Adeno-associated viral vectors for delivery of nucleic acids to retinal cells

Abstract

This document relates to AAV vectors (e.g., AAV2 vectors), e.g., AAV vectors (e.g., AAV2 vectors) that contain an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A, such AAV capsid polypeptides, nucleic acid molecules encoding such vectors, nucleic acid molecules encoding such AAV capsid polypeptides, host cells containing and / or expressing such nucleic acid molecules, and methods and materials for making or using such vectors and / or AAV capsid polypeptides.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Patent Application No. 63 / 325,541 filed on March 30, 2022, U.S. Patent Application No. 63 / 325,543 filed on March 30, 2022, U.S. Patent Application No. 63 / 325,553 filed on March 30, 2022, U.S. Patent Application No. 63 / 325,562 filed on March 30, 2022, U.S. Patent Application No. 63 / 325,540 filed on March 30, 2022, U.S. Patent Application No. 63 / 325,542 filed on March 30, 2022, U.S. Patent Application No. This application claims the benefit of U.S. Patent Application No. 63 / 325,544, U.S. Patent Application No. 63 / 325,548 filed on March 30, 2022, U.S. Patent Application No. 63 / 325,550 filed on March 30, 2022, U.S. Patent Application No. 63 / 325,551 filed on March 30, 2022, U.S. Patent Application No. 63 / 325,555 filed on March 30, 2022, U.S. Patent Application No. 63 / 325,558 filed on March 30, 2022, and U.S. Patent Application No. 63 / 325,559 filed on March 30, 2022. The disclosures of the prior applications are deemed to be part of (and are incorporated by reference into) the disclosure of this application.

[0002] Federal Funding Statement This invention was made with Government support under award MH120094 from the National Institutes of Health. The Government has certain rights in this invention.

[0003] This document relates to adeno-associated virus (AAV) vectors. For example, this document describes AAV vectors that have the following features: (a) the ability to deliver nucleic acid to foveal cones; (b) increased efficiency of delivering nucleic acid to retinal cells; (c) the ability to deliver nucleic acid to retinal cells and drive high expression levels of the nucleic acid in retinal cells; (d) the ability to deliver nucleic acid to retinal cells across retinal regions (e.g., across at least two retinal regions); (e) the ability to deliver nucleic acid to retinal cells in the parafoveal region of the eye; (f) the ability to deliver nucleic acid to two or more different retinal cell types within the eye; (g) the ability to deliver nucleic acid to retinal pigment epithelial (RPE) cells; (h) increased efficiency of delivering nucleic acid to photoreceptor cells of the retina, (i) increased efficiency of delivering nucleic acid to retinal ganglion cells of the retina, (j) increased efficiency of delivering nucleic acid to bipolar cells of the retina, (k) increased efficiency of delivering nucleic acid to off retinal ganglion cells, and / or (l) increased packaging efficiency, and methods and materials for making and using AAV vectors (e.g., AAV2 vectors) having the ability to deliver nucleic acid to cells (e.g., retinal cells). [Background technology]

[0004] Viral vectors, such as AAV vectors, are efficient vehicles for in vivo nucleic acid delivery, and their use in the clinic is expanding. Improved AAV vector and AAV manufacturing techniques for generating effective AAV vector preparations should further expand the use of AAV vectors in the laboratory and clinic. Summary of the Invention

[0005] This document provides AAV vectors (e.g., AAV2 vectors). For example, this document provides AAV vectors (e.g., AAV2 vectors) that contain a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vectors described herein (e.g., AAV2 vectors) that contain a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) in vivo and delivering an exogenous nucleic acid to the infected retinal cells, thereby allowing the infected retinal cells to express the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) capable of delivering a nucleic acid to a retinal cell.

[0006] As described herein, the AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) in vivo and delivering exogenous nucleic acid to the infected retinal cells, such that the infected retinal cells express the exogenous nucleic acid. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 2% higher, at least 2.5% higher, at least 5% higher, at least 7.5% higher, at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in retinal cells of a control mammal (e.g., a control human or control non-human primate). In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver nucleic acid to retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells).

[0007] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector). For example, this document provides an AAV vector (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vector described herein (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can infect foveal cones in vivo and deliver exogenous nucleic acid to the infected foveal cones, such that the infected foveal cones are capable of expressing the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) capable of delivering nucleic acid to foveal cones.

[0008] As described herein, AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting foveal cones in vivo and delivering an exogenous nucleic acid to the infected foveal cones such that the infected foveal cones express the exogenous nucleic acid. In some cases, AAV vectors provided herein (e.g., AAV2 vectors) can be capable of infecting and driving mRNA expression of an exogenous nucleic acid in cone cells present in the fovea of ​​a mammal (e.g., a human or non-human primate). In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in the foveal cones of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in the foveal cones of a control mammal (e.g., a control human or control non-human primate). In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver nucleic acid to the foveal cones.

[0009] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vectors described herein (e.g., AAV2 vectors) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) in vivo and delivering an exogenous nucleic acid to the infected retinal cells such that the infected retinal cells express the exogenous nucleic acid at high levels. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) capable of delivering a nucleic acid to a retinal cell and driving high expression levels of the nucleic acid in the retinal cell.

[0010] As described herein, AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) in vivo and delivering an exogenous nucleic acid to the infected retinal cells such that the infected retinal cells express the exogenous nucleic acid at high levels. In some cases, AAV vectors provided herein (e.g., AAV2 vectors) can be capable of infecting and driving mRNA expression of an exogenous nucleic acid in at least 2% (e.g., at least 2.5%, at least 5%, at least 7.5%, at least 10%, or at least 25%) of retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) in the eye of a mammal (e.g., a human or non-human primate). In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 2% higher, at least 2.5% higher, at least 5% higher, at least 7.5% higher, at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in retinal cells of a control mammal (e.g., a control human or control non-human primate).In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver nucleic acid to retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells).

[0011] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vectors described herein (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can in vivo infect retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) across a retinal region (e.g., across at least two retinal regions) and deliver an exogenous nucleic acid to the infected retinal cells, such that the infected retinal cells express the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) capable of delivering a nucleic acid to retinal cells across a retinal region and driving expression of the delivered nucleic acid in the retinal cells. For example, an AAV vector described herein (e.g., an AAV2 vector) can deliver nucleic acid to at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal cells in the foveal region, at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal cells in the parafoveal region, at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal cells in the vascular arcade region, and / or at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal cells in the peripheral region.

[0012] As described herein, the AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) across a retinal region (e.g., across at least two retinal regions) in vivo and delivering exogenous nucleic acid to the infected retinal cells, such that the infected retinal cells are capable of expressing the exogenous nucleic acid. In some cases, an AAV vector provided herein (e.g., an AAV2 vector) can be capable of infecting and driving mRNA expression of an exogenous nucleic acid in at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal cells in the foveal region, at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal cells in the parafoveal region, at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal cells in the vascular arcade region, and / or at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal cells in the peripheral region of a mammalian (e.g., human or non-human primate) eye. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in retinal cells in the foveal region, parafoveal region, vascular arcade region, and / or peripheral region of the eye of a mammal (e.g., a human or non-human primate) that is higher than the level of mRNA expression of the exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in retinal cells in these regions in a control mammal (e.g., a control human or control non-human primate).In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver nucleic acid to retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells).

[0013] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vector described herein (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can in vivo infect retinal cells in the parafoveal region of the eye and deliver an exogenous nucleic acid to the infected retinal cells in the parafoveal region, such that the infected retinal cells express the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) capable of delivering nucleic acid to retinal cells in the parafoveal region of the eye.

[0014] As described herein, the AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting retinal cells in the parafoveal region of the eye in vivo and delivering an exogenous nucleic acid to the infected retinal cells such that the infected retinal cells express the exogenous nucleic acid. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of infecting and driving mRNA expression of an exogenous nucleic acid in at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal cells present in the parafoveal region of a mammalian (e.g., human or non-human primate) eye. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in retinal cells of the parafoveal region of the eye of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in retinal cells of the parafoveal region of the eye of a control mammal (e.g., a control human or control non-human primate).In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver nucleic acid to retinal cells (e.g., retinal ganglion cells, photoreceptor cells, and bipolar cells) in the parafoveal region of the eye.

[0015] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) that contains a capsid polypeptide that includes an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. The AAV vector described herein (e.g., an AAV2 vector) that contains a capsid polypeptide that includes an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting two or more (e.g., two or more, three or more, four or more, five or more, six or more, or seven or more) different retinal cell types in the eye in vivo and delivering an exogenous nucleic acid to the infected retinal cells such that the infected retinal cells express the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) that are capable of delivering a nucleic acid to two or more (e.g., two or more, three or more, four or more, five or more, six or more, or seven or more) different retinal cell types in the eye and driving expression of the delivered nucleic acid in these retinal cells. For example, an AAV vector described herein (e.g., an AAV2 vector) can deliver a nucleic acid to two, three, four, five, six, or seven of the following retinal cell types of the eye: retinal ganglion cells, axonal cells, horizontal cells, bipolar cells, Müller glial cells, photoreceptor cells, and retinal pigment epithelial (RPE) cells. In some cases, an AAV vector described herein (e.g., an AAV2 vector) can deliver a nucleic acid to at least some (e.g., at least 2%, at least 2.5%, at least 5%, at least 10%, or at least 25%) of the retinal ganglion cells, axonal cells, horizontal cells, bipolar cells, Müller glial cells, photoreceptor cells, and RPE cells of a mammalian (e.g., human or non-human primate) eye following intravitreal administration.

[0016] As described herein, the AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting two or more (e.g., two or more, three or more, four or more, five or more, six or more, or seven or more) different retinal cell types in the eye in vivo and delivering exogenous nucleic acid to the infected retinal cells, such that the infected retinal cells express the exogenous nucleic acid. In some cases, an AAV vector provided herein (e.g., an AAV2 vector) can, for example, after intravitreal administration, affect at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal ganglion cells, at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of axonal cells, at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of horizontal cells, at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of bipolar cells in a mammalian (e.g., human or non-human primate) eye. In one embodiment, the method comprises the steps of: infecting and driving mRNA expression of an exogenous nucleic acid in at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of human retinal cells; at least about 2% of human mitochondrial cells (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of human mitochondrial cells; at least about 2% of human mitochondrial cells (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of human mitochondrial cells; at least about 2% of human mitochondrial cells (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of human mitochondrial cells;In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in retinal ganglion cells, axonal cells, horizontal cells, bipolar cells, Muller glial cells, photoreceptor cells, and / or RPE cells of the eye of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in these retinal cells of a control mammal (e.g., a control human or control non-human primate). In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver a nucleic acid to two or more (e.g., two or more, three or more, four or more, five or more, six or more, or seven or more) different retinal cell types within the eye of a mammal (e.g., a human or non-human primate).

[0017] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) that contains a capsid polypeptide that includes an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vectors described herein (e.g., AAV2 vectors) that contain a capsid polypeptide that includes an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can infect RPE cells in vivo and deliver an exogenous nucleic acid to the infected RPE cells, such that the infected RPE cells express the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) that are capable of delivering a nucleic acid to RPE cells and driving expression of the delivered nucleic acid in the RPE cells. For example, an AAV vector described herein (e.g., an AAV2 vector) can deliver nucleic acid to at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of RPE retinal cells of a mammalian eye, e.g., after intravitreal administration.

[0018] As described herein, the AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting RPE cells in vivo and delivering an exogenous nucleic acid to the infected RPE cells such that the infected RPE cells express the exogenous nucleic acid. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of infecting and driving mRNA expression of an exogenous nucleic acid in at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of RPE cells in a mammalian (e.g., human or non-human primate) eye. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in an RPE cell of the eye of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in an RPE cell of a control mammal (e.g., a control human or control non-human primate). In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver a nucleic acid to an RPE cell.

[0019] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vectors described herein (e.g., AAV2 vectors) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can infect photoreceptor cells of the retina in vivo and deliver exogenous nucleic acid to the infected photoreceptor cells, such that the infected photoreceptor cells express the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) capable of delivering nucleic acid to photoreceptor cells of the retina.

[0020] As described herein, AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting photoreceptor cells of the retina in vivo and delivering an exogenous nucleic acid to the infected photoreceptor cells such that the infected photoreceptor cells express the exogenous nucleic acid. In some cases, AAV vectors provided herein (e.g., AAV2 vectors) can be capable of infecting and driving mRNA expression of an exogenous nucleic acid in at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of photoreceptor cells in a mammalian (e.g., human or non-human primate) eye. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in photoreceptor cells of the retina of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide composed of the amino acid sequence of SEQ ID NO:1 in photoreceptor cells of the retina of a control mammal (e.g., a control human or control non-human primate). In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver nucleic acid to photoreceptor cells of the retina.

[0021] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vectors described herein (e.g., AAV2 vectors) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting retinal ganglion cells in vivo and delivering an exogenous nucleic acid to the infected retinal ganglion cells, such that the infected retinal ganglion cells express the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) capable of delivering a nucleic acid to a retinal ganglion cell.

[0022] As described herein, the AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting retinal ganglion cells in vivo and delivering an exogenous nucleic acid to the infected retinal ganglion cells such that the infected retinal ganglion cells express the exogenous nucleic acid. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of infecting and driving mRNA expression of an exogenous nucleic acid in at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of retinal ganglion cells in a mammalian (e.g., human or non-human primate) eye. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in a retinal ganglion cell of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in a retinal ganglion cell of the eye of a control mammal (e.g., a control human or control non-human primate). In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver nucleic acid to photoreceptor cells of the retina.

[0023] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vector described herein (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting bipolar cells of the retina in vivo and delivering an exogenous nucleic acid to the infected bipolar cells, such that the infected retinal bipolar cells express the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) capable of delivering nucleic acid to bipolar cells of the retina.

[0024] As described herein, the AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting bipolar cells of the retina in vivo and delivering an exogenous nucleic acid to the infected bipolar cells such that the infected bipolar cells express the exogenous nucleic acid. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of infecting and driving mRNA expression of an exogenous nucleic acid in at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of the bipolar cells of the retina of a mammal (e.g., a human or non-human primate). In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in bipolar cells of the retina of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide composed of the amino acid sequence of SEQ ID NO:1 in bipolar cells of the retina of a control mammal (e.g., a control human or control non-human primate). In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver nucleic acid to photoreceptor cells of the retina.

[0025] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vector described herein (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can infect an OFF retinal ganglion cell in vivo and deliver an exogenous nucleic acid to the infected OFF retinal ganglion cell, such that the infected OFF retinal ganglion cell expresses the exogenous nucleic acid. This document also provides methods and materials for making and using an AAV vector (e.g., an AAV2 vector) capable of delivering a nucleic acid to an OFF retinal ganglion cell.

[0026] As described herein, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting an OFF retinal ganglion cell in vivo and delivering an exogenous nucleic acid to the infected OFF retinal ganglion cell such that the infected OFF retinal ganglion cell expresses the exogenous nucleic acid. In some cases, an AAV vector provided herein (e.g., an AAV2 vector) can be capable of infecting and driving mRNA expression of an exogenous nucleic acid in at least about 2% (e.g., at least about 2.5%, at least about 5%, at least about 7.5%, at least about 10%, or at least about 25%) of OFF retinal ganglion cells of a mammal (e.g., a human or non-human primate). In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of mRNA expression of an exogenous nucleic acid in an OFF retinal ganglion cell of a mammal (e.g., a human or non-human primate) that is higher (e.g., at least 10% higher, at least 25% higher, at least 50% higher, at least 75% higher, or at least 100% higher) than the level of mRNA expression of an exogenous nucleic acid driven by a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1 in an OFF retinal ganglion cell of a control mammal (e.g., a control human or control non-human primate). At least 50% higher, at least 75% higher, or at least 100% higher).In some cases, an AAV vector provided herein (e.g., an AAV2 vector) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be used in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)) or in place of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)) to deliver nucleic acid to photoreceptor cells of the retina.

[0027] In another aspect, this document provides an AAV vector (e.g., an AAV2 vector) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. The AAV vectors described herein (e.g., AAV2 vectors) containing a capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A can have increased packaging efficiency and the ability to infect cells (e.g., retinal cells) in vivo or in vitro and deliver an exogenous nucleic acid to the infected cell such that the infected RPE cell expresses the exogenous nucleic acid. This document also provides methods and materials for making and using AAV vectors (e.g., AAV2 vectors) having increased packaging efficiency and the ability to deliver a nucleic acid to cells (e.g., retinal cells) in vivo or in vitro and drive expression of the delivered nucleic acid in the cell. For example, the AAV vectors described herein (e.g., AAV2 vectors) can have packaging efficiency that is greater than the packaging efficiency of a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1.

[0028] As described herein, the AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can have increased packaging efficiency (e.g., a packaging efficiency greater than that of a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1), the ability to infect cells (e.g., retinal cells) in vivo or in vitro, and the ability to deliver the exogenous nucleic acid to the infected cell such that the infected cell expresses the exogenous nucleic acid. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can have a packaging efficiency that is greater (e.g., at least 10% greater, at least 25% greater, at least 50% greater, at least 75% greater, or at least 100% greater) than that of a corresponding AAV vector (e.g., a wild-type AAV2 vector) having an AAV capsid polypeptide consisting of the amino acid sequence of SEQ ID NO:1. In some cases, AAV vectors provided herein (e.g., AAV2 vectors) having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can have a packaging efficiency in place of the 7m8 AAV2 vector (Dalkara et al., Sci. Transl. Med., 5(189):189ra76 (2013) and Bennett et al., J. Struct. Biol., 209(2):107433 (2020)), or higher than the packaging efficiency of the K912 AAV2 vector (Ozturk et al., eLife, 10:e64175 (2021)).

[0029] In general, one embodiment of this document relates to an adeno-associated virus (AAV) vector (e.g., an AAV2 vector) that includes an AAV capsid polypeptide, where the capsid polypeptide comprises the amino acid sequence of any one of SEQ ID NOs:2-5. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO:10, or is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acid sequence of any one of SEQ ID NOs:2-5 is located between amino acid positions 587 and 588 of SEQ ID NO:1 (or an alternative sequence appropriate amino acid position). The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acid sequence of SEQ ID NO:5 is located between amino acid positions 587 and 588 (or an appropriate amino acid position in an alternative sequence) of SEQ ID NO:1. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acid sequence at positions 585-590 (or an appropriate amino acid position in an alternative sequence) of SEQ ID NO:1 is replaced with the amino acid sequence of any one of SEQ ID NOs:2-5. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or an alternative sequence which is the amino acid sequence set forth in SEQ ID NO:10, or which is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that amino acids at positions 585-590 of SEQ ID NO:1 (or appropriate amino acid positions of an alternative sequence) are replaced with the amino acid sequence of SEQ ID NO:2. The vector can be an AAV2 vector. 7When a titer of the vector is administered intravitreally into a human (or non-human primate) eye, the vector can infect more than 2.5% of retinal cells. The vector can include an exogenous nucleic acid encoding an RNA or a polypeptide. The exogenous nucleic acid can encode an RNA. The RNA can be an siRNA or a microRNA. The exogenous nucleic acid can encode a polypeptide. The polypeptide can be an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, or an NR2E3 polypeptide. The vector can express a nucleic acid in retinal cells to a greater extent than from a corresponding AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1.

[0030] In another aspect, this document relates to an AAV capsid polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-5. The polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence set forth in SEQ ID NO: 10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of any one of SEQ ID NOs: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of SEQ ID NO: 5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1). The polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions of the alternative sequence) of SEQ ID NO:1 are replaced with the amino acid sequence of any one of SEQ ID NOs:2-5. The polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions of the alternative sequence, e.g., SEQ ID NO:10) of SEQ ID NO:1 are replaced with the amino acid sequence of SEQ ID NO:2. In some cases, the capsid polypeptide can comprise or consist of the amino acid sequence of any of SEQ ID NOs:11-26. 7When a titer of the vector is administered intravitreally into a human (or non-human primate) eye, the AAV vector containing the polypeptide can infect greater than 2.5% of retinal cells. The AAV vector containing the polypeptide can express a nucleic acid in retinal cells to a greater extent than from a corresponding AAV vector containing a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1.

[0031] In another aspect, this document relates to a nucleic acid molecule encoding an adeno-associated virus (AAV) vector comprising an AAV capsid polypeptide, the capsid polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-5. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO: 10, or is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of any one of SEQ ID NOs: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or an alternative sequence, e.g., an appropriate amino acid position of SEQ ID NO: 10). In some cases, the capsid polypeptide can comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 11-26. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acid sequence of SEQ ID NO:5 is located between amino acid positions 587 and 588 (or an appropriate amino acid position in an alternative sequence) of SEQ ID NO:1. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acid sequence at positions 585-590 (or an appropriate amino acid position in an alternative sequence) of SEQ ID NO:1 is replaced with the amino acid sequence of any one of SEQ ID NOs:2-5. In some cases, the capsid polypeptide can comprise or consist of the amino acid sequence of any of SEQ ID NOs:27-42.The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO:10, or is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that amino acids at positions 585-590 of SEQ ID NO:1 (or appropriate amino acid positions of an alternative sequence) are replaced with the amino acid sequence of SEQ ID NO:2. The vector can be an AAV2 vector. At least 1 x 10 7 When a titer of the vector is administered intravitreally into a human (or non-human primate) eye, the vector can infect more than 2.5% of retinal cells. The vector can include an exogenous nucleic acid encoding an RNA or a polypeptide. The exogenous nucleic acid can encode an RNA. The RNA can be an siRNA or a microRNA. The exogenous nucleic acid can encode a polypeptide. The polypeptide can be an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, or an NR2E3 polypeptide. The vector can express a nucleic acid in retinal cells to a greater extent than from a corresponding AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1. The nucleic acid molecule can be DNA.

[0032] In another aspect, this document relates to a nucleic acid molecule encoding an AAV capsid polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-5. The polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence set forth in SEQ ID NO: 10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of any one of SEQ ID NOs: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of SEQ ID NO: ...). The polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions in the alternative sequence) of SEQ ID NO:1 are replaced with the amino acid sequence of any one of SEQ ID NOs:2-5. The capsid polypeptide can comprise or consist of the amino acid sequence of any of SEQ ID NOs:11-42. The polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions in the alternative sequence) of SEQ ID NO:1 are replaced with the amino acid sequence of SEQ ID NO:2. 7When a titer of the vector is administered intravitreally into a human (or non-human primate) eye, the AAV vector comprising the polypeptide can infect more than 2% of retinal cells in two or more retinal regions. The AAV vector comprising the polypeptide can express the nucleic acid in retinal cells to a greater extent than from a corresponding AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1. The nucleic acid molecule can be DNA.

[0033] In another aspect, this document relates to a host cell comprising a nucleic acid molecule of any of the preceding two paragraphs. The host cell is capable of expressing the vector. The host cell is capable of expressing the polypeptide.

[0034] In another aspect, this document relates to a host cell comprising an AAV vector comprising an AAV capsid polypeptide, the capsid polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-5. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence set forth in SEQ ID NO: 10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of any one of SEQ ID NOs: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of SEQ ID NO: ...). The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence set forth in SEQ ID NO: 10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions of the alternative sequence) of SEQ ID NO: 1 are replaced with the amino acid sequence of any one of SEQ ID NOs: 2-5. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence set forth in SEQ ID NO: 10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions of the alternative sequence) of SEQ ID NO: 1 are replaced with the amino acid sequence of SEQ ID NO: 2. The capsid polypeptide can comprise or consist of the amino acid sequence of any of SEQ ID NOs: 11-42. The vector can be an AAV2 vector.At least 1×10. 7 When a titer of the vector is administered intravitreally into a human (or non-human primate) eye, the vector can infect more than 2.5% of retinal cells. The vector can include an exogenous nucleic acid encoding an RNA or a polypeptide. The exogenous nucleic acid can encode an RNA. The RNA can be an siRNA or a microRNA. The exogenous nucleic acid can encode a polypeptide. The polypeptide can be an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, or an NR2E3 polypeptide. The vector can express a nucleic acid in retinal cells to a greater extent than from a corresponding AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1. The host cell can be a retinal cell.

[0035] In another aspect, this document relates to a host cell comprising an AAV capsid polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-5. The polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence set forth in SEQ ID NO: 10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of any one of SEQ ID NOs: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of SEQ ID NO: ...). The polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions of the alternative sequence) of SEQ ID NO:1 are replaced with the amino acid sequence of any one of SEQ ID NOs:2-5. The polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions of the alternative sequence) of SEQ ID NO:1 are replaced with the amino acid sequence of SEQ ID NO:5. In some cases, the capsid polypeptide can comprise or consist of the amino acid sequence of any of SEQ ID NOs:11-42. 7When a titer of the vector is administered intravitreally into a human (or non-human primate) eye, the AAV vector comprising the polypeptide can infect more than 2.5% of retinal cells. The AAV vector comprising the polypeptide can express a nucleic acid in retinal cells to a greater extent than from a corresponding AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1. The host cell can be a retinal cell.

[0036] In another aspect, this document relates to a composition comprising an AAV vector comprising an AAV capsid polypeptide, the capsid polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 2-5. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence set forth in SEQ ID NO: 10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of any one of SEQ ID NOs: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of SEQ ID NO: ...). The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence set forth in SEQ ID NO: 10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions of the alternative sequence) of SEQ ID NO: 1 are replaced with the amino acid sequence of any one of SEQ ID NOs: 2-5. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO: 1 (or the amino acid sequence set forth in SEQ ID NO: 10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acids at positions 585-590 (or the appropriate amino acid positions of the alternative sequence) of SEQ ID NO: 1 are replaced with the amino acid sequence of SEQ ID NO: 2. The capsid polypeptide can comprise or consist of the amino acid sequence of any of SEQ ID NOs: 11-42. The vector can be an AAV2 vector.At least 1×10. 7 When a titer of the vector is administered intravitreally into a human (or non-human primate) eye, the vector can infect greater than 2.5% of retinal cells. The vector can include an exogenous nucleic acid encoding an RNA or a polypeptide. The exogenous nucleic acid can encode an RNA. The RNA can be an siRNA or a microRNA. The exogenous nucleic acid can encode a polypeptide. The polypeptide can be an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, or an NR2E3 polypeptide. The vector can express a nucleic acid in retinal cells at a level greater than that from a corresponding AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1. The composition can be at a concentration of about 1×10 7 ~Approx. 1×10 14 The composition may include phosphate buffered saline, Hank's balanced salt solution, or Pluronic® F68.

[0037] In another aspect, this document relates to a method of delivering an exogenous nucleic acid sequence to a mammalian retinal cell. The method comprises (or consists essentially of, or consists of) contacting a retinal cell with an AAV vector comprising an AAV capsid polypeptide and an exogenous nucleic acid sequence, wherein the capsid polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 2-5, and the AAV vector infects the retinal cell, thereby delivering the exogenous nucleic acid sequence to the retinal cell. The capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO: 10, or is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1), except that the amino acid sequence of any one of SEQ ID NOs: 2-5 is located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1). The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acid sequence of SEQ ID NO:5 is located between amino acid positions 587 and 588 (or an appropriate amino acid position in an alternative sequence) of SEQ ID NO:1. The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acid sequence at positions 585-590 (or an appropriate amino acid position in an alternative sequence) of SEQ ID NO:1 is replaced with the amino acid sequence of any one of SEQ ID NOs:2-5.The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or an alternative sequence which is the amino acid sequence set forth in SEQ ID NO:10, or is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that amino acids at positions 585-590 of SEQ ID NO:1 (or appropriate amino acid positions of an alternative sequence) are replaced with the amino acid sequence of SEQ ID NO:2. The capsid polypeptide can comprise or consist of the amino acid sequence of any of SEQ ID NOs:11-42. The mammal can be a human (or a non-human primate). The vector can be an AAV2 vector. At least 1 x 10 7 When a vector with a titer of 1×10 is administered intravitreally to a human (or non-human primate) eye, the vector can infect more than 2.5% of retinal cells. The exogenous nucleic acid sequence can encode an RNA or a polypeptide. The exogenous nucleic acid can encode an RNA. The RNA can be an siRNA or a microRNA. The exogenous nucleic acid can encode a polypeptide. The polypeptide can be an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, or an NR2E3 polypeptide. The vector can express the exogenous nucleic acid sequence in retinal cells to a greater extent than the expression in retinal cells from a comparable AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1. The method can include intravitreally administering a composition comprising the vector to a mammal, thereby contacting retinal cells with the vector. The composition can be about 1×10 7 ~Approx. 1×10 14 The vector may include

[0038] In another aspect, this document relates to a method of treating a retinal pathology. The method comprises (or consists essentially of, or consists of) contacting a retinal cell of an eye of a mammal having a retinal pathology with an AAV vector comprising a retinal AAV capsid polypeptide and an exogenous nucleic acid sequence, the capsid polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 2-5, and the AAV vector infects the retinal cell and drives expression of the exogenous nucleic acid sequence in the retinal cell, thereby treating the retinal pathology. The mammal can be a human (or a non-human primate). The retinal pathology can be selected from the group consisting of LCA, OCA1, retinitis pigmentosa, rod / cone dystrophy, cone dystrophy, Stargardt's disease, Usher syndrome, XLRP, and XLRS. The capsid polypeptide can include the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acid sequence of any one of SEQ ID NOs:2-5 is located between amino acid positions 587 and 588 of SEQ ID NO:1 (or an appropriate amino acid position for the alternative sequence). The capsid polypeptide can include the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acid sequence of SEQ ID NO:5 is located between amino acid positions 587 and 588 of SEQ ID NO:1 (or an appropriate amino acid position for the alternative sequence). The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or the amino acid sequence set forth in SEQ ID NO:10, or an alternative sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that the amino acids at positions 585 to 590 of SEQ ID NO:1 (or appropriate amino acid positions of an alternative sequence) are replaced with the amino acid sequences of any one of SEQ ID NOs:2 to 5.The capsid polypeptide can comprise the amino acid sequence of SEQ ID NO:1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO:10, or is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that amino acids at positions 585-590 of SEQ ID NO:1 (or appropriate amino acid positions of an alternative sequence) are replaced with the amino acid sequence of SEQ ID NO:5. In some cases, the capsid polypeptide can comprise or consist of the amino acid sequence of any of SEQ ID NOs:11-42. The vector can be an AAV2 vector. At least 1 x 10 7 When a titer of the vector is administered intravitreally to a mammalian eye, the vector can infect greater than 2.5% of retinal cells. The exogenous nucleic acid sequence can encode an RNA. The RNA can be an siRNA or a microRNA. The exogenous nucleic acid can encode a polypeptide. The polypeptide can be an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, and an NR2E3 polypeptide. The vector can express the exogenous nucleic acid sequence in retinal cells of at least two retinal regions to a greater extent than the expression in retinal cells from a comparable AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1. The method can include intravitreally administering a composition comprising the vector to a mammal, thereby contacting retinal cells with the vector. The composition can be greater than about 1×10 7 ~Approx. 1×10 14 The vector may include

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, but the preferred methods and methods are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0040] The details of one or more embodiments of the invention are set forth in the accompanying description below. Other features, objects, and advantages of the invention will become apparent from the description and the claims. [Brief description of the drawings]

[0041] [Figure 1-1] This is a table of sequence numbers 11 to 76. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 1-3] This is a continuation of Figure 1-2. [Figure 1-4] This is a continuation of Figure 1-3. [Figure 1-5] This is a continuation of Figure 1-4. [Figure 1-6] This is a continuation of Figure 1-5. [Figure 1-7] This is a continuation of Figure 1-6. [Figure 1-8] This is a continuation of Figure 1-7. [Figure 1-9] This is a continuation of Figure 1-8. [Figure 1-10] This is a continuation of Figure 1-9. [Figure 1-11] This is a continuation of Figure 1-10. [Figure 1-12] This is a continuation of Figure 1-11. [Figure 1-13] This is a continuation of Figure 1-12. [Figure 1-14] This is a continuation of Figure 1-13. [Figure 1-15] This is a continuation of Figure 1-14. [Figure 1-16] This is a continuation of Figure 1-15. [Figure 1-17] This is a continuation of Figure 1-16. [Figure 1-18] This is a continuation of Figure 1-17. [Figure 1-19] This is a continuation of Figure 1-18. [Figure 1-20] This is a continuation of Figure 1-19. [Figure 1-21] This is a continuation of Figure 1-20. [Figure 1-22] This is a continuation of Figure 1-21. [Figure 1-23] This is a continuation of Figure 1-22. [Figure 1-24] This is a continuation of Figure 1-23. [Figure 1-25] This is a continuation of Figure 1-24. [Figure 1-26] This is a continuation of Figure 1-25. [Figure 1-27] This is a continuation of Figure 1-26. [Figure 1-28] This is a continuation of Figure 1-27. [Figure 1-29] This is a continuation of Figure 1-28. [Figure 1-30] This is a continuation of Figure 1-29. [Figure 1-31] This is a continuation of Figure 1-30. [Figure 1-32] This is a continuation of Figure 1-31. [Figure 1-33] This is a continuation of Figure 1-32. [Diagram 2] FIG. 1 is a diagram of an AAV vector comprising a wild-type AAV2 Rep polypeptide and the indicated AAV2 capsid polypeptide that has been modified to include an insertion sequence (e.g., any one of SEQ ID NOs: 2-5, or a sequence of Formula A) located between positions 587 and 588 (using the numbering of SEQ ID NO: 1), according to some embodiments. [Diagram 3]FIG. 1 is a diagram of an AAV vector comprising a mutant AAV2 Rep polypeptide (AAV2-M1T-REP) and the indicated AAV2 capsid polypeptide modified to include an insertion sequence (e.g., any one of SEQ ID NOs: 2-5, or a sequence of Formula A) located between positions 587 and 588 (using the numbering of SEQ ID NO: 1), according to some embodiments. [Figure 4] FIG. 1 is a diagram of an AAV vector comprising a wild-type AAV2 Rep polypeptide and an indicated AAV2 capsid polypeptide that has been modified to include an inserted sequence (e.g., any one of SEQ ID NOs:2-5, or the sequence of Formula A) as a substitution of amino acid residues at positions 585-590 (using the numbering of SEQ ID NO:1), according to some embodiments. [Diagram 5] FIG. 1 is a diagram of an AAV vector comprising a mutant AAV2 Rep polypeptide (AAV2-M1T-REP) and an indicated AAV2 capsid polypeptide modified to include an inserted sequence (e.g., any one of SEQ ID NOs:2-5, or the sequence of Formula A) as a substitution of an amino acid residue at positions 585-590 (using the numbering of SEQ ID NO:1), according to some embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] This document provides an AAV vector (e.g., an AAV2 vector). For example, this document provides an AAV vector (e.g., an AAV2 vector) that contains a capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. Any suitable AAV vector can be primer-designed to include a capsid polypeptide described herein (e.g., a capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A). For example, AAV2, AAV8, and AAV9 can be designed to include a capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. In some cases, AAV2 having an ACG start codon for an AAV Rep polypeptide (e.g., an AAV2 Rep78 and Rep68 polypeptide; see, e.g., SEQ ID NOs: 75-76) instead of an ATG start codon (e.g., AAV2-M1T-REP) can be designed to include a capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A.

[0043] Any suitable AAV capsid polypeptide can be designed to comprise an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. For example, AAV2, AAV6, AAV8, and AAV9 capsid polypeptides can be designed to comprise an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. In some cases, an AAV2 capsid polypeptide can be designed to comprise an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. In some cases, an AAV2 capsid polypeptide having the following amino acid sequence can be designed to comprise an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A: [ka] The two bold amino acid residues are at positions 587 and 588, and the underlined amino acids are at positions 585-590.

[0044] In some cases, an AAV capsid polypeptide having the following amino acid sequence (e.g., an AAV2 capsid polypeptide) can be designed to include an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. [ka]

[0045] In some cases, an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1 can be designed to include an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A.

[0046] In some cases, certain AAV2 sequences contemplated herein can include modifications or mutations of SEQ ID NO:1, such as V708I and / or E67A substitutions.

[0047] When an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) is configured to include an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A, the included amino acid sequence can be located at any suitable position along the AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide). For example, an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A, such as any one of SEQ ID NOs: 2-5, can be located between the naturally occurring amino acid residues at positions 587 and 588 of an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide), can be located between the naturally occurring amino acid residues at positions 452 and 453 of an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide), or can be located between the naturally occurring amino acid residues at positions 453 and 454 of an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide).

[0048] [Table 1]

[0049] As described herein, an AAV vector can be designed to have an AAV capsid polypeptide comprising an amino acid sequence insert of Formula A. For example, an AAV vector can be designed to have an AAV capsid polypeptide of SEQ ID NO: 1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO: 10, or is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 1) comprising an amino acid sequence insert of Formula A located between amino acid positions 587 and 588 of SEQ ID NO: 1 (or an appropriate amino acid position of an alternative sequence). Formula A can be as follows: -L1-EGSGRN (SEQ ID NO: 2)-L2- wherein L1 and L2 are each independently an optimal amino acid linker having one, two, or three amino acids. In some cases, L1, L2, or both L1 and L2 may be absent. In some cases, L1 can be one amino acid X1, two amino acids X2-X1, or three amino acids X3-X2-X1. If X1 is present, X1 can be an amino acid residue selected from the group consisting of A, V, I, and L. If X2 is present, X2 can be an amino acid residue selected from the group consisting of A, V, I, and L. If X3 is present, X3 can be an amino acid residue selected from the group consisting of A, V, I, and L. In some cases, L2 can be one amino acid Z1, two amino acids Z1-Z2, or three amino acids Z1-Z2-Z3. If Z1 is present, Z1 can be an amino acid residue selected from the group consisting of A, V, I, and L. When Z2 is present, it can be an amino acid residue selected from the group consisting of A, V, I, and L. When Z3 is present, it can be an amino acid residue selected from the group consisting of A, V, I, and L. Examples of L1 linkers include, but are not limited to, A, V, I, L, AA, AV, AI, AL, VA, VV, VI, VL, IA, IV, II, IL, LA, LV, LI, LL, AAA, AAV, AAI, AAL, AVA, AVV, AVI, AVL, AIA, AIV, AII, AIL, ALA, ALV, ALI, ALL, VAA, VAV, VAI, VAL, VVA, VVV, VVI , VVL, VIA, VIV, VII, VIL, VLA, VLV, VLI, VLL, IAA, IAV, IAI, IAL, IVA, IVV, IVI, IVL, IIA, IIV, III, IIL, I LA, ILV, ILI, ILL, LAA, LAV, LAI, LAL, LVA, LVV, LVI, LVL, LIA, LIV, LII, LIL, LLA, LLV, LLI, and LLL.Examples of L2 linkers include, but are not limited to, A, V, I, L, AA, AV, AI, AL, VA, VV, VI, VL, IA, IV, II, IL, LA, LV, LI, LL, AAA, AAV, AAI, AAL, AVA, AVV, AVI, AVL, AIA, AIV, AII, AIL, ALA, ALV, ALI, ALL, VAA, VAV, VAI, VAL, VVA, VVV, VVI , VVL, VIA, VIV, VII, VIL, VLA, VLV, VLI, VLL, IAA, IAV, IAI, IAL, IVA, IVV, IVI, IVL, IIA, IIV, III, IIL, I LA, ILV, ILI, ILL, LAA, LAV, LAI, LAL, LVA, LVV, LVI, LVL, LIA, LIV, LII, LIL, LLA, LLV, LLI, and LLL.

[0050] In some cases, the AAV2 capsid polypeptides provided herein can have a sequence set forth in SEQ ID NO:1 (or an alternative sequence that is an amino acid sequence set forth in SEQ ID NO:10, or is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), including an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A, inserted between asparagine-587 and arginine-588 (or at an appropriate amino acid position in an alternative sequence) (see, e.g., Figures 2-3). In some cases, the AAV2 capsid polypeptides provided herein can have a sequence set forth in SEQ ID NO:1 (or an alternative sequence that is an amino acid sequence set forth in SEQ ID NO:10, or is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), including an amino acid sequence set forth in any one of SEQ ID NOs:2-5, inserted between asparagine-587 and arginine-588 (or at an appropriate amino acid position in an alternative sequence).

[0051] In some cases, when an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) is configured to contain an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A, the contained amino acid sequence can be used to replace one or more naturally occurring amino acid residues located at any suitable position along the AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide). For example, an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A, such as any one of SEQ ID NOs:2-5, can be used to replace the naturally occurring amino acid residues at positions 585-590 of an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) (see, e.g., Figures 4-5).

[0052] In some cases, the AAV2 capsid polypeptides provided herein can have the sequence set forth in SEQ ID NO:1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO:10, or that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that amino acid residues at positions 585-590 (or the appropriate amino acid positions of the alternative sequence) are replaced with an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. In some cases, the AAV2 capsid polypeptides provided herein can have the sequence set forth in SEQ ID NO:1 (or an alternative sequence that is the amino acid sequence set forth in SEQ ID NO:10, or that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:1), except that amino acid residues 585-590 (or the appropriate amino acid positions of the alternative sequence) are replaced with an amino acid sequence set forth in any one of SEQ ID NOs:2-5 (or a variant thereof).

[0053] In some cases, an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) can be designed to include two or more amino acid sequences set forth in Table 1 (or a variant thereof) or Formula A. For example, an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) can be designed to include two or three amino acid sequences set forth in Table 1 (or a variant thereof) or Formula A.

[0054] As described herein, an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) can be designed to include an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. A variant of an amino acid sequence set forth in Table 1 refers to an amino acid sequence that is identical to the amino acid sequence set forth in Table 1, except that the amino acid sequence has one, two, or three amino acid additions, deletions, substitutions, or a combination thereof. For example, a variant of SEQ ID NO: 2 can be SEQ ID NO: 2, except that it has one, two, or three amino acid additions, deletions, substitutions, or a combination thereof. In some cases, a variant provided herein can be an amino acid sequence set forth in any one of SEQ ID NOs: 2-5, except that it contains one, two, or three amino acid additions. In some cases, a variant provided herein can be an amino acid sequence set forth in any one of SEQ ID NOs: 2-5, except that it contains one, two, or three amino acid deletions. In some cases, a variant provided herein can be an amino acid sequence set forth in any one of SEQ ID NOs: 2-5, except that it contains one, two, or three amino acid deletions. In some cases, a variant provided herein can be an amino acid sequence set forth in any one of SEQ ID NOs: 2-5, except that it contains one, two, or three amino acid substitutions. In some cases, the variants provided herein can be the amino acid sequence set forth in any one of SEQ ID NOs: 2-5, except that they contain one amino acid addition, deletion, or substitution. In some cases, the variants provided herein can be the amino acid sequence set forth in any one of SEQ ID NOs: 2-5, except that they contain two amino acid additions, deletions, substitutions, or combinations thereof. In some cases, the variants provided herein can be the amino acid sequence set forth in any one of SEQ ID NOs: 2-5, except that they contain three amino acid additions, deletions, substitutions, or combinations thereof.

[0055] In some cases, the amino acid substitutions present in the variants can be conservative amino acid substitutions. For example, conservative amino acid substitutions can be made by substituting one amino acid residue for another amino acid residue with a similar side chain. Families of amino acid residues with similar side chains can include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0056] In some cases, the amino acid substitutions present in the variants can be non-conservative amino acid substitutions. Non-conservative amino acid substitutions can be made by substituting one amino acid residue for another amino acid residue with a dissimilar side chain. Examples of non-conservative substitutions include, but are not limited to, (a) hydrophilic residues (e.g., serine or threonine) for hydrophobic residues (e.g., leucine, isoleucine, phenylalanine, valine, or alanine), (b) cysteine ​​or proline for any other residue, (c) residues with basic side chains (e.g., lysine, arginine, or histidine) for residues with acidic side chains (e.g., aspartic acid or glutamic acid), and (d) residues with bulky side chains (e.g., phenylalanine) for glycine or other residues with small side chains.

[0057] The percent sequence identity of a particular amino acid to an amino acid sequence referenced by a particular sequence identifier is determined as follows: First, the amino acid sequence is compared to the sequence set forth in the particular sequence identifier using the BLAST 2 sequence (Bl2seq) program from the standalone version of BLAST, including BLASTP version 2.0.14. This standalone version of BLASTZ can be obtained from the Fish & Richardson website (e.g., www.fr.com / blast / ) or the U.S. government's National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov). Instructions explaining how to use the Bl2seq program can be found in the readme file that accompanies BLASTZ. Bl2seq uses either the BLASTN or BLASTP algorithm to perform a comparison of two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two amino acid sequences, the Bl2seq options are set as follows: -i to the file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt), -j to the file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt), -p to blastp, -o to any desired file name (e.g., C:\output.txt), and all other options are left at their default settings. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -ic:\seq1.txt -jc:\seq2.txt -p blastp -oc:\output.txt. If the two compared sequences share homology, the specified output file will present these regions of homology as aligned sequences. If the two compared sequences do not share homology, the specified output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where an identical amino acid residue is represented in both sequences. By matched positions is meant positions where an identical amino acid residue occurs at the same position in the aligned sequences.The percent sequence identity is determined by dividing the number of matches by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO:1), and then multiplying the resulting value by 100. For example, an amino acid sequence having 725 matches when aligned with the sequence set forth in SEQ ID NO:1 is 98.6% identical to the sequence set forth in SEQ ID NO:1 (i.e., 725÷735×100=98.6). Note that percent sequence identity values ​​are rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 8.17, 78.18, and 78.19 are rounded up to 78.2. Note also that length values ​​are always integers.

[0058] Methods for generating amino acid sequence variants can include site-directed (e.g., by PCR) or random mutagenesis of a nucleic acid encoding an AAV capsid polypeptide. See, e.g., Zoller, Curr. Opin. Biotechnol. 3: 348-354 (1992).

[0059] The AAV vectors described herein (e.g., AAV2 vectors) can be designed to include one or more exogenous nucleic acid sequences. For example, the AAV vectors described herein (e.g., AAV2 vectors) can be designed to include an exogenous nucleic acid sequence that encodes an RNA of interest and / or a polypeptide of interest. The exogenous nucleic acid sequence can be designed to encode any suitable RNA of interest. Examples of RNA of interest that can be encoded by an exogenous nucleic acid sequence designed to be included in the AAV vectors provided herein include, but are not limited to, siRNA, RNA components for gene editing, and microRNA. In some cases, the RNA of interest, which can be encoded by an exogenous nucleic acid sequence contained in an AAV vector provided herein, can be any of a variety of nucleic acids, including, for example, SIRNA-027 for treating subfoveal CNVM associated with age-related macular degeneration (see, e.g., NCT00363714), Cand5 / bevasiranib for treating diabetic macular edema (see, e.g., NCT00306904), PF-04523655 for treating diabetic macular edema (see, e.g., NCT01445899), QPI-1007 for treating optic nerve atrophy in NAION (see, e.g., NCT01064505), and other nucleic acids, including, for example, PI-1007 for treating ischemic CRVO (see, e.g., NCT01064505). The therapeutic agent can be aganirsen (see, e.g., NCT02947867), for example, for treating retinitis pigmentosa / Usher syndrome type 2 (see, e.g., NCT03780257), QR-1123 (see, e.g., NCT04123626), for example, for treating autosomal dominant retinitis pigmentosa (see, e.g., NCT04123626), IONIS-FB-LRx (see, e.g., NCT03815825), for example, for treating geographic atrophy associated with age-related macular degeneration, or sepofarsen / QR-110 (see, e.g., NCT03913143), for example, for treating Leber's congenital amaurosis.

[0060] The exogenous nucleic acid sequence can be designed to encode any suitable polypeptide of interest. Examples of polypeptides of interest that can be encoded by the exogenous nucleic acid sequence designed to be included in the AAV vectors provided herein include, but are not limited to, therapeutic polypeptides, trophic factor polypeptides, gene editing polypeptides (e.g., Cas9 polypeptides, TALEN polypeptides, or zinc finger polypeptides), enzymes, light-generating tool polypeptides (e.g., ChR polypeptides, NhpR polypeptides, or ReachR polypeptides), antibodies, antibody domains (e.g., VH domains), cytokines, anti-angiogenic polypeptides, and neuroprotective polypeptides. Examples of polypeptides of interest that can be encoded by exogenous nucleic acid sequences designed to be included in the AAV vectors provided herein include, but are not limited to, an ABCA4 polypeptide, a CRB1 polypeptide, an NPHP5 polypeptide, an NR2E3 polypeptide, a PDE6A polypeptide, a PDE6B polypeptide, a PDE6C polypeptide, a PRPF31 polypeptide, an RPE65 polypeptide, an RPGR polypeptide, an RS1 polypeptide, a TYR polypeptide, a polypeptide, a USH2A polypeptide, a polypeptide, a MYO7A polypeptide, a polypeptide, a REP1 polypeptide, a polypeptide, an OPN1LW polypeptide, a polypeptide, an OPN1MW polypeptide, a CNGA3 polypeptide, a CNGA4 polypeptide, a CNGA5 polypeptide, a CNGA6 polypeptide, a CNGA7 polypeptide, a CNGA8 polypeptide, a CNGA9 polypeptide, a CNGA10 polypeptide, a CNGA11 polypeptide, a CNGA12 polypeptide, a CNGA13 polypeptide, a CNGA14 polypeptide, a CNGA15 polypeptide, a CNGA16 polypeptide, a CNGA17 polypeptide, a CNGA18 polypeptide, a CNGA19 polypeptide, a CNGA110 polypeptide, a CNGA111 polypeptide, a CNGA12 polypeptide, a CNGA13 polypeptide, a CNGA14 polypeptide, a CNGA15 polypeptide, a CNGA16 polypeptide, a CNGA17 polypeptide, a CNGA18 polypeptide, a CNGA19 polypeptide, a CNGA19 polypeptide, a CNGA110 polypeptide, a CNGA111 polypeptide, a CNGA111 polypeptide, a CNGA12 polypeptide, a CNGA13 polypeptide, a CNGA14 polypeptide, a CNGA15 polypeptide, a CNGA16 polypeptide, a CNGA17 polypeptide, a CNGA18 polypeptide, a CNGA19 polypeptide, a CNGA19 polypeptide, a CNGA19 polypeptide, a CNGA19 polypeptide, a CNGA19 polypeptide, a CN tide, CNGB3 polypeptide, GUCY2D polypeptide, GACA1A polypeptide, GNAT2 polypeptide, PDE6H polypeptide, PROM1 polypeptide, PRPH2 polypeptide, CRX polypeptide, NPHP5 polypeptide, EYS polypeptide, ND4 polypeptide, CLN1-14 polypeptide (e.g., CLN3 polypeptide, CLN5 polypeptide, CLN6 polypeptide, or CLN8 polypeptide), NYX polypeptide, GRM6 polypeptide, TRPM1 polypeptide, GPR179 polypeptide, LRIT3 polypeptide, glial cell-derived neurotrophic factor (GDNF) polypeptide, brain-derived neurotrophic factor (BNDF) polypeptide, fibroblast growth factor (FGF) polypeptide,truncated rod-derived cone viability factor (RdCVF) polypeptide, full-length rod-derived cone viability factor (RdCVFL) polypeptide, X-linked inhibitor of apoptosis (XIAP) polypeptide, soluble fms-related receptor tyrosine kinase 1 (sFLT) polypeptide, CYP4V2 polypeptide, palmitoyl protein thioesterase 1 polypeptide, tripeptidyl peptidase 1 polypeptide, DNAJC5 polypeptide, MFSDOPAC8 polypeptide, cathepsin D polypeptide, granulin polypeptide, ATP13A2 polypeptide, cathepsin F polypeptide, KCTD7 polypeptide, "P" gene polypeptide, TRP1 polypeptide, MATP (SLC45A2) polypeptide, SLC24A5 polypeptide, LRMDA polypeptide, GPR143 polypeptide, RPGR-exon1-ORF15 polypeptide, USH2b polypeptide, USH1C polypeptide, CDH23 polypeptide, PCDH15 polypeptide, SANS polypeptide, USH1H polypeptide, CIB2 polypeptide, USH1K polypeptide, ADGRV1 polypeptide, WHRN polypeptide, PDZD7 polypeptide, CLRN1 polypeptide, HARS polypeptide, RP2 polypeptide, FAM161 polypeptide, DLK polypeptide, RHO polypeptide, CHM polypeptide, BEST1 polypeptide, RP1 polypeptide, OPA1 polypeptide, CEP290 polypeptide, R DH12 polypeptide, CACNA1 polypeptide, BBS1 polypeptide, FAM161A polypeptide, CERKL polypeptide, PRPF8 polypeptide, RP1L1 polypeptide, SNRNP200 polypeptide, IMPG2 polypeptide, CDHR1 polypeptide, IMPDH1 polypeptide, CNGB1 polypeptide, MERTK polypeptide, KCNV2 polypeptide, AIPL1 polypeptide, RPGRIP1 polypeptide, TULP1 polypeptide, C2ORF71 (also known as PCARE) polypeptide, MAK polypeptide, TIMP3 polypeptide, GUCA1A polypeptide, ALMS1 polypeptide, BBS10 polypeptide, IFT140 polypeptide, CNGA1 polypeptide, NMNAT1 polypeptide, COL2A1 polypeptide, EFEMP1 polypeptide, WFS1 polypeptide,RDH5 polypeptide, PRPF3 polypeptide, LRP5 polypeptide, TOPORS polypeptide, DHDDS polypeptide, LCA5 polypeptide, IQCB1 polypeptide, RP9 polypeptide, ATXN7 polypeptide, BBS2 polypeptide, SAG RLBP1 polypeptide, ND6 (MT-ND6) polypeptide, C1QTNF5 polypeptide, VPS13B polypeptide, KIF11 polypeptide, MT-TL1 polypeptide, KLHL7 polypeptide, ACO2 polypeptide, C21orf2 (also known as CFAP410) polypeptide, AHI1 polypeptide, KIZ polypeptide, SPATA7 polypeptide, TTLL5 polypeptide, HGSNAT polypeptide, NRL polypeptide, OAT polypeptide, FLVCR1 polypeptide, ABCC6 polypeptide, LRAT polypeptide, CEP78 polypeptide, CDH3 polypeptide, FZD4 polypeptide, BBS12 polypeptide, HK1 polypeptide, PRDM13 polypeptide, ADAM9 polypeptide, BBS7 polypeptide, CABP4 polypeptide, ABHD12 polypeptide, COL18A1 polypeptide, MFRP polypeptide, RIMS1 polypeptide, ROM1 polypeptide, BBS4 polypeptide, IMPG1 polypeptide, INPP5 E polypeptide, VCAN polypeptide, POC1B polypeptide, RAX2 polypeptide, TSPAN12 polypeptide, CACNA2D4 polypeptide, JAG1 polypeptide, MKKS polypeptide, NPHP4 polypeptide, BBS9 polypeptide, COL11A1 polypeptide, ELOVL4 polypeptide, NDP polypeptide, NPHP1 polypeptide, RGR polypeptide, BBS5 polypeptide, WDR19 polypeptide, C8ORF37 polypeptide, CTNNA1 polypeptide, LAMP2 polypeptide, PEX1 polypeptide, PHYH polypeptide, ATF6 polypeptide, PRPS1 polypeptide, SEMA4A polypeptide, ARL6 polypeptide, CNNM4 polypeptide, OTX2 polypeptide, PRPF6 polypeptide, RBP3 polypeptide, PNPLA6 polypeptide, SLC24A1 polypeptide, USH1G polypeptide, PITPNM3 polypeptide, TTC8 polypeptide, ARSG polypeptide, CWC27 polypeptide,DRAM2 polypeptide, PRCD polypeptide, REEP6 polypeptide, SSBP1 polypeptide, LAMA1 polypeptide, RAB28 polypeptide, ZNF408 polypeptide, GNAT1 polypeptide, IDH3A polypeptide, PDE6G polypeptide, PEX6 polypeptide, TUB polypeptide, CEP250 polypeptide, FSCN2 polypeptide, GRK1 polypeptide, RBP4 polypeptide, RD3 polypeptide, AGBL5 polypeptide, CAPN5 polypeptide, IFT172 polypeptide, KCNJ13 polypeptide, PAX2 polypeptide, CC2D2A polypeptide, HMCN1 polypeptide, MT-ATP6 polypeptide, RCBTB1 polypeptide, ARL2BP polypeptide, CA4 polypeptide, DFNB31 polypeptide, GNB3 polypeptide, MMACHC polypeptide, PRPF4 polypeptide, RGS9 polypeptide, ARHGEF18 polypeptide, KIAA1549 polypeptide, MKS1 polypeptide, MTTP (not MT-TP) polypeptide, PLK4 polypeptide, RPGRIP1L polypeptide, SDCCAG8 polypeptide, SRD5A3 polypeptide, TUBB4B polypeptide, ADAMTS18 polypeptide, ARL3 polypeptide, COL11A2 polypeptide, MVK polypeptide, NBAS polypeptide, OFD1 polypeptide, P3H2 polypeptide, RGS9BP polypeptide, CSPP1 polypeptide, ITM2B polypeptide, PANK2 polypeptide, PEX7 polypeptide, POMGNT1 polypeptide, SLC4A7 polypeptide, TMEM231 polypeptide, T RNT1 polypeptide, TUBGCP6 polypeptide, ZNF513 polypeptide, AFG3L2 polypeptide, ARL13B polypeptide, C5ORF42 (also known as CPLANE1) polypeptide, COL9A1 polypeptide, CTSD polypeptide, DTHD1 polypeptide, DYNC2H1 polypeptide, IFT81 polypeptide, KIAA0586 polypeptide, MFN2 polypeptide, NPHP3 polypeptide, PCYT1A polypeptide, PEX12 polypeptide, PLA2G5 polypeptide, POC5 polypeptide, SCAPER polypeptide, SLC25A46 polypeptide, PCYT1A polypeptide, PEX12 polypeptide,PLA2G5 polypeptide, POC5 polypeptide, SCAPER polypeptide, SLC25A46 polypeptide, CEP164 polypeptide, CLCC1 polypeptide, COL9A2 polypeptide, CTNNB1 polypeptide, DHX38 polypeptide, GNPTG polypeptide, GRN polypeptide, GUCA1B polypeptide, IFT27 polypeptide, IFT74 polypeptide, KIAA0556 polypeptide, LRP2 polypeptide, MAPKAPK3 polypeptide, MIR204 polypeptide, MT-ND3 polypeptide, MT-RNR1 polypeptide, MT-TS2 polypeptide, ND5 (MT-ND5) polypeptide, NEK2 polypeptide, OPN1SW polypeptide, PEX13 polypeptide, PEX2 polypeptide, RHBDD2 polypeptide, SAMD11 polypeptide, SCLT1 polypeptide, SLC7A14 polypeptide, TCTN1 polypeptide, TCTN2 polypeptide, TLCD3B polypeptide, TREX1 polypeptide, TTPA polypeptide, UNC119 polypeptide, WDPCP polypeptide, ACBD5 polypeptide, AHR polypeptide, ARMC9 polypeptide, ASRGL1 polypeptide, ATOH7 polypeptide, B9D1 polypeptide, B9D2 polypeptide, BBIP1 polypeptide, C12ORF65 polypeptide, C2CD3 polypeptide, C5AR2 polypeptide, CCDC188 polypeptide, CCT2 polypeptide, CEP104 polypeptide, CEP120 polypeptide, CEP19 polypeptide, CEP41 polypeptide, CISD2 polypeptide, CLUAP1 polypeptide, COL9A3 polypeptide, CRB2 polypeptide, CTC1 polypeptide, DACT2 polypeptide, DDR1 polypeptide, ENSA polypeptide, ESPN polypeptide, EXOSC2 polypeptide, FBN3 polypeptide, GDF6 polypeptide, GPR125 polypeptide, HKDC1 polypeptide, HMX1 polypeptide, IDH3B polypeptide, IFT43 polypeptide, IFT80 polypeptide, INVS polypeptide, KIAA0753 polypeptide, KIF3B polypeptide, KIF7 polypeptide, LRRTM4 polypeptide, LZTFL1 polypeptide, MT-ATP8 polypeptide, MT-CO1 polypeptide, MT-CO2 polypeptide, MT-CO3 polypeptide, MT-CYB polypeptide, MT-ND2 polypeptide, MT-ND4L polypeptide, MT-RNR2 polypeptide, MT-TA polypeptide, MT-TC polypeptide, MT-TD polypeptide, MT-TE polypeptide, MT-TF polypeptide, MT-TG polypeptide, MT-TH polypeptide, MT-TI polypeptide, MT-TK polypeptide, MT-TL2 polypeptide, MT-TM polypeptide, MT-TN polypeptide, MT-TP (not MTTP) polypeptide, MT-TQ polypeptide, MT-TR polypeptide, MT-TS1 polypeptide, MT-TT polypeptide, MT-TV polypeptide, MT-TW polypeptide, MT-TY polypeptide, NEU Examples of such polypeptides include a ROD1 polypeptide, a PDE6D polypeptide, a PEX10 polypeptide, a PEX11B polypeptide, a PEX14 polypeptide, a PEX16 polypeptide, a PEX19 polypeptide, a PEX26 polypeptide, a PEX3 polypeptide, a PEX5 polypeptide, a PGK1 polypeptide, a PISD polypeptide, a PPP2R3C polypeptide, a PROS1 polypeptide, a PSEN1 polypeptide, a RDH11 polypeptide, a RRM2B polypeptide, a SMARCA4 polypeptide, a SPP2 polypeptide, a TCTN3 polypeptide, a TEAD1 polypeptide, a TMEM107 polypeptide, a TMEM138 polypeptide, a TMEM216 polypeptide, a TMEM67 polypeptide, a TPP1 polypeptide, a TRIM32 polypeptide, a USP45 polypeptide, and a ZNF423 polypeptide.

[0061] In some cases, one or more AAV vectors provided herein can be designed to perform gene editing in one or more cells (e.g., retinal cells). Such gene editing can result in genomic modification of one or more cells. Examples of such genomic modification include, but are not limited to, targeted insertion of a nucleic acid encoding an RNA and / or a polypeptide of interest into one or more cells, targeted modification (e.g., targeted inactivation or knockout) of a genomic sequence of one or more cells, and targeted replacement of a nucleic acid (e.g., a nucleic acid encoding an RNA, a regulatory nucleic acid sequence, and / or a nucleic acid encoding a polypeptide of interest) in one or more.

[0062] Any suitable gene editing components can be engineered into one or more AAV vectors provided herein, such that the one or more AAV vectors can be used to deliver the genetic editing components to target cells (e.g., one or more retinal cells) of a mammal (e.g., a human or non-human primate) for efficient editing of the genome of these cells. Typically, gene editing components include, but are not limited to, components capable of cleaving genomic nucleic acid at a desired location, and any donor nucleic acid designed to be capable of being inserted into the desired location once cleaved. Any suitable rare-cutting endonuclease can be used to cleave genomic nucleic acid at a desired location. Examples of such rare-cutting endonucleases include, but are not limited to, meganucleases, Transcription Activation-Like Effector (TALE) nucleases (TALEN™; Cellectis, Paris, France), zinc finger nucleases (ZFNs), and endonucleases of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas system (e.g., endonucleases of the CRISPR / Cas9 system). See, e.g., Baker, Nature Methods, 9:23-26 (2012); International PCT Patent Application Publication No. WO2004 / 067736; International PCT Patent Application Publication No. WO2011 / 072246; U.S. Patent No. 8,586,363; Porteus and Carroll, Nature Biotechnol., 23:967-973 (2005); Jinek et al., Science, 337:816-821 (2012); Mali et al., Science, 339:823-826 (2013); Li et al., Nature Biotechnology, 31(8):688-691 (2013); and Makarova et al., Nat. Rev. Microbiol., 9(6):467-477 (2011)).

[0063] In some cases, two sequences (one on either side of the sequence to be removed) in the genomic nucleic acid of a cell (e.g., a retinal cell) can be targeted for endonuclease cleavage to facilitate gene replacement. For example, a first target sequence adjacent to the 5' end of the sequence to be removed and a second target sequence adjacent to the 3' end of the sequence to be removed can be targeted by guide RNA to allow Cas9 cleavage, or can be targeted by TALENs designed to specifically recognize these targets. Delivery of (a) an endonuclease targeted to genomic DNA and (b) a donor nucleic acid construct using one or more AAV vectors provided herein can allow cleavage at both genomic targets, removal of sequences between the genomic targets, and insertion of a donor sequence into the deleted location.

[0064] The AAV vectors provided herein (e.g., AAV2 vectors) can include any suitable promoter and / or other regulatory sequences (e.g., enhancers, transcription initiation sites, translation initiation sites, and termination signals) operably linked to the exogenous nucleic acid sequence designed to be expressed. In some cases, the promoter used to drive expression can be a constitutive promoter, a controllable promoter, a tissue-specific promoter, or a viral promoter. Examples of constitutive promoters that can be used as described herein include, but are not limited to, SV40 promoter, CMV promoter, and E1ALPHA promoter. Examples of controllable promoters that can be used as described herein include, but are not limited to, inducible promoters and repressible promoters. Examples of tissue-specific promoters that can be used as described herein include, but are not limited to, rhodopsin promoter, cone arrestin promoter, and synapsin promoter. Examples of viral promoters that can be used as described herein include, but are not limited to, adenovirus promoter, vaccinia virus promoter, CMV promoter (e.g., immediate early CMV promoter), and AAV promoter.

[0065] In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can contain a total of up to about 5 kb of nucleotides. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can contain a total of about 1 kb to about 5 kb, about 1 kb to about 4 kb, about 1 kb to about 3 kb, about 2 kb to about 5 kb, about 2 kb to about 4 kb, about 2 kb to about 3 kb, about 3 kb to about 5 kb, about 3 kb to about 4 kb, or about 4 kb to about 5 kb of nucleotides.

[0066] An AAV vector described herein (e.g., an AAV2 vector) containing an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A can be capable of infecting retinal cells (e.g., retinal ganglion cells) across a retinal region (e.g., across two, three, or four retinal regions) in vivo and delivering an exogenous nucleic acid sequence to the infected retinal cells, causing the infected retinal cells to express the exogenous nucleic acid sequence. In some cases, an AAV vector provided herein (e.g., an AAV2 vector) can be capable of infecting and driving RNA expression of an exogenous nucleic acid sequence in more retinal cells of a mammal (e.g., a human or non-human primate) when compared to wild-type AAV2. In some cases, the AAV vectors provided herein (e.g., AAV2 vectors) can be capable of driving a level of expression of an exogenous nucleic acid sequence in a retinal cell of a mammal (e.g., a human or non-human primate) that is greater than the level of RNA expression of the exogenous nucleic acid sequence driven by a control AAV vector (e.g., wild-type AAV2) having an AAV capsid polypeptide composed of the amino acid sequence set forth in SEQ ID NO:1 in a retinal cell of a control mammal (e.g., a control human or control non-human primate).

[0067] Examples of retinal cells that can be infected by an AAV vector described herein (e.g., an AAV2 vector) containing an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A include, but are not limited to, retinal ganglion cells, retinal pigment epithelial cells, photoreceptor cells, bipolar cells, axonal cells, Muller glia, and horizontal cells.

[0068] This document also provides compositions containing one or more AAV vectors provided herein (e.g., one or more AAV2 vectors provided herein). For example, one or more AAV vectors provided herein (e.g., one or more AAV2 vectors provided herein) can be administered to a mammal (e.g., a human or non-human primate) and formulated as a pharmaceutical composition for treating the mammal. In some cases, one or more AAV vectors provided herein (e.g., one or more AAV2 vectors provided herein) can be formulated as a pharmaceutical composition for administration to a mammal (e.g., a human or non-human primate) to deliver an exogenous nucleic acid sequence to a retinal cell (e.g., a retinal ganglion cell) for expression in the retinal cell. For example, the AAV vectors provided herein (e.g., AAV2 vectors) can be formulated as pharmaceutical compositions for administration to a mammal (e.g., a human or non-human primate NI). In some cases, the pharmaceutical compositions provided herein can include a pharma- ceutically acceptable carrier, such as a buffer, a salt, a surfactant, a sugar, a tonicity adjuster, or a combination thereof, as described elsewhere (Gervasi, et al., Eur. J. Pharmaceutics and Biopharmaceutics, 131:8-24 (2018)). Examples of pharma- ceutically acceptable carriers that can be used to make the pharmaceutical compositions provided herein include, but are not limited to, water, lactic acid, citric acid, sodium chloride, sodium citrate, sodium succinate, sodium phosphate, a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), dextran 40, or a sugar (e.g., sorbitol, mannitol, sucrose, dextrose, or trehalose), or a combination thereof.For example, pharmaceutical compositions designed to contain an AAV vector provided herein (e.g., an AAV2 vector) can be formulated to include a buffer (e.g., acetate, citrate, histidine, succinate, phosphate, or hydroxymethyl-aminomethane (Tris) buffer), a surfactant (e.g., polysorbate 20, polysorbate 80, or poloxamer 188), and a sugar such as sucrose. Other components that can be included in the pharmaceutical compositions provided herein include, but are not limited to, amino acids such as glycine or arginine, antioxidants such as ascorbic acid, methionine, or ethylenediaminetetraacetic acid (EDTA), or combinations thereof.

[0069] In some cases, when a pharmaceutical composition is formulated to include one or more AAV vectors provided herein (e.g., one or more AAV2 vectors), any suitable titer of AAV vector can be used. For example, the pharmaceutical compositions provided herein can be formulated to include one or more AAV vectors (e.g., one or more AAV2 vectors) at a titer of 1×10 7 Greater than (e.g., 1×10 8 Greater than 1×10 9 Greater than 1×10 10 Greater than 1×10 11 Greater than 1×10 12 Greater than 1×10 13 Greater than or equal to 1×10 14 In some cases, the pharmaceutical compositions provided herein can be formulated to have an AAV vector provided herein (e.g., an AAV2 vector) at a titer of about 1×10 7 ~Approx. 1×10 14 (For example, about 1×10 7 ~Approx. 1×10 13 , about 1×10 7 ~Approx. 1×10 12 , about 1×10 7 ~Approx. 1×10 11 , about 1×10 7 ~Approx. 1×10 10 , about 1×10 8 ~Approx. 1×10 14 , about 1×10 9 ~Approx. 1×1014 , about 1×10 10 ~Approx. 1×10 14 , about 1×10 8 ~Approx. 1×10 12 , or about 1 × 10 9 ~Approx. 1×10 11 ) with an AAV vector provided herein (e.g., an AAV2 vector).

[0070] The pharmaceutical compositions provided herein can be in any suitable form. For example, the pharmaceutical compositions provided herein can be designed to be liquid, semi-solid, or solid. In some cases, the pharmaceutical compositions provided herein can be a liquid solution (e.g., an injectable and / or infusible solution), a dispersion, a suspension, a tablet, a pill, a powder, a microemulsion, a liposome, or a suppository. In some cases, the pharmaceutical compositions provided herein can be lyophilized. In some cases, the pharmaceutical compositions provided herein (e.g., a pharmaceutical composition comprising one or more AAV vectors provided herein, such as one or more AAV2 vectors provided herein) can be formulated with a carrier or coating designed to protect against rapid release. For example, the pharmaceutical compositions provided herein can be formulated as controlled release or sustained release formulations, as described elsewhere (U.S. Patent Publication Nos. 2019 / 0241667; 2019 / 0233522; and 2019 / 0233498).

[0071] This document also provides nucleic acid molecules encoding an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. In some cases, the nucleic acid molecule can be designed to encode an AAV capsid polypeptide comprising an amino acid sequence encoded by a DNA sequence set forth in Table 1 (e.g., any one of SEQ ID NOs:6-9).

[0072] This document also provides nucleic acid molecules encoding the AAV vectors described herein (e.g., AAV2 vectors). For example, isolated nucleic acid molecules can be designed to encode one or more AAV vectors provided herein (e.g., AAV having an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A). In some cases, the nucleic acid molecule can be designed to encode an AAV vector having an AAV capsid polypeptide comprising an amino acid sequence encoded by a DNA sequence set forth in Table 1 (e.g., any one of SEQ ID NOs: 6-9).

[0073] This document also provides a host cell containing the nucleic acid molecule provided herein. For example, a host cell can be designed to contain a nucleic acid molecule encoding an AAV capsid polypeptide described herein and / or a nucleic acid molecule encoding an AAV vector described herein. In some cases, a host cell can be designed to contain a nucleic acid molecule encoding an AAV capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. In some cases, a host cell can be designed to contain a nucleic acid molecule encoding an AAV vector having an AAV capsid polypeptide comprising an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. Examples of host cells that can be designed to contain a nucleic acid molecule encoding an AAV capsid polypeptide described herein and / or a nucleic acid molecule encoding an AAV vector described herein include, but are not limited to, HEK293T cells (ATCC), 293AAV cells (Cell Biolabs), NEB 5-α cells, TakaraBio Stellar cells, and MegaX cells. Any suitable method can be used to introduce the nucleic acid molecules provided herein (e.g., nucleic acid molecules encoding the AAV capsid polypeptides and / or AAV vectors described herein) into cells. For example, viral transfection, electroporation, transient transfection, and gene gun techniques can be used to introduce the nucleic acid molecules provided herein into cells.

[0074] This document also provides methods and materials for making the AAV vectors provided herein (e.g., AAV2 vectors). For example, this document provides methods and materials for making AAV vectors (e.g., AAV2 vectors) containing an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. As described herein, AAV vectors can be constructed to include an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A. Any suitable method can be used to construct an AAV vector having an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) provided herein (e.g., a capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A). For example, molecular cloning and AAV vector production techniques, such as those described elsewhere, can be used to construct and produce AAV vectors having the AAV capsid polypeptides provided herein (e.g., AAV2 capsid polypeptides) (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and John Wiley & Sons, New York, NY (1994); Grieger et al., Nat. Protoc., 1(3):1412-28 (2006); and Flannery et al., Methods Mol. Biol., 935:351-69 (2013)).In some cases, AAV vectors can be produced in HEK293T cells (ATCC) or 293AAV cells (Cell Biolabs) using double or triple transfection methods (see, e.g., Grieger et al., Nat. Protoc., 1(3):1412-28 (2006); and Flannery et al., Methods Mol. Biol., 935:351-69 (2013)).

[0075] This document also provides methods and materials for using the AAV vectors provided herein (e.g., AAV2 vectors). For example, this document provides methods and materials for using AAV vectors (e.g., AAV2 vectors) that contain an AAV capsid polypeptide that includes an amino acid sequence as set forth in Table 1 (or a variant thereof) or Formula A. As described herein, the AAV vectors provided herein can be used to infect retinal cells (e.g., retinal ganglion cells) in vivo across the retinal region and deliver exogenous nucleic acid sequences to the infected retinal cells, allowing the infected retinal cells to express the exogenous nucleic acid sequences.

[0076] In some cases, AAV vectors provided herein (e.g., AAV2 vectors) (e.g., AAV vectors containing an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A) can be used to treat a retinal pathology (e.g., a retinal disease). For example, an AAV vector provided herein (e.g., AAV2 vectors) designed to contain and drive expression of an exogenous nucleic acid sequence encoding an RNA and / or polypeptide capable of treating a retinal pathology (e.g., a retinal disease) can be administered to a mammal (e.g., a human or non-human primate) having a retinal pathology in such a manner that the AAV vector (a) infects retinal cells (e.g., retinal ganglion cells) and (b) drives expression of the delivered exogenous nucleic acid in the infected retinal cells, thereby reducing the severity of one or more symptoms of the retinal pathology and / or slowing the progression of the retinal pathology.

[0077] As described herein, the AAV vectors provided herein (e.g., AAV2 vectors) can be designed to contain and drive expression of any suitable RNA of interest and / or exogenous nucleic acid sequence encoding a polypeptide of interest. When the AAV vectors provided herein are designed to treat a retinal pathology (e.g., a retinal disease), an exogenous nucleic acid sequence encoding an RNA and / or polypeptide capable of treating the retinal pathology can be included in the AAV vector. Examples of RNA designed to treat a retinal pathology (e.g., a retinal disease) and that can be encoded by an exogenous nucleic acid sequence designed to be included in the AAV vectors provided herein include, but are not limited to, SIRNA-027, e.g., for treating subfoveal CNVM associated with age-related macular degeneration (see, e.g., NCT00363714), Cand5 / bevasiranib, e.g., for treating diabetic macular edema (see, e.g., NCT00306904), PF-04523655, e.g., for treating diabetic macular edema (see, e.g., NCT01445899), QPI-1007, e.g., for treating optic nerve atrophy in NAION (see, e.g., NCT01445899), and IFN-γ-binding domain (IFN-γ-binding domain) for treating retinal pathology (e.g., retinal disease). T01064505), aganirsen, e.g., for treating ischemic CRVO and preventing neovascular glaucoma (see, e.g., NCT02947867), QR-421a, e.g., for treating retinitis pigmentosa / Usher syndrome type 2 (see, e.g., NCT03780257), QR-1123, e.g., for treating autosomal dominant retinitis pigmentosa (see, e.g., NCT04123626), IONIS-FB-LRx, e.g., for treating geographic atrophy associated with age-related macular degeneration (see, e.g., NCT03815825), and sepofarsen / QR-110, e.g., for treating Leber's congenital amaurosis (see, e.g., NCT03913143). Examples of polypeptides that are designed to treat retinal pathologies (e.g., retinal diseases) and that can be encoded by exogenous nucleic acid sequences designed to be included in the AAV vectors provided herein include, but are not limited to, ABCA4 polypeptides,CRB1 polypeptide, NPHP5 polypeptide, NR2E3 polypeptide, PDE6A polypeptide, PDE6B polypeptide, PDE6C polypeptide, PRPF31 polypeptide, RPE65 polypeptide, RPGR polypeptide, RS1 polypeptide, TYR polypeptide, polypeptide, USH2A polypeptide, polypeptide, MYO7A polypeptide, polypeptide, REP1 polypeptide, polypeptide, OPN1LW polypeptide, polypeptide, OPN1MW polypeptide, CNGA3 polypeptide, CNGB3 polypeptide, GUCY2D polypeptide, GACA1A polypeptide, GNAT2 polypeptide, PDE6H polypeptide, PROM1 polypeptide, PRPH2 polypeptide, CRX polypeptide, NPHP5 polypeptide, EYS polypeptide, ND4 polypeptide, CLN1-14 polypeptide (e.g., CLN3 polypeptide, CLN5 polypeptide, CLN6 polypeptide tide or CLN8 polypeptide), NYX polypeptide, GRM6 polypeptide, TRPM1 polypeptide, GPR179 polypeptide, LRIT3 polypeptide, glial cell line-derived neurotrophic factor (GDNF) polypeptide, brain-derived neurotrophic factor (BNDF) polypeptide, fibroblast growth factor (FGF) polypeptide, truncated rod-derived cone viability factor (RdCVF) polypeptide, full-length rod-derived cone viability factor (RdCVFL) polypeptide, X-linked inhibitor of apoptosis (XIAP) polypeptide, soluble fms-related receptor tyrosine kinase 1 (sFLT) polypeptide, CYP4V2 polypeptide, palmitoyl protein thioesterase 1 polypeptide, tripeptidyl peptidase 1 polypeptide, DNAJC5 polypeptide, MFSDOPAC8 polypeptide, cathepsin D polypeptide, granulin polypeptide, ATP13A2 polypeptide, cathepsin F polypeptide, KCTD7 Polypeptides, "P" gene polypeptides, TRP1 polypeptides, MATP (SLC45A2) polypeptides, SLC24A5 polypeptides, LRMDA polypeptides, GPR143 polypeptides, RPGR-exon 1-ORF15 polypeptides, USH2b polypeptides, USH1C polypeptides, CDH23 polypeptides, PCDH15 polypeptides, SANS polypeptides,USH1H polypeptide, CIB2 polypeptide, USH1K polypeptide, ADGRV1 polypeptide, WHRN polypeptide, PDZD7 polypeptide, CLRN1 polypeptide, HARS polypeptide, RP2 polypeptide, FAM161 polypeptide, DLK polypeptide, RHO polypeptide, CHM polypeptide, BEST1 polypeptide, RP1 polypeptide, OPA1 polypeptide, CEP290 polypeptide, RDH12 polypeptide, CACNA1 polypeptide, BBS1 polypeptide, FAM161A polypeptide, CERKL polypeptide, PRPF8 polypeptide, RP1L1 polypeptide, SNRNP200 polypeptide, IMPG2 polypeptide, CDHR1 polypeptide, IMPDH1 polypeptide, CNGB1 polypeptide tide, MERTK polypeptide, KCNV2 polypeptide, AIPL1 polypeptide, RPGRIP1 polypeptide, TULP1 polypeptide, C2ORF71 (also known as PCARE) polypeptide, MAK polypeptide, TIMP3 polypeptide, GUCA1A polypeptide, ALMS1 polypeptide, BBS10 polypeptide, IFT140 polypeptide, CNGA1 polypeptide, NMNAT1 polypeptide, COL2A1 polypeptide, EFEMP1 polypeptide, WFS1 polypeptide, RDH5 polypeptide, PRPF3 polypeptide, LRP5 polypeptide, TOPORS polypeptide, DHDDS polypeptide, LCA5 polypeptide, IQCB1 polypeptide, RP9 polypeptide, ATXN7 polypeptide, BBS2 polypeptide, SAG RLBP1 polypeptide, ND6 (MT-ND6) polypeptide, C1QTNF5 polypeptide, VPS13B polypeptide, KIF11 polypeptide, MT-TL1 polypeptide, KLHL7 polypeptide, ACO2 polypeptide, C21orf2 (also known as CFAP410) polypeptide, AHI1 polypeptide, KIZ polypeptide, SPATA7 polypeptide, TTLL5 polypeptide, HGSNAT polypeptide, NRL polypeptide, OAT polypeptide, FLVCR1 polypeptide, ABCC6 polypeptide, LRAT polypeptide, CEP78 polypeptide, CDH3 polypeptide, FZD4 polypeptide, BBS12 polypeptide, HK1 polypeptide, PRDM13 polypeptide,ADAM9 polypeptide, BBS7 polypeptide, CABP4 polypeptide, ABHD12 polypeptide, COL18A1 polypeptide, MFRP polypeptide, RIMS1 polypeptide, ROM1 polypeptide, BBS4 polypeptide, IMPG1 polypeptide, INPP5E polypeptide, VCAN polypeptide, POC1B polypeptide, RAX2 polypeptide, TSPAN12 polypeptide, CACNA2D4 polypeptide, JAG1 polypeptide, MKKS polypeptide, NPHP4 polypeptide, BBS9 polypeptide, COL11A1 polypeptide, ELOVL4 polypeptide, NDP polypeptide, NPHP1 polypeptide, RGR polypeptide, BBS5 polypeptide, WDR19 polypeptide, C8ORF37 polypeptide, CTNNA1 polypeptide, LAMP2 polypeptide, PEX1 polypeptide, PHYH polypeptide, ATF6 polypeptide, PRPS1 polypeptide, SEMA4A polypeptide, ARL6 polypeptide, CNNM4 polypeptide, OTX2 polypeptide, PRPF6 polypeptide, RBP3 polypeptide, PNPLA6 polypeptide, S LC24A1 polypeptide, USH1G polypeptide, PITPNM3 polypeptide, TTC8 polypeptide, ARSG polypeptide, CWC27 polypeptide, DRAM2 polypeptide, PRCD polypeptide, REEP6 polypeptide, SSBP1 polypeptide, LAMA1 polypeptide, RAB28 polypeptide, ZNF408 polypeptide, GNAT1 polypeptide, IDH3A polypeptide, PDE6G polypeptide, PEX6 polypeptide, TUB polypeptide, CEP250 polypeptide, FSCN2 polypeptide, GRK1 polypeptide, RBP4 polypeptide, RD3 polypeptide, AGBL5 polypeptide, CAPN5 polypeptide, IFT172 polypeptide, KCNJ13 polypeptide, PAX2 polypeptide, CC2D2A polypeptide, HMCN1 polypeptide, MT-ATP6 polypeptide, RCBTB1 polypeptide, ARL2BP polypeptide, CA4 polypeptide, DFNB31 polypeptide, GNB3 polypeptide, MMACHC polypeptide, PRPF4 polypeptide, RGS9 polypeptide, ARHGEF18 polypeptide, KIAA1549 polypeptide, MKS1 polypeptide,MTTP (not MT-TP) polypeptide, PLK4 polypeptide, RPGRIP1L polypeptide, SDCCAG8 polypeptide, SRD5A3 polypeptide, TUBB4B polypeptide, ADAMTS18 polypeptide, ARL3 polypeptide, COL11A2 polypeptide, MVK polypeptide, NBAS polypeptide, OFD1 polypeptide, P3H2 polypeptide, RGS9BP polypeptide, CSPP1 polypeptide, ITM2B polypeptide, PANK2 polypeptide, PEX7 polypeptide, POMGNT1 polypeptide, SLC4A7 polypeptide, TMEM231 polypeptide, TRNT1 polypeptide, TUBGCP6 polypeptide, ZNF513 polypeptide, AFG3L2 polypeptide, ARL13B polypeptide, C5ORF42 (aka CPLANE1) polypeptide, COL9A1 polypeptide, CTSD polypeptide, DTHD1 polypeptide, DYNC2H1 polypeptide, IFT81 polypeptide, KIAA0586 polypeptide, MFN2 polypeptide, NPHP3 polypeptide, PCYT1A polypeptide, PEX12 polypeptide, PLA2G5 polypeptide , POC5 polypeptide, SCAPER polypeptide, SLC25A46 polypeptide, PCYT1A polypeptide, PEX12 polypeptide, PLA2G5 polypeptide, POC5 polypeptide, SCAPER polypeptide, SLC25A46 polypeptide, CEP164 polypeptide, CLCC1 polypeptide, COL9A2 polypeptide, CTNNB1 polypeptide, DHX38 polypeptide, GNPTG polypeptide, GRN polypeptide, GUCA1B polypeptide, IFT27 polypeptide, IFT74 polypeptide, KIAA0556 polypeptide, LRP2 polypeptide, MAPKAPK3 polypeptide, MIR204 polypeptide, MT-ND3 polypeptide, MT-RNR1 polypeptide, MT-TS2 polypeptide, ND5 (MT-ND5) polypeptide, NEK2 polypeptide, OPN1SW polypeptide, PEX13 polypeptide, PEX2 polypeptide, RHBDD2 polypeptide, SAMD11 polypeptide, SCLT1 polypeptide, SLC7A14 polypeptide, TCTN1 polypeptide, TCTN2 polypeptide, TLCD3B polypeptide, TREX1 polypeptide,TTPA polypeptide, UNC119 polypeptide, WDPCP polypeptide, ACBD5 polypeptide, AHR polypeptide, ARMC9 polypeptide, ASRGL1 polypeptide, ATOH7 polypeptide, B9D1 polypeptide, B9D2 polypeptide, BBIP1 polypeptide, C12ORF65 polypeptide, C2CD3 polypeptide, C5AR2 polypeptide, CCDC188 polypeptide, CCT2 polypeptide, CEP104 polypeptide, CEP120 polypeptide, CEP19 polypeptide, CEP41 polypeptide, CISD2 polypeptide, CLUAP1 polypeptide, COL9A3 polypeptide, CRB2 polypeptide, CTC1 polypeptide, DACT2 polypeptide, DDR1 polypeptide, ENSA polypeptide, ESPN polypeptide, EXOSC2 polypeptide, FBN3 polypeptide, GDF6 polypeptide, GPR125 polypeptide, HKDC1 polypeptide, HMX1 polypeptide, IDH3B polypeptide, IFT43 polypeptide, IFT80 polypeptide, INVS polypeptide, KIAA0753 polypeptide, KIF3B polypeptide, KIF7 polypeptide, LRRTM4 polypeptide, LZTFL1 polypeptide, MT-A TP8 polypeptide, MT-CO1 polypeptide, MT-CO2 polypeptide, MT-CO3 polypeptide, MT-CYB polypeptide, MT-ND2 polypeptide, MT-ND4L polypeptide, MT-RNR2 polypeptide, MT-TA polypeptide, MT-TC polypeptide, MT-TD polypeptide, MT-TE polypeptide, MT-TF polypeptide, MT-TG polypeptide, MT-TH polypeptide, MT-TI polypeptide, MT-TK polypeptide, MT-TL2 polypeptide, MT-TM polypeptide, MT-TN polypeptide, MT-TP (but not MTTP) polypeptide, MT-TQ polypeptide, MT-TR polypeptide, MT-TS1 polypeptide, MT-TT polypeptide, MT-TV polypeptide, MT-TW polypeptide, MT-TY polypeptide, NEUROD1 polypeptide, PDE6D polypeptide, PEX10 polypeptide, PEX11B polypeptide, PEX14 polypeptide, PEX16 polypeptide, PEX19 polypeptide, PEX26 polypeptide, PEX3 polypeptide, PEX5 polypeptide, PGK1 polypeptide, PISD polypeptide,PPP2R3C polypeptide, PROS1 polypeptide, PSEN1 polypeptide, RDH11 polypeptide, RRM2B polypeptide, SMARCA4 polypeptide, SPP2 polypeptide, TCTN3 polypeptide, TEAD1 polypeptide, TMEM107 polypeptide, TMEM138 polypeptide, TMEM216 polypeptide, TMEM67 polypeptide, TPP1 polypeptide, TRIM32 polypeptide, USP45 polypeptide, and ZNF423 polypeptide.

[0078] The AAV vectors provided herein (e.g., AAV2 vectors) (e.g., AAV vectors containing an AAV capsid polypeptide comprising an amino acid sequence set forth in Table 1 (or a variant thereof) or Formula A and an exogenous nucleic acid sequence encoding a therapeutic RNA and / or polypeptide) can be used to treat any suitable retinal pathology (e.g., a retinal disease). Examples of such retinal conditions include, but are not limited to, Leber's congenital amaurosis (LCA), Leber's hereditary optic neuropathy (LHON), oculocutaneous albinism type 1 (OCA1), retinitis pigmentosa, rod / cone dystrophy, cone dystrophy, rod dystrophy, Stargardt disease, Usher syndrome, X-linked retinitis pigmentosa (XLRP), X-linked retinoschisis (XLRS), choroideremia, achromatopsia, blue-cone monochromatism, color vision deficiency, glaucoma, optic atrophy, Batten disease, congenital stationary night blindness (CSNB), macular degeneration, CRB1-related retinal dystrophy, and foveal cone dystrophy.

[0079] Examples of therapeutic RNAs and polypeptides that can be delivered using the AAV vectors provided herein to treat specific retinal pathologies are listed in Tables 2 and 3. Examples of genomic nucleic acids that can be inactivated and / or knocked out to treat specific retinal pathologies using one or more AAV vectors provided herein designed to deliver gene editing components are listed in Table 3. Examples of genomic nucleic acids of disease-causing alleles that can be replaced with healthy alleles to treat specific retinal pathologies using one or more AAV vectors provided herein designed to deliver gene editing components are listed in Table 3.

[0080] [Table 2-1]

[0081] [Table 2-2]

[0082] [Table 3-1]

[0083] [Table 3-2]

[0084] [Table 3-3]

[0085] In some cases, the AAV vectors provided herein are designed to express one or more polypeptides capable of inhibiting angiogenesis, and can be used to treat retinal pathologies. Examples of polypeptides capable of inhibiting angiogenesis that can be used as described herein include, but are not limited to, monoclonal anti-VEGF antibody polypeptides, angiostatin polypeptides, siRNA polypeptides, and endostatin polypeptides. In some cases, the AAV vectors provided herein are designed to express monoclonal anti-VEGF antibody polypeptides, angiostatin polypeptides, siRNA, and / or endostatin polypeptides, and can be used to treat wet AMD. In some cases, the AAV vectors provided herein are designed to express monoclonal anti-VEGF antibody polypeptides, angiostatin polypeptides, siRNA, and / or endostatin polypeptides, and can be used to treat diabetic retinopathy. In some cases, the AAV vectors provided herein are designed to express monoclonal anti-VEGF antibody polypeptides, angiostatin polypeptides, siRNA, and / or endostatin polypeptides, and can be used to treat diabetic macular edema.

[0086] In some cases, the AAV vectors provided herein are designed to express one or more polypeptides with neuroprotective capabilities, and can be used to treat retinal pathologies. Examples of polypeptides with the ability to confer neuroprotective capabilities that can be used as described herein include, but are not limited to, GDNF polypeptides, CNTF polypeptides, IGF-1 polypeptides, VEGF polypeptides, and BNDF polypeptides. In some cases, the AAV vectors provided herein are designed to express GDNF polypeptides, CNTF polypeptides, IGF-1 polypeptides, VEGF polypeptides, and / or BNDF polypeptides, and can be used to treat wet AMD. In some cases, the AAV vectors provided herein are designed to express GDNF polypeptides, CNTF polypeptides, IGF-1 polypeptides, VEGF polypeptides, and / or BNDF polypeptides, and can be used to treat dry AMD. In some cases, the AAV vectors provided herein are designed to express GDNF polypeptides, CNTF polypeptides, IGF-1 polypeptides, VEGF polypeptides, and / or BNDF polypeptides, and can be used to treat diabetic retinopathy. In some cases, diabetic macular edema can be treated using the AAV vectors provided herein that are designed to express a GDNF polypeptide, a CNTF polypeptide, an IGF-1 polypeptide, a VEGF polypeptide, and / or a BNDF polypeptide.

[0087] In some cases, the AAV vectors provided herein are designed to express one or more polypeptides that confer optogenetic capabilities and can be used to treat retinal pathologies. Examples of polypeptides capable of conferring optogenetic capabilities that can be used as described herein include, but are not limited to, ChR polypeptides, ChR2 polypeptides, ArchT polypeptides, NpHR polypeptides, and ChrimsonR polypeptides. In some cases, the AAV vectors provided herein are designed to express ChR polypeptides, ChR2 polypeptides, ArchT polypeptides, NpHR polypeptides, and / or ChrimsonR polypeptides and can be used to treat wet AMD. In some cases, the AAV vectors provided herein are designed to express ChR polypeptides, ChR2 polypeptides, ArchT polypeptides, NpHR polypeptides, and / or ChrimsonR polypeptides and can be used to treat dry AMD. In some cases, the AAV vectors provided herein are designed to express ChR polypeptides, ChR2 polypeptides, ArchT polypeptides, NpHR polypeptides, and / or ChrimsonR polypeptides and can be used to treat diabetic retinopathy. In some cases, diabetic macular edema can be treated using the AAV vectors provided herein designed to express a ChR polypeptide, a ChR2 polypeptide, an ArchT polypeptide, an NpHR polypeptide, and / or a ChrimsonR polypeptide.

[0088] In some cases, the AAV vectors provided herein are designed to express one or more polypeptides capable of inhibiting apoptosis, and can be used to treat retinal pathologies. Examples of polypeptides capable of inhibiting apoptosis that can be used as described herein include, but are not limited to, XIAP polypeptide, cIAP1 polypeptide, C-IAP2 polypeptide, Livin polypeptide, and Survivin polypeptide. In some cases, the AAV vectors provided herein are designed to express XIAP polypeptide, cIAP1 polypeptide, C-IAP2 polypeptide, Livin polypeptide, and / or Survinin polypeptide, and can be used to treat wet AMD. In some cases, the AAV vectors provided herein are designed to express XIAP polypeptide, cIAP1 polypeptide, C-IAP2 polypeptide, Livin polypeptide, and / or Survinin polypeptide, and can be used to treat diabetic retinopathy. In some cases, the AAV vectors provided herein are designed to express XIAP polypeptide, cIAP1 polypeptide, C-IAP2 polypeptide, Livin polypeptide, and / or Survinin polypeptide, and can be used to treat diabetic macular edema.

[0089] In some cases, the AAV vectors provided herein are designed to express one or more polypeptides capable of inhibiting complement and can be used to treat retinal pathologies. Examples of polypeptides capable of inhibiting complement that can be used as described herein include, but are not limited to, complement factor I polypeptides, complement factor H polypeptides, and sCD59 polypeptides. In some cases, the AAV vectors provided herein are designed to express complement factor I polypeptides, complement factor H polypeptides, and / or sCD59 polypeptides and can be used to treat wet AMD. In some cases, the AAV vectors provided herein are designed to express complement factor I polypeptides, complement factor H polypeptides, and / or sCD59 polypeptides and can be used to treat dry AMD. In some cases, the AAV vectors provided herein are designed to express complement factor I polypeptides, complement factor H polypeptides, and / or sCD59 polypeptides and can be used to treat diabetic retinopathy. In some cases, diabetic macular edema can be treated using the AAV vectors provided herein that are designed to express a complement factor I polypeptide, a complement factor H polypeptide, and / or a sCD59 polypeptide.

[0090] In some cases, the AAV vector provided herein is designed to express one or more polypeptides capable of inducing survival factors, and can be used to treat retinal pathology. Examples of polypeptides capable of inducing survival factors that can be used as described herein include, but are not limited to, RdCVF polypeptide, RdCVFL polypeptide, HIF-1 polypeptide, IAP family polypeptide, and BCL-2 family polypeptide. In some cases, the AAV vector provided herein is designed to express RdCVF polypeptide, RdCVFL polypeptide, HIF-1 polypeptide, IAP family polypeptide, and / or BCL-2 family polypeptide, and can treat wet AMD. In some cases, the AAV vector provided herein is designed to express RdCVF polypeptide, RdCVFL polypeptide, HIF-1 polypeptide, IAP family polypeptide, and / or BCL-2 family polypeptide, and can treat dry AMD. In some cases, AAV vectors provided herein designed to express RdCVF polypeptides, RdCVFL polypeptides, HIF-1 polypeptides, IAP family polypeptides, and / or BCL-2 family polypeptides can be used to treat diabetic retinopathy. In some cases, AAV vectors provided herein designed to express RdCVF polypeptides, RdCVFL polypeptides, HIF-1 polypeptides, IAP family polypeptides, and / or BCL-2 family polypeptides can be used to treat diabetic macular edema.

[0091] Any suitable method can be used to administer the AAV vectors provided herein or the compositions (e.g., pharmaceutical compositions) provided herein to a mammal (e.g., a human or non-human primate). For example, the compositions provided herein (e.g., pharmaceutical compositions containing one or more AAV vectors provided herein) can be administered to a mammal (e.g., a human or non-human primate) intravitreally, intravenously (e.g., by intravenous injection or infusion), subcutaneously (e.g., by subcutaneous injection), intraperitoneally (e.g., by intraperitoneal injection), orally, by inhalation, intramuscularly (e.g., by intramuscular injection), subretinal, intravitreal, systemic, or suprachoroidally. In some cases, the route and / or mode of administration of the composition (e.g., pharmaceutical compositions provided herein) can be adjusted for the mammal being treated.

[0092] In some cases, an effective amount of a composition (e.g., a pharmaceutical composition provided herein) containing an AAV vector provided herein for treating a retinal pathology can be an amount that reduces the severity of one or more symptoms of the retinal pathology and / or slows the progression of the retinal pathology without producing significant toxicity to a mammal. For example, an effective amount of an AAV vector provided herein can be about 1×10 7 viral genome ~ approx. 1 × 10 14 10 viral genome (e.g., approximately 1 × 10 7 viral genome ~ approx. 1 × 10 13 viral genome, approximately 1 × 10 7 viral genome ~ approx. 1 × 10 12 viral genome, approximately 1 × 10 7 viral genome ~ approx. 1 × 10 11 viral genome, approximately 1 × 10 7 viral genome ~ approx. 1 × 10 10 viral genome, approximately 1 × 10 8 viral genome ~ approx. 1 × 10 14 viral genome, approximately 1 × 10 9 viral genome ~ approx. 1 × 10 14 viral genome, approximately 1 × 10 10 viral genome ~ approx. 1 × 1014 viral genome, approximately 1 × 10 8 viral genome ~ approx. 1 × 10 12 viral genome, or approximately 1 × 10 9 viral genome ~ approx. 1 × 10 11 10 viral genomes). In some cases, an effective amount of an AAV vector provided herein can be about 1 x 10 10 1 viral genome / kg body weight ~ approx. 1 x 10 14 viral genomes / kg body weight (e.g., approximately 1 × 10 10 1 viral genome / kg body weight ~ approx. 1 x 10 13 viral genomes / kg body weight, approximately 1 x 10 10 1 viral genome / kg body weight ~ approx. 1 x 10 12 viral genomes / kg body weight, approximately 1 x 10 10 1 viral genome / kg body weight ~ approx. 1 x 10 11 The effective dose can be a constant amount, or can be adjusted on a sliding scale or as a variable dose depending on the mammal's response to treatment. Various factors may affect the actual effective amount used for a particular application. For example, the severity of the retinal pathology, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of concurrent use with other therapeutic or prophylactic treatments, such as the use of other retinal drugs, and the judgment of the treating physician may require an increase or decrease in the actual effective amount of the compositions provided herein (e.g., pharmaceutical compositions containing AAV vectors provided herein) administered.

[0093] In some cases, the effective administration frequency of the compositions containing the AAV vectors provided herein (e.g., pharmaceutical compositions provided herein) can be a frequency that reduces the severity of one or more symptoms of a retinal pathology and / or slows the progression of a retinal pathology without producing significant toxicity to the mammal. Various factors may affect the actual effective frequency used for a particular application. For example, the severity of the retinal pathology, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of concurrent use with other therapeutic or prophylactic treatments, such as the use of other retinal drugs, and the judgment of the treating physician may require an increase or decrease in the actual effective administration frequency of the compositions provided herein.

[0094] In some cases, the effective administration period of a composition containing an AAV vector provided herein (e.g., a pharmaceutical composition provided herein) can be a period that reduces the severity of one or more symptoms of a retinal pathology and / or slows the progression of a retinal pathology without producing significant toxicity to the mammal. For example, the effective administration period of a pharmaceutical composition provided herein can range from a single administration to several weeks to several months (e.g., 4 to 12 weeks). In some cases, the period can be for the life of the mammal. Several factors can affect the actual effective period used for a particular application. For example, the severity of the retinal pathology, the route of administration, the age and general health of the mammal, the use of excipients, the possibility of concurrent use with other therapeutic or prophylactic treatments, such as the use of other retinal drugs, and the judgment of the treating physician may require an increase or decrease in the actual effective administration period of a composition provided herein (e.g., a pharmaceutical composition containing an AAV vector provided herein).

[0095] In some cases, an effective amount of a composition containing an AAV vector provided herein (e.g., a pharmaceutical composition provided herein) for treating a retinal condition can be administered once or twice to a mammal (e.g., a human or non-human primate) to treat the mammal.

[0096] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0097] Example 1 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0098] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0099] An AAV vector having a capsid polypeptide comprising SEQ ID NO:14 (SEQ ID NO:5 located between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) demonstrated the ability to infect and drive mRNA expression in retinal cells in a manner similar to the 7m8 AAV vector. Thus, an AAV vector having a capsid polypeptide comprising SEQ ID NO:14 has a higher ability to infect retinal cells than a wild-type AAV2 vector containing an AAV2 capsid polypeptide consisting of SEQ ID NO:1.

[0100] Taken together, these results demonstrate that AAV vectors comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression in retinal cells.

[0101] Example 2 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0102] Example 3 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated, and specific AAV vectors with the ability to exhibit high efficiency and / or specificity for infection of the foveal cones in the retina were screened from these generated AAV vectors. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0103] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0104] AAV vectors with capsid polypeptides containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (18 vectors) or an amino acid sequence insert as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (1 vector) mediated expression in foveal cells. AAV vectors were ranked based on an overall ranking, with +++ indicating that it was performed in the top third of the vectors tested, ++ indicating that it was performed in the middle third of the vectors tested, and ++ indicating that it was performed in the bottom third of the vectors tested. These were judged in terms of the overall degree of gene expression in foveal cells. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) gave a "+". No expression was detected within the detection limit when using wild-type AAV2 vectors.

[0105] Taken together, these results demonstrate that an AAV vector comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression in the foveal cone following intravitreal injection.

[0106] Example 4 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0107] Example 5 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0108] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0109] AAV vectors with capsid polypeptides containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (305 vectors in total, less than three unique vectors in total present more than once) or an amino acid sequence insert as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (19 vectors) mediated expression in retinal cells. AAV vectors were ranked based on an overall ranking, with +++ indicating that it was performed in the top third of vectors tested, ++ indicating that it was performed in the middle third of vectors tested, and ++ indicating that it was performed in the bottom third of vectors tested. These were judged in terms of the overall degree of gene expression in retinal cells. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) gave one "+++", four "++", and one "+". When the wild-type AAV2 vector was used, no expression was detected within the detection limits.

[0110] Taken together, these results demonstrate that an AAV vector comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression (e.g., high expression) in retinal cells following intravitreal injection.

[0111] Example 6 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0112] Example 7 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0113] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0114] AAV vectors with capsid polypeptides containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (44 vectors, some overlapping) or an amino acid sequence insert as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (2 vectors) mediated expression in retinal cells across two or more retinal regions. AAV vectors were ranked based on an overall ranking, with +++ indicating that it was performed in the top third of the vectors tested, ++ indicating that it was performed in the middle third of the vectors tested, and ++ indicating that it was performed in the bottom third of the vectors tested. These were judged in terms of the overall degree of gene expression across retinal regions. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) yielded two "++". No expression was detected within the detection limits when using wild-type AAV2 vectors.

[0115] Taken together, these results demonstrate that an AAV vector comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of delivering a nucleic acid to and expressing a nucleic acid in retinal cells in at least two different retinal regions.

[0116] Example 8 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0117] Example 9 - Construction of AAV vectors containing mutant capsid polypeptides A high-throughput method was used to generate AAV vectors with mutant capsid polypeptides and screen for specific AAV vectors with the ability of these generated AAV vectors to show high efficiency and / or specificity for infecting retinal cells in the parafoveal region of the eye. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of the mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were repackaged by polymerase chain reaction (PCR) amplification to obtain a "repacked" library. In another version of the library, AAV vectors were injected into the retina of a primate, and then nucleic acids encoding AAV capsid polypeptides were amplified from the foveal cells to obtain an "enriched" library. Each replicate of the AAV library (eg, the original library, the repacked library, and the enriched library) was injected intravitreally into a primate eye.

[0118] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0119] AAV vectors with capsid polypeptides containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (79 vectors in total, less than three unique vectors in total present more than once) or an amino acid sequence insert as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (2 vectors) mediated expression in retinal cells in the parafoveal region. AAV vectors were ranked based on an overall ranking, with +++ indicating that it was performed in the top third of the vectors tested, ++ indicating that it was performed in the middle third of the vectors tested, and ++ indicating that it was performed in the bottom third of the vectors tested. These were judged in terms of the overall degree of gene expression in the parafoveal region of the retina. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) yielded one "++" and one "+". When the wild-type AAV2 vector was used, no expression was detected within the detection limits.

[0120] Taken together, these results demonstrate that an AAV vector comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression in retinal cells in the parafoveal region following intravitreal injection.

[0121] Example 10 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0122] Example 11 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0123] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0124] AAV vectors having a capsid polypeptide containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (76 vectors in total, less than three unique vectors in total occurring more than once in total) or as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (8 vectors) mediate the ability to deliver and express nucleic acids to multiple different retinal cell types in the eye, thereby providing an efficient method of delivering nucleic acids to many different retinal cell types. AAV vectors were ranked based on an overall ranking, with +++ indicating that it performed in the top third of the vectors tested, ++ indicating that it performed in the middle third of the vectors tested, and ++ indicating that it performed in the bottom third of the vectors tested. These were judged in terms of the overall degree of gene expression across cell types. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) received a "+". When the wild-type AAV2 vector was used, no expression was detected within the detection limits.

[0125] Taken together, these results demonstrate that AAV vectors comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression in multiple different retinal cells within the eye following intravitreal injection.

[0126] Example 12 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0127] Example 13 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0128] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0129] The amino acid sequence inserts located between amino acid residues 587 and 588 of SEQ ID NO:1 (10 vectors) mediated expression in RPE cells. The AAV vectors were ranked based on an overall ranking, with +++ indicating that it performed in the top third of the vectors tested, ++ indicating that it performed in the middle third of the vectors tested, and ++ indicating that it performed in the bottom third of the vectors tested. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) gave a "+". These were judged in terms of the overall gene expression level in RPE cells. No expression was detected within the detection limit when using wild-type AAV2 vectors.

[0130] Taken together, these results demonstrate that AAV vectors comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression in RPE cells following intravitreal injection.

[0131] Example 14 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0132] Example 15 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0133] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0134] AAV vectors with capsid polypeptides containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (72 vectors in total, less than three unique vectors in total present more than once in total) or an amino acid sequence insert as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (7 vectors) preferentially mediated expression in photoreceptor cells. AAV vectors were ranked based on an overall ranking, with +++ indicating that it was performed in the top third of vectors tested, ++ indicating that it was performed in the middle third of vectors tested, and ++ indicating that it was performed in the bottom third of vectors tested. These were judged in terms of the overall degree of gene expression in retinal cells. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) yielded one "+++", one "++", and one "+". When the wild-type AAV2 vector was used, no expression was detected within the detection limits.

[0135] Taken together, these results demonstrate that AAV vectors comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression preferentially in photoreceptor cells following intravitreal injection.

[0136] Example 16 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0137] Example 17 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0138] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0139] AAV vectors with capsid polypeptides containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (248 vectors in total, less than three unique vectors in total present more than once in total) or an amino acid sequence insert as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (15 vectors) mediated preferential expression in retinal ganglion cells. AAV vectors were ranked based on an overall ranking, with +++ indicating that it was performed in the top third of the vectors tested, ++ indicating that it was performed in the middle third of the vectors tested, and ++ indicating that it was performed in the bottom third of the vectors tested. These were judged in terms of the overall degree of gene expression in retinal cells. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) yielded one "+++", two "++", and one "+". When the wild-type AAV2 vector was used, no expression was detected within the detection limits.

[0140] Taken together, these results demonstrate that AAV vectors comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression preferentially in retinal ganglion cells following intravitreal injection.

[0141] Example 18 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0142] Example 19 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0143] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0144] AAV vectors with capsid polypeptides containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (67 vectors) or an amino acid sequence insert as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (7 vectors) mediated preferential expression in bipolar cells. AAV vectors were ranked based on an overall ranking, with +++ indicating that it was performed in the top third of the vectors tested, ++ indicating that it was performed in the middle third of the vectors tested, and ++ indicating that it was performed in the bottom third of the vectors tested. These were judged in terms of the overall degree of gene expression in retinal cells. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) yielded a "+". No expression was detected within the detection limits when using wild-type AAV2 vectors.

[0145] Taken together, these results demonstrate that AAV vectors comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression preferentially in bipolar cells following intravitreal injection.

[0146] Example 20 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0147] Example 21 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library. In another version of the library, an AAV vector was injected into the retina of a primate, and nucleic acid encoding an AAV capsid polypeptide was then amplified from the foveal cells to obtain an "enriched" library. Each iteration of the AAV library (e.g., the original library, the repacked library, and the enriched library) was injected intravitreally into the eye of a primate.

[0148] After injection, the AAV vectors competed with each other in vivo. Successful infection of the AAV vectors resulted in expression of the DNA barcode. Single cell suspensions were generated from isolated retinal tissues, and cDNA libraries of individual cells were generated using single cell microfluidic technology (10X Genomics). Based on the presence and amount of DNA barcodes in the transcriptomes from thousands of different cells of multiple parallel cell types, computational analysis was performed to identify optimal vectors according to cell specificity, expression levels, and / or other desirable characteristics. The performance of AAV capsid polypeptides was evaluated based on the level of mRNA transcripts rather than the presence of DNA, reflecting the ability of the AAV vector to drive expression of AAV vector nucleic acid, as opposed to simply having the ability to enter cells.

[0149] AAV vectors with capsid polypeptides containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (61 vectors) or an amino acid sequence insert as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (3 vectors) mediated preferential expression in OFF retinal ganglion cells. AAV vectors were ranked based on an overall ranking, with +++ indicating that it was performed in the top third of the vectors tested, ++ indicating that it was performed in the middle third of the vectors tested, and ++ indicating that it was performed in the bottom third of the vectors tested. These were judged in terms of the overall degree of gene expression in retinal cells. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) gave a "+++". No expression was detected within the detection limit when using wild-type AAV2 vectors.

[0150] Taken together, these results demonstrate that AAV vectors comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating transgene expression preferentially in OFF retinal ganglion cells following intravitreal injection.

[0151] Example 22 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0152] Example 23 - Construction of AAV vectors containing mutant capsid polypeptides Using a high-throughput method, AAV vectors with mutant capsid polypeptides were generated and screened for specific AAV vectors that have the ability to show high efficiency and / or specificity for infected retinal cells. See, for example, Ozturk et al., bioRxiv, 2020.10.01.323196(2020) and Ozturk et al., eLife, 10:e64175(2021). Briefly, highly complex libraries of AAV mutants were generated and injected into the eyes of primates (cynomolgus or rhesus macaques). These libraries were generated such that each AAV vector in the library contained a unique DNA barcode, which allowed tracking of mutant AAV capsid polypeptides. In one version of the library, successfully packaged AAV vectors were polymerase chain reaction (PCR) amplified and repackaged to obtain a "repacked" library.

[0153] The packaging performance of the AAV capsid polypeptides was assessed based on successful packaging in the original library and in the "repacked" library, and reflected the ability of the AAV vector to be packaged.

[0154] AAV vectors with capsid polypeptides containing an amino acid sequence insert located between amino acid residues 587 and 588 of SEQ ID NO:1 (19 vectors) or as a substitution of amino acid residues 585-590 of SEQ ID NO:1 (8 vectors) mediated efficient packaging. AAV vectors were ranked based on overall ranking, with +++ indicating performance in the top third of vectors tested, ++ indicating performance in the middle third of vectors tested, and ++ indicating performance in the bottom third of vectors tested. SEQ ID NO:14 (SEQ ID NO:5 inserted between amino acid residues 587 and 588 of SEQ ID NO:1, see e.g., FIG. 1) yielded a "+++".

[0155] Taken together, these results demonstrate that AAV vectors comprising an AAV capsid polypeptide (e.g., an AAV2 capsid polypeptide) having an amino acid sequence set forth in Table 1 (or Formula A) can be capable of mediating efficient packaging.

[0156] Example 24 - Treatment of retinal pathologies with AAV vectors An AAV vector was constructed to include an AAV2 capsid polypeptide having an amino acid sequence set forth in Table 1 (or Formula A) (e.g., SEQ ID NO: 2 or 5) and an exogenous nucleic acid sequence encoding a therapeutic polypeptide. The constructed AAV vector was then incubated at 37° C. for 1 h at 4° C. for 2 h at 4 ... 7 ~Approx. 1×10 14 The amount of AAV vector is administered intravitreally to a human identified as having a retinal pathology, where following administration, the severity of one or more symptoms of the retinal pathology is reduced and / or the progression of the retinal pathology is slowed.

[0157] Example 25 - AAV vectors containing mutant capsid polypeptides A highly diverse library of AAV variants (~1E+5 to ~1E+6 libraries) was pooled and injected into the eyes of rhesus and cynomolgus macaque non-human primates (n=4) via intravitreal injection. AAV was packaged with the ubiquitous CMV promoter driving expression of a GFP transgene. A barcode identifying the unique AAV variant was included after the GFP transgene. Thirty to sixty days after injection, single-cell RNA-Seq was used to quantitate GFP expression as a baseline for the performance of variants in the pool. The variant with sequence number 5 emerged as the top performer in this screen in four non-human primates. Performance was quantified according to the number of cells expressing the transgene.

[0158] In a separate experiment, AAV variants, including the variant with SEQ ID NO: 66, were cloned, packaged, and pooled together. The AAV variants were pooled and injected into the eyes of rhesus and cynomolgus non-human primates (n=3) by intravitreal injection. AAV was packaged with the ubiquitous CAG promoter driving expression of a GFP transgene. A barcode identifying the unique AAV variant was included after the GFP transgene. 30-60 days after injection, expression of GFP was quantified using single-cell RNA-Seq as a baseline for the performance of the variants in the pool. AAV2 (scientific name: adeno-associated virus 2 (isolate Srivastava / 1982); UniProt Taxon ID number 648242) was included in the mixture as a benchmark control in the screen. The performance of each variant was quantified according to the number of cells expressing the transgene. The variant containing SEQ ID NO:66 outperformed the naturally occurring serotype control and performed similarly to the recombinant serotype across all cell types in all non-human primates. Injection of the variant containing SEQ ID NO:5 also resulted in increased transgene expression per cell compared to that observed with the naturally occurring serotype (Table 4).

[0159] [Table 4]

[0160] These results demonstrate that AAV vectors having an AAV capsid polypeptide comprising the amino acid sequence set forth in Table 1 (or Formula A) efficiently infect retinal cells and result in high levels of expression of the delivered nucleic acid in these infected cells.

[0161] Example 26 - Additional Embodiments

[0162] Embodiment 1. An AAV capsid polypeptide comprising any one of the amino acid sequences of SEQ ID NOs: 2 to 5.

[0163] Embodiment 2. The polypeptide of embodiment 1, wherein the cell comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:10, except that the amino acid sequence of any one of SEQ ID NOs:2-5 is located between amino acid positions 587 and 588 of SEQ ID NO:1 or SEQ ID NO:10.

[0164] Embodiment 3. The polypeptide of embodiment 1, wherein the cell comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:10, except that the amino acid sequence of SEQ ID NO:5 is located between amino acid positions 587 and 588 of SEQ ID NO:1 or SEQ ID NO:10.

[0165] Embodiment 4. The polypeptide according to embodiment 1, comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that the amino acids at positions 585 to 590 of SEQ ID NO: 1 or SEQ ID NO: 10 are substituted with any one of the amino acid sequences of SEQ ID NOs: 2 to 5.

[0166] Embodiment 5. The polypeptide according to embodiment 1, comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 10, except that the amino acids at positions 585 to 590 of SEQ ID NO: 1 or SEQ ID NO: 10 are substituted with the amino acid sequence of SEQ ID NO: 2.

[0167]

[0023] Embodiment 6. The AAV vector comprising the polypeptide is at least 1 x 10 7 6. The polypeptide of any one of embodiments 1 to 5, wherein said vector at a titer of 0.1 to 0.5 infects more than 2.5% of retinal cells when administered intravitreally to a human eye.

[0168] Embodiment 7. The polypeptide according to any one of embodiments 1 to 6, wherein the AAV vector comprising said polypeptide expresses a nucleic acid in a retinal cell to a greater extent than from a corresponding AAV vector comprising a capsid polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:1.

[0169] Embodiment 8. A nucleic acid molecule encoding a polypeptide according to any one of embodiments 1 to 7.

[0170] Embodiment 9. The nucleic acid molecule of embodiment 8, wherein said nucleic acid molecule is DNA.

[0171] Embodiment 10. A host cell comprising the nucleic acid molecule of embodiment 8 or 9.

[0172] Embodiment 11. The host cell of embodiment 10, wherein the host cell expresses a vector comprising the polypeptide.

[0173] Embodiment 12. The host cell of embodiment 10, wherein the host cell expresses the polypeptide.

[0174] Embodiment 13. A host cell comprising a polypeptide according to any one of embodiments 1 to 7.

[0175] Embodiment 14. The host cell of any one of embodiments 10 to 13, wherein the host cell is a retinal cell.

[0176] Embodiment 15. A non-naturally occurring AAV capsid polypeptide, said capsid polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:10, including an amino acid sequence insert of Formula A located between amino acid positions 587 and 588 of SEQ ID NO:1 or SEQ ID NO:10, said Formula A comprising: -L1-EGSGRN (SEQ ID NO: 2)-L2- wherein L1 and L2 are each independently an optimal amino acid linker having 1, 2, or 3 amino acids.

[0177] Embodiment 16. The capsid polypeptide of embodiment 15, wherein L1 is one amino acid X1.

[0178] Embodiment 17. The capsid polypeptide of embodiment 17, wherein X1 is selected from the group consisting of amino acid residues consisting of A, V, I, and L.

[0179] Embodiment 18. The capsid polypeptide of embodiment 17, wherein X1 is A.

[0180] Embodiment 19. The capsid polypeptide of embodiment 15, wherein L1 is the two amino acids X2-X1.

[0181] Embodiment 20. The capsid polypeptide of embodiment 19, wherein X1 is selected from the group consisting of amino acid residues consisting of A, V, I, and L.

[0182] Embodiment 21. The capsid polypeptide of embodiment 19, wherein X1 is A.

[0183] Embodiment 22. The capsid polypeptide according to any one of embodiments 19 to 21, wherein X2 is selected from the group consisting of amino acid residues consisting of A, V, I, and L.

[0184] Embodiment 23. The capsid polypeptide of embodiment 22, wherein X2 is L.

[0185] Embodiment 24. The capsid polypeptide of embodiment 19, wherein X2-X1 is LA.

[0186] Embodiment 25. The capsid polypeptide of embodiment 15, wherein L1 is the three amino acids X3-X2-X1.

[0187] Embodiment 26. The capsid polypeptide of embodiment 25, wherein X1 is selected from the group consisting of amino acid residues consisting of A, V, I, and L.

[0188] Embodiment 27. The capsid polypeptide of embodiment 26, wherein X1 is A.

[0189] Embodiment 28. The capsid polypeptide according to any one of embodiments 25 to 27, wherein X2 is selected from the group consisting of amino acid residues consisting of A, V, I, and L.

[0190] Embodiment 29. The capsid polypeptide of embodiment 28, wherein X2 is L.

[0191] Embodiment 30. The capsid polypeptide of embodiment 25, wherein X2-X1 is LA.

[0192] Embodiment 31. The capsid polypeptide according to any one of embodiments 25 to 30, wherein X3 is selected from the group consisting of amino acid residues consisting of A, V, I, and L.

[0193] Embodiment 32. The capsid polypeptide of embodiment 15, wherein said L1 is absent.

[0194] Embodiment 33. The capsid polypeptide according to any one of embodiments 15 to 32, wherein L2 is one amino acid Z1.

[0195] Embodiment 34. The capsid polypeptide of embodiment 33, wherein Z1 is selected from the group consisting of amino acid residues A, V, I, and L.

[0196] Embodiment 35. The capsid polypeptide of embodiment 34, wherein Z1 is A.

[0197] Embodiment 36. The capsid polypeptide according to any one of embodiments 15 to 32, wherein L2 is two amino acids Z1-Z2.

[0198] Embodiment 37. The capsid polypeptide of embodiment 36, wherein Z1 is selected from the group consisting of amino acid residues A, V, I, and L.

[0199] Embodiment 38. The capsid polypeptide of embodiment 37, wherein Z1 is A.

[0200] Embodiment 39. The capsid polypeptide according to any one of embodiments 36 to 38, wherein Z2 is selected from the group consisting of amino acid residues consisting of A, V, I, and L.

[0201] Embodiment 40. The capsid polypeptide of embodiment 39, wherein Z2 is L.

[0202] Embodiment 41. The capsid polypeptide of embodiment 36, wherein Z1-Z2 is AL.

[0203] Embodiment 42. The capsid polypeptide according to any one of embodiments 15 to 32, wherein L2 is the three amino acids Z1-Z2-Z3.

[0204] Embodiment 43. The capsid polypeptide of embodiment 42, wherein Z1 is selected from the group consisting of amino acid residues A, V, I, and L.

[0205] Embodiment 44. The capsid polypeptide of embodiment 43, wherein Z1 is A.

[0206] Embodiment 45. The capsid polypeptide according to any one of embodiments 42 to 44, wherein Z2 is selected from the group consisting of amino acid residues A, V, I, and L.

[0207] Embodiment 46. The capsid polypeptide of embodiment 45, wherein Z2 is L.

[0208] Embodiment 47. The capsid polypeptide of embodiment 42, wherein Z1-Z2 is AL.

[0209] Embodiment 48. The capsid polypeptide according to any one of embodiments 42 to 47, wherein Z3 is selected from the group consisting of amino acid residues consisting of A, V, I, and L.

[0210] Embodiment 49. The capsid polypeptide of any one of embodiments 15 to 32, wherein said L2 is absent.

[0211] Embodiment 50. The capsid polypeptide of embodiment 15, wherein the amino acid sequence insert comprises any one of SEQ ID NOs: 2-5.

[0212] Embodiment 51. A viral particle comprising a capsid polypeptide according to any one of embodiments 15 to 51.

[0213] Other embodiments While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An adeno-associated virus (AAV) capsid polypeptide having an amino acid sequence containing one of sequence numbers 2 through 5.

2. AAV capsid polypeptide has an amino acid sequence containing SEQ ID NO: 1 or SEQ ID NO: 10, and furthermore, one of the amino acid sequences of SEQ ID NOs: 2 to 5 is (i) Located between amino acid residues corresponding to positions 587 and 588 of SEQ ID NO: 1 or SEQ ID NO: 10, or (ii) Substituting any or all of the amino acid residues corresponding to positions 585-590 in SEQ ID NO: 1 or 10, AAV capsid polypeptide.

3. (i) The AAV capsid according to claim 1 or 2, (ii) An exogenous nucleic acid encoding RNA or polypeptide, (iii) Pharmaceutically acceptable excipients, A composition containing particles that include [a certain component].

4. A composition comprising an AAV capsid for use in a method for delivering exogenous nucleic acid sequences to mammalian retinal cells, AAV particles containing an AAV capsid are administered to the eye of a mammal containing retinal cells. The particle comprises (i) an AAV capsid polypeptide having an amino acid sequence containing any one of sequence numbers 2 to 5, and (ii) the exogenous nucleic acid sequence. The AAV capsid infects the retinal cells, thereby delivering the exogenous nucleic acid sequence to the retinal cells. The aforementioned composition.

5. The AAV capsid polypeptide is (i) Except that any one of the amino acid sequences of Sequence IDs 2 to 5 is located between the amino acid residues corresponding to positions 587 and 588 of Sequence ID 1 or Sequence ID 10, (ii) Except that any or all of the amino acid residues corresponding to positions 585-590 of SEQ ID NO: 1 or SEQ ID NO: 10 are replaced by any one of the amino acid sequences of SEQ ID NOs: 2-5, The composition according to claim 4, having an amino acid sequence including SEQ ID NO: 1 or SEQ ID NO:

10.

6. The composition according to claim 4, wherein a mammal has a retinal disease, and the AAV particles infect the retinal cells of the eye, drive the expression of the exogenous nucleic acid sequence within the retinal cells, thereby treating the retinal disease.

7. The composition according to claim 6, wherein the mammal is a human.

8. The composition according to claim 6 or 7, wherein the retinal condition is selected from the group consisting of LCA, OCA1, pigmentary retinitis, rod / cone dystrophy, cone dystrophy, Stargard disease, Usher syndrome, XLRP, and XLRS.

9. It has an amino acid sequence containing SEQ ID NO: 1 or SEQ ID NO: 10, and further, an amino acid sequence insert of formula A is, (i) Located between amino acid residues corresponding to positions 587 and 588 of SEQ ID NO: 1 or SEQ ID NO: 10, or (ii) One or all of the amino acid residues corresponding to positions 585-590 of SEQ ID NO: 1 or SEQ ID NO: 10 are substituted, and formula A is, -L1-EGSGRN (Sequence ID 2)-L2- [In the formula, L1 and L2 are each independently optional amino acid linkers having 0, 1, 2, or 3 amino acids.] Adeno-associated virus (AAV) capsid polypeptide.

10. L1 is a single amino acid X1, and X1 is optionally selected from the group consisting of amino acid residues A, V, I, and L; or L1 is two amino acids X2-X1, wherein X1 is optionally selected from the group consisting of amino acid residues A, V, I, and L, and X2 is optionally selected from the group consisting of amino acid residues A, V, I, and L; or L1 is three amino acids X3-X2-X1, and X3 is optionally selected from the group consisting of amino acid residues A, V, I, and L; or The capsid polypeptide according to claim 9, wherein L1 is absent.

11. The L2 is a single amino acid Z1, and Z1 is optionally selected from the group consisting of amino acid residues A, V, I, and L; or The L2 is two amino acids Z1-Z2, and optionally, the Z2 is selected from the group consisting of amino acid residues A, V, I, and L; or L2 is three amino acids Z1-Z2-Z3, and Z3 is optionally selected from the group consisting of amino acid residues A, V, I, and L; or The aforementioned L2 does not exist. The capsid polypeptide according to claim 9 or 10.

12. A composition comprising AAV particles for use in a method of treating retinal disease, comprising administering an effective amount of AAV particles to the eye of a patient in need, wherein the AAV particles comprise an AAV capsid polypeptide and an exogenous nucleic acid sequence, and the AAV capsid polypeptide has an amino acid sequence comprising an amino acid insert represented by the following formula A: -L1-EGSGRN (Sequence ID 2)-L2- [In the formula, L1 and L2 are each independently optional amino acid linkers having 0, 1, 2, or 3 amino acids.] The aforementioned composition.

13. The composition according to claim 12, wherein the administration is intravitreous administration.

14. The composition according to claim 12 or 13, wherein the retinal disorder is a disease selected from the group consisting of LCA, OCA1, retinitis pigmentosa, rod / cone dystrophy, cone dystrophy, Stargard disease, Usher syndrome, XLRP, and XLRS.

15. A composition comprising AAV particles, wherein the AAV particles comprise a capsid having an amino acid sequence comprising SEQ ID NO: 1 or 10, and further comprises an amino acid sequence insert represented by formula A, wherein the insert is located between amino acid residues corresponding to amino acid positions 587 and 588 of SEQ ID NO: 1 or 10, The above formula A is, -L1-EGSGRN (Sequence ID 2)-L2- The composition, wherein L1 and L2 are each independently an optional amino acid linker having 0, 1, 2, or 3 amino acids.

16. AAV particles comprising a capsid polypeptide having an amino acid sequence including SEQ ID NO: 1 or 10, wherein the sequence of LAEGSGRNA (SEQ ID NO: 5) is located between amino acid residues corresponding to amino acid positions 587 and 588 of SEQ ID NO: 1 or 10.

17. An AAV vector comprising a polynucleotide encoding an AAV capsid polypeptide, wherein the capsid polypeptide has an amino acid sequence including one of sequence numbers 2 to 5.

18. The AAV vector according to claim 17, wherein the AAV capsid polypeptide has an amino acid sequence including SEQ ID NO: 1 or 10, and one of the sequences of SEQ ID NOs: 2 to 5 is located between amino acid residues corresponding to amino acid positions 587 and 588 of SEQ ID NO: 1 or 10.

19. The AAV vector according to claim 17, wherein the capsid polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or 10, and further comprises one of the amino acid sequences of SEQ ID NOs: 2 to 5 between the amino acid residues corresponding to amino acid positions 587 and 588 of SEQ ID NO: 1 or 10.

20. The AAV vector according to claim 17 for use in the production of AAV particles.

21. Mammalian cells comprising the AAV vector according to any one of claims 15 to 20.

22. A mammalian cell comprising a first AAV vector according to any one of claims 15 to 20, and a second AAV vector comprising an exogenous polynucleotide encoding RNA or polypeptide.

23. AAV particles produced using mammalian cells according to claim 21, which include an AAV vector.

24. An adeno-associated virus (AAV) capsid polypeptide, wherein the AAV capsid polypeptide comprises an amino acid sequence selected from any one of SEQ ID NOs: 2, 3, 4, or 5, and the amino acid sequence of any one of SEQ ID NOs: 2 to 5 is located within the capsid polypeptide between residues corresponding to amino acid positions 587 and 588 of SEQ ID NO: 1.

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