Capsid variants and methods of using the same

Abstract

The disclosure is directed in part to variant capsid polypeptides that can be used to deliver payloads.

Description

[0001] Atorney Docket No. 14851-008-228

[0002] CAPSID VARIANTS AND METHODS OF USING THE SAME

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 729,827, filed on December 9, 2024, U.S. Provisional Application No. 63 / 787,514, filed on April 11, 2025, and U.S. Provisional Application No. 63 / 806,504, filed on May 15, 2025, the disclosure of each of which is incorporated by reference herein in its entirety.

[0005] SEQUENCE LISTING

[0006] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14851-008-228_SEQLISTING.xml”, was created on December 5, 2025, and is 74,865 bytes in size.

[0007] BACKGROUND

[0008] Dependoparvoviruses, e.g. adeno-associated dependoparvoviruses, e.g. adeno- associated viruses (AAVs), are of interest as vectors for delivering various payloads to cells, including in human subjects.

[0009] SUMMARY

[0010] The present disclosure provides, in part, improved variant dependoparvovirus capsid polypeptides (e.g. variants of AAV2), such as VP1, methods of producing a dependoparvovirus, compositions for use in the same, as well as viral particles comprising such capsid polypeptides. In some embodiments, the viral particles that comprise the capsid polypeptides have increased ocular transduction as compared to viral particles without the mutations in the capsid proteins. In some embodiments, the viral particles that comprise the capsid polypeptides have preferential transduction in cells of the inner nuclear layer (INL) of

[0011] NAI-5007748234vl retinae (e.g., human retina), relative to transduction in cells of the outer nuclear layer (ONL) of retinae (e.g., human retina).

[0012] In some embodiments, the disclosure is directed, in part, to a nucleic acid comprising a sequence encoding a variant capsid protein as provided for herein. In some embodiments, the dependoparvovirus is an adeno-associated dependoparvovirus (AAV). In some embodiments, the AAV is an AAV2 variant.

[0013] In some embodiments, the disclosure is directed, in part, to a capsid polypeptide described herein.

[0014] In some embodiments, the disclosure is directed, in part, to a dependoparvovirus particle comprising capsid polypeptides described herein.

[0015] In some embodiments, the disclosure is directed, in part, to a vector, e.g., a plasmid, comprising a nucleic acid described herein.

[0016] In some embodiments, the disclosure is directed, in part, to a dependoparvovirus particle comprising a nucleic acid described herein (e.g., a nucleic acid comprising a sequence encoding a capsid polypeptide, such as VP1, wherein the encoding sequence comprises a change or mutation as provided herein.

[0017] In some embodiments, the disclosure is directed, in part, to a variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), wherein the variant capsid polypeptide comprises an insertion of an amino acid sequence comprising FTRP at a position corresponding to the position between positions 586 and 587 of SEQ ID NO: 1, or an insertion of an amino acid sequence comprising FTRP at a position corresponding to the position between positions 587 and 588 of SEQ ID NO: 1.

[0018] In some embodiments, the variant capsid polypeptide further comprises an amino acid substitution at a position corresponding to position 587 of SEQ ID NO: 1.

[0019] In some embodiments, the amino acid substitution is an N587A substitution.

[0020] In some embodiments, the disclosure is directed, in part, to a variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID

[0021] NAI-5007748234vl NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an insertion between positions 586 and 587, wherein the insertion comprises an amino acid sequence of a consensus formula X1-X2-X3-X4-X5-F-T-R-P (SEQ ID NO: 12), wherein

[0022] XI is absent or selected from S, T, C, Y, N, or Q;

[0023] X2 is absent or selected from G, A, V, L, I, F, W, M, or P;

[0024] X3 is absent or selected from D or E;

[0025] X4 is absent or selected from S, T, C, Y, N, or Q;

[0026] X5 is absent or selected from G, A, V, L, I, F, W, M, or P.

[0027] In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids thereof, and wherein the variant capsid polypeptide further comprises an N587A mutation.

[0028] In some embodiments, the disclosure is directed, in part, to a variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an amino acid substitution at position 547, 548, 549, 550, 555, 561, 575, 578, 580, 581, 587, 589, 591, 593, or any combination thereof.

[0029] In some embodiments, the amino acid substitution comprises an N587A substitution.

[0030] In some embodiments, the variant capsid polypeptide further comprises an insertion of an amino acid sequence SLDQAFTRP (SEQ ID NO: 23) between positions 586 and 587.

[0031] In some embodiments, the disclosure is directed, in part, to a virus particle (e.g., adeno-associated virus (“AAV”) particle) comprising the variant capsid polypeptide of any one of the embodiments disclosed herein.

[0032] In some embodiments, the virus particle exhibits increased ocular transduction, e.g., as measured in a mouse or in a non-human primate (NHP), relative to wild-type AAV2 (e.g., a virus particle comprising capsid polypeptides of SEQ ID NO: 1).

[0033] NAI-5007748234vl In some embodiments, the virus particle exhibits at least a 1.5-fold preferential transduction in a cell of an inner nuclear layer (INL) of a retina of a NHP, relative to transduction in a cell of an outer nuclear layer (ONL) of a retina of a NHP.

[0034] In some embodiments, the virus particle exhibits increased ocular transduction, e.g., as measured in a mouse or in a non-human primate (NHP), relative to an AAV2 variant identified from the ocular literature (e.g., a virus particle comprising an externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24).

[0035] In some embodiments, the increased ocular transduction is increased transduction in the retina. In some embodiments, the increased transduction in the retina is increased transduction in the inner nuclear layer (INL) of the retina. In some embodiments, the increased transduction in the INL of the retina is increased transduction of bipolar cells of the INL.

[0036] In some embodiments, the disclosure is directed, in part, to a nucleic acid molecule comprising a sequence encoding a variant capsid polypeptide disclosed herein.

[0037] In some embodiments, the disclosure is directed, in part, to a vector comprising the nucleic acid molecule disclosed herein.

[0038] In some embodiments, the disclosure is directed, in part, to a host cell transformed with the vector disclosed herein.

[0039] In some embodiments, the disclosure is directed, in part, to a method of producing a virus particle comprising a variant capsid polypeptide, comprising introducing the nucleic acid molecule, or the vector disclosed herein into a host cell, and harvesting said virus particle therefrom.

[0040] In some embodiments, the disclosure is directed, in part, to a cell, cell-free system or other translation system, comprising a first and a second nucleic acid molecule, wherein the first nucleic acid molecule is the nucleic acid molecule disclosed herein and the second nucleic acid molecule comprises a payload (e.g., a nucleic acid sequence encoding a therapeutic product).

[0041] In some embodiments, the disclosure is directed, in part, to a method of making a dependoparvovirus particle, comprising cultivating the cell, cell-free system, or other translation system disclosed herein, under conditions suitable for the production of the

[0042] NAI-5007748234vl dependoparvovirus particle. In some embodiments, the first nucleic acid mediates the production of a dependoparvovirus particle, and the second nucleic acid molecule is packaged within said dependoparvovirus particle. In some embodiments, the first nucleic acid mediates the production of a dependoparvovirus particle at a level at least 10%, at least 20%, at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, or greater than the production level mediated by the nucleic acid of SEQ ID NO: 2.

[0043] In some embodiments, the disclosure is directed, in part, to a method of delivering a payload (e.g., a nucleic acid) to a cell, comprising contacting the cell with the virus particle disclosed herein, and the payload; or with the dependoparvovirus particle produced by the method disclosed herein. In some embodiments, the cell is an ocular cell. In some embodiments, the virus particle or dependoparvovirus particle delivers the payload with increased transduction in an ocular cell of one or more regions of an eye as compared to a virus particle comprising capsid polypeptides of SEQ ID NO: 1 or the externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24. In some embodiments, the increase is 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold, 16-fold, 18-fold, 19-fold, 20-fold, 21-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 34-fold, 35-fold, 36-fold, 37- fold, 39-fold, 42-fold, 45-fold, 46-fold, 47-fold, 49-fold, 51-fold, 52-fold, 58-fold, 59-fold, 61 -fold, 65-fold, 72-fold. In some embodiments, the region of an eye is the retina, the macula, or the trabecular meshwork, or any combination thereof. In some embodiments, the retina comprises non-macular retina. In some embodiments, the region of an eye is the parafovea, the parafoveal nerve fibers, the peripheral nerve fibers, or any combination thereof.

[0044] In some embodiments, the disclosure is directed, in part, to a method of delivering a payload (e.g., a nucleic acid) to a subject comprising administering to the subject the virus particle disclosed herein, and the payload; or the dependoparvovirus particle produced by the method disclosed herein. In some embodiments, the virus particle or dependoparvovirus particle delivers the payload to an eye. In some embodiments, the virus particle or dependoparvovirus particle delivers the payload to an eye with increased transduction in one or more regions of an eye as compared to a virus particle comprising capsid polypeptides of SEQ ID NO: 1 or the externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24. In some embodiments, the increase is 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold, 16- fold, 18-fold, 19-fold, 20-fold, 21 -fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold,

[0045] NAI-5007748234vl 30-fold, 34-fold, 35-fold, 36-fold, 37-fold, 39-fold, 42-fold, 45-fold, 46-fold, 47-fold, 49- fold, 51-fold, 52-fold, 58-fold, 59-fold, 61-fold, 65-fold, 72-fold. In some embodiments, the region of an eye is the retina, the macula, the trabecular meshwork, or any combination thereof. In some embodiments, the retina comprises non-macular retina. In some embodiments, the region of an eye is the parafovea, the parafoveal nerve fibers, the peripheral nerve fibers, or any combination thereof. In some embodiments, the administration is via an intravitreal injection, or an intracameral injection.

[0046] In some embodiments, the disclosure is directed, in part, to a pharmaceutical composition comprising the virus particle disclosed herein, and a payload; or the dependoparvovirus particle produced by the method disclosed herein, and a pharmaceutically acceptable carrier.

[0047] In some embodiments, the disclosure is directed, in part, to a method of treating a disease or condition in a subject, comprising administering to the subject the virus particle disclosed herein, and a payload, in an amount effective to treat the disease or condition; or the dependoparvovirus particle produced by the method disclosed herein, in an amount effective to treat the disease or condition; or the pharmaceutical composition disclosed herein, in an amount effective to treat the disease or condition.

[0048] In some embodiments, the disclosure is directed, in part, to a variant capsid polypeptide, the nucleic acid molecule, the vector, or the virus particle disclosed herein; or the dependoparvovirus particle produced by the method disclosed herein; or the pharmaceutical composition disclosed herein, for use in treating a disease or condition in a subject.

[0049] In some embodiments, the disclosure is directed, in part, to a variant capsid polypeptide, the nucleic acid molecule, the vector, or the virus particle disclosed herein; or the dependoparvovirus particle produced by the method disclosed herein; or the pharmaceutical composition disclosed herein, for use in the manufacture of a medicament for use in treating a disease or condition in a subject.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] FIG. 1. Diagram of tissues collected in each region of the eye. In the retina (left and center figures), peripheral and central retina samples from each of the superior, nasal, inferior and

[0052] NAI-5007748234vl temporal regions of the retina were separately collected, macula was also separately collected. In each region, neural retina and choroid / RPE layers (center figure) were separately collected. In the trabecular meshwork / Schl emm’s canal (TM / SC) region (right figure), superior, temporal, nasal and inferior samples were separately collected.

[0053] FIGS. 2A - 2C. Multisequence alignment of representative reference capsid VP1 polypeptides. Such alignment can be used to determine the amino acid positions which correspond to positions within different reference capsid polypeptides.

[0054] FIG. 3. Schematic of the study design for histology -based Variant 1 transduction measurement in NHP and mouse.

[0055] FIG. 4. Schematic of the study design for biofactory-based (Aflibercept) Variant 1 transduction measurement in NHP.

[0056] FIG. 5. Immunofluorescent staining of NHP retinae transduced with Variant 1 (GFP and mCherry). The white lines indicate the areas of transduction in the NHP retina.

[0057] FIGS. 6A-6C. Immunofluorescent staining of NHP retinae transduced with Variant 1 (mCherry), segmented in retinal layers (INL, ONL, and GCL). FIG 6A shows peripheral and central retina; FIG. 6B. shows macula, peripheral and central retina; FIG. 6C represents what fraction of all the mCherry-positive cells were found in the INL, the ONL, and the GCL.

[0058] FIG. 6D. Immunofluorescent co-labelling of NHP retinae transduced with Variant 1 (GFP / mCherry+ cells) with retinal cell markers for PKCa bipolar cells of the INL. PKCa- positive bipolar cells are shown in green (lighter) and Variant- 1 -transduced cells are shown in purple (darker). Overlapping staining of bipolar cells transduced with Variant-1 are indicated with white arrows.

[0059] FIGS. 7A-7D. Immunofluorescent staining of mouse retinae transduced with Variant 1 (GFP). FIGS. 7A-7B show whole retinae; FIG. 7C shows retinal layers (INL, ONL, and GCL); FIG. 7D represents what fraction of all the GFP-positive cells were found in the INL, the ONL, and the GCL.

[0060] FIG. 8. Bar plot showing relative Aflibercept expression in the vitreous humor (VH) and the aqueous humor (AH) of NHP retina transduced with Variant 1, as measured by ELISA.

[0061] FIG. 9. Schematic of the study design for histology-based Variant 1 transduction measurement in comparison to externally engineered capsid (EEC) in NHP and mouse.

[0062] NAI-5007748234vl FIG. 10. Schematic of the study design for biofactory -based (Aflibercept) Variant 1 transduction measurement in comparison to externally engineered capsid (EEC) in NHP.

[0063] FIG. 11. Schematic of QuPATH script quantification and normalization of mCherry-postive and eGFP-positive nuclei in Variant- 1 and EEC transduced cells.

[0064] FIG. 12. Bar plot showing relative Aflibercept expression in the vitreous humor and the aqueous humor of NHP retina transduced with Variant 1 or EEC, as measured by ELISA.

[0065] FIG. 13. Bar plot showing the fold-change of NHP retinal cells transduced with Variant 1, across the whole retina and in retinal layers (INL, ONL, and GCL) compared to NHP retinal cells transduced with EEC.

[0066] FIG. 14. Bar plot representing what fraction of all the GFP-positive cells were found in the INL, the ONL, and the GCL of mouse retinae transduced with Variant 1, with EEC, or with a negative control.

[0067] FIG. 15. Immunofluorescent co-labelling of mouse retinae transduced with Variant 1 or EEC (GFP+ cells) with retinal cell markers for ChxlO bipolar cells of the INL. Chx 10-positive bipolar cells are shown in purple (darker) and Variant-l-transduced or EEC -transduced cells are shown in green (lighter). Overlapping staining of bipolar cells transduced with Variant-1 or EEC are indicated with white arrows.

[0068] FIG. 16. A chart showing uveitis scores at each time point (pre-test, week 1, week 2, week 3, and week 4) for non-human primates (NHP) that received Aflibercept via Variant- 1 or EEC. Scoring includes aqueous cell, aqueous flare, vitreous cell, and vitreous haze, assessed according to the Standardization of Uveitis Nomenclature (SUN) criteria. A blank space indicates a score of 0.

[0069] FIG. 17. Confocal scanning laser ophthalmoscopy (cSLO) images showing GFP fluorescence in the retina of the eyes of NHP transduced with Variant 1 or with AAV2 capsids packaged with a CAG-GFP genome.

[0070] DETAILED DESCRIPTION

[0071] The present disclosure is directed, in part, to the capsid polypeptides and dependoparvovirus particles comprising the same. In some embodiments, the dependoparvovirus particles have increased ocular transduction and can be used to deliver a

[0072] NAI-5007748234vl transgene or molecule of interest to an eye with higher transduction efficiency in the eye as compared to a dependoparvovirus particle without the variant capsid polypeptides. In some embodiments, the viral particles that comprise the capsid polypeptides have preferential transduction in cells of the inner nuclear layer (INL) of retinae (e.g., human retina), relative to transduction in cells of the outer nuclear layer (ONL) of retinae (e.g., human retina). In some embodiments, the viral particles that comprise the capsid polypeptides have preferential transduction in cells of the inner nuclear layer (INL) of the retina of a non-human primate (NHP), relative to transduction in cells of the outer nuclear layer (ONL) of the retina of an NHP. Accordingly, provided herein are capsid polypeptides, nucleic acid molecules encoding the same, viral particles comprising the variant capsid polypeptides, and methods of making and using the same.

[0073] Definitions

[0074] A, An, The: As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0075] About, Approximately: As used herein, the terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 15 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0076] Dependoparvovirus capsid: As used herein, the term “dependoparvovirus capsid” refers to an assembled viral capsid comprising dependoparvovirus polypeptides. In some embodiments, a dependoparvovirus capsid is a functional dependoparvovirus capsid, e.g., is fully folded and / or assembled, is competent to infect a target cell, or remains stable (e.g., folded / assembled and / or competent to infect a target cell) for at least a threshold time.

[0077] Dependoparvovirus particle: As used herein, the term “dependoparvovirus particle” refers to an assembled viral capsid comprising dependoparvovirus polypeptides and a packaged nucleic acid, e.g., comprising a payload, one or more components of a dependoparvovirus genome (e.g., a whole dependoparvovirus genome), or both. In some embodiments, a dependoparvovirus particle is a functional dependoparvovirus particle, e.g., comprises a desired payload, is fully folded and / or assembled, is competent to infect a target

[0078] NAI-5007748234vl cell, or remains stable (e.g., folded / assembled and / or competent to infect a target cell) for at least a threshold time.

[0079] Dependoparvovirus X particle / capsid: As used herein, the term “dependoparvovirus X particle / capsid” refers to a dependoparvovirus particle / capsid comprising at least one polypeptide or polypeptide encoding nucleic acid sequence derived from a naturally occurring dependoparvovirus X species. For example, a dependoparvovirus B particle refers to a dependoparvovirus particle comprising at least one polypeptide or polypeptide encoding nucleic acid sequence derived from a naturally occurring dependoparvovirus B sequence. Derived from, as used in this context, means having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the sequence in question. Correspondingly, an AAVX particle / capsid, as used herein, refers to an AAV particle / capsid comprising at least one polypeptide or polypeptide encoding nucleic acid sequence derived from a naturally occurring AAVX serotype. For example, an AAV2 particle refers to an AAV particle comprising at least one polypeptide or polypeptide encoding nucleic acid sequence derived from a naturally occurring AAV2 sequence.

[0080] Exogenous: As used herein, the term “exogenous” refers to a feature, sequence, or component present in a circumstance (e.g., in a nucleic acid, polypeptide, or cell) that does not naturally occur in said circumstance. For example, a nucleic acid sequence comprising a mutant capsid polypeptide or a nucleic acid molecule encodes the same may comprise a capsid polypeptide. Use of the term exogenous in this fashion means that the polypeptide or the nucleic acid molecule encodes a polypeptide comprising the mutation in question at this position does not occur naturally, e.g., is not present in AAV2, e.g., is not present in SEQ ID NO: 1.

[0081] Functional: As used herein in reference to a polypeptide component of a dependoparvovirus capsid (e.g., Cap (e.g., VP1, VP2, and / or VP3) or Rep), the term “functional” refers to a polypeptide which provides at least 50, 60, 70, 80, 90, or 100% of the activity of a naturally occurring version of that polypeptide component (e.g., when present in a host cell). For example, a functional VP1 polypeptide may stably fold and assemble into a dependoparvovirus capsid (e.g., that is competent for packaging and / or secretion). As used herein in reference to a dependoparvovirus capsid or particle, “functional” refers to a capsid or particle comprising one or more of the following production characteristics: comprises a desired payload, is fully folded and / or assembled, is competent to infect a target cell, or

[0082] NAI-5007748234vl remains stable (e.g., folded / assembled and / or competent to infect a target cell) for at least a threshold time.

[0083] Nucleic acid: As used herein, in its broadest sense, the term “nucleic acid” refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to an individual nucleic acid monomer (e.g., a nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid monomers or a longer polynucleotide chain comprising many individual nucleic acid monomers. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid is, comprises, or consists of one or more modified, synthetic, or non-naturally occurring nucleotides. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded.

[0084] Variant: As used herein, a "variant capsid polypeptide" refers to a polypeptide that differs from a reference sequence (e.g. SEQ ID NO: 1). The variant can, for example, comprise a mutation (e.g. substitution, deletion, or insertion). In some embodiments, the variant is about, or at least, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%., 97%, 98%, or 99% identical to the reference sequence. In some embodiments, the reference sequence is a polypeptide comprising SEQ ID NO: 1.

[0085] NAI-5007748234vl Identity: As used herein, the terms “identical” or percent “identity” in the context of two or more polypeptides or polynucleotides, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acids or nucleotides residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence. The percent identity can be measured by visual inspection or using sequence comparison software or algorithms. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. A particularly preferred sequence alignment tool is Clustal Omega (Sievers F., et al., Mol. Syst. Biol. 7:359, 2011, DOI: 10.1038 / msb.2011.75, incorporated herein by reference in its entirety). In certain embodiments, identity exists over a region of the sequences that is at least about 5, at least about 10, at least about 20, at least about 20-40, at least about 40-60, at least about 60-80 nucleotides or amino acids in length, or any integral value there between. In certain embodiments, identity exists over a longer region than 60-80 nucleotides or amino acids, such as at least about 80-100 nucleotides or more, and in certain embodiments the sequences are substantially identical over the full length of the sequences being compared, for example, the coding region of a nucleotide sequence or an amino acid sequence.

[0086] Capsid Polypeptides and Nucleic Acids Encoding the Same

[0087] The disclosure is directed, in part, to capsid polypeptides comprising a mutation (insertion, deletion, or substitution) as compared to the wild-type sequence, viral particles comprising variant capsid polypeptides, such as those described here, nucleic acid molecules, and nucleic acid molecules encoding capsid polypeptides such as those described herein. In some embodiments, the wild-type sequence is SEQ ID NO: 1. The disclosure is directed, in part, to variant capsid polypeptides comprising SEQ ID NO: 1 with one or more mutations as compared to SEQ ID NO: 1. The mutation can be, for example, an insertion, deletion, or substitution as compared to the wild-type sequence. In some embodiments, the wild-type sequence is SEQ ID NO: 1. The disclosure is directed, in part, to a variant capsid polypeptide comprising SEQ ID NO: 13 (e.g., Variant 1). The disclosure is directed, in part, to a variant capsid polypeptide comprising a VP1 sequence corresponding the amino acid sequence of position 1-744 of SEQ ID NO: 13 (e.g., Variant 1). The disclosure is directed, in part, to a variant capsid polypeptide comprising a VP2 sequence corresponding the amino acid sequence of position 138-744 of SEQ ID NO: 13 (e.g., Variant 1). The disclosure is directed,

[0088] NAI-5007748234vl in part, to a variant capsid polypeptide comprising a VP3 sequence corresponding the amino acid sequence of position 203-744 of SEQ ID NO: 13 (e.g., Variant 1).

[0089] In some embodiments, the capsid polypeptide comprises a mutation within the 547- 593 amino acid region of SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that is a substitution, e.g., a substitution of at least 1 or more residues, e.g., at least 1 residues, e.g., at least 2 residues, e.g., at least 3 residues, e.g., at least 4 residues, e.g., at least 5 residues, e.g., at least 6 residues, e.g., at least 7 residues, e.g., at least 8 residues, e.g., at least 9 residues, e.g., at least 10 residues, e.g., at least 11 residues, e.g., at least 12 residues, e.g., at least 13 residues, e.g., at least 14 residues, e.g., at least 15 residues, e.g., at least 16 residues, e.g., at least 17 residues, e.g., at least 18 residues, e.g., at least 19 residues, e.g., at least 20 residues, e.g., between 2-20 residues, e.g., between 2-19 residues, e.g., between 2-18 residues, e.g., between 2-17 residues, e.g., between 2-16 residues, e.g., between 2-15 residues, e.g., between 2-14 residues, e.g., between 2-13 residues, e.g., between 2-12 residues, e.g., between 2-11 residues, e.g., between 2-10 residues, e.g., between 2-9 residues, e.g., between 2-8 residues, e.g., between 2-7 residues, e.g., between 2-6 residues, e.g., between 2-5 residues, e.g., between 2-4 residues, e.g., between 2-3 residues, that correspond to a substitution at positions between 547-593, as compared to SEQ ID NO: 1. In some embodiments, the mutation is a substitution and further comprises at least one other mutation at positions 586, 587 and / or 588 wherein the mutations are substitutions, insertions, or deletions. In some embodiments, the mutation between positions 586 and 587, or 587 and 588 is an insertion, e.g., an insertion of 1 or more amino acids, e.g., 1 amino acid, e.g., 1-10 amino acids, e.g., 2-10 amino acids, e.g., 3-10 amino acids, e.g., 4-10 amino acids, e.g., 5-10 amino acids, e.g., 6-10 amino acids, e.g., 7-10 amino acids, e.g., 8-10 amino acids, e.g., 9-10 amino acids, e.g., 10 amino acids, that correspond to an insertion between positions 586 and 587, or 587 and 588, as compared to SEQ ID NO. 1.

[0090] According to some embodiments, variant capsid polypeptides described herein comprise a phenylalanine-threonine-arginine-proline-alanine (“F-T-R-P-A”) sequence. In some embodiments, the variant capsid polypeptide comprises an amino acid sequence insertion (relative to SEQ ID NO: 1) comprising at least some of the FTRPA sequence (e.g., 1, 2, 3, or 4 amino acids forming the FTRPA sequence). In some embodiments, the alanine of the FTRPA sequence is C -terminally juxtaposed to position R588 as compared to SEQ ID NO: 1. In some embodiments, the alanine of the FTRP sequence is inserted between position

[0091] NAI-5007748234vl 586 and 587, and the asparagine residue at position 587 is substituted for an alanine residue (N587A) as compared to SEQ ID NO: 1.

[0092] In some embodiments, the variant capsid polypeptide comprises an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), wherein the variant capsid polypeptide comprises an insertion of an amino acid sequence comprising FTRP at a position corresponding to the position between positions 586 and 587 of SEQ ID NO: 1, or an insertion of an amino acid sequence comprising FTRP at a position corresponding to the position between positions 587 and 588 of SEQ ID NO: 1.

[0093] In some embodiments, the variant capsid polypeptide further comprises an amino acid substitution at a position corresponding to position 587 of SEQ ID NO: 1. In some embodiments, the amino acid substitution is an N587A substitution.

[0094] In some embodiments, variant capsid polypeptides comprising the FTRPA sequence comprise:

[0095] (a) an insertion between positions 587 and 588 as compared to SEQ ID NO: 1, wherein the insertion comprises an amino acid sequence of a consensus formula (X)n-F-T-R- P-A (SEQ ID NO:28), wherein X is any amino acid, and wherein n is 1, 2, 3, 4, or more; or

[0096] (b) an insertion between positions 586 and 587 as compared to SEQ ID NO: 1, and wherein the insertion comprises an amino acid sequence of a consensus formula (X)n-F-T-R- P (SEQ ID NO:29), wherein X is any amino acid, and wherein n is 1, 2, 3, 4, or more; and a substitution of N587A, as compared to SEQ ID NO: 1.

[0097] In some embodiments, variant capsid polypeptides comprising the FTRPA (SEQ ID NO:27) sequence comprise:

[0098] (a) an insertion between positions 587 and 588 as compared to SEQ ID NO: 1, wherein the insertion comprises an amino acid sequence of a consensus formula (X)n-F-T-R- P-A (SEQ ID NO:28), wherein X is any amino acid, and wherein n is 1, 2, 3, 4, or more; or

[0099] (b) an insertion between positions 586 and 587 as compared to SEQ ID NO: 1, and wherein the insertion comprises an amino acid sequence of a consensus formula (X)n-F-T-R- P (SEQ ID NO:29), wherein X is any amino acid, and wherein n is 1, 2, 3, 4, or more; and a

[0100] NAI-5007748234vl substitution of N587A, as compared to SEQ ID NO: 1, wherein the capsid polypeptide has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to a capsid polypeptide of a dependoparvovirus other than wild-type AAV2 (e.g., wild-type AAV5, AAV8, AAV9, AAVrh74 or another dependoparvovirus).

[0101] In some embodiments, a variant capsid polypeptide comprises a sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an insertion between positions 586 and 587, wherein the insertion comprises an amino acid sequence of a consensus formula XI- X2-X3-X4-X5-F-T-R-P (SEQ ID NO:31), wherein:

[0102] XI is absent or is a polar and uncharged amino acid;

[0103] X2 is absent or is a non-polar amino acid;

[0104] X3 is absent or is an acidic amino acid;

[0105] X4 is absent or is a polar and uncharged amino acid;

[0106] X5 is absent or a non-polar amino acid.

[0107] In some embodiments, a variant capsid polypeptide comprises a sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an insertion between positions 586 and 587, wherein the insertion comprises an amino acid sequence of a consensus formula XI- X2-X3-X4-X5-F-T-R-P (SEQ ID NO: 12), wherein:

[0108] XI is absent or selected from S, T, C, Y, N, or Q;

[0109] X2 is absent or selected from G, A, V, L, I, F, W, M, or P;

[0110] X3 is absent or selected from D or E;

[0111] X4 is absent or selected from S, T, C, Y, N, or Q;

[0112] X5 is absent or selected from G, A, V, L, I, F, W, M, or P.

[0113] In some embodiments, XI is absent; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is NAI-5007748234vl S; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is T; X2 is absent or selected from G, A,

[0114] V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S,

[0115] T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is C; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is Y; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F,

[0116] W, M, or P. In some embodiments, XI is N; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P.

[0117] In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is G; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is A; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is V; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is V; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is L; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is I; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is

[0118] NAI-5007748234vl absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is F; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is W; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is M; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P.

[0119] In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is D; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P.

[0120] In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is S; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is T; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is C; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is Y; X5 is absent or selected from G, A, V, L, I, F, W, M,

[0121] NAI-5007748234vl or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is N; X5 is absent or selected from G, A, V, L, I, F, W, M, or P. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is Q; X5 is absent or selected from G, A, V, L, I, F, W, M, or P.

[0122] In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is absent. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is G. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is A. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is V. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is L. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is I. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is F. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is W. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is M. In some embodiments, XI is absent or selected from S, T, C, Y, N, or Q; X2 is absent or selected from G, A, V, L, I, F, W, M, or P; X3 is absent or selected from D or E; X4 is absent or selected from S, T, C, Y, N, or Q; X5 is P.

[0123] NAI-5007748234vl In some embodiments, a variant capsid polypeptide comprises a sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an insertion between positions 586 and 587, wherein the insertion comprises an amino acid sequence of a consensus formula XI- X2-X3-X4-X5-F-T-R-P (SEQ ID NO:32), wherein:

[0124] XI is absent or is an amino acid with an hydroxyl side chain;

[0125] X2 is absent or is an amino acid with an aliphatic side chain;

[0126] X3 is absent or is an amino acid with a negatively charged side chain;

[0127] X4 is absent or is an amino acid with a simple amide side chain;

[0128] X5 is absent or is an amino acid with an aliphatic side chain.

[0129] In some embodiments, a variant capsid polypeptide comprises a sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an insertion between positions 586 and 587, wherein the insertion comprises an amino acid sequence of a consensus formula XI- X2-X3-X4-X5-F-T-R-P (SEQ ID NO:30), wherein:

[0130] XI is absent or selected from S, T, or Y;

[0131] X2 is absent or selected from A, V, L, I, or P;

[0132] X3 is absent or selected from D or E;

[0133] X4 is absent or selected from N or Q;

[0134] X5 is absent or selected from A, V, L, I, or P.

[0135] In some embodiments, the insertion (e.g., the insertion between positions 586 and 587 of SEQ ID NO:1, e.g., the insertion between positions 587 and 588 of SEQ ID NO: 1) comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids thereof. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 4 amino acids thereof. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 5 amino acids

[0136] NAI-5007748234vl thereof. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 6 amino acids thereof. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 7 amino acids thereof. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 8 amino acids thereof.

[0137] In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids thereof, and the variant capsid polypeptide further comprises an N587A mutation. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 4 amino acids thereof, and the variant capsid polypeptide further comprises an N587A mutation. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 5 amino acids thereof, and the variant capsid polypeptide further comprises an N587A mutation. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 6 amino acids thereof, and the variant capsid polypeptide further comprises an N587A mutation. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 7 amino acids thereof, and the variant capsid polypeptide further comprises an N587A mutation. In some embodiments, the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 8 amino acids thereof, and the variant capsid polypeptide further comprises an N587A mutation.

[0138] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 547, 548, 549, 550, 555, 561, 575, 578, 580, 581, 586, 587, 589, 591, 593, or any combination thereof, according to SEQ ID NO: 1, optionally wherein the mutation comprises an insertion, a deletion or a substitution.

[0139] In some embodiments, the variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an amino acid substitution at position 547, 548, 549, 550, 555, 561, 575, 578, 580, 581, 587, 589, 591, 593, or any combination thereof.

[0140] NAI-5007748234vl In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 547 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds toan amino acid substitution at position 548 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 549 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 550 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 555 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 561 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 575 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 578 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 580 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 581 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 587 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 589 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 591 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an amino acid substitution at position 593 as compared to SEQ ID NO: 1.

[0141] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to an insertion between positions 586 and 587, or 587 and 588 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to an insertion between positions 587 and 588 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to an insertion between positions 586 and 587 as compared to SEQ ID NO: 1.

[0142] NAI-5007748234vl In some embodiments, the variant capsid polypeptide comprises a mutation that comprises an insertion, e.g., an insertion of 1 or more amino acids, e.g., 1 amino acid, e.g., 1- 10 amino acids, e.g., 2-10 amino acids, e.g., 3-10 amino acids, e.g., 4-10 amino acids, e.g., 5- 10 amino acids, e.g., 6-10 amino acids, e.g., 7-10 amino acids, e.g., 8-10 amino acids, e.g., 9- 10 amino acids, e.g., 10 amino acids that correspond to an insertion between positions 586 and 587, or 587 and 588 as compared to SEQ ID NO: 1.

[0143] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 547, 548, 549, 550, 555, 561, 575, 578, 580, 581, 586, 587, 589, 591, 593, or any combination thereof, an insertion between positions 586 and 587, or 587 and 588, or any combination thereof according to SEQ ID NO: 1, and wherein the mutation comprises an insertion, a deletion or a substitution.

[0144] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 580, 586 (e.g., an insertion of one or more amino acids after position 586), 587, 589, and 593, as compared to SEQ ID NO: 1.

[0145] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 547, 555, 575, 586 (e.g., an insertion of one or more amino acids after position 586), 587, and 589, as compared to SEQ ID NO: 1.

[0146] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 586 (e.g., an insertion of one or more amino acids after position 586), 587, and 589, as compared to SEQ ID NO: 1.

[0147] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 547, 575, 586 (e.g., an insertion of one or more amino acids after position 586), 587, 589, and 591, as compared to SEQ ID NO: 1.

[0148] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0149] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 548, 578, 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0150] NAI-5007748234vl In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 548, 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0151] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 549, 561, 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0152] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 581, 587 (e.g., an insertion of one or more amino acids after position 587), and 589, as compared to SEQ ID NO: 1.

[0153] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 550, 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0154] In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 580, 587, 589, 593, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 547, 555, 575, 587, 589, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 587, 589, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 547, 575, 587, 589, 591, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 548, 578, 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 548, 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 549, 561, 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a

[0155] NAI-5007748234vl mutation at position 581, 589, and an insertion between positions 587 and 588 as compared to SEQ ID NO: 1. In some embodiments, the variant capsid polypeptide comprises a mutation that corresponds to a mutation at position 550, 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1.

[0156] In some embodiments, the capsid polypeptide comprises a mutation that corresponds to an insertion between residues 586 and 587 as compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, an amino acid sequence SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence that has at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence that has at least 1, 2, 3, 4, or 5 mutations as compared to SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 4 amino acids of SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 5 amino acids of SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 6 amino acids of SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 7 amino acids of SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 8 amino acids of SLDQAFTRP (SEQ ID NO: 23).

[0157] In an aspect, described herein is a variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), and comprising one or more (e.g., all) of the mutations consisting of an N587A amino acid substitution, and an insertion of an amino acid sequence SLDQAFTRP (SEQ ID NO: 23) between position 586 and position 587, with all numbering according to SEQ ID NO: 1). In an aspect, described herein is a variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), and comprising one or more (e.g., all) of the mutations consisting of an N587A amino acid substitution, and an insertion of an amino acid sequence SLDQAFTRP (SEQ ID NO: 23) between position 586 and position 587, with all

[0158] NAI-5007748234vl numbering according to SEQ ID NO: 1), and comprising a sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, but fewer than 40, 39, 38, 37, 36, 35, 34, 33, 32, 32, 31, 29, 28, 27, 26, or 25 additional mutations as compared to SEQ ID NO: 1. In an aspect, described herein is a variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), and comprising one or more (e.g., all) of the mutations consisting of an N587A amino acid substitution, and an insertion of an amino acid sequence SLDQAFTRP (SEQ ID NO: 23) between position 586 and position 587, with all numbering according to SEQ ID NO: 1), wherein the variant capsid polypeptide comprises a sequence that has at least 95, 96, 97, 98, 99% identity to VP1, VP2, or VP3 sequence of a reference sequence other than SEQ ID NO: 1 (e.g., a reference sequence corresponding to wild-type AAV5, AAV8, AAV9, or AAVrh74, e.g., a reference sequence of SEQ ID NO: 3, 5, 7, 9 or 10).

[0159] In some embodiments, a nucleic acid molecule is provided. In some embodiments, the nucleic acid molecule has the sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid molecule comprises a sequence encoding the VP1 (amino acids 1-744), VP2 (amino acids 138-744) or VP3 (amino acids 203-744) polypeptide of SEQ ID NO: 13.

[0160] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 547, 548, 549, 550, 555, 561, 575, 578, 580, 581, 586, 587, 589, 591, 593, or any combination thereof, according to SEQ ID NO: 1, optionally wherein the mutation comprises an insertion, a deletion or a substitution. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 547, 548, 549, 550, 555, 561, 575, 578, 580, 581, 587, 589, 591, 593, or any combination thereof, according to SEQ ID NO: 1, optionally wherein the mutation comprises a substitution, or comprises a deletion, or comprises a combination of substitutions and deletions. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises an insertion between positions 586 and 587, or 587 and 588, or any combination thereof, according to SEQ ID NO: 1.

[0161] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 547 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid

[0162] NAI-5007748234vl polypeptide that comprises a mutation that corresponds to a mutation at position 548 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 549 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 550 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 555 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 561 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 575 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 578 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 580 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 581 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 589 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 591 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 593 as compared to SEQ ID NO: 1.

[0163] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to an insertion between positions 586 and 587, or 587 and 588 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to an insertion between positions 587 and 588 as compared to SEQ ID NO: 1. In some

[0164] NAI-5007748234vl embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to an insertion between positions 586 and 587 as compared to SEQ ID NO: 1.

[0165] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 547, 548, 549, 550, 555, 561, 575, 578, 580, 581, 586, 587, 589, 591, 593, or any combination thereof, an insertion between positions 586 and 587, or 587 and 588, or any combination thereof according to SEQ ID NO: 1, and wherein the mutation comprises an insertion, a deletion or a substitution.

[0166] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 580, 586 (e.g., an insertion of one or more amino acids after position 586), 587, 589, and 593, as compared to SEQ ID NO: 1.

[0167] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 547, 555, 575, 586 (e.g., an insertion of one or more amino acids after position 586), 587, and 589, as compared to SEQ ID NO: 1.

[0168] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 586 (e.g., an insertion of one or more amino acids after position 586), 587, and 589, as compared to SEQ ID NO: 1.

[0169] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 547, 575, 586 (e.g., an insertion of one or more amino acids after position 586), 587, 589, and 591, as compared to SEQ ID NO: 1.

[0170] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0171] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 548, 578, 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0172] NAI-5007748234vl In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 548, 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0173] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 549, 561, 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0174] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 581, 587 (e.g., an insertion of one or more amino acids after position 587), and 589, as compared to SEQ ID NO: 1.

[0175] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 550, 586 (e.g., an insertion of one or more amino acids after position 586), and 587, as compared to SEQ ID NO: 1.

[0176] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that comprises an insertion, e.g., an insertion of 1 or more amino acids, e.g., 1 amino acid, e.g., 1-2 amino acids, e.g., 1-3 amino acids, e.g., 1-4 amino acids, e.g., 1-5 amino acids, e.g., 1-6 amino acids, e.g., 1-7 amino acids, e.g., 1-8 amino acids, e.g., 1-9 amino acids, e.g., 1-10 amino acids, that correspond to an insertion between positions 586 and 587, or 587 and 588 as compared to SEQ ID NO: 1.

[0177] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that comprises an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that comprises an insertion between positions 587 and 588 as compared to SEQ ID NO: 1.

[0178] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 580, 587, 589, 593, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises

[0179] NAI-5007748234vl a mutation that corresponds to a mutation at position 547, 555, 575, 587, 589, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 587, 589, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 547, 575, 587, 589, 591, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 548, 578, 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 548, 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 549, 561, 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 581, 589, and an insertion between positions 587 and 588 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to a mutation at position 550, 587, and an insertion between positions 586 and 587 as compared to SEQ ID NO: 1.

[0180] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO:1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an amino acid substitution at position 547, 548, 549, 550, 555, 561, 575, 578, 580, 581, 587, 589, 591, 593, or any combination thereof.

[0181] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that comprises a mutation that corresponds to an insertion between residues 586 and 587 as NAI-5007748234vl compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, an amino acid sequence SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence that has at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence that has at least 1, 2, 3, 4, or 5 mutations as compared to SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 4 amino acids of SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 5 amino acids of SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 6 amino acids of SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 7 amino acids of SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the insertion comprises an amino acid sequence comprising a fragment of at least 8 amino acids of SLDQAFTRP (SEQ ID NO: 23). In some embodiments, the mutation further corresponds to a N587A mutation as compared to SEQ ID NO: 1.

[0182] In an aspect, described herein is a variant nucleic acid molecule encoding a variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO:1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), and comprising one or more (e.g., all) of the mutations consisting of an N587A amino acid substitution, and an insertion of an amino acid sequence SLDQAFTRP (SEQ ID NO: 23) between position 586 and position 587, with all numbering according to SEQ ID NO: 1). In an aspect, described herein is a variant nucleic acid molecule encoding a variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO:1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), and comprising one or more (e.g., all) of the mutations consisting of an N587A amino acid substitution, and an insertion of SLDQAFTRP (SEQ ID NO: 23) between position 586 and position 587, with all numbering according to SEQ ID NO: 1), and comprising a sequence having at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, but fewer than 40, 39, 38, 37, 36, 35, 34, 33, 32, 32, 31, 29, 28, 27, 26, or 25 additional mutations as compared to SEQ ID NO: 1. In an aspect, described herein is a variant nucleic acid molecule encoding a

[0183] NAI-5007748234vl variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), and comprising one or more (e.g., all) of the mutations consisting of an N587A amino acid substitution, and an insertion of an amino acid sequence SLDQAFTRP (SEQ ID NO: 23) between position 586 and position 587, with all numbering according to SEQ ID NO: 1), wherein the variant capsid polypeptide comprises a sequence that has at least 95, 96, 97, 98, 99% identity to VP1, VP2, or VP3 sequence of a reference sequence other than SEQ ID NO: 1 (e.g., a reference sequence corresponding to wild-type AAV5, AAV8, AAV9, or AAVrh74, e.g., a reference sequence of SEQ ID NO: 3, 5, 7, 9 or 10).

[0184] In some embodiments, including in the embodiments described above, a capsid polypeptide is provided that comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence of a capsid polypeptide provided herein.

[0185] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide as provided herein. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide as provided herein.

[0186] In some embodiments, including in the embodiments described above, a capsid polypeptide is provided that comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 identical to SEQ ID NO: 1.

[0187] In some embodiments, including in the embodiments described above, a capsid polypeptide is provided that comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 identical to any one of SEQ ID NO: 3, 5, 7, 9, or 10.

[0188] In some embodiments, the reference polypeptide for purposes of % identity, comprises a sequence of SEQ ID NOs: 1, 3, 5, 7, 9, or 10.

[0189] In some embodiments, the reference nucleic acid for purposes of % identity, comprises a sequence of SEQ ID NOs: 2, 4, 6, 8, or 11.

[0190] In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 13.

[0191] NAI-5007748234vl In some embodiments, the nucleic acid molecules encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 14.

[0192] In some embodiments, the nucleic acid molecules encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 14 that encodes a sequence of SEQ ID NO: 13.

[0193] In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises a sequence of SEQ ID NO: 13 that is encoded by a nucleotide sequence of SEQ ID NO: 14.

[0194] In some embodiments, the capsid polypeptide comprises a sequence that includes all of the mutation differences described herein, and further includes no more than 30, no more than 20, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 additional mutations relative to SEQ ID NO: 1.

[0195] In some embodiments, the capsid polypeptide is a VP1 capsid polypeptide. In some embodiments, the capsid polypeptide is a VP2 capsid polypeptide. In some embodiments, the capsid polypeptide is a VP3 capsid polypeptide. With respect to reference sequence SEQ ID NO: 1, a VP1 capsid polypeptide comprises amino acids 1-735 of SEQ ID NO: 1. With respect to reference sequence SEQ ID NO: 1, a VP2 capsid polypeptide comprises amino acids 138-735 of SEQ ID NO: 1. With respect to reference sequence SEQ ID NO: 1, a VP3 capsid polypeptide comprises amino acids 203-735 of SEQ ID NO: 1.

[0196] Reference (e.g., wild type AAV2) and exemplary (e.g., Variants 1-5) sequences of capsid polypeptides and nucleic acid molecules encoding the same are provided in Table 1. Table 1 illustrates the VP1, VP2 and VP3 polypeptide starting amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21. The nucleic acid sequences provided in Table 1 include a stop codon at the 3’-end of the sequence (e.g., the TAA stop codon). It will be understood by a skilled artisan that in some embodiments, the TAA stop codon is removed or replaced with a different stop codon (e g., TGA or TAG).

[0197] NAI-5007748234vl Table 1

[0198] NAI-5007748234vl

[0199] NAI-5007748234vl

[0200] NAI-5007748234vl

[0201] NAI-5007748234vl

[0202] NAI-5007748234vl

[0203] NAI-5007748234vl

[0204] In some embodiments, the variant capsid polypeptide has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence as provided in Table 1. In some embodiments, the variant capsid polypeptide has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to an amino acid sequence of positions 1-735 of

[0205] SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO:1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3). In some embodiments, the variant capsid polypeptide has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to an amino acid sequence of positions 1-744 of SEQ ID NO: 13 (VP1), positions 138-744 of SEQ ID NO: 13(VP2), or positions 203-744 of SEQ ID NO: 13 (VP3).

[0206] In some embodiments, the variant capsid polypeptide has a sequence of SEQ ID NO: 13.

[0207] In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide that has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence as provided in Table 1. In some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to an amino acid sequence of positions 1-735 of SEQ ID NO:1 (VP1), positions 138-735 of SEQ ID NO:1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3). In

[0208] NAI-5007748234vl some embodiments, the nucleic acid molecule encodes a variant capsid polypeptide has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to an amino acid sequence of positions 1-744 of SEQ ID NO: 13 (VP1), positions 138-744 of SEQ ID NO: 13 (VP2), or positions 203-744 of SEQ ID NO: 13 (VP3).

[0209] In some embodiments, the capsid polypeptide comprises a reference capsid sequence, such as SEQ ID NO: 1, and at least, or about, 80%, 85%, 90%, or 95%, or 100% of the mutations (insertions, deletions, or substitutions). In some embodiments, the reference capsid sequence comprises at least, about, or exactly, 80% of the mutations (insertions, deletions, or substitutions). In some embodiments, the reference capsid sequence comprises at least, about, or exactly, 85% of the mutations (insertions, deletions, or substitutions). In some embodiments, the reference capsid sequence comprises at least, about, or exactly, 90% of the mutations (insertions, deletions, or substitutions). In some embodiments, the reference capsid sequence comprises at least, about, or exactly, 95% of the mutations (insertions, deletions, or substitutions). In some embodiments, the reference capsid sequence comprises 100% of the mutations (insertions, deletions, or substitutions).

[0210] In some embodiments, the capsid polypeptide comprises a reference capsid sequence, such as SEQ ID NO: 1, and at least, or about, or exactly, 80%, 85%, 90%, or 95%, or 100% of the following mutations: 586_9aa_587_SLDQAFTRP, N587A.

[0211] In some embodiments, the capsid polypeptide comprises at least 7, 8, or all of the amino acid residues of the 9 amino acid insertion SLDQAFTRP (SEQ ID NO: 23).

[0212] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence that has 95% or more amino acid sequence identity with an amino acid sequence of SEQ ID NO: 13; and has at least 80% of the mutations described herein compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence that has less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO: 13; and has at least 80% of the mutations described herein compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence that has 95% or more amino acid sequence identity with SEQ ID NO: 13; and has less than 80% of the mutations described herein compared to the amino acid sequence of SEQ ID NO: 1.

[0213] As used herein, the phrase “80% of the mutations” in reference to a variant capsid sequence means that the variant has at least 80% of the mutations present in the variant

[0214] NAI-5007748234vl capsid sequence, wherein the total number of mutations are based on a comparison to a reference sequence, such as a wild-type sequence. If a variant capsid polypeptide is a mixture of an insertion and substitution or deletion, then each amino acid residue of the insert is counted in the total number of mutations. For example, if the variant capsid polypeptide has a mutation that is a mixture of substitutions of N587A and an insertion of a polypeptide comprising the sequence of SLDQAFTRP (SEQ ID NO: 23) then the total number of mutations is 10, which is the 9 amino acid insertion and the 1 amino acid substitution, and the variant capsid having “at least 80% of the mutations” will comprise at least 8, 9, or all of the mutations.

[0215] Variant Capsids (Corresponding Positions)

[0216] The mutations to capsid polypeptide sequences described herein are described in relation to a position and / or amino acid at a position within a reference sequence, e.g., SEQ ID NO: 1. Thus, in some embodiments, the capsid polypeptides described herein are variant capsid polypeptides of the reference sequence, e.g., SEQ ID NO: 1, e.g., include capsid polypeptides comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the reference capsid polypeptide sequence (e.g., reference capsid polypeptide VP1, VP2 and / or VP3 sequence), e.g., SEQ ID NO: 1 (or VP2 or VP3 sequence comprised therein) and further including one or more mutations described herein.

[0217] It will be understood by the skilled artisan, and without being bound by theory, that each amino acid position within a reference sequence corresponds to a position within the sequence of other capsid polypeptides such as capsid polypeptides derived from dependoparvoviruses with different serotypes. Such corresponding positions are identified using sequence alignment tools known in the art. A particularly preferred sequence alignment tool is Clustal Omega (Sievers F., et al., Mol. Syst. Biol. 7:359, 2011, DOI: 10.1038 / msb.2011.75, incorporated herein by reference in its entirety). An alignment of exemplary reference capsid polypeptides is shown in FIG. 2A-2C. Thus, in some embodiments, the variant capsid polypeptides of the invention include variants of reference capsid polypeptides that include one or more mutations described herein in such reference capsid polypeptides at positions corresponding to the position of the mutation described herein in relation to a different reference capsid polypeptide. Thus, for example, a mutation

[0218] NAI-5007748234vl described as XnnnY relative to SEQ ID NO: 1 (where X is the amino acid present at position nnn in SEQ ID NO: 1 and Y is the amino acid mutation at that position, e.g., described herein), the disclosure provides variant capsid polypeptides comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99 identity to the reference capsid polypeptide sequence (e.g., reference capsid polypeptide VP1, VP2 and / or VP3 sequence) other than SEQ ID NO: 1 (or VP2 or VP3 sequence comprised therein) and further comprising the disclosed mutation at a position corresponding to position nnn of SEQ ID NO: 1 (e.g., comprising Y at the position in the new variant capsid polypeptide sequence that corresponds to position nnn of SEQ ID NO: 1). As described above, such corresponding position is determined using a sequence alignment tool, such as, for example, the clustal omega tool described above. Examples of corresponding amino acid positions of exemplary known AAV serotypes is provided in FIG. 2A-2C. In some embodiments, the variant is a variant of the AAV2 capsid polypeptide, which can be referred to as a “variant AAV2 capsid polypeptide.”

[0219] Thus, in some embodiments, the disclosure provides capsid polypeptide sequences that are variants of a reference sequence other than SEQ ID NO: 1, e.g., a reference sequence other than SEQ ID NO: 1 as described herein, which include one or more mutations corresponding to the mutations described herein.

[0220] As used herein, the term “corresponds to” as used in reference to a position in a sequence, such as an amino acid or nucleic acid sequence, can be used in reference to an entire capsid polypeptide or polynucleotide sequence, such as the full length sequence of the capsid polypeptide that comprises a VP1, VP2, and VP3 polypeptide, or a nucleic acid molecule encodes the same. In some embodiments, the term “corresponds to” can be used in reference to a region or domain of the capsid polypeptide. For example, a position that corresponds to a position in the VP1 section of the reference capsid polypeptide can correspond to the VP1 portion of the polypeptide of the variant capsid polypeptide. Thus, when aligning the two sequences to determine whether a position corresponds to another position the full length polypeptide can be used or domains (regions) can be used to determine whether a position corresponds to a specific position. In some embodiments, the region is the VP1 polypeptide. In some embodiments, the region is the VP2 polypeptide. In some embodiments, the region is the VP3 polypeptide. In some embodiments, when the reference polypeptide is the wild-type sequence (e.g., full length or region) of a certain serotype of AAV, the variant polypeptide can be of the same serotype with a mutation made

[0221] NAI-5007748234vl at such corresponding position as compared to the reference sequence (e.g., full length or region). In some embodiments, the variant capsid polypeptide is a different serotype as compared to the reference sequence.

[0222] Table 2 below describes the preferred insertion sites in the listed AAV serotypes, which are homologous to the insertion sites between positions 586 and 587, or between positions 587 and 588 in the AAV2 reference capsid disclosed herein.

[0223] Table 2

[0224] The variant capsid polypeptides described herein are optionally variants of reference capsids serotypes known in the art. Non-limiting examples of such reference AAV serotypes include AAV1, AAVrhlO, AAV-DJ, AAV-DJ8, AAV5, AAVPHP.B (PHP.B), AAVPHP.A (PHP. A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1- 35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B- DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B- SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B- EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B- STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A12, AAVG2A15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9, AAV9 K449R (or K449R AAV9), AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42- lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42- 4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2-

[0225] NAI-5007748234vl 15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.5O, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-1 l / rh.53, AAV4- 8 / rl 1.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5- 3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.lO, AAV16.12 / hu.l 1, AAV29.3 / bb. l, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.4O, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV33.12 / hu.l7, AAV33.4 / hu.l5, AAV33.8 / hu. l6, AAV52 / hu. l9, AAV52.1 / hu.2O, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAVC1, AAVC2, AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. l, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH- 1 / hu.l, AAVH-5 / hu.3, AAVLG- 10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721- 8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.l, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.l 1, AAVhu.13, AAVhu. l 5, AAVhu.l 6, AAVhu.l 7, AAVhu. l 8, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.lO, AAVrh.12, AAVrh.13, AAVrh. l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74 (also referred to as AAVrh74), AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl. l, AAVhErl.5, AAVhER1.14, AAVhErl.8, AAVhErl.16, AAVhErl.18, AAVhErl.35, AAVhErl.7, AAVhErl.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV- PAEC, AAV-LK01, AAV-LK02, AAV- LK03, AAV-LK04, AAV-LK05, AAV-LK06,

[0226] NAI-5007748234vl AAV-LK07, AAV-LK08, AAV-LK09, AAV- LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV- LK17, AAV-LK18, AAV-LK19, AAV- PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV- PAEC12, AAV-2-pre-miRNA-101 , AAV-8h, AAV- 8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100- 2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.5O, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.l l, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr- E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd- B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLv 1-2, AAV CLv-12, AAV CLv 1-3, AAV CLv-13, AAV CLv 1-4, AAV Civ 1-7, AAV Civ 1-8, AAV Civ 1-9, AAV CLv- 2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv- D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv- Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv- Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv- M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12,

[0227] NAI-5007748234vl AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9, 7m8, SparklOO, AAVMYO and variants thereof.

[0228] In some embodiments, the reference AAV capsid sequence comprises an AAV2 sequence. In some embodiments, the reference AAV capsid sequence comprises an AAV5 sequence. In some embodiments, the reference AAV capsid sequence comprises an AAV8 sequence. In some embodiments, the reference AAV capsid sequence comprises an AAV9 sequence. In some embodiments, the reference AAV capsid sequence comprises an AAVrh74 sequence. While not wishing to be bound by theory, it is understood that a reference AAV capsid sequence comprises a VP1 region. In certain embodiments, a reference AAV capsid sequence comprises a VP1, VP2 and / or VP3 region, or any combination thereof. A reference VP1 sequence may be considered synonymous with a reference AAV capsid sequence.

[0229] An exemplary reference sequence of SEQ ID NO: 1 (wild-type AAV2) is as follows:

[0230] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEH DKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSP VEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSS GNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQY GYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMAS HKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGV LPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYST GQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL . ( SEQ ID NO : 1 )

[0231] Unless otherwise noted, SEQ ID NO: 1 is the reference sequence. In the sequence above, the sequence found in VP1, VP2 and VP3 is underlined (e.g., a VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 203-735 of SEQ ID NO: 1), the sequence found in both VP1 and VP2 is in bold (e.g., a VP2 capsid polypeptide includes, e.g., consists of, the sequence corresponding to amino acids 138-735 of SEQ ID NO: 1) and the sequence that is not underlined or bold is found only in VP1 (e.g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-735 of SEQ ID NO: 1).

[0232] An example nucleic acid sequence encoding SEQ ID NO: 1 is SEQ ID NO: 2:

[0233] NAI-5007748234vl ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGT GGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCA GGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGC CGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGAC AGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAA GAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTG AACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGC ACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAA AAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACA GCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAAT GGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTC CACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAA CAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCT ACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGG CAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACAT TCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACG GTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGAT GCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAA CGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGC GTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCT CACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAG AACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGT

[0234] GACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAA AGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAAT GGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAG TTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACAT TGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAG TATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACA CACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTG GGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTT AAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTT CAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATC GAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACT ACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCC ATTGGCACCAGATACCTGACTCGTAATCTGTAA (SEQ ID NO: 2)

[0235] An exemplary reference sequence of wild type AAV5, SEQ ID NO: 3 (wild-type

[0236] AAV5), is as follows:

[0237] MSFVDHPPDWLEEVGEGLREFLGLEAGPPKPKPNQQHQDQARGLVLPGYNYLGPGNGLDRGEPVNRADEVAREHD

[0238] ISYNEQLEAGDNPYLKYNHADAEFQEKLADDTSFGGNLGKAVFQAKKRVLEPFGLVEEGAKTAPTGKRIDDHFPK

[0239] RKKARTEEDSKPSTSSDAEAGPSGSQQLQIPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWM

[0240] GDRVVTKSTRTWVLPSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRPR

[0241] SLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQVFTLPQYGYATLNRDN

[0242] TENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAPSQNLFKLANPLVDQYLYRFVSTNNTGGVQFN

[0243] KNLAGRYANTYKNWFPGPMGRTQGWNLGSGVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALEN

[0244] TMIFNSQPANPGTTATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPATGTYNLQEIVPGSVWMERD

[0245] VYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFITQYSTGQVTVEMEWEL

[0246] KKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRYLTRPL ( SEQ ID NO : 3 )

[0247] NAI-5007748234vl In the sequence above, the sequence found in VP1, VP2 and VP3 is underlined (e.g., a VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 193-724 of SEQ ID NO: 3), the sequence found in both VP1 and VP2 is in bold (e.g., a VP2 capsid polypeptide includes, e.g., consists of, the sequence corresponding to amino acids 138- 724 of SEQ ID NO: 3) and the sequence that is not underlined or bold is found only in VP1 (e.g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-724 of SEQ ID NO: 3).

[0248] An example nucleic acid sequence encoding SEQ ID NO: 3 is SEQ ID NO: 4:

[0249] ATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCGCGAGTTTTT

[0250] GGGCCTTGAAGCGGGCCCACCGAAACCAAAACCCAATCAGCAGCATCAAGATCAAGCCCGTGG

[0251] TCTTGTGCTGCCTGGTTATAACTATCTCGGACCCGGAAACGGGCTCGATCGAGGAGAGCCTGTC

[0252] AACAGGGCAGACGAGGTCGCGCGAGAGCACGACATCTCGTACAACGAGCAGCTTGAGGCGGGA

[0253] GACAACCCCTACCTCAAGTACAACCACGCGGACGCCGAGTTTCAGGAGAAGCTCGCCGACGAC

[0254] ACATCCTTCGGGGGAAACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTT

[0255] TTGGCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTC

[0256] CAAAAAGAAAGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAG

[0257] CTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGGAGCTG

[0258] ATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAATAACCAAGGTGCCGATGGAGTGG

[0259] GCAATGCCTCGGGAGATTGGCATTGCGATTCCACGTGGATGGGGGACAGAGTCGTCACCAAGT

[0260] CCACCCGAACCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGATCAAAAGCGGCT

[0261] CCGTCGACGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTT

[0262] TAACCGCTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGC

[0263] TTCAGACCCCGGTCCCTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGG

[0264] ACTCCACCACCACCATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACTA

[0265] CCAGCTGCCCTACGTCGTCGGCAACGGGACCGAGGGATGCCTGCCGGCCTTCCCTCCGCAGGT

[0266] CTTTACGCTGCCGCAGTACGGTTACGCGACGCTGAACCGCGACAACACAGAAAATCCCACCGAG

[0267] AGGAGCAGCTTCTTCTGCCTAGAGTACTTTCCCAGCAAGATGCTGAGAACGGGCAACAACTTTG

[0268] AGTTTACCTACAACTTTGAGGAGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAACCTGTTC

[0269] AAGCTGGCCAACCCGCTGGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCG

[0270] GAGTCCAGTTCAACAAGAACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCGGG

[0271] GCCCATGGGCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTCAACCGCGCCAGTGTCAGCG

[0272] CCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTTACCAGGTGCCCCCGCAGCCGA

[0273] ACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAACACTATGATCTTCAA

[0274] CAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGGGCAACATGCTCATCACCAG

[0275] CGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCGGCGGGCAGATGGCCACCA

[0276] ACAACCAGAGCTCCACCACTGCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCG

[0277] GCAGCGTGTGGATGGAGAGGGACGTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGA

[0278] CGGGGGCGCACTTTCACCCCTCTCCGGCCATGGGCGGATTCGGACTCAAACACCCACCGCCCA

[0279] TGATGCTCATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGTGCCCGTCAG

[0280] CAGCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTCAAGAA

[0281] GGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCCAGTTT

[0282] GTGGACTTTGCCCCGGACAGCACCGGGGAATACAGAACCACCAGACCTATCGGAACCCGATAC

[0283] CTTACCCGACCCCTTTAA (SEQ ID NO: 4)

[0284] NAI-5007748234vl An exemplary reference sequence of wild-type AAV8, SEQ ID NO: 5 (wild-type

[0285] AAV8), is as follows:

[0286] MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEH DKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSP

[0287] QRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSS SGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ RLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMI PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSR

[0288] TQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIA MATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNS QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL ( SEQ ID NO :

[0289] 5 )

[0290] In the sequence above, the sequence found in VP1, VP2 and VP3 is underlined (e.g., a VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 204-738 of SEQ ID NO: 5), the sequence found in both VP1 and VP2 is in bold (e.g., a VP2 capsid polypeptide includes, e.g., consists of, the sequence corresponding to amino acids 138- 738 of SEQ ID NO: 5) and the sequence that is not underlined or bold is found only in VP1 (e.g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-738 of SEQ ID NO: 5).

[0291] An example nucleic acid sequence encoding SEQ ID NO: 5 is SEQ ID NO: 6:

[0292] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGT

[0293] GGTGGGCGCTGAAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGC

[0294] CGGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAG

[0295] CCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTGCA

[0296] GGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTGCA

[0297] AGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTTCT

[0298] CGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGA

[0299] GCCATCACCCCAGCGTTCTCCAGACTCCTCTACGGGCATCGGCAAGAAAGGCCAACAGCCCGC

[0300] CAGAAAAAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGTTCCAGACCCTCAACCTCTCG

[0301] GAGAACCTCCAGCAGCGCCCTCTGGTGTGGGACCTAATACAATGGCTGCAGGCGGTGGCGCAC

[0302] CAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCG

[0303] ATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCT

[0304] ACAACAACCACCTCTACAAGCAAATCTCCAACGGGACATCGGGAGGAGCCACCAACGACAACAC

[0305] CTACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTTTCAC

[0306] CACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAA

[0307] GCTCTTCAACATCCAGGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAAC

[0308] CTCACCAGCACCATCCAGGTGTTTACGGACTCGGAGTACCAGCTGCCGTACGTTCTCGGCTCTG

[0309] CCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTGTTCATGATTCCCCAGTACGGCTACCT

[0310] AACACTCAACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTACTGCCTGGAATACTTTCCTT

[0311] CGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCAC AGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGT

[0312] NAI-5007748234vl ACTACTTGTCTCGGACTCAAACAACAGGAGGCACGGCAAATACGCAGACTCTGGGCTTCAGCCA

[0313] AGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCTGCCAGGACCCTGTTACCGCCAA

[0314] CAACGCGTCTCAACGACAACCGGGCAAAACAACAATAGCAACTTTGCCTGGACTGCTGGGACCA

[0315] AATACCATCTGAATGGAAGAAATTCATTGGCTAATCCTGGCATCGCTATGGCAACACACAAAGAC GACGAGGAGCGTTTTTTTCCCAGTAACGGGATCCTGATTTTTGGCAAACAAAATGCTGCCAGAGA CAATGCGGATTACAGCGATGTCATGCTCACCAGCGAGGAAGAAATCAAAACCACTAACCCTGTG GCTACAGAGGAATACGGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTG

[0316] GAACTGTCAACAGCCAGGGGGCCTTACCCGGTATGGTCTGGCAGAACCGGGACGTGTACCTGC

[0317] AGGGTCCCATCTGGGCCAAGATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGCTGATGG GCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGA TCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACGCAATACAGCACCGGACAGG TCAGCGTGGAAATTGAATGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAGATCC

[0318] AGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTAC

[0319] TCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA (SEQ ID NO: 6)

[0320] An exemplary reference sequence of wild-type AAV9, SEQ ID NO: 7 (wild-type

[0321] AAV9), is as follows:

[0322] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEH DKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSP

[0323] QEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSS

[0324] GNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIP QYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKT INGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMA

[0325] SHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQG ILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYS TGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL ( SEQ ID NO : 7 )

[0326] In the sequence above, the sequence found in VP1, VP2 and VP3 is underlined (e.g., a VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 203-736 of SEQ ID NO: 7), the sequence found in both VP1 and VP2 is in bold (e.g., a VP2 capsid polypeptide includes, e.g., consists of, the sequence corresponding to amino acids 138- 736 of SEQ ID NO: 7) and the sequence that is not underlined or bold is found only in VP1 (e.g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-736 of SEQ ID NO: 7).

[0327] An example nucleic acid sequence encoding SEQ ID NO: 7 is SEQ ID NO: 8:

[0328] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGTATTCGCGAGT GGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCG AGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCC GGTCAACGCAGCAGACGCGGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCAAGG

[0329] NAI-5007748234vl CCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAG

[0330] AAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGA

[0331] ACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCA

[0332] GTCTCCTCAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAA

[0333] GAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAA

[0334] CCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTGGTGGCGCACCAGTG

[0335] GCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCGGGAAATTGGCATTGCGATTCCC

[0336] AATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACA

[0337] ATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTC

[0338] GGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGA

[0339] CTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTC

[0340] AACATTCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAG

[0341] CACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGTCGGCTCACGA

[0342] GGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGTACGGGTATCTGACGCTT

[0343] AATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTCCCGTCGCAAAT

[0344] GCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGCT

[0345] ACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTC

[0346] TCAAAGACTATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAG

[0347] CAACATGGCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCA

[0348] ACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAA

[0349] TGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCAGCCACAAAGAAGGAGAGGACCGT

[0350] TTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGAGACAACGTGGATGC

[0351] GGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAGTCCT

[0352] ATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAA

[0353] ACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTG

[0354] GGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATG

[0355] AAGCACCCGCCTCCTCAGATCCTCATCAAAAACACACCTGTACCTGCGGATCCTCCAACGGCCT

[0356] TCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCTACTGGCCAAGTCAGCGTGGAGATC

[0357] GAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAAC

[0358] TATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCC

[0359] ATTGGCACCAGATACCTGACTCGTAATCTGTAA (SEQ ID NO: 8)

[0360] An exemplary reference sequence of wild-type AAVrh74, SEQ ID NO: 9 (wild-type

[0361] AAVrh74), is as follows:

[0362] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEH DKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSP

[0363] QRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSS SGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMI PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSR

[0364] TQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVA MATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNS QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL ( SEQ ID NO :

[0365] 9 )

[0366] An alternative exemplary reference sequence of SEQ ID NO: 10 (alternate wild-type AAVrh74) is as follows:

[0367] NAI-5007748234vl MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEH

[0368] DKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSP

[0369] QRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSS

[0370] SGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ

[0371] RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMI

[0372] PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSR

[0373] TQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVA

[0374] MATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNS

[0375] QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFTKAKLASFITQ

[0376] YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL ( SEQ ID

[0377] NO : 10 )

[0378] In the sequences above (SEQ ID NO: 9 or SEQ ID NO: 10), the sequence found in VP1, VP2 and VP3 is underlined (e.g., a VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 204-738 of SEQ ID NO: 9), the sequence found in both VP1 and VP2 is in bold (e.g., a VP2 capsid polypeptide includes, e.g., consists of, the sequence corresponding to amino acids 138-738 of SEQ ID NO: 9) and the sequence that is not underlined or bold is found only in VP1 (e.g., a VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-738 of SEQ ID NO: 9).

[0379] An example nucleic acid sequence encoding SEQ ID NO: 9 is SEQ ID NO: 11.

[0380] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGT

[0381] GGTGGGACCTGAAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACAACGGCC

[0382] GGGGTCTGGTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGC

[0383] CCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTCCAA

[0384] GCGGGTGACAATCCGTACCTGCGGTATAATCACGCCGACGCCGAGTTTCAGGAGCGTCTGCAA

[0385] GAAGATACGTCTTTTGGGGGCAACCTCGGGCGCGCAGTCTTCCAGGCCAAAAAGCGGGTTCTC

[0386] GAACCTCTGGGCCTGGTTGAATCGCCGGTTAAGACGGCTCCTGGAAAGAAGAGGCCGGTAGAG

[0387] CCATCACCCCAGCGCTCTCCAGACTCCTCTACGGGCATCGGCAAGAAAGGCCAGCAGCCCGCA

[0388] AAAAAGAGACTCAATTTTGGGCAGACTGGCGACTCAGAGTCAGTCCCCGACCCTCAACCAATCG

[0389] GAGAACCACCAGCAGGCCCCTCTGGTCTGGGATCTGGTACAATGGCTGCAGGCGGTGGCGCTC

[0390] CAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAGTTCCTCAGGAAATTGGCATTGCGA

[0391] TTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGCACCTGGGCCCTGCCCACCTA

[0392] CAACAACCACCTCTACAAGCAAATCTCCAACGGGACCTCGGGAGGAAGCACCAACGACAACACC

[0393] TACTTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACC

[0394] ACGTGACTGGCAGCGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGGCTCAACTTCAAG

[0395] CTCTTCAACATCCAAGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACC

[0396] TTACCAGCACGATTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGTACGTGCTCGGCTCGGC

[0397] GCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGGTACCTG

[0398] ACTCTGAACAATGGCAGTCAGGCTGTGGGCCGGTCGTCCTTCTACTGCCTGGAGTACTTTCCTT

[0399] CTCAAATGCTGAGAACGGGCAACAACTTTGAATTCAGCTACAACTTCGAGGACGTGCCCTTCCAC

[0400] AGCAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGATGAACCCTCTCATCGACCAGTACTTGT

[0401] ACTACCTGTCCCGGACTCAAAGCACGGGCGGTACTGCAGGAACTCAGCAGTTGCTATTTTCTCA

[0402] GGCCGGGCCTAACAACATGTCGGCTCAGGCCAAGAACTGGCTACCCGGTCCCTGCTACCGGCA

[0403] GCAACGTGTCTCCACGACACTGTCGCAGAACAACAACAGCAACTTTGCCTGGACGGGTGCCACC

[0404] AAGTATCATCTGAATGGCAGAGACTCTCTGGTGAATCCTGGCGTTGCCATGGCTACCCACAAGG

[0405] NAI-5007748234vl ACGACGAAGAGCGATTTTTTCCATCCAGCGGAGTCTTAATGTTTGGGAAACAGGGAGCTGGAAA AGACAACGTGGACTATAGCAGCGTGATGCTAACCAGCGAGGAAGAAATAAAGACCACCAACCCA GTGGCCACAGAACAGTACGGCGTGGTGGCCGATAACCTGCAACAGCAAAACGCCGCTCCTATT GTAGGGGCCGTCAATAGTCAAGGAGCCTTACCTGGCATGGTGTGGCAGAACCGGGACGTGTAC

[0406] CTGCAGGGTCCCATCTGGGCCAAGATTCCTCATACGGACGGCAACTTTCATCCCTCGCCGCTGA TGGGAGGCTTTGGACTGAAGCATCCGCCTCCTCAGATCCTGATTAAAAACACACCTGTTCCCGC

[0407] GGATCCTCCGACCACCTTCAATCAGGCCAAGCTGGCTTCTTTCATCACGCAGTACAGTACCGGC CAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAACGCTGGAACCCAGAG ATTCAGTACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAATACTGAGGGTACT TATTCCGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA (SEQ ID NO: 11)

[0408] An exemplary reference sequence of an externally engineered capsid (EEC) AAV2 variant identified from the ocular literature, polypeptide of SEQ ID NO: 24 is as follows:

[0409] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEH DKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSP VEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSS GNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLI NNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQY GYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNT PSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMAS HKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPARQA ATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAK FASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL ( SEQ ID NO : 24 )

[0410] An example nucleic acid sequence encoding SEQ ID NO: 24 is SEQ ID NO: 25:

[0411] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGT GGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCA GGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGC CGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGAC AGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAA GAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTG AACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGC ACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAA AAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACA GCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAAT GGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTC

[0412] CACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAA CAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCT ACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGG CAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACAT TCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACG GTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGAT GCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAA CGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGC GTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCT CACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAG AACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGT

[0413] GACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAA

[0414] NAI-5007748234vl AGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAAT GGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAG TTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACAT TGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAG TATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACTTGGCTCTTGGGGAAACTACGCGTCCTG CAAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTACCAGGCATGGTCTGGCAGGA CAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCAC CCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACAC CCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGT ACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCT GGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGAC ACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA (SEQ ID NO: 25)

[0415] In some embodiments, described herein are capsid polypeptides, e.g., as described in Table 1, that when included in a virus particle comprising a payload, provide increased delivery of such payload to one or more tissues or cell types of the eye (such as, for example, the neural retina, the macula, and / or the choroid / RPE), e.g., after intravitreal administration, relative to an otherwise identical virus particle comprising the capsid polypeptides of SEQ ID NO: 1 or the externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24. In some embodiments, described herein are capsid polypeptides, e.g., as described in Table 1, that when included in a virus particle comprising a payload, provide increased delivery of such payload to one or more tissues or cell types of the eye (such as, for example, tissues or cells of the trabecular meshwork and Schl emm’s canal), e.g., after intravitreal or intracameral administration, relative to an otherwise identical virus particle comprising the capsid polypeptides of SEQ ID NO: 1 or the externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24.

[0416] The present disclosure refers to structural capsid proteins (including VP1, VP2 and VP3) which are encoded by capsid (Cap) genes. These capsid proteins form an outer protein structural shell (i.e., capsid) of a viral vector such as AAV. VP capsid proteins synthesized from Cap polynucleotides generally include a methionine as the first amino acid in the polypeptide sequence (Metl), which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for a first-methionine (Metl) residue or generally any first amino acid (AA1) to be cleaved off after or during polypeptide synthesis by protein processing enzymes such as Met-aminopeptidases. This “Met / AA-clipping” process often correlates with a corresponding acetylation of the second amino acid in the polypeptide sequence (e.g., alanine, valine, serine, threonine, etc.). Met-

[0417] NAI-5007748234vl clipping commonly occurs with VP1 and VP3 capsid proteins but can also occur with VP2 capsid proteins. Where the Met / AA-clipping is incomplete, a mixture of one or more (one, two or three) VP capsid proteins comprising the viral capsid can be produced, some of which include a Metl / AAl amino acid (Met+ / AA+) and some of which lack a Metl / AAl amino acid as a result of Met / AA-clipping (Met- / AA-). For further discussion regarding Met / AA- clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods.2017 Oct.28(5):255-267; Hwang, et al. N- Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 February 19.327(5968): 973-977; the contents of which are each incorporated herein by reference in its entirety. According to the present disclosure, references to capsid polypeptides is not limited to either clipped (Met- / AA-) or unclipped (Met+ / AA+) and, in context, also refer to independent capsid polypeptides, viral capsids comprised of a mixture of capsid proteins, and / or polynucleotide sequences (or fragments thereof) which encode, describe, produce or result in capsid polypeptides of the present disclosure. A direct reference to a “capsid polypeptide” (such as VP1, VP2 or VP3) also comprise VP capsid proteins which include a Metl / AAl amino acid (Met+ / AA+) as well as corresponding VP capsid polypeptide which lack the Metl / AAl amino acid e.g. as a result of Met / AA-clipping (Met- / AA-). Further according to the present disclosure, a reference to a specific SEQ ID NO: (whether a protein or nucleic acid) which comprises or encodes, respectively, one or more capsid polypeptides which include a Metl / AAl amino acid (Met+ / AA+) should be understood to teach the VP capsid polypeptides which lack the Metl / AAl amino acid as upon review of the sequence, it is readily apparent that the first listed amino acid (whether or not Metl / AAl) may be absent. As a non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes a “Metl” amino acid (Met+) encoded by the AUG / ATG start codon is also understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “Metl” amino acid (Met-) of the 736 amino acid Met+ sequence. As a second non-limiting example, reference to a VP1 polypeptide sequence which is 736 amino acids in length and which includes an “AA1” amino acid (AA1+) encoded by any NNN initiator codon can also be understood to teach a VP1 polypeptide sequence which is 735 amino acids in length and which does not include the “AA1” amino acid (AA1-) of the 736 amino acid AA1+ sequence. References to viral capsids formed from VP capsid proteins (such as reference to specific AAV capsid serotypes), can incorporate VP capsid proteins which include a Metl / AAl NAI-5007748234vl amino acid (Met+ / AA1+), corresponding VP capsid proteins which lack the Metl / AAl amino acid e.g. as a result of Met / AA1 -clipping (Met- / AA1-), and combinations thereof (Met+ / AA1+ and Met- / AA1-). As a non-limiting example, an AAV capsid serotype can include VP1 (Met+ / AA1+), VP1 (Met- / AA1-), or a combination of VP1 (Met+ / AA1+) and VP1 (Met- / AA1-). An AAV capsid serotype can also include VP3 (Met+ / AA1+), VP3 (Met- / AA1-), or a combination of VP3 (Met+ / AA1+) and VP3 (Met- / AA1-); and can also include similar optional combinations of VP2 (Met+ / AA1) and VP2 (Met- / AA1-).

[0418] In some embodiments, the reference AAV capsid sequence comprises an amino acid sequence with 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%, or 100% identity to any of the those described above.

[0419] In some embodiments, the reference AAV capsid sequence is encoded by a nucleotide sequence with 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%, or 100% identity to any of those described above. In certain embodiments, the reference sequence is not an AAV capsid sequence and is instead a different vector (e.g., lentivirus, plasmid, etc.).

[0420] In some embodiments, a nucleic acid of the disclosure (e.g., encoding an AAV2 variant capsid protein) comprises conventional control elements or sequences which are operably linked to the nucleic acid molecule in a manner which permits transcription, translation and / or expression in a cell transfected with the nucleic acid (e.g., a plasmid vector comprising said nucleic acid) or infected with a virus comprising said nucleic acid. As used herein, “operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0421] Expression control sequences include efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; appropriate transcription initiation, termination, promoter and enhancer sequences; sequences that stabilize cytoplasmic mRNA; sequences that enhance protein stability; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); and in some embodiments, sequences that enhance secretion of the

[0422] NAI-5007748234vl encoded transgene product. Expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized with the compositions and methods disclosed herein.

[0423] In some embodiments, the native promoter for the transgene may be used. Without wishing to be bound by theory, the native promoter may mimic native expression of the transgene, or provide temporal, developmental, or tissue-specific expression, or expression in response to specific transcriptional stimuli. In some embodiment, the transgene may be operably linked to other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences, e.g., to mimic the native expression.

[0424] In some embodiments, the transgene is operably linked to a tissue-specific promoter.

[0425] In some embodiments, a vector, e.g., a plasmid, carrying a transgene may also include a selectable marker or a reporter gene. Such selectable reporters or marker genes can be used to signal the presence of the vector, e.g., plasmid, in bacterial cells. Other components of the vector, e.g., plasmid, may include an origin of replication. Selection of these and other promoters and vector elements are conventional, and many such sequences are available (see, e.g., Sambrook et al, and references cited therein).

[0426] Viral Particles

[0427] The disclosure is also directed, in part, to a viral particle, such as but not limited to, a dependoparvovirus particle (e.g., a functional dependoparvovirus particle) comprising a nucleic acid or polypeptide described herein or produced by a method described herein.

[0428] Dependoparvovirus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided, e.g., by a co-infecting helper virus. Several species of dependoparvovirus are known, including dependoparvovirus A and dependoparvovirus B, which include serotypes known in the art as adeno-associated viruses (AAV). At least thirteen serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, Handbook of Parvoviruses, Vol. 1, pp. 169-228 (1989), and Berns, Virology, pp. 1743-1764, Raven Press, (New York, 1990). AAV serotypes, and to a degree, dependoparvovirus species, are significantly interrelated structurally and functionally. (See, for example, Blacklowe, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed. (1988); and Rose, Comprehensive Virology 3: 1-61

[0429] NAI-5007748234vl (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins. In addition, heteroduplex analysis reveals extensive cross-hybridization between serotypes along the length of the genome, further suggesting interrelatedness. Dependoparvoviruses genomes also comprise self-annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs).

[0430] The genomic organization of naturally occurring dependoparvoviruses, e.g., AAV serotypes, is very similar. For example, the genome of AAV is a linear, single-stranded DNA molecule that is approximately 5,000 nucleotides (nt) in length or less. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non-structural replication (Rep) proteins and the structural capsid (Cap) proteins. Three different viral particle (VP) proteins form the capsid. The terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. The Rep genes encode the Rep proteins: Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the pl9 promoter. The cap genes encode the VP proteins, VP1, VP2, and VP3. The cap genes are transcribed from the p40 promoter.

[0431] In some embodiments, a dependoparvovirus particle of the disclosure comprises a nucleic acid comprising a capsid polypeptide provided for herein. In some embodiments, the particle comprises a polypeptide as provided for herein.

[0432] In some embodiments, the dependoparvovirus particle of the disclosure may be an AAV2 particle or a variant thereof. In some embodiments, the AAV2 particle comprises a capsid polypeptide as provided for herein or a nucleic acid molecule encodes the same.

[0433] In some embodiments the dependoparvovirus particle comprises a capsid comprising a variant capsid polypeptide described herein. In some embodiments, the dependoparvovirus particle comprises variant capsid polypeptide described herein and a nucleic acid molecule. In some embodiments, the dependoparvovirus particle comprises variant capsid polypeptide described herein and a nucleic acid molecule comprising one or more inverted terminal repeat sequences (ITRs), for example, ITRs derived from an AAV2 dependoparvovirus, one or more regulatory elements (for example, a promoter), and a payload (e.g., as described herein). In some embodiments, at least one of the ITRs is modified. In some embodiments, the nucleic

[0434] NAI-5007748234vl acid molecule is single-stranded. In some embodiments, the nucleic acid molecule is self- complementary.

[0435] In some embodiments, the viral particle comprises a variant capsid polypeptide such as those provided herein. In some embodiments, the viral particle comprises a variant capsid polypeptide having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence as provided in Table 1. In some embodiments, the viral particle comprises a variant capsid polypeptide having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to to the amino acid sequence of positions 1-744 of SEQ ID NO: 13 (VP1), positions 138-744 of SEQ ID NO: 13 (VP2), or positions 203-744 of SEQ ID NO: 13 (VP3).

[0436] In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 13.

[0437] Increased Ocular Transduction Characteristics

[0438] The disclosure is directed, in part, to nucleic acids, polypeptides, cells, cell free systems, translation systems, viral particles, and methods associated with making the same to produce virus particles that have increased ocular transduction as compared to a virus particle having capsid polypeptides of a reference sequence, e.g., with a wild-type sequence of SEQ ID NO: 1 or with an externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24. In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the eye, and, therefore, expression of the transgene in the eye. In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the retina, and, therefore, expression of the transgene in the retina. In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the non-macular retina, and, therefore, expression of the transgene in the non- macular retina. In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the macula, and, therefore, expression of the transgene in the macula. In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the trabecular meshwork, and, therefore, expression of the transgene in the trabecular meshwork. In some embodiments, a use of a viral particle comprising the variant NAI-5007748234vl capsid polypeptide leads to increased ocular transduction of a transgene in the parafovea. In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the parafoveal nerve fibers. In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the peripheral nerve fibers.

[0439] In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the front third of the eye, which includes the structures in front of the vitreous humor. Examples of structures in front of the vitreous humor, include the cornea, iris, ciliary body, lens, trabecular meshwork, and Schlemm’s canal. Accordingly, in some embodiments, use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm’s canal, or any combination thereof. In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene posterior to the lens, such as in the anterior hyaloid membrane and all of the optical structures behind it, such as the vitreous humor, retina, choroid or optic nerve, or any combination thereof. Accordingly, in some embodiments, use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the anterior hyaloid membrane and all of the optical structures behind it, such as the vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, a use of a viral particle comprising the variant capsid polypeptide leads to increased ocular transduction of a transgene in the front third of the eye and posterior to the lens.

[0440] In some embodiments, the increase in ocular transduction is about 1.5 time to about 72 times better (e.g., about 1.5 times better, e.g., about 10 times, e.g., about 20 times, e.g., about 30 times, e.g., about 40 times, e.g., about 50 times, e.g., about 60 times, e.g., about 72 times better) than a virus particle having a reference sequence capsid polypeptide, e.g., having the wild-type capsid polypeptide of SEQ ID NO: 1 or the externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24.

[0441] In some embodiments, a virus particle comprising the variant capsid polypeptide has increased ocular transduction of a transgene in the front third of the eye, which includes the structures in front of the vitreous humor, such as the structures described herein, including, but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm’s

[0442] NAI-5007748234vl canal, or any combination thereof. In some embodiments, a virus particle comprising the variant capsid polypeptide has increased ocular transduction of a transgene posterior to the lens, such as in the anterior hyaloid membrane and all of the optical structures behind it, such as the vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 7-fold, at least 10-fold, at least 11 -fold, at least 15-fold, at least 15-fold, at least 21 -fold, at least 24-fold, at least 26-fold, at least 38-fold, or at least 39-fold over the transduction of a virus particle comprising capsid polypeptides of a reference sequence, e.g., SEQ ID NO: 1 or SEQ ID NO: 24. In some embodiments, the increased transduction is measured as described in the Examples, e.g., by NGS sequencing of viral RNA in cells of the target tissue. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracameral injection.

[0443] In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the eye as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the retina as compared to a viral particle with a reference capsid polypeptide, for example, with the wildtype capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the non-macular retina as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the macula as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the trabecular meshwork as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the parafovea as compared to

[0444] NAI-5007748234vl a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the parafoveal nerve fibers as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the peripheral nerve fibers as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the macula relative to retina as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the macula relative to non- macular retina as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the macula relative to trabecular meshwork as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the macula relative to non- macular retina and trabecular meshwork as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the macula relative to retina and trabecular meshwork as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the variant capsid polypeptide present in a viral particle increases transduction in the retina relative to macula and trabecular meshwork as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24).

[0445] NAI-5007748234vl In some embodiments, the variant capsid polypeptide present in a viral particle increases ocular transduction at least 1.5-fold to at least 72-fold, e.g., as compared to a viral particle with a reference capsid polypeptide, for example, with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24).

[0446] In some embodiments, the increased ocular transduction is measured in the trabecular meshwork, e.g., as shown in Tables 3A and 3B.

[0447] In some embodiments, the variant capsid polypeptide is an isolated or purified polypeptide (e.g., isolated or purified from a cell, other biological component, or contaminant). In some embodiments, the variant polypeptide is present in a dependoparvovirus particle, e.g., described herein. In some embodiments, the variant capsid polypeptide is present in a cell, cell-free system, or translation system, e.g., described herein.

[0448] In some embodiments, the cell is a non-human cell. In other embodiments, the cell is not a human pluripotent stem cell, e.g. it is not a human embryonic stem cell.

[0449] In some embodiments, the variant capsid polypeptide is present in a dependoparvovirus (e.g., AAV2) particle.

[0450] In some embodiments, a dependoparvovirus particle comprises an amino acid sequence that has at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the amino acid sequences provided for herein (e.g., SEQ ID NO: 13). In some embodiments, the variant capsid polypeptide comprises an amino acid sequence that differs by no more than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids from the amino acid sequence of a variant capsid polypeptide provided for herein.

[0451] In some embodiments, the additional alteration improves a production characteristic of a viral particle, such as but not limited to, a dependoparvovirus particle or method of making the same. In some embodiments, the additional alteration improves or alters another characteristic of a viral particle, such as but not limited to, a dependoparvovirus particle, e.g., tropism.

[0452] In some embodiments, the improved transduction is as measured by quantification of viral RNA from the target tissue. In some embodiments, the improved biodistribution is as measured by quantification of viral DNA from the target tissue. In some embodiments, the

[0453] NAI-5007748234vl improved transduction is as measured following production from HEK293 cells, for example as described in the Examples.

[0454] Table 3A and Table 3B, list information regarding exemplary variant dependoparvovirus particles comprising a variant capsid (e.g., Variant 1), and describing the ocular transduction properties and production characteristics of said non-limiting exemplary variants. Table 3 A and Table 3B represent data produced in a first (Table 3 A) high throughput experiment (Library Experiment 1) and second (Table 3B) high throughput experiment (Library Experiment 2) showing transduction and virus production of exemplary variant dependoparvovirus particles comprising a variant capsid polypeptide (e.g., Variant 1). Injection route is by intravitreal injection, as described in the Examples.

[0455] Trabecular Meshwork Transduction refers to transduction of tissue samples collected from the trabecular meshwork and / or Schlemm’s canal. Macular Neural Retina Transduction refers to transduction of tissue samples collected from the neural retina layer of the macula. Non-Macul ar Neural Retina Transduction refers to transduction of tissue samples collected from the neural retina layer of the retina excluding the macula, which may also be referred to herein as the “non-macular” region. Neural Retina Transduction refers to transduction of tissue samples collected from the macular and non-macular regions of the neural retina layer. Macular Choroid Transduction refers to transduction of tissue samples collected from the macular region of the choroid and / or retinal pigment epithelium (RPE). Non-Macular Choroid Transduction refers to transduction of tissue samples collected from the non-macular region of the choroid and / or RPE. Choroid Transduction refers to transduction of tissue samples collected from the macular and non-macular regions of the choroid and / or RPE. Measurements are made relative to wild-type AAV2 (SEQ ID NO: 1).

[0456] Table 3A

[0457] NAI-5007748234vl Table 3B

[0458] In some embodiments, the capsid polypeptide present in a viral particle increases transduction without increasing the biodistribution of the variant capsid polypeptide in the eye relative to SEQ ID NO: 1. In some embodiments, the capsid polypeptide present in a viral particle increases transduction without increasing the biodistribution of the variant capsid polypeptide in the retina relative to SEQ ID NO: 1. In some embodiments, the capsid polypeptide present in a viral particle increases transduction without increasing the biodistribution of the variant capsid polypeptide in the trabecular meshwork relative to SEQ ID NO: 1.

[0459] Tables 4A and 4B list information regarding biodistribution of variant dependoparvovirus particles comprising capsid polypeptides (e.g., Variant Qin the different layers, structures, and / or parts of the eye. Table 4A include biodistribution data measured for Library Experiment 1. Table 4B include biodistribution data measured for Library Experiment 2. Macular Choroid Biodistribution refers to biodistribution of tissue samples collected from the macular region of the choroid and / or retinal pigment epithelium (RPE). Non-Macular Choroid Biodistribution refers to biodistribution of tissue samples collected from the non-macular region of the choroid and / or RPE. Choroid Biodistribution refers to biodistribution of tissue samples collected from the macular and non-macular regions of the choroid and / or RPE. Trabecular Meshwork Biodistribution refers to biodistribution of tissue samples collected from the trabecular meshwork and / or Schlemm’s canal. Macular Neural Retina Biodistribution refers to biodistribution of tissue samples collected from the neural retina layer of the macula. Non-Macular Neural Retina Biodistribution refers to biodistribution of tissue samples collected from the non-macular region of the neural retina

[0460] NAI-5007748234vl layer. Neural Retina Biodistribution refers to biodistribution of tissue samples collected from the macular and non-macular regions of the neural retina layer.

[0461] Table 4A Table 4B

[0462] Category A (Neural Retina Transduction): In some aspects of the disclosure, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased neural retina transduction as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased neural retina transduction is as defined as any one of embodiments A-l through A-35.

[0463] Embodiment A-l : In an embodiment of Category A, the transduction is about (or at least about) 1.5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0464] Embodiment A-2: In an embodiment of Category A, the transduction is about (or at least about) 2 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0465] NAI-5007748234vl Embodiment A- 3 : In an embodiment of Category A, the transduction is about (or at least about) 3 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0466] Embodiment A-4: In an embodiment of Category A, the transduction is about (or at least about) 4 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0467] Embodiment A-5: In an embodiment of Category A, the transduction is about (or at least about) 5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0468] Embodiment A-6: In an embodiment of Category A, the transduction is about (or at least about) 7 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0469] Embodiment A-7: In an embodiment of Category A, the transduction is about (or at least about) 9 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0470] Embodiment A-8: In an embodiment of Category A, the transduction is about (or at least about) 11 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0471] Embodiment A-9: In an embodiment of Category A, the transduction is about (or at least about) 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0472] Embodiment A-10: In an embodiment of Category A, the transduction is about (or at least about) 16 times better relative to a virus particle comprising a variant capsid polypeptide

[0473] NAI-5007748234vl having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0474] Embodiment A-l 1 : In an embodiment of Category A, the transduction is about (or at least about) 18 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0475] Embodiment A-12: In an embodiment of Category A, the transduction is about (or at least about) 19 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0476] Embodiment A- 13 In an embodiment of Category A, the transduction is about (or at least about) 20 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0477] Embodiment A-14: In an embodiment of Category A, the transduction is about (or at least about) 21 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0478] Embodiment A-l 5: In an embodiment of Category A, the transduction is about (or at least about) 24 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0479] Embodiment A-16: In an embodiment of Category A, the transduction is about (or at least about) 25 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0480] Embodiment A- 17: In an embodiment of Category A, the transduction is about (or at least about) 26 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0481] NAI-5007748234vl Embodiment A-18: In an embodiment of Category A, the transduction is about (or at least about) 27 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0482] Embodiment A-19: In an embodiment of Category A, the transduction is about (or at least about) 28 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0483] Embodiment A-20: In an embodiment of Category A, the transduction is about (or at least about) 29 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0484] Embodiment A-21 : In an embodiment of Category A, the transduction is about (or at least about) 30 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0485] Embodiment A-22: In an embodiment of Category A, the transduction is about (or at least about) 35 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0486] Embodiment A-23: In an embodiment of Category A, the transduction is about (or at least about) 36 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0487] Embodiment A-24: In an embodiment of Category A, the transduction is about (or at least about) 37 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0488] Embodiment A-25: In an embodiment of Category A, the transduction is about (or at least about) 38 times better relative to a virus particle comprising a variant capsid polypeptide

[0489] NAI-5007748234vl having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0490] Embodiment A-26: In an embodiment of Category A, the transduction is about (or at least about) 39 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0491] Embodiment A-27: In an embodiment of Category A, the transduction is about (or at least about) 42 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0492] Embodiment A-28: In an embodiment of Category A, the transduction is about (or at least about) 45 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0493] Embodiment A-29: In an embodiment of Category A, the transduction is about (or at least about) 47 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0494] Embodiment A-30: In an embodiment of Category A, the transduction is about (or at least about) 49 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0495] Embodiment A- 31 : In an embodiment of Category A, the transduction is about (or at least about) 51 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0496] Embodiment A-32: In an embodiment of Category A, the transduction is about (or at least about) 58 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0497] NAI-5007748234vl Embodiment A-33: In an embodiment of Category A, the transduction is about (or at least about) 59 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0498] Embodiment A-34: In an embodiment of Category A, the transduction is about (or at least about) 61 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0499] Embodiment A-35: In an embodiment of Category A, the transduction is about (or at least about) 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0500] In some embodiments, the improved transduction is in a range bounded by any two values set forth in embodiments A-l to A-35. Exemplary ranges are set forth in embodiments A-36 to A-69 below.

[0501] Embodiment A-36: In an embodiment of Category A, the transduction ranges are between about 1.5 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0502] Embodiment A-37: In an embodiment of Category A, the transduction ranges are between about 2 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0503] Embodiment A-38: In an embodiment of Category A, the transduction ranges are between about 3 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0504] Embodiment A-39: In an embodiment of Category A, the transduction ranges are between about 4 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0505] NAI-5007748234vl Embodiment A-40: In an embodiment of Category A, the transduction ranges are between about 5 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0506] Embodiment A-41 : In an embodiment of Category A, the transduction ranges are between about 7 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0507] Embodiment A-42: In an embodiment of Category A, the transduction ranges are between about 9 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0508] Embodiment A-43 : In an embodiment of Category A, the transduction ranges are between about 11 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0509] Embodiment A-44: In an embodiment of Category A, the transduction ranges are between about 15 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0510] Embodiment A-45: In an embodiment of Category A, the transduction ranges are between about 16 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0511] Embodiment A-46: In an embodiment of Category A, the transduction ranges are between about 18 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0512] Embodiment A-47: In an embodiment of Category A, the transduction ranges are between about 19 and about 65 times better relative to a virus particle comprising a variant

[0513] NAI-5007748234vl capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0514] Embodiment A-48: In an embodiment of Category A, the transduction ranges are between about 20 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0515] Embodiment A-49: In an embodiment of Category A, the transduction ranges are between about 21 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0516] Embodiment A-50: In an embodiment of Category A, the transduction ranges are between about 24 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0517] Embodiment A- 51 : In an embodiment of Category A, the transduction ranges are between about 25 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0518] Embodiment A-52: In an embodiment of Category A, the transduction ranges are between about 26 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0519] Embodiment A-53: In an embodiment of Category A, the transduction ranges are between about 27 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0520] Embodiment A-54: In an embodiment of Category A, the transduction ranges are between about 28 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0521] NAI-5007748234vl Embodiment A-55: In an embodiment of Category A, the transduction ranges are between about 29 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0522] Embodiment A-56: In an embodiment of Category A, the transduction ranges are between about 30 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0523] Embodiment A-57: In an embodiment of Category A, the transduction ranges are between about 35 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0524] Embodiment A-58: In an embodiment of Category A, the transduction ranges are between about 36 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0525] Embodiment A-59: In an embodiment of Category A, the transduction ranges are between about 37 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0526] Embodiment A-60: In an embodiment of Category A, the transduction ranges are between about 38 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0527] Embodiment A-61 : In an embodiment of Category A, the transduction ranges are between about 39 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0528] Embodiment A-62: In an embodiment of Category A, the transduction ranges are between about 42 and about 65 times better relative to a virus particle comprising a variant

[0529] NAI-5007748234vl capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0530] Embodiment A-63 : In an embodiment of Category A, the transduction ranges are between about 45 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0531] Embodiment A-64: In an embodiment of Category A, the transduction ranges are between about 47 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0532] Embodiment A-65: In an embodiment of Category A, the transduction ranges are between about 49 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0533] Embodiment A-66: In an embodiment of Category A, the transduction ranges are between about 51 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0534] Embodiment A-67: In an embodiment of Category A, the transduction ranges are between about 58 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0535] Embodiment A-68: In an embodiment of Category A, the transduction ranges are between about 59 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0536] Embodiment A-69: In an embodiment of Category A, the transduction ranges are between about 61 and about 65 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0537] NAI-5007748234vl Category B (Choroid Transduction): In some aspects of the disclosure, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased choroid transduction as compared to a viral particle with the wildtype capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased choroid transduction is as defined as any one of embodiments B-l through B-24.

[0538] Embodiment B-l : In an embodiment of Category B, the transduction is about (or at least about) 1.5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0539] Embodiment B-2: In an embodiment of Category B, the transduction is about (or at least about) 2 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0540] Embodiment B-3 : In an embodiment of Category B, the transduction is about (or at least about) 3 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0541] Embodiment B-4: In an embodiment of Category B, the transduction is about (or at least about) 4 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0542] Embodiment B-5: In an embodiment of Category B, the transduction is about (or at least about) 7 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0543] Embodiment B-6: In an embodiment of Category B, the transduction is about (or at least about) 6 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0544] Embodiment B-7: In an embodiment of Category B, the transduction is about (or at least about) 9 times better relative to a virus particle comprising a variant capsid polypeptide

[0545] NAI-5007748234vl having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0546] Embodiment B-8: In an embodiment of Category B, the transduction is about (or at least about) 10 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0547] Embodiment B-9: In an embodiment of Category B, the transduction is about (or at least about) 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0548] Embodiment B-10: In an embodiment of Category B, the transduction is about (or at least about) 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0549] Embodiment B-l 1 : In an embodiment of Category B, the transduction is about (or at least about) 16 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0550] Embodiment B-12: In an embodiment of Category B, the transduction is about (or at least about) 17 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0551] Embodiment B-l 3 : In an embodiment of Category B, the transduction is about (or at least about) 19 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0552] Embodiment B-14: In an embodiment of Category B, the transduction is about (or at least about) 24 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0553] NAI-5007748234vl Embodiment B-15: In an embodiment of Category B, the transduction is about (or at least about) 25 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0554] Embodiment B-16: In an embodiment of Category B, the transduction is about (or at least about) 27 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0555] Embodiment B-17: In an embodiment of Category B, the transduction is about (or at least about) 28 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0556] Embodiment B-18: In an embodiment of Category B, the transduction is about (or at least about) 35 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0557] Embodiment B-19: In an embodiment of Category B, the transduction is about (or at least about) 36 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0558] Embodiment B-20: In an embodiment of Category B, the transduction is about (or at least about) 45 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0559] Embodiment B-21 : In an embodiment of Category B, the transduction is about (or at least about) 47 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0560] Embodiment B-22: In an embodiment of Category B, the transduction is about (or at least about) 48 times better relative to a virus particle comprising a variant capsid polypeptide

[0561] NAI-5007748234vl having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0562] Embodiment B-23 : In an embodiment of Category B, the transduction is about (or at least about) 49 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0563] Embodiment B-24: In an embodiment of Category B, the transduction is about (or at least about) 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0564] In some embodiments, the improved transduction is in a range bounded by any two values set forth in embodiments B-l to B-24. Exemplary ranges are set forth in embodiments B-25 to B-47 below.

[0565] Embodiment B-25: In an embodiment of Category B, the transduction ranges are between about 1.5 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0566] Embodiment B-26: In an embodiment of Category B, the transduction ranges are between about 2 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0567] Embodiment B-27: In an embodiment of Category B, the transduction ranges are between about 3 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0568] Embodiment B-28: In an embodiment of Category B, the transduction ranges are between about 4 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0569] NAI-5007748234vl Embodiment B-29: In an embodiment of Category B, the transduction ranges are between about 6 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0570] Embodiment B-30: In an embodiment of Category B, the transduction ranges are between about 9 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0571] Embodiment B-31 : In an embodiment of Category B, the transduction ranges are between about 10 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0572] Embodiment B-32: In an embodiment of Category B, the transduction ranges are between about 12 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0573] Embodiment B-33: In an embodiment of Category B, the transduction ranges are between about 15 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0574] Embodiment B-34: In an embodiment of Category B, the transduction ranges are between about 16 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0575] Embodiment B-35: In an embodiment of Category B, the transduction ranges are between about 17 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0576] Embodiment B-36: In an embodiment of Category B, the transduction ranges are between about 19 and about 72 times better relative to a virus particle comprising a variant

[0577] NAI-5007748234vl capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0578] Embodiment B-37: In an embodiment of Category B, the transduction ranges are between about 24 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0579] Embodiment B-38: In an embodiment of Category B, the transduction ranges are between about 25 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0580] Embodiment B-39: In an embodiment of Category B, the transduction ranges are between about 27 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0581] Embodiment B-40: In an embodiment of Category B, the transduction ranges are between about 28 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0582] Embodiment B-41 : In an embodiment of Category B, the transduction ranges are between about 29 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0583] Embodiment B-42: In an embodiment of Category B, the transduction ranges are between about 35 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0584] Embodiment B-43 : In an embodiment of Category B, the transduction ranges are between about 36 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0585] NAI-5007748234vl Embodiment B-44: In an embodiment of Category B, the transduction ranges are between about 45 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0586] Embodiment B-45: In an embodiment of Category B, the transduction ranges are between about 47 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0587] Embodiment B-46: In an embodiment of Category B, the transduction ranges are between about 48 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0588] Embodiment B-47: In an embodiment of Category B, the transduction ranges are between about 49 and about 72 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0589] Category C (Trabecular Transduction): In some aspects of the disclosure, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased trabecular transduction as compared to a viral particle with the capsid polypeptide of SEQ ID NO: 1. In some embodiments, the increased trabecular transduction is as defined as any one of embodiment C-l through C-9.

[0590] Embodiment C-l : In an embodiment of Category C, the transduction is about (or at least about) 1.5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0591] Embodiment C-2: In an embodiment of Category C, the transduction is about (or at least about) 2 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0592] Embodiment C-3: In an embodiment of Category C, the transduction is about (or at least about) 3 times better relative to a virus particle comprising a variant capsid polypeptide

[0593] NAI-5007748234vl having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0594] Embodiment C-4: In an embodiment of Category C, the transduction is about (or at least about) 4 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0595] Embodiment C-5: In an embodiment of Category C, the transduction is about (or at least about) 5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0596] Embodiment C-6: In an embodiment of Category C, the transduction is about (or at least about) 6 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0597] Embodiment C-7: In an embodiment of Category C, the transduction is about (or at least about) 8 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0598] Embodiment C-8: In an embodiment of Category C, the transduction is about (or at least about) 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0599] Embodiment C-9: In an embodiment of Category C, the transduction is about (or at least about) 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0600] In some embodiments, the improved transduction is in a range bounded by any two values set forth in embodiments C-l to C-9. Exemplary ranges are set forth in embodiments C-10 to C-17 below.

[0601] NAI-5007748234vl Embodiment C- 10: In an embodiment of Category C, the transduction ranges are between about 1.5 and about 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0602] Embodiment C-l 1 : In an embodiment of Category C, the transduction ranges are between about 2 and about 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0603] Embodiment C- 12: In an embodiment of Category C, the transduction ranges are between about 3 and about 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0604] Embodiment C-13: In an embodiment of Category C, the transduction ranges are between about 4 and about 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0605] Embodiment C- 14: In an embodiment of Category C, the transduction ranges are between about 5 and about 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0606] Embodiment C-l 5: In an embodiment of Category C, the transduction ranges are between about 6 and about 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0607] Embodiment C-l 6: In an embodiment of Category C, the transduction ranges are between about 8 and about 15 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0608] Embodiment C-l 7: In an embodiment of Category C, the transduction ranges are between about 12 and about 15 times better relative to a virus particle comprising a variant

[0609] NAI-5007748234vl capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0610] Category D (Virus Production): In some aspects of the disclosure, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased virus production as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased virus production is as defined as any one of embodiments D-l and D-4.

[0611] Embodiment D-l : In an embodiment of Category D, the virus production is about (or at least about) 1.5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0612] Embodiment D-2: In an embodiment of Category D, the virus production is about (or at least about) 2 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0613] Embodiment D-3 : In an embodiment of Category D, the virus production is about (or at least about) 3 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0614] Embodiment D-4: In an embodiment of Category D, the virus production is about (or at least about) 4 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0615] In some embodiments, the improved transduction is in a range bounded by any two values set forth in embodiments D-l to D-4. Exemplary ranges are set forth in embodiments D-5 to D-7 below.

[0616] Embodiment D-5: In an embodiment of Category D, the production ranges are between about 1.5 and about 4 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0617] NAI-5007748234vl Embodiment D-6: In an embodiment of Category D, the production ranges are between about 2 and about 4 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0618] Embodiment D-7: In an embodiment of Category D, the production ranges are between about 3 and about 4 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0619] Category E (Neural Retina Biodistribution): In some aspects of the disclosure, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased neural retina biodistribution as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased neural retina biodistribution is as defined as any one of embodiments E-l through E-13.

[0620] Embodiment E-l : In an embodiment of Category E, the biodistribution is about (or at least about) 1.5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0621] Embodiment E-2: In an embodiment of Category E, the biodistribution is about (or at least about) 2 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0622] Embodiment E-3 : In an embodiment of Category E, the biodistribution is about (or at least about) 3 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0623] Embodiment E-4: In an embodiment of Category E, the biodistribution is about (or at least about) 4 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0624] NAI-5007748234vl Embodiment E-5: In an embodiment of Category E, the biodistribution is about (or at least about) 3 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0625] Embodiment E-6: In an embodiment of Category E, the biodistribution is about (or at least about) 5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0626] Embodiment E-7: In an embodiment of Category E, the biodistribution is about (or at least about) 6 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0627] Embodiment E-8: In an embodiment of Category E, the biodistribution is about (or at least about) 7 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0628] Embodiment E-9: In an embodiment of Category E, the biodistribution is about (or at least about) 8 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0629] Embodiment E-10: In an embodiment of Category E, the biodistribution is about (or at least about) 9 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0630] Embodiment E-l 1 : In an embodiment of Category E, the biodistribution is about (or at least about) 10 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0631] Embodiment E-12: In an embodiment of Category E, the biodistribution is about (or at least about) 11 times better relative to a virus particle comprising a variant capsid

[0632] NAI-5007748234vl polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0633] Embodiment E-13 : In an embodiment of Category E, the biodistribution is about (or at least about) 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0634] In some embodiments, the improved biodistribution is in a range bounded by any two values set forth in embodiments E-l to E-13. Exemplary ranges are set forth in embodiments E-14 and E-24 below.

[0635] Embodiment E-14: In an embodiment of Category E, the improved biodistribution is in a range between about 1.5 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0636] Embodiment E-l 5: In an embodiment of Category E, the improved biodistribution is in a range between about 2 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0637] Embodiment E-16: In an embodiment of Category E, the improved biodistribution is in a range between about 3 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0638] Embodiment E-l 7: In an embodiment of Category E, the improved biodistribution is in a range between about 4 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0639] Embodiment E-l 8: In an embodiment of Category E, the improved biodistribution is in a range between about 5 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0640] NAI-5007748234vl Embodiment E-19: In an embodiment of Category E, the improved biodistribution is in a range between about 6 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0641] Embodiment E-20: In an embodiment of Category E, the improved biodistribution is in a range between about 7 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0642] Embodiment E-21 : In an embodiment of Category E, the improved biodistribution is in a range between about 8 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0643] Embodiment E-22: In an embodiment of Category E, the improved biodistribution is in a range between about 9 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0644] Embodiment E-23 : In an embodiment of Category E, the improved biodistribution is in a range between about 10 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0645] Embodiment E-24: In an embodiment of Category E, the improved biodistribution is in a range between about 11 and about 12 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0646] Category F (Choroid Biodistribution): In some aspects of the disclosure, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased choroid biodistribution as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased choroid biodistribution is as defined as any one of embodiments F-l through F-5.

[0647] Embodiment F-l : In an embodiment of Category F, the biodistribution is about (or at least about) 1.5 times better relative to a virus particle comprising a variant capsid

[0648] NAI-5007748234vl polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0649] Embodiment F-2: In an embodiment of Category F, the biodistribution is about (or at least about) 2 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0650] Embodiment F-3 : In an embodiment of Category F, the biodistribution is about (or at least about) 3 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0651] Embodiment F-4: In an embodiment of Category F, the biodistribution is about (or at least about) 4 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0652] Embodiment F-5: In an embodiment of Category F, the biodistribution is about (or at least about) 5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0653] In some embodiments, the improved biodistribution is in a range bounded by any two values set forth in embodiments F-l to F-5. Exemplary ranges are set forth in embodiments F-6 and F-8 below.

[0654] Embodiment F-5: In an embodiment of Category F, the improved biodistribution is in a range between about 1.5 and about 5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0655] Embodiment F-6: In an embodiment of Category F, the improved biodistribution is in a range between about 2 and about 5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0656] NAI-5007748234vl Embodiment F-7: In an embodiment of Category F, the improved biodistribution is in a range between about 3 and about 5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0657] Embodiment F-8: In an embodiment of Category F, the improved biodistribution is in a range between about 4 and about 5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0658] Category G (Trabecular Biodistribution): In some aspects of the disclosure, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased trabecular biodistribution as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased trabecular biodistribution is as defined in embodiment G-l.

[0659] Embodiment G-l : In an embodiment of Category G, the biodistribution is about (or at least about) 1.5 times better relative to a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having the wild-type capsid protein, e.g., having capsid polypeptides of SEQ ID NO: 1.

[0660] According to some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of one or more cell types as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, the cell type(s) is an ocular cell type found in the retinal tissue. In some embodiments, the cell type(s) is an ocular cell type found in the trabecula meshwork tissue. Examples of cell types include amacrine cells, bipolar cells, cones, horizontal cells, microglia, Muller glia, retinal ganglion cells, rods, corneal epithelium, ciliary muscle, melanocytes, Schwann cells, beam cells, juxtacanalicular tissue (JCT), fibroblasts, and pericytes.

[0661] In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction in the cells of the ganglion cell layer (GCL) of the retina as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid NAI-5007748234vl polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction in the cells of the inner nuclear layer (INL) of the retina as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction in the cells of the outer nuclear layer (ONL) of the retina as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24).

[0662] In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of retinal ganglion cells as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of amacrine cells as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of bipolar cells as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of cones as compared to a viral particle with the wildtype capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of Muller glia as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of rods as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of beam cells as compared to a viral particle

[0663] NAI-5007748234vl with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of ciliary muscle as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of corneal epithelium as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of juxtacanalicular tissue (JCT) as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of melanocytes as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits increased transduction of Schwann cells as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein, exhibits decreased transduction of ciliary muscle as compared to a viral particle with the wild-type capsid polypeptide (SEQ ID NO: 1) or the externally engineered capsid (EEC) polypeptide (SEQ ID NO: 24).

[0664] In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variants 1-5), exhibits preferential transduction in cells of the inner nuclear layer (INL) of retinae (e.g., human retina), relative to transduction of the same viral particle comprising the variant capsid polypeptide in cells of the outer nuclear layer (ONL) of retinae (e.g., human retina). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variants 1-5), exhibits preferential transduction in cells of the inner nuclear layer (INL) of retinae (e.g., human retina), relative to transduction of the same viral

[0665] NAI-5007748234vl particle comprising the variant capsid polypeptide in cells of the outer nuclear layer (ONL) of retinae (e.g., human retina), if administered by intravitreal injection.

[0666] In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variants 1-5), exhibits preferential transduction in cells of the INL of the retina of a mouse, relative to transduction of the same viral particle comprising the variant capsid polypeptide in cells of the ONL of the retina of a mouse. In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variants 1-5), exhibits preferential transduction in cells of the INL of the retina of a non-human primate (NHP), relative to transduction of the same viral particle comprising the variant capsid polypeptide in cells of the ONL of the retina of an NHP.

[0667] In some embodiments, the transduction in cells of the INL of a retina is at least 1.1- fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, or at least 4-fold over the transduction of the same viral particle comprising the variant capsid polypeptide in cells of the ONL of a retina.

[0668] In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variants 1-5), exhibits preferential transduction in cells of the inner nuclear layer (INL) of retinae (e.g., human retina), relative to transduction of the same viral particle comprising the variant capsid polypeptide in cells of the ganglion cell layer (GCL) of retinae (e.g., human retina). In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variants 1-5), exhibits preferential transduction in cells of the inner nuclear layer (INL) of retinae (e.g., human retina), relative to transduction of the same viral particle comprising the variant capsid polypeptide in cells of the ganglion cell layer (GCL) of retinae (e.g., human retina), if administered by intravitreal injection.

[0669] In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variants 1-5), exhibits preferential transduction in cells of the INL of the retina of a mouse, relative to transduction of the same viral particle comprising the variant capsid polypeptide in cells of the GCL of the retina of a mouse. In some embodiments, a viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variants 1-5), exhibits preferential transduction in cells of the INL of the retina of a non-human primate (NHP), relative to

[0670] NAI-5007748234vl transduction of the same viral particle comprising the variant capsid polypeptide in cells of the GCL of the retina of an NHP.

[0671] In some embodiments, the transduction in cells of the INL of a retina is at least 1.1- fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 2-fold, or at least 3-fold over the transduction of the same viral particle comprising the variant capsid polypeptide in cells of the GCL of a retina.

[0672] In some embodiments, the increased transduction is measured as described in Example 3 and Example 4, e.g., using fluorescent reporters (e.g., GFP or mCherry) and immunofluorescence staining of tissue prepared for histology. In some embodiments, the viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variants 1) is produced as described in the Example 3 and Example 4, to carry a fluorescent reporter transgene (e.g., GFP or mCherry) with a nuclear localization signal (NLS) tag. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, ocular tissue is collected, after intravitreal administration of the viral particle comprising the variant capsid polypeptide, e.g., the variant capsid polypeptide described herein (e.g., Variant 1) that contains a fluorescent reporter (e.g., GFP or mCherry), and is histologically processed in tissue sections and stained for fluorescent markers (e.g., GFP or mCherry). In some embodiments, the stained tissue sections are segmented into three different retinal layers including the ganglion cell layer (GCL), inner nuclear layer (INL) and outer nuclear layer (ONL), using an Al-trained biodock self-trained model (htp s : / / w w w .bi od ock . ai / ) . In some embodiments, the cells positive for fluorescent staining (e.g., GFP or mCherry) are quantified using a custom QuPATH script and averages are reported for each retinal layer (e.g., GCL, INL, or ONL).

[0673] Methods of Making Compositions Described Herein

[0674] The disclosure is directed, in part, to a method of making a capsid polypeptide described herein or a virus particle, such as but not limited to a dependoparvovirus particle, e.g., a dependoparvovirus particle described herein. In some embodiments, a method of making dependoparvovirus particle comprises providing a cell, cell-free system, or other translation system, comprising a nucleic acid described herein encoding a variant capsid polypeptide provided for herein, or a polypeptide provided for herein (e.g., a variant capsid polypeptide); and cultivating the cell, cell-free system, or other translation system under NAI-5007748234vl conditions suitable for the production of the dependoparvovirus particle, thereby making the dependoparvovirus particle.

[0675] In some embodiments, providing a cell comprising a nucleic acid described herein comprises introducing the nucleic acid to the cell, e.g., transfecting or transforming the cell with the nucleic acid. The nucleic acids of the disclosure may be situated as a part of any genetic element (vector) which may be delivered to a host cell, e.g., naked DNA, a plasmid, phage, transposon, cosmid, episome, a protein in a non-viral delivery vehicle (e.g., a lipid- based carrier), virus, etc. which transfer the sequences carried thereon. Such a vector may be delivered by any suitable method, including transfection, liposome delivery, electroporation, membrane fusion techniques, viral infection, high velocity DNA- coated pellets, and protoplast fusion. A person of skill in the art possesses the knowledge and skill in nucleic acid manipulation to construct any embodiment of this invention and said skills include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0676] In some embodiments, a vector of the disclosure comprises sequences encoding a viral particle, such as but not limited to, a dependoparvovirus variant capsid polypeptide as provided for herein or a fragment thereof. In some embodiments, vectors of the disclosure comprises sequences encoding a viral particle, such as but not limited to, a dependoparvovirus rep protein or a fragment thereof. In some embodiments, such vectors may contain sequence encoding both dependoparvovirus cap (e.g., a variant capsid polypeptide described herein) and rep proteins. In vectors in which both AAV rep and cap are provided, the dependoparvovirus rep and dependoparvovirus cap sequences may both be of the same dependoparvovirus species or serotype origin, such as AAV2. Alternatively, the present disclosure also provides vectors in which the rep sequences are from a dependoparvovirus species or serotype which differs from that from which the cap sequences are dervied. In some embodiments, the rep and cap sequences are expressed from separate sources (e.g., separate vectors, or a host cell genome and a vector). In some embodiments, the rep sequences are fused in frame to cap sequences of a different dependoparvovirus species or serotype to form a chimeric dependoparvovirus vector. In some embodiments, the vectors of the invention further contain a payload, e.g., a minigene comprising a selected transgene (e.g., a payload as described herein), e.g., flanked by dependoparvovirus 5' ITR and dependoparvovirus 3' ITR.

[0677] NAI-5007748234vl The vectors described herein, e.g., a plasmid, are useful for a variety of purposes, but are particularly well suited for use in production of recombinant viral particles, such as but not limited to, dependoparvovirus particles comprising dependoparvovirus sequences or a fragment thereof, and in some embodiments, a payload.

[0678] In some embodiments, the disclosure provides a method of making a viral particle, such as but not limited to, a dependoparvovirus particle (e.g., a dependoparvovirus B particle, e.g., an AAV2 particle or particle comprising a variant capsid polypeptide as described herein), or a portion thereof. In some embodiments, the method comprises culturing a host cell which contains a nucleic acid sequence encoding a dependoparvovirus variant capsid polypeptide as provided for herein, or fragment thereof; a functional rep gene; a payload (e.g., as described herein), e.g., a minigene comprising dependoparvovirus inverted terminal repeats (ITRs) and a transgene, optionally under the control of a regulatory element such as a promoter; and sufficient helper functions to promote packaging of the payload, e.g., minigene, into the dependoparvovirus capsid. The components necessary to be cultured in the host cell to package a payload, e.g., minigene, in a dependoparvovirus capsid may be provided to the host cell in trans. In some embodiments, any one or more of the required components (e.g., payload (e.g., minigene), rep sequences, cap sequences, and / or helper functions) may be provided by a host cell which has been engineered to stably comprise one or more of the required components using methods known to those of skill in the art. In some embodiments, a host cell which has been engineered to stably comprise the required component s) comprises it under the control of an inducible promoter. In some embodiments, the required component may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein and further examples are known to those of skill in the art. In some embodiments, a selected host cell which has been engineered to stably comprise one or more components may comprise a component under the control of a constitutive promoter and another component under the control of one or more inducible promoters. For example, a host cell which has been engineered to stably comprise the required components may be generated from 293 cells (e.g., which comprise helper functions under the control of a constitutive promoter), which comprises the rep and / or cap proteins under the control of one or more inducible promoters.

[0679] The payload (e.g., minigene), rep sequences, cap sequences, and helper functions required for producing a viral particle, such as but not limited to, a dependoparvovirus particle of the disclosure may be delivered to the packaging host cell in the form of any

[0680] NAI-5007748234vl genetic element which transfers the sequences carried thereon (e.g., in a vector or combination of vectors). The genetic element may be delivered by any suitable method, including those described herein. Methods used to construct genetic elements, vectors, and other nucleic acids of the disclosure are known to those with skill and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present invention. See, e.g., K. Fisher et al, J. Virol, 70:520-532 (1993) and US Patent 5,478,745. Unless otherwise specified, the dependoparvovirus ITRs, and other selected dependoparvovirus components described herein, may be readily selected from among any dependoparvovirus species and serotypes, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV9. ITRs or other dependoparvovirus components may be readily isolated using techniques available to those of skill in the art from a dependoparvovirus species or serotype. Dependoparvovirus species and serotypes may be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, VA). In some embodiments, the dependoparvovirus sequences may be obtained through synthetic or other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank or PubMed.

[0681] The viral particles, such as but not limited to, dependoparvovirus particles (e.g., including a variant capsid polypeptide and, for example, a payload) of the disclosure may be produced using any invertebrate cell type which allows for production of dependoparvovirus or biologic products and which can be maintained in culture. In some embodiments, an insect cell may be used in production of the compositions described herein or in the methods of making a dependoparvovirus particle described herein. For example, an insect cell line used can be from Spodoptera frugiperda, such as Sf9, SF21, SF900+, drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g. Bombyxmori cell lines, Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such s Ascalapha odorata cell lines. In some embodiments, the insect cells are susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCRl, SP900+, Sf2I, BTLTN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5 and Ao38.

[0682] In some embodiments, the methods of the disclosure can be carried out with any mammalian cell type which allows for replication of dependoparvovirus or production of

[0683] NAI-5007748234vl biologic products, and which can be maintained in culture. In some embodiments, the mammalian cells used can be HEK293, HEK293T, HeLa, CHO, NSO, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19 or MRC-5 cells. In some embodiments the culture is an adherent cell culture. In some embodiments, the culture is a suspension cell culture.

[0684] Methods of expressing proteins (e.g., recombinant or heterologous proteins, e.g., viral polypeptides, such as but not limited to, dependoparvovirus polypeptides) in insect cells are well documented, as are methods of introducing nucleic acids, such as vectors, e.g., insectcell compatible vectors, into such cells and methods of maintaining such cells in culture. See, for example, METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, N J (1995); O'Reilly et al., BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et al., J. Vir. 63:3822-8 (1989); Kajigaya et al., Proc. Nat'l. Acad. Sci. USA 88:4646-50 (1991); Ruffing et al., J. Vir. 66:6922-30 (1992); Kirnbauer et al., Vir. 219:37-44 (1996); Zhao et al., Vir. 272:382-93 (2000); and Samulski et al., U.S. Pat. No. 6,204,059. In some embodiments, a nucleic acid construct encoding dependoparvovirus polypeptides (e.g., a dependoparvovirus genome) in insect cells is an insect cell-compatible vector. An “insect cell-compatible vector” as used herein refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be employed as long as it is insect cell-compatible. The vector may integrate into the insect cell's genome or remain present extra-chromosomally. The vector may be present permanently or transiently, e.g., as an episomal vector. Vectors may be introduced by any means known in the art. Such means include but are not limited to chemical treatment of the cells, electroporation, or infection. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid.

[0685] In some embodiments, a nucleic acid sequence encoding a viral polypeptide, such as but not limited to, a dependoparvovirus polypeptide is operably linked to regulatory expression control sequences for expression in a specific cell type, such as Sf9 or HEK cells. Techniques known to one skilled in the art for expressing foreign genes in insect host cells or mammalian host cells can be used with the compositions and methods of the disclosure. Methods for molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith. A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No.

[0686] NAI-5007748234vl 7555, College Station, Tex. (1986); Luckow. 1991. In Prokop et al., Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DN A Technology and Applications, 97-152 (1986); King, L. A. and R. D. Possee, The baculovirus expression system, Chapman and Hall, United Kingdom (1992); O'Reilly, D. R., L. K. Miller,

[0687] V. A. Luckow, Baculovirus Expression Vectors: A Laboratory Manual, New York (1992);

[0688] W. H. Freeman and Richardson, C. D., Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39 (1995); U.S. Pat. No. 4,745,051; US2003148506; and WO 03 / 074714. Promoters suitable for transcription of a nucleotide sequence encoding a dependoparvovirus polypeptide include the polyhedron, plO, p35 or IE-1 promoters and further promoters described in the above references are also contemplated.

[0689] In some embodiments, providing a cell comprising a nucleic acid described herein comprises acquiring a cell comprising the nucleic acid.

[0690] Methods of cultivating cells, cell-free systems, and other translation systems are known to those of skill in the art. In some embodiments, cultivating a cell comprises providing the cell with suitable media and incubating the cell and media for a time suitable to achieve viral particle production.

[0691] In some embodiments, a method of making a viral particle, such as but not limited to a dependoparvovirus particle, further comprises a purification step comprising isolating the dependoparvovirus particle from one or more other components (e.g., from a cell or media component).

[0692] In some embodiments, production of the viral particle, such as but not limited to, the dependoparvovirus particle comprises one or more (e.g., all) of: expression of dependoparvovirus polypeptides, assembly of a dependoparvovirus capsid (e.g., a capsid comprising a variant capsid polypeptide provided for herein), expression (e.g., duplication) of a dependoparvovirus genome, and packaging of the dependoparvovirus genome into the dependoparvovirus capsid to produce a dependoparvovirus particle. In some embodiments, production of the dependoparvovirus particle further comprises secretion of the dependoparvovirus particle.

[0693] In some embodiments, and as described elsewhere herein, the nucleic acid molecule encodes the variant capsid polypeptide is disposed in a dependoparvovirus genome. In some embodiments, and as described elsewhere herein, the nucleic acid molecule encodes the variant capsid polypeptide is packaged into a dependoparvovirus particle along with the NAI-5007748234vl dependoparvovirus genome as part of a method of making a dependoparvovirus particle described herein. In other embodiments, the nucleic acid molecule encodes the variant capsid polypeptide is not packaged into a dependoparvovirus particle made by a method described herein.

[0694] In some embodiments, a method of making a viral particle, such as but not limited to, a dependoparvovirus particle described herein produces a dependoparvovirus particle comprising a payload (e.g., a payload described herein) and the variant capsid polypeptide. In some embodiments, the payload comprises a second nucleic acid (e.g., in addition to the dependoparvovirus genome), and production of the dependoparvovirus particle comprises packaging the second nucleic acid into the dependoparvovirus particle. In some embodiments, a cell, cell-free system, or other translation system for use in a method of making a dependoparvovirus particle comprises the second nucleic acid. In some embodiments, the second nucleic acid comprises an exogenous sequence (e.g., exogenous to the dependoparvovirus, the cell, or to a target cell or subject who will be administered the dependoparvovirus particle). In some embodiments, the exogenous sequence encodes an exogenous polypeptide. In some embodiments, the exogenous sequence encodes a therapeutic product.

[0695] In some embodiments, the composition of the invention is a pharmaceutical composition.

[0696] In some embodiments, a nucleic acid or polypeptide described herein is produced by a method known to one of skill in the art. The nucleic acids, polypeptides, and fragments thereof of the disclosure may be produced by any suitable means, including recombinant production, chemical synthesis, or other synthetic means. Such production methods are within the knowledge of those of skill in the art and are not a limitation of the present invention.

[0697] In some embodiments, a method of making a virus particle described herein provides increased production of the virus particle relative to a similar production system, e.g., producing virus particles comprising wild-type AAV2 capsid polypeptides from the same producer cell type (e.g., from HEK293 cells). In some embodiments, a method of making a virus particle described herein produces the virus particle at least 1.3-fold better than a similar production system, e.g., producing virus particles comprising wild-type AAV2 capsid polypeptides from the same producer cell type (e.g., from HEK293 cells).

[0698] NAI-5007748234vl Applications

[0699] The disclosure is directed, in part, to compositions comprising a nucleic acid, polypeptide, or particles described herein. The disclosure is further directed, in part, to methods of utilizing a composition, nucleic acid, polypeptide, or particles described herein. As will be apparent based on the disclosure, nucleic acids, polypeptides, particles, and methods disclosed herein have a variety of utilities.

[0700] The disclosure is directed, in part, to a vector comprising a nucleic acid described herein, e.g., a nucleic acid encoding a variant capsid polypeptide. Many types of vectors are known to those of skill in the art. In some embodiments, a vector comprises a plasmid. In some embodiments, the vector is an isolated vector, e.g., removed from a cell or other biological components.

[0701] The disclosure is directed, in part to a cell, cell-free system, or other translation system, comprising a nucleic acid or vector described herein, e.g., a nucleic acid or vector comprising a nucleic acid molecule encodes a variant capsid polypeptide. In some embodiments, the cell, cell-free system, or other translation system is capable of producing dependoparvovirus particles comprising the variant capsid polypeptides. In some embodiments, the cell, cell-free system, or other translation system comprises a nucleic acid comprising a dependoparvovirus genome or components of a dependoparvovirus genome sufficient to promote production of dependoparvovirus particles comprising the variant capsid polypeptides.

[0702] In some embodiments, the cell, cell-free system, or other translation system further comprises one or more non-dependoparvovirus nucleic acid sequences that promote dependoparvovirus particle production and / or secretion. Said sequences are referred to herein as helper sequences. In some embodiments, a helper sequence comprises one or more genes from another virus, e.g., an adenovirus or herpes virus. In some embodiments, the presence of a helper sequence is necessary for production and / or secretion of a dependoparvovirus particle. In some embodiments, a cell, cell-free system, or other translation system comprises a vector, e.g., plasmid, comprising one or more helper sequences.

[0703] In some embodiments, a cell, cell-free system, or other translation system comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a

[0704] NAI-5007748234vl sequence encoding one or more dependoparvovirus genes (e.g., a Cap gene, a Rep gene, or a complete dependoparvovirus genome) and a helper sequence, and wherein the second nucleic acid comprises a payload. In some embodiments, a cell, cell-free system, or other translation system comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a sequence encoding one or more dependoparvovirus genes (e.g., a Cap gene, a Rep gene, or a complete dependoparvovirus genome) and a payload, and wherein the second nucleic acid comprises a helper sequence. In some embodiments, a cell, cell-free system, or other translation system comprises a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises a helper sequence and a payload, and wherein the second nucleic acid comprises a sequence encoding one or more dependoparvovirus genes (e.g., a Cap gene, a Rep gene, or a complete dependoparvovirus genome). In some embodiments, a cell, cell- free system, or other translation system comprises a first nucleic acid, a second nucleic acid, and a third nucleic acid, wherein the first nucleic acid comprises a sequence encoding one or more dependoparvovirus genes (e.g., a Cap gene, a Rep gene, or a complete dependoparvovirus genome), the second nucleic acid comprises a helper sequence, and the third nucleic acid comprises a payload.

[0705] In some embodiments, the first nucleic acid, second nucleic acid, and optionally third nucleic acid are situated in separate molecules, e.g., separate vectors or a vector and genomic DNA. In some embodiments, one, two, or all of the first nucleic acid, second nucleic acid, and optionally third nucleic acid are integrated (e.g., stably integrated) into the genome of a cell.

[0706] A cell of the disclosure may be generated by transfecting a suitable cell with a nucleic acid described herein. In some embodiments, a method of making a dependoparvovirus particle comprising a variant capsid polypeptide as provided for herein or improving a method of making a dependoparvovirus particle comprises providing a cell described herein. In some embodiments, providing a cell comprises transfecting a suitable cell with one or more nucleic acids described herein.

[0707] In some embodiments, the virus particle comprising the variant capsid is produced at a level at least 10%, at least 20%, at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, or greater than the production level of wt AAV2 from the same producer cell type, e.g., from HEK293 cells, e.g., from adherent culture of HEK293 cells. In some embodiments, a nucleic acid of the variant capsid mediates the

[0708] NAI-5007748234vl production of a dependoparvovirus particle at a level at least 10%, at least 20%, at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, or greater than the production level mediated by the nucleic acid of SEQ ID NO: 2.

[0709] Many types and kinds of cells suitable for use with the nucleic acids and vectors described herein are known in the art. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immortalized cell or a cell from a cell line known in the art. In some embodiments, the cell is an HEK293 cell.

[0710] Methods of delivering a payload

[0711] The disclosure is directed, in part, to a method of delivering a payload to a cell, e.g., a cell in a subject or in a sample. In some embodiments, a method of delivering a payload to a cell comprises contacting the cell with a dependoparvovirus particle comprising a variant capsid polypeptide (e.g., described herein) comprising the payload. In some embodiments, the dependoparvovirus particle is a dependoparvovirus particle described herein and comprises a payload described herein. In some embodiments, the cell is an ocular cell. In some embodiments, the ocular cell is in the retina, macula, or trabecular meshwork. In some embodiments, the ocular cell is in the retina. In some embodiments, the ocular cell is in the macula. In some embodiments, the ocular cell is in the trabecular meshwork.

[0712] In some embodiments, the ocular cell is in the front third of the eye, which includes the structures in front of the vitreous humor. Examples of structures in front of the vitreous humor, include the cornea, iris, ciliary body, lens, trabecular meshwork, and Schl emm’s canal. Accordingly, in some embodiments, the cell is in the cornea, iris, ciliary body, lens, trabecular meshwork, or Schl emm’s canal, or any combination thereof.

[0713] In some embodiments, the ocular cell is posterior to the lens, such as in the anterior hyaloid membrane and all of the optical structures behind it, such as the vitreous humor, retina, choroid or optic nerve, or any combination thereof. Accordingly, in some embodiments, the cell is in the anterior hyaloid membrane and all of the optical structures behind it, such as the vitreous humor, retina, choroid or optic nerve, or any combination thereof.

[0714] In some embodiments, the cell type is an ocular cell such as, for example, a neural retinal cell, a photoreceptive retinal ganglion cell, a bipolar cell, a horizontal cell, an

[0715] NAI-5007748234vl amacrine cell, a photoreceptor (e.g., a rod or a cone cell), an endothelial cell (e.g., a retinal pigmented epithelial cell), and endothelial-like cell, and the like.

[0716] The disclosure is further directed in part to a virus particle comprising a capsid polypeptide described herein. In some embodiments, the virus particle comprises a capsid polypeptide described herein and a nucleic acid expression construct. In some embodiments the nucleic acid expression construct of the virus particle comprises a payload.

[0717] In some embodiments, the payload comprises a transgene. In some embodiments, the transgene is a nucleic acid sequence heterologous to the vector sequences flanking the transgene which encodes a polypeptide, RNA (e.g., a miRNA or siRNA) or other product of interest. The nucleic acid of the transgene may be operatively linked to a regulatory component in a manner sufficient to promote transgene transcription, translation, and / or expression in a host cell.

[0718] A transgene may be any polypeptide or RNA encoding sequence and the transgene selected will depend upon the use envisioned. In some embodiments, a transgene comprises a reporter sequence, which upon expression produces a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding colorimetric reporters (e.g., P-lactamase, P-galactosidase (LacZ), alkaline phosphatase), cell division reporters (e.g., thymidine kinase), fluorescent or luminescence reporters (e.g., green fluorescent protein (GFP) or luciferase), resistance conveying sequences (e.g., chloramphenicol acetyltransferase (CAT)), or membrane bound proteins including to which high affinity antibodies directed thereto exist or can be produced by conventional means, e.g., comprising an antigen tag, e.g., hemagglutinin or Myc.

[0719] In some embodiments, a reporter sequence operably linked with regulatory elements which drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.

[0720] In some embodiments, the transgene encodes a product which is useful in biology and medicine, such as RNA, proteins, peptides, enzymes, dominant negative mutants. In some embodiments, the RNA comprises a tRNA, ribosomal RNA, dsRNA, catalytic RNAs, small hairpin RNA, siRNA, trans-splicing RNA, and antisense RNAs. In some embodiments, the

[0721] NAI-5007748234vl RNA inhibits or abolishes expression of a targeted nucleic acid sequence in a treated subject (e.g., a human or animal subject).

[0722] In some embodiments, the transgene may be used to correct or ameliorate gene deficiencies. In some embodiments, gene deficiencies include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed. In some embodiments, the transgene encodes a therapeutic protein or polypeptide which is expressed in a host cell. In some embodiments, a dependoparvovirus particle may comprise or deliver multiple transgenes, e.g., to correct or ameliorate a gene defect caused by a multi-subunit protein. In some embodiments, a different transgene (e.g., each situated / delivered in a different dependoparvovirus particle, or in a single dependoparvovirus particle) may be used to encode each subunit of a protein, or to encode different peptides or proteins, e.g., when the size of the DNA encoding the protein subunit is large, e.g., for immunoglobulin, platelet-derived growth factor, or dystrophin protein. In some embodiments, different subunits of a protein may be encoded by the same transgene, e.g., a single transgene encoding each of the subunits with the DNA for each subunit separated by an internal ribozyme entry site (IRES). In some embodiments, the DNA may be separated by sequences encoding a 2 A peptide, which self-cleaves in a post-translational event. See, e.g., Donnelly et al, J. Gen. Virol., 78(Pt 1): 13-21 (January 1997); Furler, et al, Gene Ther., 8(11):864-873 (June 2001); Klump et al., Gene Ther 8(10):811-817 (May 2001).

[0723] In some embodiments, virus particles comprising a genome are provided, wherein the genome includes a nucleic acid expression construct. The nucleic acid expression construct can include a heterologous transgene and one or more regulatory elements.

[0724] In some embodiments, the regulatory elements include a promotor. In some embodiments, the promoter is a ubiquitous or constitutive promoter active in a mammalian cell, for example a human cell, for example, in a human cell type of interest.

[0725] Examples of ubiquitous promoters include, but are not limited, to a CAG promoter (hybrid from a cytomegalovirus early enhancer element, a chicken-beta actin promoter, e.g., the first exon and the first intron of the chicken beta actin gene, and optionally the splice acceptor of the rabbit beta globin gene), chicken-beta actin promoter, CBA promoter, CMV promoter, human PGK promoter, ubiquitin promoter, human EFl -alpha promoter and fragments thereof. In some embodiments, the promoter is a tissue-specific promoter, for example, a promoter specific in ocular tissue or cells of the eye. Examples of ocular tissue-

[0726] NAI-5007748234vl specific promoters include but are not limited to TBG promoters, hAAT promoters, CK8 promoters and SPc5-12 promoters, rho promoters, which are active in rods, or opsin promoters, which are active in cones. In some embodiments, the regulatory element includes a photoreceptor cell-specific regulatory element (e.g., promoter) such as, e.g., a rhodopsin promoter; a rhodopsin kinase promoter; a beta phosphodiesterase gene promoter; a retinitis pigmentosa gene promoter; an interphotoreceptor retinoid-binding protein (IRBP) gene enhancer; an IRBP gene promoter, an opsin gene promoter, a retinoschisin gene promoter, a CRX homeodomain protein gene promoter, a guanine nucleotide binding protein alpha transducing activity polypeptide 1 (GNAT1) gene promoter, a neural retina-specific leucine zipper protein (NRL) gene promoter, human cone arrestin (hCAR) promoter, and the PR2.1, PR1.7, PR1.5, and PR1.1 promoters. In some embodiments, the regulatory element includes, a retinal pigment epithelia (RPE) cell-specific regulatory element (e.g., a RPE-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked gene in a RPE cell, such as, e.g., an RPE65 gene promoter, a cellular retinaldehyde-binding protein (CRALBP) gene promoter, a pigment epithelium-derived factor (PEDF aka serpin Fl) gene promoter, and a vitelliform macular dystrophy (VMD2) promoter. In some embodiments, the regulatory element includes a promoter specific to a glial cell, e.g., a regulatory element that confers selective expression of the operably linked payload in a retinal glial cell, such as, e.g., a glial fibrillary acidic protein (GFAP) promoter. In some instances, the regulatory element includes a promoter that is specific to a bipolar cell (e.g., a bipolar-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked payload in a bipolar cell, such as, e.g., a GRM6 promoter. In some embodiments, the promoter sequence is between 100 and 1000 nucleotides in length. In some embodiments, the promoter sequence is about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900 or about 1000 nucleotides in length. As used in the preceding sentence, “about” refers to a value within 50 nucleotides of the recited length. Suitable regulatory elements, e.g., promoters, may be readily selected by persons of skill in the art, such as those, but not limited to, those described herein.

[0727] In some embodiments, the nucleic acid expression construct comprises an intron. The intron may be disposed between the promoter and the heterologous transgene. In some aspects, the intron is disposed 5’ to the heterologous transgene on the expression construct, for example immediately 5’ to the heterologous transgene or 100 nucleotides or less 5’ to the heterologous transgene. In some aspects, the intron is a chimeric intron derived from human

[0728] NAI-5007748234vl b-globin and Ig heavy chain (also known as b- globin splice donor / immunoglobulin heavy chain splice acceptor intron, or b-globin / IgG chimeric intron; Reed, R., et al. Genes and Development, 1989, incorporated herein by reference in its entirety). In other aspects, the intron is a VH4 intron or a SV40 intron.

[0729] As provided herein, in some embodiments, virus particles comprising a payload, wherein the payload includes a nucleic acid that includes a heterologous transgene are provided. In some embodiments, the heterologous transgene encodes an RNA interference agent, for example a siRNA, shRNA or other interfering nucleic acid.

[0730] In some embodiments, the payload includes a heterologous transgene that encodes a therapeutic polypeptide. In some aspects, the heterologous transgene is a human gene or fragment thereof. In some aspects, the therapeutic polypeptide is a human protein. In some embodiments, the heterologous transgene of the virus particle encodes a molecule useful in treating a disease, and the virus particle is administered to a patient in need thereof to treat said disease. Examples of diseases (and heterologous transgenes or molecules encoded by said heterologous transgenes) according to the present disclosure include: MPSI (alpha-L- iduronidase (IDUA)); MPS II - Hunter syndrome (iduronate-2-sulfatase (IDS)); Ceroid lipofuscinosis-Batten disease (CLN1, CLN2, CLN10, CLN13, CLN5, CLN11, CLN4, CNL14, CLN3, CLN6, CLN7, CLN8, CLN12); MPS Illa - Sanfilippo Type A syndrome (heparin sulfate sulfatase (also called N-sulfoglucosamine sulfohydrolase (SGSH)); MPS IIIB - Sanfilippo Type b syndrome (N-acetyl-alpha-D-glucosaminidase (NAGLU)); MPS VI - Maroteaux-Lamy syndrome (aryl sulfatase B); MPS IV A - Morquio syndrome type A (GALNS); MPS IV B - Morquio syndrome type B (GLB1); Osteogenesis Imperfecgta Type I, II, III or IV (C0L1A1 and / or C0L1A2); hereditary angioedema (SERPING1, C1NH); Osteogenesis Imperfecta Type V (IFITM5); Osteogenesis Imperfecta Type VI (SERPINF1); Osteogenesis Imperfecta Type VII (CRTAP); Osteogenesis Imperfecta Type VIII (LEPRE1 and / or P3H1); Osteogenesis Imperfecta Type IX (PPIB); Gaucher disease type I, II and III (Glucocerebrosidase; GBA1); Parkinson's Disease (Glucocerebrosidase; GBA1 and / or dopamine decarboxylase); Pompe (acid maltase; GAA; hGAA); Metachromatic leukodystrophy (Aryl sulfatase A); MPS VII - Sly syndrome (beta-glucuronidase); MPS VIII (glucosamine-6-sulfate sulfatase); MPS IX (Hyaluronidase); maple syrup urine disease (BCKDHA, BCKDHB, and / or DBT); Niemann-Pick disease (Sphingomyelinase); Parkinson’s disease (anti-alpha synuclein RNAi); Alzheimer’s disease (anit-mutant APP RNAi); Niemann-Pick disease without sphingomyelinase deficiency (NPC1 or NPC gene NAI-5007748234vl encoding a cholesterol metabolizing enzyme); Tay-Sachs disease (alpha subunit of betahexosaminidase); Sandhoff disease (both alpha and beta subunit of beta-hexosaminidase); Fabry Disease (alpha-galactosidase); Fucosidosis (fucosidase (FUCA1)); Alpha-mannosidosis (alpha-mannosidase); Beta-mannosidosis (beta-mannosidase); Wolman disease (cholesterol ester hydrolase); Dravet syndrome (SCN1A, SCN1B, SCN2A, GABRG2); Parkinson's disease (Neurturin); Parkinson's disease (glial derived growth factor (GDGF)); Parkinson's disease (tyrosine hydroxylase); Parkinson's disease (glutamic acid decarboxylase; FGF-2; BDGF); Spinal Muscular Atrophy (SMN, including SMN1 or SMN2); Friedreich's ataxia (Frataxin); Amyotrophic lateral sclerosis (ALS) (SOD1 inhibitor, e.g., anti-SODl RNAi); Glycogen Storage Disease la (Glucose-6-phosphatase); XLMTM (MTM1); Crigler Najjar (UGT1A1); CPVT (CASQ2); spinocerebellar ataxia (ATXN2; ATXN3 or other ATXN gene; anti-mutant Machado-Joseph disease / SCA3 allele RNAi); Rett syndrome (MECP2 or fragment thereof); Achromatopsia (CNGB3, CNGA3, GNAT2, PDE6C); Choroidermia (CDM); Danon Disease (LAMP2); Cystic Fibrosis (CFTR or fragment thereof); Duchenne Muscular Dystrophy (Mini- / Micro-Dystrophin Gene); SARS-Cov-2 infection (anti-SARS- Cov-2 RNAi, SARS-Cov-2 genome fragments or S protein (including variants)); Limb Girdle Muscular Dystrophy Type 2C - Gamma-sarcoglycanopathy (human-alpha-sarcoglycan); Advanced Heart Failure (SERCA2a); Rheumatoid Arthritis (TNFR:Fc Fusion; anti-TNF antibody or fragment thereof); Leber Congenital Amaurosis (GAA); X-linked adrenoleukodystrophy (ABCD1); Limb Girdle Muscular Dystrophy Type 2C - Gamma- sarcoglycanopathy (gamma-sarcoglycan); Angelman syndrome (UBE3A); Retinitis Pigmentosa (hMERTK); Age-Related Macular Degeneration (sFLTOl); Phelan-McDermid syndrome (SHANK3; 22ql3.3 replacement); Becker Muscular Dystrophy and Sporadic Inclusion Body Myositis (huFollistatin344); Parkinson's Disease (GDNF); Metachromatic Leukodystrophy - MED (cuARSA); Hepatitis C (anti-HCV RNAi); Limb Girdle Muscular Dystrophy Type 2D (hSGCA); Human Immunodeficiency Virus Infections; (PG9DP); Acute Intermittant Porphyria (PBGD); Leber's Hereditary Optical Neuropathy (PIND4v2); Alpha- 1 Antitrypsin Deficiency (alphalAT); X-linked Retinoschisis (RSI); Choroideremia (hCHM); Giant Axonal Neuropathy (GAN); Hemophilia B (Factor IX); Homozygous FH (hLDLR); Dysferlinopathies (DYSF); Achromatopsia (CNGA3 or CNGB3); Progressive supranuclear palsy (MAPT; anti-Tau; anti-MAPT RNAi); Ornithine Transcarbamylase deficiency (OTC); Hemophilia A (Factor VIII); Age-related macular degeneration (AMD), including wetAMD (anti-VEGF antibody or RNAi); X-Linked Retinitis Pigmentosa (RPGR); Myotonic dystrophy Type 1 (DMPK; anti-DMPK RNAi, including anti-CTG trinucleotide repeat NAI-5007748234vl RNAi); Myotonic dystrophy Type 2 (CNBP); Facioscapulohumeral muscular dystrophy (D4Z4 DNA); oculopharynggeal muscular dystrophy (PABPN1; mutated PABPN1 inhibitor (e.g., RNAi)); Mucopolysaccharidosis Type VI (hARSB); Leber Hereditary Optic Neuropathy (ND4); X-Linked myotubular Myopathy (MTM1); Crigler-Najjar Syndrome (UGT1A1); Retinitis Pigmentosa (hPDE6B); Mucopolysaccharidosis Type 3B (hNAGLU); Duchenne Muscular Dystrophy (GALGT2); Alzheimer's Disease (NGF; ApoE4; ApoE2; ApoE3; Anti-ApoE RNAi); Familial Lipoprotein Lipase Deficiency (LPL); Alpha- 1 Antitrypsin Deficiency (hAAT); Leber Congenital Amaurosis 2 (hRPE65v2); Batten Disease; Late Infantile Neuronal Lipofuscinosis (CLN2); Huntington’s disease (HTT; anti- HTT RNAi); Fragile X syndrome (FMRI); Leber's Hereditary Optical Neuropathy (PlND4v2); Aromatic Amino Acid Decarboxylase Deficiency (hAADC); Retinitis Pigmentosa (hMERKTK); and Retinitis Pigmentosa (RLBP1).

[0731] In some aspects, the heterologous transgene encodes an antibody or fragment thereof (for example an antibody light chain, an antibody heavy chain, a Fab or an scFv). Examples of antibodies or fragments thereof that are encoded by the heterologous transgene include but are not limited to: an anti-Ab antibody (e.g. solanezumab, GSK933776, and lecanemab), anti- sortilin ( e.g. AL-001), anti-Tau (e.g. ABBV-8E12, UCB-0107, and NI- 105), anti-SEMA4D (e.g. VX15 / 2503), anti-alpha synuclein (e.g. prasinezumab, NI-202, and MED-1341), anti- SOD1 (e.g. NI-204), anti-CGRP receptor (e.g. eptinezumab, fremanezumab, or galcanezumab), anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651, ), anti-ALKl (e.g., ascrinvacumab), anti-C5 (e.g., tesidolumab, ravulizumab, and eculizumab), anti-CD105 (e.g., carotuximab), anti-CQQ (e.g., ANX-007), anti-TNFa (e.g., adalimumab, infliximab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamrevlumab), anti- IL6R (e.g., satralizumab, tocilizumab, and sarilumab), anti-IL6 (e.g. siltuximab, clazakizumab, sirukumab, olokizumab, and gerilimzumab), anti-IL4R (e.g., dupilumab), anti- IL17A (e.g., ixekizumab and secukinumab), anti-IL5R (e.g. reslizumab), anti-IL-5 (e.g., benralizumab and mepolizumab), anti-IL13 (e.g. tralokinumab), anti-IL12 / IL23 (e.g., ustekinumab), anti-CD 19 (e.g., inebilizumab), anti-IL31RA (e.g. nemolizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-IgE (e.g. omalizumab), anti-TSLP (e.g. nemolizumab), anti-pKal mAb (e.g., lanadelumab), anti-ITGA4 (e.g., natalizumab), anti- ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., denosumab), anti-PCSK9 (e.g.,

[0732] NAI-5007748234vl alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab), optionally wherein the heavy chain (Fab and Fc region) and the light chain are separated by a self-cleaving furin (F) / F2A or furin (F) / T2A, IRES site, or flexible linker, for example, ensuring expression of equal amounts of the heavy and the light chain polypeptides.

[0733] In some embodiments, the payload comprises a nucleic acid encoding a gene product linked to a disorder of the eye, or a fragment thereof. Exemplary gene products linked to a disorder of the eye include, for example, ADP-ribosylation factor-like 6 (ARL6); BBSome interacting protein 1 (BBIP1); BBSome protein 1 (BBS1); BBSome protein 2 (BBS2); BBSome protein 4 (BBS4); BBSome protein 5 (BBS5); BBSome protein 7 (BBS7); BBSome protein 9 (BBS9); BBSome protein 10 (BBS 10); BBSome protein 12 (BBS 12); centrosomal protein 290 kDa (CEP290); intraflagellar transport protein 172 (IFT172); intraflagellar transport protein 27 (IFT27); inositol polyphosphate-5-phosphatase E (INPP5E); inwardly- rectifying potassium channel subfamily J member 13 (KCNJ13); leucine zipper transcription factor like-1 (LZTFL1); McKusick-Kaufman syndrome protein (MKKS); Meckel syndrome type 1 protein (MKS1); nephronophthisis 3 protein (NPHP1); serologically-defined colon cancer antigen 8 (SDCCAG8); tripartite motif-containing protein 32 (TRIM32); tetratricopeptide repeat domain 8 (TTC8); Batten disease protein (CLN3); cytochrome P450 4V2 (CYP4V2); Rab escort protein 1 (CHM); PR (positive regulatory) domain-containing 13 protein (PRDM13); RPE-retinal G protein-coupled receptor (RGR); TEA domain family member 1 (TEAD1); arylhydrocarbon-interacting receptor protein-like 1 (AIPL1); cone-rod otx-like photoreceptor homeobox transcription factor (CRX); guanylate cyclase activating protein 1 A (GUCA1 A); retinal-specific guanylate cyclase (GUCY2D); phosphatidylinositol transfer membrane-associated family member 3 (PITPNM3); prominin 1 (PR0M1); peripherin (PRPH); peripherin 2 (PRPH2); regulating synaptic membrane exocytosis protein 1 (RIMS1); semaphorin 4A (SEMA4A); human homolog of C. elegans uncl 19 protein (UNCI 19); ATP -binding cassette transporter — retinal (ABCA4); ADAM metallopeptidase domain 9 (ADAM9); activating transcription factor 6 (ATF6); chromosome 21 open reading frame 2 (C21orf2); chromosome 8 open reading frame 37 (C8orf37); calcium channel; voltage-dependent; alpha 2 / delta subunit 4 (CACNA2D4); cadherin-related family member 1 (protocadherin 21) (CDHR1); ceramide kinase-like protein (CERKL); cone photoreceptor cGMP -gated cation channel alpha subunit (CNGA3); cone cyclic nucleotide-gated cation channel beta 3 subunit (CNGB3); cyclin M4 (CNNM4); guanine nucleotide binding protein

[0734] NAI-5007748234vl (G protein); alpha transducing activity polypeptide 2 (GNAT2); potassium channel subfamily V member 2 (KCNV2); Phosphodiesterase 6C (PDE6C); Phosphodiesterase 6H (PDE6H); proteome of centriole 1 centriolar protein B (P0C1B); RAB28 member of RAS oncogene family (RAB28); retina and anterior neural fold homeobox 2 transcription factor (RAX2); 11- cis retinol dehydrogenase 5 (RDH5); RP GTPase regulator-interacting protein 1 (RPGRIP1); tubulin tyrosine ligase-like family member 5 (TTLL5); L-type voltage-gated calcium channel alpha- 1 subunit (CACNA1F); retinitis pigmentosa GTPase regulator (RPGR); rod transducin alpha subunit (GNAT1); rod cGMP phosphodiesterase beta subunit (PDE6B); rhodopsin (RHO); calcium binding protein 4 (CABP4); G protein-coupled receptor 179 (GPR179); rhodopsin kinase (GRK1); metabotropic glutamate receptor 6 (GRM6); leucine-rich repeat immunoglobulin-like transmembrane domains protein 3 (LRIT3); arrestin (s-antigen) (SAG); solute carrier family 24 (SLC24A1); transient receptor potential cation channel, subfamily M, member 1 (TRPM1); nyctalopin (NYX); green cone opsin (0PN1LW); red cone opsin (0PN1MW); blue cone opsin (0PN1SW); frataxin (FXN); inosine monophosphate dehydrogenase 1 (IMPDH1); orthodenticle homeobox 2 protein (0TX2); crumbs homolog 1 (CRB1); death domain containing protein 1 (DTHD1); growth differentiation factor 6 (GDF6); intraflagellar transport 140 Chlamydomonas homolog protein (IFT140); IQ motif containing B protein (IQCB1); lebercilin (LCA5); lecithin retinol acyltransferase (LRAT); nicotinamide nucleotide adenylyltransferase 1 (NMNAT1); RD3 protein (RD3); retinol dehydrogenase 12 (RDH12); retinal pigment epithelium-specific 65 kD protein (RPE65); spermatogenesis associated protein 7 (SPATA7); tubby-like protein 1 (TULP1); mitochondrial genes (KSS, LHON, MT-ATP6, MT-TH, MT-TL1, MT-TP, MT-TS2, mitochondrially encoded NADH dehydrogenases [MT-ND]); bestrophin 1 (BEST1); Clq and tumor necrosis-related protein 5 collagen (C1QTNF5); EGF-containing flbrillin-like extracellular matrix protein 1 (EFEMP1); elongation of very long fatty acids protein (ELOVL4); retinal fascin homolog 2, actin bundling protein (FSCN2); guanylate cyclase activating protein IB (GUCAB); hemicentin 1 (HMCN1); interphotoreceptor matrix proteoglycan 1 (IMPG1); retinitis pigmentosa 1 -like protein 1 (RP1L1); tissue inhibitor of metalloproteinases-3 (TIMP3); complement factor H (CFH); complement factor D (CFD); complement component 2 (C2); complement component 3(C3); complement factor B (CFB); DNA-damage regulated autophagy modulator 2 (DRAM2); chondroitin sulfate proteoglycan 2 (VCAN); mitofusin 2 (MFN2); nuclear receptor subfamily 2 group F member 1 (NR2F1); optic atrophy 1 (OPA1); transmembrane protein 126A (TMEM126A); inner mitochondrial membrane translocase 8 homolog A (TIMM8A); carbonic anhydrase IV (CA4); hexokinase 1 NAI-5007748234vl (HK1); kelch-like 7 protein (KLHL7); nuclear receptor subfamily 2 group E3 (NR2E3); neural retina lucine zipper (NRL); olfactory receptor family 2 subfamily W member 3 (OR2W3); pre-mRNA processing factor 3 (PRPF3); pre-mRNA processing factor 4 (PRPF4); pre-mRNA processing factor 6 (PRPF6); pre-mRNA processing factor 8 (PRPF8); pre- mRNA processing factor 31 (PRPF31); retinal outer segment membrane protein 1 (R0M1); retinitis pigmentosa protein 1 (RP1); PIM-kinase associated protein 1 (RP9); small nuclear ribonucleoprotein 200 kDa (SNRNP200); secreted phosphoprotein 2 (SPP2); topoisomerase I binding arginine / serine rich protein (TOPORS); ADP-ribosylation factor-like 2 binding protein (ARL2BP); chromosome 2 open reading frame 71 (C2orf71); clarin-1 (CLRN1); rod cGMP -gated channel alpha subunit (CNGA1); rod cGMP -gated channel beta subunit (CNGB1); cytochrome P450 4V2 (CYP4V2); dehydrodolichyl diphosphate synthetase (DHDDS); DEAH box polypeptide 38 (DHX38); ER membrane protein complex subunit 1 (EMC1); eyes shut / spacemaker homolog (EYS); family with sequence similarity 161 member A (FAM161 A); G protein-coupled receptor 125 (GPR125); heparan-alpha-glucosaminide N- acetyltransferase (HGSNAT); NAD(+)-specific isocitrate dehydrogenase 3 beta (IDH3B); interphotoreceptor matrix proteoglycan 2 (IMPG2); KIAA1549 protein (KIAA1549); kizuna centrosomal protein (KIZ); male germ-cell associated kinase (MAK); c-mer protooncogene receptor tyrosine kinase (MERTK); mevalonate kinase (MVK); NIMA (never in mitosis gene A)-related kinase 2 (NEK2); neuronal differentiation protein 1 (NEURODI); cGMP phosphodiesterase alpha subunit (PDE6A); phosphodiesterase 6G cGMP-specific rod gamma (PDE6G); progressive rod-cone degeneration protein (PRCD); retinol binding protein 3 (RBP3); retinaldehyde-binding protein 1 (RLBP1); solute carrier family 7 member 14 (SLC7A14); usherin (USH2A); zinc finger protein 408 (ZNF408); zinc finger protein 513 (ZNF513); oral-facial-digital syndrome 1 protein (OFD1); retinitis pigmentosa 2 (RP2); retinoschisin (RSI); abhydrolase domain containing protein 12 (ABHD12); cadherin-like gene 23 (CDH23); centrosomal protein 250 kDa (CEP250); calcium and integrin binding family member 2 (CIB2); whirlin (DFNB31); monogenic audiogenic seizure susceptibility 1 homolog (GPR98); histidyl-tRNA synthetase (HARS); myosin VIIA (MY07A); protocadherin 15 (PCDH15); harmonin (USH1C); human homolog of mouse scaffold protein containing ankyrin repeats and SAM domain (USH1G); dystrophin (DMD); norrin (NDP); phosphoglycerate kinase (PGK1); calpain 5 (CAPN5); frizzled-4 Wnt receptor homolog (FZD4); integral membrane protein 2B (ITM2B); low density lipoprotein receptor-related protein 5 (LRP5); micro RNA 204 (MIR204); retinoblastoma protein 1 (RBI); tetraspanin 12 (TSPAN12); chromosome 12 open reading frame 65 (C12orf65); cadherin 3 (CDH3); NAI-5007748234vl membrane-type frizzled-related protein (MFRP); ornithine aminotransferase (OAT); phospholipase A2 group V (PLA2G5); retinol-binding protein 4 (RBP4); regulator of G- protein signaling 9 (RGS9); regulator of G-protein signaling 9-binding protein (RGS9BP); ARMS2; excision repair cross-complementing rodent repair deficiency complementation group 6 protein (ERCC6); fibulin 5 (FBLN5); HtrA serine peptidase 1 (HTRA1); toll-like receptor 3 (TLR3); and toll-like receptor 4 (TLR4), opsin; rhodopsin; channel rhodopsin; halo rhodopsin, and the like.

[0735] In some embodiments, the virus particle comprises a heterologous transgene encoding a genome editing system. Examples include a CRISPR genome editing system (e.g., one or more components of a CRISPR genome editing system such as, for example, a guide RNA molecule and / or a RNA-guided nuclease such as a Cas enzyme such as Cas9, Cpfl and the like), a zinc finger nuclease genome editing system, a TALEN genome editing system or a meganuclease genome editing system. In some embodiments, the genome editing system targets a mammalian, e.g., human, genomic target sequence. In some embodiments, the virus particle includes a heterologous transgene encoding a targetable transcription regulator. Examples include a CRISPR-based transcription regulator (for example, one or more components of a CRISPR-based transcription regulator, for example, a guide RNA molecule and / or a enzymatically-inactive RNA-guided nuclease / transcription factor (“TF”) fusion protein such as a dCas9-TF fusion, dCpfl-TF fusion and the like), a zinc finger transcription factor fusion protein, a TALEN transcription regulator or a meganuclease transcription regulator.

[0736] In some embodiments, components of a therapeutic molecule or system are delivered by more than one unique virus particle (e.g., a population that includes more than one unique virus particles). In other embodiments, the therapeutic molecule or components of a therapeutic molecule or system are delivered by a single unique virus particle (e.g., a population that includes a single unique virus particle).

[0737] The transgene may also encode any biologically active product or other product, e.g., a product desirable for study. Suitable transgenes may be readily selected by persons of skill in the art, such as those, but not limited to, those described herein.

[0738] Other examples of proteins encoded by the transgene include, but are not limited to, colony stimulating factors (CSF); blood factors, such as P-globin, hemoglobin, tissue plasminogen activator, and coagulation factors; interleukins; soluble receptors, such as

[0739] NAI-5007748234vl soluble TNF-a. receptors, soluble VEGF receptors, soluble interleukin receptors (e.g., soluble IL-1 receptors and soluble type II IL-1 receptors), or ligand-binding fragments of a soluble receptor; growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), or fibroblast growth factor (FGF, such as basic FGF and acidic FGF); enzymes; chemokines,; enzyme activators, such as tissue plasminogen activator; angiogenic agents, such as vascular endothelial growth factors, glioma-derived growth factor, angiogenin, or angiogenin-2; anti- angiogenic agents, such as a soluble VEGF receptor; a protein vaccine; neuroactive peptides, such as nerve growth factor (NGF) or oxytocin; thrombolytic agents;; tissue factors; macrophage activating factors; tissue inhibitors of metalloproteinases; or IL-1 receptor antagonists.

[0740] The disclosure is further directed, in part, to a method of delivering a payload to a subject, e.g., an animal or human subject. In some embodiments, a method of delivering a payload to a subject comprises administering to the subject a virus particle, such as but not limited to a dependoparvovirus particle, comprising a variant polypeptide (e.g., described herein) comprising the payload, e.g., in a quantity and for a time sufficient to deliver the payload. In some embodiments, the dependoparvovirus particle is a dependoparvovirus particle described herein and comprises a payload described herein. In some embodiments, the particle delivers the payload to the eye. In some embodiments, the delivery to the eye is increased as compared to a particle without the variant capsid polypeptide or as compared to a wild-type capsid polypeptide.

[0741] Methods of treatment

[0742] In some embodiments, the methods of the invention are not methods for treatment of the human or animal body by therapy. In other embodiments, the methods of the invention are used for therapy.

[0743] The disclosure is directed, in part, to a method of treating a disease or condition in a subject, e.g., an animal or human subject. As used herein, the term “treating a disease or condition” refers to treating a manifest disease or condition, for example, where the subject is already suffering from one or more symptoms of the disease or condition, or refers to treating a pre-manifest disease or condition, for example, where the subject is identified as having a disease or condition but is not yet exhibiting one or more symptoms of the disease or condition. Pre-manifest conditions may be identified by, for example, genetic testing. In some NAI-5007748234vl embodiments, a method of treating a disease or condition in a subject comprises administering to the subject a virus particle, such as but not limited to a dependoparvovirus particle comprising a variant polypeptide described herein, e.g., comprising a payload described herein. In some embodiments, the dependoparvovirus particle, which comprises a variant polypeptide, comprising a payload described herein is administered in an amount and / or time effective to treat the disease or condition. In some embodiments, the payload is a therapeutic product. In some embodiments, the payload is a nucleic acid, e.g., encoding an exogenous polypeptide.

[0744] The viral particles, such as but not limited to, the dependoparvovirus particles comprising a variant polypeptide described herein or produced by the methods described herein can be used to express one or more therapeutic proteins to treat various diseases or disorders. In some embodiments, the disease or disorder is a cancer, e.g., a cancer such as carcinoma, sarcoma, leukemia, lymphoma; or an autoimmune disease, e.g., multiple sclerosis. Non-limiting examples of carcinomas include esophageal carcinoma; bronchogenic carcinoma; colon carcinoma; colorectal carcinoma; gastric carcinoma; hepatocellular carcinoma; basal cell carcinoma, squamous cell carcinoma (various tissues); bladder carcinoma, including transitional cell carcinoma; lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung; adrenocortical carcinoma; sweat gland carcinoma; sebaceous gland carcinoma; thyroid carcinoma; pancreatic carcinoma; breast carcinoma; ovarian carcinoma; prostate carcinoma; adenocarcinoma; papillary carcinoma; papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; renal cell carcinoma; uterine carcinoma; testicular carcinoma; osteogenic carcinoma; ductal carcinoma in situ or bile duct carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; Wilm's tumor; cervical carcinoma; epithelieal carcinoma; and nasopharyngeal carcinoma. Non-limiting examples of sarcomas include fibrosarcoma, myxosarcoma, liposarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Non-limiting examples of solid tumors include ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, menangioma, melanoma, neuroblastoma, and retinoblastoma. Non-limiting examples of leukemias include chronic myeloproliferative syndromes; T-cell CLL prolymphocytic leukemia, acute myelogenous leukemias; chronic lymphocytic leukemias, including B-cell

[0745] NAI-5007748234vl CLL, hairy cell leukemia; and acute lymphoblastic leukemias. Examples of lymphomas include, but are not limited to, B-cell lymphomas, such as Burkitt's lymphoma; and Hodgkin's lymphoma.

[0746] In some embodiments, the disease or disorder is a genetic disorder. In some embodiments, the genetic disorder is sickle cell anemia, Glycogen storage diseases (GSD, e.g., GSD types I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and XIV), cystic fibrosis, lysosomal acid lipase (LAL) deficiency 1, Tay-Sachs disease, Phenylketonuria, Mucopolysaccharidoses, Galactosemia, muscular dystrophy (e.g., Duchenne muscular dystrophy), hemophilia such as hemophilia A (classic hemophilia) or hemophilia B (Christmas Disease), Wilson's disease, Fabry Disease, Gaucher Disease hereditary angioedema (HAE), and alpha 1 antitrypsin deficiency. Examples of other diseases or disorders are provided above in the “Methods of delivering a payload” section.

[0747] The viral particles, such as but not limited to, the dependoparvovirus particles comprising a variant polypeptide described herein or produced by the methods described herein can be used to express one or more therapeutic proteins to treat various diseases or disorders. In some embodiments, the disease or disorder is a disease or disorder of the eye, for example, retinitis pigmentosa; macular degeneration (e.g.; wet age-related macular degeneration), optic neuritis; Leber’s congenital amaurosis; Leber’s hereditary optic neuropathy; achromatopsia; X-linked retinoschisis; optic neuritis; choroideremia; optic atrophy; retinal cone dystrophy; retinopathy; retinoblastoma; glaucoma; Bardet-Biedl syndrome; Usher syndrome; aniridia; Friedreich’s ataxia; vitelliform macular dystrophy; retinoblastoma; Stargardt disease; Charcot-Marie-Tooth disease; Fuch’s dystrophy; propionic acidemia; or color blindness; corneal dystrophy; keratoconus; night blindness; dry eye; Bardet-Biedl syndrome; Batten's Disease; Bietti's Crystalline Dystrophy; chorioretinal atrophy; chorioretinal degeneration; cone or cone-rod dystrophies (autosomal dominant, autosomal recessive, and X-linked), congenital stationary night blindness (autosomal dominant, autosomal recessive, and X-linked); disorders of color vision, including achromatopsia (including ACHM2, ACHM3, ACHM4, and ACHM5), protanopia, deuteranopia, and tritanopia; Friedreich's ataxia; Leber's congenital amaurosis (autosomal dominant and autosomal recessive), including, but not limited to, LCA1, LCA2, LCA3, LCA4, LCA6, LCA7, LCA8, LCA12, and LCA15; Leber's Hereditary Optic Neuropathy; macular dystrophy (autosomal dominant and autosomal recessive), including, but not limited to, acute macular degeneration, Best vitelliform macular dystrophy, pattern dystrophy, North

[0748] NAI-5007748234vl Carolina Macular Dystrophy, inherited drusen, Sorsby's fundus dystrophy, malattia levantanese, and genetically-determined retinopathy of prematurity; ocular-retinal developmental disease; ocular albinism; optic atrophies (autosomal dominant, autosomal recessive, and X-linked); retinitis pigmentosa (autosomal dominant, autosomal recessive, X- linked, and mitochondrially-inherited traits), examples of which include RP1, RP2, RP3, RP10, RP20, RP38, RP40, and RP43; X-linked retinoschisis; Stargardt disease; and Usher syndrome, including, but not limited to, USH1B, USH1C, USH1D, USH1F, USH1G, USH2A, USH2C, USH2D, AND USH3. Examples of complex genetic diseases include, but are not limited to, glaucoma (open angle, angle-closure, low-tension, normal-tension, congenital, neovascular, pigmentary, pseudoexfoliation); age-related and other forms of macular degeneration, both exudative and non-exudative forms (autosomal dominant and autosomal recessive), such as acute macular degeneration, vitelliform macular degeneration; retinopathy of prematurity; and Vogt Koyanagi-Harada (VKH) syndrome. Examples of acquired diseases include, but are not limited to, acute macular neuroretinopathy; anterior ischemic optic neuropathy and posterior ischemic optic neuropathy; Behcet's disease; branch retinal vein occlusion; choroidal neovascularization; diabetic retinopathy, including proliferative diabetic retinopathy and associated complications; diabetic uveitis; edema, such as macular edema, cystoid macular edema and diabetic macular edema; epiretinal membrane disorders; macular telangiectasia; multifocal choroiditis; non-retinopathy diabetic retinal dysfunction; ocular tumors; optic atrophies; retinal detachment; retinal disorders, such as central retinal vein occlusion, proliferative vitreoretinopathy (PVR), retinal arterial and venous occlusive disease, vascular occlusion, uveitic retinal disease; uveal effusion; retinal infective and infiltrative disease; optic nerve diseases such as acquired optic atrophy. Examples of traumatic injuries include, but are not limited to, histoplasmosis; optic nerve trauma; ocular trauma which affects a posterior ocular site or location; retinal trauma; viral infection of the eye; viral infection of the optic nerve; a posterior ocular condition caused by or influenced by an ocular laser treatment; posterior ocular conditions caused by or influenced by a photodynamic therapy; photocoagulation, radiation retinopathy; and sympathetic ophthalmia.

[0749] In some embodiments, administration of a viral particle, such as but not limited to, a dependoparvovirus particle comprising a variant polypeptide and comprising a payload (e.g., a transgene) to a subject induces expression of the payload (e.g., transgene) in a subject. In some embodiments, the expression is induced in the eye. In some embodiments, the

[0750] NAI-5007748234vl production is increased in the eye as compared to an analogous particle with the wild-type capsid protein. The amount of a payload, e.g., transgene, e.g., heterologous protein, e.g., therapeutic polypeptide, expressed in a subject (e.g., the serum of the subject) can vary. For example, in some embodiments the payload, e.g., protein or RNA product of a transgene, can be expressed in the serum of the subject in the amount of less than about 5 pg / ml. For example, in some embodiments the payload, e.g., protein or RNA product of a transgene, can be expressed in the serum of the subject in the amount of at least about 9 pg / ml, at least about 10 pg / ml, at least about 50 pg / ml, at least about 100 pg / ml, at least about 200 pg / ml, at least about 300 pg / ml, at least about 400 pg / ml, at least about 500 pg / ml, at least about 600 pg / ml, at least about 700 pg / ml, at least about 800 pg / ml, at least about 900 pg / ml, or at least about 1000 pg / ml. In some embodiments, the payload, e.g., protein or RNA product of a transgene, is expressed in the serum of the subject in the amount of about 9 pg / ml, about 10 pg / ml, about 50 pg / ml, about 100 pg / ml, about 200 pg / ml, about 300 pg / ml, about 400 pg / ml, about 500 pg / ml, about 600 pg / ml, about 700 pg / ml, about 800 pg / ml, about 900 pg / ml, about 1000 pg / ml, about 1500 pg / ml, about 2000 pg / ml, about 2500 pg / ml, or a range between any two of these values.

[0751] In some embodiments, the viral particle, such as but not limited to, the dependoparvovirus particle comprising a variant polypeptide and comprising a payload (e.g., a transgene) is administered to a subject via an injection. In some embodiments, the injection is a systemic injection, for example, intravenous, intraarterial, intramuscular, or subcutaneous injection. In some embodiments, the injection is an injection to the eye. In some embodiments, the injection is an intravitreal injection, intraorbital injection, retro-orbital injection, suprachoroidal injection, subretinal injection, sub conjuncti vital injection, or intracameral injection. In some embodiments, the injection is an intravitreal injection. In some embodiments, the injection is an intraorbital injection. In some embodiments, the injection is a retro-orbital injection. In some embodiments, the injection is a suprachoroidal injection. In some embodiments, the injection is a subretinal injection. In some embodiments, the injection is a sub conjuncti vital injection. In some embodiments, the injection is an intracameral injection.

[0752] Sequences disclosed herein may be described in terms of percent identity. A person of skill will understand that such characteristics involve alignment of two or more sequences. Alignments may be performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs, such as “Clustal W”, accessible via the Internet. As NAI-5007748234vl another example, nucleic acid sequences may be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent identity between nucleic acid sequences may be determined using FASTA with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Similar programs are available for amino acid sequences, e.g., the “Clustal X” program. Additional sequence alignment tools that may be used are provided by (protein sequence alignment; (http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / )) and (nucleic acid alignment; http: / / www.ebi.ac.uk / Tools / psa / emboss_needle / nucleotide.html)). Generally, any of these programs may be used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. Sequences disclosed herein may further be described in terms of edit distance. The minimum number of sequence edits (i.e., additions, substitutions, or deletions of a single base or nucleotide) which change one sequence into another sequence is the edit distance between the two sequences. In some embodiments, the distance between two sequences is calculated as the Levenshtein distance.

[0753] All publications, patent applications, patents, and other publications and references (e.g., sequence database reference numbers) cited herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referred to herein, e.g., in any Table herein, are incorporated by reference. Unless otherwise specified, the sequence accession numbers specified herein, including in any Table herein, refer to the database entries current as of August 21, 2020. When one gene or protein references a plurality of sequence accession numbers, all of the sequence variants are encompassed.

[0754] The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only and are not to be construed as limiting the scope or content of the invention in any way.

[0755] NAI-5007748234vl EXAMPLES

[0756] Library Experiment 1

[0757] Two libraries of 2.5e5 capsid variants of wild-type AAV2 per library were designed and cloned into plasmids to create two libraries of plasmids encoding the capsid variants (libraries for Library Experiment 1). Variants in one library were designed to maximize posterior eye transduction (including for example retina, macula, non-macular retina, neural retina and choroid / RPE) (posterior eye library) and variants in the other library were designed to maximize anterior eye transduction (including for example tissues of the trabecular meshwork and Schl emm’s canal) (anterior eye library). Both libraries were designed to include variants which would produce virus particles. A library of AAV variant genomes encoding each variant’s capsid and a unique capsid variant barcode identifier was cloned into two ITR plasmid backbones (for posterior or anterior administration) as described previously (Ogden et al. 2019). Each plasmid backbone contained a unique genomic identifier enabling analysis of biodistribution and transduction efficiencies via different routes of administration. The libraries were produced via transient triple transfection of adherent HEK293T cells followed by iodixanol gradient purification. Transfections were completed at a 1 : 1 : 1 / 150 ratio of Helper, Rep, and Cap-inside-lTR plasmid, which has been optimized with these plasmids to limit cross packaging. Cells were harvested, lysed, and purified by a sequence of steps: (1) clarification via depth and sterilizing filtration, (2) ultrafiltration and diafiltration via tangential flow filtration (TFF), (3) iodixanol gradient purification, and (4) ultrafiltration and diafiltration via molecular weight cutoff spin columns or TFF. The produced virus was tested for suitable sterility and endotoxin, and titer performed by ddPCR.

[0758] Evaluation of Library Packaging Efficiency

[0759] To measure each variant’s packaging efficiency (or ‘"production”), barcodes from vector genomes in the plasmid and produced AAV library' were prepared for illumina sequencing using two rounds of PCR. Production efficiency, normalized for presence in the input plasmid library, for each variant is expressed by comparing barcode sequencing levels for each variant in the produced vector pool to the barcode sequence levels for each variant in the input plasmid library used to create the vector pool. The measurements of variant

[0760] NAI-5007748234vl frequency in the vector library' also enable downstream normalization of biodistribution and transduction measurements by variant frequency in the input vector library. Data was prepared as described below. Production efficiency for variants in Library' Experiment 1 is reported in Table 1 A, and each reported value is reported relative to the production of wildtype AAV2.

[0761] In Vivo Evaluation of Library in Non-Human Primate

[0762] All NHP experiments were conducted in accordance with institutional policies and NIH guidelines. One young adult male and two young adult female Cynomolgus macaques (Macaca fascicularis) weighing 2-5 kg seronegative for serum anti-AAV2 neutralizing antibodies (serum NAb titers <=1 :4 based on in vitro NAb assay) were selected for the study. Prior to test article administrations samples of blood, aqueous humor (50 pL) and vitreous humor (up to 50 pL) were collected. The animals were anesthetized with ketamine and dexmedetomidine and received intravitreal (IVT; 3.48-4.09el 1 vg / eye in 50 pL) and intracameral (IC; 4.73-4.75el 1 vg / eye in 50 pL) injections of the vector libraries. During the in-life period the animals were monitored for signs of ocular inflammation via indirect ophthalmoscopy and slit-lamp biomicroscopy and treated with weekly IM injections of steroids (methylprednisolone, 40-80 mg), and topical steroids and atropine as needed according to the animal facility’s SOPs and recommendations from the veterinarian. Serum samples were collected 1, 24 and 48 hours post-dose, and at days 5, 8, 15, 22 and 29 postdose. The animals were sacrificed 4 weeks after the injections and tissues were collected for biodistribution and transduction analyses.

[0763] Tissue Processing and Data Analysis for Library Experiment 1

[0764] Posterior and anterior regions of the eyes were dissected as shown in FIG. 1. A list of other tissue samples collected is shown in Table 5. All samples were collected into RNAlater® (Sigma-Aldrich) and incubated overnight at RT, after which the RNAlater® was drained and samples were frozen at -80°C. In addition, samples of aqueous humor, vitreous humor, serum, and cerebrospinal fluid were collected at necropsy and stored at -80°C.

[0765] NAI-5007748234vl Table 5. List of tissues collected.

[0766] NAI-5007748234vl

[0767] For biodistribution and transduction analyses, total DNA and RNA was extracted from tissue samples with Trizol / chloroform followed by the use of the DNeasy 96 Blood & Tissue Kit (Qiagen) and Zymo RNA Clean & Concentrator 96 kit (Zymo). RNA samples were treated with TURBO DNase (Invitrogen). Reverse transcription was done with Protoscript II Reverse Transcriptase (NEB) with primers that were specific to the vector transgene and included unique molecular identifiers (UMIs). Control reactions lacking the reverse transcriptase enzyme (-RT control) were also prepared. Quantification of biodistribution and transduction was done...

Claims

CLAIMS1. A variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), wherein the variant capsid polypeptide comprises an insertion of an amino acid sequence comprising FTRP at a position corresponding to the position between positions 586 and 587 of SEQ ID NO: 1, or an insertion of an amino acid sequence comprising FTRP at a position corresponding to the position between positions 587 and 588 of SEQ ID NO: 1.

2. The variant capsid polypeptide of claim 1, wherein said variant capsid polypeptide further comprises an amino acid substitution at a position corresponding to position 587 of SEQ ID NO: 1.

3. The variant capsid polypeptide of claim 2, wherein said amino acid substitution is an N587A substitution.

4. A virus particle (e.g., adeno-associated virus (“AAV”) particle) comprising the variant capsid polypeptide of any one of claims 1-3.

5. The virus particle of claim 4, wherein said virus particle exhibits increased ocular transduction as measured in a mouse or in a non-human primate (NHP) relative to wildtype AAV2.

6. The virus particle of claim 5, wherein the wild-type AAV2 is a virus particle comprising capsid polypeptides of SEQ ID NO: 1.

7. The virus particle of claim 6, wherein the increased ocular transduction is increased transduction in the retina.

8. The virus particle of claim 4, wherein said virus particle exhibits at least a 1.5-fold preferential transduction in a cell of an inner nuclear layer (INL) of a retina of a NHP, relative to transduction in a cell of an outer nuclear layer (ONL) of a retina of a NHP.NAI-5007748234vl9. The virus particle of claim 4, wherein said virus particle exhibits increased ocular transduction as measured in a mouse or in a non-human primate (NHP) relative to a virus particle comprising an externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24.

10. The virus particle of claim 9, wherein the increased ocular transduction is increased transduction in the retina.

11. The virus particle of claim 10, wherein the increased transduction in the retina is increased transduction in the inner nuclear layer (INL) of the retina.

12. The virus particle of claim 11, wherein the increased transduction in the INL of the retina is increased transduction of bipolar cells of the INL.

13. A variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an insertion between positions 586 and 587, wherein the insertion comprises an amino acid sequence of a consensus formula X1-X2-X3-X4-X5-F-T-R-P (SEQ ID NO: 12), whereinXI is absent or selected from S, T, C, Y, N, or Q;X2 is absent or selected from G, A, V, L, I, F, W, M, or P;X3 is absent or selected from D or E;X4 is absent or selected from S, T, C, Y, N, or Q;X5 is absent or selected from G, A, V, L, I, F, W, M, or P.

14. The variant capsid polypeptide of claim 13, wherein the insertion comprises the amino acid sequence SLDQAFTRP (SEQ ID NO: 23), or a fragment of at least 4, at least 5, at least 6, at least 7, or at least 8 amino acids thereof, and wherein the variant capsid polypeptide further comprises an N587A substitution.NAI-5007748234vl15. A virus particle (e.g., adeno-associated virus (“AAV”) particle) comprising the variant capsid polypeptide of any one of claims 13 to 14.

16. The virus particle of claim 15, wherein said virus particle exhibits increased ocular transduction as measured in a mouse or in a non-human primate (NHP) relative to wildtype AAV2.

17. The virus particle of claim 16, wherein the wild-type AAV2 is a virus particle comprising capsid polypeptides of SEQ ID NO: 1.

18. The virus particle of claim 17, wherein the increased ocular transduction is increased transduction in the retina.

19. The virus particle of claim 15, wherein said virus particle exhibits at least a 1.5-fold preferential transduction in a cell of an inner nuclear layer (INL) of a retina of a NHP, relative to transduction in a cell of an outer nuclear layer (ONL) of a retina of a NHP.

20. The virus particle of claim 15, wherein said virus particle exhibits increased ocular transduction as measured in a mouse or in a non-human primate (NHP) relative to a virus particle comprising an externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24.

21. The virus particle of claim 20, wherein the increased ocular transduction is increased transduction in the retina.

22. The virus particle of claim 21, wherein the increased transduction in the retina is increased transduction in the inner nuclear layer (INL) of the retina.

23. The virus particle of claim 22, wherein the increased transduction in the INL of the retina is increased transduction of bipolar cells of the INL24. A variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identical to the amino acid sequence of positions 1-735 of SEQ ID NO: 1 (VP1), positions 138-735 of SEQ ID NO: 1 (VP2), or positions 203-735 of SEQ ID NO: 1 (VP3), comprising an amino acid substitution atNAI-5007748234vlposition 547, 548, 549, 550, 555, 561, 575, 578, 580, 581, 587, 589, 591, 593, or any combination thereof.

25. The variant capsid polypeptide of claim 24, wherein the amino acid substitution comprises an N587A substitution.

26. The variant capsid polypeptide of claim 25, wherein the variant capsid polypeptide further comprises an insertion of an amino acid sequence SLDQAFTRP (SEQ ID NO: 23) between positions 586 and 587.

27. A virus particle (e.g., adeno-associated virus (“AAV”) particle) comprising the variant capsid polypeptide of any one of claims 24 to 26.

28. The virus particle of claim 27, wherein said virus particle exhibits increased ocular transduction as measured in a mouse or in non-human primate (NHP) relative to wildtype AAV2.

29. The virus particle of claim 28, wherein the wild-type AAV2 is a virus particle comprising capsid polypeptides of SEQ ID NO: 1.

30. The virus particle of claim 29, wherein the increased ocular transduction is increased transduction in the retina.

31. The virus particle of claim 27, wherein said virus particle exhibits at least a 1.5-fold preferential transduction in a cell of an inner nuclear layer (INL) of a retina of a NHP, relative to transduction in a cell of an outer nuclear layer (ONL) of a retina of a NHP.

32. The virus particle of claim 27, wherein said virus particle exhibits increased ocular transduction as measured in a mouse or in a non-human primate (NHP) relative to a virus particle comprising an externally engineered capsid (EEC) polypeptide of SEQ ID NO: 24.

33. The virus particle of claim 32, wherein the increased ocular transduction is increased transduction in the retina.NAI-5007748234vl34. The virus particle of claim 33, wherein the increased transduction in the retina is increased transduction in the inner nuclear layer (INL) of the retina.

35. The virus particle of claim 34, wherein the increased transduction in the INL of the retina is increased transduction of bipolar cells of the INL. A nucleic acid molecule comprising a sequence encoding a variant capsid polypeptide of any one of claims 1-3, 13, 14, or 24-26.

36. A vector comprising the nucleic acid molecule of claim 35.

37. A host cell transformed with the vector of claim 36.

38. A method of producing a virus particle comprising a variant capsid polypeptide, comprising introducing the nucleic acid molecule of claim 35, or the vector of claim 36 into a host cell, and harvesting said virus particle therefrom.

39. A cell, cell-free system, or other translation system, comprising a first and a second nucleic acid molecule, wherein the first nucleic acid molecule is the nucleic acid molecule of claim 35 and the second nucleic acid molecule comprises a payload (e.g., a nucleic acid sequence encoding a therapeutic product).

40. A method of making a dependoparvovirus particle, comprising cultivating the cell, cell- free system, or other translation system of claim 39, under conditions suitable for the production of the dependoparvovirus particle.

41. The method of claim 40, wherein the first nucleic acid mediates the production of a dependoparvovirus particle, and the second nucleic acid molecule is packaged within said dependoparvovirus particle.

42. The method of claim 40 or 41, wherein the first nucleic acid mediates the production of a dependoparvovirus particle at a level at least 10%, at least 20%, at least 50%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, or greater than the production level mediated by the nucleic acid of SEQ ID NO: 2.

43. A method of delivering a payload (e.g., a nucleic acid) to a cell, comprising contactingNAI-5007748234vlthe cell with the virus particle of any of claims 4-9, 15-23, or 27-34, and the payload; or with the dependoparvovirus particle produced by the method of any one of claims 23-25.

44. The method of claim 43, wherein the cell is an ocular cell.

45. The method of claims 43 or 44, wherein the virus particle or dependoparvovirus particle delivers the payload with increased transduction in an ocular cell of one or more regions of an eye as compared to a virus particle comprising capsid polypeptides of SEQ ID NO: 1.

46. The method of claim 45, wherein the increase is 2-fold, 4-fold, 6-fold, 8-fold, 10-fold,12-fold, 15-fold, 16-fold, 18-fold, 19-fold, 20-fold, 21-fold, 24-fold, 25-fold, 26-fold,27-fold, 28-fold, 29-fold, 30-fold, 34-fold, 35-fold, 36-fold, 37-fold, 39-fold, 42-fold,45-fold, 46-fold, 47-fold, 49-fold, 51-fold, 52-fold, 58-fold, 59-fold, 61-fold, 65-fold,72-fold.

47. The method of claim 45 or 46, wherein the region of an eye is the retina, the macula, or the trabecular meshwork, or any combination thereof.

48. The method of claim 47, wherein the retina comprises non-macular retina.

49. The method of claim 45 or 46, wherein the region of an eye is the parafovea, the parafoveal nerve fibers, the peripheral nerve fibers, or any combination thereof.

50. A method of delivering a payload (e.g., a nucleic acid) to a subject comprising administering to the subject the virus particle of any one of claims 4-9, 15-23, or 27- 34, and the payload; or the dependoparvovirus particle produced by the method of any one of claims 40-42.

51. The method of claim 50, wherein the virus particle or dependoparvovirus particle delivers the payload to an eye.

52. The method of claim 50 or 51, wherein the virus particle or dependoparvovirus particle delivers the payload to an eye with increased transduction in one or more regions of anNAI-5007748234vleye as compared to a virus particle comprising capsid polypeptides of SEQ ID NO: 1.

53. The method of claim 52, wherein the increase is 2-fold, 4-fold, 6-fold, 8-fold, 10-fold,12-fold, 15-fold, 16-fold, 18-fold, 19-fold, 20-fold, 21-fold, 24-fold, 25-fold, 26-fold,27-fold, 28-fold, 29-fold, 30-fold, 34-fold, 35-fold, 36-fold, 37-fold, 39-fold, 42-fold,45-fold, 46-fold, 47-fold, 49-fold, 51-fold, 52-fold, 58-fold, 59-fold, 61-fold, 65-fold,72-fold.

54. The method of claim 52 or 53, wherein the region of an eye is the retina, the macula, the trabecular meshwork, or any combination thereof.

55. The method of claim 54, wherein the retina comprises non-macular retina.

56. The method of claim 52 or 53, wherein the region of an eye is the parafovea, the parafoveal nerve fibers, the peripheral nerve fibers, or any combination thereof.

57. The method of any one of claims 50-55, wherein the administration is via an intravitreal injection, or an intracam eral injection.

58. The virus particle of any one of claims 4-12, 15-23, and 27-34, or the method of any one claims 45-48, and 52-57, wherein the transduction is measured by next-generation sequencing (NGS) or by immunofluorescence staining.

59. A pharmaceutical composition comprising the virus particle of any one of claims 4-9, 15-23, or 27-34, and a payload; or the dependoparvovirus particle produced by the method of any one of claims 40-42, and a pharmaceutically acceptable carrier.

60. A method of treating a disease or condition in a subject, comprising administering to the subject the virus particle of any one of claims 4-9, 15-23, or 27-34, and a payload, in an amount effective to treat the disease or condition; or the dependoparvovirus particle produced by the method of any one of claims 40-42, in an amount effective to treat the disease or condition; or the pharmaceutical composition of claim 59, in an amount effective to treat the disease or condition.

61. The variant capsid polypeptide of any of claims 1-3, 13, 14, or 24-26, the nucleic acidNAI-5007748234vlmolecule of claim 35, the vector of claim 36, the virus particle of any of claims 4-9, 15- 23, or 27-34; the dependoparvovirus particle produced by the method of any one of claims 40-42; or the pharmaceutical composition of claim 59, for use in treating a disease or condition in a subject.

62. The variant capsid polypeptide of any of claims 1-3, 13, 14, or 24-26, the nucleic acid molecule of claim 35, the vector of claim 36, the virus particle of any of claims 4-9, 15- 23, or 27-34; the dependoparvovirus particle produced by the method of any one of claims 40-42; or the pharmaceutical composition of claim 59, for use in the manufacture of a medicament for use in treating a disease or condition in a subject.NAI-5007748234vl

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