Engineered adeno-associated virus capsids

Abstract

Provided herein are engineered adeno-associated virus (AAV) capsids having increased phospholipase activity and methods of using the same.

Description

[0001] ENGINEERED ADENO-ASSOCIATED VIRUS CAPSIDS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Patent Application No. 63 / 644,770, filed on May 9, 2024, the entire contents of which are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 24, 2025, is named 50474-357WO2_Sequence_Listing_4_24_25 and is 8,028 bytes in size.

[0006] FIELD OF THE INVENTION

[0007] Provided herein are engineered adeno-associated virus (AAV) capsids having increased phospholipase activity and methods of using the same.

[0008] BACKGROUND

[0009] Adeno-associated viruses (AAVs) are non-pathogenic single-stranded DNA (ssDNA) viruses that may be used, e.g., as vectors for gene therapy. AAVs have a non-enveloped icosahedral capsid composed of major capsid protein VP1 , minor capsid protein VP2, and minor capsid protein VP3, which are encoded by overlapping genes. The major capsid protein VP1 comprises a VP1 -unique region (VP1 u) at its N-terminus. The VP1 u region comprises a phospholipase A2 (PLA2) domain that is essential for infection. PLA2 enzymes catalyze the hydrolysis of membrane phospholipids from the sn-2 position. The VP1 u PLA2 domain is believed to facilitate endosomal escape of AAVs, which is an essential step for transduction of target cells. It is thus hypothesized that AAVs comprising VP1 u sequences with increased PLA2 domain catalytic activity would have improved endosomal escape and greater transduction efficiency.

[0010] Therefore, the development of AAV capsids having improved PLA2 domain catalytic activity represents an important unmet need.

[0011] SUMMARY OF THE INVENTION

[0012] In one aspect, the disclosure features an adeno-associated virus (AAV) having a capsid comprising a major capsid protein VP1 comprising a VP1 -unique region (VP1 u) comprising one or more of the amino acid substitution mutations provided in Table 1 , wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0013] In some aspects, the VP1 u comprises one or more of (a) a S42A amino acid substitution mutation; (b) a D80Q amino acid substitution mutation; (c) a Q82A amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; and (f) a Q101 V amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In some aspects, the VP1 u comprises only one amino acid substitution mutation selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0014] In some aspects, the VP1 u further comprises an A98E amino acid substitution mutation.

[0015] In some aspects, the VP1 u comprises two amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, A98E, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In some aspects, the VP1 u comprises S42A and Q101 V amino acid substitution mutations. In some aspects, the VP1 u comprises A98E and Q101 V amino acid substitution mutations. In some aspects, the VP1 u comprises D80Q and D87K amino acid substitution mutations.

[0016] In some aspects, the VP1 u comprises three, four, or five amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, A98E, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0017] In some aspects, the VP1 u comprises six amino acid substitution mutations selected from S42A, Q82A, D80Q, D84R, D87K, A98E, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In some aspects, the VP1 u comprises S42A, D80Q, Q82A, D87K, A98E, and Q101 V amino acid substitution mutations. In some aspects, the VP1 u comprises S42A, Q82A, D84R, D87K, A98E, and Q101 V amino acid substitution mutations.

[0018] In some aspects, the VP1 u further comprises an engineered disulfide bond. In some aspects, the VP1 u comprises (a) L53C and G1 1 1 C amino acid substitution mutations; or (b) L53C and A72C amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0019] In another aspect, the disclosure features an AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising (a) L53C and G1 1 1 C amino acid substitution mutations; or (b) L53C and A72C amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In some aspects, the VP1 u further comprises one or more of (a) a S42A amino acid substitution mutation; (b) a D80Q amino acid substitution mutation; (c) a Q82A amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; (f) a Q101 V amino acid substitution mutation, and (g) an A98E amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0020] In some aspects, the capsid comprises (a) a major capsid protein VP1 comprising an amino acid substitution mutation that increases the affinity of the AAV for AAVR; (b) a minor capsid protein VP2 comprising an amino acid substitution mutation that increases the affinity of the AAV for AAVR; and / or (c) a minor capsid protein VP3 comprising an amino acid substitution mutation that increases the affinity of the AAV for AAVR.

[0021] In some aspects, the capsid comprises a major capsid protein VP1 , minor capsid protein VP2, and / or minor capsid protein VP3 comprising one or more further amino acid substitution mutations and / or comprising one or more amino acid insertions or deletions. In some aspects, the serotype of the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , or AAV12. In some aspects, the serotype of the AAV is AAV2 or a variant thereof.

[0022] In some aspects, the AAV has an increased rate of phospholipase activity relative to an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0023] In some aspects, the AAV has an increased rate of endosomal escape relative to an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0024] In some aspects, the AAV has an increased rate of delivery to the nucleus of a target cell relative to an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0025] In some aspects, the AAV transduces a target cell at a lower dose than an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

[0026] In another aspect, the disclosure features a method of improving transduction efficiency of an AAV, the method comprising providing the AAV of any one of the above aspects. In some aspects, the method comprises contacting a target cell with the AAV, thereby improving transduction efficiency. In some aspects, the transduction efficiency is improved relative to the transduction efficiency of an AAV having a capsid not comprising any of the indicated amino acid substitution mutations.

[0027] In another aspect, the disclosure features a method of delivering a cargo to a cell, the method comprising contacting a target cell with the AAV of any one of the above aspects. In some aspects, the cargo is a nucleic acid.

[0028] In another aspect, the disclosure features a method of increasing the rate of delivery of an AAV to the nucleus of a target cell, the method comprising providing the AAV of any one of the above aspects. In some aspects, the method comprises contacting the target cell with the AAV.

[0029] In another aspect, the disclosure features a method of transduction of a target cell by an AAV at a lower dose of the AAV, the method comprising providing the AAV of any one of the above aspects. In some aspects, the method comprises contacting the target cell with the AAV.

[0030] In some aspects, 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.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Fig. 1 is a schematic diagram showing an assay used to measure phospholipase A2 (PLA2) catalytic reaction rates of wild-type AAV2 major capsid protein VP1 -unique region (VP1 u) polypeptides and of VP1 u variants. Purified wild-type and VP1 u variant polypeptides generated by a comprehensive substitution for multidimensional optimization (COSMO) workflow were provided in a plate, and their ability to cleave a PLA2 substrate was assessed. Cleavage of the PLA2 substrate at the cleavage site denoted by the arrow releases a fluorogenic cleavage product, resulting in a fluorescent signal.

[0033] Fig. 2 is a chart showing the level of fluorescence (presented as relative fluorescence units (RU)) over time (minutes) in reactions containing wild-type VP1 u or VP1 u variants with the amino acid substitution mutations A98E, D87K, D84R, D80Q, or Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). Fig. 3 is a diagram showing the normalized PLA2 reaction rate of VP1 u variants comprising the indicated amino acid substitution mutations (presented as normalized relative fluorescence units per minute (RU / [min*pg])), as measured using an ENZCHEK™ Phospholipase A2 Assay Kit. Reaction rates were normalized to the amount of protein in each well. An X denotes wild-type or a mutant that was not tested.

[0034] Fig. 4 is a diagram showing the normalized PLA2 reaction rate of VP1 u variants comprising the indicated amino acid substitution mutations (presented as RU / [min*pg]), as measured using an ENZCHEK™ Phospholipase A2 Assay Kit. Reaction rates were normalized to the amount of protein in each well. An X denotes wild-type or a mutant that was not tested.

[0035] Fig. 5 is a heatmap showing the normalized PLA2 reaction rate of VP1 u variants comprising the indicated amino acid substitution mutations (presented as RU / [min*pg]), as measured using an ENZCHEK™ Phospholipase A2 Assay Kit. Reaction rates were normalized to the amount of protein in each well. An X denotes wild-type or a mutant that was not tested.

[0036] Fig. 6 is a heatmap showing the normalized PLA2 reaction rate of VP1 u variants comprising the indicated amino acid substitution mutations (presented as RU / [min*pg]), as measured using an ENZCHEK™ Phospholipase A2 Assay Kit. Reaction rates were normalized to the amount of protein in each well. An X denotes wild-type or a mutant that was not tested.

[0037] Fig. 7 is a diagram showing the yield of VP1 u variants comprising the indicated amino acid substitution mutations in an assay for protein production of the VP1 u variants relative to the yield of wildtype VP1 u. An X denotes wild-type or a mutant that was not tested.

[0038] Fig. 8 is a diagram showing the yield of VP1 u variants comprising the indicated amino acid substitution mutations in an assay for protein production of the VP1 u variants relative to the yield of wildtype VP1 u. An X denotes wild-type or a mutant that was not tested.

[0039] Fig. 9 is a diagram showing the structure of an AAV2 VP1 u variant containing the amino acid substitution mutations L53C and A72C (“cysteine substitutions”), as predicted by AlphaFold. The locations of the two cysteine substitutions are shown as black spheres.

[0040] Fig. 10 is a diagram showing the structure of an AAV2 VP1 u variant containing the amino acid substitution mutations L53C and G1 1 1 C (“cysteine substitutions”), as predicted by AlphaFold. The locations of the two cysteine substitutions are shown as black spheres.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] I. DEFINITIONS

[0043] Unless otherwise defined, all terms of art, notations, and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0044] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se. 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.

[0045] Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0046] An “effective amount” refers to an amount of an agent (e.g., a therapeutic agent) that is effective to bring about a therapeutic / prophylactic benefit (e.g., as described herein) that is not outweighed by unwanted / undesirable side effects.

[0047] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient or ingredients to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be 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.

[0048] A “native sequence” protein herein refers to a protein comprising the amino acid sequence of a protein found in nature, including naturally occurring variants of the protein. The term as used herein includes the protein as isolated from a natural source thereof or as recombinantly produced.

[0049] The term “protein,” as used herein, refers to any native protein from any source, including viruses (e.g., adeno-associated viruses (AAVs), mammals such as primates (e.g., humans), and rodents (e.g., mice and rats). The term encompasses “full-length,” unprocessed protein any form of the protein that results from processing, e.g., processing in a cell. The term also encompasses naturally occurring variants of the protein, e.g., splice variants or allelic variants, e.g., amino acid substitution mutations or amino acid deletion mutations. The term also includes isolated regions or domains of the protein, e.g., the extracellular domain (ECD).

[0050] An “isolated” protein or peptide is one which has been separated from a component of its natural environment. In some aspects, a protein or peptide is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse phase HPLC).

[0051] An “isolated” nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0052] 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 term encompasses full-length AAV9 VP1 and isolated regions or domains of AAV9 VP1 that comprise at least a portion of the AAV9 VP1 unique N-terminus (i.e., the AAV9 VP1 N-terminal sequence not present in wild-type VP2 or VP3). The term also encompasses naturally occurring variants of AAV9 VP1 , e.g., splice variants or allelic variants. The amino acid sequence of an exemplary AAV9 VP1 is provided as SEQ ID NO: 1 and as GenBank accession number AAS99264.1 . Minor sequence variations, especially conservative amino acid substitutions of AAV9 VP1 that do not affect AAV9 VP1 function and / or activity, are also contemplated by the invention.

[0053] 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 term encompasses full-length AAV2 VP1 and isolated regions or domains of AAV2 VP1 that comprise at least a portion of the AAV2 VP1 unique N-terminus (i.e., the AAV2 VP1 N-terminal sequence not present in wild-type VP2 or VP3). The term also encompasses naturally occurring variants of AAV2 VP1 , e.g., splice variants or allelic variants. The amino acid sequence of an exemplary AAV2 VP1 is provided as SEQ ID NO: 2, in NCBI Reference Sequence YP_680426.1 , and in Uniprot sequence P03135-1 . Minor sequence variations, especially conservative amino acid substitutions of AAV2 VP1 that do not affect AAV2 VP1 function and / or activity, are also contemplated by the invention.

[0054] The terms “AAV2 major capsid protein VP1 -unique region” and “AAV2 VP1 u,” as used herein, refer to the N-terminal unique region of any native VP1 from adeno-associated virus serotype 2 (AAV2), unless otherwise indicated. The amino acid sequence of an exemplary AAV2 VP1 u is provided as SEQ ID NO: 4, which corresponds to M1 -P135 of Uniprot sequence P03135-1 . Minor sequence variations, especially conservative amino acid substitutions of AAV2 VP1 that do not affect AAV2 VP1 function and / or activity, are also contemplated by the invention.

[0055] The terms “AAV receptor” and “AAVR,” as used herein, refer to any native AAVR from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses full-length AAVR and isolated regions or domains thereof. The term also encompasses naturally occurring variants of AAVR, e.g., 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 variations, especially conservative amino acid substitutions of AAVR that do not affect AAVR function and / or activity, are also contemplated by the invention.

[0056] The term “virus-like particle” or “VLP,” as used herein, refers to an AAV-like particle having a capsid comprising (e.g., consisting essentially of) only one or two of the AAV major capsid proteins, e.g., comprising only VP1 ; comprising only VP1 and VP2; or comprising only VP1 and VP3.

[0057] “Affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1 :1 interaction between members of a binding pair (e.g., receptor and ligand). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0058] “Complex” or “complexed” as used herein refers to the 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 aspect, a complex is heteromultimeric. It should be understood that the term “protein complex” or “polypeptide complex” as used herein includes complexes that have a non-protein entity conjugated to a protein in the protein complex (e.g., including, but not limited to, chemical molecules such as a toxin or a detection agent). The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transfected cells,” “transformed cells,” and “transformants,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but 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 aspects, the host cell is stably transformed with the exogenous nucleic acid. In other aspects, the host cell is transiently transformed with the exogenous nucleic acid.

[0059] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0060] “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 with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0061] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

[0062] 100 times the fraction X / Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically 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.

[0063] II. ENGINEERED AAV CAPSIDS

[0064] Adeno-associated viruses (AAV) and virus-like particles (VLPs) having a capsid comprising a major capsid protein VP1 with a VP1 -unique region (VP1 u) that includes one or more amino acid substitution mutations relative to a wild-type VP1 u (e.g., the VP1 u of AAV2, e.g., as presented in SEQ ID NO: 4) are provided herein.

[0065] The VP1 u region comprises a phospholipase A2 (PLA2) domain that catalyzes the hydrolysis of membrane phospholipids and is believed to facilitate endosomal escape of AAVs (thus providing the AAV access to the cytoplasm of a cell and allowing downstream activity, e.g., transduction of the cell). The VP1 u regions comprised by the AAVs and VLPs of the present invention have increased PLA2 domain catalytic activity relative to wild-type VP1 u regions. Thus, in some embodiments, an AAV or VLP of the present invention may have an increased rate of phospholipase activity; an increased rate of endosomal escape; and / or an increased rate of delivery to the nucleus of a target cell and / or may transduce a target cell at a lower dose relative to a comparator AAV or VLP (e.g., an AAV or VLP having a capsid that does not comprise any of the indicated amino acid substitution mutations).

[0066] AAVs have a capsid comprising all three of major capsid protein VP1 (comprising the VP1 u region), minor capsid protein VP2, and minor capsid protein VP3. VLPs, as a general principle, have a capsid comprising only one or two of the capsid proteins VP1 , VP2, and VP3. VLPs of the present invention may comprise only VP1 ; comprise only VP1 and VP2; or comprising only VP1 and VP3. As is discussed in further detail below, an AAV or VLP of the invention may comprise one or more additional mutations (e.g., amino acid substitutions, insertions, or deletions) in one, two, or all three of VP1 , VP2, and VP3.

[0067] As described in further detail below, the AAV or VLP may be of any appropriate serotype. A skilled artisan is able to identify corresponding residues in the VP1 , VP2, and / or VP3 proteins of two or more AAV serotypes (e.g., in the VP1 u region of two or more AAV serotypes), e.g., is able to determine the residues of a given serotype that correspond to the recited residues of the VP1 u region of AAV2 (SEQ ID NO: 4), e.g., by creating a sequence alignment. Thus, a skilled artisan would understand how to modify the VP1 u region of any appropriate AAV serotype to comprise the amino acid substitution mutations provided herein. Additionally, in some aspects, the AAV or VLP provided herein is of a serotype other than AAV2, and the wild-type VP1 u region of the AAV or VLP has been replaced with an AAV2 VP1 u region comprising one or more of the amino acid substitution mutations provided herein.

[0068] Further provided herein are one or more isolated nucleic acids encoding a modified VP1 u of the invention or a major capsid protein VP1 comprising the same; one or more vectors comprising the one or more isolated nucleic acids; and one or more host cells (e.g., mammalian host cells (e.g., Chinese hamster ovary (CHO) host cells) or prokaryotic host cells (e.g., E. coli host cells) comprising the one or more vectors. Also provide herein is a method of producing a modified VP1 u of the invention or a major capsid protein VP1 , AAV, or VLP comprising the same, the method comprising culturing the one or more host cells in a culture medium. The method may further comprise recovering the modified VP1 u, major capsid protein VP1 , AAV, or VLP from the one or more host cells or the culture medium.

[0069] In some aspects, the yield and / or titer in an expression system (e.g., EXPI293™ cells) of any of the modified AAVs or VLPs provided herein is not substantially reduced relative to the yield and / or titer of wild-type AAVs or VLPs in the expression system (e.g., is similar to, equal to, or greater than the yield and / or titer of wild-type AAVs or VLPs in the expression system).

[0070] In some aspects, the AAV or VLP has a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising one or more of the amino acid substitution mutations provided in Table 1 (e.g., comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the amino acid substitution mutations provided in Table 1 ), wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0071] In some aspects, the disclosure provides an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising one or more of the amino acid substitution mutations provided in Table 1 (e.g., comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the amino acid substitution mutations provided in Table 1 ), wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4) (e.g., a modified AAV or VLP).

[0072] Table 1. AAV2 VP1u variants having increased PLA2catalytic activity (numbered relative to the

[0073] AAV2 VP1u (SEQ ID NO: 4)) A. Engineered AA V capsids comprising S42A, D80Q, Q82A, D84R, D87K, and / or Q1011 / amino acid substitution mutations

[0074] In some aspects, the disclosure provides an adeno-associated virus (AAV) or virus-like particle (VLP) having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising one or more of (a) a S42A amino acid substitution mutation; (b) a D80Q amino acid substitution mutation; (c) a Q82A amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; and (f) a Q101 V amino acid substitution mutation (e.g., comprising one, two, three, four, five, or all six of a S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutation), wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4) (e.g., a modified AAV or VLP).

[0075] In some aspects, the VP1 u of the AAV or VLP comprises a single amino acid substitution mutation selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutation is numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). Thus, in some aspects, the disclosure provides (a) an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising a S42A amino acid substitution mutation; (b) an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising a D80Q amino acid substitution mutation; (c) an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising a Q82A amino acid substitution mutation; (d) an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising a D84R amino acid substitution mutation; (e) an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising a D87K amino acid substitution mutation; and (f) an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising a Q101 V amino acid substitution mutation, wherein the amino acid substitution mutation is numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0076] In some aspects, the disclosure features an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising two amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), e.g., an AAV or VLP comprising S42A and D80Q, S42A and Q82A, S42A and D84R, S42A and D87K, S42A and Q101 V, D80Q and Q82A, D80Q and D84R, D80Q and D87K, D80Q and Q101 V, Q82A and D84R, Q82A and D87K, Q82A and Q101 V, D84R and D87K, D84R and Q101 V, or D87K and Q101 V amino acid substitution mutations. For example, in some aspects, the disclosure features an AAV or VLP having a capsid comprising S42A and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In other aspects, the disclosure features an AAV or VLP having a capsid comprising D80Q and D87K amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0077] In some aspects, the disclosure features an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising three, four, or five amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In some aspects, the disclosure features an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising all six of S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0078] In some aspects, the VP1 u further comprises one or more of the amino acid substitution mutations provided in Table 1 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the amino acid substitution mutations provided in Table 1 ).

[0079] Engineered AA V capsids with an A98E mutation

[0080] In some aspects, the disclosure features an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u that:

[0081] (i) comprises one or more of (a) a S42A amino acid substitution mutation; (b) a D80Q amino acid substitution mutation; (c) a Q82A amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; and (f) a Q101 V amino acid substitution mutation (e.g., comprises one, two, three, four, five, or all six of a S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutation), and

[0082] (ii) comprises an A98E amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0083] In some aspects, the disclosure features an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising an A98E amino acid substitution mutation and one amino acid substitution mutation selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), e.g., an AAV or VLP comprising A98E and S42A, A98E and D80Q, A98E and Q82A, A98E and D84R, A98E and D87K, or A98E and Q101 V amino acid substitution mutations. For example, in some aspects, the disclosure features an AAV or VLP having a capsid comprising A98E and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0084] In some aspects, the disclosure features an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising an A98E amino acid substitution mutation and two, three, or four amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0085] In some aspects, the disclosure features an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising an A98E amino acid substitution mutation and five amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). For example, in some aspects, the disclosure features an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising S42A, D80Q, Q82A, D87K, A98E, and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In other aspects, the disclosure features an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising S42A, Q82A, D84R, D87K, A98E, and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0086] Engineered AA V capsids with an engineered disulfide bond

[0087] In some aspects, the VP1 u further comprises one or more engineered disulfide bonds (e.g., comprises one or more amino acid substitution mutations that result in a disulfide bond not present in a wild-type VP1 u (e.g., not present in an AAV2 VP1 u (e.g., SEQ ID NO: 4)). Exemplary VP1 u polypeptides comprising engineered disulfide bonds are presented in Section I l(B), below.

[0088] For example, in some embodiments, the VP1 u further comprises (a) L53C and G1 1 1 C amino acid substitution mutations; or (b) L53C and A72C amino acid substitution mutations.

[0089] B. Engineered AA V capsids comprising engineered disulfide bonds

[0090] In some aspects, the disclosure provides an AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising one or more engineered disulfide bonds (e.g., comprising one or more amino acid substitution mutations that result in a disulfide bond not present in a wild-type VP1 u (e.g., not present in an AAV2 VP1 u (e.g., SEQ ID NO: 4)).

[0091] For example, in some aspects, the disclosure provides an AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising L53C and G1 1 1 C amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0092] In other aspects, the disclosure provides an AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising L53C and A72C amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0093] In any of the above aspects, the VP1 u may comprise one or more of the amino acid substitution mutations presented in presented in Section ll(A), above. For example, in some aspects, the VP1 u comprising an engineered disulfide bond further comprises one or more (e.g., one, two, three, four, five, six, or all seven) of (a) a S42A amino acid substitution mutation; (b) a Q82A amino acid substitution mutation; (c) a D80Q amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; (f) a Q101 V amino acid substitution mutation; and (g) an A98E amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0094] In some aspects, the VP1 u further comprises one or more of the amino acid substitution mutations provided in Table 1 (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 of the amino acid substitution mutations provided in Table 1 ).

[0095] C. Capsid mutations that increase affinity for AA VR

[0096] In some embodiments of any of the AAVs and VLPs provided herein, the AAV or VLP comprises (a) a major capsid protein VP1 comprising one or more amino acid substitution mutations that increase the affinity of the AAV for AAVR; (b) a minor capsid protein VP2 comprising one or more amino acid substitution mutations that increase the affinity of the AAV for AAVR; and / or (c) a minor capsid protein VP3 comprising one or more amino acid substitution mutations that increase the affinity of the AAV for AAVR.

[0097] In some aspects, 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 comprising one or more of an 1451 T, 1451 Y, or 1451 M 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, a W503H amino acid substitution mutation, a T592D amino acid substitution mutation, and an A593E amino acid substitution mutation, wherein the 1451 , S454, A472, P504, E500, A273, Q387, S263, and W503 amino acid substitution mutations are 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).

[0098] In some aspects, 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 comprising (i) a T592D amino acid substitution mutation (ii), an A593E amino acid substitution mutation, or (iii) T592D and A593E amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 major capsid protein VP1 (SEQ ID NO: 2).

[0099] In some aspects, 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 comprising (i) an A273N amino acid substitution mutation (ii), a Q387K amino acid substitution mutation, (iii) an E500R amino acid substitution mutation, or (iii) two or all three of an A273N, Q387K, and 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 ).

[0100] In some aspects, 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 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 ).

[0101] In some aspects, the AAV or VLP and has an increased affinity for AAVR relative to a control AAV or VLP derivative thereof, e.g., an AAV or a VLP derivative thereof 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 AAV or VLP of interest. For example, in some aspects, affinity of the AAV or VLP for the 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., is improved by 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, 100%-150%, or 150%-200% relative to the affinity of a control AAV or VLP. In some aspects, the AAV or VLP is AAV2 or a variant thereof. In some aspects, the AAV or VLP is AAV9 or a variant thereof. D. Further capsid mutations

[0102] In some aspects, the major capsid protein VP1 , minor capsid protein VP2, and / or minor capsid protein VP3 of any one of the AAVs or VLPs provided herein comprises one or more additional amino acid substitution mutations and / or comprise one or more amino acid insertions or deletions.

[0103] In some aspects, the one or more additional amino acid substitution mutations, amino acid insertions, or amino acid deletions are substitutions, insertions, or deletions that affect binding of the AAV capsid to one or more attachment receptors. Exemplary mutations that affect (e.g., attenuate) binding to attachment receptors are provided, e.g., in 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.

[0104] In some aspects, the AAV or VLP has improved transduction into liver cells (e.g., human liver cells). For example, in some aspects, one or more of the mutations provided herein is combined with a T503A or N596D amino acid substitution mutation (numbered relative to the AAV2 major capsid protein VP1 ) (Cabanes-Creus et al., Molecular Therapy: Methods and Clinical Development, 17: 1139-1154, 2020). In some aspects, the AAV has decreased affinity for heparin.

[0105] In some aspects, the AAV or VLP has decreased transduction into liver cells (e.g., human liver cells). For example, in some aspects, one or more of the mutations provided herein is combined with a R585A, R585S, R588A, or R588T amino acid substitution mutation (numbered relative to the AAV2 major capsid protein VP1 ) (Cabanes-Creus et al., Molecular Therapy: Methods and Clinical Development, 17: 1139-1154, 2020).

[0106] In some aspects, the AAV or VLP has decreased affinity for glycans. For example, in some aspects, one or more of the mutations provided herein is 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).

[0107] In some aspects, the AAV or VLP has decreased affinity for integrin a5p1 . For example, in some aspects, one or more of the mutations provided herein is combined with a 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).

[0108] In some aspects, the AAV or VLP has decreased sialic acid binding. For example, in some aspects, one or more of the mutations provided herein is combined with a L587T amino acid substitution mutation and / or a M569V amino acid substitution mutation (see Afione et al., Journal of Virology, 89(3), 2015, which is incorporated herein by reference in its entirety).

[0109] In some aspects, the AAV or VLP comprises a N272A amino acid substitution mutation (e.g., as described in Bell et al., Journal of Virology, 86(13), 2012, which is incorporated herein by reference in its entirety). E. Exemplary AA 1 / serotypes

[0110] As used herein, the serotype of an AAV or virus-like particle (VLP) refers to the serotype of the capsid protein(s) of the AAV or VLP. The serotype of a VP1 u region refers to the serotype of the AAV from which the VP1 u region is derived.

[0111] An AAV or VLP of the disclosure may be, e.g., of a natural AAV serotype (e.g., AAV1 , AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, or AAVrhI O), an engineered AAV serotype, or any variant, derivative, or pseudotype thereof. For example, the AAV may be a self-complementary AAV (scAAV), a chimeric AAV, a hybrid AAV, AAVrh74, AAVanc80L65, AAVrh.10, AAVrh.74, AAV2 / 1 , AAV2 / 5, AAV2 / 6, AAV2 / 8, AAV2 / 9, an AAV2-AAV3 hybrid, 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, 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 may be used in the invention are described, e.g., in Asokan et al., Mol Ther., 20(4): 699-708, 2012.

[0112] In some aspects, the serotype of the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , or AAV12.

[0113] In some aspects, the serotype of the AAV is AAV2 or a variant thereof.

[0114] A skilled artisan is able to identify corresponding residues in the VP1 , VP2, and / or VP3 proteins of two or more AAV serotypes (e.g., in the VP1 u region of two or more AAV serotypes), e.g., is able to determine the residues of a given serotype that correspond to the recited residues of the VP1 u region of AAV2 (SEQ ID NO: 4), e.g., by creating a sequence alignment. Thus, a skilled artisan would understand how to modify the VP1 u region of any appropriate AAV serotype to comprise the amino acid substitution mutations provided herein. Additionally, in some aspects, the AAV or VLP provided herein is of a serotype other than AAV2, and the wild-type VP1 u region of the AAV or VLP has been replaced with an AAV2 VP1 u region comprising one or more of the amino acid substitution mutations provided herein

[0115] AAVs and VLPs comprising VP1 u regions having the amino acid substitution mutations provided herein may be generated using molecular biology techniques that are known in the art.

[0116] AAVs comprising capsids having differences among the VP1 , VP2, and / or VP3 sequences may be generated using molecular biology techniques that are known in the art. For example, the VP1 , VP2, and / or VP3 may be expressed on separate plasmids or separate open reading frames (ORFs) on the same plasmid.

[0117] F. Target diseases

[0118] In some aspects of any of the AAVs, VLPs, and methods provided herein, the AAV or VLP is for use in treatment of a disease or condition in a subject, e.g., a human subject. Exemplary diseases and conditions that may be treated with the AAVs and VLPs presented herein include ophthalmological, neurological, or neurodegenerative diseases or conditions and proliferative diseases or conditions (e.g., cancer). For example, in some aspects, an AAVs or VLP provided herein is used in the treatment of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), a disease or condition (e.g., ALS) associated with the ataxin 2 (ATXN2) gene, age-related macular degeneration (AMD) (e.g., dry AMD or wet AMD), cystic fibrosis (CF), a disease or condition (e.g., CF) associated with the cystic fibrosis transmembrane conductance regulator (CFTR) gene, neuronal ceroid lipofuscinosis 2 (CLN2) disease, frontotemporal dementia (FTD), GRN-related 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 (e.g., PD) associated with the glucocerebrosidase (GBD) gene, Rett syndrome, spinal muscular atrophy (SMA), or a disease or condition associated with a Tau gene. In some embodiments, the AAV or VLP is for use in treatment of a cancer, and delivers a pro-inflammatory cytokine. In other aspects, the AAV or VLP is for use in an in vitro system (e.g., is for delivery to an in vitro cell).

[0119] III. METHODS OF USING ENGINEERED AAV CAPSIDS

[0120] A. Methods of improving transduction efficiency using engineered AA V capsids

[0121] In some aspects, the disclosure provides a method of improving transduction efficiency of an adeno-associated virus (AAV) or virus-like particle (VLP), the method comprising providing an AAV or VLP provided herein (e.g., an AAV or VLP provided in Section II, above), e.g., providing such an AAV to a cell, system, or subject. In some aspects, the method comprises contacting a target cell with the AAV or VLP, thereby improving transduction efficiency.

[0122] The disclosure further provides a method of improving transduction efficiency of an AAV or VLP, the method comprising contacting a target cell with an AAV or VLP provided herein (e.g., an AAV or VLP provided in Section II, above), thereby improving transduction efficiency.

[0123] The disclosure also provides an AAV or VLP as provided in Section II, above, for use in transducing a target cell, wherein the transduction efficiency of the AAV or VLP is improved (e.g., is improved relative to the transduction efficiency of an AAV having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (see Section II), or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method).

[0124] The disclosure also provides use of an AAV or VLP as provided in Section II, above, in the manufacture of a medicament for transducing a target cell, wherein the transduction efficiency of the AAV or VLP is improved (e.g., is improved relative to the transduction efficiency of an AAV having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (see Section II), or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method).

[0125] Improved transduction efficiency may be measured relative to the transduction efficiency of a control AAV or VLP. For example, an improved transduction efficiency may be a transduction efficiency that is greater than that of an AAV or VLP (e.g., an AAV or VLP of the same serotype) having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (see Section II), or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method. For example, in some aspects, the AAV for use in the method is AAV2 and comprises one or more of the amino acid substitutions provided in Section II above, and the control AAV is wild-type AAV2. In some aspects, transduction efficiency of the AAV or VLP is improved 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., is improved by 5%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, 100%-150%, or 150%- 200%, relative to the transduction efficiency of a control AAV or VLP, e.g., an AAV or VLP having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (e.g., an AAV or VLP of the same serotype that does not comprise any of the indicated amino acid substitution mutations or comprises fewer of the indicated amino acid substitution mutations than the AAV or VLP of interest).

[0126] In some aspects, the AAV or VLP has an increased rate of delivery to the nucleus of a target cell relative to a control AAV or VLP, e.g., an AAV or VLP having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (see Section II), or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (e.g., an AAV or VLP of the same serotype that does not comprise any of the indicated amino acid substitution mutations or comprises fewer of the indicated amino acid substitution mutations than the AAV or VLP of interest).

[0127] For example, in some aspects, the rate of delivery to the nucleus 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., is improved by 5%-10%, 10%-20%, 20%- 30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-100%, 100%-150%, or 150%-200%, relative to the rate of delivery of a control AAV or VLP.

[0128] Accordingly, in some aspects, the disclosure features a method of increasing the rate of delivery of an AAV to the nucleus of a target cell, the method comprising providing an AAV or VLP provided herein (e.g., an AAV or VLP provided in Section II, above), e.g., providing such an AAV to a cell, system, or subject. In some aspects, the method comprises contacting a target cell with the AAV, thereby increasing the rate of delivery to the nucleus.

[0129] In some aspects, the AAV transduces a target cell at a lower dose than a control AAV or VLP, e.g., an AAV or VLP having a capsid that that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (see Section II), or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (e.g., an AAV or VLP of the same serotype that does not comprise any of the indicated amino acid substitution mutations or comprises fewer of the indicated amino acid substitution mutations than the AAV or VLP of interest), e.g., transduces the target cell at a rate sufficient to achieve a desired effect, e.g., a therapeutic effect, at a lower dose.

[0130] For example, in some aspects, the AAV or VLP transduces the target cell at a dose that is at least 1 .1 -fold lower, 1 .2-fold lower, 1 .3-fold lower, 1 .4-fold lower, 1 .5-fold lower, 1 .6-fold lower, 1 .7-fold lower, 1 .8-fold lower, 1 .9-fold lower, 2-fold lower, 2.5-fold lower, 3-fold lower, 3.5-fold lower, 4-fold lower, 4.5-fold lower, 5-fold lower, 5.5-fold lower, 6-fold lower, 6.5-fold lower, 7-fold lower, 7.5-fold lower, 8-fold lower, 8.5-fold lower, 9-fold lower, 9.5-fold lower, 10-fold lower, or more than 10-fold lower, e.g., 1.1 -1 .5- fold lower, 1 .5-2-fold lower, 2-3-fold lower, 3-4-fold lower, 4-5-fold lower, 5-6-fold lower, 6-7-fold lower, 7- 8-fold lower, 8-9-fold lower, or 9-10-fold lower than the dose of a control AAV or VLP. Accordingly, in some aspects, the AAV or VLP has a lower effective dose than a control AAV or VLP, e.g., an AAV or VLP that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (see Section II), or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method

[0131] Accordingly, in some aspects, the disclosure features a method of transduction of a target cell by an AAV at a lower dose of the AAV, the method comprising providing an AAV or VLP provided herein (e.g., an AAV or VLP provided in Section II, above), e.g., providing such an AAV to a cell, system, or subject. In some aspects, the method comprises contacting a target cell with the AAV, thereby transducing the target cell at a lower AAV dose.

[0132] The improvement in transduction efficiency and / or rate of delivery to the nucleus may be in one or more target cells. Exemplary target cells include, without limitation, an endothelial cell (e.g., a brain 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, and a liver cell. In some aspects, the target cell is an in vitro cell. In other aspects, the target cell is a cell in an organism (e.g., a mammal, e.g., a human).

[0133] In some aspects, the serotype of the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , or AAV12. In some aspects, the serotype of the AAV is AAV2 or a variant thereof. Further serotypes that may be used in the invention are provided, e.g., in Section I l(E), above.

[0134] Methods of improving transduction efficiency using engineered AA V capsids comprising S42A, D80Q, Q82A, D84R, D87K, and / or Q101 V amino acid substitution mutations

[0135] In some aspects, the disclosure features a method of improving transduction efficiency of an AAV or VLP, the method comprising providing an AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 -unique region (VP1 u) comprising one or more of (a) a S42A amino acid substitution mutation; (b) a Q82A amino acid substitution mutation; (c) a D80Q amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; and (f) a Q101 V amino acid substitution mutation (e.g., comprising one, two, three, four, five, or all six of a S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutation), wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4) (e.g., providing such an AAV to a cell, system, or subject). In some aspects, the method comprises contacting a target cell with the AAV or VLP, thereby improving transduction efficiency.

[0136] In some aspects, the disclosure features a method of improving transduction efficiency of an AAV or VLP, the method comprising contacting a target cell with an AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising one or more of (a) a S42A amino acid substitution mutation; (b) a Q82A amino acid substitution mutation; (c) a D80Q amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; and (f) a Q101 V amino acid substitution mutation (e.g., comprising one, two, three, four, five, or all six of a S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutation), wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), thereby improving transduction efficiency.

[0137] The disclosure also provides use of such AAVs or VLPs in the manufacture of a medicament for transducing a target cell and such AAVs or VLPs for use in transducing a target cell.

[0138] In some aspects, the VP1 u of the AAV or VLP comprises a single amino acid substitution mutation selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutation is numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0139] In some aspects, the VP1 u of the AAV or VLP comprises a VP1 u comprising two amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), e.g., comprises S42A and D80Q, S42A and Q82A, S42A and D84R, S42A and D87K, S42A and Q101 V, D80Q and Q82A, D80Q and D84R, D80Q and D87K, D80Q and Q101 V, Q82A and D84R, Q82A and D87K, Q82A and Q101 V, D84R and D87K, D84R and Q101 V, or D87K and Q101 V amino acid substitution mutations. For example, in some aspects, the VP1 u of the AAV or VLP comprises S42A and Q101 V amino acid substitution mutations or D80Q and D87K amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0140] In some aspects, the VP1 u of the AAV or VLP comprises three, four, or five amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0141] In some aspects, the VP1 u of the AAV or VLP comprises all six of S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0142] / '. AA V capsids with an A98E mutation

[0143] In some aspects, the disclosure features a method of improving transduction efficiency of an AAV or VLP, the method comprising providing an AAV having a capsid comprising a major capsid protein VP1 that (i) comprises one or more of (a) a S42A amino acid substitution mutation; (b) a D80Q amino acid substitution mutation; (c) a Q82A amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; and (f) a Q101 V amino acid substitution mutation (e.g., comprises one, two, three, four, five, or all six of a S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutation), and (ii) comprises an A98E amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4) (e.g., providing such an AAV to a cell, system, or subject). In some aspects, the method comprises contacting a target cell with the AAV or VLP, thereby improving transduction efficiency.

[0144] In some aspects, the disclosure features a method of improving transduction efficiency of an AAV or VLP, the method comprising contacting a target cell with an AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 u that (i) comprises one or more of (a) a S42A amino acid substitution mutation; (b) a D80Q amino acid substitution mutation; (c) a Q82A amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; and (f) a Q101 V amino acid substitution mutation (e.g., comprises one, two, three, four, five, or all six of a S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutation), and (ii) comprises an A98E amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), thereby improving transduction efficiency.

[0145] In some aspects, the VP1 u of the AAV or VLP comprises an A98E amino acid substitution mutation and one amino acid substitution mutation selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), e.g., comprises A98E and S42A, A98E and D80Q, A98E and Q82A, A98E and D84R, A98E and D87K, or A98E and Q101 V amino acid substitution mutations. For example, in some aspects, the VP1 u of the AAV or VLP comprises A98E and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0146] In some aspects, the VP1 u of the AAV or VLP comprises an A98E amino acid substitution mutation and two, three, or four amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0147] In some aspects, the VP1 u of the AAV or VLP comprises an A98E amino acid substitution mutation and five amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). For example, in some aspects, the VP1 u of the AAV or VLP comprises S42A, D80Q, Q82A, D87K, A98E, and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In other aspects, the VP1 u of the AAV or VLP comprises S42A, Q82A, D84R, D87K, A98E, and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). ii. Engineered AA V capsids with an engineered disulfide bond

[0148] In some aspects, the VP1 u of the AAV or VLP further comprises one or more engineered disulfide bonds (e.g., comprises one or more amino acid substitution mutations that result in a disulfide bond not present in a wild-type VP1 u (e.g., not present in an AAV2 VP1 u (e.g., SEQ ID NO: 4)). For example, in some embodiments, the VP1 u further comprises (a) L53C and G111 C amino acid substitution mutations; or (b) L53C and A72C amino acid substitution mutations.

[0149] Methods of improving transduction efficiency using engineered AA V capsids comprising engineered disulfide bonds

[0150] In some aspects, the disclosure features a method of improving transduction efficiency of an AAV or VLP, the method comprising providing an AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising (a) L53C and G11 C amino acid substitution mutations or (b) L53C and A72C amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4) (e.g., providing such an AAV to a cell, system, or subject). In some aspects, the method comprises contacting a target cell with the AAV or VLP, thereby improving transduction efficiency. In some aspects, the disclosure features a method of improving transduction efficiency of an AAV or VLP, the method comprising contacting a target cell with an AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising (a) L53C and G11 C amino acid substitution mutations or (b) L53C and A72C amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), thereby improving transduction efficiency.

[0151] The disclosure also provides use of such AAVs or VLPs in the manufacture of a medicament for transducing a target cell and such AAVs or VLPs for use in transducing a target cell.

[0152] In some aspects, the VP1 u further comprises one or more (e.g., one, two, three, four, five, six, or all seven) of (a) a S42A amino acid substitution mutation; (b) a Q82A amino acid substitution mutation; (c) a D80Q amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; (f) a Q101 V amino acid substitution mutation; and (g) an A98E amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0153] B. Methods of delivering a cargo to a cell

[0154] In some aspects, the disclosure provides a method of delivering a cargo to a cell (e.g., a target cell), the method comprising contacting a target cell with an AAV or VLP provided herein (e.g., an AAV or VLP provided in Section II, above), e.g., wherein the AAV or VLP has an increased rate of transduction into a target cell relative to an AAV or VLP having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (see Section II), or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method).

[0155] The disclosure also provides an AAV or VLP as provided in Section II, above, for use in delivering a cargo to a target cell, e.g., wherein the AAV or VLP has an increased rate of transduction into a target cell relative to an AAV or VLP having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (see Section II), or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method).

[0156] The disclosure also provides use of an AAV or VLP as provided in Section II, above, in the manufacture of a medicament delivering a cargo to a target cell, e.g., wherein the AAV or VLP has an increased rate of transduction into a target cell relative to an AAV or VLP having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method (see Section II), or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method).

[0157] The improvement in transduction efficiency may be in one or more target cells.

[0158] Exemplary target cells include, without limitation, an endothelial cell (e.g., a brain 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, and a liver cell. In some aspects, the target cell is an in vitro cell. In other aspects, the target cell is a cell in an organism (e.g., a mammal, e.g., a human). In some aspects, the cargo of the AAV or VLP is a nucleic acid, e.g., a single-stranded RNA (ssRNA). In some aspects, the cargo is a therapeutic nucleic acid. In some aspects, the cargo is a nucleic acid that is useful as a gene therapy. In some aspects, the cargo of the AAV or VLP is an antisense oligonucleotide or an siRNA. In some aspects, the inverted terminal repeat (ITR) sequences of the AAV genome are retained for packaging, and the viral genes are replaced with a sequence comprising or encoding the cargo.

[0159] In some aspects, the cargo of the AAV or VLP is a cytokine, e.g., a pro-inflammatory cytokine.

[0160] In some aspects, the serotype of the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , or AAV12. In some aspects, the serotype of the AAV is AAV2 or a variant thereof. Further serotypes that may be used in the invention are provided, e.g., in Section I l(E), above.

[0161] Methods of delivering a cargo using engineered AA V capsids comprising S42A, D80Q, Q82A, D84R, D87K, and / or Q101V amino acid substitution mutations

[0162] In some aspects, the disclosure provides a method of delivering a cargo to a cell (e.g., a target cell), the method comprising contacting a target cell with an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 -unique region (VP1 u) comprising one or more of (a) a S42A amino acid substitution mutation; (b) a Q82A amino acid substitution mutation; (c) a D80Q amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; and (f) a Q101 V amino acid substitution mutation (e.g., comprising one, two, three, four, five, or all six of a S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutation), wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), e.g., wherein the AAV or VLP has an increased rate of transduction into a target cell relative to an AAV or VLP having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method, or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method).

[0163] The disclosure also provides use of such AAVs or VLPs in the manufacture of a medicament for delivering a cargo to a target cell and such AAVs or VLPs for use in delivering a cargo to a target cell.

[0164] In some aspects, the VP1 u of the AAV or VLP comprises a single amino acid substitution mutation selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutation is numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0165] In some aspects, the VP1 u of the AAV or VLP comprises a VP1 u comprising two amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), e.g., comprises S42A and D80Q, S42A and Q82A, S42A and D84R, S42A and D87K, S42A and Q101 V, D80Q and Q82A, D80Q and D84R, D80Q and D87K, D80Q and Q101 V, Q82A and D84R, Q82A and D87K, Q82A and Q101 V, D84R and D87K, D84R and Q101 V, or D87K and Q101 V amino acid substitution mutations. For example, in some aspects, the VP1 u of the AAV or VLP comprises S42A and Q101 V amino acid substitution mutations or D80Q and D87K amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In some aspects, the VP1 u of the AAV or VLP comprises three, four, or five amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0166] In some aspects, the VP1 u of the AAV or VLP comprises all six of S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0167] / '. Methods of delivering a cargo using engineered AAV capsids with an A98E mutation In some aspects, the disclosure provides a method of delivering a cargo to a cell (e.g., a target cell), the method comprising contacting a target cell with an AAV or VLP having a capsid comprising a major capsid protein VP1 that (i) comprises one or more of (a) a S42A amino acid substitution mutation; (b) a D80Q amino acid substitution mutation; (c) a Q82A amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; and (f) a Q101 V amino acid substitution mutation (e.g., comprises one, two, three, four, five, or all six of a S42A, D80Q, Q82A, D84R, D87K, and Q101 V amino acid substitution mutation), and (ii) comprises an A98E amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), e.g., wherein the AAV or VLP has an increased rate of transduction into a target cell relative to an AAV or VLP having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method, or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method).

[0168] In some aspects, the VP1 u of the AAV or VLP comprises an A98E amino acid substitution mutation and one amino acid substitution mutation selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), e.g., comprises A98E and S42A, A98E and D80Q, A98E and Q82A, A98E and D84R, A98E and D87K, or A98E and Q101 V amino acid substitution mutations. For example, in some aspects, the VP1 u of the AAV or VLP comprises A98E and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0169] In some aspects, the VP1 u of the AAV or VLP comprises an A98E amino acid substitution mutation and two, three, or four amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0170] In some aspects, the VP1 u of the AAV or VLP comprises an A98E amino acid substitution mutation and five amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). For example, in some aspects, the VP1 u of the AAV or VLP comprises S42A, D80Q, Q82A, D87K, A98E, and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). In other aspects, the VP1 u of the AAV or VLP comprises S42A, Q82A, D84R, D87K, A98E, and Q101 V amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4). / ' / . Engineered AA V capsids with an engineered disulfide bond

[0171] In some aspects, the VP1 u of the AAV or VLP further comprises one or more engineered disulfide bonds (e.g., comprises one or more amino acid substitution mutations that result in a disulfide bond not present in a wild-type VP1 u (e.g., not present in an AAV2 VP1 u (e.g., SEQ ID NO: 4)). For example, in some embodiments, the VP1 u further comprises (a) L53C and G1 1 1 C amino acid substitution mutations; or (b) L53C and A72C amino acid substitution mutations.

[0172] Methods of delivering a cargo using engineered AA V capsids comprising engineered disulfide bonds

[0173] In some aspects, the disclosure provides a method of delivering a cargo to a cell (e.g., a target cell), the method comprising contacting a target cell with an AAV or VLP having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising (a) L53C and G1 1 C amino acid substitution mutations or (b) L53C and A72C amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4), e.g., wherein the AAV or VLP has an increased rate of transduction into a target cell relative to an AAV or VLP having a capsid that (a) does not comprise any of the amino acid substitution mutations comprised by the AAV or VLP for use in the method, or (b) comprises only a subset of the amino acid substitution mutations comprised by the AAV or VLP for use in the method).

[0174] The disclosure also provides use of such AAVs or VLPs in the manufacture of a medicament for delivering a cargo to a target cell and such AAVs or VLPs for use in delivering a cargo to a target cell.

[0175] In some aspects, the VP1 u further comprises one or more (e.g., one, two, three, four, five, six, or all seven) of (a) a S42A amino acid substitution mutation; (b) a Q82A amino acid substitution mutation; (c) a D80Q amino acid substitution mutation; (d) a D84R amino acid substitution mutation; (e) a D87K amino acid substitution mutation; (f) a Q101 V amino acid substitution mutation; and (g) an A98E amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

[0176] C. Methods of delivery

[0177] The AAVs and VLPs provided herein can be administered by any suitable method, including, for example, intravenously, intramuscularly, subcutaneously, intradermally, percutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subconjunctivally, intravesicularly, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, transdermally, intravitreally (e.g., by intravitreal injection), by eye drop, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, by catheter, by lavage, in cremes, or in lipid compositions. The AAVs described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein. In some aspects, the AAVs and / or VLPs provided herein are administered to a cell in an in vitro system.

[0178] An AAV or VLP described herein (and any additional therapeutic agent) may be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The AAV or VLP need not be, but is optionally formulated with and / or administered concurrently with, one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of the AAV present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinical ly determined to be appropriate.

[0179] IV. EXAMPLES

[0180] Example 1. Method for generating AAV2 VP1u variants

[0181] A. Background

[0182] Adeno-associated viruses (AAVs) are non-pathogenic single-stranded DNA (ssDNA) viruses that may be used, e.g., as vectors for gene therapy. AAVs have a non-enveloped icosahedral capsid composed of major capsid protein VP1 , minor capsid protein VP2, and minor capsid protein VP3, which are encoded by overlapping genes.

[0183] The VP1 -unique (VP1 u) region of parvovirus capsid proteins contains a phospholipase A2 (PLA2) domain that is essential for infection. PLA2 enzymes catalyze the hydrolysis of membrane phospholipids from the sn-2 position (Burke and Dennis, J Lipid Res., 50 Suppl(Suppl): S237-S242, 2009). Important domains for catalytic activity are conserved between AAVs and other parvoviruses. The PLA2 domain is thought to facilitate endosomal escape of parvovirus virions (e.g., AAVs), which is an essential step for transduction of target cells (Zadori et al., Dev Cell., 1 (2): 291 -302, 2001 ; Girod et al., J Gen Virol., 83(Pt 5): 973-978, 2002).

[0184] In the present Examples, the VP1 u region of AAV2 was engineered to improve catalytic activity of the PLA2 domain. These engineered VP1 u sequences are expected to have improved endosomal escape and transduction efficiency. B. Workflow for generating AAV2 VP1u variants

[0185] Comprehensive amino acid screening was performed across regions of the AAV2 VP1 u region, which corresponded to amino acid residues 1 -135 of AAV2 VP1 capsid protein (SEQ ID NO: 1 ). A high- throughput method (COSMO workflow) was developed to identify AAV2 VP1 u variants having improved transduction efficiency. The comprehensive substitution for multidimensional optimization (COSMO) workflow is a high-throughput site-directed mutagenesis protocol for antibody engineering applications (see Sampei et al., PloS ONE, 13(12): e0209509, 2018, which is incorporated herein by reference in its entirety). The COSMO workflow was adapted for the high-throughput production of VLP1 u variants.

[0186] To generate VP1 u constructs, a wild-type AAV2 VP1 u template plasmid was used that comprised an N-terminal His-tag and a VP1 u region consisting of amino acid residues 2-135 of the AAV2 VP1 capsid protein (SEQ ID NO: 5; A12 is the start of the VP1 u sequence). Mutant DNAs were generated in 96-well plates using the wild-type AAV2 VP1 u template plasmid and a two-step site-directed mutagenesis protocol. Adapter primers that were complementary to the vector backbone and mutant primers containing nucleotide sequences encoding amino acid substitution mutations of interest were used to amplify the template plasmid sequences in two overlapping fragments. The fragments were annealed together and the entire sequence was amplified using the adapter primers in a second round of PCR to generate linear PCR products encoding the desired VP1 u variants.

[0187] The linear PCR products were transfected into HEK293 cells, and VP1 u variant proteins were purified from conditioned media by immobilized metal affinity chromatography after 7 days.

[0188] Example 2. Screening the enzymatic activity of VP1u variants

[0189] To assess enzymatic activity of wild-type VP1 u and VP1 u variants, proteins purified from the COSMO workflow provided in Example 1 or from transfected E. coli host cells were added to 384-well plates. PLA2 catalytic activity of the VP1 u variants was evaluated using an ENZCHEK ™ Phospholipase A2 Assay Kit (Invitrogen Cat: E10217). Activity was analyzed according to the kit instructions. A schematic of the assay is shown in Fig. 1 . Briefly, in this assay, a PLA2 substrate was added to each well. Upon PLA2 catalytic activity of VP1 u or VP1 u variants, the PLA2 substrate was cleaved, which released a fluorogenic cleavage product and resulted in an increase in fluorescence. Fluorescence was measured over time using a Tecan INFINITE® M1000 plate reader.

[0190] Raw data for PLA2 catalytic activity of wild-type VP1 u and of five exemplary VP1 u variant proteins (each containing one amino acid substitution mutation selected from A98E, D87K, D84R, D80Q, and Q101 V) are shown in Fig. 2. To calculate relative reaction rate, the slope (relative fluorescence units (RU) / minute) was calculated to provide a reaction rate for each VP1 u variant. The reaction rate was then normalized to the amount of protein in each well, to give a value of RU / (min*pg). Finally, the relative reaction rate of each VP1 u variant compared to wild-type VP1 u was calculated.

[0191] Numerical values of each VP1 u variant’s PLA2 reaction rate are shown in Figs. 3 and 4; grayscale heatmaps of the reaction rates are shown in Figs. 5 and 6.

[0192] Several VP1 u variants with increased enzymatic activity compared to the wild-type VP1 u were identified. VP1 u variants comprising single amino acid substitution mutations (including S42A, D80Q, Q82A, D84R, D87K, A98E, and Q101 V single amino acid substitution mutations) showed an approximately 1 -10-fold increase in enzymatic activity (Figs. 3-6).

[0193] PLA2 reaction rates of VP1 u variants comprising a single amino acid substitution mutation selected from S42A, Q82A, D84R, D87K, A98E, and Q101 V were further validated and found to be superior to wild-type VP1 u (Table 2).

[0194] Table 2. PLA2reaction rates of selected AAV2 VP1u variants

[0195] Further, two VP1 u variants comprising a set of six high-activity amino acid substitution mutations identified in the screen were generated. Variant A comprised S42A, D80Q, Q82A, D87K, A98E, and Q101 V amino acid substitution mutations; Variant B comprised S42A, D84R, Q82A, D87K, A98E, and Q101 V amino acid substitution mutations (Table 2). Combining these high-activity amino acid substitution mutations resulted in VP1 u variants with a 42-fold (for Variant A) and 144-fold (for Variant B) increase in PLA2 reaction rate compared to wild-type VP1 u (Table 2).

[0196] Additionally, the relative yield of each VP1 u variant protein compared to wild-type VP1 u protein after purification was determined (Figs. 7 and 8).

[0197] Example 3. Generation and analysis of AAV2 VP1u variants with engineered disulfide bonds

[0198] AAV2 VP1 u variants with engineered disulfide bonds were designed using an AlphaFold predicted structure and the disulfide scan tool on Molecular Operating Environment (MOE) software. Five distinct pairs of amino acid substitution mutations ((i) L53C and G111 C; (ii) L53C and A72C; (iii) Y79C and P89C, (iv) G44C and F56C, and (v) A71 C and R103C) were generated as described above. Figs. 9 and 10 show the structures, as predicted by AlphaFold, for the pairs (i) L53C and G111 C and (ii) L53C and A72C. Relative PLA2 enzymatic activity of the variants with engineered disulfide bonds was evaluated using the kit and method described in Example 2, above. As is shown in Table 3, for two of the variants, the addition of disulfide bonds resulted in a more than 200-fold increase in enzymatic activity relative to wild-type VP1 u (a 204-fold increase for the L53C and G111 C variant and a 293-fold increase for the L53C and A72C variant). Table 3. Reaction rates of AAV2 VP1u disulfide mutants

[0199] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.

Claims

WHAT IS CLAIMED IS:1 . An adeno-associated virus (AAV) having a capsid comprising a major capsid protein VP1 comprising a VP1 -unique region (VP1 u) comprising one or more of the amino acid substitution mutations provided in Table 1 , wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

2. The AAV of claim 1 , wherein the VP1 u comprises one or more of:(a) a S42A amino acid substitution mutation;(b) a D80Q amino acid substitution mutation;(c) a Q82A amino acid substitution mutation;(d) a D84R amino acid substitution mutation;(e) a D87K amino acid substitution mutation; and(f) a Q101 V amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

3. The AAV of claim 1 or 2, wherein the VP1 u comprises only one amino acid substitution mutation selected from S42A, D80Q, Q82A, D84R, D87K, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

4. The AAV of claim 1 or 2, wherein the VP1 u further comprises an A98E amino acid substitution mutation.

5. The AAV of claim 1 or 2, wherein the VP1 u comprises two amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, A98E, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

6. The AAV of claim 5, wherein the VP1 u comprises S42A and Q101 V amino acid substitution mutations.

7. The AAV of claim 5, wherein the VP1 u comprises A98E and Q101 V amino acid substitution mutations.

8. The AAV of claim 5, wherein the VP1 u comprises D80Q and D87K amino acid substitution mutations.

9. The AAV of claim 1 or 2, wherein the VP1 u comprises three, four, or five amino acid substitution mutations selected from S42A, D80Q, Q82A, D84R, D87K, A98E, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

10. The AAV of claim 1 or 2, wherein the VP1 u comprises six amino acid substitution mutations selected from S42A, Q82A, D80Q, D84R, D87K, A98E, and Q101 V, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).1 1 . The AAV of claim 10, wherein the VP1 u comprises S42A, D80Q, Q82A, D87K, A98E, and Q101 V amino acid substitution mutations.

12. The AAV of claim 10, wherein the VP1 u comprises S42A, Q82A, D84R, D87K, A98E, and Q101 V amino acid substitution mutations.

13. The AAV of any one of claims 1 -12, wherein the VP1 u further comprises an engineered disulfide bond.

14. The AAV of claim 13, wherein the VP1 u comprises:(a) L53C and G1 1 1 C amino acid substitution mutations; or(b) L53C and A72C amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

15. An AAV having a capsid comprising a major capsid protein VP1 comprising a VP1 u comprising:(a) L53C and G1 1 1 C amino acid substitution mutations; or(b) L53C and A72C amino acid substitution mutations, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ ID NO: 4).

16. The AAV of claim 15, wherein the VP1 u further comprises one or more of:(a) a S42A amino acid substitution mutation;(b) a D80Q amino acid substitution mutation;(c) a Q82A amino acid substitution mutation;(d) a D84R amino acid substitution mutation;(e) a D87K amino acid substitution mutation;(f) a Q101 V amino acid substitution mutation; and(g) an A98E amino acid substitution mutation, wherein the amino acid substitution mutations are numbered relative to the AAV2 VP1 u (SEQ IDNO: 4).

17. The AAV of any one of claims 1 -16, wherein the capsid comprises:(a) a major capsid protein VP1 comprising an amino acid substitution mutation that increases the affinity of the AAV for AAVR;(b) a minor capsid protein VP2 comprising an amino acid substitution mutation that increases the affinity of the AAV for AAVR; and / or(c) a minor capsid protein VP3 comprising an amino acid substitution mutation that increases the affinity of the AAV for AAVR.

18. The AAV of any one of claims 1 -17, wherein the capsid comprises a major capsid protein VP1 , minor capsid protein VP2, and / or minor capsid protein VP3 comprising one or more further amino acid substitution mutations and / or comprising one or more amino acid insertions or deletions.

19. The AAV of any one of claims 1 -18, wherein the serotype of the AAV is AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1 , or AAV12.

20. The AAV of any one of claims 1 -19, wherein the serotype of the AAV is AAV2 or a variant thereof.21 . The AAV of any one of claims 1 -20, wherein the AAV has an increased rate of phospholipase activity relative to an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

22. The AAV of any one of claims 1 -21 , wherein the AAV has an increased rate of endosomal escape relative to an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

23. The AAV of any one of claims 1 -22, wherein the AAV has an increased rate of delivery to the nucleus of a target cell relative to an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

24. The AAV of any one of claims 1 -23, wherein the AAV transduces a target cell at a lower dose than an AAV having a capsid that does not comprise any of the indicated amino acid substitution mutations.

25. A method of improving transduction efficiency of an AAV, the method comprising providing the AAV of any one of claims 1 -24.

26. The method of claim 25, wherein the method comprises contacting a target cell with the AAV, thereby improving transduction efficiency.

27. The method of claim 25 or 26, wherein the transduction efficiency is improved relative to the transduction efficiency of an AAV having a capsid not comprising any of the indicated amino acid substitution mutations.

28. A method of delivering a cargo to a cell, the method comprising contacting a target cell with the AAV of any one of claims 1 -24.

29. The method of claim 28, wherein the cargo is a nucleic acid.

30. A method of increasing the rate of delivery of an AAV to the nucleus of a target cell, the method comprising providing the AAV of any one of claims 1 -24.31 . A method of transduction of a target cell by an AAV at a lower dose of the AAV, the method comprising providing the AAV of any one of claims 1 -24.

32. The method of claim 30 or 31 , wherein the method comprises contacting the target cell with the AAV.

33. The method of any one of claims 23, 24, 26, and 28-32, 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.