ITR-modified AAV vectors

Abstract

Provided herein are modified AAV genomes, AAV particles comprising the modified genomes, compositions thereof, and methods of their use.

Description

T19325 Attorney Docket No. U1202.70155WO00 ITR-MODIFIED AAV VECTORS RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63 / 633,592, entitled “ITR-MODIFIED AAV VECTORS”, filed on April 12, 2024, the entire contents of which are incorporated herein by reference. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (U120270155WO00-SEQ-COB.xml; Size: 107,618 bytes; and Date of Creation: April 10, 2025) are herein incorporated by reference in their entirety. FIELD OF THE INVENTION

[0003] The application relates to AAV based gene therapy vector and modifications thereto. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant No. R21 AR081018, awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0005] Gene therapy has the potential to treat subjects suffering from or at risk of suffering from various diseases. Improved adeno-associated virus (AAV) vectors for carrying genetic payloads would be beneficial to the development of gene therapies. Many AAV vectors are limited by low transduction efficiency and transgene expression. SUMMARY

[0006] Provided are nucleic acid vectors with modified adeno-associated virus (AAV) inverted terminal repeats (ITRs) that comprise a transcription factor binding site sequence introduced in a D-sequence of an ITR and nucleotides that are complementary to the transcription factor (TF)T19325 Attorney Docket No. U1202.70155WO00 binding site sequence introduced in a stem A-domain of the ITR such that the stability of the terminal resolution site (trs) of the ITR is maintained.

[0007] In some aspects, the ITR comprises a stem A-domain and a D-sequence, wherein the D- sequence comprises about 20 nucleotides and is modified to introduce a sequence of a TF binding site and the stem A-domain contains a wild-type 6-nucleotide sequence comprising a terminal resolution site and the stem A-domain is modified to comprise nucleotides complementary to the sequence of the TF binding site of the D-sequence.

[0008] In some aspects, the ITR is a 5’ ITR or a 3’ ITR.

[0009] In some aspects, the ITR is an AAV 5’ ITR.

[0010] In some aspects, the TF binding site is inserted into the D-sequence. In some aspects, the TF binding site replaces the D-sequence. In some aspects, one or more nucleotides are replaced in the D-sequence to create the TF binding site.

[0011] In some aspects, the stem A-domain comprises the terminal resolution site upstream (5’ relative) to the sequence comprising nucleotides complementary to the sequence of the TF binding site.

[0012] In some aspects, the nucleic acid vector comprises about 10 nucleotides of the D- sequence. In some aspects, the nucleic acid vector comprises about 5 nucleotides of the D- sequence.

[0013] In some aspects, the D-sequence is modified to introduce at least two sequences of TF binding sites. In some aspects, the at least two sequences of TF binding sites are the same. In some aspects, the at least two sequences of TF binding sites are different.

[0014] In some aspects, the stem A-domain is modified to comprise a sequence complementary to the sequence of one of the at least two sequences of TF binding sites. In some aspects, the stem A-domain is modified to comprise the sequence complementary to the sequence of one of the at least two sequences of TF binding sites directly upstream (5’ relative) to the terminal resolution site.

[0015] In some aspects, the nucleic acid vector further comprising a heterologous sequence comprising a gene of interest.

[0016] In some aspects, the heterologous sequence further comprises a promoter.

[0017] In some aspects, at least one TF binding site binds a TF that is active in a target cell type. In some aspects, the target cell type is a target cell type in which the promoter is active.

[0018] In some aspects, the target cell type is a muscle cell, liver cell, CNS cell, or retinal cell.T19325 Attorney Docket No. U1202.70155WO00

[0019] In some aspects, the TF is active in a muscle cell. In some aspects, the TF is selected from a myocyte determination protein 1 (MyoD) and a myocyte enhancer factor 2C (MEF2C). In some aspects, the TF is a glucocorticoid receptor (GRE).

[0020] In some aspects, the TF is active in a retinal cell. In some aspects, the TF is selected from a Cone-Rod Homeobox (CRX), Neural Retina Leucine Zipper (NRL), and Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3).

[0021] In some aspects, the TF is active in a CNS cell. In some aspects, the TF is selected from a Hes Family BHLH Transcription Factor (HES) 1, HES5, Recombinant Signal Binding Protein for Immunoglobulin Kappa J Region (CBF-1), SYR-Box Transcription Factor 2 (SOX2), SOX1, SOX3, High Mobility Group AT-Hook 2 (HGMA2), BMI Proto-Oncogene, Polycomb Ring Finger (BMI1), GLI Family Zinc Finger (GLI) 2, GLI3, Inhibitor Of DNA Binding (ID) 2, and ID4.

[0022] In some aspects, the TF is active in a liver cell. In some aspects, the TF is selected from a Hepatocyte Specific Cis-Acting Regulatory Module (HS-CRM) 8 and a HS-CRM14.

[0023] Further provided are AAV particles comprising a nucleic acid vector described herein.

[0024] In some aspects, the AAV particle comprises a capsid protein selected from AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVvrh74, their various tyrosine, serine, threonine, and lysine mutants and combinations thereof.

[0025] Also provided are compositions comprising a nucleic acid vector described herein or a AAV particle described herein.

[0026] In some aspects, the composition is a pharmaceutical composition. In some aspects, the composition further comprises a pharmaceutically acceptable excipient.

[0027] Provided are methods comprising contacting a cell with a AAV particle described herein or a composition described herein.

[0028] In some aspects, the cell is a human cell.

[0029] In some aspects, the cell expresses the gene of interest at least 2-times higher compared to a cell contacted with an AAV particle or a composition comprising an AAV particle that does not contain a modified D-sequence and a modified stem A-domain.

[0030] Further provided are methods comprising administering an AAV particle described herein or a composition described herein to a subject in need thereof.

[0031] In some aspects, the subject is a human.T19325 Attorney Docket No. U1202.70155WO00

[0032] In some aspects, the subject expresses the gene of interest at least 2-times higher compared to a subject administered an AAV particle or a composition comprising an AAV particle that does not contain a modified D-sequence and a modified stem A-domain. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0034] FIGs.1A-1D: FIGs.1A-1B show schematic structures of the WT AAV ITR (FIG.1A) and an AAV ITR modified with a MyoD and a MEF transcription factor binding site (GenM) (FIG.1B). FIGs.1C-1D show transduction efficiency of WT and GenM AAVrh74 vectors in differentiated mouse C2C12 (FIG.1C) and primary human skeletal muscle (FIG.1D) cells.

[0035] FIGs.2A-2D: FIG.2A shows a schematic structure of an AAV-ITR modified with a GRE transcription factor binding site sequence (DeltA ITR). FIG.2B shows secondary structures of the trs sites in the WT and the DeltA ITRs. FIG.2C shows transduction efficiency of WT and DeltA AAVrh74 vectors in primary human skeletal muscle cells in vitro. FIG.2D shows transduction efficiency of WT and DeltA AAVrh74 vectors in mouse gastrocnemius skeletal muscle cells in vivo following intramuscular injections.

[0036] FIGs.3A-3C show schematic structures of AAV-ITRs modified with a CRX transcription factor binding site sequence (FIG.3A), a NRL transcription factor binding site sequence (FIG.3B) and a NR2E3 transcription factor binding site sequence (FIG.3C).

[0037] FIG.4 shows a schematic structure of an AAV-ITR modified with a GRE binding site sequence in which three nucleotides (bolded) have been modified to be complementary to nucleotides between the RBE and the A-domain (modified DeltA ITR).

[0038] FIGs.5A-5C show schematic structures of stem-loops formed from portions of wild- type ITR (FIG.5A), DeltA ITR (FIG.5B), or modified DeltA ITR (FIG.5C), showing the additional complementarity of the modified DeltA ITR relative to the DeltA ITR. DETAILED DESCRIPTION

[0039] The present disclosure is based at least in part on the development of adeno-associated virus (AAV) genomes and particles useful in the delivery of various cargoes to cells,T19325 Attorney Docket No. U1202.70155WO00 facilitating efficient transgene expression therein. The disclosure relates, at least in part, to the finding that incorporation of sequence modifications into AAV genomes results in improvements in various characteristics of AAVs, such as packaging, transduction efficiency, transgene expression, etc. These improvements may result from increased second-strand synthesis of the AAV genome and / or increased activation of transgene expression from a promoter present in an expression cassette, resulting from modifications described therein. In some aspects, the increased expression of a transgene from an expression cassette is provided by the inclusion of one or more transcription factor binding sites, e.g., binding sites for muscle, liver, CNS, or retina-specific transcription factors, in a D-sequence of an AAV ITR and inclusion of a sequence complementary to such transcription factor binding site in a stem A- domain thereby maintaining and stabilizing the AAV ITR trs site. The ITR described herein and its complement may be present in a plasmid. The nucleic acid sequences comprising modified ITRs as described herein may be present in a plasmid. The nucleic acid sequences comprising modified ITRs as described herein may be present in a single stranded AAV genome. The nucleic acid sequences comprising modified ITRs as described herein may be present in a double-stranded AAV genome. In some aspects, a wildtype AAV genome, e.g., a genome of pSub201 disclosed herein, can comprise modified ITRs as described herein. In some aspects, a wildtype AAV genome is replaced with an expression cassette comprising a transgene operably linked to a promoter to prepare a recombinant AAV genome that comprises modified ITRs as described herein. In some aspects, the transcription factor binding site sequences introduced into the D-sequence and stem A-domain of the ITR of an AAV genome increase expression of a transgene present in the expression cassette in a cell in which the transcription factor is present. In some aspects, the recombinant AAV vectors comprising modified ITRs comprising transcription factor binding site sequences in a D-sequence and a stem A-domain express a transgene in a specific cell or tissue in which the transcription factor is present. The AAV genomes, particles, etc., described herein may be used in a variety of applications including but not limited to compositions and methods (e.g., therapeutic and diagnostic methods). Therapeutic and diagnostic methods disclosed herein include those useful in the treatment and diagnosis of various diseases, disorders, and conditions, in subjects in need thereof.

[0040] Provided herein are AAV genomes (e.g., genomes comprising sequence modifications), AAV particles comprising the AAV genomes described herein, compositions comprising AAV particles (e.g., infectious AAV particles), and methods of using the AAV particles and / orT19325 Attorney Docket No. U1202.70155WO00 compositions for transducing cells of interest (e.g., for treating or diagnosing a disease or condition in a subject. Nucleic Acid Vectors (e.g., AAV Genomes)

[0041] Provided are nucleic acid vectors comprising at least one inverted terminal repeat (ITR) comprising a modification that increases transduction efficiency of an AAV vector containing the nucleic acid vector. In some aspects, the nucleic acid vectors (e.g., AAV genomes, e.g., recombinant AAV (rAAV) genomes) described herein may be encapsidated within wild-type AAV capsids of various AAV serotypes as described herein, or any one of the AAV capsids variants (e.g., comprising a capsid protein comprising one or more amino acid substitutions) described herein.

[0042] In some aspects, a nucleic acid vector comprises an expression cassette. In some aspects, an expression cassette comprises a promoter operably linked to a transgene. In some aspects, a nucleic acid vector comprises an AAV ITR modified to increase transgene expression. In some aspects, a nucleic acid vector (e.g., an AAV genome) comprises an AAV ITR modified to improve (e.g., increase the rate, efficiency, etc.) second-strand synthesis and / or activity of a promoter present in an expression cassette of the nucleic acid vector. In some aspects, an ITR as provided herein is a 5' ITR, i.e. an ITR that is 5' from a transgene in a nucleic acid vector (e.g., an AAV genome). In some aspects, an ITR as provided herein is a 3' ITR, i.e. an ITR that is 3' from a transgene in a nucleic acid vector (e.g., an AAV genome). An ITR serves as an origin of replication and is comprised of two arm palindromes (B-B' and C- C') embedded in a larger stem palindrome (A-A', stem A-domain), and a single stranded sequence (D-sequence). An AAV ITR can be in flip or flop configurations. See e.g., Mroske, et al., Human Gene Therapy Methods 23(2):128-36 (2012). In some aspects, an ITR has the B- B' and the C-C' palindrome closest to the 3' end. In wild-type ITRs, the D-sequence is present only once at each end of the genome thus remaining single-stranded. The D-sequence is also referred to as the “D-element” in the art. The D-sequence consists of 20 or approximately 20 (e.g., 19, 20, 21, 22, 23, 24, etc.) nucleotides adjacent to the terminal resolution site (trs), and generally consists or consists essentially of the medial 20 (or approximately 20) nucleotides of the ITR (where “medial” indicates the segment of the ITR that is adjacent to the center of the AAV genome). The first nucleotide of the D-sequence in a wildtype AAV2 is generally at position 126 or approximately position 126 from the terminus of the AAV genome. See, e.g., Yan, et al., “Inverted Terminal Repeat Sequences Are Important for IntermolecularT19325 Attorney Docket No. U1202.70155WO00 Recombination and Circularization of Adeno-Associated Virus Genomes” J Virol.79(1):364- 379 (2005); Earley, et al., “Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression” Hum Gene Ther.31(3-4):151-162 (2020); Shitik, et al., “AAV- based vector improvements unrelated to capsid protein modification” Front Med.10:1106085 (2023), doi: 10.3389 / fmed.2023.1106085; Savy, et al., “Impact of Inverted Terminal Repeat Integrity on rAAV8 Production Using the Baculovirus / Sf9 Cells System” Hum Gene Ther Methods 28(5): 277-289 (2017); Wilmott, et al., “A User's Guide to the Inverted Terminal Repeats of Adeno-Associated Virus” Hum Gene Ther Methods 30(6): 206-213 (2019); and Zhou, et al., “Deletion of the B-B’ and C-C’ regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression” Sci Rep.7:5432 (2017), doi: 10.1038 / s41598-017-04054-4; the entire contents of each of which are herein incorporated by reference.

[0043] In some aspects, a nucleic acid vector (e.g., an AAV genome) as provided herein comprises a 5’ITR and a 3’ ITR. Modified AAV ITRs

[0044] In some aspects, a nucleic acid vector (e.g., an AAV genome) comprises a modification (e.g., a deletion, a substitution, or an insertion) of either a left 5’ ITR or a right 3’ ITR, but not both (i.e., the nucleic acid vector comprises a modification of only one ITR). In some aspects, a nucleic acid vector (e.g., an AAV genome) comprises a modification (e.g., a deletion, a substitution, or an insertion) of both a 5’ ITR and a 3’ ITR.

[0045] In some aspects, a 5’ AAV ITR is modified. In some aspects, a 3’ AAV ITR is modified. In some aspects, a 5’ ITR and a 3’ ITR are modified.

[0046] In some aspects, a modification of an ITR comprises a substitution, a deletion and / or an insertion. In some aspects, a modification of an ITR comprises a substitution of at least one nucleotide of a D-sequence. In some aspects, a modification of an ITR comprises a substitution of at least one nucleotide of a stem A-domain sequence. In some aspects, a modification of an ITR comprises a deletion of at least one nucleotide of a D-sequence. In some aspects, a modification of an ITR comprises a deletion of at least one nucleotide of a stem A-domain sequence. In some aspects, a modification of an ITR comprises an insertion of at least one nucleotide of a D-sequence. In some aspects, a modification of an ITR comprises an insertion of at least one nucleotide of a stem A-domain sequence. In some aspects, a modification of an ITR comprises a substitution of at least one nucleotide of a D-sequence and a deletion of atT19325 Attorney Docket No. U1202.70155WO00 least one nucleotide of a D-sequence. In some aspects, a modification of an ITR comprises an insertion of at least one nucleotide of a D-sequence and a deletion of at least one nucleotide of a D-sequence. In some aspects, a modification of an ITR comprises a substitution of at least one nucleotide of a D-sequence and an insertion of at least one nucleotide of a D-sequence. In some aspects, a modification of an ITR comprises a substitution of at least one nucleotide of a stem A-domain sequence and a deletion of at least one nucleotide of a stem A-domain sequence. In some aspects, a modification of an ITR comprises an insertion of at least one nucleotide of a stem A-domain sequence and a deletion of at least one nucleotide of a stem A- domain sequence. In some aspects, a modification of an ITR comprises a substitution of at least one nucleotide of a stem A-domain sequence and an insertion of at least one nucleotide of a stem A-domain sequence.

[0047] In some aspects, a modification of an ITR comprises a substitution of at least one nucleotide of a D-sequence and a substitution of at least one nucleotide of a stem A-domain sequence. In some aspects, a modification of an ITR comprises an insertion of at least one nucleotide of a D-sequence and an insertion of at least one nucleotide of a stem A-domain sequence. In some aspects, a modification of an ITR comprises a deletion of at least one nucleotide of a D-sequence and a deletion of at least one nucleotide of a stem A-domain sequence. In some aspects, a modification of an ITR comprises a substitution and / or insertion and / or deletion of at least one nucleotide of a D-sequence and a substitution and / or deletion and / or deletion of at least one nucleotide of a stem A-domain sequence.

[0048] In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of the 5’ ITR D-sequence located directly 5’ to the terminal resolution site are modified. In some aspects, about 5 to about 10 nucleotides, e.g., 7 nucleotides of the 5’ ITR D- sequence located directly 5’ to the terminal resolution site are modified. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 nucleotides of the 5’ ITR stem A-domain sequence located 3’ to the terminal resolution site are modified. In some aspects, 4-11, 3-11, or 2-11 nucleotides of the 5’ ITR stem A-domain sequence located 3’ to the terminal resolution site are modified, e.g., 3-7, 4-7, 5-7, or 6-7 nucleotides of the 5’ ITR stem A-domain sequence located 3’ to the terminal resolution site are modified.

[0049] In some aspects, a modification of an ITR comprises a deletion or substitution or insertion of part of a D-sequence (e.g., the proximal 10 nucleotides of the ITR, located 5’ relative to the terminal resolution site). In some aspects,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of the 5’ ITR D-sequence located directly 5’ to theT19325 Attorney Docket No. U1202.70155WO00 terminal resolution site of the nucleic acid vector are deleted and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides are inserted in the 5’ ITR located directly 5’ to the terminal resolution site. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of the 5’ ITR D-sequence located directly 5’ to the terminal resolution site of the nucleic acid vector are substituted. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides of the 3’ ITR D-sequence located directly 3’ to the terminal resolution site of the nucleic acid vector are deleted and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides are inserted in the 3’ ITR located directly 3’ to the terminal resolution site. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of the 3’ ITR D-sequence located directly 3’ to the terminal resolution site of the nucleic acid vector are substituted.

[0050] In some aspects, a modification in a D-sequence and a modification in a stem A- domain comprise an introduction of nucleotides that are complementary to each other. As used herein, “complementary” refers to two sequences comprising matching nucleotides that can form a double-stranded structure, where “matching nucleotides,” refers to nucleotides with guanine and cytosine bases and nucleotides with adenine and thymine bases. The term “completely complementary” refers to two sequences comprising complementary nucleotides along the entire length of the two sequences. In some aspects, the nucleotides introduced in the D-sequence and the nucleotides introduced in the stem A-domain are completely complementary over a length of about 3, 4, 5, 6, or 7 nucleotides. The term “about,” as used herein, refers to a value that is similar to a stated value and within a range of values that fall within 25% in either direction (greater than or less than). In some aspects, the nucleotides introduced in the D-sequence and the nucleotides introduced in the stem A-domain are incompletely complementary to each other over a length of about 3, 4, 5, 6, or 7 nucleotides. The term “incompletely complementary” refers to two sequences comprising nucleotides that are complementary to each other and nucleotides that are not complementary to each other along the length of the two sequences. For example, some of the nucleotides introduced in the D-sequence and some of the nucleotides introduced in the stem A-domain can be complementary to each other and other nucleotides introduced in the D-sequence and the stem A-domain can be non-complementary. In some aspects, more of the nucleotides introduced in the D-sequence are complementary to the nucleotides introduced in the stem A-domain than non-complementary. For example, a D-sequence and a stem A-domain can comprise a 7- nucleotide modification, wherein 6 nucleotides are complementary and one nucleotide is non-T19325 Attorney Docket No. U1202.70155WO00 complementary. In some aspects, the non-complementary nucleotides are internal within a modified sequence. For example, a non-complementary nucleotide can be located in the third, fourth, or fifth position of a 7-nucleotide modification. In some aspects, a modification in a D- sequence and a modification in a stem A-domain comprise more than one non-complementary nucleotide. For example, a D-sequence can comprise a 11-nucleotide modification and a stem A-domain comprises a 7-nucleotide modification, wherein two non-complementary nucleotides are present, e.g., at the fifth and sixth position of the D-sequence modification, or at any two nucleotide positions between the second and tenth position of the modified D- sequence such that at least 1 nucleotide at each end of the modified sequences is complementary. In some aspects, a complementarity between nucleotides introduced in a D- sequence and a stem A-domain sequence of an AAV ITR is similar in extent to the complementarity between the D-sequence and the stem A-domain sequence of the ITR in the wildtype AAV. For example, if a wildtype AAV ITR comprises 7 incompletely complementary nucleotides between its D-sequence and its stem A-domain, the nucleotides introduced in the D-sequence and stem A-domain of a modified ITR of such AAV can also comprise 7 incompletely complementary nucleotides between a modified D-sequence and a modified stem A-domain.

[0051] In some aspects, different numbers of nucleotides are introduced in a D-sequence and a stem A-domain. For example, a 16-nucleotide sequence can be introduced in the D-sequence and a 7-nucleotide sequence can be introduced in the stem A-domain. In some aspects, a 20- nucleotide sequence is introduced in the D-sequence and a 7-nucleotide sequence is introduced in the stem A-domain. In some aspects, a 14-nucleotide sequence is introduced in the D- sequence and a 6-nucleotide sequence is introduced in the stem A-domain.

[0052] In some aspects, the same number of nucleotides are introduced in the D-sequence and the stem A-domain. In some aspects, a 7-nucleotide sequence is introduced in the D-sequence and a 7-nucleotide sequence is introduced in the stem A-domain.

[0053] In some aspects, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides are introduced in the 5’ ITR D-sequence directly 5’ to the terminal resolution site and about 3, 4, 5, 6, or 7 nucleotides that are complementary to the nucleotides introduced in the D-sequence are introduced in the stem A-domain.

[0054] In some aspects, about 3, 4, 5, 6, or 7 nucleotides are introduced in the stem A-domain located at least 6 nucleotides 3’ to the terminal resolution site.T19325 Attorney Docket No. U1202.70155WO00

[0055] In some aspects, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides are introduced in the 3’ ITR D-sequence directly 3’ to the terminal resolution site and about 3, 4, 5, 6, or 7 nucleotides that are complementary to the introduced nucleotides in the D-sequence are introduced in the stem A-domain.

[0056] In some aspects, the introduced nucleotides are introduced into the D-sequence in a discontinuous manner such that the modified sequence comprises a mixture of wildtype D- sequence nucleotides and introduced nucleotides.

[0057] In some aspects, the introduced nucleotides are introduced into the stem A-domain sequence in a discontinuous manner such that the modified sequence comprises a mixture of wildtype stem A-domain sequence nucleotides and introduced nucleotides.

[0058] In some aspects, nucleotides introduced in the 5’ ITR D-sequence directly 5’ to the terminal resolution site comprise a combination of non-consecutive substituted and non- consecutive wildtype D-sequence nucleotides. In some aspects, nucleotides introduced in the 5’ ITR D-sequence directly 5’ to the terminal resolution site comprise a combination of consecutive substituted and consecutive wildtype D-sequence nucleotides. For example, a combination can comprise consecutive substituted-consecutive wildtype-consecutive substituted nucleotides, wherein two sequences of consecutive substituted nucleotides are different. In some aspects, a combination comprises consecutive substituted-consecutive substituted-consecutive wildtype nucleotides, wherein the two sequence of consecutive substituted nucleotides are different. In some aspects, a combination comprises consecutive wildtype-consecutive substituted-consecutive wildtype nucleotides. In some aspects, the combination comprises consecutive wildtype- consecutive substituted- consecutive substituted nucleotides, wherein the two sequences of consecutive substituted nucleotides are different.

[0059] In some aspects, a nucleic acid vector comprising a modified ITR comprising a modification in the D-sequence and a stem A-domain comprises an intact wildtype AAV terminal resolution site. In some aspects, the modified ITR comprising a modification in the D- sequence and a modification in a stem A-domain comprises at least 6 nucleotides that are wildtype ITR nucleotides and include the terminal resolution site. In some aspects, the modified ITR comprises about 6 to about 14 nucleotides that are wildtype ITR nucleotides and include the terminal resolution site.

[0060] In some aspects, a D-sequence comprises the sequence CTCCATCACTAGGGGTTCCT (SEQ ID NO: 1) of the wild-type AAV2 ITR, or a corresponding sequence of a different serotype ITR. In some aspects, a D-sequence is definedT19325 Attorney Docket No. U1202.70155WO00 by the sequence CTCCATCACTAGGGGTTCCT (SEQ ID NO: 1). In some aspects, a sequence substituted in an ITR as described herein may be a sequence that is a “heterologous nucleotide sequence.”

[0061] As used herein, a “heterologous” nucleotide sequence or nucleic acid sequence refers to a sequence that is not native to an AAV or naturally-occurring in an AAV. In some aspects, nucleotides that are introduced in a D-sequence and a stem A-domain of an AAV ITR form heterologous nucleotide sequences. In some aspects, a heterologous sequence introduced directly 5’ to the terminal resolution site replaces 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ,15, 16, 17, 18, 19 or 20 nucleotides of a 5’ ITR D-sequence. The terms “replaces” and “substitutes” are used interchangeably herein.

[0062] In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14 ,15,16, 17, 18, 19 or 20 nucleotides of a 5’ ITR D-sequence are deleted and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides of a heterologous sequence are introduced. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ,15, 16, 17, 18, 19 or 20 nucleotides of a 3’ ITR D-sequence are deleted and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ,15, 16, 17, 18, 19 or 20 nucleotides of a heterologous sequence are introduced.

[0063] A heterologous sequence can be a sequence comprising e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 3536, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length. In some aspects, a heterologous sequence introduced in a D-sequence can be about 1 to about 500 nucleotides, about 5 to about 400 nucleotides, about 10 to about 300 nucleotides, about 20 to about 200 nucleotides, about 25 and about 150 nucleotides, about 30 and about 100 nucleotides, about 40 and about 80 nucleotides or can be a longer sequence. In some aspects, a heterologous sequence introduced in a D-sequence can be about 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 nucleotides in length, or longer. In some aspects, a sequence introduced in a D- sequence can be of any length such that the length of the entire AAV genome including, e.g., an expression cassette or a nuclei acid of interest, is within a length that is effectively packaged into an AAV capsid. In some aspects, a nucleic acid comprises a short (e.g., 1 nucleotide to 500 nucleotides or longer) heterologous sequence to be expressed in a cell and the nucleic acid further comprises a sequence introduced in a D-sequence that can be of a length such that theT19325 Attorney Docket No. U1202.70155WO00 length of the entire AAV genome is within a length that is effectively packaged into an AAV capsid. In some aspects, a sequence introduced in a D-sequence can be of any length as described herein as long as the first 7 nucleotides directly 5’ to the trs site are complementary to 7 nucleotides of the stem A-domain.

[0064] In some aspect, a heterologous sequence comprises a transcription factor binding site sequence and further comprises a sequence comprising an expression cassette (e.g., encoding a gene of interest) or a non-coding sequence (e.g., inserted in or replacing a regulatory or structural portion of an AAV nucleic acid, such as an AAV genome). In some aspects, a heterologous sequence comprises more than one transcription factor binding site sequence. In some aspects, one transcription factor binding site sequence is introduced in a D-sequence of an ITR and nucleotides complementary to such transcription factor binding site sequence are introduced in a stem A-domain of the ITR. In some aspects, a sequence of between about 6 to about 14 nucleotides of wildtype ITR sequence including a wildtype terminal resolution site (trs) are located between the transcription factor binding site sequence introduced in the D- sequence and nucleotides complementary to such transcription factor binding site sequence introduced in the stem A-domain. In some aspects, more than one transcription factor binding site sequence is introduced in the D-sequence and nucleotides complementary to one such transcription factor binding site sequence are introduced in the stem A-domain sequence. In some aspects, more than one transcription factor binding site sequence is introduced in the D- sequence and nucleotides complementary to more than one such transcription factor binding site sequence are introduced in the stem A-domain sequence, wherein a sequence of at least 6 nucleotides of wildtype ITR sequence including a wildtype trs site are located between the transcription factor binding site sequence introduced in a D-sequence and the nucleotides complementary to the transcription factor binding site sequence introduced in the stem A- domain. In some aspects, more than one transcription factor binding site sequence is introduced in the D-sequence and nucleotides complementary to more than one such transcription factor binding site sequence are introduced in the stem A-domain sequence, wherein about 7 nucleotides of the more than one transcription factor binding site sequence introduced in the D-sequence and about 7 nucleotides of the more than one such transcription factor binding site sequence introduced in the stem A-domain sequence are complementary. In some aspects, the about 7 complementary nucleotides of the D-sequence are located directly 5’ of the trs site and the about 7 complementary nucleotides of the stem A-domain sequence are located about 6 to about 14 nucleotides 3’ of the trs site.T19325 Attorney Docket No. U1202.70155WO00

[0065] In some aspects, a transcription factor binding site sequence can be a transcription factor binding site that binds a transcription factor in a specific cell type.

[0066] In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a muscle cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a skeletal muscle cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a cardiac muscle cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a liver cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a hepatocyte. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a liver sinusoidal endothelial cell.

[0067] In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a CNS cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a neuron. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in an astrocyte. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a glial cell.

[0068] In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a retinal cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a retinal pigment epithelial cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in an outer segment layer cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in an inner segment layer cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in an inner plexiform layer cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in an outer plexiform layer cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in a ganglion cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in an inner nuclear layer cell. In some aspects, a transcription factor binding site sequence can be a sequence that binds a transcription factor in an outer nuclear layer cell.

[0069] In some aspects, a transcription factor binding site sequence comprises a Myogenic Differentiation 1 (MyoD1) (UniprotKB / Swiss-Prot: P15172) binding site sequence. In someT19325 Attorney Docket No. U1202.70155WO00 aspects, a MyoD transcription factor binding site sequence comprises a sequence of AGCAGCTGCT (SEQ ID NO: 2).

[0070] In some aspects, a transcription factor binding site sequence comprises a myocyte enhancer factor 2C (MEF2C) (UniprotKB / Swiss-Prot: Q02078) binding site sequence. In some aspects, a MEF2C transcription factor binding site sequence comprises a sequence of CTAAAAATAG (SEQ ID NO: 3).

[0071] In some aspects, a transcription factor binding site sequence comprises a glucocorticoid receptor-binding element (GRE) binding site sequence. In some aspects, a GRE transcription factor binding site sequence comprises a sequence of GTCTTGTCCTACAAGA (SEQ ID NO: 4). In some aspects, a GRE transcription factor binding site sequence comprises a sequence of GTCTTGTAGAACAAGA (SEQ ID NO: 67).

[0072] In some aspects, a transcription factor binding site sequence comprises a Cone-Rod Homeobox (CRX) (UniprotKB / Swiss-Prot: O43186) binding site sequence. In some aspects, a CRX transcription factor binding site sequence comprises a sequence of GATTAGG.

[0073] In some aspects, a transcription factor binding site sequence comprises a Neural Retina Leucine Zipper (NRL) (UniprotKB / Swiss-Prot: P54845) binding site sequence. In some aspects, a NRL transcription factor binding site sequence comprises a sequence of GACGGCTTAGTCGG (SEQ ID NO: 6).

[0074] In some aspects, a transcription factor binding site sequence comprises a Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3) (UniprotKB / Swiss-Prot: Q9Y5X4) binding site sequence. In some aspects, a NR2E3 transcription factor binding site sequence comprises a sequence of GCTCCGATTTCCGAT (SEQ ID NO: 7).

[0075] In some aspects, a transcription factor binding site sequence comprises a Hes Family BHLH Transcription Factor 1 (HES1) (UniprotKB / Swiss-Prot: Q14469) binding site sequence comprises a sequence of GGCACGTGGC (SEQ ID NO: 8).

[0076] In some aspects, a transcription factor binding site sequence comprises a Hes Family BHLH Transcription Factor 5 (HES5) (UniprotKB / Swiss-Prot: Q5TA89) binding site sequence comprises a sequence of CGGCACGTGCCA (SEQ ID NO: 9).

[0077] In some aspects, a transcription factor binding site sequence comprises a Recombinant Signal Binding Protein for Immunoglobulin Kappa J Region (CBF-1) (UniprotKB / Swiss-Prot: Q06330) binding site sequence comprises a sequence of CCGTGGGAAAA (SEQ ID NO: 10).T19325 Attorney Docket No. U1202.70155WO00

[0078] In some aspects, a transcription factor binding site sequence comprises a SYR-Box Transcription Factor 2 (SOX2) (UniprotKB / Swiss-Prot: P48431) binding site sequence comprises a sequence of GAACAATAACATTGTTC (SEQ ID NO: 11).

[0079] In some aspects, a transcription factor binding site sequence comprises a High Mobility Group AT-Hook 2 (HGMA2) (UniprotKB / Swiss-Prot: P52926) binding site sequence comprises a sequence of CCGCAATAAA (SEQ ID NO: 12).

[0080] In some aspects, a transcription factor binding site sequence comprises a BMI Proto- Oncogene, Polycomb Ring Finger (BMI1) (UniprotKB / Swiss-Prot: P35226) binding site sequence comprises a sequence of TCCCAGCTACTTTGGGA (SEQ ID NO: 13).

[0081] In some aspects, a transcription factor binding site sequence comprises a GLI Family Zinc Finger 2 (GLI2) (UniprotKB / Swiss-Prot: P10070) binding site sequence comprises a sequence of GTGGGTGGTCT (SEQ ID NO: 14).

[0082] In some aspects, a transcription factor binding site sequence comprises a GLI Family Zinc Finger 3 (GLI3) (UniprotKB / Swiss-Prot: P10071) binding site sequence comprises a sequence of GTGGGTGGTCT (SEQ ID NO: 15).

[0083] In some aspects, a transcription factor binding site sequence comprises a SYR-Box Transcription Factor 1 (SOX1) (UniprotKB / Swiss-Prot: O00570) binding site sequence comprises a sequence of AACAATAACATTGTT (SEQ ID NO: 16).

[0084] In some aspects, a transcription factor binding site sequence comprises a SYR-Box Transcription Factor 3 (SOX3) (UniprotKB / Swiss-Prot: P41225) binding site sequence comprises a sequence of AGAACAATGG (SEQ ID NO: 17).

[0085] In some aspects, a transcription factor binding site sequence comprises a Inhibitor Of DNA Binding 2 (ID2) (UniprotKB / Swiss-Prot: Q02363) binding site sequence comprises a sequence of GCACGTGA.

[0086] In some aspects, a transcription factor binding site sequence comprises a Inhibitor Of DNA Binding 4 (ID4) (UniprotKB / Swiss-Prot: P47928) binding site sequence comprises a sequence of TACACCTGTC (SEQ ID NO: 19).

[0087] In some aspects, a transcription factor binding site sequence comprises a Hepatocyte Specific Cis-Acting Regulatory Module 8 (HS-CRM8) (NM_000295.4) binding site sequence. In some aspects, a HS-CRM8 comprises the sequence GGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAG GAGCAAACAGGGGCTAAGTCCAC (SEQ ID NO: 20).T19325 Attorney Docket No. U1202.70155WO00

[0088] In some aspects, a transcription factor binding site sequence comprises a Hepatocyte Specific Cis-Acting Regulatory Module 14 (HS-CRM14) (NM_000295.4) binding site sequence. In some aspects, a HS-CRM14 comprises the sequence ATCGCCAGGTCACCTGAGGAGTTAATGAATACATATCTCCT (SEQ ID NO: 21).

[0089] In some aspects, a nucleic acid vector (e.g., an AAV genome) is encapsidated within an AAV capsid forming an AAV particle. In some aspects, a nucleic acid vector disclosed herein is encapsidated within a wild-type AAV capsid disclosed herein or another AAV capsid disclosed herein, such as an AAV capsid comprising one or more amino acid substitutions.

[0090] In some aspects, a nucleic acid vector (e.g., an AAV genome) comprises native AAV genes or native AAV nucleotide sequences. In some aspects, one or more native AAV genes or native AAV nucleotide sequences may be removed from a nucleic acid vector (e.g., an AAV genome). In some aspects, one or more native AAV genes or native AAV nucleotide sequences may be removed from a nucleic acid vector (e.g., an AAV genome) and replaced with a gene of interest, e.g., an expression cassette comprising a gene of interest operably linked to a promoter.

[0091] In some aspects, a nucleic acid vector (e.g., an AAV genome) can be of any AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74, or a combination of serotypes. In some aspects, a nucleic acid vector (e.g., an AAV genome) encapsidated within an AAV capsid forms a pseudotyped AAV particle, such that the genome is of a serotype distinct from the capsid in which it is encapsidated. For example, a nucleic acid vector (e.g., an AAV genome) of serotype AAV2 may be encapsidated within a capsid of serotype AAVrh74.

[0092] As disclosed herein, the 5’ ITR refers to the ITR at the 5' terminus of the nucleic acid vector (e.g., AAV genome), and the 3’ ITR refers to the ITR at the 3' terminus of the nucleic acid vector (e.g., AAV genome). Each ITR in its native or wild-type form is or is about 145 nucleotides in length (e.g., about 140 nucleotides, about 145 nucleotides, about 150 nucleotides, about 155 nucleotides, about 160 nucleotides, or about 165 nucleotides) and comprises a D-sequence and a stem A-domain. Each ITR can independently be of any AAV serotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74), or both ITRs may be of the same serotype. ITRs are described, for example, in Grimm et al. J. Virol.80(1):426-439 (2006). Exemplary 5’ ITR sequences are provided below. In each ITR sequence, the D-sequence is underlined. A 3’ ITR has a nucleotide sequence which is the reverse complement of theT19325 Attorney Docket No. U1202.70155WO00 corresponding 5’ ITR (e.g., the AAV23’ ITR has a nucleotide sequence which is the reverse complement of the AAV25’ ITR).

[0093] Example of wild-type AAV15’ ITR: TTGCCCACTCCCTCTCTGCGCGCTCGCTCGCTCGGTGGGGCCTGCGGACCAAAGGTCCGCAGACGGCAG AGGTCTCCTCTGCCGGCCCCACCGAGCGAGCGAGCGCGCAGAGAGGGAGTGGGCAACTCCATCACTAGG GGTAA (SEQ ID NO: 22)

[0094] Example of wild-type AAV25’ ITR: TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCG GGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGG GGTTCCT (SEQ ID NO: 23)

[0095] Example of wild-type AAV35’ ITR: TTGGCCACTCCCTCTATGCGCACTCGCTCGCTCGGTGGGGCCTGGCGACCAAAGGTCGCCAGACGGACG TGCTTTGCACGTCCGGCCCCACCGAGCGAGCGAGTGCGCATAGAGGGAGTGGCCAACTCCATCACTAGA GGTATGGC (SEQ ID NO: 24)

[0096] Example of wild-type AAV45’ ITR: TTGGCCACTCCCTCTATGCGCGCTCGCTCACTCACTCGGCCCTGGAGACCAAAGGTCTCCAGACTGCCG GCCTCTGGCCGGCAGGGCCGAGTGAGTGAGCGAGCGCGCATAGAGGGAGTGGCCAACTCCATCATCTAG GTTTGCCC (SEQ ID NO: 25)

[0097] Example of wild-type AAV55’ ITR: CTCTCCCCCCTGTCGCGTTCGCTCGCTCGCTGGCTCGTTTGGGGGGGTGGCAGCTCAAAGAGCTGCCAG ACGACGGCCCTCTGGCCGTCGCCCCCCCAAACGAGCCAGCGAGCGAGCGAACGCGACAGGGGGGAGAGT GCCACACTCTCAAGCAAGGGGGTTTTGTA (SEQ ID NO: 26)

[0098] Example of wild-type AAV65’ ITR: TTGCCCACTCCCTCTATGCGCGCTCGCTCGCTCGGTGGGGCCTGCGGACCAAAGGTCCGCAGACGGCAG AGCTCTGCTCTGCCGGCCCCACCGAGCGAGCGAGCGCGCATAGAGGGAGTGGGCAACTCCATCACTAGG GGTA (SEQ ID NO: 27)

[0099] Example of wild-type AAVrh745’ ITR: TTGCCCACTCCCTCTCTGCGCGCTCGCTCGCTCGGTGGGGCCTGCGGACCAAAGGTCCGCAGACGGCAG AGGTCTCCTCTGCCGGCCCCACCGAGCGAGCGAGCGCGCAGAGAGGGAGTGGGCAACTCCATCACTAGG GGTAA (SEQ ID NO: 28)

[0100] In some aspects, an AAV1, 2, 3, 4, 5, 6, or AAVrh74 ITR (e.g., a 5’ ITR or a 3’ ITR) comprises a heterologous sequence in a D-sequence, a heterologous sequence in a stem A- domain and a wildtype AAV1, 2, 3, 4, 5, 6, or AAV rh74 terminal resolution site. In some aspects, the heterologous sequence is a transcription factor binding site sequence. In someAttorney Docket No. U1202.70155WO00 aspects, an AAV1 ITR (e.g., a 5’ ITR or a 3’ ITR) comprises a transcription factor binding site sequence in a D-sequence, a transcription factor binding site sequence in a stem A-domain and a wildtype AAV1 terminal resolution site.

[0101] In some aspects, an AAV2 ITR (e.g., a 5’ ITR or a 3’ ITR) comprises a transcription factor binding site sequence in a D-sequence, a transcription factor binding site sequence in a stem A-domain and a wildtype AAV2 terminal resolution site.

[0102] In some aspects, an AAV3 ITR (e.g., a 5’ ITR or a 3’ ITR) comprises a transcription factor binding site sequence in a D-sequence, a transcription factor binding site sequence in a stem A-domain and a wildtype AAV3 terminal resolution site.

[0103] In some aspects, an AAV4 ITR (e.g., a 5’ ITR or a 3’ ITR) comprises a transcription factor binding site sequence in a D-sequence, a transcription factor binding site sequence in a stem A-domain and a wildtype AAV4 terminal resolution site.

[0104] In some aspects, an AAV5 ITR (e.g., a 5’ ITR or a 3’ ITR) comprises a transcription factor binding site sequence in a D-sequence, a transcription factor binding site sequence in a stem A-domain and a wildtype AAV5 terminal resolution site.

[0105] In some aspects, an AAV6 ITR (e.g., a 5’ ITR or a 3’ ITR) comprises a transcription factor binding site sequence in a D-sequence, a transcription factor binding site sequence in a stem A-domain and a wildtype AAV6 terminal resolution site.

[0106] In some aspects, an AAVrh74 ITR (e.g., a 5’ ITR or a 3’ ITR) comprises a transcription factor binding site sequence in a D-sequence, a transcription factor binding site sequence in a stem A-domain and a wildtype AAVrh74 terminal resolution site.

[0107] In some aspects, an AAV1, 2, 3, 4, 5, 6, or AAVrh74 ITR (e.g., a 5’ ITR or a 3’ ITR) comprises more than one transcription factor binding site sequences in a D-sequence, more than one transcription factor binding site sequences in a stem A-domain and a wildtype AAV1, 2, 3, 4, 5, 6, or AAV rh74 terminal resolution site.

[0108] In some aspects, a modified AAV1, 2, 3, 4, 5, 6, or rh74 ITR sequence differs from a wildtype AAV1, 2, 3, 4, 5, 6, or rh74 ITR sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some aspects, a modified AAV1, 2, 3, 4, 5, 6, or rh74 ITR sequence differs from a wildtype AAV1, 2, 3, 4, 5, 6, or rh74 ITR sequence by about 1-10 nucleotides, e.g., 7 nucleotides. In some aspects, the heterologous nucleotide sequence comprises, consists essentially of, or consists of the nucleic acid sequence 5'-AGCAGCTGCT-3' (SEQ ID NO: 2), 5'-CTAAAAATAG-3' (SEQ ID NO: 3), 5’-GTCTTGTCCTACAAGA-3’ (SEQ ID NO: 4), 5’-Attorney Docket No. U1202.70155WO00 GATTAGG-3’, 5’-GACGGCTTAGTCGG-3’ (SEQ ID NO: 6), GCTCCGATTTCCGAT (SEQ ID NO: 7), 5’-GGCACGTGGC-3’ (SEQ ID NO: 8), 5’-CGGCACGTGCCA-3’ (SEQ ID NO: 9), 5’-CCGTGGGAAAA-3’ (SEQ ID NO: 10), 5’-GAACAATAACATTGTTC-3’ (SEQ ID NO: 11), 5’-CCGCAATAAA-3’ (SEQ ID NO: 12), 5’-TCCCAGCTACTTTGGGA- 3’ (SEQ ID NO: 13), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 14), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 15), 5’-AACAATAACATTGTT-3’ (SEQ ID NO: 16), 5’-AGAACAATGG-3’ (SEQ ID NO: 17), 5’-GCACGTGA-3’, 5’-TACACCTGTC-3’ (SEQ ID NO: 19), 5’- GGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAG GAGCAAACAGGGGCTAAGTCCAC-3’ (SEQ ID NO: 20), 5’- ATCGCCAGGTCACCTGAGGAGTTAATGAATACATATCTCCT-3’ (SEQ ID NO: 21) or a reverse or reverse complement of SEQ ID NO: 2-21. In some aspects, the heterologous nucleotide sequence comprises, consists essentially of, or consists of the nucleic acid sequence 5'-GTCTTGTAGAACAAGA-3' (SEQ ID NO: 67) or a reverse or reverse complement of SEQ ID NO: 67. In some aspects, the heterologous nucleotide sequence has at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity) with the sequence 5'-AGCAGCTGCT-3' (SEQ ID NO: 2), 5'-CTAAAAATAG-3' (SEQ ID NO: 3), 5’- GTCTTGTCCTACAAGA-3’ (SEQ ID NO: 4), 5’-GATTAGG-3’, 5’- GACGGCTTAGTCGG-3’ (SEQ ID NO: 6), 5’-GCTCCGATTTCCGAT-3’ (SEQ ID NO: 7), 5’-GGCACGTGGC-3’ (SEQ ID NO: 8), 5’-CGGCACGTGCCA-3’ (SEQ ID NO: 9), 5’- CCGTGGGAAAA-3’ (SEQ ID NO: 10), 5’-GAACAATAACATTGTTC-3’ (SEQ ID NO: 11), 5’-CCGCAATAAA-3’ (SEQ ID NO: 12), 5’-TCCCAGCTACTTTGGGA-3’ (SEQ ID NO: 13), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 14), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 15), 5’-AACAATAACATTGTT-3’ (SEQ ID NO: 16), 5’-AGAACAATGG-3’ (SEQ ID NO: 17), 5’-GCACGTGA-3’, 5’-TACACCTGTC-3’ (SEQ ID NO: 19), 5’- GGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAG GAGCAAACAGGGGCTAAGTCCAC-3’ (SEQ ID NO: 20), 5’- ATCGCCAGGTCACCTGAGGAGTTAATGAATACATATCTCCT-3’ (SEQ ID NO: 21), or a reverse or reverse complement of SEQ ID NO: 2-21. In some aspects, the heterologous nucleotide sequence has at least 70% identity (e.g., at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, at least 95% identity, at least 96% identity, at least 97%T19325 Attorney Docket No. U1202.70155WO00 identity, at least 98% identity, or at least 99% identity) with the sequence 5'-GTCTTGTAGAACAAGA-3' (SEQ ID NO: 67) or a reverse or reverse complement of SEQ ID NO: 67. In some aspects, the heterologous nucleotide sequence has less than 95% identity (e.g., less than 90% identity, less than 85% identity, less than 80% identity, less than 75% identity, or less than 70% identity) with the sequence 5'-AGCAGCTGCT-3' (SEQ ID NO: 2), 5'-CTAAAAATAG-3' (SEQ ID NO: 3), 5’-GTCTTGTCCTACAAGA-3’ (SEQ ID NO: 4), 5’- GATTAGG-3’, 5’- GACGGCTTAGTCGG-3’ (SEQ ID NO: 6), 5’-GCTCCGATTTCCGAT- 3’ (SEQ ID NO: 7), 5’-GGCACGTGGC-3’ (SEQ ID NO: 8), 5’-CGGCACGTGCCA-3’ (SEQ ID NO: 9), 5’-CCGTGGGAAAA-3’ (SEQ ID NO: 10), 5’-GAACAATAACATTGTTC-3’ (SEQ ID NO: 11), 5’-CCGCAATAAA-3’ (SEQ ID NO: 12), 5’-TCCCAGCTACTTTGGGA- 3’ (SEQ ID NO: 13), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 14), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 15), 5’-AACAATAACATTGTT-3’ (SEQ ID NO: 16), 5’-AGAACAATGG-3’ (SEQ ID NO: 17), 5’-GCACGTGA-3’, 5’-TACACCTGTC-3’ (SEQ ID NO: 19), 5’- GGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAG GAGCAAACAGGGGCTAAGTCCAC-3’ (SEQ ID NO: 20), 5’- ATCGCCAGGTCACCTGAGGAGTTAATGAATACATATCTCCT-3’ (SEQ ID NO: 21), or a reverse or reverse complement of SEQ ID NO: 2-21. In some aspects, the heterologous nucleotide sequence has less than 95% identity (e.g., less than 90% identity, less than 85% identity, less than 80% identity, less than 75% identity, or less than 70% identity) with the sequence 5'-GTCTTGTAGAACAAGA-3' (SEQ ID NO: 67) or a reverse or reverse complement of SEQ ID NO: 67. In some aspects, the heterologous nucleotide sequence has about 70% to about 95% identity (e.g., about 95% identity, about 90% identity, about 85% identity, about 80% identity, about 75% identity, or about 70% identity) with the sequence 5'-AGCAGCTGCT-3' (SEQ ID NO: 2), 5'-CTAAAAATAG-3' (SEQ ID NO: 3), 5’- GTCTTGTCCTACAAGA-3’ (SEQ ID NO: 4), 5’-GATTAGG-3’, 5’-GACGGCTTAGTCGG- 3’ (SEQ ID NO: 6), 5’-GCTCCGATTTCCGAT-3’ (SEQ ID NO: 7), 5’-GGCACGTGGC-3’ (SEQ ID NO: 8), 5’-CGGCACGTGCCA-3’ (SEQ ID NO: 9), 5’-CCGTGGGAAAA-3’ (SEQ ID NO: 10), 5’-GAACAATAACATTGTTC-3’ (SEQ ID NO: 11), 5’-CCGCAATAAA-3’ (SEQ ID NO: 12), 5’-TCCCAGCTACTTTGGGA-3’ (SEQ ID NO: 13), 5’-GTGGGTGGTCT- 3’ (SEQ ID NO: 14), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 15), 5’-AACAATAACATTGTT- 3’ (SEQ ID NO: 16), 5’-AGAACAATGG-3’ (SEQ ID NO: 17), 5’-GCACGTGA-3’, 5’- TACACCTGTC-3’ (SEQ ID NO: 19), 5’- GGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGT19325 Attorney Docket No. U1202.70155WO00 GAGCAAACAGGGGCTAAGTCCAC-3’ (SEQ ID NO: 20), 5’- ATCGCCAGGTCACCTGAGGAGTTAATGAATACATATCTCCT-3’ (SEQ ID NO: 21), or a reverse or reverse complement of SEQ ID NO: 2-21. In some aspects, the heterologous nucleotide sequence has about 70% to about 95% identity (e.g., about 95% identity, about 90% identity, about 85% identity, about 80% identity, about 75% identity, or about 70% identity) with the sequence 5'-GTCTTGTAGAACAAGA-3' (SEQ ID NO: 67) or a reverse or reverse complement of SEQ ID NO: 67. In some aspects, the heterologous nucleotide sequence has fewer than 6 mismatches (e.g., fewer than 5, fewer than 4, fewer than 3, fewer than 2, 1, or no mismatches) relative to the sequence 5'-AGCAGCTGCT-3' (SEQ ID NO: 2), 5'-CTAAAAATAG-3' (SEQ ID NO: 3), 5’-GTCTTGTCCTACAAGA-3’ (SEQ ID NO: 4), 5’- GATTAGG-3’, 5’-GACGGCTTAGTCGG-3’ (SEQ ID NO: 6), 5’-GCTCCGATTTCCGAT-3’ (SEQ ID NO: 7), 5’-GGCACGTGGC-3’ (SEQ ID NO: 8), 5’-CGGCACGTGCCA-3’ (SEQ ID NO: 9), 5’-CCGTGGGAAAA-3’ (SEQ ID NO: 10), 5’-GAACAATAACATTGTTC-3’ (SEQ ID NO: 11), 5’-CCGCAATAAA-3’ (SEQ ID NO: 12), 5’-TCCCAGCTACTTTGGGA- 3’ (SEQ ID NO: 13), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 14), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 15), 5’-AACAATAACATTGTT-3’ (SEQ ID NO: 16), 5’-AGAACAATGG-3’ (SEQ ID NO: 17), 5’-GCACGTGA-3’, 5’-TACACCTGTC-3’ (SEQ ID NO: 19), 5’- GGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAG GAGCAAACAGGGGCTAAGTCCAC-3’ (SEQ ID NO: 20), 5’- ATCGCCAGGTCACCTGAGGAGTTAATGAATACATATCTCCT-3’ (SEQ ID NO: 21), or a reverse or reverse complement of SEQ ID NO: 2-21. In some aspects, the heterologous nucleotide sequence has fewer than 6 mismatches (e.g., fewer than 5, fewer than 4, fewer than 3, fewer than 2, 1, or no mismatches) relative to the sequence 5'-GTCTTGTAGAACAAGA-3' (SEQ ID NO: 67) or a reverse or reverse complement of SEQ ID NO: 67. In some aspects, the heterologous nucleotide sequence has 1, 2, 3, 4, 5, or 6 mismatches relative to the sequence 5'-AGCAGCTGCT-3' (SEQ ID NO: 2), 5'-CTAAAAATAG-3' (SEQ ID NO: 3), 5’- GTCTTGTCCTACAAGA-3’ (SEQ ID NO: 4), 5’-GATTAGG-3’, 5’-GACGGCTTAGTCGG- 3’ (SEQ ID NO: 6), 5’-GCTCCGATTTCCGAT-3’ (SEQ ID NO: 7), 5’-GGCACGTGGC-3’ (SEQ ID NO: 8), 5’-CGGCACGTGCCA-3’ (SEQ ID NO: 9), 5’-CCGTGGGAAAA-3’ (SEQ ID NO: 10), 5’-GAACAATAACATTGTTC-3’ (SEQ ID NO: 11), 5’-CCGCAATAAA-3’ (SEQ ID NO: 12), 5’-TCCCAGCTACTTTGGGA-3’ (SEQ ID NO: 13), 5’-GTGGGTGGTCT- 3’ (SEQ ID NO: 14), 5’-GTGGGTGGTCT-3’ (SEQ ID NO: 15), 5’-AACAATAACATTGTT- 3’ (SEQ ID NO: 16), 5’-AGAACAATGG-3’ (SEQ ID NO: 17), 5’-GCACGTGA-3’, 5’-T19325 Attorney Docket No. U1202.70155WO00 TACACCTGTC-3’ (SEQ ID NO: 19), 5’- GGGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAG GAGCAAACAGGGGCTAAGTCCAC-3’ (SEQ ID NO: 20), 5’- ATCGCCAGGTCACCTGAGGAGTTAATGAATACATATCTCCT-3’ (SEQ ID NO: 21), or a reverse or reverse complement of SEQ ID NO: 2-21. In some aspects, the heterologous nucleotide sequence has 1, 2, 3, 4, 5, or 6 mismatches relative to the sequence 5'-GTCTTGTAGAACAAGA-3' (SEQ ID NO: 67) or a reverse or reverse complement of SEQ ID NO: 67. In some aspects, the heterologous nucleotide sequence has a length of about 10 nucleotides (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides).

[0109] In some aspects, modification of a nucleic acid vector (e.g., an AAV genome) as provided herein results in the generation of a nucleic acid vector with increased capacity. For example, wild-type AAV genomes are approximately 4.7 kilobases (kb) in length; recombinant AAV genomes have typically been limited to approximately this same length. See, e.g., Wu, et al., Mol Ther. (2010) 18(1): 80-86. Self-complementary AAV genomes are often even more limited, with maximum packaging capacities of about 2.3 kb. The modifications provided herein can, in some aspects, enable substantially larger AAV genomes (comprising the modification(s)) to be useful in generating AAV particles for delivery of genes of interest.

[0110] In some aspects, an AAV genome disclosed herein comprising a modification (e.g., one or more ITR modifications) and also optionally including a recombinant AAV genome comprising an expression cassette can be generated having a length greater than a corresponding AAV genome not comprising the modification, without substantial negative impacts on AAV genome rescue, replication, and / or packaging.

[0111] In some aspects, an AAV genome disclosed herein is about 4 kilobases (kb), 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb, 14 kb, 14.5 kb, 15 kb, 15.5 kb, 16 kb, 16.5 kb, 17 kb, 17.5 kb, or more in length. In some aspects, an AAV genome disclosed herein is at least 4 kilobases (kb), 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb, 14 kb, 14.5 kb, 15 kb, 15.5 kb, 16 kb, 16.5 kb, 17 kb, 17.5 kb, or more in length. In some aspects, an AAV genome disclosed herein is less than 4 kilobases (kb), 4.5 kb, 5 kb, 5.5 kb, 6 kb, 6.5 kb, 7 kb, 7.5 kb, 8 kb, 8.5 kb, 9 kb, 9.5 kb, 10 kb, 10.5 kb, 11 kb, 11.5 kb, 12 kb, 12.5 kb, 13 kb, 13.5 kb, 14 kb, 14.5 kb, 15 kb, 15.5 kb, 16 kb, 16.5 kb, 17 kb, or 17.5 kb in length. It should be understood that an AAV genome disclosed herein can be of any size or size range within the kb values disclosed herein (e.g., anT19325 Attorney Docket No. U1202.70155WO00 AAV genome can be 4-17.5 kb in length, 7-10 kb in length, 6-9.5 kb in length, or any range or combination of lengths disclosed herein).

[0112] A nucleic acid vector (e.g., an AAV genome) as disclosed herein in some aspects comprises an expression cassette comprising one or more regulatory elements, such as regulatory elements operably linked to a transgene. In some aspects, a regulatory element is located between two ITRs, a 5' ITR and a 3' ITR. In some aspects, a regulatory element is located upstream of or 5' relative to a transgene. In some aspects, a regulatory element is located downstream of or 3' relative to the 5' ITRs as described herein. In some aspects, a regulatory element is located upstream of or 5' relative to a transgene and downstream of or 3' relative to a 5' ITR. A regulatory element refers to a nucleotide sequence or structural component of a nucleic acid vector which is involved in the regulation of expression of components of the nucleic acid vector (e.g., a gene of interest comprised therein). Regulatory elements include, but are not limited to, promoters, enhancers, silencers, insulators, response elements, initiation sites, termination signals, and ribosome binding sites.

[0113] Promoters include constitutive promoters, inducible promoters, tissue-specific promoters, cell type-specific promoters, and synthetic promoters. For example, a nucleic acid vector disclosed herein may include viral promoters or promoters from mammalian genes that are generally active in promoting transcription. Non-limiting examples of constitutive viral promoters include the Herpes Simplex virus (HSV), thymidine kinase (TK), Rous Sarcoma Virus (RSV), Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV), Ad E1A and cytomegalovirus (CMV) promoters. Non-limiting examples of constitutive mammalian promoters include various housekeeping gene promoters, as exemplified by the β-actin promoter.

[0114] Inducible promoters or other inducible regulatory elements may also be used to achieve desired expression levels of a gene of interest (e.g., a protein or polypeptide of interest). Non- limiting examples of suitable inducible promoters include those from genes such as cytochrome P450 genes, heat shock protein genes, metallothionein genes, and hormone- inducible genes, such as the estrogen gene promoter. Another example of an inducible promoter is the tetVP16 promoter that is responsive to tetracycline.

[0115] Tissue-specific promoters or other tissue-specific regulatory elements are also contemplated herein.Attorney Docket No. U1202.70155WO00

[0116] Synthetic promoters are also contemplated herein. A synthetic promoter may comprise, for example, regions of known promoters, regulatory elements, transcription factor binding sites, enhancer elements, repressor elements, and the like.

[0117] In some aspects, a nucleic acid vector (e.g., an AAV genome) provided herein comprises a nucleotide sequence encoding a product (e.g., a protein or polypeptide product). In some aspects, a nucleotide sequence comprises an expression cassette comprising a nucleotide sequence of a gene of interest. In some aspects, a gene of interest encodes a therapeutic and / or diagnostic agent (e.g., protein or polypeptide). In some aspects, a therapeutic or diagnostic agent is an antibody, a peptibody, a growth factor, a clotting factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on cell surface receptors or ion channels, a cell-permeant peptide targeting intracellular processes, a thrombolytic agent, an enzyme, a bone morphogenetic protein, a nuclease, a protein used for gene editing, an Fc-fusion protein, an anticoagulant, or a protein or polypeptide that can be detected using a laboratory test. In some aspects, a nucleic acid vector (e.g., an AAV genome) provided herein comprises a nucleotide sequence encoding a guide RNA or other nucleic acid used for gene editing, optionally in addition to a protein used for gene editing.

[0118] In some aspects, a product encoded by a nucleic acid vector (e.g., an AAV genome) disclosed herein is a detectable molecule. A detectable molecule is a molecule that can be visualized (e.g., using a naked eye, under a microscope, or using a light detection device such as a camera). In some aspects, the detectable molecule is a fluorescent molecule, a bioluminescent molecule, or a molecule that provides color (e.g., β-galactosidase, β-lactamase, β-glucuronidase, or spheroidenone). In some aspects, the detectable molecule is a fluorescent, bioluminescent or enzymatic protein or functional peptide or polypeptide thereof.

[0119] In some aspects, fluorescent protein is a blue fluorescent protein, a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, a red fluorescent protein, or a functional peptide or polypeptide thereof. A blue fluorescent protein may be azurite, EBFP, EBFP2, mTagBFP, or Y66H. A cyan fluorescent protein may be ECFP, AmCyan1, Cerulean, CyPet, mECFP, Midori-ishi Cyan, mTFP1, or TagCFP. A Green fluorescent protein may be AcGFP, Azami Green, EGFP, Emarald, GFP or a mutated form of GFP (e.g., GFP-S65T, mWasabi, Stemmer, Superfolder GFP, TagGFP, TurboGFP, or ZsGreen). A yellow fluorescent protein may be EYFP, mBanana, mCitrine, PhiYFp, TagYFP, Topaz, Venus, YPet, or ZsYellow1. An orange fluorescent protein may be DsRed, RFP, DsRed2, DsRed-Express, Ds-Red-monomer, Tomato, tdTomato, Kusabira Orange, mKO2,Attorney Docket No. U1202.70155WO00 mOrange, mOrange2, mTangerine, TagRFP, or TagRFP-T. A red fluorescent protein may be AQ142, AsRed2, dKeima-Tandem, HcRed1, tHcRed, Jred, mApple, mCherry, mPlum, mRasberry, mRFP1, mRuby or mStrawberry.

[0120] In some aspects, a detectable molecule is a bioluminescent protein or a functional peptide or polypeptide thereof. Non-limiting examples of bioluminescent proteins are firefly luciferase, click-beetle luciferase, Renilla luciferase, and luciferase from Oplophorus gracilirostris.

[0121] In some aspects, a detectable molecule may be any polypeptide or protein that can be detected using methods known in the art. Non-limiting methods of detection are fluorescence imaging, luminescent imaging, bright filed imaging, and include imaging facilitated by immunofluorescence or immunohistochemical staining.

[0122] Additional features of AAV particles, nucleic acid vectors, and capsid proteins are described in Patent Application Publication No. US2017 / 0356009, the contents of which are incorporated herein by reference in their entirety.

[0123] In some aspects, a nucleic acid vector (e.g., an AAV genome) is comprised within or encoded by a plasmid.

[0124] Nucleic acid vectors as disclosed herein, e.g., comprising modified ITRs, can be prepared by one of ordinary skill in the art by known methods. AAV Particles

[0125] According to some aspects, provided herein are AAV particles that comprise any of the nucleic acid vectors (e.g., AAV genomes) disclosed herein. An AAV particle is a supramolecular assembly of 60 individual capsid protein subunits forming a non-enveloped T- 1 icosahedral lattice capable of protecting a single-stranded DNA genome. A mature AAV particle is approximately 20 nm in diameter, and its capsid is formed from three structural capsid proteins VP1, VP2, and VP3, with molecular masses of 87, 73, and 62 kDa, respectively, in a ratio of approximately 1:1:18. The 60 capsid proteins are arranged in an anti- parallel β-strand barreloid arrangement, resulting in a defined tropism and a high resistance to degradation.

[0126] In some aspects, an AAV particle comprises an empty capsid (e.g., a capsid without a cargo). In some aspects, an AAV particle comprises a capsid encapsidating a nucleic acid (e.g., a nucleic acid vector that comprises a gene of interest, such as a nucleic acid vector disclosedT19325 Attorney Docket No. U1202.70155WO00 herein). In some aspects, a nucleic acid encapsidated within an AAV capsid to generate an AAV particle comprises a nucleic acid vector disclosed herein. In some aspects, an AAV particle disclosed herein comprises a capsid protein comprising one or more mutations, e.g., one or more amino acid substitutions.

[0127] It is contemplated herein that any capsid protein mutations disclosed herein (e.g., amino acid substitutions) can be combined with any nucleic acid vector modifications disclosed herein (e.g., sequence deletions, substitutions, or insertions). For example, an AAV particle described herein may have an AAV capsid protein (e.g., a wild-type AAV capsid protein or one comprising one or more amino acid substitutions) and an AAV nucleic acid vector comprising a modification as described herein.

[0128] In some aspects, an AAV particle disclosed herein comprises a capsid protein comprising amino acid substitutions at one or more positions corresponding to T491, Y444, Y500, and / or Y730 of SEQ ID NO: 31. In some aspects, an AAV particle disclosed herein comprises a capsid protein comprising one or more amino acid substitutions corresponding to T491V, Y444F, Y500F, and / or Y730F substitutions in SEQ ID NO: 31.

[0129] In some aspects, an AAV particle disclosed herein comprises a capsid protein comprising amino acid substitutions at one or more positions corresponding to T491, Y444, Y500, and / or Y730 of SEQ ID NO: 31 and further comprises a nucleic acid vector comprising modification as described herein (e.g., a deletion, a substitution, or an insertion in a D- sequence and a stem A-domain of an ITR, e.g., in a 5’ITR, a 3’ITR or both.

[0130] In some aspects, the AAV particle comprises a capsid protein comprising amino acid substitutions at one or more positions corresponding to T491, Y444, Y500, and / or Y730 of SEQ ID NO: 31 and a nucleic acid vector comprising a substitution of a portion of a D- sequence of an ITR with a heterologous nucleotide sequence. In some aspects, the amino acid substitutions correspond to T491V, Y444F, Y500F, and / or Y730F substitutions in SEQ ID NO: 31.

[0131] In some aspects, an AAV particle disclosed herein is replicative. A replicative AAV particle is capable of replicating within a host cell (e.g., a host cell within a subject or a host cell in culture). In some aspects, an AAV particle disclosed herein is non-replicating. A non- replicating AAV particle is not capable of replicating within a host cell (e.g., a host cell within a subject or a host cell in culture), but can infect the host and incorporate a genetic components into the host’s genome for expression. In some aspects, an AAV particle disclosed herein is capable of infecting a host cell. In some aspects, an AAV particle disclosed herein is capable ofT19325 Attorney Docket No. U1202.70155WO00 facilitating stable integration of genetic components into the genome of a host cell. In some aspects, an AAV particle disclosed herein is not capable of facilitating integration of genetic components into the genome of a host cell.

[0132] In some aspects, an AAV particle disclosed herein comprises a nucleic acid vector (e.g., an AAV genome) provided herein. In some aspects, a nucleic acid vector (e.g., AAV genome) comprises two ITRs adjacent to the ends of a sequence encoding a gene of interest. In some aspects, the nucleic acid vector (e.g., AAV genome) is a single-stranded DNA vector. In some aspects, the nucleic acid vector (e.g., AAV genome) is a double-stranded DNA vector. In some aspects, an AAV particle disclosed herein comprises one single-stranded DNA. In some aspects, an AAV particle disclosed herein comprises two complementary DNA strands, forming a self-complementary AAV (scAAV).

[0133] In some aspects, a nucleic acid vector that may be comprised in an AAV particle (e.g., a WT particle or particle comprising a capsid comprising any one or more mutations as disclosed herein) comprises an ITR comprising a modification (e.g., a deletion, substitution, or insertion) of part or all of the ITR’s D-sequence. In some aspects, part or all of the ITR’s D- sequence is substituted with a heterologous nucleotide sequence. In some aspects, part or all of the ITR’s D-sequence is deleted. Further description of such modifications (e.g., deletions, substitutions, and insertions) is provided elsewhere herein. In some aspects, an ITR comprising a substitution, insertion, or deletion of a nucleic acid vector as disclosed herein comprises one or more additional modifications, such as an additional substitution, modification, or deletion in another portion of the ITR.

[0134] An AAV particle disclosed herein may be of any AAV serotype (e.g., AAV serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13), including any derivative (including non-naturally occurring variants of a serotype) or pseudotype. Non-limiting examples of derivatives and pseudotypes include AAV2-AAV3 hybrid, AAVrh.10, 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, AAV2.5T, AAV-HAE1 / 2, AAV clone 32 / 83, AAVShH10, AAV2.15, AAV2.4, AAVM41, and AAVr3.45. Such AAV serotypes and derivatives / pseudotypes, and methods of producing such derivatives / pseudotypes are known in the art (see, e.g., Mol. Ther.2012 Apr; 20(4):699-708. doi: 10.1038 / mt.2011.287. Epub 2012 Jan 24. The AAV vector toolkit: poised at the clinical crossroads. Asokan A, Schaffer DV, Samulski RJ.). In some aspects, the AAV particle is a pseudotyped AAV particle, which comprises a nucleic acid vector comprising ITRs from one serotype (e.g., AAV2 or AAV3)T19325 Attorney Docket No. U1202.70155WO00 and a capsid comprised of capsid proteins derived from another serotype (i.e., a serotype other than AAV2 or AAV3, respectively). SEQ ID NOs: 1-14 provide examples of amino acid sequences of AAV capsid proteins of different serotypes. Methods for producing and using pseudotyped rAAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods, 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet., 10:3075-3081 (2001)).

[0135] In some aspects, an AAV particle disclosed herein is a recombinant AAV (rAAV) particle, e.g., comprising a recombinant nucleic acid or transgene.

[0136] Any combination of modifications described herein (e.g., capsid protein modifications, a deletion, insertion, or substitution of a D-sequence, a deletion, insertion, or substitution of a stem A-domain sequence and / or an insertion of a non-AAV (heterologous) sequence into an AAV genome (e.g., in a D-sequence or adjacent to a D-sequence and in a stem A-domain sequence) may result in an additive or synergistic effect, in which the beneficial properties of the resulting combination are equal to or greater than, respectively, the sum of the effects of the individual modifications. For example, an AAV particle comprising a modified capsid protein and a modified genome may have improvements in transduction efficiency, transgene expression, and / or packaging efficiency relative to a corresponding wildtype AAV particle that are equal to the sum of the improvements conferred by the individual capsid protein modification and the genome modification, or that are greater than the sum of the improvements conferred by the individual modifications.

[0137] AAV particles as disclosed herein, e.g., comprising modified ITRs, can be prepared by one of ordinary skill in the art by known methods.

[0138] In some aspects, AAV particles (e.g., comprising modified genome and / or capsid protein) disclosed herein can be generated using a plasmid, such as a pSub201 plasmid, which includes sequences encoding AAV2 Rep and capsid proteins, and in which a gene of interest can be inserted in place of the AAV2 Rep and capsid protein sequences to prepare a recombinant AAV genome. The pSub201 sequence is provided below, in which the open reading frame encoding ampicillin-resistance marker is bolded and the D-sequences are underlined. The portions of the underlined D-sequences that are also bolded are the preferred portion that can be replaced by a heterologous nucleotide sequence. A corresponding position for replacement by a heterologous nucleotide sequence in a different sequence can be identified by methods known in the art.T19325 Attorney Docket No. U1202.70155WO00 CAGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGT CGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTG TAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGAGTCCTGTATTAGAGGTCA CGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGC AGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCATGCCGGGGTTTTACGAGATTGTGAT TAAGGTCCCCAGCGACCTTGACGGGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGA GAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGT GGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTTTT CTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAA ATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGAT CGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGT GGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGAC TAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCT GACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGAT CAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGA GAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCA AATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGT GGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGA TCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTG GCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTA CGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTG GGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCG CGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAA CATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTT CAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTT TTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAA GAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCC ATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGT GGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTT CACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAA AAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTG CGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTG CCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCA AACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTG GGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGG CCCTCGAGCACGTCAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACC ACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAG TCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGG GAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCC AGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGC CTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCAC CAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCgGGAAATTGGCATTGCGATTCCA CATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACC TCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTT GGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACA ACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGC AGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGT ACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCA TGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACT GCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGG ACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACC AGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTT CTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGC AGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCT19325 Attorney Docket No. U1202.70155WO00 ACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGGCCCGCCATGGCAAGCCACAAGGACGATGAAGAAA AGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGAACATTG AAAAGGTCATGATTACAGACGAAGAGGAAATCGGAACAACCAATCCCGTGGCTACGGAGCAGTATGGTT CTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTC TTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACA CGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTC TCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCA TCACACAGTACTCCACGGGACACGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAA CGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATCGTGGACTTACCGTGGAT ACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATTGCTT GTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAGT TTCCATGCTCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAACCC CTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCCAGCTGGCGTAATAGCGA AGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGAATTCCAGACGATT GAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCAATGGCTGGCGGTAATATTGTTCTG GATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGA AGTATTGCGACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAA AACACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCC CGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAG CGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGC GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAA TCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGG TGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTT CTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTT ATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA TTTTAACAAAATATTAACGTTTACAATTTAAATATTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTT CTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGT TTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGAGACCTCTCAAAAATAGCTACCCTCTCC GGCATGAATTTATCAGCTAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCT CACCCGTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAAT TTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACA ACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTATGAT TTATTGGATGTTGGAATTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCAT ATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACC CGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGG GAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACG CCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAA TGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCT TATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGA TGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGA GAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATT ATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGA GTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCAT AACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGC TTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT ACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGG CGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACC ACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGG GAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTG GTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAG GATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAttorney Docket No. U1202.70155WO00 AGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTT TCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGG CCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCG GTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATA CCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAG CGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCC TGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATG GAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT TCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCG CAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCC TCTCCCCGCGCGTTGGCCGATTCATTAATG (SEQ ID NO: 29) Capsid Proteins

[0139] AAV particles disclosed herein in some aspects comprise capsid proteins having one or more modifications characterized by amino acid substitutions. In some aspects, an AAV capsid protein disclosed herein comprises amino acid substitutions at one or more positions corresponding to T491, Y444, Y500, and / or Y730 of SEQ ID NO: 31. In some aspects, the amino acid substitutions correspond to T491V, Y444F, Y500F, and / or Y730F substitutions in SEQ ID NO: 31. It should be understood that an amino acid substitution at a position corresponding to a position of SEQ ID NO: 31 can be an amino acid substitution in a capsid protein of any serotype. The corresponding position in a capsid protein having a different baseline amino acid sequence can be determined by methods known in the art, such as by constructing structural alignments of the amino acid sequences and identifying corresponding amino acids. A “corresponding” amino acid to be substituted is one which is at the corresponding position, and may have the same amino acid identity (i.e., the amino acid at the corresponding position in the second capsid protein sequence is the same as the amino acid in the reference capsid protein sequence), or may be an amino acid with similar properties (e.g., similar hydrophobicity, size, charge, etc.) as the amino acid in the reference capsid protein. For example, an amino acid substitution at a position corresponding to T491 of SEQ ID NO: 31, or corresponding to a T491V substitution in SEQ ID NO:31 may be a substitution at a position corresponding to position 491 of SEQ ID NO: 31 in a second capsid protein, which may also be a threonine, or which may be a similar amino acid (e.g., another amino acid with a polar uncharged side chain, such as serine, asparagine, or glutamine).

[0140] In some aspects, an AAV capsid protein disclosed herein comprises amino acid substitutions as described in Patent Application Publication Nos. US2010 / 0104561, US2014 / 0050701, US2016 / 0333372, US2015 / 0133530, US2014 / 0341852, andAttorney Docket No. U1202.70155WO00 WO2022 / 226289, the contents of each of which are herein incorporated by reference in their entireties for this purpose.

[0141] In some aspects, an AAV capsid protein as disclosed herein is a VP1 protein, a VP2 protein, or a VP3 protein. The VP1, VP2, and VP3 capsid proteins are each encoded from the same segment of the AAV genome, and differ in their N termini based on alternative mRNA splicing.

[0142] The different capsid proteins VP1, VP2, and VP3 are defined according to numbering of the full-length VP1 protein. In some aspects, for AAV2 capsid proteins, a VP1 capsid protein is defined by amino acids 1-735 of SEQ ID NO: 31; a VP2 capsid protein is defined by amino acids 138-735 of SEQ ID NO: 31; and a VP3 capsid protein is defined by amino acids 203-735 of SEQ ID NO: 31. Numbering of AAV capsid proteins is provided according to the VP1 sequence. For example, T491 refers to the threonine at position 491 of SEQ ID NO: 2 in a VP1 protein or the corresponding threonine in a VP2 or VP3 protein. Similarly, Y444, Y500, and Y730 refer to the tyrosines at positions 444, 500, and 730 of SEQ ID NO: 2, respectively, in a VP1 protein, or the corresponding tyrosines in a VP2 or VP3 protein.

[0143] An AAV capsid protein disclosed herein can be of any serotype, or can be a chimeric capsid protein (i.e., comprising segments from capsid proteins of two or more serotypes). In some aspects, a capsid protein disclosed herein is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74 capsid protein. In some aspects, an AAV capsid protein as provided herein is of serotype 2, serotype 3, serotype 6, or serotype rh74. Amino acid sequences of capsid proteins of other AAV serotypes are known and can be aligned with SEQ ID NO: 31 (AAV2 capsid protein) using techniques known in the art. Examples of amino acid sequences of AAV capsid proteins of various serotypes are provided below.

[0144] Example of an amino acid sequence of wild-type AAV1 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY 51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKTAP GKKRPVEQSP 151 QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE SVPDPQPLGE PPATPAAVGP 201 TTMASGGGAP MADNNEGADG VGNASGNWHC DSTWLGDRVI TTSTRTWALP 251 TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 301 INNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ 351 LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP 401 SQMLRTGNNF TFSYTFEEVP FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ 451 NQSGSAQNKD LLFSRGSPAG MSVQPKNWLP GPCYRQQRVS KTKTDNNNSN 501 FTWTGASKYN LNGRESIINP GTAMASHKDD EDKFFPMSGV MIFGKESAGA 551 SNTALDNVMI TDEEEIKATN PVATERFGTV AVNFQSSSTD PATGDVHAMG 601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KNPPPQILIK 651 NTPVPANPPA EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQT19325 Attorney Docket No. U1202.70155WO00 701 YTSNYAKSAN VDFTVDNNGL YTEPRPIGTR YLTRPL (SEQ ID NO: 30)

[0145] Example of an amino acid sequence of wild-type AAV2 capsid protein 1 MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY 51 KYLGPFNGLD KGEPVNEADA AALEHDKAYD RQLDSGDNPY LKYNHADAEF 101 QERLKEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEPVKTAP GKKRPVEHSP 151 VEPDSSSGTG KAGQQPARKR LNFGQTGDAD SVPDPQPLGQ PPAAPSGLGT 201 NTMATGSGAP MADNNEGADG VGNSSGNWHC DSTWMGDRVI TTSTRTWALP 251 TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI 301 NNNWGFRPKR LNFKLFNIQV KEVTQNDGTT TIANNLTSTV QVFTDSEYQL 351 PYVLGSAHQG CLPPFPADVF MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS 401 QMLRTGNNFT FSYTFEDVPF HSSYAHSQSL DRLMNPLIDQ YLYYLSRTNT 451 PSGTTTQSRL QFSQAGASDI RDQSRNWLPG PCYRQQRVSK TSADNNNSEY 501 SWTGATKYHL NGRDSLVNPG PAMASHKDDE EKFFPQSGVL IFGKQGSEKT 551 NVDIEKVMIT DEEEIRTTNP VATEQYGSVS TNLQRGNRQA ATADVNTQGV 601 LPGMVWQDRD VYLQGPIWAK IPHTDGHFHP SPLMGGFGLK HPPPQILIKN 651 TPVPANPSTT FSAAKFASFI TQYSTGQVSV EIEWELQKEN SKRWNPEIQY 701 TSNYNKSVNV DFTVDTNGVY SEPRPIGTRY LTRNL (SEQ ID NO: 31)

[0146] Example of an amino acid sequence of wild-type AAV3 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWALKPGVP QPKANQQHQD NRRGLVLPGY 51 KYLGPGNGLD KGEPVNEADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRILEPLG LVEEAAKTAP GKKGAVDQSP 151 QEPDSSSGVG KSGKQPARKR LNFGQTGDSE SVPDPQPLGE PPAAPTSLGS 201 NTMASGGGAP MADNNEGADG VGNSSGNWHC DSQWLGDRVI TTSTRTWALP 251 TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI 301 NNNWGFRPKK LSFKLFNIQV RGVTQNDGTT TIANNLTSTV QVFTDSEYQL 351 PYVLGSAHQG CLPPFPADVF MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS 401 QMLRTGNNFQ FSYTFEDVPF HSSYAHSQSL DRLMNPLIDQ YLYYLNRTQG 451 TTSGTTNQSR LLFSQAGPQS MSLQARNWLP GPCYRQQRLS KTANDNNNSN 501 FPWTAASKYH LNGRDSLVNP GPAMASHKDD EEKFFPMHGN LIFGKEGTTA 551 SNAELDNVMI TDEEEIRTTN PVATEQYGTV ANNLQSSNTA PTTGTVNHQG 601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQIMIK 651 NTPVPANPPT TFSPAKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ 701 YTSNYNKSVN VDFTVDTNGV YSEPRPIGTR YLTRNL (SEQ ID NO: 32)

[0147] Example of an amino acid sequence of wild-type AAV4 capsid protein 1 MTDGYLPDWL EDNLSEGVRE WWALQPGAPK PKANQQHQDN ARGLVLPGYK 51 YLGPGNGLDK GEPVNAADAA ALEHDKAYDQ QLKAGDNPYL KYNHADAEFQ 101 QRLQGDTSFG GNLGRAVFQA KKRVLEPLGL VEQAGETAPG KKRPLIESPQ 151 QPDSSTGIGK KGKQPAKKKL VFEDETGAGD GPPEGSTSGA MSDDSEMRAA 201 AGGAAVEGGQ GADGVGNASG DWHCDSTWSE GHVTTTSTRT WVLPTYNNHL 251 YKRLGESLQS NTYNGFSTPW GYFDFNRFHC HFSPRDWQRL INNNWGMRPK 301 AMRVKIFNIQ VKEVTTSNGE TTVANNLTST VQIFADSSYE LPYVMDAGQE 351 GSLPPFPNDV FMVPQYGYCG LVTGNTSQQQ TDRNAFYCLE YFPSQMLRTG 401 NNFEITYSFE KVPFHSMYAH SQSLDRLMNP LIDQYLWGLQ STTTGTTLNA 451 GTATTNFTKL RPTNFSNFKK NWLPGPSIKQ QGFSKTANQN YKIPATGSDS 501 LIKYETHSTL DGRWSALTPG PPMATAGPAD SKFSNSQLIF AGPKQNGNTA 551 TVPGTLIFTS EEELAATNAT DTDMWGNLPG GDQSNSNLPT VDRLTALGAV 601 PGMVWQNRDI YYQGPIWAKI PHTDGHFHPS PLIGGFGLKH PPPQIFIKNT 651 PVPANPATTF SSTPVNSFIT QYSTGQVSVQ IDWEIQKERS KRWNPEVQFT 701 SNYGQQNSLL WAPDAAGKYT EPRAIGTRYL THHL (SEQ ID NO: 33)

[0148] Example of an amino acid sequence of wild-type AAV5 capsid protein 1 MSFVDHPPDW LEEVGEGLRE FLGLEAGPPK PKPNQQHQDQ ARGLVLPGYNT19325 Attorney Docket No. U1202.70155WO00 51 YLGPGNGLDR GEPVNRADEV AREHDISYNE QLEAGDNPYL KYNHADAEFQ 101 EKLADDTSFG GNLGKAVFQA KKRVLEPFGL VEEGAKTAPT GKRIDDHFPK 151 RKKARTEEDS KPSTSSDAEA GPSGSQQLQI PAQPASSLGA DTMSAGGGGP 201 LGDNNQGADG VGNASGDWHC DSTWMGDRVV TKSTRTWVLP SYNNHQYREI 251 KSGSVDGSNA NAYFGYSTPW GYFDFNRFHS HWSPRDWQRL INNYWGFRPR 301 SLRVKIFNIQ VKEVTVQDST TTIANNLTST VQVFTDDDYQ LPYVVGNGTE 351 GCLPAFPPQV FTLPQYGYAT LNRDNTENPT ERSSFFCLEY FPSKMLRTGN 401 NFEFTYNFEE VPFHSSFAPS QNLFKLANPL VDQYLYRFVS TNNTGGVQFN 451 KNLAGRYANT YKNWFPGPMG RTQGWNLGSG VNRASVSAFA TTNRMELEGA 501 SYQVPPQPNG MTNNLQGSNT YALENTMIFN SQPANPGTTA TYLEGNMLIT 551 SESETQPVNR VAYNVGGQMA TNNQSSTTAP ATGTYNLQEI VPGSVWMERD 601 VYLQGPIWAK IPETGAHFHP SPAMGGFGLK HPPPMMLIKN TPVPGNITSF 651 SDVPVSSFIT QYSTGQVTVE MEWELKKENS KRWNPEIQYT NNYNDPQFVD 701 FAPDSTGEYR TTRPIGTRYL TRPL (SEQ ID NO: 34)

[0149] Example of an amino acid sequence of wild-type AAV6 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY 51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPFG LVEEGAKTAP GKKRPVEQSP 151 QEPDSSSGIG KTGQQPAKKR LNFGQTGDSE SVPDPQPLGE PPATPAAVGP 201 TTMASGGGAP MADNNEGADG VGNASGNWHC DSTWLGDRVI TTSTRTWALP 251 TYNNHLYKQI SSASTGASND NHYFGYSTPW GYFDFNRFHC HFSPRDWQRL 301 INNNWGFRPK RLNFKLFNIQ VKEVTTNDGV TTIANNLTST VQVFSDSEYQ 351 LPYVLGSAHQ GCLPPFPADV FMIPQYGYLT LNNGSQAVGR SSFYCLEYFP 401 SQMLRTGNNF TFSYTFEDVP FHSSYAHSQS LDRLMNPLID QYLYYLNRTQ 451 NQSGSAQNKD LLFSRGSPAG MSVQPKNWLP GPCYRQQRVS KTKTDNNNSN 501 FTWTGASKYN LNGRESIINP GTAMASHKDD KDKFFPMSGV MIFGKESAGA 551 SNTALDNVMI TDEEEIKATN PVATERFGTV AVNLQSSSTD PATGDVHVMG 601 ALPGMVWQDR DVYLQGPIWA KIPHTDGHFH PSPLMGGFGL KHPPPQILIK 651 NTPVPANPPA EFSATKFASF ITQYSTGQVS VEIEWELQKE NSKRWNPEVQ 701 YTSNYAKSAN VDFTVDNNGL YTEPRPIGTR YLTRPL (SEQ ID NO: 35)

[0150] Example of an amino acid sequence of wild-type AAV7 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD NGRGLVLPGY 51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKTAP AKKRPVEPSP 151 QRSPDSSTGI GKKGQQPARK RLNFGQTGDS ESVPDPQPLG EPPAAPSSVG 201 SGTVAAGGGA PMADNNEGAD GVGNASGNWH CDSTWLGDRV ITTSTRTWAL 251 PTYNNHLYKQ ISSETAGSTN DNTYFGYSTP WGYFDFNRFH CHFSPRDWQR 301 LINNNWGFRP KKLRFKLFNI QVKEVTTNDG VTTIANNLTS TIQVFSDSEY 351 QLPYVLGSAH QGCLPPFPAD VFMIPQYGYL TLNNGSQSVG RSSFYCLEYF 401 PSQMLRTGNN FEFSYSFEDV PFHSSYAHSQ SLDRLMNPLI DQYLYYLART 451 QSNPGGTAGN RELQFYQGGP STMAEQAKNW LPGPCFRQQR VSKTLDQNNN 501 SNFAWTGATK YHLNGRNSLV NPGVAMATHK DDEDRFFPSS GVLIFGKTGA 551 TNKTTLENVL MTNEEEIRPT NPVATEEYGI VSSNLQAANT AAQTQVVNNQ 601 GALPGMVWQN RDVYLQGPIW AKIPHTDGNF HPSPLMGGFG LKHPPPQILI 651 KNTPVPANPP EVFTPAKFAS FITQYSTGQV SVEIEWELQK ENSKRWNPEI 701 QYTSNFEKQT GVDFAVDSQG VYSEPRPIGT RYLTRNL (SEQ ID NO: 36)

[0151] Example of an amino acid sequence of wild-type AAV8 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP KPKANQQKQD DGRGLVLPGY 51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLQAGDNPY LRYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKTAP GKKRPVEPSP 151 QRSPDSSTGI GKKGQQPARK RLNFGQTGDS ESVPDPQPLG EPPAAPSGVG 201 PNTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV ITTSTRTWAL 251 PTYNNHLYKQ ISNGTSGGAT NDNTYFGYST PWGYFDFNRF HCHFSPRDWQT19325 Attorney Docket No. U1202.70155WO00 301 RLINNNWGFR PKRLSFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE 351 YQLPYVLGSA HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY 401 FPSQMLRTGN NFQFTYTFED VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR 451 TQTTGGTANT QTLGFSQGGP NTMANQAKNW LPGPCYRQQR VSTTTGQNNN 501 SNFAWTAGTK YHLNGRNSLA NPGIAMATHK DDEERFFPSN GILIFGKQNA 551 ARDNADYSDV MLTSEEEIKT TNPVATEEYG IVADNLQQQN TAPQIGTVNS 601 QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL 651 IKNTPVPADP PTTFNQSKLN SFITQYSTGQ VSVEIEWELQ KENSKRWNPE 701 IQYTSNYYKS TSVDFAVNTE GVYSEPRPIG TRYLTRNL (SEQ ID NO: 37)

[0152] Example of an amino acid sequence of wild-type AAV9 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY 51 KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF 101 QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP 151 QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS 201 LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP 251 TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR 301 LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY 351 QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF 401 PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT 451 INGSGQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE 501 FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR 551 DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG 601 ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK 651 NTPVPADPPT AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ 701 YTSNYYKSNN VEFAVNTEGV YSEPRPIGTR YLTRNL (SEQ ID NO: 38)

[0153] Example of an amino acid sequence of wild-type AAV10 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY 51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKTAP GKKRPVEPSP 151 QRSPDSSTGI GKKGQQPAKK RLNFGQTGDS ESVPDPQPIG EPPAGPSGLG 201 SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV ITTSTRTWAL 251 PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFR PKRLNFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE 351 YQLPYVLGSA HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY 401 FPSQMLRTGN NFEFSYQFED VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR 451 TQSTGGTAGT QQLLFSQAGP NNMSAQAKNW LPGPCYRQQR VSTTLSQNNN 501 SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS GVLMFGKQGA 551 GKDNVDYSSV MLTSEEEIKT TNPVATEQYG VVADNLQQQN AAPIVGAVNS 601 QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL 651 IKNTPVPADP PTTFSQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE 701 IQYTSNYYKS TNVDFAVNTD GTYSEPRPIG TRYLTRNL (SEQ ID NO: 39)

[0154] Example of an amino acid sequence of wild-type AAV11 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY 51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKTAP GKKRPLESPQ 151 EPDSSSGIGK KGKQPARKRL NFEEDTGAGD GPPEGSDTSA MSSDIEMRAA 201 PGGNAVDAGQ GSDGVGNASG DWHCDSTWSE GKVTTTSTRT WVLPTYNNHL 251 YLRLGTTSSS NTYNGFSTPW GYFDFNRFHC HFSPRDWQRL INNNWGLRPK 301 AMRVKIFNIQ VKEVTTSNGE TTVANNLTST VQIFADSSYE LPYVMDAGQE 351 GSLPPFPNDV FMVPQYGYCG IVTGENQNQT DRNAFYCLEY FPSQMLRTGN 401 NFEMAYNFEK VPFHSMYAHS QSLDRLMNPL LDQYLWHLQS TTSGETLNQG 451 NAATTFGKIR SGDFAFYRKN WLPGPCVKQQ RFSKTASQNY KIPASGGNAL 501 LKYDTHYTLN NRWSNIAPGP PMATAGPSDG DFSNAQLIFP GPSVTGNTTTT19325 Attorney Docket No. U1202.70155WO00 551 SANNLLFTSE EEIAATNPRD TDMFGQIADN NQNATTAPIT GNVTAMGVLP 601 GMVWQNRDIY YQGPIWAKIP HADGHFHPSP LIGGFGLKHP PPQIFIKNTP 651 VPANPATTFT AARVDSFITQ YSTGQVAVQI EWEIEKERSK RWNPEVQFTS 701 NYGNQSSMLW APDTTGKYTE PRVIGSRYLT NHL (SEQ ID NO: 40)

[0155] Example of an amino acid sequence of wild-type AAV12 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NGRGLVLPGY 51 KYLGPFNGLD KGEPVNEADA AALEHDKAYD KQLEQGDNPY LKYNHADAEF 101 QQRLATDTSF GGNLGRAVFQ AKKRILEPLG LVEEGVKTAP GKKRPLEKTP 151 NRPTNPDSGK APAKKKQKDG EPADSARRTL DFEDSGAGDG PPEGSSSGEM 201 SHDAEMRAAP GGNAVEAGQG ADGVGNASGD WHCDSTWSEG RVTTTSTRTW 251 VLPTYNNHLY LRIGTTANSN TYNGFSTPWG YFDFNRFHCH FSPRDWQRLI 301 NNNWGLRPKS MRVKIFNIQV KEVTTSNGET TVANNLTSTV QIFADSTYEL 351 PYVMDAGQEG SFPPFPNDVF MVPQYGYCGV VTGKNQNQTD RNAFYCLEYF 401 PSQMLRTGNN FEVSYQFEKV PFHSMYAHSQ SLDRMMNPLL DQYLWHLQST 451 TTGNSLNQGT ATTTYGKITT GDFAYYRKNW LPGACIKQQK FSKNANQNYK 501 IPASGGDALL KYDTHTTLNG RWSNMAPGPP MATAGAGDSD FSNSQLIFAG 551 PNPSGNTTTS SNNLLFTSEE EIATTNPRDT DMFGQIADNN QNATTAPHIA 601 NLDAMGIVPG MVWQNRDIYY QGPIWAKVPH TDGHFHPSPL MGGFGLKHPP 651 PQIFIKNTPV PANPNTTFSA ARINSFLTQY STGQVAVQID WEIQKEHSKR 701 WNPEVQFTSN YGTQNSMLWA PDNAGNYHEL RAIGSRFLTH HL (SEQ ID NO: 41)

[0156] Example of an amino acid sequence of wild-type AAVrh10 capsid protein 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD DGRGLVLPGY 51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LRYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEGAKTAP GKKRPVEPSP 151 QRSPDSSTGI GKKGQQPAKK RLNFGQTGDS ESVPDPQPIG EPPAGPSGLG 201 SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV ITTSTRTWAL 251 PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFR PKRLNFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE 351 YQLPYVLGSA HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY 401 FPSQMLRTGN NFEFSYQFED VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR 451 TQSTGGTAGT QQLLFSQAGP NNMSAQAKNW LPGPCYRQQR VSTTLSQNNN 501 SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS GVLMFGKQGA 551 GKDNVDYSSV MLTSEEEIKT TNPVATEQYG VVADNLQQQN AAPIVGAVNS 601 QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL 651 IKNTPVPADP PTTFSQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE 701 IQYTSNYYKS TNVDFAVNTD GTYSEPRPIG TRYLTRNL (SEQ ID NO: 42)

[0157] Example of an amino acid sequence of wild-type AAVrh74 capsid protein: 1 MAADGYLPDW LEDNLSEGIR EWWDLKPGAP KPKANQQKQD NGRGLVLPGY 51 KYLGPFNGLD KGEPVNAADA AALEHDKAYD QQLQAGDNPY LRYNHADAEF 101 QERLQEDTSF GGNLGRAVFQ AKKRVLEPLG LVESPVKTAP GKKRPVEPSP 151 QRSPDSSTGI GKKGQQPAKK RLNFGQTGDS ESVPDPQPIG EPPAGPSGLG 201 SGTMAAGGGA PMADNNEGAD GVGSSSGNWH CDSTWLGDRV ITTSTRTWAL 251 PTYNNHLYKQ ISNGTSGGST NDNTYFGYST PWGYFDFNRF HCHFSPRDWQ 301 RLINNNWGFR PKRLNFKLFN IQVKEVTQNE GTKTIANNLT STIQVFTDSE 351 YQLPYVLGSA HQGCLPPFPA DVFMIPQYGY LTLNNGSQAV GRSSFYCLEY 401 FPSQMLRTGN NFEFSYNFED VPFHSSYAHS QSLDRLMNPL IDQYLYYLSR 451 TQSTGGTAGT QQLLFSQAGP NNMSAQAKNW LPGPCYRQQR VSTTLSQNNN 501 SNFAWTGATK YHLNGRDSLV NPGVAMATHK DDEERFFPSS GVLMFGKQGA 551 GKDNVDYSSV MLTSEEEIKT TNPVATEQYG VVADNLQQQN AAPIVGAVNS 601 QGALPGMVWQ NRDVYLQGPI WAKIPHTDGN FHPSPLMGGF GLKHPPPQIL 651 IKNTPVPADP PTTFNQAKLA SFITQYSTGQ VSVEIEWELQ KENSKRWNPE 701 IQYTSNYYKS TNVDFAVNTE GTYSEPRPIG TRYLTRNL (SEQ ID NO: 43)T19325 Attorney Docket No. U1202.70155WO00

[0158] Also provided herein are nucleic acids encoding capsid proteins. A nucleic acid may comprise a sequence that encodes a capsid protein disclosed here (e.g., a capsid protein comprising one or more amino acid substitutions). A sequence encoding a capsid protein disclosed herein can be determined by one of ordinary skill in the art by known methods. A nucleic acid encoding a capsid protein may comprise a promoter or other regulatory sequence operably linked to the coding sequence. A nucleic acid encoding a capsid protein may be in the form of a plasmid, an mRNA, or another nucleic acid capable of being used by enzymes or machinery of a host cell to produce a capsid protein. Nucleic acids encoding capsid proteins as provided herein can be used to make AAV particles that can be used for delivering a gene to a cell. Methods of making AAV particles are known in the art. For example, see Scientific Reports volume 9, Article number: 13601 (2019); Methods Mol Biol.2012; 798: 267–284; and thermofisher.com / us / en / home / clinical / cell-gene-therapy / gene-therapy / aav-production- workflow.html. Example sequences of nucleic acids encoding capsid proteins are provided below.

[0159] Example of a nucleotide sequence encoding AAV1 capsid protein: atggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttga aacctggagccccgaagcccaaagccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaa gtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggacgcagcggccctcgagcacgac aaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcggtataaccacgccgacgccgagtttcagg agcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaagaagcgggttctcga acctctcggtctggttgaggaaggcgctaagacggctcctggaaagaaacgtccggtagagcagtcgccacaagag ccagactcctcctcgggcatcggcaagacaggccagcagcccgctaaaaagagactcaattttggtcagactggcg actcagagtcagtccccgatccacaacctctcggagaacctccagcaacccccgctgctgtgggacctactacaat ggcttcaggcggtggcgcaccaatggcagacaataacgaaggcgccgacggagtgggtaatgcctcaggaaattgg cattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgcacctgggccttgcccacctacaata accacctctacaagcaaatctccagtgcttcaacgggggccagcaacgacaaccactacttcggctacagcacccc ctgggggtattttgatttcaacagattccactgccacttttcaccacgtgactggcagcgactcatcaacaacaat tggggattccggcccaagagactcaacttcaaactcttcaacatccaagtcaaggaggtcacgacgaatgatggcg tcacaaccatcgctaataaccttaccagcacggttcaagtcttctcggactcggagtaccagcttccgtacgtcct cggctctgcgcaccagggctgcctccctccgttcccggcggacgtgttcatgattccgcaatacggctacctgacg ctcaacaatggcagccaagccgtgggacgttcatccttttactgcctggaatatttcccttctcagatgctgagaa cgggcaacaactttaccttcagctacacctttgaggaagtgcctttccacagcagctacgcgcacagccagagcct ggaccggctgatgaatcctctcatcgaccaatacctgtattacctgaacagaactcaaaatcagtccggaagtgcc caaaacaaggacttgctgtttagccgtgggtctccagctggcatgtctgttcagcccaaaaactggctacctggac cctgttatcggcagcagcgcgtttctaaaacaaaaacagacaacaacaacagcaattttacctggactggtgcttc aaaatataacctcaatgggcgtgaatccatcatcaaccctggcactgctatggcctcacacaaagacgacgaagac aagttctttcccatgagcggtgtcatgatttttggaaaagagagcgccggagcttcaaacactgcattggacaatg tcatgattacagacgaagaggaaattaaagccactaaccctgtggccaccgaaagatttgggaccgtggcagtcaa tttccagagcagcagcacagaccctgcgaccggagatgtgcatgctatgggagcattacctggcatggtgtggcaa gatagagacgtgtacctgcagggtcccatttgggccaaaattcctcacacagatggacactttcacccgtctcctc ttatgggcggctttggactcaagaacccgcctcctcagatcctcatcaaaaacacgcctgttcctgcgaatcctcc ggcggagttttcagctacaaagtttgcttcattcatcacccaatactccacaggacaagtgagtgtggaaattgaa tgggagctgcagaaagaaaacagcaagcgctggaatcccgaagtgcagtacacatccaattatgcaaaatctgcca acgttgattttactgtggacaacaatggactttatactgagcctcgccccattggcacccgttaccttacccgtcc cctgtaa (SEQ ID NO: 44)

[0160] Example of a nucleotide sequence encoding AAV2 capsid protein:T19325 Attorney Docket No. U1202.70155WO00 atggctgccgatggttatcttccagattggctcgaggacactctctctgaaggaataagacagtggtggaagctca aacctggcccaccaccaccaaagcccgcagagcggcataaggacgacagcaggggtcttgtgcttcctgggtacaa gtacctcggacccttcaacggactcgacaagggagagccggtcaacgaggcagacgccgcggccctcgagcacgac aaagcctacgaccggcagctcgacagcggagacaacccgtacctcaagtacaaccacgccgacgcggagtttcagg agcgccttaaagaagatacgtcttttgggggcaacctcggacgagcagtcttccaggcgaaaaagagggttcttga acctctgggcctggttgaggaacctgttaagacggctccgggaaaaaagaggccggtagagcactctcctgtggag ccagactcctcctcgggaaccggaaaggcgggccagcagcctgcaagaaaaagattgaattttggtcagactggag acgcagactcagtacctgacccccagcctctcggacagccaccagcagccccctctggtctgggaactaatacgat ggctacaggcagtggcgcaccaatggcagacaataacgagggcgccgacggagtgggtaattcctccggaaattgg cattgcgattccacatggatgggcgacagagtcatcaccaccagcacccgaacctgggccctgcccacctacaaca accacctctacaaacaaatttccagccaatcaggagcctcgaacgacaatcactactttggctacagcaccccttg ggggtattttgacttcaacagattccactgccacttttcaccacgtgactggcaaagactcatcaacaacaactgg ggattccgacccaagagactcaacttcaagctctttaacattcaagtcaaagaggtcacgcagaatgacggtacga cgacgattgccaataaccttaccagcacggttcaggtgtttactgactcggagtaccagctcccgtacgtcctcgg ctcggcgcatcaaggatgcctcccgccgttcccagcagacgtcttcatggtgccacagtatggatacctcaccctg aacaacgggagtcaggcagtaggacgctcttcattttactgcctggagtactttccttctcagatgctgcgtaccg gaaacaactttaccttcagctacacttttgaggacgttcctttccacagcagctacgctcacagccagagtctgga ccgtctcatgaatcctctcatcgaccagtacctgtattacttgagcagaacaaacactccaagtggaaccaccacg cagtcaaggcttcagttttctcaggccggagcgagtgacattcgggaccagtctaggaactggcttcctggaccct gttaccgccagcagcgagtatcaaagacatctgcggataacaacaacagtgaatactcgtggactggagctaccaa gtaccacctcaatggcagagactctctggtgaatccgggcccggccatggcaagccacaaggacgatgaagaaaag ttttttcctcagagcggggttctcatctttgggaagcaaggctcagagaaaacaaatgtggacattgaaaaggtca tgattacagacgaagaggaaatcaggacaaccaatcccgtggctacggagcagtatggttctgtatctaccaacct ccagagaggcaacagacaagcagctaccgcagatgtcaacacacaaggcgttcttccaggcatggtctggcaggac agagatgtgtaccttcaggggcccatctgggcaaagattccacacacggacggacattttcacccctctcccctca tgggtggattcggacttaaacaccctcctccacagattctcatcaagaacaccccggtacctgcgaatccttcgac caccttcagtgcggcaaagtttgcttccttcatcacacagtactccacgggacaggtcagcgtggagatcgagtgg gagctgcagaaggaaaacagcaaacgctggaatcccgaaattcagtacacttccaactacaacaagtctgttaatg tggactttactgtggacactaatggcgtgtattcagagcctcgccccattggcaccagatacctgactcgtaatct gtaa (SEQ ID NO: 45)

[0161] Example of a nucleotide sequence encoding AAV3 capsid protein: atggctgctgacggttatcttccagattggctcgaggacaacctttctgaaggcattcgtgagtggtgggctctga aacctggagtccctcaacccaaagcgaaccaacaacaccaggacaaccgtcggggtcttgtgcttccgggttacaa atacctcggacccggtaacggactcgacaaaggagagccggtcaacgaggcggacgcggcagccctcgaacacgac aaagcttacgaccagcagctcaaggccggtgacaacccgtacctcaagtacaaccacgccgacgccgagtttcagg agcgtcttcaagaagatacgtcttttgggggcaaccttggcagagcagtcttccaggccaaaaagaggatccttga gcctcttggtctggttgaggaagcagctaaaacggctcctggaaagaagggggctgtagatcagtctcctcaggaa ccggactcatcatctggtgttggcaaatcgggcaaacagcctgccagaaaaagactaaatttcggtcagactggag actcagagtcagtcccagaccctcaacctctcggagaaccaccagcagcccccacaagtttgggatctaatacaat ggcttcaggcggtggcgcaccaatggcagacaataacgagggtgccgatggagtgggtaattcctcaggaaattgg cattgcgattcccaatggctgggcgacagagtcatcaccaccagcaccagaacctgggccctgcccacttacaaca accatctctacaagcaaatctccagccaatcaggagcttcaaacgacaaccactactttggctacagcaccccttg ggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactcattaacaacaactgg ggattccggcccaagaaactcagcttcaagctcttcaacatccaagttagaggggtcacgcagaacgatggcacga cgactattgccaataaccttaccagcacggttcaagtgtttacggactcggagtatcagctcccgtacgtgctcgg gtcggcgcaccaaggctgtctcccgccgtttccagcggacgtcttcatggtccctcagtatggatacctcaccctg aacaacggaagtcaagcggtgggacgctcatccttttactgcctggagtacttcccttcgcagatgctaaggactg gaaataacttccaattcagctataccttcgaggatgtaccttttcacagcagctacgctcacagccagagtttgga tcgcttgatgaatcctcttattgatcagtatctgtactacctgaacagaacgcaaggaacaacctctggaacaacc aaccaatcacggctgctttttagccaggctgggcctcagtctatgtctttgcaggccagaaattggctacctgggc cctgctaccggcaacagagactttcaaagactgctaacgacaacaacaacagtaactttccttggacagcggccag caaatatcatctcaatggccgcgactcgctggtgaatccaggaccagctatggccagtcacaaggacgatgaagaa aaatttttccctatgcacggcaatctaatatttggcaaagaagggacaacggcaagtaacgcagaattagataatg taatgattacggatgaagaagagattcgtaccaccaatcctgtggcaacagagcagtatggaactgtggcaaataa cttgcagagctcaaatacagctcccacgactggaactgtcaatcatcagggggccttacctggcatggtgtggcaa gatcgtgacgtgtaccttcaaggacctatctgggcaaagattcctcacacggatggacactttcatccttctcctc tgatgggaggctttggactgaaacatccgcctcctcaaatcatgatcaaaaatactccggtaccggcaaatcctccT19325 Attorney Docket No. U1202.70155WO00 gacgactttcagcccggccaagtttgcttcatttatcactcagtactccactggacaggtcagcgtggaaattgag tgggagctacagaaagaaaacagcaaacgttggaatccagagattcagtacacttccaactacaacaagtctgtta atgtggactttactgtagacactaatggtgtttatagtgaacctcgccctattggaacccggtatctcacacgaaa cttgtga (SEQ ID NO: 46)

[0162] Example of a nucleotide sequence encoding AAV4 capsid protein: atgactgacggttaccttccagattggctagaggacaacctctctgaaggcgttcgagagtggtgggcgctgcaac ctggagcccctaaacccaaggcaaatcaacaacatcaggacaacgctcggggtcttgtgcttccgggttacaaata cctcggacccggcaacggactcgacaagggggaacccgtcaacgcagcggacgcggcagccctcgagcacgacaag gcctacgaccagcagctcaaggccggtgacaacccctacctcaagtacaaccacgccgacgcggagttccagcagc ggcttcagggcgacacatcgtttgggggcaacctcggcagagcagtcttccaggccaaaaagagggttcttgaacc tcttggtctggttgagcaagcgggtgagacggctcctggaaagaagagaccgttgattgaatccccccagcagccc gactcctccacgggtatcggcaaaaaaggcaagcagccggctaaaaagaagctcgttttcgaagacgaaactggag caggcgacggaccccctgagggatcaacttccggagccatgtctgatgacagtgagatgcgtgcagcagctggcgg agctgcagtcgagggcggacaaggtgccgatggagtgggtaatgcctcgggtgattggcattgcgattccacctgg tctgagggccacgtcacgaccaccagcaccagaacctgggtcttgcccacctacaacaaccacctctacaagcgac tcggagagagcctgcagtccaacacctacaacggattctccaccccctggggatactttgacttcaaccgcttcca ctgccacttctcaccacgtgactggcagcgactcatcaacaacaactggggcatgcgacccaaagccatgcgggtc aaaatcttcaacatccaggtcaaggaggtcacgacgtcgaacggcgagacaacggtggctaataaccttaccagca cggttcagatctttgcggactcgtcgtacgaactgccgtacgtgatggatgcgggtcaagagggcagcctgcctcc ttttcccaacgacgtctttatggtgccccagtacggctactgtggactggtgaccggcaacacttcgcagcaacag actgacagaaatgccttctactgcctggagtactttccttcgcagatgctgcggactggcaacaactttgaaatta cgtacagttttgagaaggtgcctttccactcgatgtacgcgcacagccagagcctggaccggctgatgaaccctct catcgaccagtacctgtggggactgcaatcgaccaccaccggaaccaccctgaatgccgggactgccaccaccaac tttaccaagctgcggcctaccaacttttccaactttaaaaagaactggctgcccgggccttcaatcaagcagcagg gcttctcaaagactgccaatcaaaactacaagatccctgccaccgggtcagacagtctcatcaaatacgagacgca cagcactctggacggaagatggagtgccctgacccccggacctccaatggccacggctggacctgcggacagcaag ttcagcaacagccagctcatctttgcggggcctaaacagaacggcaacacggccaccgtacccgggactctgatct tcacctctgaggaggagctggcagccaccaacgccaccgatacggacatgtggggcaacctacctggcggtgacca gagcaacagcaacctgccgaccgtggacagactgacagccttgggagccgtgcctggaatggtctggcaaaacaga gacatttactaccagggtcccatttgggccaagattcctcataccgatggacactttcacccctcaccgctgattg gtgggtttgggctgaaacacccgcctcctcaaatttttatcaagaacaccccggtacctgcgaatcctgcaacgac cttcagctctactccggtaaactccttcattactcagtacagcactggccaggtgtcggtgcagattgactgggag atccagaaggagcggtccaaacgctggaaccccgaggtccagtttacctccaactacggacagcaaaactctctgt tgtgggctcccgatgcggctgggaaatacactgagcctagggctatcggtacccgctacctcacccaccacctgta ataacctgttaatcaataaaccggtttattcgtttcagttgaactttggtctccgtgtccttcttatcttatctcg tttcc (SEQ ID NO: 47)

[0163] Example of a nucleotide sequence encoding AAV5 capsid protein: atgtcttttgttgatcaccctccagattggttggaagaagttggtgaaggtcttcgcgagtttttgggccttgaag cgggcccaccgaaaccaaaacccaatcagcagcatcaagatcaagcccgtggtcttgtgctgcctggttataacta tctcggacccggaaacggtctcgatcgaggagagcctgtcaacagggcagacgaggtcgcgcgagagcacgacatc tcgtacaacgagcagcttgaggcgggagacaacccctacctcaagtacaaccacgcggacgccgagtttcaggaga agctcgccgacgacacatccttcgggggaaacctcggaaaggcagtctttcaggccaagaaaagggttctcgaacc ttttggcctggttgaagagggtgctaagacggcccctaccggaaagcggatagacgaccactttccaaaaagaaag aaggctcggaccgaagaggactccaagccttccacctcgtcagacgccgaagctggacccagcggatcccagcagc tgcaaatcccagcccaaccagcctcaagtttgggagctgatacaatgtctgcgggaggtggcggcccattgggcga caataaccaaggtgccgatggagtgggcaatgcctcgggagattggcattgcgattccacgtggatgggggacaga gtcgtcaccaagtccacccgaacctgggtgctgcccagctacaacaaccaccagtaccgagagatcaaaagcggct ccgtcgacggaagcaacgccaacgcctactttggatacagcaccccctgggggtactttgactttaaccgcttcca cagccactggagcccccgagactggcaaagactcatcaacaactactggggcttcagaccccggtccctcagagtc aaaatcttcaacattcaagtcaaagaggtcacggtgcaggactccaccaccaccatcgccaacaacctcacctcca ccgtccaagtgtttacggacgacgactaccagctgccctacgtcgtcggcaacgggaccgagggatgcctgccggc cttccctccgcaggtctttacgctgccgcagtacggttacgcgacgctgaaccgcgacaacacagaaaatcccacc gagaggagcagcttcttctgcctagagtactttcccagcaagatgctgagaacgggcaacaactttgagtttacct acaactttgaggaggtgcccttccactccagcttcgctcccagtcagaacctgttcaagctggccaacccgctggt ggaccagtacttgtaccgcttcgtgagcacaaataacactggcggagtccagttcaacaagaacctggccgggagaT19325 Attorney Docket No. U1202.70155WO00 tacgccaacacctacaaaaactggttcccggggcccatgggccgaacccagggctggaacctgggctccggggtca accgcgccagtgtcagcgccttcgccacgaccaataggatggagctcgagggcgcgagttaccaggtgcccccgca gccgaacggcatgaccaacaacctccagggcagcaacacctatgccctggagaacactatgatcttcaacagccag ccggcgaacccgggcaccaccgccacgtacctcgagggcaacatgctcatcaccagcgagagcgagacgcagccgg tgaaccgcgtggcgtacaacgtcggcgggcagatggccaccaacaaccagagctccaccactgcccccgcgaccgg cacgtacaacctccaggaaatcgtgcccggcagcgtgtggatggagagggacgtgtacctccaaggacccatctgg gccaagatcccagagacgggggcgcactttcacccctctccggccatgggcggattcggactcaaacacccaccgc ccatgatgctcatcaagaacacgcctgtgcccggaaatatcaccagcttctcggacgtgcccgtcagcagcttcat cacccagtacagcaccgggcaggtcaccgtggagatggagtgggagctcaagaaggaaaactccaagaggtggaac ccagagatccagtacacaaacaactacaacgacccccagtttgtggactttgccccggacagcaccggggaataca gaaccaccagacctatcggaacccgataccttacccgacccctttaa (SEQ ID NO: 48)

[0164] Example of a nucleotide sequence encoding AAV6 capsid protein: atggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttga aacctggagccccgaaacccaaagccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaa gtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggatgcagcggccctcgagcacgac aaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcggtataaccacgccgacgccgagtttcagg agcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaagaagagggttctcga accttttggtctggttgaggaaggtgctaagacggctcctggaaagaaacgtccggtagagcagtcgccacaagag ccagactcctcctcgggcattggcaagacaggccagcagcccgctaaaaagagactcaattttggtcagactggcg actcagagtcagtccccgacccacaacctctcggagaacctccagcaacccccgctgctgtgggacctactacaat ggcttcaggcggtggcgcaccaatggcagacaataacgaaggcgccgacggagtgggtaatgcctcaggaaattgg cattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgaacatgggccttgcccacctataaca accacctctacaagcaaatctccagtgcttcaacgggggccagcaacgacaaccactacttcggctacagcacccc ctgggggtattttgatttcaacagattccactgccatttctcaccacgtgactggcagcgactcatcaacaacaat tggggattccggcccaagagactcaacttcaagctcttcaacatccaagtcaaggaggtcacgacgaatgatggcg tcacgaccatcgctaataaccttaccagcacggttcaagtcttctcggactcggagtaccagttgccgtacgtcct cggctctgcgcaccagggctgcctccctccgttcccggcggacgtgttcatgattccgcagtacggctacctaacg ctcaacaatggcagccaggcagtgggacggtcatccttttactgcctggaatatttcccatcgcagatgctgagaa cgggcaataactttaccttcagctacaccttcgaggacgtgcctttccacagcagctacgcgcacagccagagcct ggaccggctgatgaatcctctcatcgaccagtacctgtattacctgaacagaactcagaatcagtccggaagtgcc caaaacaaggacttgctgtttagccgggggtctccagctggcatgtctgttcagcccaaaaactggctacctggac cctgttaccggcagcagcgcgtttctaaaacaaaaacagacaacaacaacagcaactttacctggactggtgcttc aaaatataaccttaatgggcgtgaatctataatcaaccctggcactgctatggcctcacacaaagacgacaaagac aagttctttcccatgagcggtgtcatgatttttggaaaggagagcgccggagcttcaaacactgcattggacaatg tcatgatcacagacgaagaggaaatcaaagccactaaccccgtggccaccgaaagatttgggactgtggcagtcaa tctccagagcagcagcacagaccctgcgaccggagatgtgcatgttatgggagccttacctggaatggtgtggcaa gacagagacgtatacctgcagggtcctatttgggccaaaattcctcacacggatggacactttcacccgtctcctc tcatgggcggctttggacttaagcacccgcctcctcagatcctcatcaaaaacacgcctgttcctgcgaatcctcc ggcagagttttcggctacaaagtttgcttcattcatcacccagtattccacaggacaagtgagcgtggagattgaa tgggagctgcagaaagaaaacagcaaacgctggaatcccgaagtgcagtatacatctaactatgcaaaatctgcca acgttgatttcactgtggacaacaatggactttatactgagcctcgccccattggcacccgttacctcacccgtcc cctgtaat (SEQ ID NO: 49)

[0165] Example of a nucleotide sequence encoding AAV7 capsid protein: atggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacctga aacctggagccccgaaacccaaagccaaccagcaaaagcaggacaacggccggggtctggtgcttcctggctacaa gtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggacgcagcggccctcgagcacgac aaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcggtataaccacgccgacgccgagtttcagg agcgtctgcaagaagatacgtcatttgggggcaacctcgggcgagcagtcttccaggccaagaagcgggttctcga acctctcggtctggttgaggaaggcgctaagacggctcctgcaaagaagagaccggtagagccgtcacctcagcgt tcccccgactcctccacgggcatcggcaagaaaggccagcagcccgccagaaagagactcaatttcggtcagactg gcgactcagagtcagtccccgaccctcaacctctcggagaacctccagcagcgccctctagtgtgggatctggtac agtggctgcaggcggtggcgcaccaatggcagacaataacgaaggtgccgacggagtgggtaatgcctcaggaaat tggcattgcgattccacatggctgggcgacagagtcattaccaccagcacccgaacctgggccctgcccacctaca acaaccacctctacaagcaaatctccagtgaaactgcaggtagtaccaacgacaacacctacttcggctacagcac cccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactcatcaacaacT19325 Attorney Docket No. U1202.70155WO00 aactggggattccggcccaagaagctgcggttcaagctcttcaacatccaggtcaaggaggtcacgacgaatgacg gcgttacgaccatcgctaataaccttaccagcacgattcaggtattctcggactcggaataccagctgccgtacgt cctcggctctgcgcaccagggctgcctgcctccgttcccggcggacgtcttcatgattcctcagtacggctacctg actctcaacaatggcagtcagtctgtgggacgttcctccttctactgcctggagtacttcccctctcagatgctga gaacgggcaacaactttgagttcagctacagcttcgaggacgtgcctttccacagcagctacgcacacagccagag cctggaccggctgatgaatcccctcatcgaccagtacttgtactacctggccagaacacagagtaacccaggaggc acagctggcaatcgggaactgcagttttaccagggcgggccttcaactatggccgaacaagccaagaattggttac ctggaccttgcttccggcaacaaagagtctccaaaacgctggatcaaaacaacaacagcaactttgcttggactgg tgccaccaaatatcacctgaacggcagaaactcgttggttaatcccggcgtcgccatggcaactcacaaggacgac gaggaccgctttttcccatccagcggagtcctgatttttggaaaaactggagcaactaacaaaactacattggaaa atgtgttaatgacaaatgaagaagaaattcgtcctactaatcctgtagccacggaagaatacgggatagtcagcag caacttacaagcggctaatactgcagcccagacacaagttgtcaacaaccagggagccttacctggcatggtctgg cagaaccgggacgtgtacctgcagggtcccatctgggccaagattcctcacacggatggcaactttcacccgtctc ctttgatgggcggctttggacttaaacatccgcctcctcagatcctgatcaagaacactcccgttcccgctaatcc tccggaggtgtttactcctgccaagtttgcttcgttcatcacacagtacagcaccggacaagtcagcgtggaaatc gagtgggagctgcagaaggaaaacagcaagcgctggaacccggagattcagtacacctccaactttgaaaagcaga ctggtgtggactttgccgttgacagccagggtgtttactctgagcctcgccctattggcactcgttacctcacccg taatctgtaa (SEQ ID NO: 50)

[0166] Example of a nucleotide sequence encoding AAV8 capsid protein: atggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggcgctga aacctggagccccgaagcccaaagccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaa gtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggacgcagcggccctggagcacgac aaggcctacgaccagcagctgcaggcgggtgacaatccgtacctgcggtataaccacgccgacgccgagtttcagg agcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaagaagcgggttctcga acctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagccatcaccccagcgt tctccagactcctctacgggcatcggcaagaaaggccaacagcccgccagaaaaagactcaattttggtcagactg gcgactcagagtcagttccagaccctcaacctctcggagaacctccagcagcgccctctggtgtgggacctaatac aatggctgcaggcggtggcgcaccaatggcagacaataacgaaggcgccgacggagtgggtagttcctcgggaaat tggcattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgaacctgggccctgcccacctaca acaaccacctctacaagcaaatctccaacgggacatcgggaggagccaccaacgacaacacctacttcggctacag caccccctgggggtattttgactttaacagattccactgccacttttcaccacgtgactggcagcgactcatcaac aacaactggggattccggcccaagagactcagcttcaagctcttcaacatccaggtcaaggaggtcacgcagaatg aaggcaccaagaccatcgccaataacctcaccagcaccatccaggtgtttacggactcggagtaccagctgccgta cgttctcggctctgcccaccagggctgcctgcctccgttcccggcggacgtgttcatgattccccagtacggctac ctaacactcaacaacggtagtcaggccgtgggacgctcctccttctactgcctggaatactttccttcgcagatgc tgagaaccggcaacaacttccagtttacttacaccttcgaggacgtgcctttccacagcagctacgcccacagcca gagcttggaccggctgatgaatcctctgattgaccagtacctgtactacttgtctcggactcaaacaacaggaggc acggcaaatacgcagactctgggcttcagccaaggtgggcctaatacaatggccaatcaggcaaagaactggctgc caggaccctgttaccgccaacaacgcgtctcaacgacaaccgggcaaaacaacaatagcaactttgcctggactgc tgggaccaaataccatctgaatggaagaaattcattggctaatcctggcatcgctatggcaacacacaaagacgac gaggagcgtttttttcccagtaacgggatcctgatttttggcaaacaaaatgctgccagagacaatgcggattaca gcgatgtcatgctcaccagcgaggaagaaatcaaaaccactaaccctgtggctacagaggaatacggtatcgtggc agataacttgcagcagcaaaacacggctcctcaaattggaactgtcaacagccagggggccttacccggtatggtc tggcagaaccgggacgtgtacctgcagggtcccatctgggccaagattcctcacacggacggcaacttccacccgt ctccgctgatgggcggctttggcctgaaacatcctccgcctcagatcctgatcaagaacacgcctgtacctgcgga tcctccgaccaccttcaaccagtcaaagctgaactctttcatcacgcaatacagcaccggacaggtcagcgtggaa attgaatgggagctgcagaaggaaaacagcaagcgctggaaccccgagatccagtacacctccaactactacaaat ctacaagtgtggactttgctgttaatacagaaggcgtgtactctgaaccccgccccattggcacccgttacctcac ccgtaatctgtaa (SEQ ID NO: 51)

[0167] Example of a nucleotide sequence encoding AAV9 capsid protein: atggctgccgatggttatcttccagattggctcgaggacaaccttagtgaaggaattcgcgagtggtgggctttga aacctggagcccctcaacccaaggcaaatcaacaacatcaagacaacgctcgaggtcttgtgcttccgggttacaa ataccttggacccggcaacggactcgacaagggggagccggtcaacgcagcagacgcggcggccctcgagcacgac aaggcctacgaccagcagctcaaggccggagacaacccgtacctcaagtacaaccacgccgacgccgagttccagg agcggctcaaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaaaaagaggcttcttgaT19325 Attorney Docket No. U1202.70155WO00 acctcttggtctggttgaggaagcggctaagacggctcctggaaagaagaggcctgtagagcagtctcctcaggaa ccggactcctccgcgggtattggcaaatcgggtgcacagcccgctaaaaagagactcaatttcggtcagactggcg acacagagtcagtcccagaccctcaaccaatcggagaacctcccgcagccccctcaggtgtgggatctcttacaat ggcttcaggtggtggcgcaccagtggcagacaataacgaaggtgccgatggagtgggtagttcctcgggaaattgg cattgcgattcccaatggctgggggacagagtcatcaccaccagcacccgaacctgggccctgcccacctacaaca atcacctctacaagcaaatctccaacagcacatctggaggatcttcaaatgacaacgcctacttcggctacagcac cccctgggggtattttgacttcaacagattccactgccacttctcaccacgtgactggcagcgactcatcaacaac aactggggattccggcctaagcgactcaacttcaagctcttcaacattcaggtcaaagaggttacggacaacaatg gagtcaagaccatcgccaataaccttaccagcacggtccaggtcttcacggactcagactatcagctcccgtacgt gctcgggtcggctcacgagggctgcctcccgccgttcccagcggacgttttcatgattcctcagtacgggtatctg acgcttaatgatggaagccaggccgtgggtcgttcgtccttttactgcctggaatatttcccgtcgcaaatgctaa gaacgggtaacaacttccagttcagctacgagtttgagaacgtacctttccatagcagctacgctcacagccaaag cctggaccgactaatgaatccactcatcgaccaatacttgtactatctctcaaagactattaacggttctggacag aatcaacaaacgctaaaattcagtgtggccggacccagcaacatggctgtccagggaagaaactacatacctggac ccagctaccgacaacaacgtgtctcaaccactgtgactcaaaacaacaacagcgaatttgcttggcctggagcttc ttcttgggctctcaatggacgtaatagcttgatgaatcctggacctgctatggccagccacaaagaaggagaggac cgtttctttcctttgtctggatctttaatttttggcaaacaaggaactggaagagacaacgtggatgcggacaaag tcatgataaccaacgaagaagaaattaaaactactaacccggtagcaacggagtcctatggacaagtggccacaaa ccaccagagtgcccaagcacaggcgcagaccggctgggttcaaaaccaaggaatacttccgggtatggtttggcag gacagagatgtgtacctgcaaggacccatttgggccaaaattcctcacacggacggcaactttcacccttctccgc tgatgggagggtttggaatgaagcacccgcctcctcagatcctcatcaaaaacacacctgtacctgcggatcctcc aacggccttcaacaaggacaagctgaactctttcatcacccagtattctactggccaagtcagcgtggagatcgag tgggagctgcagaaggaaaacagcaagcgctggaacccggagatccagtacacttccaactattacaagtctaata atgttgaatttgctgttaatactgaaggtgtatatagtgaaccccgccccattggcaccagatacctgactcgtaa tctgtaa (SEQ ID NO: 52)

[0168] Example of a nucleotide sequence encoding AAV10 capsid protein: atggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacttga aacctggagccccgaaacccaaagccaaccagcaaaagcaggacgacggccggggtctggtgcttcctggctacaa gtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggacgcagcggccctcgagcacgac aaggcctacgaccagcagctcaaagcgggtgacaatccgtacctgcggtataaccacgccgacgccgagtttcagg agcgtctgcaagaagatacgtcttttgggggcaacctcgggcgagcagtcttccaggccaagaagcgggttctcga acctctcggtctggttgaggaaggcgctaagacggctcctggaaagaagagaccggtagagccatcaccccagcgt tctccagactcctctacgggcatcggcaagaaaggccagcagcccgcgaaaaagagactcaactttgggcagactg gcgactcagagtcagtgcccgaccctcaaccaatcggagaaccccccgcaggcccctctggtctgggatctggtac aatggctgcaggcggtggcgctccaatggcagacaataacgaaggcgccgacggagtgggtagttcctcaggaaat tggcattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgaacctgggccctccccacctaca acaaccacctctacaagcaaatctccaacgggacttcgggaggaagcaccaacgacaacacctacttcggctacag caccccctgggggtattttgactttaacagattccactgccacttctcaccacgtgactggcagcgactcatcaac aacaactggggattccggcccaagagactcaacttcaagctcttcaacatccaggtcaaggaggtcacgcagaatg aaggcaccaagaccatcgccaataaccttaccagcacgattcaggtctttacggactcggaataccagctcccgta cgtcctcggctctgcgcaccagggctgcctgcctccgttcccggcggacgtcttcatgattcctcagtacgggtac ctgactctgaacaatggcagtcaggccgtgggccgttcctccttctactgcctggagtactttccttctcaaatgc tgagaacgggcaacaactttgagttcagctaccagtttgaggacgtgccttttcacagcagctacgcgcacagcca aagcctggaccggctgatgaaccccctcatcgaccagtacctgtactacctgtctcggactcagtccacgggaggt accgcaggaactcagcagttgctattttctcaggccgggcctaataacatgtcggctcaggccaaaaactggctac ccgggccctgctaccggcagcaacgcgtctccacgacactgtcgcaaaataacaacagcaactttgcctggaccgg tgccaccaagtatcatctgaatggcagagactctctggtaaatcccggtgtcgctatggcaacccacaaggacgac gaagagcgattttttccgtccagcggagtcttaatgtttgggaaacagggagctggaaaagacaacgtggactata gcagcgttatgctaaccagtgaggaagaaattaaaaccaccaacccagtggccacagaacagtacggcgtggtggc cgataacctgcaacagcaaaacgccgctcctattgtaggggccgtcaacagtcaaggagccttacctggcatggtc tggcagaaccgggacgtgtacctgcagggtcctatctgggccaagattcctcacacggacggaaactttcatccct cgccgctgatgggaggctttggactgaaacacccgcctcctcagatcctgattaagaatacacctgttcccgcgga tcctccaactaccttcagtcaagctaagctggcgtcgttcatcacgcagtacagcaccggacaggtcagcgtggaa attgaatgggagctgcagaaagaaaacagcaaacgctggaacccagagattcaatacacttccaactactacaaat ctacaaatgtggactttgctgttaacacagatggcacttattctgagcctcgccccatcggcacccgttacctcac ccgtaatctgtaa (SEQ ID NO: 53)T19325 Attorney Docket No. U1202.70155WO00

[0169] Example of a nucleotide sequence encoding AAVrh74 capsid protein: atggctgccgatggttatcttccagattggctcgaggacaacctctctgagggcattcgcgagtggtgggacctga aacctggagccccgaaacccaaagccaaccagcaaaagcaggacaacggccggggtctggtgcttcctggctacaa gtacctcggacccttcaacggactcgacaagggggagcccgtcaacgcggcggacgcagcggccctcgagcacgac aaggcctacgaccagcagctccaagcgggtgacaatccgtacctgcggtataatcacgccgacgccgagtttcagg agcgtctgcaagaagatacgtcttttgggggcaacctcgggcgcgcagtcttccaggccaaaaagcgggttctcga acctctgggcctggttgaatcgccggttaagacggctcctggaaagaagagaccggtagagccatcaccccagcgc tctccagactcctctacgggcatcggcaagaaaggccagcagcccgcaaaaaagagactcaattttgggcagactg gcgactcagagtcagtccccgaccctcaaccaatcggagaaccaccagcaggcccctctggtctgggatctggtac aatggctgcaggcggtggcgctccaatggcagacaataacgaaggcgccgacggagtgggtagttcctcaggaaat tggcattgcgattccacatggctgggcgacagagtcatcaccaccagcacccgcacctgggccctgcccacctaca acaaccacctctacaagcaaatctccaacgggacctcgggaggaagcaccaacgacaacacctacttcggctacag caccccctgggggtattttgacttcaacagattccactgccacttttcaccacgtgactggcagcgactcatcaac aacaactggggattccggcccaagaggctcaacttcaagctcttcaacatccaagtcaaggaggtcacgcagaatg aaggcaccaagaccatcgccaataaccttaccagcacgattcaggtctttacggactcggaataccagctcccgta cgtgctcggctcggcgcaccagggctgcctgcctccgttcccggcggacgtcttcatgattcctcagtacgggtac ctgactctgaacaatggcagtcaggctgtgggccggtcgtccttctactgcctggagtactttccttctcaaatgc tgagaacgggcaacaactttgaattcagctacaacttcgaggacgtgcccttccacagcagctacgcgcacagcca gagcctggaccggctgatgaaccctctcatcgaccagtacttgtactacctgtcccggactcaaagcacgggcggt actgcaggaactcagcagttgctattttctcaggccgggcctaacaacatgtcggctcaggccaagaactggctac ccggtccctgctaccggcagcaacgcgtctccacgacactgtcgcagaacaacaacagcaactttgcctggacggg tgccaccaagtatcatctgaatggcagagactctctggtgaatcctggcgttgccatggctacccacaaggacgac gaagagcgattttttccatccagcggagtcttaatgtttgggaaacagggagctggaaaagacaacgtggactata gcagcgtgatgctaaccagcgaggaagaaataaagaccaccaacccagtggccacagaacagtacggcgtggtggc cgataacctgcaacagcaaaacgccgctcctattgtaggggccgtcaatagtcaaggagccttacctggcatggtg tggcagaaccgggacgtgtacctgcagggtcccatctgggccaagattcctcatacggacggcaactttcatccct cgccgctgatgggaggctttggactgaagcatccgcctcctcagatcctgattaaaaacacacctgttcccgccga tcctccgaccaccttcaatcaggccaagctggcttctttcatcacgcagtacagtaccggtcaggtcagcgtggag atcgagtgggagctgcagaaggagaacagcaaacgctggaacccagagattcagtacacttccaactactacaaat ctacaaatgtggactttgctgtcaatactgagggtacttattccgagcctcgccccattggcacccgttacctcac ccgtaatctgtaa (SEQ ID NO: 54) Second-Strand Synthesis

[0170] According to some aspects, second-strand synthesis of a single-stranded AAV genome disclosed herein (e.g., comprising a modification) is modified relative to a corresponding wild- type AAV genome (e.g., not comprising the modification). Second-strand synthesis can be measured by one of ordinary skill in the art by known methods. In some aspects, second-strand synthesis of an AAV genome disclosed herein is increased relative to a corresponding wild- type AAV genome. In some aspects, second-strand synthesis of an AAV genome disclosed herein is increased as a result of a decrease in binding of a host-cell protein (e.g., a phosphorylated host-cell protein, such as FKBP52) to the AAV genome (e.g., to a D-sequence of the AAV genome).

[0171] In some aspects, the second-strand synthesis of an AAV genome as disclosed herein is at least 5% higher (e.g., at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher, at least 250% higher, or more) than theT19325 Attorney Docket No. U1202.70155WO00 second-strand synthesis of a corresponding wild-type AAV genome. In some aspects, the second-strand synthesis of an AAV genome as disclosed herein is at least 1.5-fold higher (e.g., at least 2-fold higher, at least 2.5-fold higher, at least 3-fold higher, at least 3.5-fold higher, at least 4-fold higher, at least 4.5-fold higher, at least 5-fold higher, at least 5.5-fold higher, at least 6-fold higher, at least 6.5-fold higher, at least 7-fold higher, at least 7.5-fold higher, at least 8-fold higher, at least 8.5-fold higher, at least 9-fold higher, at least 9.5-fold higher, at least 10-fold higher, at least 10.5-fold higher, at least 11-fold higher, at least 11.5-fold higher, at least 12-fold higher, at least 12.5-fold higher, at least 13-fold higher, at least 13.5-fold higher, at least 14-fold higher, at least 14.5-fold higher, at least 15-fold higher, at least 15.5- fold higher, at least 16-fold higher, at least 16.5-fold higher, at least 17-fold higher, at least 17.5-fold higher, at least 18-fold higher, at least 18.5-fold higher, at least 19-fold higher, at least 19.5-fold higher, at least 20-fold higher, or more) than the second-strand synthesis of a corresponding wild-type AAV genome. In some aspects, second-strand synthesis of an AAV particle as disclosed herein is not modified relative to a corresponding wild-type AAV particle. Transduction Efficiency

[0172] According to some aspects, transduction efficiency of an AAV particle disclosed herein (e.g., comprising a modification in a nucleic acid vector and / or in a capsid protein) is modified relative to a corresponding wild-type AAV particle (e.g., not comprising the modification in the nucleic acid vector and / or in the capsid protein). Transduction efficiency of an AAV particle can be determined, for example, by comparing expression of a gene of interest in a cell following contacting the cell with the AAV particle, or by measuring the number of viral genome copies per cell following contacting a population of cells with the AAV particle. In some aspects, transduction efficiency of an AAV particle as disclosed herein (e.g., an AAV particle comprising a modified capsid protein (e.g., comprising one or more amino acid substitutions), a modified nucleic acid vector (e.g., comprising one or two modified ITRs), or both a modified capsid protein (e.g., comprising one or more amino acid substitutions) and one or two modified ITRs is higher than the transduction efficiency of a corresponding wild-type AAV particle (e.g., not comprising the modified capsid protein or modified ITR(s). In some aspects, the transduction efficiency of an AAV particle as disclosed herein is at least 5% higher (e.g., at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at leastT19325 Attorney Docket No. U1202.70155WO00 150% higher, at least 200% higher, at least 250% higher, or more) than the transduction efficiency of a corresponding wild-type AAV particle. In some aspects, the transduction efficiency of an AAV particle as disclosed herein is at least 1.5-fold higher (e.g., at least 2-fold higher, at least 2.5-fold higher, at least 3-fold higher, at least 3.5-fold higher, at least 4-fold higher, at least 4.5-fold higher, at least 5-fold higher, at least 5.5-fold higher, at least 6-fold higher, at least 6.5-fold higher, at least 7-fold higher, at least 7.5-fold higher, at least 8-fold higher, at least 8.5-fold higher, at least 9-fold higher, at least 9.5-fold higher, at least 10-fold higher, at least 10.5-fold higher, at least 11-fold higher, at least 11.5-fold higher, at least 12- fold higher, at least 12.5-fold higher, at least 13-fold higher, at least 13.5-fold higher, at least 14-fold higher, at least 14.5-fold higher, at least 15-fold higher, at least 15.5-fold higher, at least 16-fold higher, at least 16.5-fold higher, at least 17-fold higher, at least 17.5-fold higher, at least 18-fold higher, at least 18.5-fold higher, at least 19-fold higher, at least 19.5-fold higher, at least 20-fold higher, or more) than the transduction efficiency of a corresponding wild-type AAV particle. In some aspects, transduction efficiency of an AAV particle as disclosed herein is not modified relative to a corresponding wild-type AAV particle. Transgene expression

[0173] According to some aspects, expression of a transgene encoded by a nucleic acid vector comprising a modification (e.g., one or more modified ITRs) disclosed herein is altered relative to expression of the transgene encoded by a nucleic acid vector that does not comprise the modification. Such alteration of transgene expression is, in some aspects, on a per nucleic acid vector copy number basis (e.g., transgene expression in a cell, when normalized to the total amount of nucleic acid vector in the cell, is altered). For example, in some aspects, a modified AAV particle as disclosed herein results in greater transgene expression relative to a corresponding AAV particle not comprising the same modification but that delivers a comparable number of viral genomes to a cell. Relative transgene expression levels can be determined, for example, by measuring expression of the transgene in a cell by methods known in the art following contacting the cell with an AAV particle comprising the modified nucleic acid vector encoding the transgene and comparing an equivalent measurement in another cell contacted with an AAV particle comprising a nucleic acid vector that does not comprise the modification.

[0174] In some aspects, transgene expression from a modified nucleic acid vector as disclosed herein is higher than the transgene expression from a corresponding nucleic acid vector thatT19325 Attorney Docket No. U1202.70155WO00 does not comprise the modification. In some aspects, the transgene expression from a modified nucleic acid vector as disclosed herein is at least 5% higher (e.g., at least 10% higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher, at least 250% higher, or more) than the transgene expression from a corresponding nucleic acid vector that does not comprise the modification.

[0175] In some aspects, the transgene expression from a modified nucleic acid vector as disclosed herein is at least 1.5-fold higher (e.g., at least 2-fold higher, at least 2.5-fold higher, at least 3-fold higher, at least 3.5-fold higher, at least 4-fold higher, at least 4.5-fold higher, at least 5-fold higher, at least 5.5-fold higher, at least 6-fold higher, at least 6.5-fold higher, at least 7-fold higher, at least 7.5-fold higher, at least 8-fold higher, at least 8.5-fold higher, at least 9-fold higher, at least 9.5-fold higher, at least 10-fold higher, at least 10.5-fold higher, at least 11-fold higher, at least 11.5-fold higher, at least 12-fold higher, at least 12.5-fold higher, at least 13-fold higher, at least 13.5-fold higher, at least 14-fold higher, at least 14.5-fold higher, at least 15-fold higher, at least 15.5-fold higher, at least 16-fold higher, at least 16.5- fold higher, at least 17-fold higher, at least 17.5-fold higher, at least 18-fold higher, at least 18.5-fold higher, at least 19-fold higher, at least 19.5-fold higher, at least 20-fold higher, or more) than the transgene expression from a corresponding nucleic acid vector that does not comprise the modification. Packaging efficiency

[0176] According to some aspects, packaging efficiency of an AAV particle disclosed herein is modified relative to a corresponding wild-type AAV particle. Packaging efficiency of an AAV particle refers to the capability of a particular AAV capsid to encapsidate a particular viral genome. Packaging efficiency can be measured by one of ordinary skill in the art, such as by quantifying the ratio of capsids to viral genomes (see, e.g., Grimm, et al. Gene Ther.6:1322- 1330 (1999)).

[0177] In some aspects, the packaging efficiency of an AAV particle as disclosed herein (e.g., an AAV particle comprising a modified capsid protein, a modified nucleic acid vector, or both a modified capsid protein and a modified nucleic acid vector) is higher than the packaging efficiency of a corresponding wild-type AAV particle. In some aspects, the packaging efficiency of an AAV particle as disclosed herein is at least 5% higher (e.g., at least 10%T19325 Attorney Docket No. U1202.70155WO00 higher, at least 15% higher, at least 20% higher, at least 25% higher, at least 30% higher, at least 35% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 100% higher, at least 150% higher, at least 200% higher, at least 250% higher, or more) than the packaging efficiency of a corresponding wild-type AAV particle. In some aspects, the packaging efficiency of an AAV particle as disclosed herein is at least 1.5-fold higher (e.g., at least 2-fold higher, at least 2.5- fold higher, at least 3-fold higher, at least 3.5-fold higher, at least 4-fold higher, at least 4.5- fold higher, at least 5-fold higher, at least 5.5-fold higher, at least 6-fold higher, at least 6.5- fold higher, at least 7-fold higher, at least 7.5-fold higher, at least 8-fold higher, at least 8.5- fold higher, at least 9-fold higher, at least 9.5-fold higher, at least 10-fold higher, at least 10.5- fold higher, at least 11-fold higher, at least 11.5-fold higher, at least 12-fold higher, at least 12.5-fold higher, at least 13-fold higher, at least 13.5-fold higher, at least 14-fold higher, at least 14.5-fold higher, at least 15-fold higher, at least 15.5-fold higher, at least 16-fold higher, at least 16.5-fold higher, at least 17-fold higher, at least 17.5-fold higher, at least 18-fold higher, at least 18.5-fold higher, at least 19-fold higher, at least 19.5-fold higher, at least 20- fold higher, or more) than the packaging efficiency of a corresponding wild-type AAV particle.

[0178] In some aspects, the packaging efficiency of an AAV particle as disclosed herein (e.g., an AAV particle comprising a modified capsid protein, a modified nucleic acid vector, or both a modified capsid protein and a modified nucleic acid vector) is lower than the packaging efficiency of a corresponding wild-type AAV particle. In some aspects, the packaging efficiency of an AAV particle as disclosed herein is decreased by at least 5% (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, or more) relative to the packaging efficiency of a corresponding wild-type AAV particle.

[0179] In some aspects, packaging efficiency of an AAV particle disclosed herein is not modified relative to a corresponding wild-type AAV particle.

[0180] In some aspects, both the transduction efficiency and the packaging efficiency of an AAV particle as disclosed herein is modified (i.e., increased or decreased) relative to a corresponding unmodified or wild-type AAV particle (e.g., of the same serotype). In some aspects, the immunogenicity of an AAV particle as disclosed herein is modified relative to a corresponding unmodified or wild-type AAV particle (e.g., of the same serotype).T19325 Attorney Docket No. U1202.70155WO00 Pharmaceutical compositions

[0181] Any one of the AAV particles, capsid proteins, or nucleic acids disclosed herein may be comprised within a pharmaceutical composition comprising a pharmaceutically-acceptable carrier or may be comprised within a pharmaceutically-acceptable carrier. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the AAV particle, capsid protein, or nucleic acid is comprised or administered to a subject. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum oil such as mineral oil, vegetable oil such as peanut oil, soybean oil, and sesame oil, animal oil, or oil of synthetic origin. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Non-limiting examples of pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, polyacrylic acids, lubricating agents (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifying agents, suspending agents, preserving agents (such as methyl-, ethyl-, and propyl-hydroxy-benzoates), and pH adjusting agents (such as inorganic and organic acids and bases), and solutions or compositions thereof. Other examples of carriers include phosphate buffered saline, HEPES-buffered saline, and water for injection, any of which may be optionally combined with one or more of calcium chloride dihydrate, disodium phosphate anhydrous, magnesium chloride hexahydrate, potassium chloride, potassium dihydrogen phosphate, sodium chloride, or sucrose. Other examples of carriers that might be used include saline (e.g., sterilized, pyrogen-free saline), saline buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. USP grade carriers and excipients are particularly useful for delivery of AAV particles to human subjects.

[0182] Typically, such compositions may contain at least about 0.1% of the therapeutic agent (e.g., AAV particle) or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of therapeutic agent(s) (e.g., AAV particle) in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, productT19325 Attorney Docket No. U1202.70155WO00 shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be designed.

[0183] Pharmaceutical compositions as disclosed herein, e.g., comprising nucleic acid vectors comprising modified ITRs and / or AAV particles comprising nucleic acid vectors comprising modified ITRs, can be prepared by one of ordinary skill in the art by known methods. Methods of contacting a cell

[0184] According to some aspects, methods of contacting a cell with an AAV particle or nucleic acid vector are provided herein. Methods of contacting a cell may comprise, for example, contacting a cell in a culture with a composition comprising an AAV particle or nucleic acid vector. In some aspects, contacting a cell comprises adding a composition comprising an AAV particle or nucleic acid vector to the supernatant of a cell culture (e.g., a cell culture on a tissue culture plate or dish) or mixing a composition comprising an AAV particle or nucleic acid vector with a cell culture (e.g., a suspension cell culture). In some aspects, contacting a cell comprises mixing a composition comprising an AAV particle or nucleic acid vector with another solution, such as a cell culture media, and incubating a cell with the mixture.

[0185] In some aspects, contacting a cell with an AAV particle or nucleic acid vector comprises administering a composition comprising an AAV particle or nucleic acid vector to a subject or device in which the cell is located. In some aspects, contacting a cell comprises injecting a composition comprising an AAV particle or nucleic acid vector into a subject in which the cell is located. In some aspects, contacting a cell comprises administering a composition comprising an AAV particle or nucleic acid vector directly to a cell, or into or substantially adjacent to a tissue of a subject in which the cell is present.

[0186] In some aspects, “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful. In some aspects, an rAAV particle is administered to a subject enterally. In some aspects, an enteral administration of the essential metal element / s is oral. In some aspects, a rAAV particle is administered to the subject parenterally. In some aspects, a rAAV particle is administered to a subject subcutaneously, intraocularly, intravitreally, subretinally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracisternally, intraperitoneally, via inhalation, topically,T19325 Attorney Docket No. U1202.70155WO00 or by direct injection to one or more cells, tissues, or organs. In some aspects, a rAAV particle is administered to the subject by injection into the hepatic artery or portal vein.

[0187] In some aspects, a compositions of AAV particles is administered to a subject to treat a disease or condition. To "treat" a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. The compositions described above or elsewhere herein are typically administered to a subject in an effective amount, that is, an amount capable of producing a desirable result. The desirable result will depend upon the active agent being administered. For example, an effective amount of rAAV particles may be an amount of the particles that are capable of transferring an expression construct to a host organ, tissue, or cell. A therapeutically acceptable amount may be an amount that is capable of treating a disease, e.g., a muscular dystrophy. As is well known in the medical and veterinary arts, dosage for any one subject depends on many factors, including the subject's size, body surface area, age, the particular composition to be administered, the active ingredient(s) in the composition, time and route of administration, general health, and other drugs being administered concurrently.

[0188] In some aspects, a cell disclosed herein is a cell isolated or derived from a subject. In some aspects, a cell is a mammalian cell (e.g., a cell isolated or derived from a mammal). In some aspects, a cell is a human cell. In some aspects, a cell is isolated or derived from a particular tissue of a subject, such as muscle tissue. In some aspects, a cell is a muscle cell. In some aspects, a cell is a skeletal muscle cell or a smooth muscle cell. In some aspects, a cell is in vitro. In some aspects, a cell is ex vivo. In some aspects, a cell in in vivo. In some aspects, a cell is within a subject (e.g., within a tissue or organ of a subject). In some aspects, a cell is a primary cell. In some aspects, a cell is from a cell line (e.g., an immortalized cell line). In some aspects a cell is a cancer cell or an immortalized cell.

[0189] In some aspects, “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful.

[0190] In certain circumstances it will be desirable to deliver an AAV particle disclosed herein in a suitably formulated pharmaceutical composition disclosed herein either subcutaneously, intraocularly, intravitreally, subretinally, parenterally, intravenously (IV), intracerebro- ventricularly, intramuscularly, intrathecally (IT), intracisternally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection. In some aspects, the administration is a route suitable for systemic delivery, such as by intravenous injection. In some aspects, the administration is a route suitable for localT19325 Attorney Docket No. U1202.70155WO00 delivery, such as by intramuscular injection. In some aspects, “administering” or “administration” means providing a material to a subject in a manner that is pharmacologically useful.

[0191] In some aspects, the concentration of AAV particles in a composition administered to a subject may be on the order ranging from 106to 1014particles / ml or 103to 1015particles / ml, or any values therebetween for either range, such as for example, about 106, 107, 108, 109, 1010, 1011, 1012, 1013, or 1014particles / ml. In some aspects, AAV particles of a higher concentration than 1013particles / ml are administered. In some aspects, the concentration of AAV particles in a composition administered to a subject may be on the order ranging from 106to 1014vector genomes (vgs) / ml or 103to 1015vgs / ml, or any values therebetween for either range (e.g., 106, 107, 108, 109, 1010, 1011, 1012, 1013, or 1014vgs / ml). In some aspects, AAV particles of higher concentration in a composition than 1013vgs / ml are administered. The AAV particles can be administered as a single dose, or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In some aspects, 0.0001 ml to 10 ml are delivered to a subject. In some aspects, the number of AAV particles administered to a subject may be on the order ranging from 106-1014vgs / kg body mass of the subject, or any values therebetween (e.g., 106, 107, 108, 109, 1010, 1011, 1012, 1013, or 1014vgs / kg). In some aspects, the dose of AAV particles administered to a subject may be on the order ranging from 1012-1014vgs / kg. In some aspects, the volume of AAVrh74 composition delivered to a subject (e.g., via one or more routes of administration as described herein) is 0.0001 ml to 10 ml.

[0192] In some aspects, a composition disclosed herein (e.g., comprising an AAV particle) is administered to a subject once. In some aspects, the composition is administered to a subject multiple times (e.g., twice, three times, four times, five times, six times, or more). Repeated administration to a subject may be conducted at a regular interval (e.g., daily, every other day, twice per week, weekly, twice per month, monthly, every six months, once per year, or less or more frequently) as necessary to treat (e.g., improve or alleviate) one or more symptoms of a disease, disorder, or condition in the subject. Subjects

[0193] Aspects of the disclosure relate to methods for use with a subject, such as human or non-human primate subjects; with a host cell in situ in a subject; or with a host cell derived from a subject (e.g., ex vivo or in vitro). Non-limiting examples of non-human primate subjectsT19325 Attorney Docket No. U1202.70155WO00 include macaques (e.g., cynomolgus or rhesus macaques), marmosets, tamarins, spider monkeys, owl monkeys, vervet monkeys, squirrel monkeys, baboons, gorillas, chimpanzees, and orangutans. In some aspects, the subject is a human subject. Other exemplary subjects include domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.

[0194] In some aspects, the subject has or is suspected of having a disease or disorder that may be treated with gene therapy. A disease or disorder that may be treated with gene therapy may be characterized by one or more mutation(s) in the genome that results in abnormal structure or function of one or more proteins associated with development, health, maintenance and / or function of a cell and / or organ. Diseases and disorders can be characterized and identified, e.g., through laboratory tests and / or evaluation by a clinician. In some aspects, the subject has or is suspected of having a disease (e.g., a disease caused by a defect, such as a genetic mutation, in one or more cells or genes). In some aspects, a nucleic acid isolated or derived from the subject (e.g., genomic DNA, mRNA, or cDNA from the subject) is identified via sequencing (e.g., Sanger or next-generation sequencing) to comprise a mutation (e.g., in a gene associated with development, health, maintenance, or function of a cell and / or organ).

[0195] In some aspects, a subject comprises a mutant form of one or more genes associated with development, health, maintenance and / or function of a cell and / or organ. In some aspects, methods disclosed herein provide a cell of a subject with a functional form of a gene. EXAMPLES Example 1.

[0196] The distal 10-nucleotides (nts) in the AAV2 D-sequence share partial homology to the consensus half-site of the glucocorticoid receptor-binding element (GRE) and the replacement of the distal 10 nts of the D-sequence with a full GRE site leads to generation Y (GenY) ssAAVrh74 vectors that mediate up to 6-fold higher transgene expression in primary human skeletal muscle cells compared with those containing the wild-type (WT) D-sequence (Mol. Ther., 30: 237-238, 2022). As described herein, an approach was evaluated to determine whether a sequence in the stem A-domain and the D-sequence could be replaced with binding site sequence for muscle cell-specific transcription factors. The myoblast determination protein 1 (MyoD) and myocyte enhancer factor 2 (MEF2C) have been previously shown to cooperate in transcription activation during myogenesis (Mol Cell Biol., 18: 69-77, 1998). A novel AAV- ITR was designed in which the entire D10-sequence (FIG.1A) was replaced with MyoD andT19325 Attorney Docket No. U1202.70155WO00 MEF2C binding site sequences (FIG.1B). Additionally, part of the stem A-domain was replaced to be complementary to the MyoD sequence to ensure that the terminal resolution site (trs) was conserved to allow AAV Rep-mediated resolution, rescue, replication, and packaging. Encapsidation of these genomes containing a muscle cell-specific promoter (MHCK7)-driven hrGFP expression cassette into AAVrh74 capsids led to the development of Generation M (GenM) AAVrh74 vectors. The transduction efficiency of WT and GenM MHCK7-hrGFP AAVrh74 vectors was evaluated in differentiated C2C12 mouse myotubules. Cells were transduced with WT and GenM AAVrh74 vectors at 37ºC for 2 hrs, and transgene expression was visualized under a fluorescence microscope following differentiation. Data were quantitated using the NIH ImageJ software.

[0197] As can be seen in FIG.1C, the GenM AAVrh74 vectors averaged ~3-fold increased transgene expression compared with transgene expression from the WT AAVrh74 vectors (p<0.01). These vectors were further evaluated in differentiated primary human skeletal muscle cells, and as shown in FIG.1D, mediated ~2-fold increased transgene expression (p<0.01), compared with transgene expression from the WT AAVrh74 vectors. These data suggest that genome-modifications involving the AAV D-sequence is a useful strategy to achieve improved transgene expression from ssAAV vectors. Example 2.

[0198] In this approach, a modified AAV ITR was designed, designated as DeltA ITR, in which the A-domain was made complementary to the GRE sequence, and the D-sequence was replaced with the GRE sequence (FIG.2A). This allowed the terminal resolution site (trs) to be conserved (FIG.2B), for AAV Rep-mediated resolution, rescue, replication of the AAV genomes. WT ITR and DeltA ITR genomes containing a muscle cell-specific promoter (CDK8)-driven hrGFP expression cassettes were packaged into AAVrh74 capsids. The extent of the transgene expression from the DeltA AAVrh74 vectors was ~34-fold higher than that from the WT AAVrh74 vectors in differentiated primary human skeletal muscle cells in vitro (FIG.2C). The transduction efficiencies of the WT and the DeltA AAVrh74 vectors were also evaluated in a mouse model in vivo.1x109vgs of WT or DeltA-CK8-hrGFP AAVrh74 vectors were administered into gastrocnemius (GA) muscles in C57 / BL6 mice (n=6 each). Cryo- sectioning and imaging of muscle tissues were performed 2 weeks post-injections. These results, shown in FIG.2D, document that DeltA-ITR AAVrh74 vectors averaged ~24-fold increased transgene expression compared to transgene expression from the WT-ITR AAVrh74T19325 Attorney Docket No. U1202.70155WO00 vectors. These data suggest that this novel ITR-modification subverts the need for the AAV D- sequence and is a useful strategy to achieve improved transgene expression from ssAAV vectors.

[0199] Additional modified AAV ITRs were designed, in which the D sequence was replaced with CRX (FIG.3A), NRL (FIG.3B), or NR2E3 (FIG.3C) transcription factor binding site sequences, while making the A-domain complementary to a portion of the transcription factor binding site sequence. Example 3.

[0200] An additional modified AAV ITR weas designed, in which the D sequence was replaced with a GRE sequence in which three nucleotides have been replaced (FIG.4). The replacement of the three nucleotides (shown in bold in FIG.4) allows for the formation of a longer stem structure, which closely resembles the length of the stem in the wild-type AAV ITR. A segment of the wild-type ITR is shown in FIG.5A. A corresponding segment of an ITR with a D-sequence substitution with a GRE, and concomitant substitution of the A-domain to be complementary to the GRE sequence is shown in FIG.5B (a DeltA ITR as described above). A corresponding segment of an ITR with a D-sequence substitution with a GRE sequence which has been modified is shown in FIG.5C (a “modified DeltA ITR”). The modification to the GRE sequence in the ITR of FIG.5C results in the formation of a longer stem.

[0201] The efficiency of rescue, replication, and packaging of AAVrh74 vectors comprising a modified DeltA ITR is tested. Results demonstrate that rescue, replication, and packaging are all increased with the use of modified DeltA ITRs relative to WT ITRs. The extent of transgene expression from the modified DeltA ITR in an AAVrh74 vectors is also tested in primary human skeletal muscle cells in vitro. The transduction efficiency of the modified DeltA ITR-containing vector is increased relative to a corresponding vector comprising only WT ITRs. EQUIVALENTS AND SCOPE

[0202] While several inventive aspects have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantagesT19325 Attorney Docket No. U1202.70155WO00 described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive aspects described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive aspects described herein. It is, therefore, to be understood that the foregoing aspects are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive aspects may be practiced otherwise than as specifically described and claimed. Inventive aspects of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0203] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0204] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0205] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0206] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one aspect, to A only (optionally including elements other than B); in another aspect, to B only (optionallyT19325 Attorney Docket No. U1202.70155WO00 including elements other than A); in yet another aspect, to both A and B (optionally including other elements); etc.

[0207] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0208] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one aspect, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another aspect, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another aspect, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0209] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.T19325 Attorney Docket No. U1202.70155WO00

[0210] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of” shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that aspects described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative aspects, as “consisting of” and “consisting essentially of” the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B,” the disclosure also contemplates the alternative aspects “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

Claims

T19325 Attorney Docket No. U1202.70155WO00 CLAIMS What is claimed is:

1. A nucleic acid vector comprising an inverted terminal repeat (ITR) comprising a stem A-domain and a D-sequence, wherein the D-sequence comprises about 20 nucleotides and is modified to introduce a sequence of a transcription factor (TF) binding site and the stem A- domain contains a wild-type 6-nucleotide sequence comprising a terminal resolution site and is modified to comprise a sequence comprising nucleotides complementary to the sequence of the TF binding site.

2. The nucleic acid vector of claim 1, wherein the ITR is a 5’ ITR or a 3’ ITR.

3. The nucleic acid vector of claim 1 or 2, wherein the ITR is an AAV 5’ ITR.

4. The nucleic acid vector of any one of claims 1 to 3, wherein the TF binding site is inserted into the D-sequence.

5. The nucleic acid vector of claim 1 or 4, wherein the TF binding site replaces the D- sequence.

6. The nucleic acid vector of claim 1 or 5, wherein one or more nucleotides are replaced in the D-sequence to create the TF binding site.

7. The nucleic acid vector of any one of claims 1 to 6, wherein the stem A-domain comprises the terminal resolution site upstream (5’ relative) to the sequence comprising nucleotides complementary to the sequence of the TF binding site.

8. The nucleic acid vector of any one of claims 1 to 7, wherein the nucleic acid vector comprises about 10 nucleotides of the D-sequence.

9. The nucleic acid vector of any one of claims 1 to 8, wherein the nucleic acid vector comprises about 5 nucleotides of the D-sequence.T19325 Attorney Docket No. U1202.70155WO00 10. The nucleic acid vector of any one of claims 1 to 9, wherein the D-sequence is modified to introduce at least two sequences of TF binding sites.

11. The nucleic acid vector of claim 10, wherein the at least two sequences of TF binding sites are the same.

12. The nucleic acid vector of claim 10, wherein the at least two sequences of TF binding sites are different.

13. The nucleic acid vector of claim 12, wherein the stem A-domain is modified to comprise a sequence complementary to the sequence of one of the at least two sequences of TF binding sites.

14. The nucleic acid vector of claim 13, wherein the stem A-domain is modified to comprise the sequence complementary to the sequence of one of the at least two sequences of TF binding sites directly upstream (5’ relative) to the terminal resolution site.

15. The nucleic acid vector of any one of claims 1 to 14, further comprising a heterologous sequence comprising a gene of interest.

16. The nucleic acid vector of claim 15, wherein the heterologous sequence further comprises a promoter.

17. The nucleic acid vector of any one of claims 1 to 16, wherein at least one TF binding site binds a TF that is active in a target cell type.

18. The nucleic acid vector of claim 17, wherein the target cell type is a target cell type in which the promoter is active.

19. The nucleic acid vector of claim 17 or 18, wherein the target cell type is a muscle cell, liver cell, CNS cell, or retinal cell.

20. The nucleic acid vector of claim 19, wherein the TF is active in a muscle cell.T19325 Attorney Docket No. U1202.70155WO00 21. The nucleic acid vector of claim 19 or 20, wherein the TF is selected from a myocyte determination protein 1 (MyoD) and a myocyte enhancer factor 2C (MEF2C).

22. The nucleic acid vector of claim 19 or 20, wherein the TF is a glucocorticoid receptor.

23. The nucleic acid vector of claim 19, wherein the TF is active in a retinal cell.

24. The nucleic acid vector of claim 19 or 23, wherein the TF is selected from a Cone- Rod Homeobox (CRX), Neural Retina Leucine Zipper (NRL), and Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3).

25. The nucleic acid vector of claim 19, wherein the TF is active in a CNS cell.

26. The nucleic acid vector of claim 19 or 25, wherein the TF is selected from a Hes Family BHLH Transcription Factor (HES) 1, HES5, Recombinant Signal Binding Protein for Immunoglobulin Kappa J Region (CBF-1), SYR-Box Transcription Factor 2 (SOX2), SOX1, SOX3, High Mobility Group AT-Hook 2 (HGMA2), BMI Proto-Oncogene, Polycomb Ring Finger (BMI1), GLI Family Zinc Finger (GLI) 2, GLI3, Inhibitor Of DNA Binding (ID) 2, and ID4.

27. The nucleic acid vector of claim 19, wherein the TF is active in a liver cell.

28. The nucleic acid vector of claim 19 or 27, wherein the TF is selected from a Hepatocyte Specific Cis-Acting Regulatory Module (HS-CRM) 8 and a HS-CRM14.

29. An AAV particle comprising a nucleic acid vector of any one of claim 1-28.

30. The AAV particle of claim 29, comprising a capsid protein selected from AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAVvrh74, their various tyrosine, serine, threonine, and lysine mutants and combinations thereof.

31. A composition comprising a nucleic acid vector of any one of claims 1-28 or a AAV particle of claim 29 or 30.T19325 Attorney Docket No. U1202.70155WO00 32. The composition of claim 31, wherein the composition is a pharmaceutical composition.

33. The composition of claim 32, further comprising a pharmaceutically acceptable excipient.

34. A method comprising contacting a cell with a AAV particle of claim 29 or 30, or a composition of claims 31 to 33.

35. The method of claim 34, wherein the cell is a human cell.

36. The method of claim 34 or 35, wherein the cell expresses the gene of interest at least 2-times higher compared to a cell contacted with an AAV particle or a composition comprising an AAV particle that does not contain a modified D-sequence and a modified stem A-domain.

37. A method comprising administering an AAV particle of claim 29 or 30, or a composition of claims 31 to 33 to a subject in need thereof.

38. The method of claim 37, wherein the subject is a human.

39. The method of claim 37 or 38, wherein the subject expresses the gene of interest at least 2-times higher compared to a subject administered an AAV particle or a composition comprising an AAV particle that does not contain a modified D-sequence and a modified stem A-domain.

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