Capsid variants and methods of use thereof

Abstract

The present disclosure relates, in part, to variant capsid polypeptides that can be used to deliver payloads.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 331,543, filed on April 15, 2022, and U.S. Provisional Application No. 63 / 443,262, filed on February 3, 2023, each of which is hereby incorporated by reference in its entirety.

[0003] Reference to a sequence listing submitted electronically

[0004] This application contains a sequence listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on April 14, 2023, named "DYO-015WOSEQ.XML", and is 119,720 bytes in size. Background Art

[0005] Dependoparvoviruses, such as adeno-associated dependoparvoviruses, such as adeno-associated virus (AAV), have gained attention as vectors for delivering various payloads to cells, including human subjects. Summary of the invention

[0006] The present disclosure provides, in part, improved variant parvovirus capsid polypeptides such as VP1 (e.g., variants of AAV2), methods of producing parvoviruses, compositions for use in the methods, and viral particles comprising such capsid polypeptides. In some embodiments, the viral particles comprising the capsid polypeptides have increased ocular transduction compared to viral particles without mutations in the capsid protein.

[0007] In some embodiments, the disclosure relates in part to a nucleic acid comprising a sequence encoding a variant capsid protein as provided herein. In some embodiments, the parvovirus is an adeno-associated parvovirus (AAV). In some embodiments, the AAV is an AAV2 variant.

[0008] In some embodiments, the present disclosure relates in part to a capsid polypeptide described herein.

[0009] In some embodiments, the present disclosure relates in part to a parvoviral particle comprising a capsid polypeptide described herein.

[0010] In some embodiments, the disclosure relates in part to a vector, such as a plasmid, comprising a nucleic acid described herein.

[0011] In some embodiments, the disclosure relates in part to a parvoviral 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 an alteration or mutation as provided herein.

[0012] In some embodiments, the present disclosure relates in part to a parvoviral particle comprising a variant capsid polypeptide described herein, which variant capsid polypeptide, for example, comprises a polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.

[0013] In some embodiments, the present disclosure relates in part to a parvoviral virus particle comprising a variant capsid polypeptide described herein, such as a polypeptide comprising the VP1, VP2 or VP3 sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.

[0014] In some embodiments, the capsid polypeptide comprises a mutation selected from the mutations associated with any one of VAR-1 to VAR- 16. In some embodiments, the capsid polypeptide comprises more than one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or all, of the mutations selected from the mutations associated with any one of VAR-1 to VAR-16.

[0015] In some embodiments, the capsid polypeptide comprises an amino acid sequence having 95% or greater amino acid sequence identity to an amino acid sequence of one of SEQ ID NOs: 12-27, and having at least 80% of the mutations in one of SEQ ID NOs: 12-27 compared to SEQ ID NO:1.

[0016] In some embodiments, the capsid polypeptide comprises an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of one of SEQ ID NOs: 12-27, and having at least 80% of the mutations in one of SEQ ID NOs: 12-27 compared to SEQ ID NO:1.

[0017] In some embodiments, the capsid polypeptide comprises an amino acid sequence having 95% or greater amino acid sequence identity to an amino acid sequence of one of SEQ ID NOs: 12-27, and having less than 80% of the mutations in one of SEQ ID NOs: 12-27 compared to SEQ ID NO:1.

[0018] In some embodiments, the disclosure relates in part to a nucleic acid molecule comprising SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, a fragment thereof, or a variant thereof having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity thereto.

[0019] In some embodiments, the disclosure relates in part to a vector comprising a nucleic acid described herein, e.g., a nucleic acid comprising a sequence encoding a capsid polypeptide (e.g., a VP1 polypeptide), wherein the coding sequence comprises an alteration or mutation as provided herein.

[0020] In some embodiments, the disclosure relates in part to a cell, cell-free system, or other translation system comprising a nucleic acid or vector as described herein, e.g., comprising a sequence encoding a capsid polypeptide such as VP1, wherein the capsid polypeptide coding sequence comprises an alteration or mutation in the coding sequence as provided herein. In some embodiments, the cell, cell-free system, or other translation system comprises a parvoviral particle as described herein, e.g., wherein the particle comprises a nucleic acid comprising a sequence encoding a capsid polypeptide such as VP1 polypeptide, wherein the coding sequence comprises an alteration or mutation as provided herein.

[0021] In some embodiments, the disclosure relates in part to a cell, cell-free system or other translation system comprising a polypeptide described herein, wherein the polypeptide coding sequence comprises an alteration or mutation as provided herein. In some embodiments, the cell, cell-free system or other translation system comprises a parvoviral particle described herein, e.g., wherein the particle comprises a nucleic acid comprising a sequence encoding a VP1 polypeptide, wherein the VP1 coding sequence comprises an alteration or mutation corresponding to as provided herein.

[0022] In some embodiments, the present disclosure relates in part to a method of delivering a payload to a cell, the method comprising contacting the cell with a parvoviral particle comprising a nucleic acid described herein. In some embodiments, the present disclosure relates in part to a method of delivering a payload to a cell, the method comprising contacting the cell with a parvoviral particle comprising a capsid polypeptide described herein.

[0023] In some embodiments, the present disclosure relates in part to a method of preparing a parvoviral particle, the method comprising providing a cell, a cell-free system, or other translation system comprising a nucleic acid described herein (e.g., a nucleic acid comprising a sequence encoding an AAV2 capsid variant as provided herein); and culturing the cell, cell-free system, or other translation system under conditions suitable for producing the parvoviral particle, thereby preparing the parvoviral particle. In some embodiments, the present disclosure relates in part to a method of preparing a parvoviral particle described herein.

[0024] In some embodiments, the present disclosure relates in part to a method of preparing a parvoviral particle, the method comprising providing a cell, a cell-free system, or other translation system comprising a polypeptide described herein; and culturing the cell, the cell-free system, or other translation system under conditions suitable for producing the parvoviral particle, thereby preparing the parvoviral particle. In some embodiments, the present disclosure relates in part to a method of preparing a parvoviral particle described herein.

[0025] In some embodiments, the disclosure relates in part to a parvovirus particle prepared in a cell, cell-free system, or other translation system, wherein the cell, cell-free system, or other translation system comprises a nucleic acid encoding a parvovirus comprising a capsid variant as provided herein.

[0026] In some embodiments, the present disclosure relates in part to a method of treating a disease or condition in a subject, the method comprising administering to the subject a parvoviral particle described herein in an amount effective to treat the disease or condition.

[0027] The invention is further described with reference to the following numbered examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 . Tissue diagrams collected in each area of ​​the eye. In the retina (left and middle), peripheral and central retinal samples from each of the upper, nasal, lower and temporal regions of the retina were collected, and the macula was also collected. In each area, the neural retina and choroid / RPE layer were collected (middle). In the trabecular meshwork / Schlemm's canal (TM / SC) area (right), upper, temporal, nasal and lower samples were collected, respectively.

[0029] FIG. 2A to FIG. 2C Multiple sequence alignment of representative reference capsid VP1 polypeptides. This alignment can be used to determine amino acid positions that correspond to positions within different reference capsid polypeptides.

[0030] FIG. 3A to FIG. 3B AAV2 wild-type ( Figure 3A ) and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8 ​​and VAR-11( Figure 3B ) single nucleus RNA sequencing results, reporting the number of unique transduction events for AAV2 wild type and each of VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11. All results were normalized to the amount of viral reads.

[0031] FIG. 4A to FIG. 4B AAV2 wild-type ( Figure 4A ) and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8 ​​and VAR-11( Figure 4B ), reporting the number of unique transduction events for AAV2 wild type and each of VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11. All results were normalized to the amount of vector genomes (vg) in the input test article.

[0032] Figure 5 Single nucleus RNA sequencing results for VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11 of intravitreally ("IVT") administered macular tissue samples from a medium throughput study (Example 3), reporting the number of unique transduction events for each of the variants. All results were normalized to the amount of vector genomes (vg) in the input test article. Results indicate zero transduction with AAV2 wild-type (not shown) for any of the listed cell types.

[0033] Figure 6 Single nucleus RNA sequencing results for VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11 of intravitreal ("IVT") administered trabecular meshwork tissue samples from a medium throughput study (Example 3), reporting the number of unique transduction events for each of the variants. All results were normalized to the amount of vector genomes (vg) in the input test article. Results indicate zero transduction for AAV2 wild-type (not shown), VAR-5, and VAR-11 for any of the listed cell types.

[0034] FIG. 7A to FIG. 7B AAV2 wild-type ( Fig. 7A ) and VAR-2, VAR-3, VAR-13 and VAR-14( Figure 7B ) single nucleus RNA sequencing results, reporting the number of unique transduction events for each of the variants. All results were normalized to the amount of vector genomes (vg) in the input test article. The results showed that VAR-2 had zero transduction for any of the listed cell types.

[0035] Figure 8 . Retinal distribution of AAV VAR-8-eGFP in cynomolgus monkeys following intravitreal ocular injection at a higher dose (2.26e11 vg). The genome packaged in the VAR-8 ​​capsid is a self-complementary genome consisting of a modified AAV2 ITR and an eGFP reporter gene under the control of the CBH promoter. Animals were sacrificed 4 weeks after dosing. Eyes were enucleated, fixed, divided into two halves (top and bottom) and embedded in paraffin for histological sectioning. (A) Schematic diagram showing the locations where cross sections of the retina were collected. Briefly, 5 μm sections of the top surface of the lower half of the eye were collected and stained for eGFP. (B) Representative images of the entire cross section of the collected eye. eGFP appears as white dots on the retina. Arrows indicate the starting position of the close-up retinal image shown in (C). (C) Close-up of retinal images from each end of the eye linearly stitched together as shown in (B). White dots represent eGFP expression delivered from VAR-8. ONL = outer nuclear layer / photoreceptor layer, INL = inner nuclear layer, GCL = ganglion cell layer.

[0036] Fig. 9. Retinal distribution of AAV VAR-8-eGFP in cynomolgus monkeys following intravitreal ocular injection at a lower dose (8.08e10vg). The genome packaged in the VAR-8 ​​capsid is a self-complementary genome consisting of a modified AAV2 ITR and an eGFP reporter gene under the control of the CBH promoter. Animals were sacrificed 4 weeks after dosing. The eyes were enucleated, fixed, divided into two halves (top and bottom) and embedded in paraffin for histological sectioning. (A) Schematic diagram showing the locations where cross sections of the retina were collected. Briefly, 5 μm sections were collected from approximately 1 / 3 of the lower half of the eye and stained for eGFP. (B) Representative images of the entire cross section of the collected eye. eGFP appears as white dots on the retina. Arrows indicate the starting position of the close-up retinal image shown in (C). (C) Close-up of retinal images from each end of the eye linearly stitched together as shown in (B). The white dots represent eGFP expression delivered from VAR-8. ONL = outer nuclear layer / photoreceptor cell layer, INL = inner nuclear layer, GCL = ganglion cell layer.

[0037] Examples of Examples

[0038] 1. A variant capsid polypeptide comprising a polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:27.

[0039] 2. The variant capsid polypeptide according to embodiment 1, wherein the polypeptide comprises:

[0040] A mutation selected from the group consisting of mutations associated with any one of VAR-1 to VAR-16.

[0041] 3. The variant capsid polypeptide of embodiment 2, wherein:

[0042] The mutation associated with any one of VAR-1 to VAR-16 includes mutations at positions corresponding to residues 550-597 compared to SEQ ID NO:1.

[0043] 4. The variant capsid polypeptide of any of the preceding embodiments, wherein the polypeptide comprises a sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1 and comprises a mutation selected from the mutations associated with any one of VAR-1 to VAR-16.

[0044] 5. The variant capsid polypeptide according to any of the preceding embodiments, wherein the polypeptide comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1, and wherein the variant capsid polypeptide comprises a mutation corresponding to a mutation at position 550, 559, 561, 586, 587, 592, 593, 597, or any combination thereof, an insertion between positions 584 and 585, between 586 and 587, or between 587 and 588, or any combination thereof, according to SEQ ID NO: 1, optionally wherein the mutation comprises an insertion, a deletion, or a substitution.

[0045] 6. The variant capsid polypeptide according to any one of the preceding embodiments, wherein the capsid polypeptide comprises a mutation corresponding to an insertion at a position between positions 587 and 588 compared to SEQ ID NO: 1.

[0046] 7. The variant capsid polypeptide according to any one of the preceding embodiments, wherein the capsid polypeptide comprises a mutation corresponding to an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1.

[0047] 8. The variant capsid polypeptide of any preceding embodiment, wherein the capsid polypeptide comprises a mutation corresponding to a mutation at position 587 compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1.

[0048] 9. The variant capsid polypeptide of any preceding embodiment, wherein the capsid polypeptide comprises mutations corresponding to mutations at positions 587 and 593 compared to SEQ ID NO: 1, and an insertion between positions 586 and 587 compared to SEQ ID NO: 1.

[0049] 10. The variant capsid polypeptide of any preceding embodiment, wherein the capsid polypeptide comprises mutations corresponding to mutations at positions 586 and 587 compared to SEQ ID NO: 1, and an insertion between positions 584 and 585 compared to SEQ ID NO: 1.

[0050] 11. The variant capsid polypeptide of any preceding embodiment, wherein the capsid polypeptide comprises mutations corresponding to mutations at positions 592 and 597 compared to SEQ ID NO: 1, and an insertion between positions 587 and 588 compared to SEQ ID NO: 1.

[0051] 12. The variant capsid polypeptide of any preceding embodiment, wherein the capsid polypeptide comprises mutations corresponding to mutations at positions 561, 587, and 597 compared to SEQ ID NO: 1, and an insertion between positions 586 and 587 compared to SEQ ID NO: 1.

[0052] 13. The variant capsid polypeptide of any preceding embodiment, wherein the capsid polypeptide comprises mutations corresponding to mutations at positions 550, 586, and 587 compared to SEQ ID NO: 1, and an insertion between positions 584 and 585 compared to SEQ ID NO: 1.

[0053] 14. The variant capsid polypeptide of any preceding embodiment, wherein the capsid polypeptide comprises mutations corresponding to mutations at positions 559 and 587 compared to SEQ ID NO: 1, and an insertion between positions 586 and 587 compared to SEQ ID NO: 1.

[0054] 15. The variant capsid polypeptide according to any of the preceding embodiments, wherein the capsid polypeptide comprises an insertion, such as an insertion of 1 or more amino acids, such as 1 amino acid, such as 1-2 amino acids, which corresponds to an insertion between 584 and 585, between 586 and 587, or between 587 and 588 compared to SEQ ID NO: 1.

[0055] 16. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises the polypeptide of LALGEQTRPA (SEQ ID NO: 44), or a fragment thereof of at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids.

[0056] 17. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises an insertion at a position between positions 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of LAIEQTRPA (SEQ ID NO: 45), or a fragment thereof of at least 5, at least 6, at least 7, or at least 8 amino acids.

[0057] 18. A variant capsid polypeptide according to any of the preceding embodiments, wherein the capsid polypeptide comprises a mutation of N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises the polypeptide of LALAEITRP (SEQ ID NO: 46), or a fragment thereof of at least 5, at least 6, at least 7 or at least 8 amino acids.

[0058] 19. A variant capsid polypeptide according to any of the preceding embodiments, wherein the capsid polypeptide comprises a mutation of N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises a polypeptide of LKNAETARP (SEQ ID NO: 47), or a fragment thereof of at least 5, at least 6, at least 7 or at least 8 amino acids.

[0059] 20. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations of N587A and A593T compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises a polypeptide of LNLAIEQTRP (SEQ ID NO: 48), or a fragment of at least 5, at least 6, at least 7, or at least 8 amino acids thereof.

[0060] 21. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises a mutation of N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises a polypeptide of MLNEQTRP (SEQ ID NO: 49), or a fragment of at least 4, at least 5, at least 6, or at least 7 amino acids thereof.

[0061] 22. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations G586P and N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 584 and 585, wherein the insertion comprises a polypeptide of RSGNRADSETA (SEQ ID NO: 50), or a fragment of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids thereof.

[0062] 23. A variant capsid polypeptide according to any of the preceding embodiments, wherein the capsid polypeptide comprises a mutation of N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises a polypeptide of TGDTRP (SEQ ID NO: 51), or a fragment thereof of at least 3, at least 4, or at least 5 amino acids.

[0063] 24. The variant capsid polypeptide of any preceding embodiment, wherein the capsid polypeptide comprises an insertion at a position between positions 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of LQGETIRPA (SEQ ID NO: 52), or a fragment of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids thereof.

[0064] 25. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises a mutation of N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises a polypeptide of QNLANPETTRP (SEQ ID NO: 53), or a fragment of at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids thereof.

[0065] 26. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations of T592A and T597W compared to SEQ ID NO: 1, and an insertion at a position between positions 587 and 588, wherein the insertion comprises a polypeptide of RAPQETTRPA (SEQ ID NO: 54), or a fragment of at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids thereof.

[0066] 27. A variant capsid polypeptide according to any of the preceding embodiments, wherein the capsid polypeptide comprises a mutation of N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises a polypeptide of ANLTTTRP (SEQ ID NO: 55), or a fragment thereof of at least 4, at least 5, at least 6 or at least 7 amino acids.

[0067] 28. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises a mutation of N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises a polypeptide of ALLAGEQTRP (SEQ ID NO: 56), or a fragment thereof of at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids.

[0068] 29. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations D561C, N587A and T597N compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises a polypeptide of GLRAEQTRP (SEQ ID NO: 57), or a fragment of at least 5, at least 6, at least 7 or at least 8 amino acids thereof.

[0069] 30. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations of T550N, G586P and N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 584 and 585, wherein the insertion comprises a polypeptide of RARLDETA (SEQ ID NO: 58), or a fragment of at least 4, at least 5, at least 6 or at least 7 amino acids thereof.

[0070] 31. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations of I559L and N587A compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587, wherein the insertion comprises a polypeptide of TNLARGETARP (SEQ ID NO: 59), or a fragment of at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids thereof.

[0071] 32. A variant capsid polypeptide comprising (a) a polypeptide of any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27, (b) a polypeptide of any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: NO:27, (c) a polypeptide comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, wherein the sequence comprises at least one (e.g., one, two, three or more, such as all) of the mutational differences associated with any one of SEQ ID NO:12 to SEQ ID NO:27 relative to SEQ ID NO:1; or (d) a polypeptide having at least 1 but no more than 20, no more than 19, no more than 18, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, 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 3 or no more than 2 amino acid mutations relative to the polypeptide of (a) or (b), wherein the polypeptide comprises a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, wherein the sequence comprises at least one (e.g., one, two, three or more, such as all) of the mutational differences associated with any one of SEQ ID NO:12 to SEQ ID NO:27 relative to SEQ ID NO:1; At least one (e.g., one, two, three or more, such as all) of the mutational differences associated with any one of NO:27.

[0072] 33. The variant capsid polypeptide according to any one of the preceding embodiments, wherein the variant capsid polypeptide is a VP1 polypeptide, a VP2 polypeptide or a VP3 polypeptide.

[0073] 34. A variant capsid polypeptide comprising:

[0074] has 95% or greater amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs: 12-27; and

[0075] An amino acid sequence having at least 80% of the mutations in the amino acid sequence of one of SEQ ID NOs: 12-27 compared to SEQ ID NO: 1.

[0076] 35. A variant capsid polypeptide comprising:

[0077] has less than 95% amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs: 12-27; and

[0078] An amino acid sequence having at least 80% of the mutations in the amino acid sequence of one of SEQ ID NOs: 12-27 compared to SEQ ID NO: 1.

[0079] 36. A variant capsid polypeptide comprising:

[0080] has 95% or greater amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs: 12-27; and

[0081] An amino acid sequence having less than 80% of the mutations in the amino acid sequence of one of SEQ ID NOs: 12-27 compared to SEQ ID NO: 1.

[0082] 37. A nucleic acid molecule comprising a sequence encoding the variant capsid polypeptide of any one of embodiments 1 to 36.

[0083] 38. The nucleic acid molecule of embodiment 37, comprising one or more regulatory elements operably linked to the sequence encoding the variant capsid polypeptide.

[0084] 39. The nucleic acid molecule of any one of embodiments 37 to 38, comprising SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 or 43, or a fragment thereof, or a variant thereof having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0085] 40. A viral particle (e.g., an adeno-associated virus ("AAV") particle) comprising a variant capsid polypeptide according to any one of embodiments 1 to 36 or comprising a variant capsid polypeptide encoded by a nucleic acid molecule according to any one of embodiments 37 to 39.

[0086] 41. The viral particle of embodiment 40, comprising a nucleic acid comprising a heterologous transgene and one or more regulatory elements.

[0087] 42. The viral particle of any one of embodiments 40 to 41, comprising a variant capsid polypeptide of any one of embodiments 1 to 36, wherein the viral particle, or a viral particle comprising the variant capsid polypeptide, or a viral particle comprising a variant capsid polypeptide encoded by a nucleic acid molecule of any one of embodiments 37 to 39, exhibits increased ocular transduction relative to wild-type AAV2 (e.g., a viral particle comprising a capsid polypeptide of SEQ ID NO: 1 or a capsid polypeptide encoded by SEQ ID NO: 2), e.g., as measured in mice or NHPs, e.g., as described herein.

[0088] 43. The nucleic acid molecule of any one of embodiments 37 to 39 or the viral particle of any one of embodiments 40 to 42, wherein the nucleic acid molecule is double-stranded or single-stranded, optionally wherein the nucleic acid molecule is linear or circular, for example, wherein the nucleic acid molecule is a plasmid.

[0089] 44. A method for producing viral particles comprising a variant AAV2 capsid polypeptide, the method comprising introducing a nucleic acid molecule according to any one of embodiments 37 to 39 or 43 into a cell (e.g., a HEK293 cell) and harvesting the viral particles therefrom.

[0090] 45. A method of delivering a payload (e.g., a nucleic acid) to a cell, the method comprising contacting the cell with a parvoviral particle comprising a variant capsid polypeptide according to any one of embodiments 1 to 36 or a viral particle according to any one of embodiments 40 to 42 and a payload.

[0091] 46. ​​The method of embodiment 45, wherein the cell is an ocular cell.

[0092] 47. The method of embodiment 46, wherein the ocular cell is located in the retina, macula, or trabecular meshwork.

[0093] 48. A method of delivering a payload (e.g., a nucleic acid) to a subject, the method comprising administering to the subject a parvoviral particle comprising a variant capsid polypeptide according to any one of embodiments 1 to 36 and the payload, or administering to the subject a viral particle according to any one of embodiments 40 to 42.

[0094] 49. The method of embodiment 48, wherein the particles deliver the payload to the eye.

[0095] 50. The method of embodiment 48, wherein the particles deliver the payload to the retina, macula, or trabecular meshwork.

[0096] 51. The method of any one of embodiments 48 to 50, wherein the particle delivers the payload to the eye with increased transduction in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1.

[0097] 52. The method of embodiment 51, wherein the one or more regions of the eye are selected from the retina, the macula, the trabecular meshwork, or any combination thereof.

[0098] 53. The method of embodiment 51, wherein the retina comprises a non-macular retina.

[0099] 54. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 128-fold, 200-fold, 300-fold, 400-fold, 500-fold, or 1000-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1.

[0100] 55. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold or 32-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to non-macular retinal tissue relative to macular tissue.

[0101] 56. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold or 128-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to macular tissue relative to non-macular retinal tissue.

[0102] 57. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to macular tissue relative to trabecular meshwork tissue.

[0103] 58. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold or 32-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to non-macular retinal tissue relative to trabecular meshwork tissue.

[0104] 59. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the (e.g., particle comprising the variant capsid polypeptide) particle delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold or 32-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to macular tissue and non-macular retinal tissue relative to trabecular meshwork tissue.

[0105] 60. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to trabecular meshwork tissue relative to macular tissue and non-macular retinal tissue.

[0106] 61. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to trabecular meshwork tissue relative to macular tissue.

[0107] 62. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold or 16-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to trabecular meshwork tissue relative to non-macular retinal tissue.

[0108] 63. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to trabecular meshwork tissue, macular tissue, and non-macular retinal tissue.

[0109] 64. The variant capsid polypeptide of any one of embodiments 1 to 36, the viral particle of any one of embodiments 40 to 42, or the method of any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, without increased biodistribution in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1.

[0110] 65. The method of any one of embodiments 45 to 53, wherein administration to the subject is via intravitreal injection or intracameral injection.

[0111] 66. A method of treating a disease or condition in a subject, the method comprising administering to the subject a parvoviral particle in an amount effective to treat the disease or condition, wherein the parvoviral particle is a particle comprising a capsid polypeptide according to any one of embodiments 1 to 36 and 54 to 64 or a capsid polypeptide encoded by a nucleic acid according to any one of embodiments 37 to 39 or 43, or is a viral particle according to any one of embodiments 40 to 42.

[0112] 67. A cell, cell-free system or other translation system comprising the capsid polypeptide, nucleic acid molecule or viral particle of any one of embodiments 1 to 43 or 54 to 64.

[0113] 68. A method of preparing parvovirus (e.g., adeno-associated parvovirus (AAV)) particles, the method comprising:

[0114] Providing a cell, cell-free system or other translation system comprising a nucleic acid according to any one of embodiments 37 to 39 or 43; and

[0115] culturing the cell, cell-free system or other translation system under conditions suitable for production of the parvoviral particle,

[0116] Thus, the parvovirus particles are prepared.

[0117] 69. The method of embodiment 68, wherein the cell, cell-free system or other translation system comprises a second nucleic acid molecule and the second nucleic acid molecule is packaged in the parvoviral particle.

[0118] 70. The method of embodiment 68, wherein the second nucleic acid comprises a payload, such as a heterologous nucleic acid sequence encoding a therapeutic product.

[0119] 71. The method of any one of embodiments 68 to 70, wherein the nucleic acid of any one of embodiments 37 to 39 or 43 mediates the production of parvoviral-dependent particles that do not comprise the nucleic acid of any one of embodiments 37 to 39 or 43.

[0120] 72. The method of any one of embodiments 68 to 71, wherein the nucleic acid of any one of embodiments 37 to 39 or 43 mediates production of parvoviral-dependent particles at a level that is at least 10%, at least 20%, at least 50%, at least 100%, at least 200% or more than the level of production mediated by the nucleic acid of SEQ ID NO: 2.

[0121] 74. A composition, e.g., a pharmaceutical composition, comprising the viral particle of any one of embodiments 40 to 42 or the viral particle produced by the method of any one of embodiments 43 or 68 to 72, and a pharmaceutically acceptable carrier.

[0122] 75. The variant capsid polypeptide of any one of embodiments 1 to 36 and 54 to 64, the nucleic acid molecule of any one of embodiments 37 to 39 or 43, or the viral particle of any one of embodiments 40 to 42 for use in treating a disease or condition in a subject.

[0123] 76. A variant capsid polypeptide according to any one of embodiments 1 to 36 and 54 to 64, a nucleic acid molecule according to any one of embodiments 37 to 39 or 43, or a viral particle according to any one of embodiments 40 to 42, for use in the manufacture of a medicament for treating a disease or condition in a subject. DETAILED DESCRIPTION

[0124] The present disclosure relates in part to capsid polypeptides and parvoviral particles comprising the same. In some embodiments, the parvoviral particles have increased ocular transduction compared to parvoviral particles without variant capsid polypeptides and can be used to deliver transgenes or molecules of interest to the eye with higher transduction efficiency in the eye. Thus, provided herein are capsid polypeptides, nucleic acid molecules encoding the same, viral particles comprising variant capsid polypeptides, and methods of making and using the same.

[0125] definition

[0126] A, An, The: As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0127] About, approximately: As used herein, the terms "about" and "approximately" generally mean an acceptable degree of error for a measured amount given the nature or precision of the measured values. Exemplary degrees of error are within 15 percent (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0128] Dependaviral capsid: As used herein, the term "dependaviral capsid" refers to an assembled viral capsid comprising a Dependaviral polypeptide. In some embodiments, the Dependaviral capsid is a functional Dependaviral capsid, e.g., fully folded and / or assembled, capable of infecting a target cell, or remains stable (e.g., folded / assembled and / or capable of infecting a target cell) for at least a threshold time.

[0129] Dependaviviral particle: As used herein, the term "dependaviviral particle" refers to an assembled viral capsid comprising a dependaviviral polypeptide and a packaged nucleic acid, e.g., comprising a payload, one or more components of a dependaviviral genome (e.g., a complete dependaviviral genome), or both. In some embodiments, the dependaviviral particle is a functional dependaviviral particle, e.g., comprising a desired payload, fully folded and / or assembled, capable of infecting a target cell, or remaining stable (e.g., folded / assembled and / or capable of infecting a target cell) for at least a threshold time.

[0130] Dependaviridovirus X particles / capsids: As used herein, the term "dependaviridovirus X particles / capsids" refers to a dependaviridovirus particle / capsid comprising at least one polypeptide or polypeptide-encoding nucleic acid sequence derived from a naturally occurring dependaviridovirus X species. For example, a dependaviridovirus B particle refers to a dependaviridovirus particle comprising at least one polypeptide or polypeptide-encoding nucleic acid sequence derived from a naturally occurring dependaviridovirus B sequence. As used herein, derived from means having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the sequence in question. Accordingly, 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 AAV X 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.

[0131] Exogenous: As used herein, the term "exogenous" refers to a feature, sequence, or component present in an environment (e.g., in a nucleic acid, polypeptide, or cell) that does not naturally occur in that environment. For example, a nucleic acid sequence comprising a mutant capsid polypeptide, or a nucleic acid molecule encoding the same, may comprise a capsid polypeptide. Using the term exogenous in this manner means that the polypeptide or nucleic acid molecule encoding the polypeptide comprising the mutation in question at that position does not naturally occur, e.g., does not exist in AAV2, e.g., does not exist in SEQ ID NO: 1.

[0132] Functionality: As used herein, with respect to a polypeptide component of a parvoviral capsid (e.g., Cap (e.g., VP1, VP2 and / or VP3) or Rep), the term "functional" refers to a polypeptide that provides at least 50%, 60%, 70%, 80%, 90%, or 100% of the activity of the naturally occurring form of the polypeptide component (e.g., when present in a host cell). For example, a functional VP1 polypeptide can stably fold and assemble into a parvoviral capsid (e.g., it is capable of packaging and / or secretion). As used herein, with respect to a parvoviral capsid or particle, "functional" refers to a capsid or particle that comprises one or more of the following production characteristics: comprises a desired payload, is fully folded and / or assembled, is capable of infecting a target cell, or remains stable (e.g., folded / assembled and / or capable of infecting a target cell) for at least a threshold time.

[0133] 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, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As is clear from the context, in some embodiments, "nucleic acid" refers to a single nucleic acid monomer (e.g., a nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising a single nucleic acid monomer or a longer polynucleotide chain comprising many single nucleic acid monomers. In some embodiments, "nucleic acid" is or comprises RNA; in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, nucleic acid is one or more natural nucleic acid residues, comprises one or more natural nucleic acid residues, or consists thereof. In some embodiments, nucleic acid is one or more nucleic acid analogs, comprises one or more nucleic acid analogs, or consists thereof. In some embodiments, nucleic acid is one or more modified, synthetic or non-naturally occurring nucleotides, comprises one or more modified, synthetic or non-naturally occurring nucleotides, or consists thereof. In some embodiments, nucleic acid analogs differ from nucleic acids in that they do not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds, and are considered to be within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite bonds instead of phosphodiester bonds. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product such as an RNA or a protein. In some embodiments, the nucleic acid is partially or entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded.

[0134] Variant: As used herein, "variant capsid polypeptide" refers to a polypeptide that is different from a reference sequence (e.g., SEQ ID NO: 1). A variant may, for example, comprise a mutation (e.g., a 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.

[0135] Capsid polypeptide and nucleic acid encoding the same

[0136] The present disclosure relates in part to capsid polypeptides comprising mutations (insertions, deletions or substitutions) compared to the wild-type sequence, viral particles comprising variant capsid polypeptides (such as those described herein), 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 present disclosure relates in part to variant capsid polypeptides comprising SEQ ID NO: 1 having one or more mutations compared to SEQ ID NO: 1. The mutation may be, for example, an insertion, deletion or substitution compared to the wild-type sequence. In some embodiments, the wild-type sequence is SEQ ID NO: 1. The present disclosure relates in part to a variant capsid polypeptide comprising any one of SEQ ID NO: 12 to SEQ ID NO: 27. The present disclosure relates in part to a variant capsid polypeptide comprising a VP1 sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 27. The present disclosure relates in part to a variant capsid polypeptide comprising a VP2 sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 27. The present disclosure relates in part to a variant capsid polypeptide comprising the VP3 sequence of any one of SEQ ID NO:12 to SEQ ID NO:27.

[0137] In some embodiments, the capsid polypeptide comprises a mutation selected from the mutation differences disclosed in any one of Tables 1A-1G, e.g., selected from the mutation differences associated with any variant disclosed in any one of Tables 1A-1G. In some embodiments, the mutation differences disclosed in any one of Tables 1A-1G, e.g., selected from the mutation differences associated with any variant disclosed in any one of Tables 1A-1G, comprise mutations at positions corresponding to residues 550-597 compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation within the 550-597 amino acid region of SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation selected from the mutation differences disclosed in any one of Tables 1A-1G, e.g., selected from the mutation differences associated with any variant disclosed in any one of Tables 1A-1G. In some embodiments, a mutation selected from the mutation differences disclosed in any one of Tables 1A-1G, e.g., a mutation selected from the mutation differences associated with any variant disclosed in any one of Tables 1A-1G, is a substitution, e.g., a substitution of 2 or more residues corresponding to a substitution at a position between 550 and 597 compared to SEQ ID NO: 1. In some embodiments, a mutation selected from the mutation differences disclosed in any one of Tables 1A-1G, e.g., a mutation selected from the mutation differences associated with any variant disclosed in any one of Tables 1A-1G, is a substitution and further comprises at least one other mutation between positions 550 and 597, wherein the mutation is a substitution, insertion, or deletion. In some embodiments, a mutation selected from the mutation differences disclosed in any one of Tables 1A-1G, for example, a mutation selected from the mutation differences associated with any variant disclosed in any one of Tables 1A-1G is: an insertion, for example, an insertion of 1 or more amino acids (e.g., 1 amino acid, for example 1-2 amino acids), corresponding to an insertion between positions 584 and 585, between 586 and 587, or between 587 and 588 compared to SEQ ID NO.1; and a substitution, for example, a substitution of 2 or more residues, corresponding to a substitution at a position between 550 and 597 compared to SEQ ID NO:1.

[0138] In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 550, 559, 561, 586, 587, 592, 593, 597, or any combination thereof, an insertion between positions 584 and 585, between 586 and 587, or between 587 and 588, or any combination thereof, according to SEQ ID NO: 1, and optionally wherein the mutation comprises an insertion, a deletion, or a substitution.

[0139] In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 550 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 559 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 561 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 586 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 587 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 592 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 593 as compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 597 as compared to SEQ ID NO: 1.

[0140] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion at a position between positions 584 and 585 compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion at a position between positions 587 and 588 compared to SEQ ID NO: 1.

[0141] In some embodiments, the capsid polypeptide comprises a mutation according to SEQ ID NO: 1 corresponding to a mutation at position 550, 559, 561, 586, 587, 592, 593, 597, or any combination thereof, an insertion between positions 584 and 585, between 586 and 587, or between 587 and 588, or any combination thereof, and wherein the mutation comprises an insertion, a deletion, or a substitution.

[0142] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 587 and 593 compared to SEQ ID NO:1.

[0143] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 586 and 587 compared to SEQ ID NO:1.

[0144] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 592 and 597 compared to SEQ ID NO:1.

[0145] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 587 and 597 compared to SEQ ID NO:1.

[0146] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 550, 586, and 587 compared to SEQ ID NO:1.

[0147] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 559 and 587 compared to SEQ ID NO:1.

[0148] In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 587, and an insertion between positions 586 and 587, compared to SEQ ID NO: 1.

[0149] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 587 and 593, and an insertion between positions 586 and 587, compared to SEQ ID NO: 1.

[0150] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 586 and 587, and an insertion between positions 584 and 585, compared to SEQ ID NO: 1.

[0151] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 592 and 597, and an insertion between positions 587 and 588, compared to SEQ ID NO: 1.

[0152] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 561, 587, and 597, and an insertion between positions 586 and 587, compared to SEQ ID NO: 1.

[0153] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 550, 586, and 587, and an insertion between positions 584 and 585, compared to SEQ ID NO: 1.

[0154] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 559 and 587, and an insertion between positions 586 and 587, compared to SEQ ID NO: 1.

[0155] In some embodiments, the mutation corresponding to position 550 compared to SEQ ID NO: 1 is a substitution. In some embodiments, the substitution is to a naturally occurring amino acid. In some embodiments, the substitution is asparagine (N). In some embodiments, the substitution at position 550 is T550N according to SEQ ID NO: 1. In some embodiments, the substitution at the position corresponding to T550 of SEQ ID NO: 1 is a substitution of asparagine (N) at the position corresponding to T550 of SEQ ID NO: 1 in a reference capsid sequence other than SEQ ID NO: 1, e.g., as described herein. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation T550N mutation compared to SEQ ID NO: 1.

[0156] In some embodiments, the mutation corresponding to position 559 compared to SEQ ID NO: 1 is a substitution. In some embodiments, the substitution is for a naturally occurring amino acid. In some embodiments, the substitution is leucine (L). In some embodiments, the substitution at position 559 is I559L according to SEQ ID NO: 1. In some embodiments, the substitution at the position corresponding to I559 of SEQ ID NO: 1 is a substitution of leucine (L) at the position corresponding to I559 of SEQ ID NO: 1 in a reference capsid sequence other than SEQ ID NO: 1, e.g., as described herein. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation I559L mutation compared to SEQ ID NO: 1.

[0157] In some embodiments, the mutation corresponding to position 561 compared to SEQ ID NO: 1 is a substitution. In some embodiments, the substitution is for a naturally occurring amino acid. In some embodiments, the substitution is cysteine ​​(C). In some embodiments, the substitution at position 561 is D561 according to SEQ ID NO: 1. In some embodiments, the substitution at a position corresponding to D561 of SEQ ID NO: 1 is a substitution of cysteine ​​(C) at a position corresponding to D561 of SEQ ID NO: 1 in a reference capsid sequence other than SEQ ID NO: 1, e.g., as described herein. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation D561C mutation compared to SEQ ID NO: 1.

[0158] In some embodiments, the mutation corresponding to position 586 compared to SEQ ID NO: 1 is a substitution. In some embodiments, the substitution is to a naturally occurring amino acid. In some embodiments, the substitution is proline (P). In some embodiments, the substitution at position 586 is G586P according to SEQ ID NO: 1. In some embodiments, the substitution at the position corresponding to G586 of SEQ ID NO: 1 is a substitution of proline (P) at the position corresponding to G586 of SEQ ID NO: 1 in a reference capsid sequence other than SEQ ID NO: 1, e.g., as described herein. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation G586P mutation compared to SEQ ID NO: 1.

[0159] In some embodiments, the mutation corresponding to position 587 compared to SEQ ID NO: 1 is a substitution. In some embodiments, the substitution is for a naturally occurring amino acid. In some embodiments, the substitution is alanine (A). In some embodiments, the substitution at position 587 is N587A according to SEQ ID NO: 1. In some embodiments, the substitution at the position corresponding to N587 of SEQ ID NO: 1 is a substitution of alanine (A) at the position corresponding to N587 of SEQ ID NO: 1 in a reference capsid sequence other than SEQ ID NO: 1, e.g., as described herein. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation N587A mutation compared to SEQ ID NO: 1.

[0160] In some embodiments, the mutation corresponding to position 592 compared to SEQ ID NO: 1 is a substitution. In some embodiments, the substitution is for a naturally occurring amino acid. In some embodiments, the substitution is alanine (A). In some embodiments, the substitution at position 592 is T592A according to SEQ ID NO: 1. In some embodiments, the substitution at the position corresponding to T592 of SEQ ID NO: 1 is a substitution of alanine (A) at a position corresponding to T592 of SEQ ID NO: 1 in a reference capsid sequence other than SEQ ID NO: 1, e.g., as described herein. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation T592A mutation compared to SEQ ID NO: 1.

[0161] In some embodiments, the mutation corresponding to position 593 compared to SEQ ID NO: 1 is a substitution. In some embodiments, the substitution is to a naturally occurring amino acid. In some embodiments, the substitution is threonine (T). In some embodiments, the substitution at position 593 is A593T according to SEQ ID NO: 1. In some embodiments, the substitution at the position corresponding to A593 of SEQ ID NO: 1 is a substitution of threonine (T) at the position corresponding to A593 of SEQ ID NO: 1 in a reference capsid sequence other than SEQ ID NO: 1, e.g., as described herein. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation A593T mutation compared to SEQ ID NO: 1.

[0162] In some embodiments, the mutation corresponding to position 597 compared to SEQ ID NO: 1 is a substitution. In some embodiments, the substitution is for a naturally occurring amino acid. In some embodiments, the substitution is asparagine (N). In some embodiments, the substitution is for a naturally occurring amino acid. In some embodiments, the substitution is tryptophan (W). In some embodiments, the substitution at position 597 is T597N according to SEQ ID NO: 1. In some embodiments, the substitution at position 597 is T597W according to SEQ ID NO: 1. In some embodiments, the substitution at the position corresponding to T597 of SEQ ID NO: 1 is a substitution of asparagine (N) at a position corresponding to T597 of SEQ ID NO: 1 in a reference capsid sequence other than SEQ ID NO: 1, e.g., as described herein. In some embodiments, the substitution at the position corresponding to T597 of SEQ ID NO: 1 is a substitution of tryptophan (W) at a position corresponding to T597 of SEQ ID NO: 1 in a reference capsid sequence other than SEQ ID NO: 1, e.g., as described herein. In some embodiments, the capsid polypeptide comprises a mutation corresponding to the mutation T597N mutation compared to SEQ ID NO: 1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a T597W mutation compared to SEQ ID NO:1.

[0163] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion between residues 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or 100% identity to LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of LALGEQTRPA (SEQ ID NO: 44).

[0164] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion between residues 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LAIEQTRPA (SEQ ID NO:45).

[0165] In some embodiments, the capsid polypeptide comprises a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of LALAEITRP (SEQ ID NO: 46), for example, consisting thereof. In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8% or 100% identity to LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4 or 5 mutations compared to LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LALAEITRP (SEQ ID NO: 46).

[0166] In some embodiments, the capsid polypeptide comprises a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8% or 100% identity to LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4 or 5 mutations compared to LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LKNAETARP (SEQ ID NO: 47).

[0167] In some embodiments, the capsid polypeptide comprises mutations corresponding to N587A and A593T mutations compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or 100% identity to LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of LNLAIEQTRP (SEQ ID NO: 48).

[0168] In some embodiments, the capsid polypeptide comprises a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide having at least 50%, 62.5%, 75%, 87.5%, or 100% identity to MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, or 4 mutations compared to MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 4 amino acids of MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of MLNEQTRP (SEQ ID NO: 49).

[0169] In some embodiments, the capsid polypeptide comprises mutations corresponding to G586P and N587A mutations compared to SEQ ID NO: 1, and an insertion between residues 584 and 585 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide having at least 45%, 54%, 63%, 72%, 81%, 90%, or 100% identity to RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, 5, or 6 mutations compared to RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 10 amino acids of RSGNRADSETA (SEQ ID NO: 50).

[0170] In some embodiments, the capsid polypeptide comprises a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of TGDTRP (SEQ ID NO: 51). In some embodiments, the insertion comprises a polypeptide having at least 50.1%, 66.8%, 83.5% or 100% identity to TGDTRP (SEQ ID NO: 51). In some embodiments, the insertion comprises a polypeptide having at least 1, 2 or 3 mutations compared to TGDTRP (SEQ ID NO: 51). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 3 amino acids of TGDTRP (SEQ ID NO: 51). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 4 amino acids of TGDTRP (SEQ ID NO: 51). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of TGDTRP (SEQ ID NO: 51).

[0171] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion between residues 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LQGETIRPA (SEQ ID NO:52).

[0172] In some embodiments, the capsid polypeptide comprises a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide having at least 45%, 54%, 63%, 72%, 81%, 90%, or 100% identity to QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, 5, or 6 mutations compared to QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 10 amino acids of QNLANPETTRP (SEQ ID NO: 53).

[0173] In some embodiments, the capsid polypeptide comprises mutations corresponding to T592A and T597W mutations compared to SEQ ID NO: 1, and an insertion between residues 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or 100% identity to RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of RAPQETTRPA (SEQ ID NO: 54).

[0174] In some embodiments, the capsid polypeptide comprises a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide having at least 50%, 62.5%, 75%, 87.5% or 100% identity to ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3 or 4 mutations compared to ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 4 amino acids of ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of ANLTTTRP (SEQ ID NO: 55).

[0175] In some embodiments, the capsid polypeptide comprises a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or 100% identity to ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of ALLAGEQTRP (SEQ ID NO: 56).

[0176] In some embodiments, the capsid polypeptide comprises mutations corresponding to D561C, N587A, and T597N mutations compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of GLRAEQTRP (SEQ ID NO: 57).

[0177] In some embodiments, the capsid polypeptide comprises mutations corresponding to T550N, G586P, and N587A mutations compared to SEQ ID NO: 1, and an insertion between residues 584 and 585 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide having at least 50%, 62.5%, 75%, 87.5%, or 100% identity to RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, or 4 mutations compared to RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 4 amino acids of RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of RARLDETA (SEQ ID NO:58).

[0178] In some embodiments, the capsid polypeptide comprises mutations corresponding to I559L and N587A mutations compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide having at least 45%, 54%, 63%, 72%, 81%, 90% or 100% identity to TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, 5 or 6 mutations compared to TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 10 amino acids of TNLARGETARP (SEQ ID NO: 59).

[0179] In some embodiments, a nucleic acid molecule is provided. In some embodiments, the nucleic acid molecule has a sequence selected from Table 2. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:28-43. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:28. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:29. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:30. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:31. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:32. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:33. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:34. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:35. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:36. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:37. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:38. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:39. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:40. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:41. In some embodiments, the nucleic acid molecule has a sequence of SEQ ID NO:42. In some embodiments, the nucleic acid molecule has the sequence of SEQ ID NO:43.

[0180] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising, according to SEQ ID NO: 1, a mutation corresponding to a mutation at position 550, 559, 561, 586, 587, 592, 593, 597, or any combination thereof, an insertion between positions 584 and 585, between 586 and 587, or between 587 and 588, or any combination thereof, and optionally wherein the mutation comprises an insertion, a deletion, or a substitution.

[0181] In some embodiments, the nucleic acid molecule comprises a sequence encoding a capsid polypeptide comprising a mutation corresponding to a mutation at position 550 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation at position 559 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation at position 561 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation at position 586 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation at position 587 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation at position 592 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation at position 593 as compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation at position 597 compared to SEQ ID NO:1.

[0182] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an insertion at a position between positions 584 and 585 compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an insertion at a position between positions 587 and 588 compared to SEQ ID NO: 1.

[0183] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation at position 550, 559, 561, 586, 587, 592, 593, 597, or any combination thereof, an insertion between positions 584 and 585, between 586 and 587, or between 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.

[0184] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 587 and 593 compared to SEQ ID NO:1.

[0185] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 586 and 587 compared to SEQ ID NO:1.

[0186] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 592 and 597 compared to SEQ ID NO:1.

[0187] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 587 and 597 compared to SEQ ID NO:1.

[0188] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 550, 586, and 587 compared to SEQ ID NO:1.

[0189] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 559 and 587 compared to SEQ ID NO:1.

[0190] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 451, 456, 457, 458, 459, and 461, and an insertion between positions 449 and 450, compared to SEQ ID NO:1.

[0191] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation at position 587 and an insertion between positions 586 and 587 compared to SEQ ID NO: 1.

[0192] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 587 and 593, and an insertion between positions 586 and 587, compared to SEQ ID NO: 1.

[0193] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 586 and 587, and an insertion between positions 584 and 585, compared to SEQ ID NO: 1.

[0194] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 592 and 597, and an insertion between positions 587 and 588, compared to SEQ ID NO: 1.

[0195] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 561, 587, and 597, and an insertion between positions 586 and 587, compared to SEQ ID NO: 1.

[0196] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 550, 586, and 587, and an insertion between positions 584 and 585, compared to SEQ ID NO: 1.

[0197] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to mutations at positions 559 and 587, and an insertion between positions 586 and 587, compared to SEQ ID NO: 1.

[0198] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a T550N mutation compared to SEQ ID NO:1.

[0199] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation I559L mutation compared to SEQ ID NO:1.

[0200] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to mutation D561C compared to SEQ ID NO:1.

[0201] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation G586P mutation compared to SEQ ID NO:1.

[0202] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation N587A mutation compared to SEQ ID NO:1.

[0203] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation T592A mutation compared to SEQ ID NO:1.

[0204] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a mutation A593T mutation compared to SEQ ID NO:1.

[0205] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a T597N mutation compared to SEQ ID NO:1.

[0206] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to a T597W mutation compared to SEQ ID NO:1.

[0207] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an insertion between residues 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of LALGEQTRPA (SEQ ID NO: 44), and wherein the nucleic acid has the sequence of SEQ ID NO: 28. In some embodiments, the insertion comprises a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or 100% identity to LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of LALGEQTRPA (SEQ ID NO: 44).

[0208] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an insertion between residues 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of LAIEQTRPA (SEQ ID NO: 45), and wherein the nucleic acid has the sequence of SEQ ID NO: 29. In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LAIEQTRPA (SEQ ID NO: 45). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LAIEQTRPA (SEQ ID NO: 45).

[0209] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of LALAEITRP (SEQ ID NO: 46), and wherein the nucleic acid has the sequence of SEQ ID NO: 30. In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8% or 100% identity to LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4 or 5 mutations compared to LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LALAEITRP (SEQ ID NO: 46).

[0210] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of LKNAETARP (SEQ ID NO: 47), and wherein the nucleic acid has the sequence of SEQ ID NO: 31. In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8% or 100% identity to LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4 or 5 mutations compared to LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LKNAETARP (SEQ ID NO: 47).

[0211] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to N587A and A593T mutations compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of LNLAIEQTRP (SEQ ID NO: 48), and wherein the nucleic acid has the sequence of SEQ ID NO: 32. In some embodiments, the insertion comprises a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or 100% identity to LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of LNLAIEQTRP (SEQ ID NO: 48).

[0212] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of MLNEQTRP (SEQ ID NO: 49), and wherein the nucleic acid has the sequence of SEQ ID NO: 33. In some embodiments, the insertion comprises a polypeptide having at least 50%, 62.5%, 75%, 87.5%, or 100% identity to MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, or 4 mutations compared to MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 4 amino acids of MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of MLNEQTRP (SEQ ID NO: 49). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of MLNEQTRP (SEQ ID NO: 49).

[0213] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to G586P and N587A mutations compared to SEQ ID NO: 1, and an insertion between residues 584 and 585 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of RSGNRADSETA (SEQ ID NO: 50), and wherein the nucleic acid has the sequence of SEQ ID NO: 34. In some embodiments, the insertion comprises a polypeptide having at least 45%, 54%, 63%, 72%, 81%, 90%, or 100% identity to RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, 5, or 6 mutations compared to RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of RSGNRADSETA (SEQ ID NO: 50). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 10 amino acids of RSGNRADSETA (SEQ ID NO: 50).

[0214] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of TGDTRP (SEQ ID NO: 51), and wherein the nucleic acid has the sequence of SEQ ID NO: 35. In some embodiments, the insertion comprises a polypeptide having at least 50.1%, 66.8%, 83.5%, or 100% identity to TGDTRP (SEQ ID NO: 51). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, or 3 mutations compared to TGDTRP (SEQ ID NO: 51). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 3 amino acids of TGDTRP (SEQ ID NO: 51). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 4 amino acids of TGDTRP (SEQ ID NO: 51). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of TGDTRP (SEQ ID NO: 51).

[0215] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an insertion between residues 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of LQGETIRPA (SEQ ID NO: 52), and wherein the nucleic acid has the sequence of SEQ ID NO: 36. In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of LQGETIRPA (SEQ ID NO: 52). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of LQGETIRPA (SEQ ID NO: 52).

[0216] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of QNLANPETTRP (SEQ ID NO: 53), and wherein the nucleic acid has the sequence of SEQ ID NO: 37. In some embodiments, the insertion comprises a polypeptide having at least 45%, 54%, 63%, 72%, 81%, 90%, or 100% identity to QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, 5, or 6 mutations compared to QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of QNLANPETTRP (SEQ ID NO: 53). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 10 amino acids of QNLANPETTRP (SEQ ID NO: 53).

[0217] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to T592A and T597W mutations compared to SEQ ID NO: 1, and an insertion between residues 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of RAPQETTRPA (SEQ ID NO: 54), and wherein the nucleic acid has the sequence of SEQ ID NO: 38. In some embodiments, the insertion comprises a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or 100% identity to RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of RAPQETTRPA (SEQ ID NO: 54).

[0218] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, for example, consists of, a polypeptide of ANLTTTRP (SEQ ID NO: 55), and wherein the nucleic acid has the sequence of SEQ ID NO: 39. In some embodiments, the insertion comprises a polypeptide having at least 50%, 62.5%, 75%, 87.5%, or 100% identity to ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, or 4 mutations compared to ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 4 amino acids of ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of ANLTTTRP (SEQ ID NO: 55). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of ANLTTTRP (SEQ ID NO: 55).

[0219] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the N587A mutation compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of ALLAGEQTRP (SEQ ID NO: 56), and wherein the nucleic acid has the sequence of SEQ ID NO: 40. In some embodiments, the insertion comprises a polypeptide having at least 50%, 60%, 70%, 80%, 90%, or 100% identity to ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of ALLAGEQTRP (SEQ ID NO: 56). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of ALLAGEQTRP (SEQ ID NO: 56).

[0220] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to D561C, N587A, and T597N mutations compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of GLRAEQTRP (SEQ ID NO: 57), and wherein the nucleic acid has the sequence of SEQ ID NO: 41. In some embodiments, the insertion comprises a polypeptide having at least 44.4%, 55.5%, 66.6%, 77.7%, 88.8%, or 100% identity to GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, or 5 mutations compared to GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of GLRAEQTRP (SEQ ID NO: 57).

[0221] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to T550N, G586P, and N587A mutations as compared to SEQ ID NO: 1, and an insertion between residues 584 and 585 as compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of RARLDETA (SEQ ID NO: 58), and wherein the nucleic acid has the sequence of SEQ ID NO: 42. In some embodiments, the insertion comprises a polypeptide having at least 50%, 62.5%, 75%, 87.5%, or 100% identity to RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, or 4 mutations as compared to RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 4 amino acids of RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of RARLDETA (SEQ ID NO: 58). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of RARLDETA (SEQ ID NO: 58).

[0222] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to I559L and N587A mutations compared to SEQ ID NO: 1, and an insertion between residues 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises, e.g., consists of, a polypeptide of TNLARGETARP (SEQ ID NO: 59), and wherein the nucleic acid has the sequence of SEQ ID NO: 43. In some embodiments, the insertion comprises a polypeptide having at least 45%, 54%, 63%, 72%, 81%, 90%, or 100% identity to TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide having at least 1, 2, 3, 4, 5, or 6 mutations compared to TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 5 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 6 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 7 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 8 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 9 amino acids of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising a fragment of at least 10 amino acids of TNLARGETARP (SEQ ID NO: 59).

[0223] In some embodiments, including in the above embodiments, 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 the sequence of the capsid polypeptide provided herein.

[0224] 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 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.

[0225] In some embodiments, including the above embodiments, a capsid polypeptide is provided, which 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.

[0226] In some embodiments, including the above embodiments, a capsid polypeptide is provided, which 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.

[0227] In some embodiments, for purposes of % identity, a reference polypeptide comprises the sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10.

[0228] In some embodiments, for purposes of % identity, a reference nucleic acid comprises the sequence of SEQ ID NO: 2, 4, 6, 8, or 11.

[0229] In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises the sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0230] In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 28. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 29. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 30. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 31. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 32. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 33. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 34. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 35. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 36. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 37. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 38. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 39. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 40. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 41. In some embodiments, for purposes of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises the sequence of SEQ ID NO: 42. In some embodiments, for purposes of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises the sequence of SEQ ID NO: 43.

[0231] In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43, which encodes a sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27, respectively. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 28, which encodes a sequence of SEQ ID NO: 12. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 29, which encodes a sequence of SEQ ID NO: 13. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 30, which encodes a sequence of SEQ ID NO: 14. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 31, which encodes a sequence of SEQ ID NO: 15. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 32, which encodes a sequence of SEQ ID NO: 16. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 33, which encodes a sequence of SEQ ID NO: 17. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 34, which encodes a sequence of SEQ ID NO: 18. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 35, which encodes a sequence of SEQ ID NO: 19. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 36, which encodes a sequence of SEQ ID NO: 20. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 37, which encodes a sequence of SEQ ID NO: 21. In some embodiments, for the purpose of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 38, which encodes a sequence of SEQ ID NO: 22.In some embodiments, for purposes of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 39, which encodes a sequence of SEQ ID NO: 23. In some embodiments, for purposes of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 40, which encodes a sequence of SEQ ID NO: 24. In some embodiments, for purposes of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 41, which encodes a sequence of SEQ ID NO: 25. In some embodiments, for purposes of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 42, which encodes a sequence of SEQ ID NO: 26. In some embodiments, for purposes of % identity, a nucleic acid molecule encoding a capsid polypeptide or a reference polypeptide comprises a sequence of SEQ ID NO: 43, which encodes a sequence of SEQ ID NO: 27.

[0232] In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27, which is encoded by a nucleotide sequence of SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43, respectively. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 12, which is encoded by a nucleotide sequence of SEQ ID NO: 28. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 13, which is encoded by a nucleotide sequence of SEQ ID NO: 29. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 14, which is encoded by a nucleotide sequence of SEQ ID NO: 30. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 15, which is encoded by a nucleotide sequence of SEQ ID NO: 31. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 16, which is encoded by a nucleotide sequence of SEQ ID NO: 32. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 17, which is encoded by a nucleotide sequence of SEQ ID NO: 33. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 18, which is encoded by a nucleotide sequence of SEQ ID NO: 34. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 19, which is encoded by a nucleotide sequence of SEQ ID NO: 35. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 20, which is encoded by a nucleotide sequence of SEQ ID NO: 36. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 21, which is encoded by a nucleotide sequence of SEQ ID NO: 37. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 22, which is encoded by a nucleotide sequence of SEQ ID NO: 38. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 23, which is encoded by a nucleotide sequence of SEQ ID NO: 39.In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 24, which is encoded by a nucleotide sequence of SEQ ID NO: 40. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 25, which is encoded by a nucleotide sequence of SEQ ID NO: 41. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 26, which is encoded by a nucleotide sequence of SEQ ID NO: 42. In some embodiments, for purposes of % identity, a capsid polypeptide or reference polypeptide comprises a sequence of SEQ ID NO: 27, which is encoded by a nucleotide sequence of SEQ ID NO: 43.

[0233] In some embodiments, the capsid polypeptide comprises a sequence that includes all of the mutational differences associated with any one of VAR-1 to VAR-16 (e.g., as shown in Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, Table 1F, and Table 1G), 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 mutation relative to SEQ ID NO: 1.

[0234] 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. Relative to the reference sequence SEQ ID NO: 1, the VP1 capsid polypeptide comprises amino acids 1-724 of SEQ ID NO: 1. Relative to the reference sequence SEQ ID NO: 1, the VP2 capsid polypeptide comprises amino acids 138-724 of SEQ ID NO: 1. Relative to the reference sequence SEQ ID NO: 1, the VP3 capsid polypeptide comprises amino acids 203-724 of SEQ ID NO: 1.

[0235] Tables 1A, 1B, 1C, 1D, 1E, 1F, and 1G list information about exemplary variant-dependent parvoviral particles comprising variant capsids and describe the ocular transduction properties and production characteristics of the non-limiting exemplary variants. Exemplary sequences of capsid polypeptides and nucleic acid molecules encoding them are provided in Table 2. Table 2 shows the VP1, VP2, and VP3 polypeptide starting amino acid sequences for each of SEQ ID NO: 12 to SEQ ID NO: 27. The exemplary nucleic acid sequences provided in Table 2 include a stop codon (e.g., a TAA stop codon) located at the 3' end of the sequence. It will be appreciated by those skilled in the art that in some embodiments, the TAA stop codon is removed or replaced with a different stop codon (e.g., TGA or TAG).

[0236] Table 1A, Table 1B, and Table 1C represent data generated in the first (Table 1A) high throughput experiment (library experiment 1) and the second (Tables 1B to 1C) high throughput experiment (library experiment 2). Table 1D, Table 1E, Table 1F, and Table 1G represent data generated in the medium throughput experiment (library experiment 3). Exemplary variants comprising variant capsid polypeptides rely on transduction of parvovirus (e.g., AVV) particles and virus production. The injection route is indicated in the column headings. Substitutions are labeled n###N, where "N" is the final amino acid, "n" is the reference amino acid, and "###" is the reference amino acid position of SEQ ID NO: 1; deletions are labeled n###-, where "-" indicates the deletion of "n" at position "###" of the reference sequence SEQ ID NO: 1; insertions are labeled ###_Naa_###_(n)y, where "###" is the amino acid position where the insertion occurs in the reference sequence SEQ ID NO: 1, "Naa" refers to the length of the insertion (with "N" amino acids) and "(n)y" provides the sequence of the insertion). Each individual mutation difference (e.g., within a row, each mutation in quotes ('') in column 8 of Table 1A, column 7 of Table 1B, column 7 of Table 1C, column 6 of Table 1D, column 6 of Table 1E, column 7 of Table 1F, column 6 of Table 1G) and combinations of such individual mutation differences are sometimes referred to herein as "mutations associated with VAR-X", where VAR-X is the variant identifier listed in the "Name Column". Macular transduction refers to the transduction of the neural retinal layer of the macula. In Table 1A and Table 1C, non-macular transduction refers to the transduction of the neural retinal layer of the retina other than the macula. Retinal transduction refers to the aggregated measurement value from macular transduction and non-macular transduction. For the purpose of Table 1A, the average retinal transduction is calculated as the average of macular transduction and non-macular retinal transduction. Trabecular transduction refers to the transduction of tissue samples collected from the trabecular meshwork and / or Schlemm's canal. For the purpose of Table 1D and Table 1E, neural retinal transduction refers to the transduction of tissue samples collected from the neural retinal layer (including the macular area). "Not measured" means that the variant was not detected in the specified sample. Unless otherwise stated, the measurement is relative to wild-type AAV2 (SEQ ID NO: 1). The trabecular transduction measurements shown in Table 1F and Table 1G are shown relative to AAV2 variants identified from the ophthalmic literature, whose capsid has a polypeptide of SEQ ID NO: 60 encoded by the exemplary nucleic acid SEQ ID NO: 61. The data shown in Table 1A, Table 1B, and Table 1C are on a log2 scale.

[0237]

[0238]

[0239]

[0240] Table 1D

[0241]

[0242] Table 1E

[0243]

[0244] Table 1F

[0245]

[0246] Table 1G

[0247]

[0248] Table 2

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267] In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence provided in Table 2. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 12. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 13. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 14. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 15. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 16. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 17. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 18. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 19. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO:20.In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 21. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 22. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 23. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 24. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 25. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO: 26. In some embodiments, the capsid polypeptide is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the VP1, VP2 or VP3 sequence of SEQ ID NO:27.

[0268] In some embodiments, the capsid polypeptide has a sequence of SEQ ID NOs: 12-27. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 12. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 13. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 14. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 15. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 16. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 17. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 18. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 19. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 20. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 21. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 22. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 23. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 24. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 25. In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 26. In some embodiments, the capsid polypeptide has the sequence of SEQ ID NO:27.

[0269] In some embodiments, the nucleic acid molecule encodes a 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 2. In some embodiments, the nucleic acid molecule encodes a 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 of SEQ ID NO: 12. In some embodiments, the nucleic acid molecule encodes a 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 of SEQ ID NO: 13. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 15. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 16. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 17. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 18. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 19.In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 20. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 21. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 22. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 23. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 24. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 25. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 26. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO: 27.

[0270] 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) shown in the mutation difference column of Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, Table 1F, and Table 1G of VAR-1, VAR-2, VAR-3, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, VAR-9, VAR-10, VAR-11, VAR-12, VAR-13, VAR-14, VAR-15, or VAR-16. 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).

[0271] In some embodiments, the capsid polypeptide comprises at least, about, or exactly 80%, 85%, 90%, or 95%, or 100% of a reference capsid sequence, such as SEQ ID NO: 1, and one of the following mutation groups (the terms of these mutation groups are provided in the legends to Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, Table 1F, and Table 1G above):

[0272] [587_10aa_588_LALGEQTRPA];

[0273] [587_9aa_588_LAIEQTRPA];

[0274] [586_9aa_587_LALAEITRP,N587A];

[0275] [586_9aa_587_LKNAETARP,N587A];

[0276] [586_10aa_587_LNLAIEQTRP,N587A,A593T];

[0277] [586_8aa_587_MLNEQTRP,N587A];

[0278] [584_11aa_585_RSGNRADSETA,G586P,N587A];

[0279] [586_6aa_587_TGDTRP,N587A];

[0280] [587_9aa_588_LQGETIRPA];

[0281] [586_11aa_587_QNLANPETTRP,N587A];

[0282] [587_10aa_588_RAPQETTRPA,T592A,T597W];

[0283] [586_8aa_587_ANLTTTRP,N587A];

[0284] [586_10aa_587_ALLAGEQTRP,N587A];

[0285] [D561C,586_9aa_587_GLRAEQTRP,N587A,T597N];

[0286] [T550N,584_8aa_585_RARLDETA,G586P,N587A]; and

[0287] [I559L,586_11aa_587_TNLARGETARP,N587A].

[0288] In some embodiments, the capsid polypeptide comprises at least, about, or exactly 80%, 85%, 90%, or 95%, or 100% of a reference capsid sequence, such as SEQ ID NO: 1, and [587_10aa_588_LALGEQTRPA]. In some embodiments, the capsid polypeptide comprises at least 8 or all of the amino acid residues of the ten amino acid insertion.

[0289] In some embodiments, the capsid polypeptide comprises at least, about, or exactly 80%, 85%, 90%, or 95%, or 100% of a reference capsid sequence, such as SEQ ID NO: 1, and [587_9aa_588_LAIEQTRPA]. In some embodiments, the capsid polypeptide comprises at least 7 or all of the amino acid residues of the nine amino acid insertion.

[0290] 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 [586_9aa_587_LALAEITRP, N587A]. In some embodiments, the capsid polypeptide comprises at least 7 or all amino acid residues of the nine amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 8 or all amino acid residues of the nine amino acid insertion and the N587A mutation.

[0291] 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 [586_9aa_587_LKNAETARP, N587A]. In some embodiments, the capsid polypeptide comprises at least 7 or all of the amino acid residues of the nine amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 8 or all of the amino acid residues of the nine amino acid insertion and the N587A mutation.

[0292] 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 [586_10aa_587_LNLAIEQTRP, N587A, A593T]. In some embodiments, the capsid polypeptide comprises at least 8 or all of the amino acid residues of the ten amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 9 or all of the amino acid residues of the ten amino acid insertion and N587A and A593T mutations.

[0293] 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 [586_8aa_587_MLNEQTRP, N587A]. In some embodiments, the capsid polypeptide comprises at least 6 or all of the amino acid residues of the eight amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 7 or all of the amino acid residues of the eight amino acid insertion and the N587A mutation.

[0294] 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 [584_11aa_585_RSGNRADSETA, G586P, N587A]. In some embodiments, the capsid polypeptide comprises at least 9 or all amino acid residues of the eleven amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 10 or all amino acid residues of the eleven amino acid insertion and the G586P and N587A mutations.

[0295] 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 [586_6aa_587_TGDTRP, N587A]. In some embodiments, the capsid polypeptide comprises at least 5 or all of the amino acid residues of the six amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 6 or all of the amino acid residues of the six amino acid insertion and the N587A mutation.

[0296] In some embodiments, the capsid polypeptide comprises at least, about, or exactly 80%, 85%, 90%, or 95%, or 100% of a reference capsid sequence, such as SEQ ID NO: 1, and [587_9aa_588_LQGETIRPA]. In some embodiments, the capsid polypeptide comprises at least 7 or all of the amino acid residues of the nine amino acid insertion.

[0297] 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 [586_11aa_587_QNLANPETTRP, N587A]. In some embodiments, the capsid polypeptide comprises at least 9 or all amino acid residues of the eleven amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 10 or all amino acid residues of the eleven amino acid insertion and the N587A mutation.

[0298] In some embodiments, the capsid polypeptide comprises at least, about, or exactly 80%, 85%, 90%, or 95%, or 100% of a reference capsid sequence, such as SEQ ID NO: 1, and [587_10aa_588_RAPQETTRPA, T592A, T597W]. In some embodiments, the capsid polypeptide comprises at least 8 or all of the amino acid residues of the ten amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 9 or all of the amino acid residues of the ten amino acid insertion and T592A and T597W mutations.

[0299] 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 [586_8aa_587_ANLTTTRP, N587A]. In some embodiments, the capsid polypeptide comprises at least 6 or all of the amino acid residues of the eight amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 7 or all of the amino acid residues of the eight amino acid insertion and the N587A mutation.

[0300] 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 [586_10aa_587_ALLAGEQTRP, N587A]. In some embodiments, the capsid polypeptide comprises at least 8 or all of the amino acid residues of the ten amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 8 or all of the amino acid residues of the ten amino acid insertion and the N587A mutation.

[0301] 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 [D561C, 586_9aa_587_GLRAEQTRP, N587A, T597N]. In some embodiments, the capsid polypeptide comprises at least 7 or all amino acid residues of the nine amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 9 or all amino acid residues of the nine amino acid insertion and D561C, N587A, and T597N mutations.

[0302] 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 [T550N, 584_8aa_585_RARLDETA, G586P, N587A]. In some embodiments, the capsid polypeptide comprises at least 6 or all amino acid residues of eight amino acid insertions. In some embodiments, the capsid polypeptide comprises at least 8 or all amino acid residues of eight amino acid insertions and T550N, G586P, and N587A mutations.

[0303] In some embodiments, the capsid polypeptide comprises at least, about, or exactly 80%, 85%, 90%, or 95%, or 100% of a reference capsid sequence, such as SEQ ID NO: 1, and [I559L, 586_11aa_587_TNLARGETARP, N587A]. In some embodiments, the capsid polypeptide comprises at least 8 or all of the amino acid residues of the eleven amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 10 or all of the amino acid residues of the eleven amino acid insertion and I559L and N587A mutations.

[0304] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 12; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 12 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO: 12; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 12 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 12; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO: 12 as compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 5-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid, or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, at least 16-fold, at least 32-fold, at least 64-fold, at least 128-fold, or at least 300-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0305] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 13; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 13 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO: 13; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 13 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 13; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO: 13 as compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the increase is at least 2-fold or at least 3-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, at least 16-fold, at least 32-fold, at least 64-fold, at least 110-fold or at least 128-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0306] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or greater amino acid sequence identity to an amino acid sequence of SEQ ID NO: 14; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 14 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO: 14; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 14 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or greater amino acid sequence identity to an amino acid sequence of SEQ ID NO: 14; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO: 14 as compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 8-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid, or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, at least 16-fold, at least 32-fold, at least 64-fold, or at least 128-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0307] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 15; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 15 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO: 15; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 15 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 15; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO: 15 as compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the increase is at least 2-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, at least 16-fold, at least 32-fold, at least 64-fold or at least 128-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increase is at least 60-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0308] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 16; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 16 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO: 16; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO: 16 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 16; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO: 16 as compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2 times, at least 4 times, at least 8 times, at least 16 times, at least 32 times, at least 64 times, at least 128 times, or at least 230 times compared to the transduction of the viral particles comprising the capsid polypeptide of the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0309] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 17; and having at least 80% of the mutations in SEQ ID NO: 17 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO: 17; and having at least 80% of the mutations in SEQ ID NO: 17 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 17; and having less than 80% of the mutations in SEQ ID NO: 17 as compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2 times, at least 4 times, at least 8 times, at least 16 times, at least 32 times, at least 64 times, at least 70 times, at least 128 times, or at least 520 times compared to the transduction of the viral particles comprising the capsid polypeptide of the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0310] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 18; and having at least 80% of the mutations in SEQ ID NO: 18 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO: 18; and having at least 80% of the mutations in SEQ ID NO: 18 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 18; and having less than 80% of the mutations in SEQ ID NO: 18 as compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2 times, at least 4 times, at least 8 times, at least 16 times, at least 32 times, at least 50 times, at least 64 times, at least 128 times, or at least 460 times compared to the transduction of the viral particles comprising the capsid polypeptide of the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0311] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 19; and having at least 80% of the mutations in SEQ ID NO: 19 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO: 19; and having at least 80% of the mutations in SEQ ID NO: 19 as compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO: 19; and having less than 80% of the mutations in SEQ ID NO: 19 as compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2 times, at least 4 times, at least 8 times, at least 16 times, at least 32 times, at least 64 times, at least 100 times, at least 128 times, or at least 1000 times compared to the transduction of the viral particles comprising the capsid polypeptide of the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0312] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:20; and having at least 80% of the mutations in SEQ ID NO:20 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO:20; and having at least 80% of the mutations in SEQ ID NO:20 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:20; and having less than 80% of the mutations in SEQ ID NO:20 as compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2 times, at least 4 times, at least 8 times, at least 16 times, at least 32 times, at least 64 times or at least 128 times compared to the transduction of the viral particles comprising the capsid polypeptide of the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0313] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:21; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:21 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO:21; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:21 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:21; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO:21 as compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2 times, at least 4 times, at least 8 times, at least 16 times, at least 32 times, at least 64 times or at least 128 times compared to the transduction of the viral particles comprising the capsid polypeptide of the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0314] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:22; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:22 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO:22; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:22 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:22; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO:22 as compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 5-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid, or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, at least 16-fold, at least 28-fold, at least 32-fold, at least 64-fold, or at least 128-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0315] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:23; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:23 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO:23; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:23 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:23; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO:23 as compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2 times, at least 4 times, at least 8 times, at least 16 times, at least 32 times, at least 64 times or at least 128 times compared to the transduction of the viral particles comprising the capsid polypeptide of the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0316] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:24; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:24 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO:24; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:24 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:24; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO:24 as compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 5-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid, or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, at least 16-fold, at least 32-fold, at least 64-fold, or at least 128-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0317] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:25; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:25 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO:25; and having at least 80% of the amino acid sequence in mutations in SEQ ID NO:25 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:25; and having less than 80% of the amino acid sequence in mutations in SEQ ID NO:25 as compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, or at least 12-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid, or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, at least 16-fold, at least 32-fold, at least 64-fold, or at least 128-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0318] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:26; and having at least 80% of the mutations in SEQ ID NO:26 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO:26; and having at least 80% of the mutations in SEQ ID NO:26 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:26; and having less than 80% of the mutations in SEQ ID NO:26 as compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2 times, at least 4 times, at least 8 times, at least 16 times, at least 32 times, at least 64 times or at least 128 times compared to the transduction of the viral particles comprising the capsid polypeptide of the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0319] In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:27; and having at least 80% of the mutations in SEQ ID NO:27 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having less than 95% amino acid sequence identity to an amino acid sequence of SEQ ID NO:27; and having at least 80% of the mutations in SEQ ID NO:27 as compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided, comprising an amino acid sequence having 95% or more amino acid sequence identity to an amino acid sequence of SEQ ID NO:27; and having less than 80% of the mutations in SEQ ID NO:27 as compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene in the anterior third of the eye, the anterior third including structures anterior to the vitreous humor, such as structures described herein, including but not limited to the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 8-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the viral particle comprising a variant capsid polypeptide has increased ocular transduction of a transgene behind the lens, such as in the anterior hyaloid membrane and all optical structures behind it, such as vitreous humor, retina, choroid, or optic nerve, or any combination thereof. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, at least 16-fold, at least 32-fold, at least 64-fold, or at least 128-fold compared to the transduction of a viral particle comprising a capsid polypeptide of a reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the increased transduction is measured, for example, by NGS sequencing of viral RNA in target tissue cells, as described in the examples. In some embodiments, transduction is measured after intravitreal administration. In some embodiments, transduction is measured after intracameral injection.

[0320] As used herein, the phrase "80% of mutations" with respect to a variant capsid sequence means that the variant has at least 80% of the mutations present in the variant capsid sequence, wherein the total number of mutations is based on a comparison with a reference sequence such as a wild-type sequence. If the variant capsid polypeptide is a mixture of insertions and substitutions or deletions, each amino acid residue of the insertion is counted in the total number of mutations. For example, if the mutations possessed by the variant capsid polypeptide are a mixture of substitutions of 'T550N, 'G586P', and 'N587A' and insertions of a polypeptide comprising the sequence of RARLDETA (SEQ ID NO: 58), the total number of mutations is 11, i.e., 8 amino acid insertions and three amino acid substitutions, and a variant capsid having "80% of mutations" will contain at least 9, 10, or all mutations.

[0321] Variant capsid (corresponding position)

[0322] The mutations of the capsid polypeptide sequences described herein are described relative to the positions and / or amino acids at the positions within the reference sequence (e.g., SEQ ID NO: 1). Therefore, in some embodiments, the capsid polypeptides described herein are variant capsid polypeptides of the reference sequence (e.g., SEQ ID NO: 1), for example, including capsid polypeptides, which contain 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 with the reference capsid polypeptide sequence (e.g., reference capsid polypeptide VP1, VP2 and / or VP3 sequence), for example, SEQ ID NO: 1 (or the VP2 or VP3 sequence contained therein), and further include one or more mutations described herein.

[0323] It will be understood by those skilled in the art, without being bound by theory, that each amino acid position within the reference sequence corresponds to a position within the sequence of another capsid polypeptide (e.g., a capsid polypeptide derived from a parvovirus having a different serotype). 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, which is incorporated herein by reference in its entirety). An alignment of an exemplary reference capsid polypeptide is shown in FIG. 2A to FIG. 2CThus, in some embodiments, 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 positions of mutations described herein relative to different reference capsid polypeptides. Thus, for example, with respect to a mutation described as XnnnY in SEQ ID NO: 1 (wherein X is an amino acid present at position nnn in SEQ ID NO: 1 and Y is an amino acid mutation at that position, e.g., as described herein), the disclosure provides a variant capsid polypeptide 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 a reference capsid polypeptide sequence other than SEQ ID NO: 1 (or a VP2 or VP3 sequence contained therein), and further comprising a disclosed mutation at a position corresponding to position nnn of SEQ ID NO: 1 (e.g., comprising Y at a position corresponding to position nnn of SEQ ID NO: 1 in the new variant capsid polypeptide sequence). As described above, such corresponding positions are determined using a sequence alignment tool, such as, for example, the clustal omega tool described above. Examples of corresponding amino acid positions for exemplary known AAV serotypes are provided in FIG. 2A to FIG. 2C In some embodiments, the variant is a variant of an AAV2 capsid polypeptide, which may be referred to as a "variant AAV2 capsid polypeptide".

[0324] Thus, in some embodiments, the disclosure provides a capsid polypeptide sequence that is a reference sequence other than SEQ ID NO: 1, e.g., a variant of a reference sequence other than SEQ ID NO: 1 as described herein, the variant comprising one or more mutations corresponding to the mutations described herein. In some embodiments, such variants comprise all of the mutations corresponding to any of the mutations associated with VAR-1 to VAR-16 according to Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, Table 1F, and Table 1G.

[0325] As used herein, the term "corresponding to" as used with respect to a position in a sequence (such as an amino acid or nucleic acid sequence) can be used to refer to an entire capsid polypeptide or polynucleotide sequence, such as a full-length sequence of a capsid polypeptide comprising VP1, VP2 and VP3 polypeptides or a nucleic acid molecule encoding the same. In some embodiments, the term "corresponding to" can be used to refer to a region or domain of a capsid polypeptide. For example, a position corresponding to a position in the VP1 segment of a reference capsid polypeptide can correspond to the VP1 portion of a polypeptide of a variant capsid polypeptide. Therefore, when comparing two sequences to determine whether a position corresponds to another position, a full-length polypeptide can be used or a domain (region) can be used to determine whether a position corresponds to a specific position. In some embodiments, the region is a VP1 polypeptide. In some embodiments, the region is a VP2 polypeptide. In some embodiments, the region is a VP3 polypeptide. In some embodiments, when the reference polypeptide is a wild-type sequence (e.g., full length or region) of a certain serotype of AAV, the variant polypeptide can be the same serotype having a mutation performed at such a corresponding position compared to the reference sequence (e.g., full length or region). In some embodiments, the variant capsid polypeptide is a different serotype compared to the reference sequence.

[0326] The variant capsid polypeptides described herein are optionally variants of reference capsid serotypes known in the art. Non-limiting examples of such reference AAV serotypes include AAV1, AAVrh10, 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.B / G2A12 , AAVG2A15 / G2A3(G2A3), AAVG2B4(G2B4), AAVG2B5(G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AA V3-3, 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, AAV42-1b, A AV42-2, 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、AA V223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3. AV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / h u.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh .70、AAVpi.1、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 .1、AAVH-5 / hu.3、AAVLG-10 / rh.40、AAVLG-4 / rh.38、AAVLG-9 / hu.39、AAVN721-8 / rh.43、AAVCh.5、AAVCh.5R1、AAVcy.2、AAVcy.3、AAVcy.4、AAVcy.5.AAVCy.5R1、AAVCy.5R2、AAVCy.5R3、AAVCy.5R4、AAVcy.6、AAVhu.1、AAVhu.2、AAVhu.3、AAVhu.4、AAVhu.5、AAVhu.6、AAVhu.7、AAVhu.9、AAVhu.10、AAVhu.11、AAVhu.13、AAVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、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.44R1、AAVhu.44R2、AAVhu.44R3、AAVhu.45、AAVhu.46、AAVhu.47、AAVhu.48、AAVhu.48R1、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.10、AAVrh.12、AAVrh.13、AAVrh.13R、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.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74 (also known as AAVrh74), AAVrh8R, AAVrh8R A586R mutant, AAVrh8RR533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhE1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, 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, 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, AAVShuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAVShuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAVSM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, 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 serotype、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-E1、AAVCBr-E2、AAV CBr-E3、AAV CBr-E4、AAV CBr-E5、AAV CBr-e5、AAV CBr-E6、AAV CBr-E7、AAVCBr-E8、AAV CHt-1、AAV CHt-2、AAV CHt-3、AAV CHt-6.1、AAV CHt-6.10、AAV CHt-6.5、AAVCHt-6.6、AAV CHt-6.7、AAV CHt-6.8、AAV CHt-P1、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、AAVCKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAVCKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAVCKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAVCLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAVCLv-1、AAV CLv1-1、AAV CLv1-10、AAV CLv1-2、AAV CLv12、AAV CLv1-3、AAV CLv-13、AAVCLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAVCLv-6、AAV CLv-8, AAV CLv-D1, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-E1, AAV CLv-K1, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-M1, AAV CLv-M11, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-R1, 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, 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, Spark100, AAVMYO, and variants thereof.

[0327] 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. Although not wishing to be bound by theory, it should be understood that the reference AAV capsid sequence comprises a VP1 region. In certain embodiments, the reference AAV capsid sequence comprises a VP1, VP2 and / or VP3 region, or any combination thereof. The reference VP1 sequence can be considered synonymous with the reference AAV capsid sequence.

[0328] An exemplary reference sequence of SEQ ID NO: 1 (wild-type AAV2) is as follows:

[0329]

[0330] Unless otherwise indicated, SEQ ID NO: 1 is a reference sequence. In the above sequences, sequences found in VP1, VP2, and VP3 are underlined (e.g., the VP3 capsid polypeptide comprises, e.g., consists of, amino acids corresponding to amino acids 203-735 of SEQ ID NO: 1), sequences found in both VP1 and VP2 are bolded (e.g., the VP2 capsid polypeptide comprises, e.g., consists of, amino acids corresponding to amino acids 138-735 of SEQ ID NO: 1), and sequences not underlined or bolded are found only in VP1 (e.g., the VP1 capsid polypeptide comprises, e.g., consists of, amino acids corresponding to amino acids 1-735 of SEQ ID NO: 1).

[0331] An exemplary nucleic acid sequence encoding SEQ ID NO: 1 is SEQ ID NO: 2:

[0332]

[0333] An exemplary reference sequence of wild-type AAV5 is SEQ ID NO:3 (wild-type AAV5) as follows:

[0334]

[0335] In the above sequences, sequences found in VP1, VP2 and VP3 are underlined (e.g., the VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 193-725 of SEQ ID NO:3), sequences found in both VP1 and VP2 are marked in bold (e.g., the VP2 capsid polypeptide includes, e.g., consists of, amino acids 137-725 of SEQ ID NO:3), and sequences not underlined or marked in bold are only found in VP1 (e.g., the VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-725 of SEQ ID NO:3).

[0336] An exemplary nucleic acid sequence encoding SEQ ID NO:3 is SEQ ID NO:4:

[0337]

[0338] An exemplary reference sequence of wild-type AAV8 is SEQ ID NO:5 (wild-type AAV8) as follows:

[0339]

[0340] In the above sequences, sequences found in VP1, VP2 and VP3 are underlined (e.g., the VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 204-739 of SEQ ID NO:5), sequences found in both VP1 and VP2 are marked in bold (e.g., the VP2 capsid polypeptide includes, e.g., consists of, amino acids 138-735 of SEQ ID NO:5), and sequences not underlined or marked in bold are only found in VP1 (e.g., the VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-739 of SEQ ID NO:5).

[0341] An exemplary nucleic acid sequence encoding SEQ ID NO:5 is SEQ ID NO:6:

[0342]

[0343] An exemplary reference sequence of wild-type AAV9 is SEQ ID NO:7 (wild-type AAV9) as follows:

[0344]

[0345] In the above sequences, sequences found in VP1, VP2 and VP3 are underlined (e.g., the VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 203-737 of SEQ ID NO:7), sequences found in both VP1 and VP2 are marked in bold (e.g., the VP2 capsid polypeptide includes, e.g., consists of, amino acids 138-737 of SEQ ID NO:7), and sequences not underlined or marked in bold are only found in VP1 (e.g., the VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 1-737 of SEQ ID NO:7).

[0346] An exemplary nucleic acid sequence encoding SEQ ID NO:7 is SEQ ID NO:8:

[0347]

[0348] An exemplary reference sequence of wild-type AAVrh74 is SEQ ID NO:9 (wild-type AAVrh74) as follows:

[0349]

[0350] An alternative exemplary reference sequence of SEQ ID NO: 10 (instead of wild-type AAVrh74) is as follows:

[0351]

[0352]

[0353] In the above sequences (SEQ ID NO:9 or SEQ ID NO:10), sequences found in VP1, VP2 and VP3 are underlined (e.g., the VP3 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 204-739 of SEQ ID NO:9), sequences found in both VP1 and VP2 are marked in bold (e.g., the VP2 capsid polypeptide includes, e.g., consists of, amino acids 137-739 of SEQ ID NO:9), and sequences not marked in underline or bold are only found in VP1 (e.g., the VP1 capsid polypeptide includes, e.g., consists of, amino acids corresponding to amino acids 9-739 of SEQ ID NO:1).

[0354] An exemplary nucleic acid sequence encoding SEQ ID NO:9 is SEQ ID NO:11.

[0355]

[0356] Exemplary reference sequences SEQ ID NO:60 of AAV2 variants identified from ophthalmic literature are as follows:

[0357] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL(SEQ IDNO:60)

[0358] An exemplary nucleic acid sequence encoding SEQ ID NO:60 is SEQ ID NO:61.

[0359]

[0360] In some embodiments, described herein are capsid polypeptides, e.g., as described in Table 2, which, when included in a viral particle comprising a payload, e.g., after intravitreal administration, provide increased delivery of such payload to one or more tissues or cell types of the eye relative to otherwise identical viral particles comprising the capsid polypeptide of SEQ ID NO: 60 (such as, e.g., neural retina, macula, and / or choroid / RPE). In some embodiments, described herein are capsid polypeptides, e.g., as described in Table 2, which, when included in a viral particle comprising a payload, e.g., after intravitreal or intracameral administration, provide increased delivery of such payload to one or more tissues or cell types of the eye relative to otherwise identical viral particles comprising the capsid polypeptide of SEQ ID NO: 60 (such as, e.g., tissues or cells of the trabecular meshwork and Schlemm's canal).

[0361] The present disclosure relates to structural capsid proteins (including VP1, VP2 and VP3) encoded by capsid (Cap) genes. These capsid proteins form the outer protein structural shell (i.e., capsid) of viral vectors (such as AAV). The VP capsid proteins synthesized by Cap polynucleotides typically contain methionine as the first amino acid (Met1) in the polypeptide sequence, which is associated with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, after or during polypeptide synthesis, the first methionine (Met1) residue or generally any first amino acid (AA1) is typically removed by a protein processing enzyme (such as Met-aminopeptidase). This "Met / AA shearing" process is typically associated with the corresponding acetylation of the second amino acid (e.g., alanine, valine, serine, threonine, etc.) in the polypeptide sequence. Met shearing typically occurs on VP1 and VP3 capsid proteins, but may also occur on VP2 capsid protein. In the case of incomplete Met / AA cleavage, a mixture of one or more (one, two or three) VP capsid proteins containing viral capsids can be produced, some of which include Met1 / AA1 amino acids (Met+ / AA+), and some of which lack Met1 / AA1 amino acids (Met- / AA-) due to Met / AA cleavage. For further discussion of Met / AA cleavage 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. October 2017. 28(5):255-267; Hwang et al. N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. February 19, 2010. 327(5968):973–977; the contents of each of which are incorporated herein by reference in their entirety. According to the present disclosure, reference to capsid polypeptides is not limited to cleaved (Met- / AA-) or uncleaved (Met+ / AA+), and in the context also refers to independent capsid polypeptides, viral capsids composed of a mixture of capsid proteins and / or polynucleotide sequences (or fragments thereof) encoding, describing, producing or leading to the capsid polypeptides of the present disclosure.Direct reference to a "capsid polypeptide" (such as VP1, VP2 or VP3) also includes VP capsid proteins comprising Met1 / AA1 amino acids (Met+ / AA+), as well as corresponding VP capsid polypeptides lacking Met1 / AA1 amino acids, for example due to Met / AA cleavage (Met- / AA-). Further in accordance with the present disclosure, reference to a specific SEQ ID NO: (whether protein or nucleic acid) that comprises or encodes one or more capsid polypeptides comprising Met1 / AA1 amino acids (Met+ / AA+) should be understood to teach VP capsid polypeptides lacking Met1 / AA1 amino acids, when it is apparent upon reviewing the sequence that the first listed amino acid (whether Met1 / AA1 or not) may not be present. As a non-limiting example, reference to a VP1 polypeptide sequence that is 736 amino acids in length and includes the "Met1" amino acid (Met+) encoded by the AUG / ATG start codon should also be understood to teach a VP1 polypeptide sequence that is 735 amino acids in length and does not include the "Met1" amino acid (Met-) of the 736 amino acid Met+ sequence. As a second non-limiting example, reference to a VP1 polypeptide sequence that is 736 amino acids in length and includes the "AA1" amino acids (AA1+) encoded by any NNN start codon may also be understood to teach a VP1 polypeptide sequence that is 735 amino acids in length and does not include the "AA1" amino acids (AA1-) of the 736 amino acid AA1+ sequence. Reference to a viral capsid formed by a VP capsid protein (such as reference to a specific AAV capsid serotype) may incorporate a VP capsid protein that includes Met1 / AA1 amino acids (Met+ / AA1+), a corresponding VP capsid protein that lacks Met1 / AA1 amino acids, for example due to Met / AA1 splicing (Met- / AA1-), and combinations thereof (Met+ / AA1+ and Met- / AA1-). As non-limiting examples, an AAV capsid serotype may include a combination of VP1 (Met+ / AA1+), VP1 (Met- / AA1-), or VP1 (Met+ / AA1+) and VP1 (Met- / AA1-). The AAV capsid serotype may also include VP3(Met+ / AA1+), VP3(Met- / AA1-), or a combination of VP3(Met+ / AA1+) and VP3(Met- / AA1-); and may also include similar optional combinations of VP2(Met+ / AA1) and VP2(Met- / AA1-).

[0362] In some embodiments, the reference AAV capsid sequence comprises an amino acid sequence that is 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% identical to any of those described above.

[0363] In some embodiments, the reference AAV capsid sequence is encoded by a nucleotide sequence that is 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% identical to any of those described above. In certain embodiments, the reference sequence is not an AAV capsid sequence, but rather a different vector (e.g., lentivirus, plasmid, etc.).

[0364] In some embodiments, nucleic acids of the present disclosure (e.g., encoding AAV2 variant capsid proteins) comprise conventional control elements or sequences that are operably linked to the nucleic acid molecule in a manner that allows transcription, translation, and / or expression in cells transfected with the nucleic acid (e.g., a plasmid vector comprising the nucleic acid) or infected with a virus comprising the nucleic acid. As used herein, "operably linked" sequences include expression control sequences that are contiguous to a gene of interest and expression control sequences that act in trans or at a distance to control a gene of interest.

[0365] Expression control sequences include efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; appropriate transcription initiation, termination, promoter and enhancer sequences; sequences that stabilize cytoplasmic mRNA; sequences that enhance protein stability; sequences that improve translation efficiency (e.g., Kozak consensus sequences); and in some embodiments, sequences that enhance secretion of the encoded transgenic product. Expression control sequences (including native, constitutive, inducible and / or tissue-specific promoters) are known in the art and can be used with the compositions and methods disclosed herein.

[0366] In some embodiments, the native promoter of the transgenic can be used. Without wishing to be bound by theory, the native promoter can mimic the native expression of the transgenic, or provide temporal, developmental or tissue-specific expression, or expression in response to a particular transcriptional stimulus. In some embodiments, the transgenic can be operably linked to other native expression control elements, such as an enhancer element, a polyadenylation site or a Kozak consensus sequence, for example to mimic native expression.

[0367] In some embodiments, the transgene is operably linked to a tissue-specific promoter.

[0368] In certain embodiments, the vector carrying the transgene, such as a plasmid, may also include a selectable marker or reporter gene. Such selectable reporter gene or marker gene may be used to signal the presence of the vector (e.g., plasmid) in a bacterial cell. Other components of the vector, such as a plasmid, may include an origin of replication. The selection of these and other promoters and vector elements is conventional and many such sequences are available (see, e.g., Sambrook et al. and references cited therein).

[0369] In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the eye compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the retina compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the non-macular retina compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the macula compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the trabecular meshwork compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the trabecular meshwork relative to the retina compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the trabecular meshwork relative to the non-macular retina compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the trabecular meshwork relative to the macula compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the macula relative to the retina compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the macula relative to the non-macular retina compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the macula relative to the trabecular meshwork compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the macula relative to the non-macular retina and trabecular meshwork compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1).In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the macula relative to the retina and trabecular meshwork compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the retina relative to the macula and trabecular meshwork compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the non-macular retina relative to the macula and trabecular meshwork compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the trabecular meshwork relative to the macula and retina compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the trabecular meshwork relative to the macula and non-macular retina compared to a viral particle with a reference capsid polypeptide, e.g., with a wild-type capsid polypeptide (SEQ ID NO: 1).

[0370] In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by at least 1-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by at least 2-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 4-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 6-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 8-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 10-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 15-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 16-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 32-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 50-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 70-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 100-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 200-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1).In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 300-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 400-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 500-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by 1000-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, increased ocular transduction is measured by comparing the level of mRNA in the target tissue (e.g., in a cell or cell population of the target tissue) produced by the nucleic acid packaged in the variant viral particles with the level of mRNA in the target tissue (e.g., in a cell or cell population of the target tissue) produced by the nucleic acid packaged in the reference viral particles (e.g., packaged in a capsid comprising the capsid polypeptide of SEQ ID NO: 1).

[0371] In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the eye compared to a viral particle with a reference capsid polypeptide, e.g., a reference capsid polypeptide with SEQ ID NO: 60. In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the trabecular meshwork compared to a viral particle with a reference capsid polypeptide, e.g., a reference capsid polypeptide of SEQ ID NO: 60. In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by at least 1-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a reference capsid polypeptide of SEQ ID NO: 60. In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by at least 1.5-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a reference capsid polypeptide of SEQ ID NO: 60. In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by at least 2-fold, e.g., compared to a viral particle with a reference capsid polypeptide, e.g., a reference capsid polypeptide of SEQ ID NO: 60. In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction by at least 2.5-fold, e.g., compared to a viral particle having a reference capsid polypeptide, e.g., a reference capsid polypeptide of SEQ ID NO: 60. In some embodiments, the increased ocular transduction is measured in the trabecular meshwork, e.g., as described in Example 3. In some embodiments, the capsid polypeptide is an isolated or purified polypeptide (e.g., isolated or purified from cells, other biological components, or contaminants). In some embodiments, the variant polypeptide is present in a parvoviral particle, e.g., as described herein. In some embodiments, the variant capsid polypeptide is present in a cell, a cell-free system, or a translation system, e.g., as described herein.

[0372] In some embodiments, the cell is a non-human cell. In other embodiments, the cell is not a human pluripotent stem cell, for example, it is not a human embryonic stem cell.

[0373] In some embodiments, the capsid polypeptide is present in a parvovirus B (eg, AAV2) particle. In some embodiments, the capsid particle has increased ocular transduction.

[0374] In some embodiments, the parvoviral particle comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence provided herein (e.g., SEQ ID NO: 12-27). In some embodiments, the variant capsid polypeptide comprises an amino acid sequence that differs from the amino acid sequence of the variant capsid polypeptide provided herein 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 acid.

[0375] In some embodiments, the additional changes improve the production characteristics of the viral particles (such as but not limited to the parvoviral particles) or the method of making them. In some embodiments, the additional changes improve or change another characteristic of the viral particles (such as but not limited to the parvoviral particles), such as tropism.

[0376] In embodiments, improved transduction is measured as by quantifying viral RNA from target tissue. In some embodiments, improved biodistribution is measured as by quantifying viral DNA from target tissue. In some embodiments, improved transduction is measured after production from HEK293 cells, e.g., as described in the Examples.

[0377] VP1 nucleic acid and peptide

[0378] The present disclosure further relates in part to a nucleic acid comprising a sequence encoding a capsid polypeptide, such as, but not limited to, a parvovirus (e.g., parvovirus B, such as AAV2) capsid polypeptide as provided herein, and to a VP1 polypeptide encoded thereby. In some embodiments, the polypeptide comprises the sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0379] Virus particles

[0380] The present disclosure also relates in part to a viral particle, such as, but not limited to, a parvoviral particle (eg, a functional parvoviral particle) comprising a nucleic acid or polypeptide described herein or produced by a method described herein.

[0381] Parvovirus is a single-stranded DNA parvovirus that grows only in cells that provide certain functions, such as by co-infection with a helper virus. Several species of parvovirus are known, including parvovirus A and parvovirus B, which include serotypes known in the art as adeno-associated viruses (AAV). At least thirteen AAV serotypes 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 some extent parvovirus species are significantly related in structure and function. (See, for example, Blacklowe, Parvoviruses and Human Disease, pp. 165-174, JRPattison, ed. (1988); and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes apparently display very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins. In addition, heteroduplex analysis revealed extensive cross-hybridization between serotypes along the length of the genome, further indicating mutual relatedness. The Dependox virus genome also contains self-annealing segments at the ends corresponding to "inverted terminal repeats" (ITRs).

[0382] The genome structures of naturally occurring parvoviruses (e.g., AAV serotypes) are very similar. For example, the genome of AAV is a linear single-stranded DNA molecule with a length of approximately 5,000 nucleotides (nt) or less. Inverted terminal repeats (ITRs) are located on both sides of the unique coding nucleotide sequences of non-structural replication (Rep) proteins and structural capsid (Cap) proteins. Three different viral particle (VP) proteins form the capsid. The terminal 145nt is self-complementary and organized into an energy-stable intramolecular duplex that can form a T-shaped hairpin. These hairpin structures serve as the starting point for viral DNA replication and are thus used as primers for cellular DNA polymerase complexes. The Rep gene encodes Rep proteins: Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed by the p5 promoter, and Rep 52 and Rep40 are transcribed by the p19 promoter. The cap gene encodes VP proteins VP1, VP2, and VP3. The cap gene is transcribed by the p40 promoter.

[0383] In some embodiments, the parvoviral virus particles of the present disclosure comprise a nucleic acid comprising a capsid polypeptide as provided herein. In some embodiments, the particles comprise a polypeptide as provided herein.

[0384] In some embodiments, the parvoviral particles of the present disclosure may be AAV2 particles or variants thereof. In some embodiments, the AAV2 particles comprise a capsid polypeptide as provided herein or a nucleic acid molecule encoding the same.

[0385] In some embodiments, the parvovirus particle comprises a capsid comprising a variant capsid polypeptide described herein. In some embodiments, the parvovirus particle comprises a variant capsid polypeptide and a nucleic acid molecule described herein. In some embodiments, the parvovirus particle comprises a variant capsid polypeptide and a nucleic acid molecule described herein, the nucleic acid molecule comprising one or more inverted terminal repeats (ITRs), such as ITRs derived from AAV2 parvovirus, one or more regulatory elements (e.g., promoters) and a payload (e.g., as described herein). In some embodiments, at least one of the ITRs is modified. In some embodiments, the nucleic acid molecule is single-stranded. In some embodiments, the nucleic acid molecule is self-complementary.

[0386] In some embodiments, the viral particles comprise variant capsid polypeptides, such as those provided herein. In some embodiments, the viral particles comprise variant capsid polypeptides having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequences provided in Table 2. In some embodiments, the viral particles comprise variant capsid polypeptides having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequences of SEQ ID NO: 12. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 13. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 14. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 15. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 16. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 17. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 18. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO:19.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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 20. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 21. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 22. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 23. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 24. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 25. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 26. 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 the VP1, VP2 or VP3 sequence of SEQ ID NO: 27.

[0387] In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 12. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 13. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 14. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 15. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 16. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 17. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 18. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 19. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 20. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 21. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 22. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 23. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 24. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 25. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 26. In some embodiments, the viral particle comprises a variant capsid polypeptide having a sequence of SEQ ID NO: 27.

[0388] Increased ocular transduction properties

[0389] The present disclosure relates in part to nucleic acids, polypeptides, cells, cell-free systems, translation systems, viral particles, and methods associated with making the same to produce viral particles having increased ocular transduction compared to viral particles having a capsid polypeptide of a reference sequence (e.g., having the wild-type sequence of SEQ ID NO: 1). In some embodiments, the use of viral particles comprising variant capsid polypeptides results in increased ocular transduction of a transgene in the eye, and thus results in increased expression of the transgene in the eye. In some embodiments, the use of viral particles comprising variant capsid polypeptides results in increased ocular transduction of a transgene in the retina, and thus results in increased expression of the transgene in the retina. In some embodiments, the use of viral particles comprising variant capsid polypeptides results in increased ocular transduction of a transgene in the non-macular retina, and thus results in increased expression of the transgene in the non-macular retina. In some embodiments, the use of viral particles comprising variant capsid polypeptides results in increased ocular transduction of a transgene in the macula, and thus results in increased expression of the transgene in the macula. In some embodiments, the use of viral particles comprising variant capsid polypeptides results in increased ocular transduction of a transgene in the trabecular meshwork, and thus results in increased expression of the transgene in the trabecular meshwork.

[0390] In some embodiments, the use of viral particles comprising variant capsid polypeptides results in increased ocular transduction of transgenes in the anterior third of the eye, which includes 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. Thus, in some embodiments, the use of viral particles comprising variant capsid polypeptides results in increased ocular transduction of transgenes in the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof. In some embodiments, the use of viral particles comprising variant capsid polypeptides results in increased ocular transduction of transgenes behind the lens, such as in the anterior vitreous membrane and all optical structures behind it, such as the vitreous humor, retina, choroid, or optic nerve, or any combination thereof. Thus, in some embodiments, the use of viral particles comprising variant capsid polypeptides results in increased ocular transduction of transgenes in the anterior vitreous membrane and all optical structures behind it, such as the vitreous humor, retina, choroid, or optic nerve, or any combination thereof. In some embodiments, use of viral particles comprising a variant capsid polypeptide results in increased ocular transduction of the transgene in the anterior third of the eye and behind the lens.

[0391] In some embodiments, the increase in ocular transduction is about 1 to 7 times better (e.g., about 2 to 5 times better, e.g., about 3 to 5 times better) on a log2 scale compared to viral particles having a reference sequence capsid polypeptide (e.g., having a wild-type capsid polypeptide SEQ ID NO: 1).

[0392] In some embodiments, the capsid polypeptide present in the 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 the 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 the viral particle increases transduction without increasing the biodistribution of the variant capsid polypeptide in the trabecular meshwork relative to SEQ ID NO: 1.

[0393] Table 3, Table 4A, Table 4B, Table 4C, and Table 4D list information about the biodistribution of variant-dependent parvoviral particles of capsid polypeptides comprising the indicated variant capsids in different layers, structures, and / or parts of the eye. In Table 3, in all cases of library experiment 1, the biodistribution in the retina was measured after IVT injection, and the biodistribution in the trabecular meshwork was measured after IC injection. In Table 4A, in all cases of library experiment 2, the biodistribution in the retina was measured after IVT injection, the biodistribution in the trabecular meshwork was measured after IC or IVT injection (as indicated in the table), and the biodistribution in the choroid was measured after IVT injection. In Table 4B, in all cases of library experiment 3, the biodistribution in neural retinal tissue was measured after IVT injection. In Table 4C, in all cases of library experiment 3, the biodistribution in neural retinal tissue, the biodistribution in macular tissue, and the biodistribution in the trabecular meshwork were measured after IVT injection. In Table 4D, in all cases of Library Experiment 3, biodistribution in the trabecular meshwork was measured after IC injection. Unless otherwise indicated, measurements were made relative to wild-type AAV2 (SEQ ID NO: 1). Trabecular biodistribution measurements shown in Table 4C and Table 4D are shown relative to an AAV2 variant identified from the ophthalmic literature having a capsid polypeptide of SEQ ID NO: 60 and encoded by a nucleic acid of SEQ ID NO: 61. The data shown in Table 3 and Table 4A are in log2 scale.

[0394] Table 3

[0395]

[0396] Table 4A

[0397]

[0398]

[0399] Table 4B

[0400]

[0401] Table 4C

[0402]

[0403] Table 4D

[0404]

[0405] According to some embodiments, the variant capsid polypeptides described herein comprise a combination of an N587 substitution mutation and an N-terminally juxtaposed insertion peptide (e.g., an insertion peptide derived from after the glycine (G) at position 586 (numbered according to WT AAV2 VP1; SEQ ID NO: 1)). In some embodiments, the peptide insertion is 6 or fewer amino acids. In some embodiments, the peptide insertion consists of 6 amino acids. In some embodiments, the peptide insertion consists of 7 amino acids. In some embodiments, the peptide insertion is 7 or fewer amino acids. In some embodiments, the peptide insertion is 7 or more amino acids, such as 7, 8, 9, 10, or 11 amino acids. In some embodiments, the peptide insertion is between 6 and 11 amino acids. In some embodiments, the peptide insertion consists of 11 amino acids. In some embodiments, the insertion peptide comprises a threonine-arginine-proline ("TRP") sequence at its C-terminus (e.g., as in VAR-3, VAR-5, VAR-6, VAR-8, VAR-11, VAR-13, and VAR-14). In some embodiments, viral particles comprising a variant capsid polypeptide comprising a N587 substitution mutation (e.g., an N587A mutation) in combination with an N-terminally juxtaposed insertion peptide exhibit increased ocular transduction and / or biodistribution relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0406] According to some embodiments, the variant capsid polypeptides described herein comprise a threonine-arginine-proline-alanine ("TRPA") sequence. In some embodiments, the variant capsid polypeptide comprises a peptide insertion (relative to SEQ ID NO: 1) comprising at least some TRPA sequences (e.g., 3 or 4 amino acids forming a TRPA sequence). For example, VAR-1, VAR-2, VAR-3, VAR-5, VAR-6, VAR-8, VAR-11, VAR-13, and VAR-14 each comprise a TRPA sequence. In some embodiments, the insertion site is after the glycine (G) at position 586 (numbered according to WT AAV2 VP1; SEQ ID NO: 1). In some embodiments, the insertion site is after the asparagine (N) at position 587 (numbered according to WT AAV2 VP1; SEQ ID NO: 1). In some embodiments, the TRPA sequence is formed by a peptide insertion comprising a C-terminal TRPA sequence. For example, both VAR-1 and VAR-2 comprise a peptide insertion comprising a TRPA sequence at their C-terminus, optionally wherein the peptide insertion is derived from after an asparagine (N) at position 587 (numbered according to WT AAV2 VP1; SEQ ID NO: 1). In some embodiments, the TRPA sequence is formed by a peptide insertion comprising a threonine-arginine-proline ("TRP") sequence at its C-terminus followed by an alanine (A) substitution (e.g., a N587A substitution). In such embodiments, an alanine (A) substitution mutation is combined with an inserted peptide juxtaposed to the N-terminus comprising a TRP sequence to form a "TRPA" sequence (e.g., as in VAR-3, VAR-5, VAR-6, VAR-8, VAR-11, VAR-13, and VAR-14). In some embodiments, the peptide insertion is after a glycine (G) at position 586 and an alanine substitution is at position 587 (numbered according to WT AAV2 VP1; SEQ ID NO: 1). In some embodiments, the peptide insertion is 6 or fewer amino acids. In some embodiments, the peptide insertion consists of 6 amino acids. In some embodiments, the peptide insertion is 7 or less amino acids. In some embodiments, the peptide insertion consists of 7 amino acids. In some embodiments, the peptide insertion is 7 or more amino acids, such as 7, 8, 9, 10 or 11 amino acids. In some embodiments, the peptide insertion is between 6 and 11 amino acids. In some embodiments, the peptide insertion consists of 7 amino acids. In some embodiments, relative to a virus particle comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1), a virus particle comprising a variant capsid polypeptide containing a TRPA sequence exhibits increased eye transduction and / or biodistribution.

[0407] According to some embodiments, the variant capsid polypeptide described herein comprises a threonine-alanine-arginine-proline-alanine ("TARPA") sequence. In some embodiments, the variant capsid polypeptide comprises a peptide insertion comprising at least some TARPA sequences (e.g., 2, 3 or 4 amino acids forming a TARPA sequence). In some embodiments, the variant capsid polypeptide comprises a peptide insertion (e.g., relative to SEQ ID NO: 1) comprising the entire TARPA sequence. In some embodiments, the insertion site is after the glycine (G) at position 586 (according to WT AAV2 VP1 numbering; SEQ ID NO: 1). In some embodiments, the insertion site is after the glutamine (Q) at position 584 (according to WT AAV2 VP1 numbering; SEQ ID NO: 1). In some embodiments, the TARPA sequence is formed as a combination of a peptide insertion and a substitution mutation (e.g., G586P mutation, N587A mutation) after the peptide insertion, for example, a peptide insertion comprising TAR at its C-terminus (according to WT AAV2 VP1 numbering; SEQ ID NO: 1). In some embodiments, the TARPA sequence is formed as a combination of a peptide insertion, a substitution mutation (e.g., G586P mutation, N587A mutation) following the peptide insertion (numbering according to WT AAV2 VP1; SEQ ID NO: 1), and an amino acid present in WT AAV2 VP1 (SEQ ID NO: 1), such as arginine (R) at position 585. In some embodiments, the variant capsid polypeptide comprises a peptide insertion comprising a threonine-alanine-arginine-proline ("TARP") sequence at its C-terminus and inserted after a glycine (G) at position 586, followed by an alanine substitution at position 587 (numbering according to WT AAV2 VP1; SEQ ID NO: 1), e.g., as in VAR-4 and VAR-16. In some embodiments, the variant capsid polypeptide comprises a peptide insertion comprising a threonine-alanine ("TA") sequence at its C-terminus and inserted after a glutamine (Q) at position 584, followed by a proline (P) substitution at position 586 and an alanine (A) substitution at position 587 (numbering according to WT AAV2 VP1; SEQ ID NO: 1), for example, as in VAR-7, wherein the arginine (R) at 585 forms a TARPA sequence. In some embodiments, the peptide insertion is 9 or fewer amino acids. In some embodiments, the peptide insertion is 9 or more amino acids, such as 10 or 11 amino acids. In some embodiments, the peptide insertion is between 9 and 11 amino acids. In some embodiments, the peptide insertion consists of 9 amino acids. In some embodiments, the peptide insertion consists of 11 amino acids.In some embodiments, viral particles comprising a variant capsid polypeptide comprising a TARPA sequence exhibit increased ocular transduction and / or biodistribution relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0408] Category A (neural retinal transduction): In some aspects of the present disclosure, a viral particle comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibits increased neural retinal transduction compared to a viral particle having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased neural retinal transduction is as defined in any one of Embodiments A-1 to A-8.

[0409] Embodiment A-1: ​​In embodiments of category A, transduction is about (or at least about) 2-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0410] Embodiment A-2: In embodiments of category A, transduction is about (or at least about) 4 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0411] Embodiment A-3: In embodiments of category A, transduction is about (or at least about) 6 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0412] Embodiment A-4: In embodiments of category A, transduction is about (or at least about) 10 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0413] Embodiment A-5: In embodiments of category A, transduction is about (or at least about) 30 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0414] Embodiment A-6: In embodiments of category A, transduction is about (or at least about) 50 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0415] Embodiment A-7: In embodiments of category A, transduction is about (or at least about) 70 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0416] Embodiment A-8: In embodiments of category A, transduction is about (or at least about) 100 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0417] In some embodiments, the improved transduction is within a range bounded by any two values ​​set forth in Example A-1 to Example A-8. Exemplary ranges are set forth below in Example A-9 to Example A-14.

[0418] Embodiment A-9: In embodiments of category A, the transduction range is between about 2-fold and about 100-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0419] Embodiment A-10: In embodiments of category A, the transduction range is between about 4-fold and about 100-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0420] Embodiment A-11: In embodiments of category A, the transduction range is between about 6-fold and about 100-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0421] Embodiment A-12: In embodiments of category A, the transduction range is between about 10-fold and about 100-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0422] Embodiment A-13: In embodiments of category A, the transduction range is between about 30-fold and about 100-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0423] Embodiment A-14: In embodiments of category A, the transduction range is between about 50-fold and about 100-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0424] Category B (macular transduction): In some aspects of the present disclosure, a viral particle comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibits increased macular transduction compared to a viral particle having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, increased macular transduction is as defined in any one of Embodiments B-1 to B-10.

[0425] Embodiment B-1: In embodiments of category B, transduction is about (or at least about) 2-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0426] Embodiment B-2: In embodiments of category B, transduction is about (or at least about) 4 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0427] Embodiment B-3: In embodiments of category B, transduction is about (or at least about) 20 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0428] Embodiment B-4: In embodiments of category B, transduction is about (or at least about) 50 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0429] Embodiment B-5: In embodiments of category B, transduction is about (or at least about) 100 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0430] Embodiment B-6: In embodiments of category B, transduction is about (or at least about) 200 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0431] Embodiment B-7: In embodiments of category B, transduction is about (or at least about) 300 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0432] Embodiment B-8: In embodiments of category B, transduction is about (or at least about) 400 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0433] Embodiment B-9: In embodiments of category B, transduction is about (or at least about) 500 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0434] Embodiment B-10: In embodiments of Class B, transduction is about (or at least about) 1000 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0435] In some embodiments, the improved transduction is within a range bounded by any two values ​​set forth in Examples B-1 to B-10. Exemplary ranges are set forth below in Examples B-11 to B-17.

[0436] Embodiment B-11: In embodiments of category B, the transduction range is between about 2-fold and about 1000-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0437] Embodiment B-12: In embodiments of category B, the transduction range is between about 4-fold and about 1000-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0438] Embodiment B-13: In embodiments of category B, the transduction range is between about 20-fold and about 1000-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0439] Embodiment B-14: In embodiments of category B, the transduction range is between about 50-fold and about 1000-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0440] Embodiment B-15: In embodiments of category B, the transduction range is between about 100-fold and about 1000-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0441] Embodiment B-16: In embodiments of category B, the transduction range is between about 200-fold and about 1000-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0442] Embodiment B-17: In embodiments of category B, the transduction range is between about 300-fold and about 1000-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0443] Category C (trabecular transduction): In some aspects of the present disclosure, a viral particle comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibits increased trabecular transduction compared to a viral particle having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, increased trabecular transduction is as defined in any one of Embodiment C-1 to Embodiment C-4.

[0444] Embodiment C-1: In embodiments of category C, transduction is about (or at least about) 2-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0445] Embodiment C-2: In embodiments of category C, transduction is about (or at least about) 4 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0446] Embodiment C-3: In embodiments of category C, transduction is about (or at least about) 8 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0447] Embodiment C-4: In embodiments of category C, transduction is about (or at least about) 12 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0448] In some embodiments, the improved transduction is within a range bounded by any two values ​​set forth in Example C-1 to Example C-4. Exemplary ranges are set forth below in Example C-5 to Example C-7.

[0449] Embodiment C-5: In embodiments of category C, the transduction range is between about 2-fold and about 12-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0450] Embodiment C-6: In embodiments of category C, the transduction range is between about 4-fold and about 12-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0451] Embodiment C-7: In embodiments of category C, the transduction range is between about 8-fold and about 12-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0452] Category D (trabecular transduction): In some aspects of the present disclosure, a viral particle comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibits increased trabecular transduction compared to a viral particle having a capsid polypeptide of SEQ ID NO: 60. In some embodiments, increased trabecular transduction is as defined in any one of Embodiment D-1 to Embodiment D-4.

[0453] Embodiment D-1: In embodiments of category D, transduction is about (or at least about) 1.4 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0454] Embodiment D-2: In embodiments of category D, transduction is about (or at least about) 1.5 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0455] Embodiment D-3: In embodiments of category D, transduction is about (or at least about) 2-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0456] Embodiment D-4: In embodiments of category D, transduction is about (or at least about) 2.7 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0457] In some embodiments, the improved transduction is within a range bounded by any two values ​​set forth in Example D-1 to Example D-4. Exemplary ranges are set forth below in Example D-5 and Example D-6.

[0458] Embodiment D-5: In embodiments of category D, the transduction range is between about 1.4-fold and about 2.7-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0459] Embodiment D-6: In embodiments of category D, the transduction range is between about 1.5-fold and about 2.7-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0460] Category E (choroidal transduction): In some aspects of the present disclosure, a viral particle comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibits increased choroidal transduction compared to a viral particle having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased choroidal transduction is as defined in any one of Embodiments E-1 to E-3.

[0461] Embodiment E-1: In embodiments of category E, transduction is about (or at least about) 1.5 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0462] Embodiment E-2: In embodiments of category E, transduction is about (or at least about) 2-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0463] Embodiment E-3: In embodiments of category E, the improved transduction is in the range of between about 1.5-fold and about 2-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0464] Category F (non-macular transduction): In some aspects of the present disclosure, a viral particle comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibits increased non-macular transduction compared to a viral particle having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, increased non-macular transduction is as defined in any one of Embodiments F-1 to F-6.

[0465] Embodiment F-1: In embodiments of category F, transduction is about (or at least about) 2-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0466] Embodiment F-2: In embodiments of category F, transduction is about (or at least about) 4 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0467] Embodiment F-3: In embodiments of category F, transduction is about (or at least about) 8 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0468] Embodiment F-4: In embodiments of category F, transduction is about (or at least about) 15-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0469] Embodiment F-5: In embodiments of category F, transduction is about (or at least about) 30 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0470] Embodiment F-6: In embodiments of category F, transduction is about (or at least about) 50 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0471] In some embodiments, the improved transduction is within a range bounded by any two values ​​set forth in Example F-1 to Example F-6. Exemplary ranges are set forth below in Example F-7 to Example F-9.

[0472] Embodiment F-7: In embodiments of category F, the transduction range is between about 2-fold and about 50-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0473] Embodiment F-8: In embodiments of category F, the transduction range is between about 4-fold and about 50-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0474] Embodiment F-9: In embodiments of category F, the transduction range is between about 15-fold and about 50-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0475] Category G (Virus Production): In some aspects of the present disclosure, virus particles comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibit increased virus production compared to virus particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, increased virus production is as defined in any one of Embodiments G-1 to G-8.

[0476] Embodiment G-1: In embodiments of category G, virus production is about (or at least about) 1.2 times better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0477] Embodiment G-2: In embodiments of category G, virus production is about (or at least about) 1.4 times better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0478] Embodiment G-3: In embodiments of category G, virus production is about (or at least about) 1.6 times better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0479] Embodiment G-4: In embodiments of category G, virus production is about (or at least about) 2-fold better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0480] Embodiment G-5: In embodiments of category G, virus production is about (or at least about) 2.5 times better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0481] Embodiment G-6: In embodiments of category G, virus production is about (or at least about) 3 times better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0482] Embodiment G-7: In embodiments of category G, virus production is about (or at least about) 4 times better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0483] Embodiment G-8: In embodiments of category G, virus production is about (or at least about) 6 times better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0484] In some embodiments, the improved virus production is within a range bounded by any two values ​​set forth in Example G-1 to Example G-8. Exemplary ranges are set forth below in Example G-9 to Example G-13.

[0485] Embodiment G-9: In embodiments of category G, virus production ranges between about 1.2-fold and about 6-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0486] Embodiment G-10: In embodiments of category G, virus production ranges between about 1.2-fold and about 4-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0487] Embodiment G-11: In embodiments of category G, virus production ranges between about 1.4-fold and about 4-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0488] Embodiment G-12: In embodiments of category G, virus production ranges between about 1.6-fold and about 4-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0489] Embodiment G-13: In embodiments of category G, virus production ranges between about 2-fold and about 4-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0490] Category H (non-macular biodistribution): In some aspects of the present disclosure, a viral particle comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibits increased non-macular biodistribution compared to a viral particle having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased non-macular biodistribution is as defined in any one of Embodiments H-1 to H-3.

[0491] Embodiment H-1: In embodiments of category H, the biodistribution is about (or at least about) 2-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0492] Embodiment H-2: In embodiments of category H, the biodistribution is about (or at least about) 4 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0493] Embodiment H-3: In embodiments of category H, the biodistribution is about (or at least about) 6 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0494] In some embodiments, the improved biodistribution is within a range bounded by any two values ​​set forth in Example H-1 to Example H-3. Exemplary ranges are set forth in Example H-4 and Example H-5 below.

[0495] Embodiment H-4: In embodiments of category H, the improved biodistribution is in the range of between about 2-fold and about 6-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0496] Embodiment H-5: In embodiments of category H, the improved biodistribution is in the range of between about 2-fold and about 4-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0497] Category I (trabecular meshwork biodistribution): In some aspects of the present disclosure, a viral particle comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibits increased trabecular meshwork biodistribution compared to a viral particle having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased trabecular meshwork biodistribution is as defined in any one of Embodiments I-1 to I-3.

[0498] Embodiment I-1: In embodiments of Class I, the biodistribution is about (or at least about) 1.1 times better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0499] Embodiment I-2: In embodiments of category H, the biodistribution is about (or at least about) 2-fold better relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0500] Embodiment I-3: In embodiments of category H, the improved biodistribution is in the range of between about 1.1-fold and about 2-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0501] Category J (neural retinal biodistribution): In some aspects of the present disclosure, viral particles comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibit increased neural retinal biodistribution compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased neural retinal biodistribution is as defined in any one of Embodiments J-1 to J-4.

[0502] Embodiment J-1: In embodiments of Class J, the biodistribution is about (or at least about) 2-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0503] Embodiment J-2: In embodiments of Class J, the biodistribution is about (or at least about) 5 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0504] Embodiment J-3: In embodiments of Class J, the biodistribution is about (or at least about) 10 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0505] Embodiment J-4: In embodiments of Class J, the biodistribution is about (or at least about) 20 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0506] In some embodiments, the improved biodistribution is within a range bounded by any two values ​​set forth in Example J-1 to Example J-4. Exemplary ranges are set forth below in Example J-5 and Example J-6.

[0507] Embodiment J-5: In embodiments of category J, the improved biodistribution is in the range of between about 2-fold and about 20-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0508] Embodiment J-6: In embodiments of category J, the improved biodistribution is in the range of between about 5-fold and about 20-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0509] Category K (macular biodistribution): In some aspects of the present disclosure, a viral particle comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibits increased macular biodistribution compared to a viral particle having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased macular biodistribution is as defined in any one of Embodiments K-1 to K-7.

[0510] Embodiment K-1: In embodiments of category K, the biodistribution is about (or at least about) 3 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0511] Embodiment K-2: In embodiments of category K, the biodistribution is about (or at least about) 10 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0512] Embodiment K-3: In embodiments of category K, the biodistribution is about (or at least about) 30 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0513] Embodiment K-4: In embodiments of category K, the biodistribution is about (or at least about) 60 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0514] Embodiment K-5: In embodiments of category K, the biodistribution is about (or at least about) 80 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0515] Embodiment K-6: In embodiments of category K, the biodistribution is about (or at least about) 100 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0516] Embodiment K-7: In embodiments of category K, the biodistribution is about (or at least about) 180 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0517] In some embodiments, the improved biodistribution is within a range bounded by any two values ​​set forth in Example K-1 to Example K-7. Exemplary ranges are set forth below in Example K-8 to Example K-12.

[0518] Embodiment K-8: In embodiments of category K, the biodistribution range is between about 3-fold and about 180-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0519] Embodiment K-9: In embodiments of category K, the biodistribution range is between about 10-fold and about 180-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0520] Embodiment K-10: In embodiments of category K, the biodistribution range is between about 30-fold and about 180-fold relative to virus particles comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0521] Embodiment K-11: In an embodiment of category K, the biodistribution range is between about 60 times and about 180 times greater relative to a virus particle comprising a variant capsid polypeptide having a reference sequence (e.g., a capsid polypeptide having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0522] Embodiment K-12: In an embodiment of category K, the biodistribution range is between about 80-fold and about 180-fold relative to a virus particle comprising a variant capsid polypeptide having a reference sequence (e.g., having a wild-type capsid protein, such as a capsid polypeptide having SEQ ID NO: 1).

[0523] Category L (trabecular biodistribution): In some aspects of the present disclosure, viral particles comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibit increased trabecular biodistribution compared to viral particles having an AAV2 capsid polypeptide of SEQ ID NO: 60. In some embodiments, the increased trabecular biodistribution is as defined in any one of Embodiment L-1 to Embodiment L-4.

[0524] Embodiment L-1: In embodiments of Class L, transduction is about (or at least about) 1.5 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0525] Embodiment L-2: In embodiments of Class L, transduction is about (or at least about) 2-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0526] Embodiment L-3: In embodiments of Class L, transduction is about (or at least about) 2.3 times better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0527] Embodiment L-4: In embodiments of Class L, transduction is about (or at least about) 3-fold better relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0528] In some embodiments, the improved transduction is within a range bounded by any two values ​​set forth in Example L-1 to Example L-4. Exemplary ranges are set forth below in Example L-5 to Example L-6.

[0529] Embodiment L-5: In embodiments of Class L, the transduction range is between about 1.5-fold and about 3-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0530] Embodiment L-6: In embodiments of Class L, the transduction range is between about 2-fold and about 3-fold relative to viral particles comprising a variant capsid polypeptide having a reference sequence (eg, a capsid polypeptide having SEQ ID NO: 60).

[0531] According to some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of one or more cell types compared to viral particles having wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, the cell type is an ocular cell type found in retinal tissue. In some embodiments, the cell type is an ocular cell type found in trabecular meshwork tissue. Examples of cell types include amacrine cells, bipolar cells, cones, horizontal cells, microglia, Müller glial cells, retinal ganglion cells, rods, corneal epithelium, ciliary muscle, melanocytes, Schwann cells, beam cells, juxtatubular tissue (JCT), fibroblasts, and pericytes.

[0532] In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of retinal ganglion cells compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of amacrine cells compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of bipolar cells compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of cone cells compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of Müller glial cells compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of rod cells compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of beam cells compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of ciliary muscle compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of corneal epithelium compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of proximal tubule tissue (JCT) compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of melanocytes compared to viral particles having wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of Schwann cells compared to viral particles having wild-type capsid polypeptides (SEQ ID NO: 1).In some embodiments, viral particles comprising a variant capsid polypeptide (eg, a variant capsid polypeptide described herein) exhibit reduced transduction of ciliary muscle compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1).

[0533] In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of one cell type relative to another cell type compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of retinal ganglion cells relative to amacrine cells, bipolar cells, cones, Müller glial cells, or rods compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, viral particles comprising variant capsid polypeptides (e.g., variant capsid polypeptides described herein) exhibit increased transduction of amacrine cells relative to bipolar cells, cones, Müller glial cells, or rods compared to viral particles with wild-type capsid polypeptides (SEQ ID NO: 1). In some embodiments, the viral particles comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibit increased transduction of cone cells relative to bipolar cells, amacrine cells, Müller glial cells, or rod cells compared to viral particles with wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the viral particles comprising a variant capsid polypeptide (e.g., a variant capsid polypeptide described herein) exhibit increased transduction of juxtatubular tissue (JCT) relative to beam cells, ciliary muscle, corneal epithelium, melanocytes, or Schwann cells compared to viral particles with wild-type capsid polypeptide (SEQ ID NO: 1).

[0534] Methods of making the compositions described herein

[0535] The present disclosure relates in part to a method for preparing a capsid polypeptide or a virus particle as described herein, such as, but not limited to, a parvoviral particle, for example, a parvoviral particle as described herein. In some embodiments, a method for preparing a parvoviral particle comprises providing a cell, a cell-free system, or other translation system comprising a nucleic acid as described herein encoding a variant capsid polypeptide as provided herein, or a polypeptide as provided herein (e.g., a variant capsid polypeptide); and culturing the cell, cell-free system, or other translation system under conditions suitable for producing the parvoviral particle, thereby preparing the parvoviral particle.

[0536] In certain embodiments, providing a cell comprising nucleic acid as described herein includes introducing nucleic acid into a cell, such as with nucleic acid transfection or transforming a cell. The nucleic acid disclosed herein can be used as a part of any genetic element (vector) that can be delivered to a host cell, such as naked DNA, plasmid, phage, transposon, cosmid, episome, protein in a non-viral delivery vehicle (e.g., lipid-based carrier), virus, etc., which transfers the sequence carried thereon. Such carriers can be delivered by any suitable method, including transfection, liposome delivery, electroporation, membrane fusion technology, viral infection, high-speed DNA-coated pellets and protoplast fusion delivery. Those skilled in the art have the knowledge and skills of constructing the nucleic acid manipulation of any embodiment of the present invention, and the skills include genetic engineering, recombinant engineering and synthetic technology. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0537] In some embodiments, the vectors of the present disclosure comprise sequences encoding viral particles, such as, but not limited to, the variant capsid polypeptides or fragments thereof provided herein. In some embodiments, the vectors of the present disclosure comprise sequences encoding viral particles, such as, but not limited to, the variant capsid polypeptides or fragments thereof provided herein. In some embodiments, the vectors of the present disclosure comprise sequences encoding viral particles, such as, but not limited to, the rep protein or fragments thereof. In some embodiments, such vectors may contain sequences encoding both the cap of the dependent parvovirus (e.g., the variant capsid polypeptides described herein) and the rep protein. In vectors in which both AAV rep and cap are provided, the dependent parvovirus rep and the dependent parvovirus cap sequences may all have the same dependent parvovirus species or serotype source, such as AAV2. Alternatively, the present disclosure also provides vectors in which the rep sequence is from a species or serotype different from the dependent parvovirus species or serotype from which the cap sequence is derived. In some embodiments, the rep and cap sequences are expressed from different sources (e.g., different vectors, or host cell genomes and vectors). In some embodiments, the rep sequence is fused in-frame to the cap sequence of different dependent parvovirus species or serotypes to form a chimeric dependent parvovirus vector. In some embodiments, the vectors of the invention further contain a payload, such as a minigene comprising a selected transgene (eg, a payload as described herein), eg, flanked by a parvoviral 5' ITR and a parvoviral 3' ITR.

[0538] The vectors described herein, such as plasmids, can be used for a variety of purposes, but are particularly suitable for use in producing recombinant viral particles, such as but not limited to parvoviral particles comprising parvoviral sequences or fragments thereof, and in some embodiments, a payload.

[0539] In some embodiments, the present disclosure provides a method for preparing a viral particle, such as but not limited to a parvovirus particle (e.g., a parvovirus B particle, such as an AAV2 particle or a particle comprising a variant capsid polypeptide as described herein), or a portion thereof. In some embodiments, the method comprises culturing a host cell containing a nucleic acid sequence encoding a parvovirus variant capsid polypeptide or a fragment thereof as provided herein; a functional rep gene; a payload (e.g., as described herein), for example, a minigene and a transgene comprising a parvovirus inverted terminal repeat (ITR), optionally under the control of a regulatory element such as a promoter; and sufficient auxiliary functions to facilitate the packaging of the payload (e.g., a minigene) into a parvovirus capsid. The components necessary for...

Claims

1. A variant capsid polypeptide comprising a polypeptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the VP1, VP2 or VP3 sequence of SEQ ID NO:19, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:

27.

2. The variant capsid polypeptide according to claim 1, wherein the polypeptide comprises: A mutation selected from the group consisting of mutations associated with any one of VAR-1 to VAR-16.

3. The variant capsid polypeptide according to claim 2, wherein: The mutations associated with any one of VAR-1 to VAR-16 include mutations at positions corresponding to residues 550-597 compared to SEQ ID NO:

1.

4. A variant capsid polypeptide according to any of the preceding claims, wherein the polypeptide comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1 and comprises a mutation selected from the mutations associated with any one of VAR-1 to VAR-16.

5. A variant capsid polypeptide according to any of the preceding claims, wherein the polypeptide comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to SEQ ID NO: 1, and wherein the variant capsid polypeptide comprises a mutation corresponding to a mutation at position 550, 559, 561, 586, 587, 592, 593, 597, or any combination thereof, an insertion between positions 584 and 585, between 586 and 587, or between 587 and 588, or any combination thereof, according to SEQ ID NO: 1, optionally wherein the mutation comprises an insertion, a deletion or a substitution.

6. The variant capsid polypeptide according to any one of the preceding claims, wherein the capsid polypeptide comprises: a mutation corresponding to an insertion at a position between positions 587 and 588 compared to SEQ ID NO: 1; a mutation corresponding to an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1; a mutation corresponding to a mutation at position 587 compared to SEQ ID NO: 1, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1; mutations corresponding to mutations at positions 587 and 593 compared to SEQ ID NO: 1, and insertions between positions 586 and 587 compared to SEQ ID NO: 1; mutations corresponding to mutations at positions 586 and 587 compared to SEQ ID NO: 1, and insertions between positions 584 and 585 compared to SEQ ID NO: 1; mutations corresponding to mutations at positions 592 and 597 compared to SEQ ID NO: 1, and insertions between positions 587 and 588 compared to SEQ ID NO: 1; mutations corresponding to mutations at positions 561, 587 and 597 compared to SEQ ID NO: 1, and insertions between positions 586 and 587 compared to SEQ ID NO: 1; mutations corresponding to mutations at positions 550, 586 and 587 compared to SEQ ID NO: 1, and insertions between positions 584 and 585 compared to SEQ ID NO: 1; mutations corresponding to mutations at positions 559 and 587 compared to SEQ ID NO: 1, and insertions between positions 586 and 587 compared to SEQ ID NO: 1; An insertion, such as an insertion of 1 or more amino acids, such as 1, 2 amino acids, which corresponds to an insertion between positions 584 and 585, between 586 and 587, and between 587 and 588 compared to SEQ ID NO: 1; an insertion, such as an insertion of 6 or fewer amino acids, such as an insertion of 6 amino acids, which corresponds to an insertion between positions 586 and 587, wherein the insertion sequence comprises Threonine-Arginine-Proline at its C-terminus; and a substitution mutation at the position corresponding to N587, such as Alanine at the position corresponding to N587, all numberings are compared to SEQ ID NO: 1; an insertion between positions 586 and 587, such as an insertion of 7 or more amino acids, such as 10 or 11 amino acids, wherein the insertion sequence comprises Threonine-Arginine-Proline at its C-terminus; and a substitution mutation at the position corresponding to N587, such as Alanine at the position corresponding to N587, all numbering compared to SEQ ID NO: 1; comprising a mutation of the sequence Threonine-Alanine-Arginine-Proline-Alanine, optionally wherein said sequence is located at the C-terminal position corresponding to position 584 and at the N-terminal position corresponding to position 590 compared to SEQ ID NO: 1, and optionally wherein said sequence Threonine-Alanine-Arginine-Proline-Alanine comprises both insertion and substitution mutations compared to SEQ ID NO: 1; or A combination of any of the foregoing.

7. The variant capsid polypeptide according to any one of the preceding claims, wherein the capsid polypeptide comprises: (a) an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises the polypeptide of LALGEQTRPA (SEQ ID NO: 44), or a fragment of at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids thereof; (b) an insertion at a position between positions 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises the polypeptide of LAIEQTRPA (SEQ ID NO: 45), or a fragment of at least 5, at least 6, at least 7, or at least 8 amino acids thereof; (c) a mutation of N587A, and an insertion at a position between positions 586 and 587, compared to SEQ ID NO: 1, wherein the insertion comprises the polypeptide of LALAEITRP (SEQ ID NO: 46), or a fragment thereof of at least 5, at least 6, at least 7, or at least 8 amino acids; (d) a mutation of N587A, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of LKNAETARP (SEQ ID NO: 47), or a fragment of at least 5, at least 6, at least 7, or at least 8 amino acids thereof; (e) mutations of N587A and A593T, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of LNLAIEQTRP (SEQ ID NO: 48), or a fragment of at least 5, at least 6, at least 7, or at least 8 amino acids thereof; (f) a mutation of N587A, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of MLNEQTRP (SEQ ID NO: 49), or a fragment thereof of at least 4, at least 5, at least 6, or at least 7 amino acids; (g) mutations of G586P and N587A, and an insertion at a position between positions 584 and 585 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of RSGNRADSETA (SEQ ID NO: 50), or a fragment thereof of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids; (h) a mutation of N587A, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of TGDTRP (SEQ ID NO: 51), or a fragment thereof of at least 3, at least 4, or at least 5 amino acids; (i) an insertion at a position between positions 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises the polypeptide of LQGETIRPA (SEQ ID NO: 52), or a fragment of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids thereof; (j) a mutation of N587A, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of QNLANPETTRP (SEQ ID NO: 53), or a fragment thereof of at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids; (k) mutations of T592A and T597W, and an insertion at a position between positions 587 and 588 compared to SEQ ID NO: 1, wherein the insertion comprises the polypeptide of RAPQETTRPA (SEQ ID NO: 54), or a fragment of at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids thereof; (l) a mutation of N587A, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of ANLTTTRP (SEQ ID NO: 55), or a fragment of at least 4, at least 5, at least 6, or at least 7 amino acids thereof; (m) a mutation of N587A, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of ALLAGEQTRP (SEQ ID NO: 56), or a fragment thereof of at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids; (n) a mutation of N587A, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of ALLAGEQTRP (SEQ ID NO: 56), or a fragment thereof of at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids; (o) mutations of D561C, N587A and T597N, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of GLRAEQTRP (SEQ ID NO: 57), or a fragment of at least 5, at least 6, at least 7 or at least 8 amino acids thereof; (p) mutations of T550N, G586P and N587A, and an insertion at a position between positions 584 and 585 compared to SEQ ID NO: 1, wherein the insertion comprises the polypeptide of RARLDETA (SEQ ID NO: 58), or a fragment of at least 4, at least 5, at least 6 or at least 7 amino acids thereof; or (q) mutations of I559L and N587A, and an insertion at a position between positions 586 and 587 compared to SEQ ID NO: 1, wherein the insertion comprises a polypeptide of TNLARGETARP (SEQ ID NO: 59), or a fragment thereof of at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids.

8. A variant capsid polypeptide comprising (a) a polypeptide of any one of SEQ ID NO: 19, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: 27, (b) a polypeptide of any one of SEQ ID NO: 19, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO: NO:27, (c) a polypeptide comprising a sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, wherein the sequence comprises at least one (e.g., one, two, three or more, such as all) of the mutational differences associated with any one of SEQ ID NO:12 to SEQ ID NO:27 relative to SEQ ID NO:1; or (d) a polypeptide having at least 1 but no more than 20, no more than 19, no more than 18, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, 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 3 or no more than 2 amino acid mutations relative to the polypeptide of (a) or (b), wherein the polypeptide comprises a sequence that is associated with any one of SEQ ID NO:12 to SEQ ID NO:27 relative to SEQ ID NO:

1. At least one (e.g., one, two, three or more, such as all) of the mutational differences associated with any one of NO:

27.

9. A variant capsid polypeptide comprising: having 95% or greater amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs: 12-27 and; An amino acid sequence having at least 80% of the mutations in said amino acid sequence of one of SEQ ID NOs: 12-27 compared to SEQ ID NO:

1.

10. A variant capsid polypeptide comprising: having less than 95% amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs: 12-27 and; An amino acid sequence having at least 80% of the mutations in said amino acid sequence of one of SEQ ID NOs: 12-27 compared to SEQ ID NO:

1.

11. A variant capsid polypeptide comprising: having 95% or greater amino acid sequence identity to the amino acid sequence of one of SEQ ID NOs: 12-27 and; An amino acid sequence having less than 80% of the mutations in said amino acid sequence of one of SEQ ID NOs: 12-27 compared to SEQ ID NO:

1.

12. A nucleic acid molecule comprising a sequence encoding the variant capsid polypeptide according to any one of claims 1 to 11.

13. The nucleic acid molecule of claim 12, comprising one or more regulatory elements operably linked to the sequence encoding the variant capsid polypeptide.

14. A nucleic acid molecule according to any one of claims 12 to 13, comprising SEQ ID NO: 35, 28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42 or 43, or a fragment thereof, or a variant thereof having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

15. A viral particle (e.g., an adeno-associated virus ("AAV") particle) comprising a variant capsid polypeptide according to any one of claims 1 to 11 or comprising a variant capsid polypeptide encoded by a nucleic acid molecule according to any one of claims 12 to 14.

16. The viral particle of claim 15, comprising a nucleic acid comprising a heterologous transgene and one or more regulatory elements.

17. The viral particle of any one of claims 15 to 16, comprising a variant capsid polypeptide according to any one of claims 1 to 11, wherein the viral particle, or a viral particle comprising the variant capsid polypeptide, or a viral particle comprising a variant capsid polypeptide encoded by a nucleic acid molecule according to any one of claims 12 to 14, exhibits increased ocular transduction relative to wild-type AAV2 (e.g., a viral particle comprising a capsid polypeptide of SEQ ID NO: 1 or a capsid polypeptide encoded by SEQ ID NO: 2), e.g., as measured in mice or NHPs, e.g., as described herein.

18. A method for producing viral particles comprising a variant AAV2 capsid polypeptide, the method comprising introducing a nucleic acid molecule according to any one of claims 12 to 14 into a cell (eg, HEK293 cell), and harvesting the viral particles therefrom.

19. A method of delivering a payload (e.g., a nucleic acid) to a cell, the method comprising contacting the cell with a parvoviral particle comprising a variant capsid polypeptide according to any one of claims 1 to 11 or a viral particle according to any one of claims 15 to 17 and a payload.

20. The method of claim 19, wherein the cell is an ocular cell.

21. The method of claim 20, wherein the ocular cell is located in the retina, macula, or trabecular meshwork.

22. A method of delivering a payload (e.g., a nucleic acid) to a subject, the method comprising administering to the subject a parvoviral particle comprising a variant capsid polypeptide according to any one of claims 1 to 11 and the payload, or administering to the subject a viral particle according to any one of claims 15 to 17.

23. The method of claim 22, wherein the particles deliver the payload to an eye.

24. The method of claim 22, wherein the particles deliver the payload to the retina, macula, or trabecular meshwork.

25. The method of any one of claims 22 to 24, wherein the particles deliver the payload to the eye with increased transduction in one or more regions of the eye compared to viral particles comprising the capsid polypeptide of SEQ ID NO: 1, wherein the one or more regions of the eye are selected from the retina, the macula, the trabecular meshwork, or any combination thereof.

26. The method of claim 25, wherein the retina comprises a non-macular retina.

27. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 100-fold, 128-fold, or 1000-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO:

1.

28. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to non-macular retinal tissue relative to macular tissue.

29. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to macular tissue relative to non-macular retinal tissue.

30. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to macular tissue relative to trabecular meshwork tissue.

31. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, or 64-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to non-macular retinal tissue relative to trabecular meshwork tissue.

32. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific for macular tissue and non-macular retinal tissue relative to trabecular meshwork tissue.

33. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to trabecular meshwork tissue relative to macular tissue and non-macular retinal tissue.

34. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to trabecular meshwork tissue relative to macular tissue.

35. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to trabecular meshwork tissue relative to non-macular retinal tissue.

36. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, wherein the increase in transduction is at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, and wherein the increase in transduction is specific to trabecular meshwork tissue, macular tissue, and non-macular retinal tissue.

37. The variant capsid polypeptide of any one of claims 1 to 11, the viral particle of any one of claims 15 to 17, or the method of any one of claims 18 to 26, wherein the particle (e.g., a particle comprising the variant capsid polypeptide) delivers the payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO: 1, without increased biodistribution in one or more regions of the eye compared to a viral particle comprising the capsid polypeptide of SEQ ID NO:

1.

38. The method of any one of claims 19 to 26, wherein administration to the subject is via intravitreal injection or intracameral injection.

39. A method of treating a disease or condition in a subject, the method comprising administering to the subject a Parvoviral particle in an amount effective to treat the disease or condition, wherein the Parvoviral particle is a particle comprising a capsid polypeptide according to any one of claims 1 to 11 and 27 to 37 or a capsid polypeptide encoded by a nucleic acid according to any one of claims 12 to 14, or is a viral particle according to any one of claims 15 to 17.

40. A cell, cell-free system or other translation system comprising a capsid polypeptide, nucleic acid molecule or viral particle according to any one of claims 1 to 17 or 27 to 37.

41. A method for preparing parvovirus (e.g., adeno-associated parvovirus (AAV)) particles, the method comprising: Providing a cell, a cell-free system or other translation system comprising a nucleic acid according to any one of claims 12 to 14; as well as culturing the cell, cell-free system or other translation system under conditions suitable for producing the parvoviral virus-dependent particle, Thus, the parvovirus particles are prepared.

42. The method of claim 41, wherein the cell, cell-free system or other translation system comprises a second nucleic acid molecule and the second nucleic acid molecule is packaged in the parvoviral particle.

43. The method of claim 42, wherein the second nucleic acid comprises a payload, such as a heterologous nucleic acid sequence encoding a therapeutic product.

44. The method of any one of claims 41 to 43, wherein the nucleic acid of any one of claims 12 to 14 mediates the production of parvoviral-dependent particles that do not comprise the nucleic acid of any one of claims 12 to 14.

45. The method of any one of claims 41 to 44, wherein the nucleic acid of any one of claims 12 to 14 mediates the production of parvoviral-dependent particles at a level that is at least 10%, at least 20%, at least 50%, at least 100%, at least 200% or higher than the level of production mediated by the nucleic acid of SEQ ID NO:

2.

46. ​​A composition, such as a pharmaceutical composition, comprising the viral particle according to any one of claims 15 to 17 or the viral particle produced by the method according to any one of claims 18 or 41 to 45, and a pharmaceutically acceptable carrier.

47. The variant capsid polypeptide of any one of claims 1 to 11 and 27 to 37, the nucleic acid molecule of any one of claims 12 to 14, or the viral particle of any one of claims 15 to 17 for use in treating a disease or condition in a subject.

48. A variant capsid polypeptide according to any one of claims 1 to 11 and 27 to 37, a nucleic acid molecule according to any one of claims 12 to 14, or a viral particle according to any one of claims 15 to 17 for use in the manufacture of a medicament for treating a disease or condition in a subject.

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