Capsid variants and methods of using same

Abstract

This disclosure is directed, in part, to variant capsid polypeptides that can be used to deliver a payload.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 331,543, filed April 15, 2022, and U.S. Provisional Patent Application No. 63 / 443,262, filed February 3, 2023, each of which is incorporated by reference in its entirety herein.

[0002] Reference to Electronically Submitted Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML file format, which is incorporated herein by reference in its entirety. The XML copy created on April 14, 2023 is named "DYO-015WOSEQ.XML" and is 119,720 bytes in size. [Background technology]

[0003] Dependoparvoviruses, such as adeno-associated dependoparvoviruses, such as adeno-associated viruses (AAV), are of interest as vectors for delivering various payloads to cells, including human subjects. Summary of the Invention

[0004] The present disclosure provides, in part, improved variant depend parvovirus capsid polypeptides (e.g., variants of AAV2), e.g., VP1, methods for generating depend parvoviruses, compositions for use therein, as well as viral particles comprising such capsid polypeptides. In some embodiments, viral particles comprising the capsid polypeptides have increased ocular transduction compared to viral particles that do not comprise a mutation in the capsid protein.

[0005] In some embodiments, the present disclosure is directed, in part, to a nucleic acid comprising a sequence encoding a variant capsid protein provided herein. In some embodiments, the dependoparvovirus is an adeno-associated dependoparvovirus (AAV). In some embodiments, the AAV is an AAV2 variant.

[0006] In some embodiments, the disclosure is directed, in part, to the capsid polypeptides described herein.

[0007] In some embodiments, the disclosure is directed, in part, to a dependoparvovirus particle comprising a capsid polypeptide described herein.

[0008] In some embodiments, the disclosure is directed, in part, to vectors, e.g., plasmids, that contain the nucleic acids described herein.

[0009] In some embodiments, the disclosure is directed, in part, to a dependoparvoviral particle comprising a nucleic acid described herein (e.g., a nucleic acid comprising a sequence encoding a capsid polypeptide, e.g., VP1), wherein the coding sequence comprises an alteration or mutation provided herein.

[0010] In some embodiments, the disclosure is directed, in part, to dependoparvovirus particles comprising a variant capsid polypeptide described herein, e.g., comprising a 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, 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.

[0011] In some embodiments, the disclosure is directed, in part, to dependoparvovirus particles comprising a variant capsid polypeptide described herein, e.g., a polypeptide that is a 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.

[0012] In some embodiments, the capsid polypeptide comprises a mutation selected from the mutations associated with any of VAR-1 through 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 of VAR-1 through VAR-16.

[0013] In some embodiments, the capsid polypeptide comprises an amino acid sequence that is 95% or more identical to the amino acid sequence of one of SEQ ID NOs: 12-27 and has at least 80% of the mutations in one of SEQ ID NOs: 12-27 compared to SEQ ID NO: 1.

[0014] In some embodiments, the capsid polypeptide comprises an amino acid sequence that is less than 95% amino acid sequence identical to the amino acid sequence of one of SEQ ID NOs: 12-27 and has at least 80% of the mutations in one of SEQ ID NOs: 12-27 compared to SEQ ID NO: 1.

[0015] In some embodiments, the capsid polypeptide comprises an amino acid sequence that is 95% or more identical to the amino acid sequence of one of SEQ ID NOs: 12-27 and has less than 80% of mutations in one of SEQ ID NOs: 12-27 compared to SEQ ID NO: 1.

[0016] In some embodiments, the disclosure is directed to a nucleic acid molecule comprising, in part, 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.

[0017] In some embodiments, the disclosure is directed, 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 provided herein.

[0018] In some embodiments, the disclosure is directed, in part, to a cell, cell-free system, or other translation system that includes a nucleic acid or vector described herein, e.g., a sequence encoding a capsid polypeptide, such as VP1, where the capsid polypeptide coding sequence includes an alteration or mutation provided herein in the coding sequence. In some embodiments, the cell, cell-free system, or other translation system includes a dependoparvovirus particle described herein, e.g., the particle includes a nucleic acid that includes a sequence encoding a capsid polypeptide, e.g., a VP1 polypeptide, where the coding sequence includes an alteration or mutation provided herein.

[0019] In some embodiments, the disclosure is directed, in part, to a cell, cell-free system, or other translation system that comprises a polypeptide described herein, where 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 dependoparvovirus particle described herein, e.g., the particle comprises a nucleic acid that comprises a sequence encoding a VP1 polypeptide, where the VP1 coding sequence comprises the corresponding alteration or mutation as provided herein.

[0020] In some embodiments, the disclosure is directed, in part, to a method of delivering a payload to a cell, comprising contacting the cell with a dependoparvovirus particle comprising a nucleic acid described herein. In some embodiments, the disclosure is directed, in part, to a method of delivering a payload to a cell, comprising contacting the cell with a dependoparvovirus particle comprising a capsid polypeptide described herein.

[0021] In some embodiments, the disclosure is directed, in part, to a method of making depend parvovirus particles, the method comprising providing a cell, 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 provided herein) and culturing the cell, cell-free system, or other translation system under conditions suitable for the production of depend parvovirus particles, thereby making depend parvovirus particles. In some embodiments, the disclosure is directed, in part, to a method of making depend parvovirus particles described herein.

[0022] In some embodiments, the disclosure is directed, in part, to a method of making depend parvovirus particles, the method comprising providing a cell, cell-free system, or other translation system comprising a polypeptide described herein, and culturing the cell, cell-free system, or other translation system under conditions suitable for the production of depend parvovirus particles, thereby making depend parvovirus particles. In some embodiments, the disclosure is directed, in part, to a method of making depend parvovirus particles described herein.

[0023] In some embodiments, the disclosure is directed, in part, to depend parvovirus particles made in a cell, cell-free system, or other translation system, where the cell, cell-free system, or other translation system comprises a nucleic acid encoding a depend parvovirus comprising a capsid variant provided herein.

[0024] In some embodiments, the disclosure is directed, in part, to a method of treating a disease or condition in a subject, comprising administering to the subject a dependoparvovirus particle described herein in an amount effective to treat the disease or condition.

[0025] The invention is further described with reference to the following numbered embodiments. [Brief description of the drawings]

[0026] [Figure 1] Figure 1. Diagram of tissues collected in each region of the eye. For the retina (left and center panels), peripheral and central retinal samples from each of the superior, nasal, inferior, and temporal regions of the retina were collected separately, as was the macula. For each region, the neural retina and choroid / RPE layers (center panel) were collected separately. For the trabecular meshwork / Schlemm's canal (TM / SC) region (right panel), superior, temporal, nasal, and inferior samples were collected separately. [Figure 2A] A multiple sequence alignment of a representative reference capsid VP1 polypeptide is shown. Such an alignment can be used to determine amino acid positions that correspond to positions in different reference capsid polypeptides. [Figure 2B] A multiple sequence alignment of a representative reference capsid VP1 polypeptide is shown. Such an alignment can be used to determine amino acid positions that correspond to positions in different reference capsid polypeptides. [Figure 2C] A multiple sequence alignment of a representative reference capsid VP1 polypeptide is shown. Such an alignment can be used to determine amino acid positions that correspond to positions in different reference capsid polypeptides. [Figure 3A]3A and 3B show single nucleus RNA sequencing results of AAV2 wild type (FIG. 3A) and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11 (FIG. 3B) from retinal tissue samples from intravitreal ("IVT") administration during a medium throughput study (Example 3), reporting the number of unique transduction events for each of AAV2 wild type, and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11, respectively. All results are normalized to the amount of viral reads. [Figure 3B] 3A and 3B show single nucleus RNA sequencing results of AAV2 wild type (FIG. 3A) and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11 (FIG. 3B) from retinal tissue samples from intravitreal ("IVT") administration during a medium throughput study (Example 3), reporting the number of unique transduction events for each of AAV2 wild type, and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11, respectively. All results are normalized to the amount of viral reads. [Figure 4A] 4A and 4B show single nucleus RNA sequencing results of AAV2 wild type (FIG. 4A) and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11 (FIG. 4B) from retinal tissue samples from intravitreal ("IVT") administration during a medium throughput study (Example 3), reporting the number of unique transduction events for each of AAV2 wild type, and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11, respectively. All results are normalized to the amount of vector genome (vg) in the input test article. [Figure 4B]4A and 4B show single nucleus RNA sequencing results of AAV2 wild type (FIG. 4A) and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11 (FIG. 4B) from retinal tissue samples from intravitreal ("IVT") administration during a medium throughput study (Example 3), reporting the number of unique transduction events for each of AAV2 wild type, and VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11, respectively. All results are normalized to the amount of vector genome (vg) in the input test article. [Diagram 5] Figure 1 shows single nucleus RNA sequencing results of VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11 from macular tissue samples from intravitreal ("IVT") administration during a medium throughput study (Example 3), reporting the number of unique transduction events for each of the variants. All results are normalized to the amount of vector genome (vg) in the input test article. Results show zero transduction of AAV2 wild type (not shown) for any of the listed cell types. [Figure 6] Figure 1 shows single nucleus RNA sequencing results of VAR-1, VAR-2, VAR-4, VAR-5, VAR-6, VAR-7, VAR-8, and VAR-11 from trabecular meshwork tissue samples from intravitreal ("IVT") administration during a medium throughput study (Example 3), reporting the number of unique transduction events for each variant. All results are normalized to the amount of vector genome (vg) in the input test article. Results show zero transduction of AAV2 wild type (not shown), VAR-5, and VAR-11 for any of the listed cell types. [Figure 7A]Figure 7 shows single nucleus RNA sequencing results of AAV2 wild type (Figure 7A) and VAR-2, VAR-3, VAR-13, and VAR-14 (Figure 7B) from trabecular meshwork tissue samples from intracavitary ("IC") administration of a medium throughput study (Example 3), reporting the number of unique transduction events for each of the variants. All results are normalized to the amount of vector genome (vg) in the input test article. The results show zero transduction of VAR-2 for any of the listed cell types. [Figure 7B] Figure 7 shows single nucleus RNA sequencing results of AAV2 wild type (Figure 7A) and VAR-2, VAR-3, VAR-13, and VAR-14 (Figure 7B) from trabecular meshwork tissue samples from intracavitary ("IC") administration of a medium throughput study (Example 3), reporting the number of unique transduction events for each of the variants. All results are normalized to the amount of vector genome (vg) in the input test article. The results show zero transduction of VAR-2 for any of the listed cell types. [Figure 8] Figure 1 shows the retinal distribution of AAV VAR-8-eGFP from a high dose (2.26e11vg) intravitreal eye injection in cynomolgus monkeys. The genome packaged in the VAR-8 ​​capsid is a self-complementary genome composed of modified AAV2 ITRs and an eGFP reporter gene under the control of the CBH promoter. Animals were euthanized 4 weeks after dosing. Eyes were enucleated, fixed, bisected into two parts (top and bottom) and embedded in paraffin to allow sectioning for histology. (A) Schematic illustrating where retinal cross sections were collected. Briefly, 5 μm sections were collected from the top surface of the bottom half of the eye and stained for eGFP. (B) Representative images of all collected cross sections of the eye. eGFP is shown as white dots within the retina. Arrows indicate the starting position of the enlarged retinal image shown in (C). (C) Enlarged view of retinal images from both ends of the eye shown in (B) linearly stitched together. White dots represent eGFP expression delivered from VAR-8. ONL = outer nuclear layer / photoreceptor layer, INL = inner nuclear layer, GCL = ganglion cell layer. [Figure 9]Figure 1 shows the retinal distribution of AAV VAR-8-eGFP from intravitreal eye injection of a low dose (8.08e10vg) in cynomolgus monkeys. The genome packaged in the VAR-8 ​​capsid is a self-complementary genome composed of modified AAV2 ITRs and an eGFP reporter gene under the control of the CBH promoter. Animals were euthanized 4 weeks after dosing. Eyes were enucleated, fixed, bisected into two parts (upper and lower), and embedded in paraffin to allow sectioning for histology. (A) Schematic illustrating where retinal cross sections were collected. Briefly, 5 μm sections were collected from approximately one-third of the lower half of the eye and stained for eGFP. (B) Representative images of all collected eye cross sections. eGFP is shown as white dots within the retina. Arrows indicate the starting position of the enlarged retinal image shown in (C). (C) Enlarged view of retinal images from both ends of the eye shown in (B) linearly stitched together. White dots represent eGFP expression delivered from VAR-8. ONL = outer nuclear layer / photoreceptor layer, INL = inner nuclear layer, GCL = ganglion cell layer.

[0027] Enumerated Embodiments 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 a 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.

[0028] 2. The variant capsid polypeptide of embodiment 1, wherein the polypeptide comprises a mutation selected from a mutation associated with any of VAR-1 to VAR-16.

[0029] 3. The variant capsid polypeptide of embodiment 2, wherein the mutation associated with any of VAR-1 to VAR-16 comprises a mutation at a position corresponding to residues 550 to 597 compared to SEQ ID NO:1.

[0030] 4. The variant capsid polypeptide of 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 comprises a mutation selected from a mutation associated with any of VAR-1 through VAR-16.

[0031] 5. The variant capsid polypeptide of 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 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 the mutation comprises an insertion, deletion or substitution.

[0032] 6. The variant capsid polypeptide of any 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.

[0033] 7. The variant capsid polypeptide of any 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.

[0034] 8. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises a mutation at position 587 compared to SEQ ID NO:1, and a mutation corresponding to an insertion at a position between positions 586 and 587 compared to SEQ ID NO:1.

[0035] 9. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations at positions 587 and 593 compared to SEQ ID NO:1, and mutations corresponding to an insertion between positions 586 and 587 compared to SEQ ID NO:1.

[0036] 10. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations at positions 586 and 587 compared to SEQ ID NO:1, and mutations corresponding to an insertion between positions 584 and 585 compared to SEQ ID NO:1.

[0037] 11. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations at positions 592 and 597 compared to SEQ ID NO:1, and mutations corresponding to an insertion between positions 587 and 588 compared to SEQ ID NO:1.

[0038] 12. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations at positions 561, 587 and 597 compared to SEQ ID NO:1, and mutations corresponding to an insertion between positions 586 and 587 compared to SEQ ID NO:1.

[0039] 13. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations at positions 550, 586 and 587 compared to SEQ ID NO:1, and mutations corresponding to an insertion between positions 584 and 585 compared to SEQ ID NO:1.

[0040] 14. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises mutations at positions 559 and 587 compared to SEQ ID NO:1, and mutations corresponding to an insertion between positions 586 and 587 compared to SEQ ID NO:1.

[0041] 15. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises an insertion, e.g., an insertion of one or more amino acids, e.g., an insertion of one amino acid, e.g., 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.

[0042] 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, and wherein the insertion comprises a 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.

[0043] 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, and wherein the insertion comprises a 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.

[0044] 18. The variant capsid polypeptide of any of the previous embodiments, wherein the capsid polypeptide comprises 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 LALAEITRP (SEQ ID NO:46), or a fragment of at least 5, at least 6, at least 7, or at least 8 amino acids thereof.

[0045] 19. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises 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.

[0046] 20. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises N587A and A593T mutations 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.

[0047] 21. The variant capsid polypeptide of any of the previous embodiments, wherein the capsid polypeptide comprises 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 of at least 4, at least 5, at least 6, or at least 7 amino acids thereof.

[0048] 22. The variant capsid polypeptide of any of the previous embodiments, wherein the capsid polypeptide comprises G586P and N587A mutations, 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 of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids thereof.

[0049] 23. The variant capsid polypeptide of any of the previous embodiments, wherein the capsid polypeptide comprises an N587A mutation 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 of at least 3, at least 4, or at least 5 amino acids thereof.

[0050] 24. 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, and 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.

[0051] 25. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises 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 of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids thereof.

[0052] 26. The variant capsid polypeptide of any of the previous embodiments, wherein the capsid polypeptide comprises mutations T592A and T597W and an insertion at a position between positions 587 and 588 compared to SEQ ID NO:1, 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.

[0053] 27. The variant capsid polypeptide of any of the previous embodiments, wherein the capsid polypeptide comprises 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.

[0054] 28. The variant capsid polypeptide of any of the preceding embodiments, wherein the capsid polypeptide comprises 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 of at least 5, at least 6, at least 7, at least 8, or at least 9 amino acids thereof.

[0055] 29. The variant capsid polypeptide of any of the previous embodiments, wherein the capsid polypeptide comprises D561C, N587A, and T597N mutations, 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.

[0056] 30. The variant capsid polypeptide of any of the previous embodiments, wherein the capsid polypeptide comprises T550N, G586P, and N587A mutations, and an insertion at a position between positions 584 and 585 compared to SEQ ID NO:1, and 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.

[0057] 31. The variant capsid polypeptide of any of the previous embodiments, wherein the capsid polypeptide comprises I559L and N587A mutations, 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 of at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acids thereof.

[0058] 32. A variant capsid polypeptide comprising: (a) any one of the polypeptides 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) any one of the VP2 or VP3 sequences 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; or (c) a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. or (d) a polypeptide having at least one but not more than 20, not more than 19, not more than 18, not more than 17, not more than 16, not more than 15, not more than 14, not more than 13, not more than 12, not more than 10, not more than 9, not more than 8, not more than 7, not more than 6, not more than 5, not more than 3, or not more than 2 amino acid mutations relative to the polypeptide of (a) or (b), wherein the polypeptide comprises at least one (e.g., 1, 2, 3 or more, for example all) of the mutational differences associated with any of SEQ ID NOs: 12 to 27 relative to SEQ ID NO: 1.

[0059] 33. The variant capsid polypeptide of any of the previous embodiments, wherein the variant capsid polypeptide is a VP1 polypeptide, a VP2 polypeptide, or a VP3 polypeptide.

[0060] 34. A variant capsid polypeptide comprising: An amino acid sequence having 95% or more amino acid sequence identity to one of the amino acid sequences of SEQ ID NOs: 12 to 27, A variant capsid polypeptide having at least 80% of mutations within the amino acid sequence of one of SEQ ID NOs: 12-27, as compared to SEQ ID NO:1.

[0061] 35. A variant capsid polypeptide comprising: An amino acid sequence having less than 95% amino acid sequence identity to one of the amino acid sequences of SEQ ID NOs: 12 to 27, A variant capsid polypeptide having at least 80% of mutations within the amino acid sequence of one of SEQ ID NOs: 12-27, as compared to SEQ ID NO:1.

[0062] 36. A variant capsid polypeptide comprising: An amino acid sequence having 95% or more amino acid sequence identity to one of the amino acid sequences of SEQ ID NOs: 12 to 27, A variant capsid polypeptide having less than 80% of mutations within the amino acid sequence of one of SEQ ID NOs: 12-27, as compared to SEQ ID NO:1.

[0063] 37. A nucleic acid molecule comprising a sequence encoding a variant capsid polypeptide according to any one of embodiments 1 to 36.

[0064] 38. The nucleic acid molecule of embodiment 37, comprising one or more regulatory elements operably linked to a sequence encoding a variant capsid polypeptide.

[0065] 39. The nucleic acid molecule of embodiment 37 or 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.

[0066] 40. A viral particle (e.g., an adeno-associated virus ("AAV") particle) comprising a variant capsid polypeptide described in any one of embodiments 1 to 36, or comprising a variant capsid polypeptide encoded by a nucleic acid molecule described in any one of embodiments 37 to 39.

[0067] 41. A viral particle according to embodiment 40, comprising a nucleic acid comprising a heterologous transgene and one or more regulatory elements.

[0068] 42. A viral particle according to embodiment 40 or 41, comprising a variant capsid polypeptide according to any one of embodiments 1 to 36, wherein the viral particle, or the viral particle comprising the variant capsid polypeptide, or the viral particle comprising a variant capsid polypeptide encoded by a nucleic acid molecule according to 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 encoded by SEQ ID NO: 2), e.g., when measured in mice or NHPs, e.g., as described herein.

[0069] 43. The nucleic acid molecule according to any one of embodiments 37 to 39, or the viral particle according to any one of embodiments 40 to 42, wherein the nucleic acid molecule is double-stranded or single-stranded, and optionally the nucleic acid molecule is linear or circular, for example, the nucleic acid molecule is a plasmid.

[0070] 44. A method for producing viral particles comprising a variant AAV2 capsid polypeptide, the method comprising introducing a nucleic acid molecule described in any one of embodiments 37-39 or 43 into a cell (e.g., a HEK293 cell) and recovering the viral particles therefrom.

[0071] 45. A method for delivering a payload (e.g., a nucleic acid) to a cell, comprising contacting the cell with a dependoparvovirus particle comprising a variant capsid polypeptide described in any one of embodiments 1 to 36, or a virus particle and a payload described in any one of embodiments 40 to 42.

[0072] 46. ​​The method of embodiment 45, wherein the cell is an ocular cell.

[0073] 47. The method of embodiment 46, wherein the ocular cell is within the retina, macula, or trabecular meshwork.

[0074] 48. A method for delivering a payload (e.g., a nucleic acid) to a subject, comprising administering to the subject a dependoparvovirus particle comprising a variant capsid polypeptide and a payload described in any one of embodiments 1 to 36, or administering to the subject a virus particle described in any one of embodiments 40 to 42.

[0075] 49. The method of embodiment 48, wherein the particles deliver a payload to the eye.

[0076] 50. The method of embodiment 48, wherein the particles deliver the payload to the retina, macula, or trabecular meshwork.

[0077] 51. The method of any one of embodiments 48 to 50, wherein the particles deliver a payload to the eye with increased transduction in one or more areas of the eye compared to viral particles comprising a capsid polypeptide of SEQ ID NO:1.

[0078] 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.

[0079] 53. The method of embodiment 51, wherein the retina comprises non-macular retina.

[0080] 54. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, and 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 a capsid polypeptide of SEQ ID NO:1.

[0081] 55. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, the increase in transduction being at least 2-fold, 4-fold, 8-fold, 16-fold, or 32-fold compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, and the increase in transduction is specific to non-macular retinal tissue relative to macular tissue.

[0082] 56. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, the increase in transduction being at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, and the increase in transduction is specific to macular tissue relative to non-macular retinal tissue.

[0083] 57. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, the increase in transduction being at least 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, or 128-fold compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, and the increase in transduction is specific to macular tissue versus trabecular meshwork tissue.

[0084] 58. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, the increase in transduction being at least 2-fold, 4-fold, 8-fold, 16-fold, or 32-fold compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, and the increase in transduction is specific to non-macular retinal tissue relative to trabecular meshwork tissue.

[0085] 59. A variant capsid polypeptide described in any one of embodiments 1 to 36, a viral particle described in any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO: 1, the increase in transduction being at least 2-fold, 4-fold, 8-fold, 16-fold, or 32-fold compared to a viral particle comprising a capsid polypeptide of SEQ ID NO: 1, and the increase in transduction is specific to macular tissue and non-macular retinal tissue relative to trabecular meshwork tissue.

[0086] 60. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO: 1, the increase in transduction being at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising a capsid polypeptide of SEQ ID NO: 1, and the increase in transduction is specific to trabecular meshwork tissue relative to macular tissue and non-macular retinal tissue.

[0087] 61. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, the increase in transduction being at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, and the increase in transduction is specific to trabecular meshwork tissue relative to macular tissue.

[0088] 62. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO: 1, the increase in transduction being at least 2-fold, 4-fold, 8-fold, or 16-fold compared to a viral particle comprising a capsid polypeptide of SEQ ID NO: 1, and the increase in transduction is specific to trabecular meshwork tissue relative to non-macular retinal tissue.

[0089] 63. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, the increase in transduction being at least 2-fold, 4-fold, or 8-fold compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, and the increase in transduction is specific to trabecular meshwork tissue, macular tissue, and non-macular retinal tissue.

[0090] 64. A variant capsid polypeptide according to any one of embodiments 1 to 36, a viral particle according to any one of embodiments 40 to 42, or a method according to any one of embodiments 44 to 53, wherein the particle (e.g., a particle comprising a variant capsid polypeptide) delivers a payload to the eye with increased transduction specificity in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1, and without increased biodistribution in one or more regions of the eye compared to a viral particle comprising a capsid polypeptide of SEQ ID NO:1.

[0091] 65. The method of any one of embodiments 45 to 53, wherein administration to the subject is via intravitreal injection or intracameral injection.

[0092] 66. A method for treating a disease or condition in a subject, comprising administering to the subject a dependoparvovirus particle in an amount effective to treat the disease or condition, wherein the dependoparvovirus particle is a particle comprising a capsid polypeptide described in any one of embodiments 1 to 36 and 54 to 64, or encoded by a nucleic acid described in any one of embodiments 37 to 39 or 43, or is a virus particle described in any one of embodiments 40 to 42.

[0093] 67. A cell, cell-free system, or other translation system comprising a capsid polypeptide, a nucleic acid molecule, or a viral particle according to any one of embodiments 1 to 43 or 54 to 64.

[0094] 68. A method of producing a dependoparvovirus (e.g., an adeno-associated dependoparvovirus (AAV) particle, comprising: 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; Cultivating the cells, cell-free system, or other translation system under conditions suitable for the production of dependoparvovirus particles; This method produces dependoparvovirus particles.

[0095] 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 into the dependoparvovirus particle.

[0096] 70. The method of embodiment 68, wherein the second nucleic acid comprises a heterologous nucleic acid sequence encoding a payload, e.g., a therapeutic product.

[0097] 71. The method according to any one of embodiments 68 to 70, wherein the nucleic acid according to any one of embodiments 37 to 39 or 43 mediates the production of dependoparvovirus particles that do not contain the nucleic acid according to any one of embodiments 37 to 39 or 43.

[0098] 72. The method of any one of embodiments 68 to 71, wherein the nucleic acid molecule of any one of embodiments 37 to 39 or 43 mediates production of dependoparvovirus particles at a level at least 10%, at least 20%, at least 50%, at least 100%, at least 200% or more higher than the production level mediated by the nucleic acid of SEQ ID NO:2.

[0099] 74. A composition, e.g., a pharmaceutical composition, comprising a viral particle according to any one of embodiments 40 to 42, or a viral particle produced by a method according to any one of embodiments 43 or 68 to 72, and a pharma- ceutically acceptable carrier.

[0100] 75. A variant capsid polypeptide according to any of embodiments 1 to 36 and 54 to 64, a nucleic acid molecule according to any of embodiments 37 to 39 or 43, or a viral particle according to any of embodiments 40 to 42, for use in treating a disease or condition in a subject.

[0101] 76. A variant capsid polypeptide according to any of embodiments 1 to 36 and 54 to 64, a nucleic acid molecule according to any of embodiments 37 to 39 or 43, or a viral particle according to any of embodiments 40 to 42, for use in the manufacture of a medicament for use in treating a disease or condition in a subject. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0102] The present disclosure is directed, in part, to capsid polypeptides and depend parvoviral particles comprising the same. In some embodiments, the depend parvoviral particles have increased ocular transduction compared to depend parvoviral particles not comprising 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 same, viral particles comprising variant capsid polypeptides, and methods of making and using same.

[0103] definition A, An, The: As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0104] About, Approximately: As used herein, the terms "about" and "approximately" are intended to mean an acceptable degree of error for the quantity measured, generally given the nature or precision of the measurement. Exemplary degrees of error are within 15 percent (%), typically within 10%, and more typically within 5% of a given value or range of values.

[0105] Depend parvovirus capsid: As used herein, the term "depend parvovirus capsid" refers to an assembled viral capsid comprising a depend parvovirus polypeptide. In some embodiments, the depend parvovirus capsid is a functional depend parvovirus capsid, e.g., fully folded and / or assembled and capable of infecting a target cell, or remains stable for at least a threshold time (e.g., folded / assembled and / or capable of infecting a target cell).

[0106] Depend parvovirus particle: As used herein, the term "depend parvovirus particle" refers to an assembled viral capsid comprising a depend parvovirus polypeptide and packaged nucleic acid, e.g., including a payload, one or more components of a depend parvovirus genome (e.g., an entire depend parvovirus genome), or both. In some embodiments, a depend parvovirus particle is a functional depend parvovirus particle, e.g., comprises a desired payload, is fully folded and / or assembled, and is capable of infecting a target cell, or remains stable for at least a threshold time (e.g., is folded / assembled and / or is capable of infecting a target cell).

[0107] Depend parvovirus X particle / capsid: As used herein, the term "Depend parvovirus X particle / capsid" refers to a Depend parvovirus particle / capsid that comprises at least one polypeptide or polypeptides encoding a nucleic acid sequence derived from a naturally occurring Depend parvovirus X species. For example, Depend parvovirus B particle refers to a Depend parvovirus particle that comprises at least one polypeptide or polypeptides encoding a nucleic acid sequence derived from a naturally occurring Depend parvovirus B sequence. As used in this context, derived from means having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to the sequence in question. Correspondingly, as used herein, AAV X particle / capsid refers to an AAV particle / capsid that comprises at least one polypeptide or polypeptides encoding a nucleic acid sequence derived from a naturally occurring AAV X serotype. For example, an AAV2 particle refers to an AAV particle that includes at least one polypeptide or polypeptides encoding a nucleic acid sequence derived from a naturally occurring AAV2 sequence.

[0108] Exogenous: As used herein, the term "exogenous" refers to a feature, sequence, or component present in a context (e.g., a nucleic acid, a polypeptide, or a cell) that does not naturally occur in that context. For example, a nucleic acid sequence that includes or encodes a mutant capsid polypeptide may include the capsid polypeptide. This use of the term exogenous means that the polypeptide or nucleic acid molecule encoding the polypeptide that includes the mutation in question at this position does not naturally occur, e.g., does not exist in AAV2, e.g., does not exist in SEQ ID NO:1.

[0109] Functional: As used herein in reference to a polypeptide component of a depend parvovirus 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 a naturally occurring version of that polypeptide component (e.g., when present in a host cell). For example, a functional VP1 polypeptide can be stably folded and assembled into a depend parvovirus capsid (e.g., compatible for packaging and / or secretion). As used herein in reference to a depend parvovirus capsid or particle, "functional" refers to a capsid or particle that contains one or more of the product characteristics of containing a desired payload, being fully folded and / or assembled, and having the ability to infect a target cell, or remaining stable for at least a threshold time (e.g., being folded / assembled and / or having the ability to infect a target cell).

[0110] Nucleic Acid: As used herein, in its broadest sense, the term "nucleic acid" refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. As will be clear from the context, in some embodiments, "nucleic acid" refers to an individual nucleic acid monomer (e.g., nucleotide and / or nucleoside), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain that includes an individual nucleic acid monomer, or a longer polynucleotide chain that includes many individual nucleic acid monomers. In some embodiments, "nucleic acid" is or comprises RNA, and in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid is, comprises, or consists of one or more modified, synthetic, or non-naturally occurring nucleotides. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, the nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," as known in the art and having peptide bonds instead of phosphodiester bonds in the backbone, and are considered 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 linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a protein. In some embodiments, the nucleic acid is partially or completely single-stranded, and in some embodiments, the nucleic acid is partially or completely double-stranded.

[0111] Variant: As used herein, a "variant capsid polypeptide" refers to a polypeptide that differs from a reference sequence (e.g., SEQ ID NO:1). A variant can include, for example, mutations (e.g., substitutions, deletions, or insertions). In some embodiments, a variant is about or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence. In some embodiments, the reference sequence is a polypeptide comprising SEQ ID NO:1.

[0112] Capsid polypeptides and nucleic acids encoding same The present disclosure is directed, in part, to capsid polypeptides comprising mutations (insertions, deletions, or substitutions) compared to a 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 is directed, in part, to variant capsid polypeptides comprising SEQ ID NO:1 having one or more mutations compared to SEQ ID NO:1. The mutations can be, for example, insertions, deletions, or substitutions compared to the wild-type sequence. In some embodiments, the wild-type sequence is SEQ ID NO:1. The present disclosure is directed, in part, to variant capsid polypeptides comprising any one of SEQ ID NOs:12-27. The present disclosure is directed, in part, to variant capsid polypeptides comprising a VP1 sequence of any one of SEQ ID NOs:12-27. The present disclosure is directed, in part, to variant capsid polypeptides comprising a VP2 sequence of any one of SEQ ID NOs:12-27. The present disclosure is directed, in part, to a variant capsid polypeptide that includes a VP3 sequence of any one of SEQ ID NO:12-SEQ ID NO:27.

[0113] In some embodiments, the capsid polypeptide comprises a mutation selected from a mutational difference disclosed in any of Tables 1A-1G, e.g., a mutation selected from a mutational difference associated with any variant disclosed in any of Tables 1A-1G. In some embodiments, the mutational difference selected from a mutational difference disclosed in any of Tables 1A-1G, e.g., a mutation selected from a mutational difference associated with any variant disclosed in any of Tables 1A-1G, comprises a mutation at a position 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 a mutational difference disclosed in any of Tables 1A-1G, e.g., a mutation selected from a mutational difference associated with any variant disclosed in any of Tables 1A-1G. In some embodiments, the mutational difference selected from those disclosed in any of Tables 1A-1G, e.g., those associated with any variant disclosed in any of Tables 1A-1G, is a substitution, e.g., a substitution of two or more residues corresponding to a substitution at positions between 550 and 597 compared to SEQ ID NO: 1. In some embodiments, the mutational difference selected from those disclosed in any of Tables 1A-1G, e.g., those associated with any variant disclosed in any of Tables 1A-1G, is a substitution, further comprising at least one other mutation between positions 550 and 597, wherein the mutation is a substitution, insertion, or deletion. In some embodiments, the mutations selected from the mutational differences disclosed in any of Tables 1A-1G, e.g., selected from the mutational differences associated with any variant disclosed in any of Tables 1A-1G, are insertions, e.g., of one or more amino acids, e.g., of one amino acid, e.g., of one to two 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 substitutions, e.g., substitutions of two or more residues corresponding to substitutions at positions between 550 and 597, compared to SEQ ID NO:1.

[0114] In some embodiments, the capsid polypeptide comprises 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 the mutation comprises an insertion, deletion, or substitution.

[0115] In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 550 compared to SEQ ID NO:1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 559 compared to SEQ ID NO:1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 561 compared to SEQ ID NO:1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 586 compared to SEQ ID NO:1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 587 compared to SEQ ID NO:1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 592 compared to SEQ ID NO:1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 593 compared to SEQ ID NO:1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to a mutation at position 597 compared to SEQ ID NO:1.

[0116] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion at a position between positions 584 and 585, as 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, as 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, as compared to SEQ ID NO:1.

[0117] In some embodiments, the capsid polypeptide comprises a mutation at position 550, 559, 561, 586, 587, 592, 593, 597, or any combination thereof, and an insertion between positions 584 and 585, 586 and 587, or 587 and 588, or any combination thereof, according to SEQ ID NO:1, wherein the mutation comprises an insertion, deletion, or substitution.

[0118] In some embodiments, the capsid polypeptide comprises mutations corresponding to the mutations at positions 587 and 593 compared to SEQ ID NO:1.

[0119] In some embodiments, the capsid polypeptide comprises mutations corresponding to the mutations at positions 586 and 587 compared to SEQ ID NO:1.

[0120] In some embodiments, the capsid polypeptide comprises mutations corresponding to the mutations at positions 592 and 597 compared to SEQ ID NO:1.

[0121] In some embodiments, the capsid polypeptide comprises mutations corresponding to the mutations at positions 587 and 597 compared to SEQ ID NO:1.

[0122] In some embodiments, the capsid polypeptide comprises mutations corresponding to mutations at positions 550, 586, and 587 compared to SEQ ID NO:1.

[0123] In some embodiments, the capsid polypeptide comprises mutations corresponding to the mutations at positions 559 and 587 compared to SEQ ID NO:1.

[0124] In some embodiments, the capsid polypeptide comprises a mutation at position 587 and a corresponding insertion between positions 586 and 587 compared to SEQ ID NO:1.

[0125] In some embodiments, the capsid polypeptide comprises mutations at positions 587 and 593, and mutations corresponding to an insertion between positions 586 and 587, compared to SEQ ID NO:1.

[0126] In some embodiments, the capsid polypeptide comprises mutations at positions 586 and 587 and mutations corresponding to an insertion between positions 584 and 585 compared to SEQ ID NO:1.

[0127] In some embodiments, the capsid polypeptide comprises mutations at positions 592 and 597 and mutations corresponding to an insertion between positions 587 and 588 compared to SEQ ID NO:1.

[0128] In some embodiments, the capsid polypeptide comprises mutations at positions 561, 587, and 597, and mutations corresponding to an insertion between positions 586 and 587, compared to SEQ ID NO:1.

[0129] In some embodiments, the capsid polypeptide comprises mutations at positions 550, 586, and 587, and mutations corresponding to an insertion between positions 584 and 585, compared to SEQ ID NO:1.

[0130] In some embodiments, the capsid polypeptide comprises mutations at positions 559 and 587, and mutations corresponding to an insertion between positions 586 and 587, compared to SEQ ID NO:1.

[0131] In some embodiments, the mutation corresponding to position 550 is a substitution compared to SEQ ID NO:1. In some embodiments, the substitution is to a naturally occurring amino acid. In some embodiments, the substitution is an 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 in SEQ ID NO:1 is for an asparagine (N) at the position corresponding to T550 in 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 mutant T550N mutation compared to SEQ ID NO:1.

[0132] In some embodiments, the mutation corresponding to position 559 is a substitution compared to SEQ ID NO:1. In some embodiments, the substitution is to 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 in SEQ ID NO:1 is for a leucine (L) at the position corresponding to I559 in 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 I559L mutation compared to SEQ ID NO:1.

[0133] In some embodiments, the mutation corresponding to position 561 is a substitution compared to SEQ ID NO:1. In some embodiments, the substitution is to a naturally occurring amino acid. In some embodiments, the substitution is a 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 in SEQ ID NO:1 is a substitution for a cysteine ​​(C) at a position corresponding to D561 in 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 D561C mutation compared to SEQ ID NO:1.

[0134] In some embodiments, the mutation corresponding to position 586 is a substitution compared to SEQ ID NO:1. In some embodiments, the substitution is to a naturally occurring amino acid. In some embodiments, the substitution is a 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 in SEQ ID NO:1 is to a proline (P) at the position corresponding to G586 in 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 G586P mutation compared to SEQ ID NO:1.

[0135] In some embodiments, the mutation corresponding to position 587 is a substitution compared to SEQ ID NO:1. In some embodiments, the substitution is to a naturally occurring amino acid. In some embodiments, the substitution is an 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 in SEQ ID NO:1 is a substitution to an alanine (A) at the position corresponding to N587 in 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 N587A mutation compared to SEQ ID NO:1.

[0136] In some embodiments, the mutation corresponding to position 592 is a substitution compared to SEQ ID NO:1. In some embodiments, the substitution is to a naturally occurring amino acid. In some embodiments, the substitution is an alanine (A). In some embodiments, the substitution at position 592 is T592A, according to SEQ ID NO:1. In some embodiments, the substitution at a position corresponding to T592 in SEQ ID NO:1 is a substitution to an alanine (A) at a position corresponding to T592 in 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 mutant T592A mutation compared to SEQ ID NO:1.

[0137] In some embodiments, the mutation corresponding to position 593 is a substitution compared to SEQ ID NO:1. 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 in SEQ ID NO:1 is for a threonine (T) at the position corresponding to A593 in 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 A593T mutation, compared to SEQ ID NO:1.

[0138] In some embodiments, the mutation corresponding to position 597 is a substitution compared to SEQ ID NO:1. 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 is to a naturally occurring amino acid. In some embodiments, the substitution is tryptophan (W). In some embodiments, the substitution at position 597 is T597N, per SEQ ID NO:1. In some embodiments, the substitution at position 597 is T597W, per SEQ ID NO:1. In some embodiments, the substitution at the position corresponding to T597 in SEQ ID NO:1 is a substitution for asparagine (N) at a position corresponding to T597 in 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 in SEQ ID NO:1 is a substitution for tryptophan (W) at a position corresponding to T597 in 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, as compared to SEQ ID NO:1. In some embodiments, the capsid polypeptide comprises a mutation corresponding to the mutation T597W mutation compared to SEQ ID NO:1.

[0139] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion between residues 587 and 588 compared to SEQ ID NO:1, and 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 at least a 5 amino acid fragment of LALGEQTRPA (SEQ ID NO:44). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of LALGEQTRPA (SEQ ID NO:44). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment of LALGEQTRPA (SEQ ID NO:44). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment 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).

[0140] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion between residues 587 and 588 compared to SEQ ID NO:1, and 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 at least a 5 amino acid fragment of LAIEQTRPA (SEQ ID NO:45). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of LAIEQTRPA (SEQ ID NO:45). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment 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).

[0141] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an N587A mutation and an insertion between residues 586 and 587 compared to SEQ ID NO:1, wherein the insertion comprises, e.g., consists of, the polypeptide of LALAEITRP (SEQ ID NO:46). 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 at least a 5 amino acid fragment of LALAEITRP (SEQ ID NO:46). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of LALAEITRP (SEQ ID NO:46). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment 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).

[0142] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an N587A mutation 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 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 at least a 5 amino acid fragment of LKNAETARP (SEQ ID NO:47). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of LKNAETARP (SEQ ID NO:47). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment 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).

[0143] 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 at least a 5 amino acid fragment of LNLAIEQTRP (SEQ ID NO:48). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of LNLAIEQTRP (SEQ ID NO:48). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment of LNLAIEQTRP (SEQ ID NO:48). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of LNLAIEQTRP (SEQ ID NO: 48).

[0144] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an N587A mutation 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). 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 at least a 4 amino acid fragment of MLNEQTRP (SEQ ID NO:49). In some embodiments, the insertion comprises a polypeptide comprising at least a 5 amino acid fragment of MLNEQTRP (SEQ ID NO:49). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of MLNEQTRP (SEQ ID NO:49). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment of MLNEQTRP (SEQ ID NO:49).

[0145] In some embodiments, the capsid polypeptide comprises mutations corresponding to G586P and N587A 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 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 as 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 at least an 8 amino acid fragment of RSGNRADSETA (SEQ ID NO:50). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of RSGNRADSETA (SEQ ID NO:50). In some embodiments, the insertion comprises a polypeptide comprising at least a 10 amino acid fragment of RSGNRADSETA (SEQ ID NO:50).

[0146] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an N587A mutation 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 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).

[0147] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an insertion between residues 587 and 588 compared to SEQ ID NO:1, and 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 at least a 5 amino acid fragment of LQGETIRPA (SEQ ID NO:52). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of LQGETIRPA (SEQ ID NO:52). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment 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).

[0148] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an N587A mutation 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 at least an 8 amino acid fragment of QNLANPETTRP (SEQ ID NO:53). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of QNLANPETTRP (SEQ ID NO:53). In some embodiments, the insertion comprises a polypeptide comprising at least a 10 amino acid fragment of QNLANPETTRP (SEQ ID NO:53).

[0149] In some embodiments, the capsid polypeptide comprises mutations corresponding to T592A and T597W as compared to SEQ ID NO:1, and an insertion between residues 587 and 588 as compared to SEQ ID NO:1, and the insertion comprises, e.g., 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 as compared to RAPQETTRPA (SEQ ID NO:54). In some embodiments, the insertion comprises a polypeptide comprising at least a 5 amino acid fragment of RAPQETTRPA (SEQ ID NO:54). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of RAPQETTRPA (SEQ ID NO:54). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment of RAPQETTRPA (SEQ ID NO:54). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of RAPQETTRPA (SEQ ID NO: 54). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of RAPQETTRPA (SEQ ID NO: 54).

[0150] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an N587A mutation 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 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 at least a 4 amino acid fragment of ANLTTTRP (SEQ ID NO:55). In some embodiments, the insertion comprises a polypeptide comprising at least a 5 amino acid fragment of ANLTTTRP (SEQ ID NO:55). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment 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).

[0151] In some embodiments, the capsid polypeptide comprises a mutation corresponding to an N587A mutation 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). 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 at least a 5 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56).

[0152] In some embodiments, the capsid polypeptide comprises mutations corresponding to D561C, N587A, and T597N, and an insertion between residues 586 and 587, compared to SEQ ID NO:1, where 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 at least a 5 amino acid fragment of GLRAEQTRP (SEQ ID NO:57). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of GLRAEQTRP (SEQ ID NO:57). In some embodiments, the insertion comprises a polypeptide comprising at least a seven amino acid fragment of GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising at least an eight amino acid fragment of GLRAEQTRP (SEQ ID NO: 57).

[0153] In some embodiments, the capsid polypeptide comprises mutations corresponding to T550N, G586P, and N587A, 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 at least a 4 amino acid fragment of RARLDETA (SEQ ID NO:58). In some embodiments, the insertion comprises a polypeptide comprising at least a 5 amino acid fragment of RARLDETA (SEQ ID NO:58). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment 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).

[0154] In some embodiments, the capsid polypeptide comprises mutations corresponding to I559L and N587A as compared to SEQ ID NO:1, and an insertion between residues 586 and 587 as compared to SEQ ID NO:1, wherein the insertion comprises, e.g., 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 as 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 at least an 8 amino acid fragment of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of TNLARGETARP (SEQ ID NO: 59). In some embodiments, the insertion comprises a polypeptide comprising at least a 10 amino acid fragment of TNLARGETARP (SEQ ID NO: 59).

[0155] 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 the sequence of SEQ ID NO: 42. In some embodiments, the nucleic acid molecule has the sequence of SEQ ID NO: 43.

[0156] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation at position 550, 559, 561, 586, 587, 592, 593, 597, or any combination thereof, and an insertion between positions 584 and 585, 586 and 587, or 587 and 588, or any combination thereof, according to SEQ ID NO:1, optionally wherein the mutation comprises an insertion, deletion, or substitution.

[0157] In some embodiments, the nucleic acid molecule comprises a sequence encoding a capsid polypeptide comprising a mutation corresponding to the mutation at position 550 compared to SEQ ID NO:1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the mutation at position 559 compared to SEQ ID NO:1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the mutation at position 561 compared to SEQ ID NO:1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the mutation at position 586 compared to SEQ ID NO:1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the mutation at position 587 compared to SEQ ID NO:1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the mutation at position 592 compared to SEQ ID NO:1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the mutation at position 593 compared to SEQ ID NO:1. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to the mutation at position 597 compared to SEQ ID NO:1.

[0158] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes 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 that includes 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 that includes a mutation corresponding to an insertion at a position between positions 587 and 588 compared to SEQ ID NO:1.

[0159] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation at position 550, 559, 561, 586, 587, 592, 593, 597, or any combination thereof, and an insertion between positions 584 and 585, 586 and 587, or 587 and 588, or any combination thereof, according to SEQ ID NO:1, wherein the mutation comprises an insertion, deletion, or substitution.

[0160] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations corresponding to the mutations at positions 587 and 593 compared to SEQ ID NO:1.

[0161] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations corresponding to the mutations at positions 586 and 587 compared to SEQ ID NO:1.

[0162] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations corresponding to the mutations at positions 592 and 597 compared to SEQ ID NO:1.

[0163] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations corresponding to the mutations at positions 587 and 597 compared to SEQ ID NO:1.

[0164] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations corresponding to the mutations at positions 550, 586, and 587 compared to SEQ ID NO:1.

[0165] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations corresponding to the mutations at positions 559 and 587 compared to SEQ ID NO:1.

[0166] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations at positions 451, 456, 457, 458, 459, and 461, and mutations corresponding to an insertion between positions 449 and 450, compared to SEQ ID NO:1.

[0167] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation at position 587 and a mutation corresponding to an insertion between positions 586 and 587 compared to SEQ ID NO:1.

[0168] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations at positions 587 and 593, and mutations corresponding to an insertion between positions 586 and 587, compared to SEQ ID NO:1.

[0169] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations at positions 586 and 587 and mutations corresponding to an insertion between positions 584 and 585 compared to SEQ ID NO:1.

[0170] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations at positions 592 and 597, and mutations corresponding to an insertion between positions 587 and 588, compared to SEQ ID NO:1.

[0171] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations at positions 561, 587, and 597, and mutations corresponding to an insertion between positions 586 and 587, compared to SEQ ID NO:1.

[0172] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations at positions 550, 586, and 587, and mutations corresponding to an insertion between positions 584 and 585, compared to SEQ ID NO:1.

[0173] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes mutations at positions 559 and 587, and mutations corresponding to an insertion between positions 586 and 587, compared to SEQ ID NO:1.

[0174] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation corresponding to the mutant T550N mutation compared to SEQ ID NO:1.

[0175] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation corresponding to the mutation I559L mutation compared to SEQ ID NO:1.

[0176] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation corresponding to the mutation D561C mutation compared to SEQ ID NO:1.

[0177] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation corresponding to the mutation G586P mutation compared to SEQ ID NO:1.

[0178] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation corresponding to the mutation N587A mutation compared to SEQ ID NO:1.

[0179] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation corresponding to the mutant T592A mutation compared to SEQ ID NO:1.

[0180] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation corresponding to the A593T mutation compared to SEQ ID NO:1.

[0181] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation corresponding to the mutation T597N mutation compared to SEQ ID NO:1.

[0182] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that includes a mutation corresponding to the mutation T597W mutation compared to SEQ ID NO:1.

[0183] 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, the insertion comprising, e.g., consisting of, a polypeptide of LALGEQTRPA (SEQ ID NO:44), the nucleic acid having a 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 at least a 5 amino acid fragment of LALGEQTRPA (SEQ ID NO:44). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of LALGEQTRPA (SEQ ID NO:44). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment of LALGEQTRPA (SEQ ID NO:44). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of LALGEQTRPA (SEQ ID NO: 44). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of LALGEQTRPA (SEQ ID NO: 44).

[0184] 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, the insertion comprising, e.g., consisting of, a polypeptide of LAIEQTRPA (SEQ ID NO:45), the nucleic acid having a 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 at least a 5 amino acid fragment of LAIEQTRPA (SEQ ID NO:45). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of LAIEQTRPA (SEQ ID NO:45). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment 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).

[0185] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising an N587A mutation compared to SEQ ID NO:1 and a corresponding mutation between residues 586 and 587 compared to SEQ ID NO:1, wherein the insertion comprises, e.g., consists of, the polypeptide of LALAEITRP (SEQ ID NO:46), and 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 at least a 7 amino acid fragment of LALAEITRP (SEQ ID NO: 46). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of LALAEITRP (SEQ ID NO: 46).

[0186] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an N587A mutation and an insertion between residues 586 and 587 compared to SEQ ID NO:1, the insertion comprising, e.g., consisting of, a polypeptide of LKNAETARP (SEQ ID NO:47), the nucleic acid having a 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 at least a 7 amino acid fragment of LKNAETARP (SEQ ID NO: 47). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of LKNAETARP (SEQ ID NO: 47).

[0187] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to N587A and A593T as compared to SEQ ID NO:1 and an insertion between residues 586 and 587 as compared to SEQ ID NO:1, the insertion comprising, e.g., consisting of, a polypeptide of LNLAIEQTRP (SEQ ID NO:48), the nucleic acid having a 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 as 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 at least a 7 amino acid fragment of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of LNLAIEQTRP (SEQ ID NO: 48). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of LNLAIEQTRP (SEQ ID NO: 48).

[0188] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an N587A mutation and an insertion between residues 586 and 587 compared to SEQ ID NO:1, the insertion comprising, e.g., consisting of, a polypeptide of MLNEQTRP (SEQ ID NO:49), the nucleic acid having a 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 at least a 7 amino acid fragment of MLNEQTRP (SEQ ID NO:49).

[0189] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising G586P and N587A mutations compared to SEQ ID NO:1, and a corresponding insertion between residues 584 and 585 compared to SEQ ID NO:1, where the insertion comprises, e.g., consists of, a polypeptide of RSGNRADSETA (SEQ ID NO:50), and 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 at least a 7 amino acid fragment of RSGNRADSETA (SEQ ID NO:50). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of RSGNRADSETA (SEQ ID NO:50). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of RSGNRADSETA (SEQ ID NO:50). In some embodiments, the insertion comprises a polypeptide comprising at least a 10 amino acid fragment of RSGNRADSETA (SEQ ID NO:50).

[0190] 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, e.g., consists of, a polypeptide of TGDTRP (SEQ ID NO:51), and 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).

[0191] 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, the insertion comprising, e.g., consisting of, a polypeptide of LQGETIRPA (SEQ ID NO:52), the nucleic acid having a 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 at least a 5 amino acid fragment of LQGETIRPA (SEQ ID NO:52). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of LQGETIRPA (SEQ ID NO:52). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment 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).

[0192] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising a mutation corresponding to an N587A mutation and an insertion between residues 586 and 587 compared to SEQ ID NO:1, the insertion comprising, e.g., consisting of, a polypeptide of QNLANPETTRP (SEQ ID NO:53), the nucleic acid having a 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 at least a 7 amino acid fragment of QNLANPETTRP (SEQ ID NO:53). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of QNLANPETTRP (SEQ ID NO:53). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of QNLANPETTRP (SEQ ID NO:53). In some embodiments, the insertion comprises a polypeptide comprising at least a 10 amino acid fragment of QNLANPETTRP (SEQ ID NO:53).

[0193] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations T592A and T597W compared to SEQ ID NO:1, and a corresponding 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 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 at least a 7 amino acid fragment of RAPQETTRPA (SEQ ID NO:54). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of RAPQETTRPA (SEQ ID NO:54). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of RAPQETTRPA (SEQ ID NO:54).

[0194] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising an N587A mutation compared to SEQ ID NO:1 and a corresponding mutation between residues 586 and 587 compared to SEQ ID NO:1, wherein the insertion comprises, e.g., consists of, a polypeptide of ANLTTTRP (SEQ ID NO:55), 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 at least a 4 amino acid fragment of ANLTTTRP (SEQ ID NO:55). In some embodiments, the insertion comprises a polypeptide comprising at least a 5 amino acid fragment of ANLTTTRP (SEQ ID NO:55). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment 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).

[0195] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising an N587A mutation compared to SEQ ID NO:1 and a corresponding insertion between residues 586 and 587 compared to SEQ ID NO:1, the insertion comprising, e.g., consisting of, a polypeptide of ALLAGEQTRP (SEQ ID NO:56), the nucleic acid having a 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 at least a 5 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56). In some embodiments, the insertion comprises a polypeptide comprising at least a 7 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of ALLAGEQTRP (SEQ ID NO:56).

[0196] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising D561C, N587A, and T597N mutations compared to SEQ ID NO:1, and a corresponding insertion between residues 586 and 587 compared to SEQ ID NO:1, where the insertion comprises, e.g., consists of, a polypeptide of GLRAEQTRP (SEQ ID NO:57), and the nucleic acid has a 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 at least a 5 amino acid fragment of GLRAEQTRP (SEQ ID NO:57). In some embodiments, the insertion comprises a polypeptide comprising at least a 6 amino acid fragment of GLRAEQTRP (SEQ ID NO:57). In some embodiments, the insertion comprises a polypeptide comprising at least a seven amino acid fragment of GLRAEQTRP (SEQ ID NO: 57). In some embodiments, the insertion comprises a polypeptide comprising at least an eight amino acid fragment of GLRAEQTRP (SEQ ID NO: 57).

[0197] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising mutations corresponding to T550N, G586P, and N587A compared to SEQ ID NO:1, and an insertion between residues 584 and 585 compared to SEQ ID NO:1, where the insertion comprises, e.g., consists of, a polypeptide of RARLDETA (SEQ ID NO:58), and 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 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 at least a six amino acid fragment of RARLDETA (SEQ ID NO:58). In some embodiments, the insertion comprises a polypeptide comprising at least a seven amino acid fragment of RARLDETA (SEQ ID NO:58).

[0198] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide comprising I559L and N587A mutations compared to SEQ ID NO:1, and a corresponding insertion between residues 586 and 587 compared to SEQ ID NO:1, where the insertion comprises, e.g., consists of, a polypeptide of TNLARGETARP (SEQ ID NO:59), and 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 at least a 7 amino acid fragment of TNLARGETARP (SEQ ID NO:59). In some embodiments, the insertion comprises a polypeptide comprising at least an 8 amino acid fragment of TNLARGETARP (SEQ ID NO:59). In some embodiments, the insertion comprises a polypeptide comprising at least a 9 amino acid fragment of TNLARGETARP (SEQ ID NO:59). In some embodiments, the insertion comprises a polypeptide comprising at least a 10 amino acid fragment of TNLARGETARP (SEQ ID NO:59).

[0199] In some embodiments, including those described above, capsid polypeptides are provided that comprise 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 a capsid polypeptide provided herein.

[0200] In some embodiments, the nucleic acid molecule encodes a capsid polypeptide provided herein. In some embodiments, the nucleic acid molecule encodes a capsid polypeptide that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a capsid polypeptide provided herein.

[0201] In some embodiments, including those described above, capsid polypeptides are provided that comprise 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.

[0202] In some embodiments, including those described above, a capsid polypeptide is provided that comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99 identical to any one of SEQ ID NOs: 3, 5, 7, 9 or 10.

[0203] In some embodiments, the reference polypeptide for purposes of % identity comprises the sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 10.

[0204] In some embodiments, the reference nucleic acid for purposes of % identity comprises the sequence of SEQ ID NO: 2, 4, 6, 8, or 11.

[0205] In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27.

[0206] In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the 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, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 28. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 29. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 30. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 31. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 32. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 33. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 34. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 35. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 36. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 37. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 38. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 39.In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 40. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 41. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 42. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 43.

[0207] In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43, which encodes the 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, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:28, which encodes the sequence of SEQ ID NO:12. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:29, which encodes the sequence of SEQ ID NO:13. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:30, which encodes the sequence of SEQ ID NO:14. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:31, which encodes the sequence of SEQ ID NO:15. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:32, which encodes the sequence of SEQ ID NO:16. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:33, which encodes the sequence of SEQ ID NO:17. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:34, which encodes the sequence of SEQ ID NO:18. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:35, which encodes the sequence of SEQ ID NO:19. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:36, which encodes the sequence of SEQ ID NO:20.In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:37, which encodes the sequence of SEQ ID NO:21. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:38, which encodes the sequence of SEQ ID NO:22. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:39, which encodes the sequence of SEQ ID NO:23. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:40, which encodes the sequence of SEQ ID NO:24. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:41, which encodes the sequence of SEQ ID NO:25. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:42, which encodes the sequence of SEQ ID NO:26. In some embodiments, the nucleic acid molecule encoding the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:43, which encodes the sequence of SEQ ID NO:27.

[0208] In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27, encoded by the 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, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 12, encoded by the nucleotide sequence of SEQ ID NO: 28. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 13, encoded by the nucleotide sequence of SEQ ID NO: 29. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 14, encoded by the nucleotide sequence of SEQ ID NO: 30. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 15, encoded by the nucleotide sequence of SEQ ID NO: 31. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 16, encoded by the nucleotide sequence of SEQ ID NO: 32. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 17, encoded by the nucleotide sequence of SEQ ID NO: 33. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 18, encoded by the nucleotide sequence of SEQ ID NO: 34. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 19, encoded by the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO: 20, encoded by the nucleotide sequence of SEQ ID NO: 36.In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:21, encoded by the nucleotide sequence of SEQ ID NO:37. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:22, encoded by the nucleotide sequence of SEQ ID NO:38. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:23, encoded by the nucleotide sequence of SEQ ID NO:39. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:24, encoded by the nucleotide sequence of SEQ ID NO:40. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:25, encoded by the nucleotide sequence of SEQ ID NO:41. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:26, encoded by the nucleotide sequence of SEQ ID NO:42. In some embodiments, the capsid polypeptide, or the reference polypeptide for purposes of % identity, comprises the sequence of SEQ ID NO:27, which is encoded by the nucleotide sequence of SEQ ID NO:43.

[0209] In some embodiments, the capsid polypeptide comprises a sequence that includes all mutational differences associated with any one of VAR-1 through VAR-16 (e.g., as shown in Tables 1A, 1B, 1C, 1D, 1E, 1F, and 1G), and further comprises 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.

[0210] In some embodiments, the capsid polypeptide is a VP1 capsid polypeptide. In some embodiments, the capsid polypeptide is a VP2 capsid polypeptide. In some embodiments, the capsid polypeptide is a VP3 capsid polypeptide. With respect to the reference sequence SEQ ID NO:1, the VP1 capsid polypeptide comprises amino acids 1-724 of SEQ ID NO:1. With respect to the reference sequence SEQ ID NO:1, the VP2 capsid polypeptide comprises amino acids 138-724 of SEQ ID NO:1. With respect to the reference sequence SEQ ID NO:1, the VP3 capsid polypeptide comprises amino acids 203-724 of SEQ ID NO:1.

[0211] Tables 1A, 1B, 1C, 1D, 1E, 1F, and 1G list information about exemplary variant depend parvovirus particles comprising variant capsids and describe the ocular transduction and production characteristics of the non-limiting exemplary variants. Exemplary sequences of capsid polypeptides and nucleic acid molecules encoding same are provided in Table 2. Table 2 illustrates the VP1, VP2, and VP3 polypeptide starting amino acid sequences of each of SEQ ID NOs: 12 to 27. The exemplary nucleic acid sequences provided in Table 2 include a stop codon (e.g., a TAA stop codon) at the 3' end of the sequence. It will be understood by one of skill 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).

[0212] Tables 1A, 1B, and 1C represent data generated in a first (Table 1A) high throughput experiment (library experiment 1) and a second (Tables 1B-1C) high throughput experiment (library experiment 2). Tables 1D, 1E, 1F, and 1G represent data generated in a medium throughput experiment (library experiment 3). Transduction and virus production of exemplary variant depend parvovirus (e.g., AVV) particles comprising variant capsid polypeptides. Injection routes are as indicated in the column headings. Substitutions are denoted as n###N, where "N" is the last amino acid, "n" is the reference amino acid and "###" is the reference amino acid position in SEQ ID NO:1; deletions are denoted as n###-, where "-" indicates a deletion of "n" at position "###" of SEQ ID NO:1 of the reference sequence; insertions are denoted as ###_Naa_###_(n)y, where "###" is the amino acid position in SEQ ID NO:1 of the reference sequence between which the insertion occurs, "Naa" refers to the length of the insertion (having "N" amino acids), and "(n)y" provides the sequence of the insertion). Each individual mutational difference (e.g., each mutation within a row in quotation marks ('') 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 mutational differences may be 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 transduction of the neuroretinal layers of tissue within the macula. In Tables 1A and 1C, non-macular transduction refers to transduction of the neuroretinal layers of the retina excluding the macula. Retinal transduction refers to the aggregated measurements from macular transduction and non-macular transduction. For purposes of Table 1A, the average retinal transduction is calculated as the average of macular transduction and non-macular retinal transduction. Trabecular meshwork transduction refers to the transduction of tissue samples collected from the trabecular meshwork and / or Schlemm's canal. For the purposes of Tables 1D and 1E, neural retinal transduction refers to transduction of tissue samples collected from the neural retinal layer, including the macular region. "Not determined" indicates that the variant was not detected in the indicated sample. Measurements are made against wild-type AAV2 (SEQ ID NO: 1) unless otherwise stated.The trabecular meshwork transduction measurements shown in Tables 1F and 1G are shown for an AAV2 variant identified from the ocular literature having a capsid having the polypeptide of SEQ ID NO: 60, encoded by exemplary nucleic acid SEQ ID NO: 61. The data shown in Tables 1A, 1B, and 1C are in log2 scale. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] TIFF2025512534000010.tif203170TIFF2025512534000011.tif248170TIFF2025512534000012.tif176170TIFF2025512534000013.tif247170TIFF2025512534000014.tif173170TIFF2025512534000015.tif248170TIFF2025512534000016.tif173170TIFF2025512534000017.tif248170TIFF2025512534000018.tif176170TIFF2025512534000019.tif248170TIFF2025512534000020.tif175170TIFF2025512534000021.tif248170TIFF2025512534000022.tif175170TIFF2025512534000023.tif248170TIFF2025512534000024.tif174170TIFF2025512534000025.tif249170TIFF2025512534000026.tif174170TIFF2025512534000027.tif248170TIFF2025512534000028.tif175170TIFF2025512534000029.tif248170TIFF2025512534000030.tif175170TIFF2025512534000031.tif249170TIFF2025512534000032.tif176170TIFF2025512534000033.tif249170TIFF2025512534000034.tif174170TIFF2025512534000035.tif249170TIFF2025512534000036.tif175170TIFF2025512534000037.tif248170TIFF2025512534000038.tif175170TIFF2025512534000039.tif249170TIFF2025512534000040.tif177170

[0213] In some embodiments, the capsid polypeptide has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence provided in Table 2. In some embodiments, the capsid polypeptide has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 12. In some embodiments, the capsid polypeptide has at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence of SEQ ID NO: 13. In some embodiments, the capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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 capsid polypeptide has 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.

[0214] In some embodiments, the capsid polypeptide has a sequence of SEQ ID NO: 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 the sequence of SEQ ID NO: 25. In some embodiments, the capsid polypeptide has the sequence of SEQ ID NO: 26. In some embodiments, the capsid polypeptide has the sequence of SEQ ID NO: 27.

[0215] 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 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.

[0216] In some embodiments, the capsid polypeptide comprises at least, or about, 80%, 85%, 90%, or 95%, or 100% of the mutations (insertions, deletions, or substitutions) as shown in the Mutation Difference column of Tables 1A, 1B, 1C, 1D, 1E, 1F, and 1G of a reference capsid sequence, such as SEQ ID NO: 1, and 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 contains at least, about, or exactly 90% of the mutations (insertions, deletions, or substitutions). In some embodiments, the reference capsid sequence contains at least, about, or exactly 95% of the mutations (insertions, deletions, or substitutions). In some embodiments, the reference capsid sequence contains 100% of the mutations (insertions, deletions, or substitutions).

[0217] 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 one of the following mutation groups (the terminology for these mutation groups is provided in the legends to Tables 1A, 1B, 1C, 1D, 1E, 1F, and 1G above): [587_10aa_588_LALGEQTRPA]; [587_9aa_588_LAIEQTRPA]; [586_9aa_587_LALAEITRP,N587A]; [586_9aa_587_LKNAETARP,N587A]; [586_10aa_587_LNLAIEQTRP,N587A,A593T]; [586_8aa_587_MLNEQTRP,N587A]; [584_11aa_585_RSGNRADSETA,G586P,N587A]; [586_6aa_587_TGDTRP,N587A]; [587_9aa_588_LQGETIRPA]; [586_11aa_587_QNLANPETTRP,N587A]; [587_10aa_588_RAPQETTRPA,T592A,T597W]; [586_8aa_587_ANLTTTRP,N587A]; [586_10aa_587_ALLAGEQTRP,N587A]; [D561C,586_9aa_587_GLRAEQTRP,N587A,T597N]; [T550N,584_8aa_585_RARLDETA,G586P,N587A]; and [I559L,586_11aa_587_TNLARGETARP,N587A].

[0218] 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 10 amino acid insertion.

[0219] 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 9 amino acid insertion.

[0220] 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 [586_9aa_587_LALAEITRP,N587A]. In some embodiments, the capsid polypeptide comprises at least 7, or all, of the amino acid residues of the 9 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 8, or all, of the amino acid residues of the 9 amino acid insertion and the N587A mutation.

[0221] 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 [586_9aa_587_LKNAETARP,N587A]. In some embodiments, the capsid polypeptide comprises at least 7, or all, of the amino acid residues of the 9 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 8, or all, of the amino acid residues of the 9 amino acid insertion and the N587A mutation.

[0222] 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 [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 10 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 9, or all, of the amino acid residues of the 10 amino acid insertion and the N587A and A593T mutations.

[0223] 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 [586_8aa_587_MLNEQTRP,N587A]. In some embodiments, the capsid polypeptide comprises at least 6, or all, of the amino acid residues of the 8 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 7, or all, of the amino acid residues of the 8 amino acid insertion and the N587A mutation.

[0224] 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 [584_11aa_585_RSGNRADSETA,G586P,N587A]. In some embodiments, the capsid polypeptide comprises at least 9, or all, of the amino acid residues of the 11 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 10, or all, of the amino acid residues of the 11 amino acid insertion and the G586P and N587A mutations.

[0225] 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 [586_6aa_587_TGDTRP,N587A]. In some embodiments, the capsid polypeptide comprises at least five, or all, of the amino acid residues of the six amino acid insertion. In some embodiments, the capsid polypeptide comprises at least six, or all, of the amino acid residues of the six amino acid insertion and the N587A mutation.

[0226] 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 9 amino acid insertion.

[0227] 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 [586_11aa_587_QNLANPETTRP,N587A]. In some embodiments, the capsid polypeptide comprises at least 9, or all, of the amino acid residues of the 11 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 10, or all, of the amino acid residues of the 11 amino acid insertion and the N587A mutation.

[0228] 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 10 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 9, or all, of the amino acid residues of the 10 amino acid insertion and the T592A and T597W mutations.

[0229] 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 [586_8aa_587_ANLTTTRP,N587A]. In some embodiments, the capsid polypeptide comprises at least 6, or all of the amino acid residues of the 8 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 7, or all of the amino acid residues of the 8 amino acid insertion and the N587A mutation.

[0230] 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 [586_10aa_587_ALLAGEQTRP,N587A]. In some embodiments, the capsid polypeptide comprises at least 8, or all, of the amino acid residues of the 10 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 8, or all, of the amino acid residues of the 10 amino acid insertion and the N587A mutation.

[0231] 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 [D561C,586_9aa_587_GLRAEQTRP,N587A,T597N]. In some embodiments, the capsid polypeptide comprises at least 7, or all, of the amino acid residues of the 9 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 9, or all, of the amino acid residues of the 9 amino acid insertion and the D561C, N587A, and T597N mutations.

[0232] 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 [T550N,584_8aa_585_RARLDETA,G586P,N587A]. In some embodiments, the capsid polypeptide comprises at least 6, or all, of the amino acid residues of the 8 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 8, or all, of the amino acid residues of the 8 amino acid insertion and the T550N, G586P, and N587A mutations.

[0233] 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 11 amino acid insertion. In some embodiments, the capsid polypeptide comprises at least 10, or all, of the amino acid residues of the 11 amino acid insertion and the I559L and N587A mutations.

[0234] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 12 and having at least 80% of the mutations in SEQ ID NO: 12 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 12 and having at least 80% of the mutations in SEQ ID NO: 12 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO: 12 and having less than 80% of the mutations in SEQ ID NO: 12 compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 5-fold over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0235] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 13 and having at least 80% of the mutations in SEQ ID NO: 13 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 13 and having at least 80% of the mutations in SEQ ID NO: 13 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO: 13 and having less than 80% of the mutations in SEQ ID NO: 13 compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the increase is at least 2-fold or at least 3-fold over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0236] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 14 and having at least 80% of the mutations in SEQ ID NO: 14 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 14 and having at least 80% of the mutations in SEQ ID NO: 14 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO: 14 and having less than 80% of the mutations in SEQ ID NO: 14 compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 8-fold over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0237] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 15 and having at least 80% of the mutations in SEQ ID NO: 15 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 15 and having at least 80% of the mutations in SEQ ID NO: 15 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO: 15 and having less than 80% of the mutations in SEQ ID NO: 15 compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the increase is at least 2-fold over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO:1. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 over transduction of viral particles comprising a reference sequence, e.g., SEQ ID NO:1 capsid polypeptide. In some embodiments, the increase is at least 60-fold over transduction of viral particles comprising a reference sequence, e.g., SEQ ID NO:1 capsid polypeptide. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration.In some embodiments, transduction is as measured following intraluminal injection.

[0238] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 16 and having at least 80% of the mutations in SEQ ID NO: 16 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 16 and having at least 80% of the mutations in SEQ ID NO: 16 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO: 16 and having less than 80% of the mutations in SEQ ID NO: 16 compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 230-fold relative to the transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0239] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 17 and having at least 80% of the mutations in SEQ ID NO: 17 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 17 and having at least 80% of the mutations in SEQ ID NO: 17 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO: 17 and having less than 80% of the mutations in SEQ ID NO: 17 compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 70-fold, at least 128-fold, or at least 520-fold relative to the transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0240] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 18 and having at least 80% of the mutations in SEQ ID NO: 18 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 18 and having at least 80% of the mutations in SEQ ID NO: 18 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO: 18 and having less than 80% of the mutations in SEQ ID NO: 18 compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 50-fold, at least 64-fold, at least 128-fold, or at least 460-fold relative to the transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0241] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 19 and having at least 80% of the mutations in SEQ ID NO: 19 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 19 and having at least 80% of the mutations in SEQ ID NO: 19 compared to SEQ ID NO: 1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO: 19 and having less than 80% of the mutations in SEQ ID NO: 19 compared to SEQ ID NO: 1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 100-fold, at least 128-fold, or at least 1000-fold relative to the transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0242] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:20 and having at least 80% of the mutations in SEQ ID NO:20 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:20 and having at least 80% of the mutations in SEQ ID NO:20 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO:20 and having less than 80% of the mutations in SEQ ID NO:20 compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 relative to the transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0243] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:21 and having at least 80% of the mutations in SEQ ID NO:21 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:21 and having at least 80% of the mutations in SEQ ID NO:21 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO:21 and having less than 80% of the mutations in SEQ ID NO:21 compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 relative to the transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0244] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:22 and having at least 80% of the mutations in SEQ ID NO:22 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:22 and having at least 80% of the mutations in SEQ ID NO:22 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO:22 and having less than 80% of the mutations in SEQ ID NO:22 compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 5-fold over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0245] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:23 and having at least 80% of the mutations in SEQ ID NO:23 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:23 and having at least 80% of the mutations in SEQ ID NO:23 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO:23 and having less than 80% of the mutations in SEQ ID NO:23 compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 relative to the transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0246] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:24 and having at least 80% of the mutations in SEQ ID NO:24 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:24 and having at least 80% of the mutations in SEQ ID NO:24 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO:24 and having less than 80% of the mutations in SEQ ID NO:24 compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 5-fold over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0247] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:25 and having at least 80% of the mutations in SEQ ID NO:25 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:25 and having at least 80% of the mutations in SEQ ID NO:25 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO:25 and having less than 80% of the mutations in SEQ ID NO:25 compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the increase is at least 2-fold, at least 4-fold, at least 8-fold, or at least 12-fold over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0248] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:26 and having at least 80% of the mutations in SEQ ID NO:26 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:26 and having at least 80% of the mutations in SEQ ID NO:26 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO:26 and having less than 80% of the mutations in SEQ ID NO:26 compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 relative to the transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0249] In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO:27 and having at least 80% of the mutations in SEQ ID NO:27 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having less than 95% amino acid sequence identity with the amino acid sequence of SEQ ID NO:27 and having at least 80% of the mutations in SEQ ID NO:27 compared to SEQ ID NO:1. In some embodiments, a variant capsid polypeptide is provided that comprises an amino acid sequence having 95% or more amino acid sequence identity with SEQ ID NO:27 and having less than 80% of the mutations in SEQ ID NO:27 compared to SEQ ID NO:1. In some embodiments, a viral particle comprising a variant capsid polypeptide has increased ocular transduction of the transgene in the anterior third of the eye, including structures anterior to the vitreous humor, such as, but not limited to, the cornea, iris, ciliary body, lens, trabecular meshwork, or Schlemm's canal, or any combination thereof, as described herein. In some embodiments, the increase is at least 2-fold, at least 4-fold, or at least 8-fold over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the viral particles comprising the variant capsid polypeptide have increased ocular transduction of the transgene posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., the 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 over transduction of viral particles comprising a capsid polypeptide of a reference sequence, e.g., SEQ ID NO: 1. In some embodiments, the increased transduction is measured, e.g., by NGS sequencing of viral RNA in cells of the target tissue, as described in the Examples. In some embodiments, the transduction is as measured after intravitreal administration. In some embodiments, the transduction is as measured after intracavitary injection.

[0250] As used herein, the phrase "80% of mutations" in reference to a variant capsid sequence means that the variant has at least 80% of the mutations present in the variant capsid sequence, where the total number of mutations is based on a comparison to 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 toward the total number of mutations. For example, if the variant capsid polypeptide has a mixture of "T550N", "G586P", and "N587A" substitutions, and an insertion of a polypeptide containing the sequence of RARLDETA (SEQ ID NO:58), the total number of mutations is 11, which is 8 amino acid insertions and 3 amino acid substitutions, and a variant capsid with "80% of mutations" would contain at least 9, 10, or all of the mutations.

[0251] Variant capsid (corresponding position) Mutations to the capsid polypeptide sequences described herein are described with reference to positions and / or amino acids at positions within a reference sequence (e.g., SEQ ID NO:1). Thus, in some embodiments, the capsid polypeptides described herein are variant capsid polypeptides of a reference sequence (e.g., SEQ ID NO:1), including, for example, capsid polypeptides having 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 (e.g., a reference capsid polypeptide VP1, VP2, and / or VP3 sequence), e.g., SEQ ID NO:1 (or a VP2 or VP3 sequence contained therein), and further comprising one or more mutations described herein.

[0252] It will be understood by those skilled in the art, without being bound by theory, that each amino acid position in the reference sequence corresponds to a position in the sequence of other capsid polypeptides, such as capsid polypeptides from dependoparvoviruses having different serotypes. Such corresponding positions are identified using sequence alignment tools known in the art. A particularly preferred sequence alignment tool is Clustal Omega (Sievers F., et al., Mol. Syst. Biol. 7:359, 2011, DOI:10.1038 / msb.2011.75, incorporated herein by reference in its entirety). Exemplary reference capsid polypeptide alignments are shown in Figures 2A-2C. Thus, in some embodiments, the variant capsid polypeptides of the invention include variants of reference capsid polypeptides that include one or more mutations described herein in such reference capsid polypeptides at positions corresponding to the positions of the mutations described herein in relation to different reference capsid polypeptides. Thus, for example, for a mutation set forth as XnnnY relative to SEQ ID NO:1 (where X is an amino acid present at position nnn of SEQ ID NO:1 and Y is an amino acid mutation at that position, e.g., as described herein), the present disclosure provides variant capsid polypeptides that comprise 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 (e.g., a reference capsid polypeptide VP1, VP2 and / or VP3 sequence) other than SEQ ID NO:1 (or a VP2 or VP3 sequence contained therein), and further comprise a disclosed mutation at a position corresponding to position nnn of SEQ ID NO:1 (e.g., comprising a Y at a position within the new variant capsid polypeptide sequence that corresponds to position nnn of SEQ ID NO:1). As noted above, such corresponding positions may be determined using, for example, a sequence alignment tool, such as the Cluster Omega tool described above. Examples of corresponding amino acid positions for exemplary known AAV serotypes are shown in Figures 2A-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."

[0253] Thus, in some embodiments, the disclosure provides capsid polypeptide sequences that are variants of a reference sequence other than SEQ ID NO: 1, e.g., a reference sequence other than SEQ ID NO: 1 described herein, that includes one or more mutations corresponding to those described herein. In some embodiments, such variants include mutations that correspond to all of the mutations associated with any one of VAR-1 through VAR-16 according to Table 1A, Table 1B, Table 1C, Table 1D, Table 1E, Table 1F, and Table 1G.

[0254] As used herein, the term "corresponding to" used in reference to a position within a sequence, such as an amino acid or nucleic acid sequence, may be used in reference to an entire capsid polypeptide or polynucleotide sequence, such as a full-length capsid polypeptide sequence, including a VP1, VP2, and VP3 polypeptide, or a nucleic acid molecule encoding the same. In some embodiments, the term "corresponding to" may be used in reference to a region or domain of a capsid polypeptide. For example, a position corresponding to a position within a VP1 section of a reference capsid polypeptide may correspond to the VP1 portion of a polypeptide of a variant capsid polypeptide. Thus, when aligning two sequences to determine whether a position corresponds to another position, the full-length polypeptide may be used, or a domain (region) may be used to determine whether a position corresponds to a particular 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, where the reference polypeptide is a wild-type sequence (e.g., full length or region) of an AAV of a particular serotype, the variant polypeptide can be of the same serotype with mutations made at such corresponding positions compared to the reference sequence (e.g., full length or region). In some embodiments, the variant capsid polypeptide is of a different serotype compared to the reference sequence.

[0255] 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), AAVP HP.N / PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAV PHP.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, AAV PHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B-TMP, AAVPHP.B-TTP, AAVPHP.S / G2A 12, AAVG2A15 / G2A3(G2A3), AAVG2B4(G2B4), AAVG2B5(G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, A AV3-3, 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、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-1 5、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 AV223.5、AAV223.6、AAV223.7、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 / rh.6、AAV3.1 / rh.61 hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / r11.64、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / rh.7、AAV16.8 / hu.1 0、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 / hu.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、AAV hu.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、AA VCh.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. AVhu.15、AAVhu.16、AAVhu.17、AAVhu.18、AAVhu.20、AAVhu.21、AAVhu.22、AAVhu.23.2、AAVhu.24、AAVhu.25、AAVhu. u.27、AAVhu.28、AAVhu.29、AAVhu.29R、AAVhu.31、AAVhu.32、AAVhu.34、AAVhu.35、AAVhu.37、AAVhu.39、AAVhu.4 0、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. u.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. rh.21、AAVrh.22、AAVrh.23、AAVrh.24、AAVrh.25、AAVrh.31、AAVrh.32、AAVrh.33、AAVrh.34、AAVrh.35、AAVrh.36、AAVrh.37、AAVrh.37、AAVrh.37、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、AA Vrh.56、AAVrh.57、AAVrh.58、AAVrh.61、AAVrh.64、AAVrh.64R1、AAVrh.64R2、AAVrh.67、AAVrh.73、AAVrh.74(also called AAVrh74)、AAVrh8R、AAVrh8R. A586R mutation、AAVrh8R R533A mutation、AAAV、BAAV、ヤギAAV、ウシAAV、AAVhE1.1、AAVhEr1.5、AAVhEr1.14、AAVhEr1.8、AAVhEr1.16、AAVhEr1.18、AAVhEr1.35、AAVhEr1.7、AAVhEr1.36、AAVhEr r2.29、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-L K04、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 BNP61 AAV、BNP62 AAV、BNP63 AAV、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、AAV CBr-E2、AAV CBr-E3、AAV CBr-E4、AAV CBr-E5、AAV CBr-E6、AAV CBr-E6 CBr-E7、AAV CBr-E8、AAV CHt-1、AAV CHt-2、AAV CHt-3、AAV CHt-6.1、AAV CHt-6.10、AAV CHt-6.5、AAV CHt-6.6、AAV CHt-6.7、AAV CHt-6.8、AAV CHt-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、AAV CKd-6、AAV CKd-7、AAV CKd-8、AAV CKd-B1、AAV CKd-B2、AAV CKd-B3、AAV CKd-B4、AAV CKd-B5、AAV CKd-B6、AAV CKd-B7、AAV CKd-B8、AAV CKd-H1、AAV CKd-H2、AAV CKd-H3、AAV CKd-H4、AAV CKd-H5、AAV CKd-H6、AAV CKd-N3、AAV CKd-N4、AAV CKd-N9、AAV CLg-F1、AAV CLg-F2、AAV CLg-F3、AAV CLg-F4、AAV CLg-F5、AAV CLg-F6、AAV CLg-F7、AAV CLg-F8、AAV CLv-1、AAV CLv1-1、AAV CLv1-10、AAV CLv1-2、AAV CLv-12、AAV CLv1-3、AAV CLv-13、AAV CLv1-4、AAV Clv1-7、AAV Clv1-8、AAV Clv1-9、AAV CLv-2、AAV CLv-3、AAV CLv-4、AAV CLv-6、AAV CLv-8、AAV CLv-D1、AAV CLv-D2、AAV CLv-D3、AAV CLv-D4、AAV CLv-D5、AAV CLv-D6、A. AV 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.

[0256] 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. Without wishing to be bound by theory, it is 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 may be considered synonymous with the reference AAV capsid sequence.

[0257] An exemplary reference sequence of SEQ ID NO:1 (wild type AAV2) is as follows: [ka]

[0258] Unless otherwise indicated, SEQ ID NO: 1 is the reference sequence. In the above sequence, 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, sequences corresponding to amino acids 138-735 of SEQ ID NO: 1), and sequences that are 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).

[0259] An exemplary nucleic acid sequence that encodes SEQ ID NO:1 is SEQ ID NO:2.

[0260] An exemplary reference sequence for wild-type AAV5, SEQ ID NO:3 (wild-type AAV5), is as follows: [ka]

[0261] 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 193-725 of SEQ ID NO:3), sequences found in both VP1 and VP2 are bolded (e.g., the VP2 capsid polypeptide comprises, e.g., consists of, sequences corresponding to amino acids 137-725 of SEQ ID NO:3), and sequences that are 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 1-725 of SEQ ID NO:3).

[0262] An exemplary nucleic acid sequence that encodes SEQ ID NO:3 is SEQ ID NO:4.

[0263] An exemplary reference sequence for wild-type AAV8, SEQ ID NO:5 (wild-type AAV8), is as follows: [ka]

[0264] 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 204-735 of SEQ ID NO:5), sequences found in both VP1 and VP2 are bolded (e.g., the VP2 capsid polypeptide comprises, e.g., consists of, sequences corresponding to amino acids 138-735 of SEQ ID NO:5), and sequences that are 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 1-739 of SEQ ID NO:5).

[0265] An exemplary nucleic acid sequence that encodes SEQ ID NO:5 is SEQ ID NO:6.

[0266] An exemplary reference sequence for wild-type AAV9, SEQ ID NO:7 (wild-type AAV9), is as follows: [ka]

[0267] 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 to 737 of SEQ ID NO:7), sequences found in both VP1 and VP2 are bolded (e.g., the VP2 capsid polypeptide comprises, e.g., consists of, sequences corresponding to amino acids 138 to 737 of SEQ ID NO:7), and sequences that are 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 to 737 of SEQ ID NO:7).

[0268] An exemplary nucleic acid sequence that encodes SEQ ID NO:7 is SEQ ID NO:8.

[0269] An exemplary reference sequence for wild-type AAVrh74, SEQ ID NO:9 (wild-type AAVrh74), is as follows: [ka]

[0270] An alternative exemplary reference sequence to SEQ ID NO:10 (alternative wild type AAVrh74) is as follows: [ka]

[0271] 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 comprises, e.g., consists of, amino acids corresponding to amino acids 204 to 739 of SEQ ID NO:9), sequences found in both VP1 and VP2 are bolded (e.g., the VP2 capsid polypeptide comprises, e.g., consists of, sequences corresponding to amino acids 137 to 739 of SEQ ID NO:9), and sequences that are 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 to 739 of SEQ ID NO:9).

[0272] An exemplary nucleic acid sequence that encodes SEQ ID NO:9 is SEQ ID NO:11.

[0273] An exemplary reference sequence for an AAV2 variant (SEQ ID NO: 60) identified from the ophthalmic literature is as follows: (SEQ ID NO:60)

[0274] An exemplary nucleic acid sequence that encodes SEQ ID NO:60 is SEQ ID NO:61.

[0275] 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, provide increased delivery of such payload to one or more tissues or cell types of the eye (e.g., neural retina, macula, and / or choroid / RPE) after, e.g., intravitreal administration, relative to an otherwise identical viral particle comprising a capsid polypeptide of SEQ ID NO: 60. 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, provide increased delivery of such payload to one or more tissues or cell types of the eye (e.g., tissues or cells of the trabecular meshwork and Schlemm's canal) after, e.g., intravitreal administration or intracameral administration, relative to an otherwise identical viral particle comprising a capsid polypeptide of SEQ ID NO: 60.

[0276] The present disclosure refers 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. VP capsid proteins synthesized from Cap polynucleotides generally contain a methionine (Met1) as the first amino acid in the polypeptide sequence that associates with the start codon (AUG or ATG) in the corresponding Cap nucleotide sequence. However, it is common for the first methionine (Met1) residue, or generally any first amino acid (AA1), to be cleaved after or during polypeptide synthesis by a protein processing enzyme such as Met-aminopeptidase. This "Met / AA-clipping" process often correlates with the corresponding acetylation of the second amino acid (e.g., alanine, valine, serine, threonine, etc.) in the polypeptide sequence. Met-clipping generally occurs in the VP1 and VP3 capsid proteins, but can also occur in the VP2 capsid protein. When Met / AA-clipping is incomplete, a mixture of one or more (one, two, or three) VP capsid proteins comprising the viral capsid can be generated, some of which contain the Met1 / AA1 amino acids (Met+ / AA+) and some of which lack the Met1 / AA1 amino acids as a result of Met / AA-clipping (Met- / AA-).For further discussion of Met / AA-clipping in capsid proteins, see Jin, et al. Direct Liquid Chromatography / Mass Spectrometry Analysis for Complete Characterization of Recombinant Adeno-Associated Virus Capsid Proteins. Hum Gene Ther Methods. 2017 Oct. 28(5):255-267; Hwang, et al. N- Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals. Science. 2010 February 19.327(5968):973-977, the contents of each of which are incorporated herein by reference in their entireties. According to the present disclosure, reference to a capsid polypeptide is not limited to either clipped (Met- / AA-) or unclipped (Met+ / AA+), but also refers in context to an individual capsid polypeptide, a viral capsid composed of a mixture of capsid proteins, and / or a polynucleotide sequence (or fragment thereof) that encodes, describes, produces, or results in a capsid polypeptide of the present disclosure. Direct reference to a "capsid polypeptide" (such as VP1, VP2, or VP3) also includes VP capsid proteins, including the Met1 / AA1 amino acids (Met+ / AA+), and the corresponding VP capsid polypeptide lacking the Met1 / AA1 amino acids (Met- / AA-), e.g., as a result of Met / AA clipping. Furthermore, in accordance with the present disclosure, reference to a particular SEQ ID NO: (whether protein or nucleic acid) that contains or encodes one or more capsid polypeptides that contain the Met1 / AA1 amino acids (Met+ / AA+), respectively, should be understood to teach a VP capsid polypeptide that lacks the Met1 / AA1 amino acids, and it is readily apparent that, upon review of the sequence, the first recited amino acid (whether Met1 / AA1 or not) may not be present.As a non-limiting example, reference to a (Met+) VP1 polypeptide sequence that is 736 amino acids in length and includes a "Met1" amino acid encoded by an AUG / ATG start codon is understood to also teach a (Met-) VP1 polypeptide sequence that is 735 amino acids in length and does not include the "Met1" amino acid of the 736 amino acid Met+ sequence. As a second non-limiting example, reference to a (AA1+) VP1 polypeptide sequence that is 736 amino acids in length and includes an "AA1" amino acid encoded by any NNN initiator codon can also be understood to teach a (AA1-) VP1 polypeptide sequence that is 735 amino acids in length and does not include the "AA1" amino acid of the 736 amino acid AA1+ sequence. Reference to a viral capsid formed from VP capsid proteins (e.g., reference to a particular AAV capsid serotype) can incorporate VP capsid proteins that include Met1 / AA1 amino acids (Met+ / AA1+), corresponding VP capsid proteins that lack Met1 / AA1 amino acids (Met- / AA1-), e.g., as a result of Met / AA1 clipping, and combinations thereof (Met+ / AA1+ and Met- / AA1-). As non-limiting examples, an AAV capsid serotype may include VP1(Met+ / AA1+), VP1(Met- / AA1-), or a combination of VP1(Met+ / AA1+) and VP1(Met- / AA1-). AAV capsid serotypes may also include VP3(Met+ / AA1+), VP3(Met- / AA1-), or combinations of VP3(Met+ / AA1+) and VP3(Met- / AA1-), as well as optional combinations of VP2(Met+ / AA1) and VP2(Met- / AA1-).

[0277] In some embodiments, the reference AAV capsid sequence comprises an amino acid sequence having 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of these above.

[0278] In some embodiments, the reference AAV capsid sequence is encoded by a nucleotide sequence having 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any of these above. In certain embodiments, the reference sequence is not the AAV capsid sequence, but instead is a different vector (e.g., a lentivirus, a plasmid, etc.).

[0279] In some embodiments, a nucleic acid of the disclosure (e.g., encoding an AAV2 variant capsid protein) comprises conventional control elements or sequences operably linked to the nucleic acid molecule in a manner that allows for transcription, translation, and / or expression in a cell transfected with the nucleic acid (e.g., a plasmid vector containing the nucleic acid) or infected with a virus containing the nucleic acid. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with a gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0280] 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 enhance translation efficiency (e.g., Kozak consensus sequences); and, in some embodiments, sequences that enhance secretion of the encoded transgene product. Expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized with the compositions and methods disclosed herein.

[0281] In some embodiments, a native promoter for the transgene may be used. Without wishing to be bound by theory, the native promoter may mimic the native expression of the transgene and may provide temporal, developmental, or tissue-specific expression, or expression in response to a specific transcriptional stimulus. In some embodiments, the transgene may be operably linked to other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, for example, to mimic native expression.

[0282] In some embodiments, the transgene is operably linked to a tissue-specific promoter.

[0283] In some embodiments, the vector (e.g., a plasmid) carrying the transgene may also include a selectable marker or reporter gene. Such a selectable reporter or marker gene can be used to signal the presence of the vector, e.g., a plasmid, in the bacterial cell. Other components of the vector, e.g., a plasmid, may include an origin of replication. 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).

[0284] In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the eye compared to a viral particle having 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 transduction in the retina compared to a viral particle having 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 transduction in the non-macular retina compared to a viral particle having 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 transduction in the macula compared to a viral particle having 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 transduction in the trabecular meshwork compared to a viral particle having 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 transduction in the trabecular meshwork relative to the retina, as compared to viral particles having a reference capsid polypeptide, e.g., 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, as compared to viral particles having a reference capsid polypeptide, e.g., 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, as compared to viral particles having a reference capsid polypeptide, e.g., 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, as compared to viral particles having a reference capsid polypeptide, e.g., 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 non-macular retina as compared to viral particles having a reference capsid polypeptide, e.g., 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 as compared to viral particles having a reference capsid polypeptide, e.g., 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 non-macular retina and trabecular meshwork as compared to viral particles having a reference capsid polypeptide, e.g., 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 as compared to viral particles having a reference capsid polypeptide, e.g., 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, as compared to viral particles having a reference capsid polypeptide, e.g., 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, as compared to viral particles having a reference capsid polypeptide, e.g., 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, as compared to viral particles having a reference capsid polypeptide, e.g., 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, as compared to viral particles having a reference capsid polypeptide, e.g., wild-type capsid polypeptide (SEQ ID NO:1).

[0285] In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction at least 1-fold, for example, as compared to a viral particle having a reference capsid polypeptide, for example, a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction at least 2-fold, for example, as compared to a viral particle having a reference capsid polypeptide, for example, a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction 4-fold, for example, as compared to a viral particle having a reference capsid polypeptide, for example, a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, the capsid polypeptide present in the viral particle increases ocular transduction 6-fold, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, compared to a viral particle having 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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, for example, as compared to a viral particle having a reference capsid polypeptide, for example, 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 from nucleic acid packaged in a variant viral particle to the level of mRNA in the target tissue (e.g., in a cell or cell population of the target tissue) produced from nucleic acid packaged in a reference viral particle (e.g., packaged in a capsid comprising the capsid polypeptide of SEQ ID NO:1).

[0286] In some embodiments, the capsid polypeptide present in the viral particle increases transduction in the eye 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 capsid polypeptide present in the viral particle increases transduction in the trabecular meshwork 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 capsid polypeptide present in the viral particle increases ocular transduction at least 1-fold 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 capsid polypeptide present in the viral particle increases ocular transduction at least 1.5-fold 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 capsid polypeptide present in the viral particle increases ocular transduction at least 2-fold 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 capsid polypeptide present in the viral particle increases ocular transduction by at least 2.5-fold 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 dependoparvoviral 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.

[0287] In some embodiments, the cell is a non-human cell, hi other embodiments, the cell is not a human pluripotent stem cell, e.g., is not a human embryonic stem cell.

[0288] In some embodiments, the capsid polypeptide is present in a dependoparvovirus B (e.g., AAV2) particle. In some embodiments, the capsid particle has increased ocular transduction.

[0289] In some embodiments, the dependoparvovirus particle comprises an amino acid sequence having at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity to an amino acid sequence provided herein (e.g., SEQ ID NOs: 12-27). In some embodiments, the variant capsid polypeptide comprises an amino acid sequence that differs from the amino acid sequence of a variant capsid polypeptide provided herein by no more than 30, no more than 29, no more than 28, no more than 27, no more than 26, no more than 25, no more than 24, no more than 23, no more than 22, no more than 21, 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 11, 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 amino acid.

[0290] In some embodiments, the additional modification improves the production characteristics of, or the method of making, the viral particle, such as, but not limited to, the dependoparvovirus particle. In some embodiments, the additional modification improves or alters another characteristic, such as, but not limited to, the tropism, of the viral particle, such as, but not limited to, the dependoparvovirus particle.

[0291] In embodiments, improved transduction is as measured by quantification of viral RNA from target tissue. In some embodiments, improved biodistribution is as measured by quantification of viral DNA from target tissue. In some embodiments, improved transduction is as measured after generation from HEK293 cells, for example, as described in the Examples.

[0292] VP1 Nucleic Acids and Polypeptides The present disclosure is further directed, in part, to a nucleic acid comprising a sequence encoding a capsid polypeptide, such as, but not limited to, a dependoparvovirus (e.g., dependoparvovirus B, e.g., AAV2) capsid polypeptide provided herein, and the 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.

[0293] Virus particles The present disclosure is also directed, in part, to viral particles comprising a nucleic acid or polypeptide described herein or produced by the methods described herein, including, but not limited to, dependoparvoviral particles (e.g., functional dependoparvoviral particles).

[0294] Dependoparvoviruses are single-stranded DNA parvoviruses that grow only in cells where certain functions are provided, for example, by a coinfecting helper virus. Several species of dependoparvoviruses are known, including dependoparvovirus A and dependoparvovirus B, which include serotypes known in the art as adeno-associated viruses (AAV). At least 13 AAV serotypes have been characterized. General information and reviews of AAV can be found, for example, in 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 dependoparvovirus species, are structurally and functionally significantly related to each other. (See, e.g., Blacklowe, pp. 165-174 of Parvoviruses and Human Disease, J.R. Puttson, ed. (1988), and Rose, Comprehensive Virology 3:1-61 (1974).) For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all have three related capsid proteins. In addition, heteroduplex analysis reveals extensive cross-hybridization between serotypes along the length of the genome, further suggesting an interrelatedness. Dependoparvovirus genomes also contain self-annealing segments at the ends that correspond to the "inverted terminal repeats" (ITRs).

[0295] The genomic organization of naturally occurring dependoparvoviruses, such as the AAV serotypes, is very similar. For example, the genome of AAV is a linear single-stranded DNA molecule that is approximately 5,000 nucleotides (nt) long or less. Inverted terminal repeats (ITRs) flank the coding nucleotide sequences specific for the nonstructural replication (Rep) proteins and the structural capsid (Cap) proteins. Three different virion (VP) proteins form the capsid. The terminal 145 nt are self-complementary and are organized in such a way that energetically stable intramolecular duplexes that form T-shaped hairpins can form. These hairpin structures serve as origins of viral DNA replication and as primers for the cellular DNA polymerase complex. The Rep genes encode the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep52 and Rep40 are transcribed from the p19 promoter. The cap gene encodes the VP proteins VP1, VP2, and VP3. The cap gene is transcribed from the p40 promoter.

[0296] In some embodiments, the dependoparvovirus particles of the present disclosure comprise a nucleic acid comprising a capsid polypeptide provided herein. In some embodiments, the particle comprises a polypeptide provided herein.

[0297] In some embodiments, the dependoparvovirus particle of the present disclosure may be an AAV2 particle or a variant thereof. In some embodiments, the AAV2 particle comprises a capsid polypeptide provided herein, or a nucleic acid molecule encoding same.

[0298] In some embodiments, the depend parvoviral particle comprises a capsid comprising a variant capsid polypeptide described herein. In some embodiments, the depend parvoviral particle comprises a variant capsid polypeptide described herein and a nucleic acid molecule. In some embodiments, the depend parvoviral particle comprises a variant capsid polypeptide described herein and a nucleic acid molecule comprising one or more inverted terminal repeats (ITRs), e.g., ITRs from an AAV2 depend 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.

[0299] In some embodiments, the viral particle comprises a variant capsid polypeptide such as those provided herein. In some embodiments, the viral particle comprises a variant capsid polypeptide having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence provided in Table 2. In some embodiments, the viral particle comprises a variant capsid polypeptide having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or 100% identity to a VP1, VP2, or VP3 sequence 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 a 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.

[0300] 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.

[0301] Increased Ocular Transduction Characteristics The present disclosure is directed, in part, to nucleic acids, polypeptides, cells, cell-free systems, translation systems, viral particles, and methods associated with making same, for generating viral particles with increased ocular transduction compared to viral particles having a capsid polypeptide of a reference sequence, e.g., having a 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, thus resulting in 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, thus resulting in 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, thus resulting in 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, thus resulting in 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 transgenes in the trabecular meshwork, thus resulting in expression of the transgene in the trabecular meshwork. 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, including structures anterior to the vitreous humor. Examples of structures anterior to 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 posterior to the lens, e.g., in the anterior hyaloid membrane and all optical structures behind it, e.g., 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 hyaloid membrane and all optical structures behind it, e.g., the vitreous humor, retina, choroid, or optic nerve, or any combination thereof. 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 and anterior to the vitreous humor.

[0302] In some embodiments, the increase in ocular transduction is about 1-7 fold better (e.g., about 2-5 fold better, e.g., about 3-5 fold better) on a log2 scale than viral particles having a reference sequence capsid polypeptide (e.g., having a wild-type capsid polypeptide of SEQ ID NO:1).

[0303] 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.

[0304] Tables 3, 4A, 4B, 4C, and 4D list information regarding the biodistribution of variant depend parvovirus particles comprising capsid polypeptides of the indicated variant capsids in different layers, structures, and / or parts of the eye. In Table 3, biodistribution in the retina is as measured after IVT injection, and biodistribution in the trabecular meshwork is as measured after IC injection in library experiment 1 in all cases. In Table 4A, biodistribution in the retina is as measured after IVT injection, biodistribution in the trabecular meshwork is as measured after IC or IVT injection (as indicated in the table), and biodistribution in the choroid is as measured after IVT injection in library experiment 2 in all cases. In Table 4B, biodistribution in the neural retina tissue is as measured after IVT injection in library experiment 3 in all cases. In Table 4C, the biodistribution in the neuroretina tissue, the biodistribution in the macular tissue, and the biodistribution in the trabecular meshwork are in all cases as measured after IVT injection in library experiment 3. In Table 4D, the biodistribution in the trabecular meshwork are in all cases as measured after IC injection in library experiment 3. Measurements are performed for wild type AAV2 (SEQ ID NO: 1) unless otherwise stated. The trabecular meshwork biodistribution measurements shown in Tables 4C and 4D are shown for an AAV2 variant identified from the ocular literature having SEQ ID NO: 60 and a capsid polypeptide encoded by nucleic acid SEQ ID NO: 61. The data shown in Tables 3 and 4A are in log2 scale. [Table 9] [Table 10] [Table 11] [Table 12] [Table 13]

[0305] According to some embodiments, the variant capsid polypeptides described herein comprise an N587 substitution mutation in combination with an inserted peptide (e.g., an inserted peptide derived after the glycine (G) at position 586 (numbering according to WT AAV2 VP1, SEQ ID NO:1)) juxtaposed to the N-terminus. In some embodiments, the peptide insertion is six or fewer amino acids. In some embodiments, the peptide insertion consists of six amino acids. In some embodiments, the peptide insertion consists of seven amino acids. In some embodiments, the peptide insertion is seven or fewer amino acids. In some embodiments, the peptide insertion is seven or more amino acids, e.g., seven, eight, nine, ten, or eleven amino acids. In some embodiments, the peptide insertion is six to eleven amino acids. In some embodiments, the peptide insertion consists of eleven amino acids. In some embodiments, the inserted 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 an N587 substitution mutation (e.g., an N587A mutation) in combination with an inserted peptide juxtaposed to the N-terminus exhibit increased ocular transduction and / or biodistribution compared to viral particles comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0306] According to some embodiments, the variant capsid polypeptides described herein include a threonine-arginine-proline-alanine ("TRPA") sequence. In some embodiments, the variant capsid polypeptide includes a peptide insertion (relative to SEQ ID NO: 1) that includes at least some of the TRPA sequence (e.g., 3 or 4 amino acids that form the TRPA sequence). For example, VAR-1, VAR-2, VAR-3, VAR-5, VAR-6, VAR-8, VAR-11, VAR-13, and VAR-14 each include a TRPA 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 asparagine (N) at position 587 (according to WT AAV2 VP1 numbering, SEQ ID NO: 1). In some embodiments, the TRPA sequence is formed by a peptide insertion that includes the C-terminal TRPA sequence. For example, both VAR-1 and VAR-2 contain a peptide insertion that includes a TRPA sequence at its C-terminus, optionally beginning after the asparagine (N) at position 587 (according to WT AAV2 VP1 numbering, SEQ ID NO:1). In some embodiments, the TRPA sequence is formed by a peptide insertion that includes a threonine-arginine-proline ("TRP") sequence at its C-terminus followed by an alanine (A) substitution (e.g., an N587A substitution). In such embodiments, the alanine (A) substitution mutation in combination with the inserted peptide juxtaposed to the N-terminus that includes the TRP sequence forms 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 the alanine substitution is at position 587 (according to WT AAV2 VP1 numbering, SEQ ID NO:1). In some embodiments, the peptide insertion is six or fewer amino acids. In some embodiments the peptide insertion consists of 6 amino acids. In some embodiments the peptide insertion is 7 or fewer amino acids. In some embodiments the peptide insertion consists of 7 amino acids.In some embodiments, the peptide insertion is 7 or more, e.g., 7, 8, 9, 10, or 11 amino acids. In some embodiments, the peptide insertion is 6-11 amino acids. In some embodiments, the peptide insertion consists of 7 amino acids. In some embodiments, a viral particle comprising a variant capsid polypeptide comprising a TRPA sequence exhibits increased ocular transduction and / or biodistribution compared to a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0307] According to some embodiments, the variant capsid polypeptides described herein comprise a threonine-alanine-arginine-proline-alanine ("TARPA") sequence. In some embodiments, the variant capsid polypeptide comprises a peptide insertion comprising at least some of the TARPA sequence (e.g., 2, 3, or 4 amino acids forming a TARPA sequence). In some embodiments, the variant capsid polypeptide comprises a peptide insertion comprising all of the TARPA sequence (e.g., relative to SEQ ID NO:1). 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 after the peptide insertion (e.g., G586P mutation, N587A mutation), e.g., a peptide insertion comprising TAR at its C-terminus (per 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 after the peptide insertion (e.g., G586P mutation, N587A mutation) (per WT AAV2 VP1 numbering, SEQ ID NO:1), and an amino acid present in WT AAV2 VP1 (SEQ ID NO:1), e.g., an 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, inserted after a glycine (G) at position 586, followed by an alanine substitution at position 587 (per WT AAV2 VP1 numbering, SEQ ID NO:1), e.g., as in VAR-4 and VAR-16.In some embodiments, the variant capsid polypeptide comprises a threonine-alanine ("TA") sequence at its C-terminus, e.g., as in VAR-7, and comprises a peptide insertion (according to WT AAV2 VP1 numbering, SEQ ID NO:1) 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, with an arginine (R) at 585 forming the TARPA sequence. In some embodiments, the peptide insertion is 9 amino acids or less. In some embodiments, the peptide insertion is 9 or more amino acids, e.g., 10, or 11 amino acids. In some embodiments, the peptide insertion is 9-11 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 compared to viral particles comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0308] Category A (Neural Retina Transduction): In some aspects of the disclosure, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased neural retina transduction as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased neural retina transduction is as defined as any one of embodiments A-1 through A-8.

[0309] Embodiment A-1: ​​In one embodiment of category A, transduction is about (or at least about) 2-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0310] Embodiment A-2: In one embodiment of category A, transduction is about (or at least about) 4 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0311] Embodiment A-3: In one embodiment of category A, transduction is about (or at least about) 6-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0312] Embodiment A-4: In one embodiment of category A, transduction is about (or at least about) 10 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0313] Embodiment A-5: In one embodiment of category A, transduction is about (or at least about) 30 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0314] Embodiment A-6: In one embodiment of category A, transduction is about (or at least about) 50 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0315] Embodiment A-7: In one embodiment of category A, transduction is about (or at least about) 70 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0316] Embodiment A-8: In one embodiment of category A, transduction is about (or at least about) 100 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0317] In some embodiments, the improved transduction is within a range bounded by any two values ​​shown in embodiments A-1 through A-8. Exemplary ranges are shown in embodiments A-9 through A-14 below.

[0318] Embodiment A-9: In one embodiment of Category A, transduction is in the range of about 2 to about 100 fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0319] Embodiment A-10: In one embodiment of Category A, transduction is in the range of about 4 to about 100 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0320] Embodiment A-11: In one embodiment of Category A, transduction is in the range of about 6 to about 100 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0321] Embodiment A-12: In one embodiment of Category A, transduction is in the range of about 10 to about 100 fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0322] Embodiment A-13: In one embodiment of Category A, transduction is in the range of about 30 to about 100 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0323] Embodiment A-14: In one embodiment of Category A, transduction is in the range of about 50 to about 100 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0324] Category B (Macular Transduction): In some aspects of the disclosure, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased macular transduction as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased macular transduction is as defined as any one of embodiments B-1 through B-10.

[0325] Embodiment B-1: In one embodiment of category B, transduction is about (or at least about) 2-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0326] Embodiment B-2: In one embodiment of category B, transduction is about (or at least about) 4 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0327] Embodiment B-3: In one embodiment of category B, transduction is about (or at least about) 20 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0328] Embodiment B-4: In one embodiment of category B, transduction is about (or at least about) 50 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0329] Embodiment B-5: In one embodiment of category B, transduction is about (or at least about) 100 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0330] Embodiment B-6: In one embodiment of category B, transduction is about (or at least about) 200 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0331] Embodiment B-7: In one embodiment of category B, transduction is about (or at least about) 300 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0332] Embodiment B-8: In one embodiment of category B, transduction is about (or at least about) 400 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0333] Embodiment B-9: In one embodiment of category B, transduction is about (or at least about) 500 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0334] Embodiment B-10: In one embodiment of category B, transduction is about (or at least about) 1000 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0335] In some embodiments, the improved transduction is within a range bounded by any two values ​​shown in embodiments B-1 through B-10. Exemplary ranges are shown in embodiments B-11 through B-17 below.

[0336] Embodiment B-11: In one embodiment of category B, transduction is in the range of about 2 to about 1000 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0337] Embodiment B-12: In one embodiment of category B, transduction is in the range of about 4 to about 1000 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0338] Embodiment B-13: In one embodiment of category B, transduction is in the range of about 20 to about 1000 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0339] Embodiment B-14: In one embodiment of category B, transduction is in the range of about 50 to about 1000 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0340] Embodiment B-15: In one embodiment of category B, transduction is in the range of about 100 to about 1000 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0341] Embodiment B-16: In one embodiment of category B, transduction is in the range of about 200 to about 1000 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0342] Embodiment B-17: In one embodiment of category B, transduction is in the range of about 300 to about 1000 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0343] Category C (Trabecular Meshwork Transduction): 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 meshwork transduction compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased trabecular meshwork transduction is as defined as any one of embodiments C-1 to C-4.

[0344] Embodiment C-1: In one embodiment of category C, transduction is about (or at least about) 2-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0345] Embodiment C-2: In one embodiment of category C, transduction is about (or at least about) 4 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0346] Embodiment C-3: In one embodiment of category C, transduction is about (or at least about) 8 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0347] Embodiment C-4: In one embodiment of category C, transduction is about (or at least about) 12 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0348] In some embodiments, the improved transduction is within a range bounded by any two values ​​shown in embodiments C-1 through C-4. Exemplary ranges are shown in embodiments C-5 through C-7 below.

[0349] Embodiment C-5: In one embodiment of category C, transduction is in the range of about 2 to about 12 fold better than virus particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0350] Embodiment C-6: In one embodiment of category C, transduction is in the range of about 4 to about 12 fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0351] Embodiment C-7: In one embodiment of category C, transduction is in the range of about 8 to about 12 fold better than virus particles containing a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0352] Category D (Trabecular Meshwork Transduction): In some aspects of the disclosure, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased trabecular meshwork transduction as compared to viral particles having a capsid polypeptide of SEQ ID NO: 60. In some embodiments, the increased trabecular meshwork transduction is as defined as any one of embodiments D-1 to D-4.

[0353] Embodiment D-1: In one embodiment of category D, transduction is about (or at least about) 1.4 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0354] Embodiment D-2: In one embodiment of category D, transduction is about (or at least about) 1.5 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0355] Embodiment D-3: In one embodiment of category D, transduction is about (or at least about) twice as good as that of a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0356] Embodiment D-4: In one embodiment of category D, transduction is about (or at least about) 2.7 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0357] In some embodiments, improved transduction is within a range bounded by any two values ​​shown in embodiments D-1 to D-4. Exemplary ranges are shown in embodiments D-5 to D-6 below.

[0358] Embodiment D-5: In one embodiment of category D, transduction is in the range of about 1.4 to about 2.7 fold better than virus particles containing a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0359] Embodiment D-6: In one embodiment of Category D, transduction is in the range of about 1.5 to about 2.7 fold better than virus particles containing a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0360] Category E (choroidal transduction): In some aspects of the present disclosure, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased choroidal transduction as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased choroidal transduction is as defined as any one of embodiments E-1 to E-3.

[0361] Embodiment E-1: In one embodiment of Category E, transduction is about (or at least about) 1.5-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0362] Embodiment E-2: In one embodiment of Category E, transduction is about (or at least about) 2-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0363] Embodiment E-3: In one embodiment of Category E, the improved transduction is in the range of about 1.5 to about 2 fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0364] Category F (Non-macular transduction): In some aspects of the disclosure, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased non-macular transduction as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased non-macular transduction is as defined as any one of embodiments F-1 to F-6.

[0365] Embodiment F-1: In one embodiment of category F, transduction is about (or at least about) 2-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0366] Embodiment F-2: In one embodiment of category F, transduction is about (or at least about) 4 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0367] Embodiment F-3: In one embodiment of category F, transduction is about (or at least about) 8 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0368] Embodiment F-4: In one embodiment of category F, transduction is about (or at least about) 15 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0369] Embodiment F-5: In one embodiment of category F, transduction is about (or at least about) 30 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0370] Embodiment F-6: In one embodiment of category F, transduction is about (or at least about) 50 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0371] In some embodiments, the improved transduction is within a range bounded by any two values ​​shown in embodiments F-1 through F-6. Exemplary ranges are shown in embodiments F-7 through F-9 below.

[0372] Embodiment F-7: In one embodiment of category F, transduction is in the range of about 2 to about 50 fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0373] Embodiment F-8: In one embodiment of category F, transduction is in the range of about 4 to about 50 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0374] Embodiment F-9: In one embodiment of category F, transduction is in the range of about 15 to about 50 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0375] Category G (Virus Production): In some aspects of the disclosure, virus particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased virus production as compared to virus particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased virus production is as defined as any one of embodiments G-1 to G-8.

[0376] Embodiment G-1: In one embodiment of category G, virus production is about (or at least about) 1.2-fold better compared to virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0377] Embodiment G-2: In one embodiment of category G, virus production is about (or at least about) 1.4-fold better compared to virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0378] Embodiment G-3: In one embodiment of category G, virus production is about (or at least about) 1.6-fold better than virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO:1.

[0379] Embodiment G-4: In one embodiment of category G, virus production is about (or at least about) 2-fold better compared to virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0380] Embodiment G-5: In one embodiment of category G, virus production is about (or at least about) 2.5 times better compared to virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0381] Embodiment G-6: In one embodiment of category G, virus production is about (or at least about) three times better than virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0382] Embodiment G-7: In one embodiment of category G, virus production is about (or at least about) 4 times better compared to virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0383] Embodiment G-8: In one embodiment of category G, virus production is about (or at least about) 6-fold better compared to virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0384] In some embodiments, the improved virus production is within a range bounded by any two values ​​shown in embodiments G-1 through G-8. Exemplary ranges are shown in embodiments G-9 through G-13 below.

[0385] Embodiment G-9: In one embodiment of category G, virus production is in the range of about 1.2 to about 6 fold better than virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0386] Embodiment G-10: In one embodiment of category G, virus production is in the range of about 1.2 to about 4 times better than virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0387] Embodiment G-11: In one embodiment of category G, virus production is in the range of about 1.4 to about 4 times better than virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0388] Embodiment G-12: In one embodiment of category G, virus production is in the range of about 1.6 to about 4 times better than virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0389] Embodiment G-13: In one embodiment of category G, virus production is in the range of about 2 to about 4 fold better than virus particles comprising a variant capsid polypeptide having a reference sequence, e.g., a wild-type capsid protein, e.g., a capsid polypeptide of SEQ ID NO: 1.

[0390] Category H (Non-macular 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 non-macular biodistribution as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased non-macular biodistribution is as defined as any one of embodiments H-1 through H-3.

[0391] Embodiment H-1: In one embodiment of category H, the biodistribution is about (or at least about) 2-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0392] Embodiment H-2: In one embodiment of category H, the biodistribution is about (or at least about) 4-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0393] Embodiment H-3: In one embodiment of category H, the biodistribution is about (or at least about) 6-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0394] In some embodiments, the improved biodistribution is within a range bounded by any two values ​​shown in embodiments H-1 through H-3. Exemplary ranges are shown in embodiments H-4 through H-5 below.

[0395] Embodiment H-4: In one embodiment of category H, the improved biodistribution is in the range of about 2 to about 6 fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0396] Embodiment H-5: In one embodiment of category H, the improved biodistribution is in the range of about 2 to about 4 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0397] Category I (Trabecular Meshwork 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 meshwork biodistribution as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased trabecular meshwork biodistribution is as defined as any one of embodiments I-1 to I-3.

[0398] Embodiment I-1: In one embodiment of Category I, the biodistribution is about (or at least about) 1.1-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0399] In one embodiment of embodiment I-2: Category H, the biodistribution is about (or at least about) 2-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0400] In one embodiment of embodiment I-3: Category H, the improved biodistribution is in the range of about 1.1 to about 2 fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0401] Category J (Neural Retina Biodistribution): In some aspects of the disclosure, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased neural retina biodistribution as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased neural retina biodistribution is as defined as any one of embodiments J-1 to J-4.

[0402] Embodiment J-1: In one category J embodiment, the biodistribution is about (or at least about) 2-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0403] Embodiment J-2: In one category J embodiment, the biodistribution is about (or at least about) 5-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0404] Embodiment J-3: In one category J embodiment, the biodistribution is about (or at least about) 10 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0405] Embodiment J-4: In one category J embodiment, the biodistribution is about (or at least about) 20 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0406] In some embodiments, the improved biodistribution is within a range bounded by any two values ​​shown in embodiments J-1 through J-4. Exemplary ranges are shown in embodiments J-5 through J-6 below.

[0407] Embodiment J-5: In one embodiment of Category J, the improved biodistribution is in the range of about 2 to about 20 times better compared to a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0408] Embodiment J-6: In one embodiment of Category J, the improved biodistribution is in the range of about 5 to about 20 times better compared to a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0409] Category K (Macular 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 macular biodistribution as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, the increased macular biodistribution is as defined as any one of embodiments K-1 through K-7.

[0410] Embodiment K-1: In one embodiment of category K, the biodistribution is about (or at least about) 3-fold better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0411] Embodiment K-2: In one embodiment of category K, the biodistribution is about (or at least about) 10 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0412] Embodiment K-3: In one embodiment of category K, the biodistribution is about (or at least about) 30 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0413] Embodiment K-4: In one embodiment of category K, the biodistribution is about (or at least about) 60 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0414] Embodiment K-5: In one embodiment of category K, the biodistribution is about (or at least about) 80 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0415] Embodiment K-6: In one embodiment of category K, the biodistribution is about (or at least about) 100 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0416] Embodiment K-7: In one embodiment of category K, the biodistribution is about (or at least about) 180 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO:1.

[0417] In some embodiments, the improved biodistribution is within a range bounded by any two values ​​shown in embodiments K-1 through K-7. Exemplary ranges are shown in embodiments K-8 through K-12 below.

[0418] Embodiment K-8: In one embodiment of category K, the biodistribution is in the range of about 3 to about 180 times better than a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0419] Embodiment K-9: In one embodiment of category K, the biodistribution is in the range of about 10 to about 180 times better than a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0420] Embodiment K-10: In one embodiment of category K, the biodistribution is in the range of about 30 to about 180 times better than a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0421] Embodiment K-11: In one embodiment of category K, the biodistribution is in the range of about 60 to about 180 times better than a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0422] Embodiment K-12: In one embodiment of category K, the biodistribution is in the range of about 80 to about 180 times better than a virus particle comprising a variant capsid polypeptide having a reference sequence, e.g., having a wild-type capsid protein, e.g., having a capsid polypeptide of SEQ ID NO: 1.

[0423] Category L (Trabecular Biodistribution): In some aspects of the 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 as any one of embodiments L-1 through L-4.

[0424] Embodiment L-1: In one embodiment of category L, transduction is about (or at least about) 1.5 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0425] Embodiment L-2: In one embodiment of category L, transduction is about (or at least about) twice as good as that of a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0426] Embodiment L-3: In one embodiment of category L, transduction is about (or at least about) 2.3 times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0427] Embodiment L-4: In one embodiment of category L, transduction is about (or at least about) three times better than a viral particle comprising a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0428] In some embodiments, the improved transduction is within a range bounded by any two values ​​shown in embodiments L-1 to L-4. Exemplary ranges are shown in embodiments L-5 to L-6 below.

[0429] Embodiment L-5: In one embodiment of category L, transduction is in the range of about 1.5 to about 3 times better than virus particles containing a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0430] Embodiment L-6: In one embodiment of category L, transduction is in the range of about 2 to about 3 times better than virus particles containing a variant capsid polypeptide having a reference sequence, e.g., a capsid polypeptide of SEQ ID NO: 60.

[0431] According to some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction of one or more cell types compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, the cell type(s) are ocular cell types found in retinal tissue. In some embodiments, the cell type(s) are ocular cell types found in trabecular meshwork tissue. Exemplary cell types include axonal cells, biopolar cells, cones, horizontal cells, microglia, Müller glia, retinal ganglion cells, rods, corneal epithelium, ciliary muscle, melanocytes, Schwann cells, beam cells, juxta-Schlemm's canal tissue (JCT), fibroblasts, and pericytes.

[0432] In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction of retinal ganglion cells compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction of axonal cells compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction of bipolar cells compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction of cones compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction of Müller glia compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction of rods compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction of beam cells compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction of ciliary muscles compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1).In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, show increased transduction of corneal epithelium compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, show increased transduction of juxta-Schlemm's canal tissue (JCT) compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, show increased transduction of melanocytes compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, show increased transduction of Schwann cells compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit reduced transduction of the ciliary muscle compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO:1).

[0433] In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction in one cell type over another cell type, as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction in retinal ganglion cells over axonal cells, biopolar cells, cones, Muller glia, or rods, as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction in axonal cells over biopolar cells, cones, Muller glia, or rods, as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction in cones over biopolar cells, axonal cells, Muller glia, or rods, as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1). In some embodiments, viral particles comprising a variant capsid polypeptide, e.g., a variant capsid polypeptide described herein, exhibit increased transduction in juxta-Schlemm's canal tissue (JCT) over beam cells, ciliary muscle, corneal epithelium, melanocytes, or Schwann cells, as compared to viral particles having a wild-type capsid polypeptide (SEQ ID NO: 1).

[0434] Methods of Making the Compositions Described Herein The present disclosure is directed, in part, to methods of making a capsid polypeptide or virus particle described herein, such as, but not limited to, a depend parvovirus particle, such as, a depend parvovirus particle described herein. In some embodiments, the method of making a depend parvovirus particle includes providing a cell, cell-free system, or other translation system that includes a variant capsid polypeptide provided herein, or a nucleic acid described herein that encodes a polypeptide provided herein (e.g., a variant capsid polypeptide), and culturing the cell, cell-free system, or other translation system under conditions suitable for the production of depend parvovirus particles, thereby making the depend parvovirus particle.

[0435] In some embodiments, providing a cell comprising a nucleic acid as described herein includes introducing the nucleic acid into a cell, e.g., transfecting or transforming a cell with the nucleic acid. The nucleic acid of the present disclosure may be located as part of any genetic element (vector) that can be delivered to a host cell, e.g., naked DNA, plasmid, phage, transposon, cosmid, episome, protein in a non-viral delivery vehicle (e.g., lipid-based carrier), virus that transfers the sequence carried thereon, and the like. Such vectors may be delivered by any suitable method, including transfection, liposome delivery, electroporation, membrane fusion techniques, viral infection, high-speed DNA-coated pellets, and protoplast fusion. Those skilled in the art have the knowledge and skill in nucleic acid manipulation to construct any embodiment of the present invention, including genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0436] In some embodiments, the vectors of the disclosure include a viral particle, such as, but not limited to, a sequence encoding a depend parvovirus variant capsid polypeptide as provided herein, or a fragment thereof. In some embodiments, the vectors of the disclosure include a viral particle, such as, but not limited to, a sequence encoding a depend parvovirus rep protein, or a fragment thereof. In some embodiments, such vectors may include sequences encoding both a depend parvovirus cap (e.g., a variant capsid polypeptide described herein) and a rep protein. In vectors in which both an AAV rep and cap are provided, the depend parvovirus rep sequence and the depend parvovirus cap sequence may both be of the same depend parvovirus species or serotype origin (e.g., AAV2). Alternatively, the disclosure also provides vectors in which the rep sequence is from a different depend parvovirus species or serotype from which the cap sequence is derived. In some embodiments, the rep sequence and the cap sequence are expressed from separate sources (e.g., separate vectors, or the host cell genome and the vector). In some embodiments, the rep sequence is fused in frame to a cap sequence of a different depend parvovirus species or serotype to form a chimeric depend parvovirus vector. In some embodiments, the vectors of the invention further contain a payload, e.g., a minigene comprising a selected transgene (e.g., a payload described herein) flanked by the depend parvovirus 5' ITR and the depend parvovirus 3' ITR.

[0437] The vectors, e.g., plasmids, described herein are useful for a variety of purposes, but are particularly well suited for use in generating recombinant viral particles, such as, but not limited to, depend parvovirus particles that contain depend parvovirus sequences or fragments thereof, and in some embodiments, a payload.

[0438] In some embodiments, the present disclosure provides a method of making a viral particle, such as, but not limited to, a depend parvovirus particle (e.g., a depend parvovirus B particle, e.g., an AAV2 particle, or a particle comprising a variant capsid polypeptide described herein), or a portion thereof. In some embodiments, the method includes culturing a host cell that includes a nucleic acid sequence encoding a depend parvovirus variant capsid polypeptide provided herein, or a fragment thereof; a functional rep gene; a payload (e.g., as described herein), e.g., a minigene comprising a depend parvovirus inverted terminal repeat (ITR) and a transgene, optionally under the control of a regulatory element such as a promoter; and sufficient helper functions to facilitate packaging of the payload, e.g., the minigene, into a depend parvovirus capsid. Components that need to be cultured in the host cell to package the payload, e.g., the minigene, into a depend parvovirus capsid can be provided to the host cell in trans. In some embodiments, any one or more of the required components (e.g., payload (e.g., minigene), rep sequences, cap sequences, and / or helper functions) may be provided by a host cell that has been engineered to stably contain one or more of the required components using methods known to those of skill in the art. In some embodiments, a host cell that has been engineered to stably contain a required component(s) contains it under the control of an inducible promoter. In some embodiments, a required component may be under the control of a constitutive promoter. Examples of suitable inducible and constitutive promoters are provided herein, and further examples will be known to those of skill in the art. In some embodiments, a selected host cell that has been engineered to stably contain one or more components may contain a component under the control of a constitutive promoter and another component under the control of one or more inducible promoters. For example, a host cell that has been engineered to stably contain a required component may be generated from a 293 cell (e.g., containing helper functions under the control of a constitutive promoter) that contains rep and / or cap proteins under the control of one or more inducible promoters.

[0439] The payload (e.g., minigene), rep sequences, cap sequences, and helper functions required for the generation of viral particles, including but not limited to, dependoparvoviral particles of the present disclosure, can be delivered to the host cell for packaging in the form of any genetic element that transfers the sequences carried thereon (e.g., in a vector or combination of vectors). The genetic element can be delivered by any suitable method, including those described herein. The methods used to construct the genetic elements, vectors, and other nucleic acids of the present disclosure are known to those of skill in the art and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for generating rAAV virions are well known, and the selection of a suitable method is not a limitation of the present invention. See, e.g., K. Fisher et al, J...

Claims

1. A variant capsid polypeptide comprising an amino acid sequence that is at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of VP1, VP2, or VP3 of SEQ ID NO: 1, The variant capsid polypeptide comprises at least one mutation at positions 550 and 597, or between positions 550 and 597.

2. The variant capsid polypeptide according to claim 1, wherein the at least one mutation is a substitution at position 550, 559, 561, 586, 587, 592, 593, or 597.

3. The variant capsid polypeptide is (a) Variation at position 587, and insertion between positions 586 and 587; (b) Insertion between positions 586 and 587; (c) Insertion between positions 584 and 585; (d) Insertion between positions 587 and 588; (e) Variations at positions 587 and 593, and insertions between positions 586 and 587; (f) Variations at positions 586 and 587, and insertions between positions 584 and 585; (g) Variations at positions 592 and 597, and insertions between positions 587 and 588; (h) Variations at positions 561, 587 and 597, and insertions between positions 586 and 587; (i) Variations at positions 550, 586 and 587, and insertions between positions 584 and 585; (j) Variations at positions 559 and 587, and insertions between positions 586 and 587; (k) Insertion, for example, the insertion of one or more amino acids, for example, one or two amino acids between positions 584 and 585, between 586 and 587, and between 587 and 588; (l) Insertions, for example, six or fewer amino acids, for example, six amino acids between positions 586 and 587, wherein the insertion sequence contains threonine-arginine-proline at its C-terminus; and substitutions at position 587, for example, alanine at position 587; (m) Insertions, for example, seven or more amino acids, for example, insertions of 10 or 11 amino acids between positions 586 and 587, wherein the insertion sequence contains threonine-arginine-proline at its C-terminus; and substitutions at position 587, for example, alanine at position 587; (n) A mutation comprising the sequence threonine-alanine-arginine-proline-alanine, wherein optionally the sequence is located at the C-terminus relative to position 584 and at the N-terminus relative to position 590, and optionally the sequence threonine-alanine-arginine-proline-alanine comprises insertion and substitution mutations, or (o) The variant capsid polypeptide according to claim 1, comprising any combination of the above.

4. The variant capsid polypeptide is (a) N587A substitution and insertion between positions 586 and 587, wherein the insertion comprises the amino acid sequence TGDTRP (SEQ ID NO: 51), or at least three, at least four, or at least five amino acid fragments thereof; (b) An insertion between positions 586 and 587, wherein the insertion comprises the amino acid sequence LALGEEQTRPA (SEQ ID NO: 44), or at least 5, at least 6, at least 7, at least 8, or at least 9 amino acid fragments thereof, (c) an insertion between positions 587 and 588, wherein the insertion comprises the amino acid sequence LAIEQTRPA (SEQ ID NO: 45), or at least 5, at least 6, at least 7, or at least 8 amino acid fragments thereof; (d) N587A substitution and insertion between positions 586 and 587, wherein the insertion comprises the amino acid sequence LALAEITRP (SEQ ID NO: 46), or at least 5, at least 6, at least 7, or at least 8 amino acid fragments thereof; (e) N587A substitution and insertion between positions 586 and 587, wherein the insertion comprises the amino acid sequence LKNAETARP (SEQ ID NO: 47), or at least 5, at least 6, at least 7, or at least 8 amino acid fragments thereof; (f) N587A and A593T substitutions, and insertions between positions 586 and 587, wherein the insertion comprises the amino acid sequence LNLAIEQTRP (SEQ ID NO: 48), or at least 5, at least 6, at least 7, or at least 8 amino acid fragments thereof; (g) N587A substitution and insertion between positions 586 and 587, wherein the insertion comprises the amino acid sequence MLNEQTRP (SEQ ID NO: 49), or at least four, at least five, at least six, or at least seven amino acid fragments thereof; (h) G586P and N587A substitutions, and insertions between positions 584 and 585, wherein the insertion comprises the amino acid sequence RSGNRADSETA (SEQ ID NO: 50), or at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid fragments thereof; (i) an insertion between positions 587 and 588, wherein the insertion comprises the amino acid sequence LQGETIRPA (SEQ ID NO: 52), or at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid fragments thereof; (j) N587A substitution and insertion between positions 586 and 587, wherein the insertion comprises the amino acid sequence QNLANPETRP (SEQ ID NO: 53), or at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid fragments thereof; (k) T592A and T597W substitutions, and insertions between positions 587 and 588, wherein the insertion comprises the amino acid sequence RAPQETTRPA (SEQ ID NO: 54), or at least 5, at least 6, at least 7, at least 8, or at least 9 amino acid fragments thereof; (l) N587A substitution and insertion between positions 586 and 587, wherein the insertion comprises the amino acid sequence ANLTTTRP (SEQ ID NO: 55), or at least four, at least five, at least six, or at least seven amino acid fragments thereof; (m) N587A substitution and insertion between positions 586 and 587, wherein the insertion comprises the amino acid sequence ALLAGEQTRP (SEQ ID NO: 56), or at least 5, at least 6, at least 7, at least 8, or at least 9 amino acid fragments thereof, (n) D561C, N587A and T597N substitutions, and insertions between positions 586 and 587, wherein the insertion comprises the amino acid sequence GLRAEQTRP (SEQ ID NO: 57), or at least 5, at least 6, at least 7, or at least 8 amino acid fragments thereof. (o) T550N, G586P, and N587A substitutions, and insertions between positions 584 and 585, wherein the insertion comprises the amino acid sequence RARLDETA (SEQ ID NO: 58), or at least 4, at least 5, at least 6, or at least 7 amino acid fragments thereof, or (p) Substitutions of I559L and N587A, and an insertion between positions 586 and 587, wherein the insertion comprises the amino acid sequence TNLARGETARP (SEQ ID NO: 59), or at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 amino acid fragments thereof, according to claim 1.

5. A variant capsid polypeptide, (a) A polypeptide comprising any one of the amino acid sequences 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) Any one of the VP2 or VP3 amino acid sequences from 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, (c) A polypeptide comprising an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of VP1, VP2, or VP3 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, wherein the amino acid sequence, compared to the amino acid sequence of SEQ ID NO: 1, comprises at least one (e.g., one, two, three or more, e.g., all) of the mutations present in one of the amino acid sequences of SEQ ID NO: 12 to SEQ ID NO: 27; (d) A polypeptide having at least one amino acid mutation relative to the polypeptide of (a) or (b), wherein the polypeptide has 20 or fewer, 19 or fewer, 18 or fewer, 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 3 or fewer, or 2 or fewer amino acid mutations, wherein the polypeptide, compared to the amino acid sequence of SEQ ID NO: 1, contains at least one mutation (for example, 1, 2, 3 or more, for example, all) present in any one of the amino acid sequences of SEQ ID NO: 12 to SEQ ID NO: 27; (e) an amino acid sequence having less than 95% amino acid sequence identity with one of the amino acid sequences of SEQ ID NOs: 12 to 27; and having at least 80% of the mutations present in one of the amino acid sequences of SEQ ID NOs: 12 to 27 compared to the amino acid sequence of SEQ ID NOs: 1; or (f) The variant capsid polypeptide comprising an amino acid sequence having 95% or more amino acid sequence identity with respect to one of the amino acid sequences of SEQ ID NOs: 12 to 27; and having less than 80% of the mutations present in one of the amino acid sequences of SEQ ID NOs: 12 to 27 compared to the amino acid sequence of SEQ ID NO:

1.

6. The variant capsid polypeptide according to claim 1, wherein the at least one mutation is an insertion between positions 584 and 585, between 585 and 586, or between 586 and 587.

7. The variant capsid polypeptide according to claim 6, wherein the insertion is located between positions 586 and 587.

8. The variant capsid polypeptide according to claim 7, wherein the insertion comprises or consists of the amino acid sequence TGDTRP (SEQ ID NO: 51).

9. The variant capsid polypeptide according to claim 8, further comprising an amino acid substitution at position 550, 559, 561, 586, 587, 592, 593, or 597.

10. The variant capsid polypeptide according to claim 9, wherein the substitution is located at position 587.

11. The variant capsid polypeptide according to claim 10, wherein the substitution is an N587A substitution.

12. A variant capsid polypeptide containing the amino acid sequence of SEQ ID NO:

19.

13. A nucleic acid molecule comprising a sequence encoding a variant capsid polypeptide according to any one of claims 1 to 12, wherein the nucleic acid molecule comprises one or more regulatory elements operably linked to the sequence encoding the variant capsid polypeptide.

14. A nucleic acid molecule according to claim 13, comprising the nucleic acid sequence of sequence number 35, 28, 29, 30, 31, 32, 33, 34, 36, 37, 38, 39, 40, 41, 42, or 43, or a fragment thereof, or variants thereof having at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% sequence identity thereto.

15. A virus particle comprising a variant capsid polypeptide according to any one of claims 1 to 12, wherein the virus particle optionally further comprises a nucleic acid containing a heterologous transgene and one or more regulatory elements.

16. The virus particle according to claim 15, wherein the virus particle is a dependent parvovirus particle.

17. The virus particle according to claim 15, wherein, when measured in mice or non-human primates (NHPs), the virus particle exhibits increased ocular transduction compared to wild-type AAV2 (for example, a virus particle comprising a capsid polypeptide of the amino acid sequence of SEQ ID NO: 1 or encoded by the nucleic acid sequence of SEQ ID NO: 2).

18. A method for generating viral particles containing a variant AAV2 capsid polypeptide, the method comprising introducing a nucleic acid molecule according to claim 13 into cells (e.g., HEK293 cells) and recovering the viral particles therefrom.

19. An in vitro method for delivering a payload to a cell, comprising contacting the cell with a dependent parvovirus particle and payload containing a variant capsid polypeptide according to any one of claims 1 to 12, wherein The aforementioned cells are eye cells, and The method wherein the eye cells are located within the retina, macula, or trabecular network.

20. A composition for use in a method of delivering a payload to a target, wherein the method comprises administering the composition to the target, the composition comprising the virus particles and the payload described in claim 15, and the composition delivers the payload to the eye.

21. The composition according to claim 20, wherein the composition delivers the payload to the eye with increased transduction in one or more regions of the eye compared to a virus particle containing 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 network, or any combination thereof.

22. The composition according to claim 20, wherein the administration to the subject is via intravitreous injection or intracavitary injection.

23. Compared to virus particles containing the capsid polypeptide of SEQ ID NO: 1, the virus particles deliver the payload to the eye with increased transduction in one or more regions of the eye, and without increased in vivo distribution in one or more regions of the eye, compared to virus particles containing the capsid polypeptide of SEQ ID NO:

1. The increase in transduction is at least 2 times, 4 times, 8 times, 16 times, 32 times, 64 times, 100 times, 128 times, or 1000 times compared to virus particles containing the capsid polypeptide of Sequence ID No. 1, and The increase in the aforementioned trait introduction, (a) Regarding macular tissue, is it specific to non-macular retinal tissue? (b) For non-macular retinal tissue, is it specific to macular tissue? (c) Is the trabecular network tissue specific to macular tissue? (d) Is the trabecular network tissue specific to non-macular retinal tissue? (e) Is the trabecular network tissue specific to macular tissue and non-macular retinal tissue? (f) For macular tissue and non-macular retinal tissue, is it specific to trabecular reticular tissue? (g) Is it specific to the trabecular reticular tissue in relation to macular tissue? (h) For non-macular retinal tissue, is it specific to trabecular retinal tissue, or (i) The virus particle according to claim 15, which is specific to trabecular retinal tissue, macular tissue, and non-macular retinal tissue.

24. A composition for use in a method of treating a disease or condition in a subject, The method comprises administering dependent parvovirus particles to the subject in an amount effective for treating the disease or condition, The composition wherein the dependent parvovirus particles are virus particles comprising a variant capsid polypeptide according to any one of claims 1 to 12.

25. A cell, a cell-free system, or another translation system comprising the nucleic acid molecule described in claim 13.

26. A method for producing dependent parvovirus particles, (a) To provide a cell, cell-free system, or other translation system containing the nucleic acid molecule described in claim 13, (b) Culturing the cells, cell-free system, or other translation system under conditions suitable for the generation of dependent parvovirus particles, This is used to produce the dependent parvovirus particles; The cell, cell-free system, or other translation system contains a second nucleic acid molecule, and the second nucleic acid molecule is packaged in the dependent parvovirus particle. The second nucleic acid molecule comprises a payload (for example, a heterogeneous nucleic acid sequence encoding a therapeutic product), The nucleic acid molecule described in claim 13 mediates the generation of dependent parvovirus particles that do not contain the nucleic acid molecule described in claim 13, and The method according to claim 13, wherein the nucleic acid molecule mediates the generation of dependent parvovirus particles at a level at least 10%, at least 20%, at least 50%, at least 100%, or at least 200% higher than the generation level mediated by the nucleic acid of SEQ ID NO:

2.

27. A pharmaceutical composition comprising the virus particles described in claim 15.

28. A variant capsid polypeptide according to any one of claims 1 to 12, for use in the treatment of therapies.

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