a target cell with a modified AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides the use of such an AAV or VLP in the manufacture of a medicament for transducing a target cell, and the use of such an AAV or VLP for use in transducing a target cell.

[0268] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for transducing target cells, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0269] In some aspects, the disclosure provides a modified AAV for use in transducing a target cell, having a capsid comprising: (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0270] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9. Additional serotypes that can be used in the present invention are provided, for example, in Section II(F) above.

[0271] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV or VLP, the method including providing a modified AAV or VLP having a capsid including: (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV or VLP, thereby improving the transduction efficiency.

[0272] In another aspect, the disclosure features a method of improving the transduction efficiency of an AAV or VLP, comprising contacting a target cell with a modified AAV or VLP having a capsid comprising (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides the use of such an AAV or VLP in the manufacture of a medicament for transducing a target cell, and the use of such an AAV or VLP for use in transducing a target cell.

[0273] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0274] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0275] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0276] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0277] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and E500R amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and E500R amino acid substitution mutations.

[0278] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, A472H, and E500R amino acid substitution mutations; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV, thereby improving the transduction efficiency.

[0279] In another aspect, the disclosure features a method of improving the transduction efficiency of an AAV, comprising contacting a target cell with a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides the use of such an AAV in the manufacture of a medicament for transducing a target cell, and the use of such an AAV or VLP for use in transducing a target cell.

[0280] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for transducing target cells, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0281] In some aspects, the disclosure provides a modified AAV for use in transducing a target cell, the modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0282] In some embodiments, the AAV is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9.

[0283] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and the AAV is AAV9 or a variant thereof, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method comprises contacting a target cell with the modified AAV, thereby improving the transduction efficiency.

[0284] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, comprising contacting a target cell with a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency. The disclosure also provides the use of such an AAV in the manufacture of a medicament for transducing a target cell, and the use of such an AAV or VLP for use in transducing a target cell.

[0285] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including A273N, Q387K, A472H, and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, A472H, and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, A472H, and E500P amino acid substitution mutations; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV, thereby improving the transduction efficiency.

[0286] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, comprising contacting a target cell with a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides the use of such an AAV in the manufacture of a medicament for transducing a target cell, and the use of such an AAV for use in transducing a target cell.

[0287] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for transducing target cells, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0288] In some aspects, the disclosure provides a modified AAV for use in transducing a target cell, having a capsid comprising: (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0289] In some embodiments of any of the above aspects, the modified AAV or VLP may comprise an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation in place of the A472H amino acid substitution mutation.

[0290] Capsid containing the E500R mutation In one aspect, the disclosure features a method of improving the transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), the method including providing a modified AAV or VLP having a capsid including: (a) a major capsid protein VP1 including an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 including an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an E500R amino acid substitution mutation; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV, thereby improving the transduction efficiency.

[0291] In another aspect, the disclosure features a method of improving the transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), comprising contacting a target cell with a modified AAV or VLP having a capsid including (a) a major capsid protein VP1 including an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 including an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an E500R amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides the use of such an AAV or VLP in the manufacture of a medicament for transducing a target cell, and the use of such an AAV or VLP for use in transducing a target cell.

[0292] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9. Additional serotypes that can be used in the present invention are provided, for example, in Section II(F) above.

[0293] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV or VLP, the method including providing a modified AAV or VLP having a capsid including: (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV or VLP, thereby improving the transduction efficiency.

[0294] In another aspect, the disclosure features a method of improving the transduction efficiency of an AAV or VLP, comprising contacting a target cell with a modified AAV or VLP having a capsid including (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides the use of such an AAV or VLP in the manufacture of a medicament for transducing a target cell, and the use of such an AAV or VLP for use in transducing a target cell.

[0295] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0296] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0297] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0298] In some embodiments, (a) the major capsid protein VP1 further comprises A273N and Q387K amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N and Q387K amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N and Q387K amino acid substitution mutations.

[0299] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and A472H amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and A472H amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and A472H amino acid substitution mutations.

[0300] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, and E500R amino acid substitution mutations; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV, thereby improving the transduction efficiency.

[0301] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, comprising contacting a target cell with a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides the use of such an AAV in the manufacture of a medicament for transducing a target cell, and the use of such an AAV for use in transducing a target cell.

[0302] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for transducing target cells, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0303] In some aspects, the disclosure provides a modified AAV for use in transducing a target cell, having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0304] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0305] In some embodiments, the AAV is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9.

[0306] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, and E500R amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV, thereby improving the transduction efficiency.

[0307] In another aspect, the disclosure features a method for improving the transduction efficiency of AAV, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 including A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including A273N, Q387K, and E500R amino acid substitution mutations; where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0308] Alternative amino acid substitution at position E500 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may comprise an E500P amino acid substitution mutation in place of the E500R amino acid substitution mutation.

[0309] Capsids containing the T592D and A593E mutations In another aspect, the disclosure features a method of improving the transduction efficiency of an AAV or VLP, the method including providing a modified AAV having a capsid including (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations, where the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2). In some aspects, the method includes contacting a target cell with the modified AAV or VLP, thereby improving the transduction efficiency.

[0310] In another aspect, the disclosure features a method of improving the transduction efficiency of an AAV or VLP, comprising contacting a target cell with a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2). The disclosure also provides the use of such an AAV or VLP in the manufacture of a medicament for transducing a target cell, and the use of such an AAV or VLP for use in transducing a target cell.

[0311] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for transducing target cells, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations; the amino acid substitution mutations are numbered relative to the AAV2 major capsid protein VP1 (SEQ ID NO: 2); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0312] In some aspects, the disclosure provides a modified AAV for use in transducing a target cell, having a capsid comprising: (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0313] In some embodiments, the AAV is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9.

[0314] In another aspect, the disclosure features a method for improving the transduction efficiency of an adeno-associated virus (AAV), the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2). In some aspects, the method includes contacting a target cell with the modified AAV, thereby improving the transduction efficiency.

[0315] In another aspect, the disclosure features a method of improving the transduction efficiency of an adeno-associated virus (AAV), comprising contacting a target cell with a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV2 major capsid protein VP1 (SEQ ID NO: 2). The disclosure also provides the use of such an AAV in the manufacture of a medicament for transducing a target cell, and the use of such an AAV for use in transducing a target cell.

[0316] Capsids containing the I451Y, S454I, and P504I mutations In another aspect, the disclosure features a method for improving the transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), the method including providing a modified AAV or VLP having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, and / or P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, and / or P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, and / or P504I amino acid substitution mutations; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV or VLP, thereby improving the transduction efficiency.

[0317] In another aspect, the disclosure features a method for improving the transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), the method including providing a modified AAV or VLP having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, and P504I amino acid substitution mutations; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV or VLP, thereby improving the transduction efficiency.

[0318] In another aspect, the disclosure features a method of improving the transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), comprising contacting a target cell with a modified AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides the use of such an AAV or VLP in the manufacture of a medicament for transducing a target cell, and the use of such an AAV or VLP for use in transducing a target cell.

[0319] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for transducing target cells, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0320] In some aspects, the disclosure provides a modified AAV for use in transducing a target cell, having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0321] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9. Additional serotypes that can be used in the present invention are provided, for example, in Section II(F) above.

[0322] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV or VLP, the method including providing a modified AAV or VLP having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, and P504I amino acid substitution mutations, where the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV or VLP, thereby improving the transduction efficiency.

[0323] In another aspect, the disclosure features a method of improving the transduction efficiency of an AAV or VLP, comprising contacting a target cell with a modified AAV or VLP having a capsid comprising (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides the use of such an AAV or VLP in the manufacture of a medicament for transducing a target cell, and the use of such an AAV or VLP for use in transducing a target cell.

[0324] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0325] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0326] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV, thereby improving the transduction efficiency.

[0327] In another aspect, the disclosure features a method for improving the transduction efficiency of AAV, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500P, and P504I amino acid substitution mutations, where the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0328] In another aspect, the disclosure features a method for improving the transduction efficiency of AAV, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500P, and P504I amino acid substitution mutations, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0329] In another aspect, the disclosure features a method for improving the transduction efficiency of an AAV, the method including providing a modified AAV having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some aspects, the method includes contacting a target cell with the modified AAV, thereby improving the transduction efficiency.

[0330] In another aspect, the disclosure features a method for improving AAV transduction efficiency, the method including contacting a target cell with a modified AAV having a capsid including: (a) a major capsid protein VP1 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500R, and P504I amino acid substitution mutations, where the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency.

[0331] In another aspect, the disclosure features a method for improving the transduction efficiency of AAV, the method including contacting a target cell with a modified AAV having a capsid including (a) a major capsid protein VP1 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 including I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including I451Y, S454I, E500R, and P504I amino acid substitution mutations, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving the transduction efficiency.

[0332] The present disclosure also provides the above-described AAV or VLP in the manufacture of a medicament for transducing target cells, and the use of such AAV or VLP for use in transducing target cells.

[0333] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for transducing target cells, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0334] In some aspects, the disclosure provides a modified AAV for use in transducing a target cell, having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0335] In some aspects, the disclosure provides use of a modified AAV in the manufacture of a medicament for transducing target cells, wherein the modified AAV has a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500R, and P504I amino acid substitution mutations; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0336] In some aspects, the disclosure provides a modified AAV for use in transducing a target cell, having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV or VLP of interest.

[0337] Alternative amino acid substitution at position I451 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may comprise an I451Y or I451M amino acid substitution mutation in place of the I451T amino acid substitution mutation.

[0338] Alternative amino acid substitution at position S454 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may comprise a S454F amino acid substitution mutation in place of the S454I amino acid substitution mutation.

[0339] Alternative amino acid substitution at position P504 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may include a P504T or P504V amino acid substitution mutation in place of the P504I amino acid substitution mutation.

[0340] Further capsid mutations In another aspect, the disclosure provides a method for improving the transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), comprising: a) introducing an amino acid substitution mutation selected from the group consisting of: an I451T, an I451Y, or an I451M amino acid substitution mutation; an S454I or an S454F amino acid substitution mutation; an A472F, an A472H, an A472K, an A472N, an A472W, or an A472Y amino acid substitution mutation; a P504I, a P504T, or a P504V amino acid substitution mutation; an E500P or an E500R amino acid substitution mutation; an A273N amino acid substitution mutation; and / or (c) minor capsid protein VP3, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some embodiments, the method includes contacting a target cell with the modified AAV, thereby improving transduction efficiency.

[0341] In another aspect, the disclosure provides a method for improving the transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), comprising: converting a target cell to one of the following amino acid substitution mutations: I451T, I451Y, or I451M; S454I or S454F; A472F, A472H, A472K, A472N, A472W, or A472Y; P504I, P504T, or P504V; E500P or E500R; A273N; Q387K; and / or (c) minor capsid protein VP3, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides a method for transducing a target cell, comprising contacting a modified AAV or VLP with a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), thereby improving transduction efficiency. The disclosure also provides a method for transducing a target cell, and ...

[0342] Thus, in some aspects, the disclosure provides a method for the manufacture of a medicament for transducing a target cell, the method comprising the step of: (a) administering to a mammalian subject a modified AAV comprising: an I451T, I451Y, or I451M amino acid substitution mutation; an S454I or S454F amino acid substitution mutation; an A472F, A472H, A472K, A472N, A472W, or A472Y amino acid substitution mutation; a P504I, P504T, or P504V amino acid substitution mutation; an E500P or E500R amino acid substitution mutation; an A273N amino acid substitution mutation; a Q387K amino acid substitution mutation; an S263A or S263Y amino acid substitution mutation; and a W503 and / or (c) minor capsid protein VP3, each of which contains one or more of the H amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1), and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest.

[0343] In some aspects, the disclosure provides a modified AAV for use in transducing a target cell, the modified AAV comprising an I451T, I451Y, or I451M amino acid substitution mutation; an S454I or S454F amino acid substitution mutation; an A472F, A472H, A472K, A472N, A472W, or A472Y amino acid substitution mutation; a P504I, P504T, or P504V amino acid substitution mutation; an E500P or E500R amino acid substitution mutation; an A273N amino acid substitution mutation; a Q387K amino acid substitution mutation; an S263A or S263Y amino acid substitution mutation; and a W503H amino acid substitution mutation. (b) minor capsid protein VP2; and / or (c) minor capsid protein VP3, each of which contains one or more of the following amino acid substitution mutations: (a) major capsid protein VP1; (b) minor capsid protein VP2; and / or (c) minor capsid protein VP3; wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest.

[0344] In some embodiments, the AAV or VLP comprises 2, 3, 4, 5, 6, 7, 8, or 9 of the listed amino acid substitution mutations. For example, in some embodiments, the AAV or VLP has a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, each of which comprises the following amino acid substitution mutations: I451T and A472H, I451Y and A472H, S454I and A472H, A472H and P504I, A472H and P504T, A472H and E500R, A273N and A472H, A472H and E500P, Q387K and A472H, A273N and Q387K, A273N and E500R, or Q387K and E500R. In some embodiments, the AAV or VLP has a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, each of which contains the following amino acid substitution mutations: Q387K, A472H, and E500P; A273N, A472H, and E500P; I451Y, S454I, and E500P; or A273N, Q387K, and E500R. In some embodiments, the AAV or VLP has a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, each of which contains the following amino acid substitution mutations: I451Y, S454I, E500P, and P504I; I451Y, S454I, E500P, and P504T; or A273N, Q387K, A472H, and E500P.

[0345] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9. Additional serotypes that can be used in the present invention are provided, for example, in Section II(F) above.

[0346] Improved transduction efficiency In some embodiments, the transduction efficiency of the modified AAV or VLP is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, 1 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or greater than 200% improvement, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% improvement.

[0347] The improvement in transduction efficiency can be in one or more target cells. Exemplary target cells include, but are not limited to, endothelial cells (e.g., brain endothelial cells), cancer cells, central nervous system (CNS) cells, eye cells, retinal cells, muscle cells, stem cells, cardiac cells, lung cells, skin cells, kidney cells, and liver cells. In some embodiments, the target cells are in vitro cells. In other embodiments, the target cells are cells in an organism (e.g., a mammal, e.g., a human).

[0348] In some embodiments, the modified AAV or VLP has increased transduction efficiency compared to a control AAV or VLP (e.g., an AAV or VLP of the same serotype that does not contain any of the indicated amino acid substitution mutations or that contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest) that (a) does not contain any of the indicated amino acid substitution mutations, or (b) has a capsid that contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest.

[0349] In some embodiments, the modified AAV or VLP has increased transduction efficiency compared to an AAV or VLP derivative thereof having a capsid that does not contain any of the A273N, Q387K, A472H, and E500R amino acid substitution mutations. In some embodiments, the modified AAV or VLP contains two of the four A273N, Q387K, A472H, and E500R amino acid substitution mutations (e.g., A273N and Q387K; A273N and A472H; A273N and E500R; Q387K and A472H; Q387K and E500R; or A472H and E500R amino acid substitution mutations), the modified AAV or VLP has increased transduction efficiency compared to a control AAV or VLP having a capsid that contains only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises A273N and A472H amino acid substitution mutations; comprises A273N and E500R amino acid substitution mutations; comprises Q387K and A472H amino acid substitution mutations; comprises Q387K and E500R amino acid substitution mutations; or comprises A472H and E500R amino acid substitution mutations, and the modified AAV or VLP has increased transduction efficiency compared to a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises three of the four A273N, Q387K, A472H, and E500R amino acid substitution mutations (e.g., A273N, Q387K, and A472H; A273N, Q387K, and E500R; A273N, A472H, and E500R; or Q387K, A472H, and E500R amino acid substitution mutations), and the modified AAV or VLP has increased transduction efficiency compared to a control AAV or VLP having a capsid containing only one or only two of the indicated amino acid substitution mutations.In some embodiments, the modified AAV or VLP comprises A273N, Q387K, and A472H amino acid substitution mutations; A273N, Q387K, and E500R amino acid substitution mutations; A273N, A472H, and E500R amino acid substitution mutations; or Q387K, A472H, and E500R amino acid substitution mutations, and the modified AAV or VLP has increased transduction efficiency compared to a control AAV or VLP having a capsid containing only one or only two of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises all four of the A273N, Q387K, A472H, and E500R amino acid substitution mutations, the modified AAV or VLP has increased transduction efficiency compared to a control AAV or VLP having a capsid containing only one, two, or three of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP is AAV9 or a variant thereof.

[0350] In some embodiments, the modified AAV or VLP is AAV2 or a variant thereof, and has increased transduction efficiency compared to AAV2 or a VLP derivative thereof having a capsid that (a) does not contain either the T592D or A593E amino acid substitution mutation, or (b) contains only one of the T592D and A593E amino acid substitution mutations.

[0351] For example, in some embodiments, transduction efficiency is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% compared to the affinity of a control AAV or VLP.

[0352] In some embodiments, the modified AAV or VLP has an increased rate of delivery to the nucleus of a target cell compared to a control AAV or VLP having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest (e.g., an AAV or VLP of the same serotype that does not contain any of the indicated amino acid substitution mutations or contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest).

[0353] In some embodiments, the modified AAV or VLP has an increased rate of delivery to the nucleus of a target cell compared to its AAV or VLP derivative having a capsid that does not contain any of the A273N, Q387K, A472H, and E500R substitution mutations. In some embodiments, the modified AAV or VLP contains two of the four A273N, Q387K, A472H, and E500R amino acid substitution mutations (e.g., A273N and Q387K; A273N and A472H; A273N and E500R; Q387K and A472H; Q387K and E500R; or A472H and E500R amino acid substitution mutations), the modified AAV or VLP has an increased rate of delivery to the nucleus compared to a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises A273N and A472H amino acid substitution mutations; comprises A273N and E500R amino acid substitution mutations; comprises Q387K and A472H amino acid substitution mutations; comprises Q387K and E500R amino acid substitution mutations; or comprises A472H and E500R amino acid substitution mutations, and the modified AAV or VLP has an increased rate of delivery to the nucleus compared to a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises three of the four A273N, Q387K, A472H, and E500R amino acid substitution mutations (e.g., A273N, Q387K, and A472H; A273N, Q387K, and E500R; A273N, A472H, and E500R; or Q387K, A472H, and E500R amino acid substitution mutations), the modified AAV or VLP has an increased rate of delivery to the nucleus compared to a control AAV or VLP having a capsid containing only one or only two of the indicated amino acid substitution mutations.In some embodiments, the modified AAV or VLP comprises A273N, Q387K, and A472H amino acid substitution mutations; A273N, Q387K, and E500R amino acid substitution mutations; A273N, A472H, and E500R amino acid substitution mutations; or Q387K, A472H, and E500R amino acid substitution mutations, and the modified AAV or VLP has an increased rate of nuclear delivery compared to a control AAV or VLP having a capsid containing only one or two of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises all four of the A273N, Q387K, A472H, and E500R amino acid substitution mutations, the modified AAV or VLP has an increased rate of nuclear delivery compared to a control AAV or VLP having a capsid containing only one, two, or three of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP is AAV9 or a variant thereof.

[0354] In some embodiments, the modified AAV or VLP is AAV2 or a variant thereof, and has an increased rate of delivery to the nucleus of a target cell compared to AAV2 or a VLP derivative thereof having a capsid that (a) does not contain either the T592D or A593E amino acid substitution mutation, or (b) contains only one of the T592D and A593E amino acid substitution mutations.

[0355] For example, in some embodiments, the rate of nuclear delivery is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, or more than 200%, e.g., 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 100% to 150%, or 150% to 200% compared to the rate of delivery of a control AAV or VLP.

[0356] In some aspects, the present disclosure provides a method for increasing the rate of delivery of an AAV to the nucleus of a target cell, comprising: (i) (a) a major capsid protein VP1 containing an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an A472H amino acid substitution mutation; (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) (a) a major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 containing A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations; providing a modified AAV having a capsid comprising The method includes contacting a target cell with a modified AAV, thereby increasing the rate of nuclear delivery. In some aspects, the present disclosure provides a method for increasing the rate of delivery of an AAV to the nucleus of a target cell, comprising: (i) (a) a major capsid protein VP1 containing an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 containing an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an E500R amino acid substitution mutation; or (ii) (a) a major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 containing A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations. providing a modified AAV having a capsid comprising: The method includes contacting a target cell with the modified AAV, thereby increasing the rate of nuclear delivery.

[0357] In some aspects, the disclosure features a method for increasing the rate of AAV delivery to the nucleus of a target cell, the method comprising: providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2). In some aspects, the method comprises contacting the target cell with the modified AAV, thereby increasing the rate of delivery to the nucleus.

[0358] In some aspects, the present disclosure provides a method for increasing the rate of delivery of an AAV to the nucleus of a target cell, comprising: (i) (a) a major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 containing I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) a major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (iii) (a) a major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations. providing a modified AAV having a capsid comprising:

[0359] The method includes contacting a target cell with the modified AAV, thereby increasing the rate of nuclear delivery.

[0360] In some embodiments, the modified AAV transduces target cells at a lower dose than a control AAV or VLP (e.g., an AAV or VLP of the same serotype that does not contain any of the indicated amino acid substitution mutations or that contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest) that (a) does not contain any of the indicated amino acid substitution mutations, or (b) has a capsid that contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest, e.g., transduces target cells at a rate sufficient to achieve a desired effect, e.g., a therapeutic effect, at a lower dose.

[0361] In some embodiments, the modified AAV or VLP transduces target cells at a lower dose than its AAV or VLP derivative having a capsid that does not contain any of the A273N, Q387K, A472H, and E500R substitution mutations. In some embodiments, the modified AAV or VLP contains two of the four A273N, Q387K, A472H, and E500R amino acid substitution mutations (e.g., A273N and Q387K; A273N and A472H; A273N and E500R; Q387K and A472H; Q387K and E500R; or A472H and E500R amino acid substitution mutations), the modified AAV or VLP transduces target cells at a lower dose than a control AAV or VLP having a capsid that contains only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises A273N and A472H amino acid substitution mutations; comprises A273N and E500R amino acid substitution mutations; comprises Q387K and A472H amino acid substitution mutations; comprises Q387K and E500R amino acid substitution mutations; or comprises A472H and E500R amino acid substitution mutations, and the modified AAV or VLP transduces target cells at a lower dose than a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, wherein the modified AAV or VLP comprises three of the four A273N, Q387K, A472H, and E500R amino acid substitution mutations (e.g., A273N, Q387K, and A472H; A273N, Q387K, and E500R; A273N, A472H, and E500R; or Q387K, A472H, and E500R amino acid substitution mutations), the modified AAV or VLP transduces target cells at a lower dose than a control AAV or VLP having a capsid containing only one or only two of the indicated amino acid substitution mutations.In some embodiments, the modified AAV or VLP comprises A273N, Q387K, and A472H amino acid substitution mutations; A273N, Q387K, and E500R amino acid substitution mutations; A273N, A472H, and E500R amino acid substitution mutations; or Q387K, A472H, and E500R amino acid substitution mutations, and the modified AAV or VLP transduces target cells at a lower dose than a control AAV or VLP having a capsid containing only one or only two of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises all four of the A273N, Q387K, A472H, and E500R amino acid substitution mutations, and the modified AAV or VLP transduces target cells at a lower dose than a control AAV or VLP having a capsid containing only one, two, or three of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP is AAV9 or a variant thereof.

[0362] In some embodiments, the modified AAV or VLP is AAV2 or a variant thereof that transduces target cells at a lower dose than AAV2 or a VLP derivative thereof having a capsid that (a) does not contain either the T592D or A593E amino acid substitution mutation, or (b) contains only one of the T592D and A593E amino acid substitution mutations.

[0363] For example, in some embodiments, the modified AAV or VLP is at least 1.1-fold less, 1.2-fold less, 1.3-fold less, 1.4-fold less, 1.5-fold less, 1.6-fold less, 1.7-fold less, 1.8-fold less, 1.9-fold less, 2-fold less, 2.5-fold less, 3-fold less, 3.5-fold less, 4-fold less, 4.5-fold less, 5-fold less, 5.5-fold less, 6-fold less than the dose of the control AAV or VLP. , 6.5-fold less, 7-fold less, 7.5-fold less, 8-fold less, 8.5-fold less, 9-fold less, 9.5-fold less, 10-fold less, or more than 10-fold less, e.g., 1.1-1.5-fold less, 1.5-2-fold less, 2-3-fold less, 3-4-fold less, 4-5-fold less, 5-6-fold less, 6-7-fold less, 7-8-fold less, 8-9-fold less, or 9-10-fold less. Thus, in some embodiments, the modified AAV or VLP has a lower effective dose than a control AAV or VLP that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest.

[0364] In some aspects, the present disclosure provides a method for transducing target cells with a low dose of AAV, comprising: (i) (a) a major capsid protein VP1 containing an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an A472H amino acid substitution mutation; (ii) (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) providing a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations; wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some embodiments, the method comprises contacting a target cell with the modified AAV, thereby transducing the target cell at a lower dose.

[0365] In some aspects, the present disclosure provides a method for transducing target cells with a low dose of AAV, comprising: (i) (a) a major capsid protein VP1 containing an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 containing an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an E500R amino acid substitution mutation; or (ii) providing a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some embodiments, the method comprises contacting a target cell with the modified AAV, thereby transducing the target cell at a lower dose.

[0366] In some aspects, the disclosure features a method for transducing target cells with a low dose of AAV, the method including providing a modified AAV having a capsid including (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2). In some aspects, the method includes contacting the target cells with the modified AAV, thereby transducing the target cells at a lower dose.

[0367] In some aspects, the present disclosure provides a method for transducing target cells with a low dose of AAV, comprising: (i) (a) a major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 containing I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) a major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (iii) providing a modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1). In some embodiments, the method comprises contacting a target cell with the modified AAV, thereby transducing the target cell at a lower dose.

[0368] B. Methods of Delivering Cargo into Cells Capsid containing the A472H mutation In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV or virus-like particle (VLP) having a capsid including (a) a major capsid protein VP1 including an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 including an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an A472H amino acid substitution mutation, wherein the cargo is encapsulated by the AAV or VLP; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV or VLP has an increased transduction rate of the target cell compared to an AAV or VLP having a capsid that (a) does not include any of the indicated amino acid substitution mutations, or (b) includes fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. The present disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0369] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid comprising fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0370] In some aspects, the disclosure provides a modified AAV for use in delivering cargo to a target cell, the modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an A472H amino acid substitution mutation; the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0371] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9. Additional serotypes that can be used in the present invention are provided, for example, in Section II(F) above.

[0372] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting the target cell with a modified AAV or VLP having a capsid including (a) a major capsid protein VP1 including an A472H amino acid substitution mutation; (b) a minor capsid protein VP2 including an A472H amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an A472H amino acid substitution mutation, wherein the cargo is encapsulated by the AAV or VLP; and the AAV or VLP is AAV9 or a variant thereof, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and wherein the AAV or VLP exhibits an increased transduction rate of the target cell compared to an AAV9 or VLP having a capsid that either (a) does not include any of the indicated amino acid substitution mutations, or (b) includes fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. The present disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0373] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0374] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0375] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0376] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0377] In some embodiments, (a) the major capsid protein VP1 further comprises A273N and Q387K amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N and Q387K amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N and Q387K amino acid substitution mutations.

[0378] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and E500R amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and E500R amino acid substitution mutations.

[0379] In some aspects, the disclosure features a method of delivering cargo to a cell, the method comprising contacting a target cell with a modified AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, and A472H amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and A472H amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and A472H amino acid substitution mutations, wherein the cargo is encapsulated by the AAV or VLP, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV or VLP has an increased transduction rate of the target cell compared to an AAV or VLP having a capsid that does not comprise any of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises two of the three A273N, Q387K, and A472H amino acid substitution mutations (e.g., A273N and Q387K; A273N and A472H; or Q387K and A472H amino acid substitution mutations), the modified AAV or VLP has an increased target cell transduction rate compared to a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises A273N and A472H amino acid substitution mutations, or Q387K and A472H amino acid substitution mutations, and the modified AAV or VLP has an increased target cell transduction rate compared to a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP contains all three of the A273N, Q387K, and A472H amino acid substitution mutations, and the modified AAV or VLP has an increased transduction rate into target cells compared to a control AAV or VLP having a capsid containing only one or only two of the indicated amino acid substitution mutations.

[0380] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

[0381] The present disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0382] Thus, in some aspects, the disclosure provides a method for the production of a medicament for delivering cargo to a target cell, the method comprising the steps of: (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations. The present invention provides a modified AAV having a capsid comprising a minor capsid protein VP3 containing substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0383] In some aspects, the disclosure provides a modified AAV for use in delivering cargo to a target cell, the modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0384] In another aspect, the disclosure features a method for delivering a cargo to a cell, comprising contacting a target cell with a modified AAV having a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations, wherein the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV has an increased transduction rate into the target cell compared to an AAV9 having a capsid that does not comprise any of the indicated amino acid substitution mutations. The disclosure also provides such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and the use of such an AAV or VLP for use in delivering a cargo to a target cell.

[0385] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0386] In some aspects, the disclosure provides a modified AAV for use in delivering cargo to a target cell, the modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A472H and E500P amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A472H and E500P amino acid substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0387] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9.

[0388] In some embodiments, the cargo of the AAV or VLP is a nucleic acid, such as a single-stranded RNA (ssRNA). In some embodiments, the cargo is a therapeutic nucleic acid. In some embodiments, the cargo is a nucleic acid useful for gene therapy. In some embodiments, the cargo of the AAV or VLP is an antisense oligonucleotide or siRNA. In some embodiments, the inverted terminal repeat (ITR) sequence of the AAV genome is retained for packaging, and the viral gene is replaced with a sequence containing or encoding the cargo.

[0389] In some embodiments, the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or one or more amino acid insertions or deletions, e.g., any of the features described in Section II(H) above.

[0390] The cells may be, for example, endothelial cells (e.g., brain endothelial cells), cancer cells, central nervous system (CNS) cells, eye cells, retinal cells, muscle cells, stem cells, cardiac cells, lung cells, skin cells, kidney cells, and liver cells. In some embodiments, the target cells are in vitro cells. In other embodiments, the target cells are cells in an organism (e.g., a mammal, e.g., a human).

[0391] In some embodiments of any of the above aspects, the modified AAV or VLP may comprise an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation in place of the A472H amino acid substitution mutation.

[0392] Capsid containing the E500R mutation In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting a target cell with a modified AAV or virus-like particle (VLP) having a capsid including (a) a major capsid protein VP1 including an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 including an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 including an E500R amino acid substitution mutation, wherein the cargo is encapsulated by the AAV or VLP; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV or VLP has an increased transduction rate of the target cell compared to an AAV or VLP having a capsid that either (a) does not include any of the indicated amino acid substitution mutations, or (b) includes fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. The present disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0393] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid comprising fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0394] In some aspects, the disclosure provides a modified AAV for use in delivering cargo to a target cell, the modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation; the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0395] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9. Additional serotypes that can be used in the present invention are provided, for example, in Section II(F) above.

[0396] In another aspect, the disclosure features a method of delivering cargo to a cell, the method comprising contacting a target cell with a modified AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising an E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 comprising an E500R amino acid substitution mutation, wherein the cargo is encapsulated by the AAV or VLP; and the AAV or VLP is AAV9 or a variant thereof, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and wherein the AAV or VLP exhibits an increased transduction rate of the target cell compared to an AAV9 or VLP having a capsid that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. The present disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0397] In some embodiments, (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

[0398] In some embodiments, (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

[0399] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0400] In some embodiments, (a) the major capsid protein VP1 further comprises A273N and Q387K amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N and Q387K amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N and Q387K amino acid substitution mutations.

[0401] In some embodiments, (a) the major capsid protein VP1 further comprises A273N, Q387K, and A472H amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and A472H amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and A472H amino acid substitution mutations.

[0402] In some aspects, the disclosure features a method of delivering cargo to a cell, the method comprising contacting a target cell with a modified AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations, wherein the cargo is encapsulated by the AAV or VLP, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV or VLP has an increased transduction rate of the target cell compared to an AAV or VLP having a capsid that does not comprise any of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises two of the three A273N, Q387K, and E500R amino acid substitution mutations (e.g., A273N and Q387K; A273N and E500R; or Q387K and E500R amino acid substitution mutations), the modified AAV or VLP has an increased transduction rate into target cells compared to a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises A273N and E500R amino acid substitution mutations, or comprises Q387K and E500R amino acid substitution mutations, and the modified AAV or VLP has an increased transduction rate into target cells compared to a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP contains all three of the A273N, Q387K, and E500R amino acid substitution mutations, and the modified AAV or VLP exhibits increased target cell transduction rates compared to a control AAV or VLP having a capsid containing only one or two of the indicated amino acid substitution mutations. The present disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0403] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0404] In some aspects, the disclosure provides a modified AAV for use in delivering cargo to a target cell, the modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, A472H, and E500R amino acid substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0405] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0406] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9.

[0407] In some aspects, the disclosure features a method of delivering cargo to a cell, the method comprising contacting a target cell with a modified AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising A273N, Q387K, and E500R amino acid substitution mutations, wherein the cargo is encapsulated by the AAV or VLP, and the AAV or VLP is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV or VLP has an increased transduction rate of the target cell compared to an AAV9 or VLP having a capsid that does not comprise any of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises two of the three A273N, Q387K, and E500R amino acid substitution mutations (e.g., A273N and Q387K; A273N and E500R; or Q387K and E500R amino acid substitution mutations), the modified AAV or VLP has an increased transduction rate into target cells compared to a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises A273N and E500R amino acid substitution mutations, or comprises Q387K and E500R amino acid substitution mutations, and the modified AAV or VLP has an increased transduction rate into target cells compared to a control AAV or VLP having a capsid containing only one of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP contains all three of the A273N, Q387K, and E500R amino acid substitution mutations, and the modified AAV or VLP has an increased transduction rate into target cells compared to a control AAV or VLP having a capsid containing only one or only two of the indicated amino acid substitution mutations.The present disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0408] In some embodiments, (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

[0409] In some embodiments, the cargo of the AAV or VLP is a nucleic acid, such as a single-stranded RNA (ssRNA). In some embodiments, the cargo is a therapeutic nucleic acid. In some embodiments, the cargo is a nucleic acid useful for gene therapy. In some embodiments, the cargo of the AAV or VLP is an antisense oligonucleotide or siRNA. In some embodiments, the inverted terminal repeat (ITR) sequence of the AAV genome is retained for packaging, and the viral gene is replaced with a sequence containing or encoding the cargo.

[0410] In some embodiments, the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or one or more amino acid insertions or deletions, e.g., any of the features described in Section II(H) above.

[0411] The cells may be, for example, endothelial cells (e.g., brain endothelial cells), cancer cells, central nervous system (CNS) cells, eye cells, retinal cells, muscle cells, stem cells, cardiac cells, lung cells, skin cells, kidney cells, and liver cells. In some embodiments, the target cells are in vitro cells. In other embodiments, the target cells are cells in an organism (e.g., a mammal, e.g., a human).

[0412] In some embodiments of any of the above aspects, the modified AAV or VLP may comprise an A472F, A472K, A472N, A472W, or A472Y amino acid substitution mutation in place of the A472H amino acid substitution mutation.

[0413] Capsids containing the T592D and A593E mutations In another aspect, the disclosure features a method of delivering cargo to a cell, the method comprising contacting a target cell with a modified AAV or VLP having a capsid including (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations, wherein the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2); and the AAV has an increased transduction rate into the target cell compared to an AAV2 that (a) does not include any of the indicated amino acid substitution mutations, or (b) has a capsid including only one of the T592D and A593E amino acid substitution mutations.

[0414] The present disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0415] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, wherein the modified AAV has a capsid comprising (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0416] In some aspects, the disclosure provides a modified AAV for use in delivering cargo to a target cell, the modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising T592D and A593E amino acid substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) has a capsid that comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0417] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9.

[0418] In another aspect, the disclosure features a method of delivering cargo to a cell, the method including contacting the target cell with a modified AAV or VLP having a capsid including (a) a major capsid protein VP1 including T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 including T592D and A593E amino acid substitution mutations; and / or (c) a minor capsid protein VP3 including T592D and A593E amino acid substitution mutations, wherein the cargo is encapsulated by the AAV or VLP; the AAV or VLP is AAV2 or a variant thereof, wherein the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2); and the AAV or VLP has an increased transduction rate of the target cell compared to AAV2 that (a) does not include any of the indicated amino acid substitution mutations, or (b) has a capsid including only one of the T592D and A593E amino acid substitution mutations. The present disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0419] In some embodiments, the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or one or more amino acid insertions or deletions, e.g., any of the features described in Section II(H) above.

[0420] The cells may be, for example, endothelial cells (e.g., brain endothelial cells), cancer cells, central nervous system (CNS) cells, eye cells, retinal cells, muscle cells, stem cells, cardiac cells, lung cells, skin cells, kidney cells, and liver cells. In some embodiments, the target cells are in vitro cells. In other embodiments, the target cells are cells in an organism (e.g., a mammal, e.g., a human).

[0421] Capsids containing the I451Y, S454I, and P504I mutations In another aspect, the disclosure provides a method for delivering cargo to a cell, comprising: treating a target cell with a modified AAV or virus having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, and / or P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and / or P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and / or P504I amino acid substitution mutations. The method includes contacting an AAV or VLP with a virus-like particle (VLP), wherein the cargo is encapsulated by the AAV or VLP; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV or VLP has an increased transduction rate into target cells compared to an AAV or VLP having a capsid that either (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest.

[0422] In another aspect, the disclosure provides a method for delivering cargo to a cell, comprising: treating a target cell with a modified AAV or virus-like particle (V) having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations. VP1 (SEQ ID NO: 1), wherein the cargo is encapsulated by the AAV or VLP; the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV or VLP has an increased target cell transduction rate compared to an AAV or VLP having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. The disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering cargo to a target cell, and for the use of such an AAV or VLP for use in delivering cargo to a target cell.

[0423] Thus, in some aspects, the disclosure provides the use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, wherein the modified AAV has a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0424] In some aspects, the disclosure provides a modified AAV for use in delivering cargo to a target cell, the modified AAV having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the specified amino acid substitution mutations than the modified AAV of interest.

[0425] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9. Additional serotypes that can be used in the present invention are provided, for example, in Section II(F) above.

[0426] In another aspect, the disclosure provides a method for delivering cargo to a cell, comprising contacting a target cell with a modified AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, and P504I amino acid substitution mutations, wherein the modified AAV or VLP delivers cargo to a cell. is encapsulated by an AAV or VLP; the AAV or VLP is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), wherein the AAV or VLP has an increased transduction rate into a target cell compared to an AAV9 or VLP having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. The disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering a cargo to a target cell, and for use in delivering a cargo to a target cell.

[0427] In some embodiments, (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

[0428] In some embodiments, (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation. In some aspects, the disclosure features a method of delivering cargo to a cell, the method comprising contacting a target cell with a modified AAV or VLP having a capsid comprising: (a) a major capsid protein VP1 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 comprising I451Y, S454I, E500P, and P504I amino acid substitution mutations, wherein the cargo is encapsulated by the AAV or VLP, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); and the AAV or VLP has an increased transduction rate of the target cell compared to an AAV or VLP having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0429] In some embodiments, the modified AAV or VLP comprises three of the four I451Y, S454I, E500P, and P504I amino acid substitution mutations, or three of the four I451Y, S454I, E500R, and P504I amino acid substitution mutations, and the modified AAV or VLP has an increased transduction rate into target cells compared to a control AAV or VLP having a capsid containing only one or two of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises the I451Y, S454I, and P504I amino acid substitution mutations, and the modified AAV or VLP has an increased transduction rate into target cells compared to a control AAV or VLP having a capsid containing only one or two of the indicated amino acid substitution mutations. In some embodiments, the modified AAV or VLP comprises all four of the I451Y, S454I, E500P, and P504I amino acid substitution mutations, or all four of the I451Y, S454I, E500R, and P504I amino acid substitution mutations, the modified AAV or VLP has an increased transduction rate into target cells compared to a control AAV or VLP having a capsid containing only one, only two, or only three of the indicated amino acid substitution mutations.

[0430] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9. In some embodiments, the AAV or VLP is AAV9 or a variant thereof.

[0431] In some embodiments, the cargo of the AAV or VLP is a nucleic acid, such as a single-stranded RNA (ssRNA). In some embodiments, the cargo is a therapeutic nucleic acid. In some embodiments, the cargo is a nucleic acid useful for gene therapy. In some embodiments, the cargo of the AAV or VLP is an antisense oligonucleotide or siRNA. In some embodiments, the inverted terminal repeat (ITR) sequence of the AAV genome is retained for packaging, and the viral gene is replaced with a sequence containing or encoding the cargo.

[0432] In some embodiments, the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or one or more amino acid insertions or deletions, e.g., any of the features described in Section II(H) above.

[0433] The cells may be, for example, endothelial cells (e.g., brain endothelial cells), cancer cells, central nervous system (CNS) cells, eye cells, retinal cells, muscle cells, stem cells, cardiac cells, lung cells, skin cells, kidney cells, and liver cells. In some embodiments, the target cells are in vitro cells. In other embodiments, the target cells are cells in an organism (e.g., a mammal, e.g., a human).

[0434] Alternative amino acid substitution at position I451 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may comprise an I451Y or I451M amino acid substitution mutation in place of the I451T amino acid substitution mutation.

[0435] Alternative amino acid substitution at position S454 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may comprise a S454F amino acid substitution mutation in place of the S454I amino acid substitution mutation.

[0436] Alternative amino acid substitution at position P504 In some embodiments of any of the aspects provided herein, the modified AAV or VLP may include a P504T or P504V amino acid substitution mutation in place of the P504I amino acid substitution mutation.

[0437] Further capsid mutations In another aspect, the disclosure provides a method of delivering cargo to a cell, comprising: treating a target cell with (a) a major capsid protein VP1; (b) a minor capsid protein VP2; (c) a minor capsid protein VP3; (d) a minor capsid protein VP4; (e) a minor capsid protein VP5; (f) a minor capsid protein VP6; (g) a minor capsid protein VP7; (h) a minor capsid protein VP8; (i) a major capsid protein VP9; (j) a minor capsid protein VP10; (j) a minor capsid protein VP11; (j) a minor capsid protein VP12; (j) a minor capsid protein VP13; (j) a minor capsid protein VP14; (j) a minor capsid protein VP15; (j) a minor capsid protein VP16; (j) a minor capsid protein VP17; (j) a minor capsid protein VP18; (j) a minor capsid protein VP19 ...9; (j) a minor capsid protein VP11; (j) a minor capsid protein VP12; (j) a minor capsid protein VP13; (j) a minor capsid protein VP14; (j) a minor capsid protein VP15; (j) a minor capsid protein VP16; (j) a minor caps and / or (c) minor capsid protein VP3, wherein the cargo is encapsulated by the AAV or VLP, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and the AAV or VLP has an increased target cell transduction rate compared to an AAV or VLP having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest. The disclosure also provides for the use of such an AAV or VLP in the manufacture of a medicament for delivering cargo to a target cell, and for the use of such an AAV or VLP for use in delivering cargo to a target cell.

[0438] Thus, in some aspects, the disclosure provides a method for the manufacture of a medicament for delivering cargo to a target cell, the method comprising the step of: modifying an AAV to one of the following amino acid substitution mutations: I451T, I451Y, or I451M; S454I or S454F; A472F, A472H, A472K, A472N, A472W, or A472Y; P504I, P504T, or P504V; E500P or E500R; A273N; Q387K; S263A or S263Y; and W503H. (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, wherein the cargo is encapsulated by the AAV, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not contain any of the indicated amino acid substitution mutations, or (b) contains fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest.

[0439] In some aspects, the disclosure provides a modified AAV for use in delivering cargo to a target cell, the modified AAV comprising one or more of: an I451T, an I451Y, or an I451M amino acid substitution mutation; an S454I or an S454F amino acid substitution mutation; an A472F, A472H, A472K, A472N, A472W, or A472Y amino acid substitution mutation; a P504I, P504T, or P504V amino acid substitution mutation; an E500P or an E500R amino acid substitution mutation; an A273N amino acid substitution mutation; a Q387K amino acid substitution mutation; an S263A or S263Y amino acid substitution mutation; and a W503H amino acid substitution mutation ( Provided is a modified AAV having a capsid comprising: (a) major capsid protein VP1; (b) minor capsid protein VP2; and / or (c) minor capsid protein VP3, wherein a cargo is encapsulated by the AAV, and wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1), and wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that (a) does not comprise any of the indicated amino acid substitution mutations, or (b) comprises fewer of the indicated amino acid substitution mutations than the modified AAV or VLP of interest.

[0440] In some embodiments, the AAV or VLP comprises 2, 3, 4, 5, 6, or 7, 8 or 9 of the listed amino acid substitution mutations. For example, in some embodiments, the AAV or VLP has a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3 that contain the following amino acid substitution mutations: I451T and A472H, I451Y and A472H, S454I and A472H, A472H and P504I, A472H and P504T, A472H and E500R, A273N and A472H, A472H and E500P, Q387K and A472H, A273N and Q387K, A273N and E500R, or Q387K and E500R. In some embodiments, the AAV or VLP has a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, each of which contains the following amino acid substitution mutations: Q387K, A472H, and E500P; A273N, A472H, and E500P; I451Y, S454I, and E500P; or A273N, Q387K, and E500R. In some embodiments, the AAV or VLP has a capsid comprising (a) a major capsid protein VP1; (b) a minor capsid protein VP2; and / or (c) a minor capsid protein VP3, each of which contains the following amino acid substitution mutations: I451Y, S454I, E500P, and P504I; I451Y, S454I, E500P, and P504T; or A273N, Q387K, A472H, and E500P.

[0441] In some embodiments, the AAV or VLP is of a serotype that interacts with the AAV receptor (AAVR) (e.g., AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12). In some embodiments, the AAV is AAV1, AAV2, or AAV9.

[0442] In some embodiments, the cargo of the AAV or VLP is a nucleic acid, such as a single-stranded RNA (ssRNA). In some embodiments, the cargo is a therapeutic nucleic acid. In some embodiments, the cargo is a nucleic acid useful for gene therapy. In some embodiments, the cargo of the AAV or VLP is an antisense oligonucleotide or siRNA. In some embodiments, the inverted terminal repeat (ITR) sequence of the AAV genome is retained for packaging, and the viral gene is replaced with a sequence containing or encoding the cargo.

[0443] In some embodiments, the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or one or more amino acid insertions or deletions, e.g., any of the features described in Section II(H) above.

[0444] The cells may be, for example, endothelial cells (e.g., brain endothelial cells), cancer cells, central nervous system (CNS) cells, eye cells, retinal cells, muscle cells, stem cells, cardiac cells, lung cells, skin cells, kidney cells, and liver cells. In some embodiments, the target cells are in vitro cells. In other embodiments, the target cells are cells in an organism (e.g., a mammal, e.g., a human).

[0445] D. Delivery method The modified AAV and VLP provided herein can be administered to target cells directly, by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subconjunctivally, intravesically, mucosally, intrapericardially, subumbilically, intraocularly, intraorbitally, orally, transdermally, intravitreally (e.g., by intravitreal injection), by eye drops, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by local perfusion bath, by catheter, by lavage solution, in cream, or in lipid composition. The modified AAV described herein can also be administered systemically or locally. The administration method can vary depending on various factors (e.g., the compound or composition to be administered and the severity of the symptoms, disease, or disorder to be treated). Administration can be by any suitable route, for example, injection, for example, intravenous injection or subcutaneous injection, depending in part on whether administration is brief or chronic.Contemplated herein are various administration schedules, including but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusion.In some embodiments, the modified AAV and / or VLP provided herein is administered to cells in an in vitro system.

[0446] The modified AAV or VLP described herein (and any additional therapeutic agents) may be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to consider in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the administration schedule, and other factors known to medical professionals. The modified AAV or VLP need not, but may, be formulated and / or administered simultaneously with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents will depend on the amount of modified AAV present in the formulation, the type of disorder or treatment, and other factors discussed above. These will generally be used in the same dosages and via any route of administration as described herein, or at dosages between about 1 and 99% of the dosages described herein, or at any dosage and via any route of administration determined empirically / clinically appropriate.

[0447] IV. Methods for Determining AAV Capsid Avidity In another aspect, the disclosure features a method for determining the affinity of an adeno-associated virus (AAV) capsid for a query protein, the method including: (a) providing a virus-like particle (VLP) having a capsid consisting essentially of (e.g., consisting of) the VP3 subunit of the AAV capsid (or a mutant thereof) (e.g., having a capsid that does not contain VP1 or VP2); (b) contacting the VLP of step (a) with the query protein under conditions that allow binding of the query protein to the VLP; and (c) quantitating the strength of the interaction between the query protein and the VLP, thereby determining the affinity of the AAV capsid for the query protein.

[0448] In some embodiments, the VP3 subunit of the AAV capsid in the VLP is a mutant VP3 subunit, e.g., a VP3 subunit that contains one or more amino acid insertion, deletion, and / or substitution mutations relative to the sequence of the wild-type VP3 subunit of the AAV capsid (e.g., relative to the wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 VP3 sequence). For example, in some cases, the VP3 subunit contains one or more amino acid substitution mutations relative to the sequence of the wild-type VP3 subunit of the AAV capsid. In some embodiments, the method includes comparing the affinity of a VLP containing the mutant VP3 subunit for a query protein with the affinity of a VLP containing a wild-type VP3 subunit, thereby determining whether the mutations (e.g., amino acid insertion, deletion, and / or substitution mutations) affect (e.g., increase or decrease) the affinity of the AAV capsid for the query protein.

[0449] The AAV can be of any serotype. In some embodiments, the AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12. Additional serotypes that can be used in the present invention are provided, for example, in Section II(F) above.

[0450] Exemplary amino acid substitution mutations that can be evaluated using this method include, but are not limited to, A273N, Q387K, A472H, or E500R amino acid substitution mutations numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1) (e.g., A273N, Q387K, A472H, and / or E500R mutations in the VP3 subunit of AAV9) and T592D or A593E amino acid substitution mutations numbered relative to the AAV2 major capsid protein VP1 (SEQ ID NO: 2) (e.g., T592D and / or A593E mutations in the VP3 subunit of AAV2).

[0451] In some embodiments, the VLP is immobilized on a solid surface (e.g., a sensor chip) and contacted with a query protein, e.g., in a soluble format. In some embodiments, the solid surface comprises an antibody (e.g., a single domain antibody) or antigen-binding fragment thereof (e.g., a Fab, Fab', Fab'-SH, Fv, single-chain variable fragment (scFv), or (Fab')2 fragment) having affinity for the VLP. In some embodiments, the antibody or antigen-binding fragment thereof binds to the VP3 subunit of the AAV capsid.

[0452] In some embodiments, VLPs are produced by cotransfecting host cells (e.g., mammalian cells, e.g., HEK293 cells) with a first plasmid containing only the wild-type VP3 subunit of the AAV capsid and a second plasmid encoding the wild-type assembly-activating protein (AAP) of AAV. In some embodiments, the VP3 subunit and AAP are derived from the same AAV serotype. In some embodiments, the plasmids are transfected at a molar ratio of 5:1 (VP3 plasmid:AAP plasmid). After an appropriate growth period (e.g., 7 days), VLPs can be harvested from the host cells (e.g., from conditioned medium and / or from lysates of pelleted host cells).

[0453] In some embodiments, the VLPs are not purified. For example, in some embodiments, the VLPs are produced in a cell line, and immobilizing the VLPs on the solid surface comprises contacting the solid surface with culture medium comprising the VLPs. In some embodiments, the culture medium comprises a lysate of the cell line in which the VLPs are produced (e.g., cells of the cell line in which the VLPs are produced are lysed, and the VLPs are not separated from the cell lysate prior to immobilizing the VLPs on the solid surface).

[0454] Quantification of the strength of the interaction between the query protein and the VLP can be performed using any suitable assay for evaluating protein-protein interactions in vitro. In some embodiments, the quantification is performed using biolayer interferometry (BLI) and / or surface plasmon resonance (SPR). In some embodiments, an initial BLI assay is performed, and an SPR assay is used to confirm or verify the results of the BLI assay and / or to quantify binding kinetics.

[0455] VI. Working Examples Example 1. Engineering AAV capsids for improved transduction A. Background Adeno-associated viruses (AAVs) are nonpathogenic single-stranded DNA (ssDNA) viruses that can be used as vectors for gene therapy, for example. AAVs have a nonenveloped icosahedral capsid composed of the major capsid protein VP1, the minor capsid protein VP2, and the minor capsid protein VP3, which are encoded by overlapping genes.

[0456] The key factor in the use of AAV is to mitigate their immunogenicity in subjects.The factors that affect the immunogenicity of AAV include the existing humoral immunity of subjects to AAV (the majority of the human population has already been exposed to at least one AAV serotype) and innate and adaptive immune response.These immune responses have been shown to be correlated with viral genome dosage.

[0457] The experiments provided herein are based on the hypothesis that improving the affinity of the AAV capsid for the AAV receptor (AAVR) can increase AAV transduction efficiency. Increasing transduction affinity could reduce dose requirements and potentially mitigate immunogenicity.

[0458] AAVR is critical for cellular transduction by most AAV serotypes. AAVR rapidly traffics between the plasma membrane and the trans-Golgi network and acts upstream of or at the stage of AAV nuclear import.

[0459] Although AAVR is required for AAV transduction, AAV attachment is independent of AAVR (Dudek et al., JVI, 92(7):e02213-17, 2018; Dudek et al., Mol Ther., 28(2):367-381, 2020). Instead, AAV attachment to host cells occurs through binding of AAV to a primary receptor, which is serotype-dependent but often associated with glycans and heparan sulfate proteoglycans (HSPGs).

[0460] B. Workflow for Screening AAV Virus-Like Particle (VLP) Mutants We developed a method for screening the affinity of AAV capsid mutants for target receptors using unpurified virus-like particles (VLPs) rather than complete AAV virions (Figure 1; image generated with BioRenderer). The VLPs used in this method are composed of only VP3. This method preserves the capsid and associated exterior of the capsid assembly; however, VLPs are easier and safer to express and screen than full virions. Sufficient amounts of VLPs for the experiments described herein can be harvested from less than 1 mL of conditioned medium. This minimal volume requirement lends itself to high-throughput screening.

[0461] Mutant VP3 proteins (e.g., VP3 proteins containing one or more amino acid substitution mutations of interest) were generated by introducing the desired mutations into a template plasmid containing only the wild-type VP3 subunit of the AAV capsid (e.g., AAV2 or AAV9). The resulting mutant plasmid was then co-transfected into mammalian cells (HEK293) at a 5:1 molar ratio (VP3:AAP) with a separate plasmid encoding the wild-type assembly activating protein (AAP) of AAV (e.g., AAV2 or AAV9 AAP). Alternatively, co-transfection can be performed at a molar ratio of 1:1 VP3:AAP to 20:1 VP3:AAP. After 7 days of growth, cells and conditioned medium were harvested. Either the conditioned medium or cell pellet lysate can be used as a source of VLPs.

[0462] Transfected cell supernatants are screened, and an initial ranking of VP3 mutants based on their affinity for AAVR is determined using biolayer interferometry (BLI). Because domains "PKD1" and "PKD2" (residues V305 to V499, numbering begins from the initiating methionine of the complete protein (SEQ ID NO: 3)) are the only domains known to interact with the AAV capsid, binding studies are performed using fragments of AAVR containing these domains. The AAVR binding kinetics of promising mut...

Claims

1. 1. A method for improving the transduction efficiency of an adeno-associated virus (AAV), comprising: (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) Minor capsid protein VP3 containing the A472H amino acid substitution mutation providing a modified AAV having a capsid comprising: The method, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

2. (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or 2. The method of claim 1, wherein (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

3. (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or 3. The method of claim 1 or 2, wherein (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

4. (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

5. (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

6. (a) the major capsid protein VP1 further comprises A273N, Q387K, and E500R amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and E500R amino acid substitution mutations; and / or 2. The method of claim 1, wherein (c) the minor capsid protein VP3 further comprises A273N, Q387K, and E500R amino acid substitution mutations.

7. 1. A method for improving AAV transduction efficiency, comprising: (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

8. 1. A method for improving AAV transduction efficiency, comprising: (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations providing a modified AAV having a capsid comprising: The method, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

9. The method of any one of claims 1 to 8, comprising contacting a target cell with said modified AAV, thereby improving transduction efficiency.

10. The method of any one of claims 1 to 9, wherein the transduction efficiency is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

11. A method for delivering cargo to a cell, comprising: (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) Minor capsid protein VP3 containing the A472H amino acid substitution mutation contacting the modified AAV with a capsid comprising: the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The method, wherein the AAV has an increased transduction rate into target cells compared to an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

12. (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or 12. The method of claim 11, wherein (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

13. (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or 13. The method of claim 11 or 12, wherein (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

14. (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

15. (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

16. (a) the major capsid protein VP1 further comprises A273N, Q387K, and E500R amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and E500R amino acid substitution mutations; and / or 12. The method of claim 11, wherein (c) the minor capsid protein VP3 further comprises A273N, Q387K, and E500R amino acid substitution mutations.

17. A method for delivering cargo to a cell, comprising: (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; contacting the modified AAV with a capsid comprising: the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The method, wherein the AAV has an increased transduction rate into target cells compared to an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

18. A method for delivering cargo to a cell, comprising: (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations contacting the modified AAV with a capsid comprising: the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The method, wherein the AAV has an increased transduction rate into target cells compared to an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

19. 19. The method of any one of claims 1 to 18, wherein the modified AAV has increased affinity for the AAV receptor (AAVR) compared to an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

20. The method of any one of claims 9 to 19, wherein the modified AAV has an increased rate of delivery to the nucleus of the target cell compared to an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

21. 21. The method of any one of claims 9 to 20, wherein the modified AAV transduces the target cell at a lower dose than an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

22. 1. A method for improving the affinity of AAV for an AAV receptor (AAVR), comprising: (i) (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the A472H amino acid substitution mutation; (ii) (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations providing a modified AAV having a capsid comprising: The method, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

23. 1. A method for increasing the rate of delivery of AAV to the nucleus of a target cell, comprising: (i) (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the A472H amino acid substitution mutation; (ii) (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations providing a modified AAV having a capsid comprising: The method, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

24. 1. A method for transducing target cells with low doses of AAV, comprising: (i) (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the A472H amino acid substitution mutation; (ii) (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations providing a modified AAV having a capsid comprising: The method, wherein the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

25. The method of any one of claims 22 to 24, comprising contacting a target cell with the modified AAV.

26. 26. The method of any one of claims 9 to 21 and 23 to 25, wherein the target cell is an endothelial cell, a cancer cell, a central nervous system (CNS) cell, an eye cell, a retinal cell, a muscle cell, a stem cell, a heart cell, a lung cell, a skin cell, a kidney cell or a liver cell.

27. 27. The method of claim 26, wherein the endothelial cells are brain endothelial cells.

28. 28. The method of any one of claims 1 to 27, wherein the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or comprise one or more amino acid insertions or deletions.

29. 29. The method of any one of claims 1 to 28, wherein the serotype of the modified AAV is AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12.

30. The method of any one of claims 1 to 29, wherein the serotype of the modified AAV is AAV9 or a mutant thereof.

31. 1. Use of a modified AAV in the manufacture of a medicament for transducing a target cell, the modified AAV comprising: (i) (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the A472H amino acid substitution mutation; (ii) (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations having a capsid comprising: the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The use, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

32. 1. Use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, the modified AAV comprising: (i) (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the A472H amino acid substitution mutation; (ii) (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations having a capsid comprising: the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The use, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

33. 1. A modified AAV for use in transducing a target cell, comprising: (i) (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the A472H amino acid substitution mutation; (ii) (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations having a capsid comprising: the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); A modified AAV, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

34. 1. A modified AAV for use in delivering cargo to a target cell, comprising: (i) (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the A472H amino acid substitution mutation; (ii) (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the A273N, Q387K, A472H, and E500R amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations having a capsid comprising: the cargo is encapsulated by the AAV; the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); A modified AAV, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

35. (a) Major capsid protein VP1 containing the A472H amino acid substitution mutation; (b) a minor capsid protein VP2 containing an A472H amino acid substitution mutation; and / or (c) Minor capsid protein VP3 containing the A472H amino acid substitution mutation An AAV having a capsid comprising: The amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

36. (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

37. (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

38. (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the AAV according to any one of claims 35 to 37, wherein the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

39. (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the AAV according to any one of claims 35 to 37, wherein the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

40. (a) the major capsid protein VP1 further comprises A273N, Q387K, and E500R amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and E500R amino acid substitution mutations.

41. (a) Major capsid protein VP1 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, A472H, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, A472H, and E500R amino acid substitution mutations; An AAV having a capsid comprising: The amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

42. (a) Major capsid protein VP1 containing A472H and E500P amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A472H and E500P amino acid substitution mutations; and / or (c) Minor capsid protein VP3 containing A472H and E500P amino acid substitution mutations An AAV having a capsid comprising: The amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

43. The AAV according to any one of claims 35 to 42, wherein the AAV serotype is AAV1, AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12.

44. The AAV according to any one of claims 35 to 42, wherein the serotype of the modified AAV is AAV9 or a mutant thereof.

45. 1. A method for improving the transduction efficiency of an adeno-associated virus (AAV), comprising: (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the E500R amino acid substitution mutation providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

46. (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or 46. ​​The method of claim 45, wherein (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

47. (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or 47. The method of claim 45 or 46, wherein (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

48. (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

49. (a) the major capsid protein VP1 further comprises A273N, Q387K, and A472H amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and A472H amino acid substitution mutations; and / or 46. ​​The method of claim 45, wherein (c) the minor capsid protein VP3 further comprises A273N, Q387K and A472H amino acid substitution mutations.

50. 1. A method for improving AAV transduction efficiency, comprising: (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

51. (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or 51. The method of claim 50, wherein (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

52. 52. The method of any one of claims 45 to 51, comprising contacting a target cell with said modified AAV, thereby improving transduction efficiency.

53. 53. The method of any one of claims 45 to 52, wherein the transduction efficiency is improved compared to the transduction efficiency of an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

54. A method for delivering cargo to a cell, comprising: (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the E500R amino acid substitution mutation contacting the modified AAV with a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The method, wherein the AAV has an increased transduction rate into target cells compared to an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

55. (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or 55. The method of claim 54, wherein (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

56. (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or 56. The method of claim 54 or 55, wherein (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

57. (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or 57. The method of any one of claims 54 to 56, wherein (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

58. (a) the major capsid protein VP1 further comprises A273N, Q387K, and A472H amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and A472H amino acid substitution mutations; and / or 55. The method of claim 54, wherein (c) the minor capsid protein VP3 further comprises A273N, Q387K and A472H amino acid substitution mutations.

59. A method for delivering cargo to a cell, comprising: (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; contacting the modified AAV with a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The method, wherein the AAV has an increased transduction rate into target cells compared to an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

60. (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or 60. The method of claim 59, wherein (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

61. 61. The method of any one of claims 45 to 60, wherein the modified AAV has increased affinity for the AAV receptor (AAVR) compared to AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

62. 61. The method of any one of claims 52 to 60, wherein the modified AAV has an increased rate of delivery to the nucleus of the target cell compared to AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

63. 63. The method of any one of claims 52 to 62, wherein the modified AAV transduces the target cells at a lower dose than AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

64. 1. A method for improving the affinity of AAV for an AAV receptor (AAVR), comprising: (i) (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an E500R amino acid substitution mutation; or (ii) (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

65. 1. A method for increasing the rate of delivery of AAV to the nucleus of a target cell, comprising: (i) (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an E500R amino acid substitution mutation; or (ii) (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

66. 1. A method for transducing target cells with low doses of AAV, comprising: (i) (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an E500R amino acid substitution mutation; or (ii) (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

67. 67. The method of any one of claims 64 to 66, comprising contacting a target cell with the modified AAV.

68. 68. The method of any one of claims 52-63 and 65-67, wherein the target cell is an endothelial cell, a cancer cell, a central nervous system (CNS) cell, an eye cell, a retinal cell, a muscle cell, a stem cell, a heart cell, a lung cell, a skin cell, a kidney cell or a liver cell.

69. 69. The method of claim 68, wherein the endothelial cells are brain endothelial cells.

70. 70. The method of any one of claims 45 to 69, wherein the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or comprise one or more amino acid insertions or deletions.

71. 1. Use of a modified AAV in the manufacture of a medicament for transducing a target cell, the modified AAV comprising: (i) (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an E500R amino acid substitution mutation; or (ii) (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; having a capsid comprising: the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The use, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

72. 1. Use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, the modified AAV comprising: (i) (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an E500R amino acid substitution mutation; or (ii) (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; having a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The use, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

73. 1. A modified AAV for use in transducing a target cell, comprising: (i) (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an E500R amino acid substitution mutation; or (ii) (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; having a capsid comprising: the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); A modified AAV, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

74. 1. A modified AAV for use in delivering cargo to a target cell, comprising: (i) (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) a minor capsid protein VP3 containing an E500R amino acid substitution mutation; or (ii) (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; having a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); A modified AAV, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

75. (a) Major capsid protein VP1 containing the E500R amino acid substitution mutation; (b) a minor capsid protein VP2 comprising an E500R amino acid substitution mutation; and / or (c) minor capsid protein VP3 containing the E500R amino acid substitution mutation An AAV having a capsid comprising: The AAV, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

76. (a) the major capsid protein VP1 further comprises an A273N amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A273N amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A273N amino acid substitution mutation.

77. (a) the major capsid protein VP1 further comprises a Q387K amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises a Q387K amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises a Q387K amino acid substitution mutation.

78. (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

79. (a) the major capsid protein VP1 further comprises A273N, Q387K, and A472H amino acid substitution mutations; (b) the minor capsid protein VP2 further comprises A273N, Q387K, and A472H amino acid substitution mutations; and / or (c) the minor capsid protein VP3 further comprises A273N, Q387K, and A472H amino acid substitution mutations.

80. (a) Major capsid protein VP1 containing A273N, Q387K, and E500R amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the A273N, Q387K, and E500R amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing A273N, Q387K, and E500R amino acid substitution mutations; An AAV having a capsid comprising: An AAV which is AAV9 or a variant thereof, wherein the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

81. (a) the major capsid protein VP1 further comprises an A472H amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an A472H amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an A472H amino acid substitution mutation.

82. 1. A method for improving the transduction efficiency of an adeno-associated virus (AAV), comprising: (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

83. 83. The method of claim 82, comprising contacting a target cell with the modified AAV, thereby improving transduction efficiency.

84. 84. The method of claim 82 or 83, wherein the transduction efficiency is improved compared to the transduction efficiency of an AAV2 having a capsid that does not contain any of the indicated amino acid substitution mutations.

85. A method for delivering cargo to a cell, comprising: (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; contacting the modified AAV with a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2); The method, wherein the AAV has an increased transduction rate into target cells compared to an AAV2 having a capsid that does not contain any of the indicated amino acid substitution mutations.

86. 86. The method of any one of claims 82-85, wherein the modified AAV has increased affinity for the AAV receptor (AAVR) compared to an AAV2 having a capsid that does not contain any of the indicated amino acid substitution mutations.

87. 87. The method of any one of claims 83 to 86, wherein the modified AAV has an increased rate of delivery to the nucleus of the target cell compared to an AAV2 having a capsid that does not contain any of the indicated amino acid substitution mutations.

88. 88. The method of any one of claims 83 to 87, wherein the modified AAV transduces the target cell at a lower dose than an AAV2 having a capsid that does not contain any of the indicated amino acid substitution mutations.

89. 1. A method for improving the affinity of AAV for an AAV receptor (AAVR), comprising: (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

90. 1. A method for increasing the rate of delivery of AAV to the nucleus of a target cell, comprising: (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

91. 1. A method for transducing target cells with low doses of AAV, comprising: (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

92. 92. The method of any one of claims 89 to 91, comprising contacting a target cell with said modified AAV.

93. 93. The method of any one of claims 83-88 and 90-92, wherein the target cell is an endothelial cell, a cancer cell, a CNS cell, an eye cell, a retinal cell, a muscle cell, a stem cell, a cardiac cell, a lung cell, a skin cell, a kidney cell, or a liver cell.

94. 94. The method of claim 93, wherein the endothelial cells are brain endothelial cells.

95. 95. The method of any one of claims 82 to 94, wherein the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or comprise one or more amino acid insertions or deletions.

96. 1. Use of a modified AAV in the manufacture of a medicament for transducing a target cell, the modified AAV comprising: (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV2 major capsid protein VP1 (SEQ ID NO: 2), and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

97. 1. Use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, the modified AAV comprising: (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; having a capsid comprising: The cargo is encapsulated by AAV; the AAV is AAV2 or a variant thereof, the amino acid substitution mutations are numbered relative to the AAV2 major capsid protein VP1 (SEQ ID NO: 2), and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

98. 1. A modified AAV for use in transducing a target cell, comprising: (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; having a capsid comprising: A modified AAV, wherein the AAV is AAV2 or a variant thereof, the amino acid substitution mutations are numbered relative to the AAV2 major capsid protein VP1 (SEQ ID NO: 2), and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

99. 1. A modified AAV for use in delivering cargo to a target cell, comprising: (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; having a capsid comprising: A modified AAV, wherein the AAV is AAV2 or a variant thereof, the amino acid substitution mutations are numbered relative to the AAV2 major capsid protein VP1 (SEQ ID NO: 2), and the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

100. (a) Major capsid protein VP1 containing T592D and A593E amino acid substitution mutations; (b) a minor capsid protein VP2 comprising T592D and A593E amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing T592D and A593E amino acid substitution mutations; An AAV having a capsid comprising: The AAV, wherein the AAV is AAV2 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV2 major capsid protein VP1 (SEQ ID NO: 2).

101. 1. A method for improving the transduction efficiency of an adeno-associated virus (AAV), comprising: (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

102. (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

103. (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

104. 1. A method for improving AAV transduction efficiency, comprising: (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

105. 1. A method for improving AAV transduction efficiency, comprising: (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

106. 106. The method of any one of claims 101 to 105, comprising contacting a target cell with said modified AAV, thereby improving transduction efficiency.

107. 107. The method of any one of claims 101 to 106, wherein the transduction efficiency is improved compared to the transduction efficiency of an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

108. A method for delivering cargo to a cell, comprising: (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; contacting the modified AAV with a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The method, wherein the AAV has an increased transduction rate into target cells compared to an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

109. (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation.

110. (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation.

111. A method for delivering cargo to a cell, comprising: (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; contacting the modified AAV with a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The method, wherein the AAV has an increased transduction rate into target cells compared to an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

112. A method for delivering cargo to a cell, comprising: (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; contacting the modified AAV with a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The method, wherein the AAV has an increased transduction rate into target cells compared to an AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

113. The method of any one of claims 101 to 112, wherein the modified AAV has increased affinity for the AAV receptor (AAVR) compared to AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

114. The method of any one of claims 106 to 113, wherein the modified AAV has an increased rate of delivery to the nucleus of the target cell compared to AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

115. The method of any one of claims 106 to 114, wherein the modified AAV transduces the target cells at a lower dose than AAV9 having a capsid that does not contain any of the indicated amino acid substitution mutations.

116. 1. A method for improving the affinity of AAV for an AAV receptor (AAVR), comprising: (i) (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

117. 1. A method for increasing the rate of delivery of AAV to the nucleus of a target cell, comprising: (i) (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

118. 1. A method for transducing target cells with low doses of AAV, comprising: (i) (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; providing a modified AAV having a capsid comprising: The method, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1).

119. 119. The method of any one of claims 116 to 118, comprising contacting a target cell with said modified AAV.

120. 120. The method of any one of claims 106-115 and 117-119, wherein the target cell is an endothelial cell, a cancer cell, a central nervous system (CNS) cell, an eye cell, a retinal cell, a muscle cell, a stem cell, a heart cell, a lung cell, a skin cell, a kidney cell or a liver cell.

121. 121. The method of claim 120, wherein the endothelial cells are brain endothelial cells.

122. 122. The method of any one of claims 101 to 121, wherein the major capsid protein VP1, the minor capsid protein VP2, and / or the minor capsid protein VP3 comprise one or more additional amino acid substitution mutations and / or comprise one or more amino acid insertions or deletions.

123. 1. Use of a modified AAV in the manufacture of a medicament for transducing a target cell, the modified AAV comprising: (i) (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; having a capsid comprising: the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The use, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

124. 1. Use of a modified AAV in the manufacture of a medicament for delivering cargo to a target cell, the modified AAV comprising: (i) (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; having a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); The use, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

125. 1. A modified AAV for use in transducing a target cell, comprising: (i) (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; having a capsid comprising: the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); A modified AAV, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

126. 1. A modified AAV for use in delivering cargo to a target cell, comprising: (i) (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; (ii) (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) a minor capsid protein VP3 containing the I451Y, S454I, E500P, and P504I amino acid substitution mutations; or (iii) (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; having a capsid comprising: the cargo is encapsulated by the AAV; the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to AAV9 major capsid protein VP1 (SEQ ID NO: 1); A modified AAV, wherein the transduction affinity of the modified AAV is improved compared to the transduction efficiency of an AAV having a capsid that does not contain any of the indicated amino acid substitution mutations.

127. (a) Major capsid protein VP1 containing I451Y, S454I, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, and P504I amino acid substitution mutations; An AAV having a capsid comprising: The AAV, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

128. (a) the major capsid protein VP1 further comprises an E500P amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500P amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500P amino acid substitution mutation. The AAV of claim 127.

129. (a) the major capsid protein VP1 further comprises an E500R amino acid substitution mutation; (b) the minor capsid protein VP2 further comprises an E500R amino acid substitution mutation; and / or (c) the minor capsid protein VP3 further comprises an E500R amino acid substitution mutation. The AAV of claim 128.

130. (a) Major capsid protein VP1 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500P, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500P, and P504I amino acid substitution mutations; An AAV having a capsid comprising: The AAV, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

131. (a) Major capsid protein VP1 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; (b) a minor capsid protein VP2 comprising the I451Y, S454I, E500R, and P504I amino acid substitution mutations; and / or (c) minor capsid protein VP3 containing I451Y, S454I, E500R, and P504I amino acid substitution mutations; An AAV having a capsid comprising: The AAV, wherein the AAV is AAV9 or a variant thereof, and the amino acid substitution mutations are numbered relative to the AAV9 major capsid protein VP1 (SEQ ID NO: 1).

132. 1. A method for determining the affinity of an AAV capsid for a query protein, comprising: (a) providing a virus-like particle (VLP) having a capsid consisting essentially of the VP3 subunit of the AAV capsid; (b) contacting the VLP of step (a) with the query protein under conditions that allow binding of the query protein to the VLP; (c) quantifying the strength of the interaction between the query protein and the VLP, thereby determining the affinity of the AAV capsid for the query protein.

133. 133. The method of claim 132, wherein the VLP is immobilized on a solid surface.

134. 134. The method of claim 133, wherein the solid surface comprises an antibody or antigen-binding fragment thereof having affinity for the VLP.

135. 135. The method of claim 134, wherein the antibody is a single domain antibody.

136. 135. The method of claim 134, wherein the antigen-binding fragment is a Fab.

137. 137. The method of any one of claims 134 to 136, wherein the antibody or antigen-binding fragment thereof binds to the VP3 subunit of the AAV capsid.

138. 138. The method of any one of claims 132 to 137, wherein the VLPs are produced in a cell line and immobilizing the VLPs on the solid surface comprises contacting the solid surface with a culture medium containing the VLPs.

139. 139. The method of claim 138, wherein the culture medium comprises a lysate of the cell line in which the VLP is produced.

140. 140. The method of any one of claims 132 to 139, wherein said quantifying is performed using biolayer interferometry (BLI) and / or surface plasmon resonance (SPR).

141. 141. The method of any one of claims 132-140, wherein the VP3 subunit of the AAV capsid comprises one or more amino acid substitution mutations relative to the sequence of a wild-type VP3 subunit of the AAV capsid.

142. The method of any one of claims 132 to 141, wherein the AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12.

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