Adeno-associated virus variant capsids and applications for inhibiting angiogenesis

Abstract

This invention relates to adeno-associated virus (AAV) variant capsids and their applications in inhibiting angiogenesis. Variant AAV capsid proteins are provided herein, having one or more modifications in the amino acid sequence relative to the parental AAV capsid protein, which, when present in AAV viral particles, confer increased infectivity to one or more types of retinal cells compared to the infectivity of AAV viral particles containing unmodified parental AAV capsid proteins. Recombinant AAV viral particles and pharmaceutical compositions thereof comprising the variant AAV capsid proteins described herein are also provided, methods for manufacturing these rAAV capsid proteins and viral particles, and methods for using these rAAV capsid proteins and viral particles in research and in clinical practice, such as in the delivery of nucleic acid sequences to one or more cells of the retina to treat retinal conditions and diseases.

Description

[0001] This application is a divisional application of the invention patent filed on November 26, 2018, with application number 201880087687.3 (PCT / US2018 / 062478) and entitled "Adeno-associated virus variant capsid and its application for inhibiting angiogenesis".

[0002] Cross-references to related applications This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 590,976, filed November 27, 2017, and 62 / 664,726, filed April 30, 2018, the entire disclosure of each of which is incorporated herein by reference. Technical Field

[0003] The invention disclosed herein generally relates to the field of adeno-associated virus (AAV) viral particles containing variant capsid proteins and to the generation of such variant capsids using directed evolution techniques. Background Technology

[0004] Hereditary retinal diseases encompass a large number of heterogeneous genetic disorders, affecting approximately 1 in 3,000 people (more than 2 million worldwide), and are a leading cause of severe vision loss or blindness. Complex multifactorial retinal diseases such as wet age-related macular degeneration (wAMD) and diabetic retinopathy (DR) affect even more individuals; currently, 1.7 million Americans suffer from severe central vision loss associated with wAMD, and nearly one-third of adults over 40 years of age with diabetes have visual impairment. These diseases are typically associated with dysfunction or death of one or more types of retinal cells, sometimes attributed to a lack of expression or function of key proteins, such as RPE65 in LCA2; in other cases to gene mutations that produce toxic gene products, such as dominant mutations affecting rhodopsin protein folding; or in still other cases to retinal physiological changes induced by ectopic protein expression, such as VEGF in wAMD. One approach to addressing this significant unmet medical need is gene-based adeno-associated virus (AAV)-mediated therapy, in which recombinant adeno-associated virus (rAAV) is used to deliver genes to one or more cell types in the retina to, for example, replace missing genes, correct dominant defective genes, or provide templates for sequential protein therapies. While AAV-based clinical gene therapies have been increasingly successful, limitations remain regarding the nature of the viral vector, including, for example, the ability to efficiently target desired retinal cells. For instance, numerous primate AAV serotypes and many non-human primate serotypes have been identified and characterized, with AAV2 being the best-characterized AAV serotype and the first suitable candidate for gene delivery in the eye. However, these AAVs (including AAV2) have not been reported to efficiently transduce deeper cell types of the retina when delivered via intravitreal administration. Therefore, there is a need in the art for novel AAV variants with superior transduction capabilities that would provide more efficient gene-based delivery to retinal cells for the treatment of eye diseases. There is a need in the art for an AAV variant that exhibits an enhanced retinal transduction spectrum—that, in some cases, is widely used for certain retinal cell types, and in others, is preferentially used for certain retinal cell types, compared to wild-type AAV and AAV variants known in the art.

[0005] Naturally occurring AAVs are single-stranded DNA viruses containing three open reading frames. rep , cap and aap The first gene rep The second gene encodes four proteins essential for genome replication (Rep78, Rep68, Rep52, and Rep40). cap The gene expresses three structural proteins (VP1-3) that assemble to form the viral capsid, and the third gene expresses the assembly activation protein (AAP), which is crucial for capsid assembly. AAV relies on the presence of helper viruses (such as adenovirus or herpesvirus) for active replication. In the absence of helper viruses, AAV establishes a latent state, in which its genome is either maintained in a free manner or integrated into the host chromosome at the AAVS1 locus.

[0006] Directed evolution techniques, both in vitro and in vivo, can be used to select AAV variants that provide improvements over current AAV-based gene delivery vectors. Such directed evolution techniques are known in the art and described, for example, in PCT Publication WO2014 / 194132 and Kotterman & Schaffer (Nature Review Genetics, AOP, May 20, 2014 online; doi:10.1038 / nrg3742), both of which are incorporated herein by reference in their entirety. Directed evolution is a capsid engineering approach that mimics natural evolution through iterative rounds of genetic diversification and selection processes, thereby enabling the accumulation of beneficial mutations that progressively improve the function of biomolecules such as AAV-based viral particles. In this approach, wild-type AAVs are selected... cap Gene diversification is used to produce large genetic libraries, which are packaged to produce viral particle libraries, and selective pressure is applied to isolate unique variants with superior phenotypes that can overcome gene delivery barriers.

[0007] AAV variants have been disclosed in the following examples: U.S. Patent Nos. 9,193,956; 9,186;419; 8,632,764; 8,663,624; 8,927,514; 8,628,966; 8,263,396; 8,734,809; 8,889,641; 8,632,764; 8,691,948; 8,299,295; 8,802,440; 8,445,267; 8,906,307; 8,574,583; 8,067,015; 7,588,772; 7,867,484; 8,163,543; 8,283,151; 8,999,678; 7,892,809 7,906,111; 7,259,151; 7,629,322; 7,220,577; 8,802,080; 7,198,951; 8,318,480; 8,962,332; 7,790,449; 7,282,199; 8,906,675; 8,524,446; 7,71 2,893; 6,491,907; 8,637,255; 7,186,522; 7,105,345; 6,759,237; 6,984,517; 6,962,815; 7,749,492; 7,259,151; and 6,156,303; U.S. Publication No. 2013 / 0295614; 2015 / 0065562; 2014 / 0364338; 2013 / 0323226; 2014 / 0359799; 2013 / 0059732; 2014 / 0037585; 2014 / 0056854; 2013 / 0296409; 2014 / 0335054 2013 / 0195801; 2012 / 0070899; 2011 / 0275529; 2011 / 0171262; 2009 / 0215879; 2010 / 0297177; 2010 / 0203083; 2009 / 0317417; 2009 / 0202490; 2012 / 0220492; 2006 / 0292117; and 2004 / 0002159; European Open No. 2692731 A1; 2383346 B1; 2359865 B1; 2359866 B1; 2359867 B1; and 2357010 B1; 1791858B1; 1668143 B1; 1660678 B1; 1664314 B1; 1496944 B1; 1456383 B1; 2341068 B1; 2338900B1; 1456419 B1; 1310571 B1; 1456383 B1; 1633772 B1; and 1135468 B1;And international (PCT) publication numbers WO 2014 / 124282; WO 2013 / 170078; WO 2014 / 160092; WO 2014 / 103957; WO 2014 / 052789; WO2013 / 174760; WO 2013 / 123503; WO 2011 / 038187; and WO 2008 / 124015; WO 2003 / 054197; however, these references do not disclose embodiments and / or compositions of the AAV variants described herein and their characteristics and / or material structures.

[0008] All references and citations in this paper and in the cited patent documents are incorporated herein by reference. Summary of the Invention

[0009] This document provides a variant adeno-associated virus (AAV) capsid protein having one or more modifications in its amino acid sequence relative to the parental AAV capsid protein, which, when present in AAV viral particles, confers increased infectivity to one or more types of retinal cells compared to the infectivity of AAV viral particles containing the unmodified parental AAV capsid protein. Also provided are recombinant AAV viral particles and pharmaceutical compositions thereof comprising the variant AAV capsid protein described herein, methods for manufacturing the rAAV capsid protein and viral particles, and methods for using these rAAV capsid proteins and viral particles in research and in clinical practice, such as in the delivery of nucleic acid sequences to one or more cells of the retina to treat retinal conditions and diseases.

[0010] In some aspects of this disclosure, variant adeno-associated virus (AAV) capsid proteins are provided that have one or more modifications in the amino acid sequence relative to the parental AAV capsid, which, when present in AAV viral particles, confer increased infectivity to one or more types of retinal cells (e.g., photoreceptor cells (e.g., rods; cones), retinal ganglion cells (RGCs), glial cells (e.g., Müller glial cells, microglia), bipolar cells, amacrine cells, horizontal cells, and / or retinal pigment epithelial (RPE) cells) compared to the infectivity of AAV viral particles containing the parental AAV capsid protein to said retinal cells, wherein the parental AAV capsid protein does not include the amino acid sequence modifications.

[0011] In some aspects of this disclosure, recombinant AAV (rAAV) viral particles are provided, which include variant capsid proteins as described herein, wherein the rAAV viral particles exhibit increased infectivity to one or more types of retinal cells (e.g., photoreceptor cells) (e.g., rods; cones), retinal ganglion cells (RGCs), glial cells (e.g., Müller glial cells, microglia), bipolar cells, amacrine cells, horizontal cells, and / or retinal pigment epithelial (RPE) cells) relative to AAV viral particles comprising the corresponding unmodified parental AAV capsid protein. In some embodiments, the rAAV viral particles exhibit increased infectivity to all retinal cells relative to AAV viral particles comprising the parental AAV capsid protein. In other embodiments, the rAAV viral particles exhibit increased infectivity to certain cell types of the retina, but not others, relative to AAV viral particles comprising the parental AAV capsid protein. In other words, the rAAV viral particles exhibit increased infectivity for certain cell types of the retina rather than for others. For example, rAAV exhibits increased infectivity preferably for one or more cell types selected from photoreceptor cells, retinal ganglion cells, glial cells, bipolar cells, amacrine cells, horizontal cells, and / or retinal pigment epithelium (RPE) cells, but does not exhibit increased infectivity for all cell types.

[0012] In some embodiments, the rAAV viral particle comprises a heterologous nucleic acid. In some such embodiments, the heterologous nucleic acid encodes RNA that encodes a polypeptide. In other such embodiments, the heterologous nucleic acid sequence encodes RNA that does not encode a polypeptide, for example, the heterologous nucleic acid sequence encodes an RNA interference agent targeting a nuclease, a guide RNA, etc.

[0013] This article also provides pharmaceutical compositions comprising infectious rAAV viral particles and pharmaceutically acceptable carriers.

[0014] Also provided is the use of rAAV viral particles comprising the variant capsid protein described herein in a method of delivering heterologous nucleic acids to target cells (e.g., retinal cells) by contacting target cells with the rAAV viral particles. In some embodiments, the target cells are in vivo, for example, in the eye of an individual requiring treatment for an eye disease. In other embodiments, the target cells are in vitro. A method for treating an eye disease by administering an effective amount of rAAV viral particles to a subject in need of such treatment, said viral particles comprising a variant capsid protein as described herein or a pharmaceutical composition comprising an effective amount of rAAV viral particles is also provided. An isolated nucleic acid and a host cell comprising the isolated nucleic acid are also provided, the isolated nucleic acid comprising a sequence encoding a variant AAV capsid protein as described herein. In yet another embodiment, the isolated nucleic acid and / or the isolated host cell comprises rAAV.

[0015] In some respects, the variant AAV capsid protein includes an insertion of about 5 to about 20 amino acids (“heteropeptide” or “peptide insertion”) in the GH-ring of the capsid protein relative to the corresponding parental AAV capsid protein, wherein, when present in AAV viral particles, the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of AAV viral particles comprising the corresponding parental AAV capsid protein to retinal cells. In some embodiments, the peptide comprises a sequence selected from the group consisting of: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), QANANEN (SEQ ID NO:22), GKSKVID (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST (SEQ ID NO:26), LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA (SEQ ID NO:20), and 5ASDSTKAA (SEQ ID NO:21). NO:30), LANQDYTKTA (SEQ ID NO:31), LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA (SEQ ID NO:33), LAHQDTTKNA (SEQ ID NO:34), LANKTTNKDA (SEQ ID NO:35), LPISNENEHA (SEQ ID NO:36), LPQANANENA (SEQ ID NO:37), LAGKSKVIDA (SEQ ID NO:38), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ ID NO:40) and LAKDRAPSTA (SEQ ID NO:41).In some embodiments, the peptide is substantially composed of sequences selected from the group consisting of: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), QANANEN (SEQ ID NO:22), GKSKVID (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST (SEQ ID NO:26), LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA (SEQ ID NO:20), and 5ASDSTKAA (SEQ ID NO:21). The variant AAV capsid protein comprises one or more amino acid substitutions relative to the corresponding parental AAV capsid protein, wherein, when present in AAV viral particles, the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of AAV viral particles containing the corresponding parental AAV capsid protein. (SEQ ID NO:30), LANQDYTKTA (SEQ ID NO:31), LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA (SEQ ID NO:33), LAHQDTTKNA (SEQ ID NO:34), LANKTTNKDA (SEQ ID NO:35), LPISNENEAHA (SEQ ID NO:36), LPQANANENA (SEQ ID NO:37), LAGKSKVIDA (SEQ ID NO:38), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ ID NO:40), and LAKDRAPSTA (SEQ ID NO:41). In some aspects, the variant AAV capsid protein contains one or more amino acid substitutions relative to the corresponding parental AAV capsid protein, wherein, when present in AAV viral particles, the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of AAV viral particles containing the corresponding parental AAV capsid protein to retinal cells.

[0016] In related aspects, the variant AAV capsid protein comprises a peptide insertion and one or more amino acid substitutions relative to the corresponding parental AAV capsid protein, wherein, when present in AAV viral particles, the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of AAV viral particles containing the corresponding parental AAV capsid protein to retinal cells. This document also discloses a variant AAV capsid protein comprising the heteropeptide LAISDQTKHA (SEQ ID NO:28) and a P34A substitution relative to AAV2. In related embodiments, this document discloses an infectious recombinant AAV comprising a variant AAV capsid protein having at least 90% identity with the sequence shown in SEQ ID NO:42 and a heteronucleotide comprising a sequence encoding a VEGF inhibitor (preferably a VEGFa inhibitor), and discloses pharmaceutical compositions comprising said infectious recombinant AAV. In some embodiments, the heteronucleotide sequence encoding a VEGF inhibitor is selected from sequences encoding aflibercept, ranibizumab, single-chain ranibizumab (sc-ranibizumab LH or HL), sc-ranibizumab fused to an IgG Fc domain (sc-ranibizumab-Fc), or bromoizumab fused to an IgG Fc domain (bromoizumab-Fc). In related embodiments, the recombinant AAV comprises a heterologous nucleic acid containing two or more sequences, each of which encodes a VEGFa inhibitor (e.g., a first sequence encoding aflibercept and a second sequence encoding bromizumab). In a preferred embodiment, the heterologous nucleic acid sequence has any one of SEQ ID NO: 65, 67, 69, 70, 72, 74, 76 or a sequence having at least 90% identity with it. In other related embodiments, a method is provided for treating a patient with an eye disease associated with elevated intraocular VEGFa levels, comprising, preferably by intravitreal injection, administering to the patient an effective amount of infectious recombinant AAV and a heterologous nucleic acid, wherein the infectious recombinant AAV comprises a variant AAV capsid protein having at least 90% identity with the sequence shown in SEQ ID NO: 42, and the heterologous nucleic acid comprises a sequence encoding a VEGF inhibitor.

[0017] This article also discloses a variant AAV capsid protein comprising the heteropeptide LAISDQTKHA (SEQ ID NO:28) and amino acid substitutions N312K, N449D, N551S, I698V and L735Q relative to AAV2.

[0018] This document also discloses methods for manufacturing and / or delivering rAAV containing a variant AAV capsid as disclosed herein. Additionally, this document provides kits containing rAAV and uses for the methods described herein, wherein the rAAV contains a variant AAV capsid as disclosed herein.

[0019] In other embodiments, AAV viral particles comprising the variant capsid protein described in the foregoing paragraphs may be incorporated into any of the foregoing or subsequently disclosed embodiments. Indeed, it should be understood that certain features of the invention described for clarity in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, different features of the invention described for brevity in the context of a single embodiment may also be provided individually or in any suitable sub-combination. The invention specifically covers all combinations of embodiments relating to the invention and is disclosed herein as if each and every combination were individually and explicitly disclosed. Furthermore, the invention also specifically covers all sub-combinations of various embodiments and their elements and is disclosed herein as if each and every such sub-combination were individually and explicitly disclosed herein.

[0020] The summary is not intended to limit the scope of the claims, nor is it intended to restrict the scope of the invention in any way.

[0021] Other features and advantages of the invention disclosed herein will become apparent from the following figures, detailed descriptions, and claims.

[0022] Before describing the methods and compositions of the present invention, it should be understood that the invention is not limited to the specific methods or compositions described herein, and therefore variations are possible. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the invention is limited only by the appended claims.

[0023] The invention disclosed herein is illustrated in the accompanying drawings and description. However, although specific embodiments are shown in the drawings, the invention is not intended to be limited to the one or more specific embodiments shown and / or disclosed. Rather, the invention disclosed herein is intended to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention. Therefore, the drawings are intended to be illustrative rather than restrictive.

[0024] Where a numerical range is provided, it should be understood that, unless the context explicitly indicates otherwise, each intermediate value between the upper and lower limits of the range, down to one-tenth of the lower limit unit, is also specifically disclosed. Every smaller range between any stated value or intermediate value within the range and any other stated value or intermediate value within the range is included in this invention. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and every range in which neither limit nor both limits are included is also included in this invention, subject to any specific exclusion limit within the range. When the range includes one or two limits, ranges excluding one or both of those included limits are also included in this invention.

[0025] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, some possible and preferred methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference to disclose and describe methods and / or materials relating to the cited publications. It should be understood that this disclosure supersedes any disclosure of the incorporated publications to the extent that there may be contradiction.

[0026] Those skilled in the art will understand upon reading this disclosure that the individual embodiments described and illustrated herein have discrete components and features that can be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the invention. Any enumerated methods may be performed in the order of the enumerated events or in any other logically possible order.

[0027] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “recombinant AAV viral particles” includes a plurality of such viral particles, and reference to “photoreceptor cells” includes reference to one or more photoreceptor cells and their equivalents known to those skilled in the art, and so on. It should also be noted that the claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a prior basis for using exclusive terms such as “solely,” “only,” etc., in relation to the recitation of the elements of the claims, or for using a “negative” designation.

[0028] The publications discussed herein provide only their disclosure prior to the filing date of this application. This document should not be construed as an admission that the invention is not entitled to rely on prior inventions preceding these publications. Furthermore, the publication dates provided may differ from the actual publication dates, which may require independent verification. Attached Figure Description

[0029] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. This patent or application contains at least one color drawing. Upon request and payment of the necessary fees, the official authority will provide copies of this patent or patent application disclosure with one or more color drawings. It should be emphasized that, by convention, the various features in the drawings are not to scale. Instead, for clarity, the dimensions of different features have been arbitrarily enlarged or reduced. The drawings include the following figures: Figure 1 The implementation of the directed evolution methodology is described. Step (a) describes the techniques including DNA mutation technology and... capThe generation of a viral capsid library by combining genes. Step (b) describes the packaging of the virus, such that each viral particle is surrounded by a capsid encoding the virus and purified... cap The mutant capsid of the gene is composed. The capsid library is then placed under selection pressure in vitro or in vivo. In this aspect of directed evolution technology, tissue or cellular material of interest is harvested to isolate AAV variants that have been successfully infected with the target, and the successful virus is recovered. Step (c) depicts stage 1 enrichment of successful clones through repeated selection. Step (d) depicts stage 2 enrichment of the selected cap gene, which undergoes re-diversification and further selection steps to iteratively increase viral fit. Step (e) depicts variants identified as hits during vector selection stages 1 and 2, which will be manufactured as recombinant AAV vectors and characterized for transduction levels across various cell types and tissue targets. Based on the nature of the AAV directed evolution process, the variants disclosed herein have demonstrated the ability to transduce retinal cells and deliver the genome (the genome encoding the variant cap gene) during the selection process.

[0030] Figure 2 A schematic diagram of a retinal plane mounting is provided, showing where samples from which the viral genome is amplified are collected over a wide area of ​​the retina.

[0031] Figure 3 This image shows PCR amplification of the viral genome from retinal tissues representing the ganglion cell layer (GCL), nuclear layer (INL), photoreceptor / outer nuclear layer (ONL), and retinal pigment epithelium (RPE) layers of representative selection rings. Libraries were injected into both the right eye (top image) and the left eye (bottom image). Sampling was performed on the inner retina (internal), middle retina (intermediate), and outer / peripheral retina (outer). The bands within the red boxes represent successful amplification of the viral genome.

[0032] Figure 4 A-4D shows the frequency of motifs within the sequencing analysis. Figure 4 A provided the third round of sequencing analysis. Figure 4 B provided the fourth round of sequencing analysis. Figure 4 C provided the fifth round of sequencing analysis. Figure 4 D provided the 6th round of sequencing analysis.

[0033] Figure 5 A representative three-dimensional model of AAV2 containing random heptamers after amino acid 587 is provided.

[0034] Figure 6A-6WA comparison of wild-type AAV SEQ ID NOS:1-11 is provided, showing the amino acid positions between and across wild-type (naturally occurring) serotypes AAV1, AAV2, AAV3A, AAV3B and AAV4-10.

[0035] Figure 7 Provided for intravitreal application of 2x10 11 Heidelberg Spectralis retina of African green monkeys after AAV2 vector genome (vg) TM Fundus fluorescence images captured by the AAV2 gene delivering a GFP transgene under the control of the CMV promoter (AAV2.CMV.GFP). Images were taken at baseline (A) and on days 14 (B), 28 (C), and 42 (D) after injection.

[0036] Figure 8 Provided for intravitreal administration of 2x10 11 A novel AAV variant, LAISDQTKHA+P34A, was used in the vector genome (vg) of the Heidelberg Spectralis retina of African green monkeys. TM Fundus fluorescence images of the AAV variant delivered under the control of the CMV promoter (LAISDQTKHA+P34A.CMV.GFP). Images were taken at baseline (A) and on days 14 (B), 28 (C), and 42 (D) after injection.

[0037] Figure 9 Heidelberg Spectralis provides a retinal image of an African green monkey after intravitreal administration of the novel AAV variant LAISDQTKHA+P34A. TM Fundus fluorescence images captured, showing the AAV variant delivering the GFP transgene (LAISDQTKHA+P34A.CAG.EGFP) under the control of the CAG promoter. (A) with 2x10 11 (A) Intravitreal injection of a vg carrier into the retina of monkeys, imaging on day 14 (A1), day 21 (A2), and day 28 (A3) post-injection. (B) Using 1x10 12 The retina of monkeys was injected intravitreal with a vg vector, and imaging was performed on day 14 (B1) and day 21 (B2) after injection.

[0038] Figures 10A to 10E Provided intravitreal injection of 1x10 at 3 weeks post-injection analysis 12Results of immunohistochemical analysis of the monkey retina of the novel AAV variant LAISDQTKHA+P34A, which delivers the GFP transgene under the control of the CAG promoter. All immunohistochemicals are provided along with the corresponding fundus fluorescence images, with red boxes indicating approximate locations in the retina where the analyses were performed. Figure 10A Robust retinal pigment epithelium (RPE) and photoreceptor transduction were observed using a GFP-specific antibody (red). Cone photoreceptors were shown as white upon immunostaining with an M / L opsin antibody. Figure 10B and Figure 10C Robust rod and cone photoreceptors were observed by direct EGFP fluorescence (green) and immunohistochemistry using a GFP-specific antibody (red). Figure 10B ) and RPE ( Figure 10C Transduction. Melanocytes in RPE appear as black in the image. Figure 10D Transduction of cone photoreceptors (recognized by M / L opsin, white) and retinal ganglion cells (RGCs) in and around the fovea was observed by direct EGFP fluorescence (green) and immunohistochemistry using a GFP-specific antibody (red). The image in the middle figure is a higher magnification (63x) of the area indicated by the white box in the left figure. Figure 10E Transduction of retinal ganglion cells (RGCs) and retinal ganglion cell layers was observed using direct EGFP fluorescence (right image, green; bottom right image is a 63x magnification of top right image); the area under bright field illumination is shown in the upper left.

[0039] Figures 11A to 11F Data are provided for in vitro transduction of human retinal pigment epithelial (RPE) cells via recombinant AAV virus containing a novel AAV variant, LAISDQTKHA+P34A, capsid under the control of the CAG promoter, and a GFP transgene. Human embryonic stem cell lines were infected with either the novel AAV variant LAISDQTKHA+P34A.CAG.GFP or the wild-type control AAV2.CAG.GFP. Figure 11A and Figure 11C ) or cells derived from induced pluripotent stem cells (FB-iPSCs) from human fibroblasts that differentiate into RPE cells ( Figure 11B and 11D ). Figure 11A and 11B Immunofluorescence imaging of cell cultures 7 days after infection with an MOI of 500 showed that the novel AAV variant capsid (left panel) transduced RPE cells better than the wild-type AAV2 capsid (right panel). Figure 11C and Figure 11DQuantitative analysis of the percentage of GFP-positive RPE cells in each culture by flow cytometry revealed that the novel AAV variant capsid provided a significant dose-dependent improvement in the number of transduced cells compared to the wild-type AAV2 capsid, regardless of cell origin. Figure 11E and Figure 11F Quantitative analysis of GFP levels in each culture using Western blotting revealed that the novel AAV variants provided a significant improvement in transgene expression compared to the wild-type AAV2 capsid, regardless of cell origin.

[0040] Figure 12A-12F The implementation methods of the anti-VEGF protein are described. Figure 12A The design of aflibercept is described, which consists of a human Fltl signal peptide fused to the Fc region of human IgG1, VEGFR1 domain 2, and VEGFR2 domain 3. Figure 12B -C describes the design of ranibizumab, which includes a flexible protein linker to convert a double-stranded antigen-binding fragment (Fab) into a single-stranded Fab (scFab). Figure 12B The light-heavy (LH) form is described, which consists of a human Igκ light chain signal peptide linked by a flexible peptide, a variable light chain of ranibizumab, a constant light chain, a variable heavy chain, and a constant heavy chain 1 domain. Figure 12C The heavy-light (HL) form was described, which is similar to the LH form except that the signal peptide is derived from the human IgG heavy chain and the heavy and light chain domains are on opposite sides of the linker. Figure 12D The design of bromoizumab is described, which includes variable light chain and variable heavy chain domains linked by flexible peptides. Figure 12E The design of sc-ranibizumab LH-Fc is described, which consists of scFab LH form fused to the Fc region of human IgG1. Figure 12F The bromizumab-Fc design is described, consisting of bromizumab fused to the Fc region of human IgG1. Red bands indicate the complementarity-determining region (CDR). The gene was codon-optimized to improve expression from human cells and synthesized via GeneArt or GenScript, then inserted between the CAG promoter and the SV40 polyA signal in the pAAV-CAG-SV40 pA vector.

[0041] Figure 13A -B shows the ELISA results for detecting proteins that bind to VEGF. Figure 13A VEGF binding activity was detected in the culture medium of HEK293T cells transfected with aflibercept (SEQ ID NO:65) or single-chain (sc) ranibizumab (SEQ ID NO:67, 69 and 70) expression plasmids, but not in cells that were simulated or transfected with GFP expression vectors. Figure 13BVEGF binding activity was detected in the culture medium from HEK293T cells transfected with aflibercept (SEQ ID NO:65), sc-ranibizumab LH1 (SEQ ID NO:69), or bromizumab (SEQ ID NO:74) expression plasmids, but not in cells transfected with the GFP expression vector. The signal was very low with bromizumab, most likely due to poor recognition by the detection antibody. Error bars represent the standard deviation of four transfection wells.

[0042] Figure 14A -B provides a representative Western blot of the culture medium from HEK293T cells transfected with an anti-VEGF construct. Figure 14A The expression plasmids for aflibercept (SEQ ID NO: 65) or sc-ranibizumab (SEQ ID NO: 67, 69, and 70) are displayed. Both clinical Eylea and aflibercept samples were reduced to half of the dimer (including glycosylation) with an apparent molecular weight of 58 kD. Clinical Lucentis was reduced to separate light and heavy chains of 24 kD, while the sc-ranibizumab protein was not separated and migrated at approximately 48 kD. Higher amounts of the LH form of protein were present compared to the HL form, consistent with protein quantification obtained by ELISA. Figure 14B The plasmids for aflibercept (SEQ ID NO:65), sc-ranibizumab LH1 (SEQ ID NO:69), or bromizumab (SEQ ID NO:74) are displayed. The bromizumab signal is low, most likely due to poor recognition by the detection antibody. The protein migrates at the correct molecular weight of 26 kD. No signal was found in any simulated transfection or GFP negative control samples.

[0043] Figure 15A -B shows the results of a competitive ELISA assay for detecting free VEGF after incubation with culture medium from HEK293T cells transfected with an anti-VEGF construct. Figure 15A The plasmids for aflibercept (SEQ ID NO: 65) or sc-ranibizumab (SEQ ID NO: 67, 69, and 70) are shown. Inhibition curves for the four anti-VEGF proteins from the transfected samples are very similar to those for the clinically comparable proteins Eylea and Lucentis. Aflibercept and Eylea are more competitive against VEGF than the sc-ranibizumab variants and Lucentis. All three forms of sc-ranibizumab are nearly identical. Figure 15B The plasmids for aflibercept (SEQ ID NO:65), ranibizumab LH1 (SEQ ID NO:69), or bromizumab (SEQ ID NO:74) are displayed. The GFP negative control sample showed no competitive activity. Error bars represent the standard deviation of two copies of the transfection wells.

[0044] Figure 16 The results of a cellular VEGF neutralization assay are displayed. This assay used HEK293 cells expressing a VEGF receptor / β-galactosidase fusion protein, which produces active β-galactosidase upon VEGF binding. Cells were incubated with a mixture of VEGF and various diluted media derived from HEK293T cells transfected with aflibercept (SEQ ID NO:65) or sc-ranibizumab (SEQ ID NO:69) expression plasmids. Inhibition curves of anti-VEGF protein from transfected samples demonstrate that the anti-VEGF protein neutralizes VEGF activity at levels equivalent to clinically comparable proteins. Error bars represent the standard deviation of a duplicate assay.

[0045] Figure 17A -B shows the results of a VEGF neutralization assay performed using an equal volume of culture medium from HEK293T cells transfected with an anti-VEGF plasmid. Figure 17A Displaying expression plasmids for GFP, aflibercept (SEQ ID NO:65), sc-ranibizumab HL (SEQ ID NO:67), or sc-ranibizumab LH1 (SEQ ID NO:69). Neutralized VEGF was evaluated in all anti-VEGF constructs. Figure 17B Displays expression plasmids for GFP, aflibercept (SEQ ID NO:65), sc-ranibizumab LH1 (SEQ ID NO:69), and bromizumab (SEQ ID NO:74). Neutralized VEGF was assessed in all anti-VEGF constructs. A slight matrix effect was observed in the GFP control sample in the assayed dilutions. Error bars represent the standard deviation of two copies of the transfection wells. Figure 18 This image shows the ELISA results for detecting VEGF in culture medium from RPE cells transfected with plasmids expressing aflibercept (SEQ ID NO: 65) or ranibizumab (SEQ ID NOs: 67, 69, and 70). Both aflibercept and ranibizumab expression were observed to decrease VEGF levels. The effects of all three ranibizumab formulations on VEGF levels were similar. Error bars represent the standard deviation of four transfection wells.

[0046] Figure 19The ELISA results for VEGF in culture medium collected from RPE cells were displayed six or ten days after transduction with R100 capsids (having the amino acid sequence shown in SEQ ID NO: 42) expressing aflibercept (SEQ ID NO: 65), sc-ranibizumab (SEQ ID NO: 67 and 69), or bromizumab (SEQ ID NO: 74). Endogenous VEGF levels in cells transduced with the GFP control vector ranged from 4,500 to 8,300 pg / ml. Transduction with all anti-VEGF vectors produced undetectable VEGF levels in the culture medium. Error bars represent the standard deviation of four transduction wells.

[0047] Figure 20 This describes the ELISA results of proteins binding to VEGF in culture medium collected from RPE cells six or ten days after transduction with an R100 capsid expressing an anti-VEGF transgene (having the amino acid sequence shown in SEQ ID NO:42). VEGF binding activity was detected in the culture medium from cells transduced with aflibercept (SEQ ID NO:65), sc-ranibizumab (SEQ ID NO:67 and 69), or bromizumab (SEQ ID NO:74) expression vectors, but not from cells transduced with a GFP expression vector. The bromizumab signal was very low, most likely due to poor recognition by the detection antibody. Error bars represent the standard deviation of four transduction wells.

[0048] Figure 21 Representative Western blots of the protein were provided from culture media collected from RPE cells six or ten days after transduction with an R100 capsid expressing an anti-VEGF gene (having the amino acid sequence shown in SEQ ID NO:42). Clinical Eylea and aflibercept (SEQ ID NO:65) samples were both reduced to half of the dimer (including glycosylation) with an apparent molecular weight of 60 kD, as indicated by the black arrows. No bands migrated correctly in the GFP negative control sample. Clinical Lucentis was reduced to separate light and heavy chains of 24 kD, while sc-ranibizumab HL and LH (SEQ ID NO:67 and 69) did not separate and migrated with an apparent molecular weight of 58 kD, as indicated by the gray arrows. Bromizumab (SEQ ID NO:74) showed a low signal, most likely due to poor recognition by the detection antibody. The protein migrated with the correct molecular weight of 26 kD, as indicated by the dotted arrows.

[0049] Figure 22This study demonstrates the results of a competitive ELISA for detecting free VEGF six or ten days after transduction with an R100 capsid expressing an anti-VEGF transgene (having the amino acid sequence shown in SEQ ID NO:42), followed by incubation with culture medium collected from RPE cells. All anti-VEGF constructs competed for VEGF. The GFP negative control sample showed no competitive activity. Free VEGF levels were higher at the lowest dilution due to endogenous VEGF produced by RPE cells. Error bars represent the standard deviation of a duplicate assay.

[0050] Figure 23 The results of a cellular VEGF neutralization assay performed six days after transduction with an R100 capsid expressing an anti-VEGF transgene (having the amino acid sequence shown in SEQ ID NO:42) using medium collected from RPE cells are shown. Neutralized VEGF was assessed in all anti-VEGF constructs. No VEGF neutralization was observed with medium transduced from the GFP control. Error bars represent the standard deviation of the assay in duplicate.

[0051] definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology pertains.

[0052] Adeno-associated virus (AAV) is a non-pathogenic parvovirus consisting of a 4.7 kb single-stranded DNA genome within a non-enveloped icosahedral capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeat (ITR) sequences that serve as the origin of viral replication and packaging signals. rep ORF encodes four non-structural proteins that play a role in viral replication, transcriptional regulation, site-specific integration, and viral particle assembly. cap The ORF encodes three structural proteins (VP1-3), which assemble to form the 60-mer viral capsid. Finally, as... cap The ORF containing the alternative reading frame within the gene produces the assembly activation protein (AAP), a viral protein that localizes the AAV capsid protein to the cell nucleus and plays a role in capsid assembly.

[0053] Several naturally occurring (“wild-type”) serotypes and over 100 known AAV variants exist, each with a different amino acid sequence, particularly in the hypervariable region of the capsid protein and therefore in its gene delivery properties. No AAV is associated with any human disease, making recombinant AAV attractive for clinical application.

[0054] For the purposes of this publication, the term "AAV" is an adeno-associated virus, including but not limited to the virus itself and its derivatives. Unless otherwise stated, the term refers to all subtypes or serotypes, as well as replicative and recombinant types. The term "AAV" includes, but is not limited to, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3A (AAV-3A or AAV3A), AAV type 3B (AAV-3B or AAV3B), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV-5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV-8 or AAV8), AAV type 9 (AAV-9 or AAV9), AAV type 10 (AAV-10 or AAV10 or AAVrh10), avian AAV, bovine AAV, canine AAV, goat AAV, horse AAV, primate AAV, non-primate AAV, and sheep AAV. "Primate AAV" refers to AAV that infects primates, "Non-primate AAV" refers to AAV that infects non-primate mammals, and "Bovine AAV" refers to AAV that infects bovine mammals, etc.

[0055] The genomic sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep protein, and capsid subunit, are known in the art. These sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank login numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3A), NC_001729.1 (AAV3A), AF028705.1 (AAV3B), NC_001829.1 (AAV4), U89790.1 (AAV4), NC_006152.1 (AA5), AF085716.1 (AAV-5), AF028704.1 (AAV6), NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8). NC_006261.1 (AAV-8), AY530579.1 (AAV9), AAT46337 (AAV10) and AAO88208 (AAVrh10); the public information thereof is incorporated herein by reference to teach AAV nucleic acid and amino acid sequences. See, for example, Srivistava et al., (1983) J. Virology 45:555; Chiorini et al., (1998) J. Virology 71:6823; Chiorini et al., (1999) J. Virology 73:1309; Bantel-Schaal et al., (1999) J. Virology 73:939; Xiao et al., (1999) J. Virology 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al. (1986) J. Virol. 58:921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al., (2004) Virology 33:375-383; International Patent Publication WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303.

[0056] The sequences of naturally occurring cap proteins associated with AAV serotypes are known in the art and include those sequence numbers disclosed herein: such as AAV1 (SEQ ID NO:1), AAV2 (SEQ ID NO:2), AAV3A (SEQ ID NO:3), AAV3B (SEQ ID NO:4), AAV4 (SEQ ID NO:5), AAV5 (SEQ ID NO:6), AAV6 (SEQ ID NO:7), AAV7 (SEQ ID NO:8), AAV8 (SEQ ID NO:9), AAV9 (SEQ ID NO:10), AAV10 (SEQ ID NO:11), and AAVrh10 (SEQ ID NO:12). The terms "variant AAV capsid protein" or "AAV variant" refer to an AAV capsid protein comprising an amino acid sequence containing at least one modification or substitution (including deletion, insertion, point mutation, etc.) relative to a naturally occurring or "wild-type" AAV capsid protein sequence (e.g., as illustrated herein as SEQ ID NO:1-12). Variant AAV capsid proteins can share approximately 80% or more amino acid sequence identity with wild-type capsid proteins, for example, over 85%, over 90%, or over 95% identity, such as 98% or 99% identity. Variant AAV capsid proteins may not be wild-type capsid proteins.

[0057] For the purposes of this publication, “AAV virus particle” or “AAV virus particle” refers to a virus particle composed of at least one AAV capsid protein and capsidated AAV polynucleotide.

[0058] For the purposes of this disclosure, the term "rAAV" is an abbreviation for recombinant adeno-associated virus. "Recombination" as applied to polynucleotides refers to the product of various combinations of processes involving cloning, restriction, or ligation steps, and other processes that result in constructs of polynucleotides different from those found in nature. A recombinant virus is a viral particle containing recombinant polynucleotides. The terms respectively encompass replication of the original polynucleotide construct and the progeny of the original viral construct.

[0059] The term “rAAV vector” encompasses rAAV viral particles (i.e., infectious rAAV viral particles), and its definition includes rAAV polynucleotides; and also encompasses polynucleotides encoding rAAV (e.g., single-stranded polynucleotides encoding rAAV (ss-rAAV); double-stranded polynucleotides encoding rAAV (ds-rAAV), such as plasmids encoding rAAV; etc.).

[0060] If an AAV viral particle contains heteropolynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as transgenes to be delivered to target cells, RNAi agents to be delivered to target cells, or CRISPR agents), it is usually referred to as a "recombinant AAV (rAAV) viral particle" or "rAAV viral particle." Typically, at least one heteropolynucleotide is side-joined, and usually two AAV inverted terminal repeat (ITR) sequences.

[0061] The term "packaging" refers to a series of intracellular events that lead to the assembly and capsid formation of AAV particles. rep "and" cap "A gene is a polynucleotide sequence that encodes adeno-associated virus (AAV) replication and capsidation proteins." rep and cap In this paper, it is referred to as the AAV “packaging gene”.

[0062] The term "helper virus" in AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. A wide variety of helper viruses used for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia virus. Adenoviruses encompass many different subgroups, although subgroup C adenovirus type 5 is the most commonly used. Many adenoviruses of humans, non-human mammals, and birds are known and available from collections such as the ATCC. Viruses in the herpesvirus family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); these are also available from collections such as the ATCC.

[0063] The term "one or more helper viral functions" refers to one or more functions encoded in the helper viral genome that allow AAV replication and packaging (in conjunction with other requirements for replication and packaging as described herein). As described herein, "helper viral functions" can be provided in a variety of ways, including by providing a helper virus or by providing trans-production cells with a polynucleotide sequence encoding one or more essential functions. For example, a plasmid or other expression vector containing a nucleotide sequence encoding one or more adenoviral proteins may be transfected into production cells along with an rAAV vector.

[0064] The term "infectious" virus or viral particle is a term that contains an efficiently assembled viral capsid and is capable of delivering polynucleotide components into cells of tropical viral species. The term does not necessarily imply any viral replication capacity. Assays for counting infectious viral particles are described in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods for determining the ratio of infectious viral particles to total viral particles are known in the art. See, for example, Grainger et al., (2005) Mol. Ther. 11: S337 (description of TCID50 infectiousness titer assay); and Zolotukhin et al., (1999) Gene Ther. 6:973. See also the examples.

[0065] As used herein, the term "tendency" refers to a virus's (e.g., AAV) preferential targeting of cells of a particular host species or specific cell types within a host species. For example, a virus that can infect heart, lung, liver, and muscle cells has a broader (i.e., increased) tendency compared to a virus that can only infect lung and muscle cells. Tendency can also include a virus's dependence on a specific type of cell surface molecule of the host. For example, some viruses are able to infect only cells with surface glycosaminoglycans, while others are able to infect only cells with sialic acid (this dependence can be tested using various cell lines lacking a particular class of molecules as potential host cells for viral infection). In some cases, viral tendency describes a relative preference of the virus. For example, a first virus may be able to infect all cell types, but is much more successful in infecting those cells with surface glycosaminoglycans. If a second virus also prefers the same trait (e.g., the second virus is also more successful in infecting those cells with surface glycosaminoglycans), then the second virus can be considered to have similar (or the same) tendency as the first virus, even if the absolute transduction efficiency is not similar. For example, a second virus may be more effective than the first virus in infecting a given cell type for each test, but if the relative preferences are similar (or the same), the second virus can still be considered to have a similar (or the same) tropism as the first virus. In some embodiments, the tropism of viral particles containing the subject variant AAV capsid protein remains unchanged relative to naturally occurring viral particles. In some embodiments, the tropism of viral particles containing the subject variant AAV capsid protein is broadened (i.e., widened) relative to naturally occurring viral particles. In some embodiments, the tropism of viral particles containing the subject variant AAV capsid protein is reduced relative to naturally occurring viral particles.

[0066] The term "replicating" virus (e.g., replicating AAV) refers to a phenotypic wild-type virus that is infectious and also capable of replicating in infected cells (i.e., in the presence of helper virus or helper virus function). In the case of AAV, replication capability typically requires the presence of a functional AAV packaging gene. Typically, rAAV vectors as described herein are not replicable in mammalian cells (particularly human cells) due to the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequence to minimize the possibility of generating replicating AAV through recombination between AAV packaging genes and the entering rAAV vector. In many embodiments, rAAV vector formulations as described herein are those containing very little (if any) replicating AAV (rcAAV, also known as RCA) (e.g., per 10... 2 Each rAAV particle is smaller than approximately 1 rcAAV, and every 10 4 Each rAAV particle is less than approximately 1 rcAAV, and every 10 rAAV particles are less than approximately 1 rcAAV. 12 (The rAAV particles are smaller than approximately 1 rcAAV, or there are no rcAAV particles).

[0067] The term "polynucleotide" refers to a polymeric form of nucleotide of any length, comprising deoxyribonucleotides or ribonucleotides or analogues thereof. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogues, and may be interrupted by non-nucleotide components. If modifications to the nucleotide structure are present, they are performed before or after polymer assembly. As used herein, the term polynucleotide is used interchangeably to refer to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of polynucleotides herein covers both double-stranded forms and each of the two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0068] A polynucleotide or polypeptide shares a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that the percentage of bases or amino acids is the same when comparing the two sequences during alignment. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using the BLAST method and computer program, which is available at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, which is available from the Genetics Computing Group (GCG), a wholly owned subsidiary of Oxford Molecular Group, Inc. in Madison, Wisconsin, USA. Other techniques for alignment are described in Methods in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that allow for gaps in the sequence. The Smith-Waterman algorithm is one that allows for sequence alignment gaps. See Meth. Mol. Biol. 70: 173-187 (1997). Additionally, the GAP program, using the Needleman and Wunsch alignment methods, can be used for sequence alignment. See J. Mol. Biol. 48: 443-453 (1970).

[0069] The term "gene" refers to a polynucleotide that performs a specific function within a cell. For example, a gene may contain an open reading frame that encodes a gene product. One example of a gene product is a protein that is transcribed and translated from a gene. Another example of a gene product is RNA that is transcribed but not translated, such as functional RNA products, including aptamers of nucleases, interfering RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), non-coding RNA (ncRNA), guide RNA, etc.

[0070] The term "gene expression product" or "gene product" refers to a molecule produced by the expression of a specific gene as defined above. Gene expression products include, for example, polypeptides, aptamers, interfering RNA, messenger RNA (mRNA), rRNA, tRNA, and non-coding RNA (ncRNA).

[0071] The term "siRNA agent" ("small interfering" or "short interfering RNA" (or siRNA)) is an RNA double-stranded RNA that targets a gene of interest ("target gene"). An "RNA double-stranded RNA" refers to a structure formed by complementary pairing between two regions of a double-stranded RNA region (dsRNA) in the formation of an RNA molecule. siRNA "targets" a gene because the nucleotide sequence of the siRNA double-stranded portion is complementary to the nucleotide sequence of the target gene. In some embodiments, the length of the siRNA double-stranded RNA is less than 30 nucleotides. In some embodiments, the length of the double-stranded RNA can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides. In some embodiments, the length of the double-stranded RNA is 19-25 nucleotides. In some embodiments, siRNA-mediated gene targeting is achieved using DNA-guided RNA interference (ddRNAi), a gene silencing technique that utilizes a DNA construct to activate the endogenous RNA interference (RNAi) pathway in animal cells. This DNA construct is designed to express a self-complementary double-stranded RNA, typically a short hairpin RNA (shRNA), that, once processed, silences one or more target genes. Any RNA, including endogenous mRNA or viral RNA, can be silenced by designing the construct to express a double-stranded RNA complementary to the desired mRNA target. Therefore, the RNA double-stranded portion of the siRNA agent can be part of a short hairpin structure referred to as shRNA. In addition to the double-stranded portion, the hairpin structure may contain a loop portion located between the two sequences forming the double strand. The length of the loop can vary. In some embodiments, the loop length is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides. The hairpin structure may also include a 3' or 5' overhang. In some embodiments, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides in length. Typically, the level of the target gene expression product (e.g., mRNA, polypeptide, etc.) is reduced by an siRNA agent (e.g., siRNA, shRNA, etc.) containing a specific double-stranded nucleotide sequence complementary to the target gene transcript of at least 19-25 nucleotides in length (e.g., 20-21 nucleotides), including a 5' untranslated (UT) region, an ORF, or a 3' UT region. In some embodiments, the short interfering RNA is about 19-25 nt in length.See, for example, PCT applications WO 00 / 44895, WO 99 / 32619, WO 01 / 75164, WO 01 / 92513, WO 01 / 29058, WO 01 / 89304, WO 02 / 16620, and WO 02 / 29858; and U.S. Patent Publication No. 2004 / 0023390 describing siRNA technology. siRNA and / or shRNA may be encoded by a nucleic acid sequence, and the nucleic acid sequence may also include a promoter. The nucleic acid sequence may also include a polyadenylation signal. In some embodiments, the polyadenylation signal is a minimally synthesized polyadenylation signal.

[0072] The term "antisense RNA" encompasses RNA that is complementary to a gene expression product. For example, antisense RNA targeting specific mRNA is an RNA-based reagent (or may be a modified RNA) that is complementary to the mRNA, wherein hybridization of the antisense RNA with the mRNA alters mRNA expression (e.g., by changing RNA stability, altering RNA translation, etc.). "Antisense RNA" also includes nucleic acids encoding the antisense RNA.

[0073] Regarding “CRISPR / Cas9 agents,” the term “CRISPR” encompasses aggregated, regularly spaced short palindromic repeats / CRISPR-associated (Cas) systems that have evolved to provide adaptive immunity against viruses and plasmids in bacteria and archaea through the use of CRISPR RNA (crRNA) to guide the silencing of invading nucleic acids. Cas9 proteins (or their functional equivalents and / or variants, i.e., Cas9-like proteins) naturally contain DNA endonuclease activity dependent on the association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also known as guide RNA). In some cases, the two molecules are covalently linked to form a single molecule (also known as a single guide RNA (“sgRNA”)). Thus, Cas9 or Cas9-like proteins are associated with DNA-targeting RNAs (the term encompasses both bimolecular and monomolecular guide RNA constructions), which activate the Cas9 or Cas9-like protein and guide the protein to the target nucleic acid sequence.

[0074] If Cas9 or Cas9-like proteins retain their native enzymatic function, they will cleave target DNA to produce double-strand breaks, which can lead to genomic alterations (i.e., editing: deletions, insertions (when donor polynucleotides are present), substitutions, etc.) that alter gene expression. Some variants of Cas9 (whose variants are covered by the term Cas9-like) have been modified to have reduced DNA-cutting activity (in some cases, they cleave a single strand instead of both strands of the target DNA, while in others, they are severely reduced to no DNA-cutting activity). Cas9-like proteins with reduced DNA-cutting activity (or even no DNA-cutting activity) can still be directed to the target DNA to block RNA polymerase activity. Alternatively, Cas9 or Cas9-like proteins can be modified to form a co-activation mediator (Cas9-SAM) complex in transcriptionally activated cells by fusing the VP64 transcription activation domain to the Cas9 protein and co-delivering with an MS2-P65-HSF1 helper protein and a single guide RNA encoding an MS2 RNA aptamer contained in a tetracyclic ring. Therefore, enzymatically inactivated Cas9-like proteins can target specific locations in the target DNA via RNA targeting the DNA, thereby blocking or activating transcription of the target DNA. As used herein, the term "CRISPR / Cas9 agent" encompasses all forms of CRISPR / Cas9 as described above or known in the art.

[0075] Detailed information about CRISPR agents can be found in the following literature, for example (a) Jinek et al., Science. 2012 Aug 17;337(6096):816-21: "A programmable dual-RNA-guided DNAendonuclease in adaptive bacterial immunity"; (b) Qi et al., Cell. 2013 Feb 28;152(5): 1173-83: "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression"; and (c) U.S. Patent Application No. 13 / 842,859 and PCT Application No. PCT / US13 / 32589, the entire contents of which are incorporated herein by reference. Therefore, the term "CRISPR agent" as used herein encompasses any agent containing naturally occurring and / or synthetic sequences that may be used in Cas9-based systems (e.g., Cas9 or Cas9-like proteins; any component of DNA-targeting RNA, such as crRNA-like RNA, tracrRNA-like RNA, single guide RNA, etc.; donor polynucleotides; etc.).

[0076] Zinc finger nucleases (ZFNs) are artificial DNA endonucleases created by fusing a zinc finger DNA-binding domain with a DNA-cutting domain. ZFNs can be engineered to target desired DNA sequences, enabling them to cleave unique target sequences. When introduced into cells, ZFNs can be used to edit target DNA within cells (e.g., the cell's genome) by inducing double-strand breaks. For more information on the use of ZFN, see, for example: Asuri et al., Mol Ther. 2012 Feb; 20(2):329-38; Bibikova et al., Science. 2003 May 2;300(5620):764; Wood et al., Science. 2011 Jul 15;333(6040):307; Ochiai et al., Genes Cells. 2010 Aug;15(8):875-85; Takasu et al., Insect Biochem Mol Biol. 2010 Oct;40(10):759-65; Ekker et al., Zebrafish 2008 Summer;5(2):121-3; Young et al., Proc Natl Acad Sci US A. 2011 Apr 26;108(17):7052-7; Goldberg et al., Cell. 2010 Mar 5;140(5):678-91; Geurts et al., Science. 2009 Jul 24;325(5939):433; Flisikowska et al., PLoS One. 2011;6(6):e21045. doi: 10.1371 / journal.pone.0021045. Epub 2011 Jun 13; Hauschild et al., Proc Natl Acad Sci US A. 2011 Jul 19;108(29): 12013-7; and Yu et al., Cell Res. 2011 Nov;21(l 1): 1638-40; All these references are incorporated herein by reference to their teachings related to ZFN. The term “ZFN agent” encompasses zinc finger nucleases and / or polynucleotides containing nucleotide sequences encoding zinc finger nucleases.

[0077] The term "transcription activator-like effector nuclease" or "TALEN" refers to a transcription activator-like effector nuclease (TALEN), an artificial DNA endonuclease created by fusing a TAL (transcription activator-like) effector DNA-binding domain with a DNA-cutting domain. TALENs can be rapidly engineered to bind virtually any desired DNA sequence, and when introduced into cells, TALENs can edit target DNA (e.g., the cell's genome) by inducing double-strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al., Nat Biotechnol. 2011 Jul 7;29(8):731-4; Wood et al., Science. 2011 Jul 15;333(6040):307; Tesson et al., Nat Biotechnol. 2011 Aug 5;29(8):695-6; and Huang et al., Nat Biotechnol. 2011 Aug 5;29(8):699-700; all of these references are incorporated herein by reference to their teachings relating to TALENs. The term “TALEN agent” encompasses TALENs and / or polynucleotides containing nucleotide sequences encoding TALENs.

[0078] The term "control element" or "control sequence" refers to a nucleotide sequence that participates in molecular interactions and contributes to the functional regulation of polynucleotides, including replication, duplication, transcription, splicing, translation, or degeneracy. This regulation can affect the frequency, rate, or specificity of the process and may be inherently enhancing or repressive. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable of binding to RNA polymerase under certain conditions and initiating transcription of a coding region typically located downstream (3' direction) of the promoter. Promoters can be universally active, i.e., active in many cell types, such as the CAG or CMV promoters; or tissue- or cell-specific, such as the rho promoter active in rods or the opsin promoter active in cones.

[0079] The term "operatively linked" or "operably linked" refers to the juxtaposition of genetic elements in a relationship that allows them to operate in the intended manner. For example, if a promoter facilitates the initiation of transcription of a coding sequence, then the promoter is operatively linked to the coding region. Intercalation residues may exist between the promoter and the coding region, as long as this functional relationship is maintained.

[0080] The term "expression vector" encompasses a vector containing a multinucleotide region encoding a polypeptide of interest and used to achieve protein expression in designated target cells. Expression vectors may also contain control elements operatively linked to the coding region to facilitate protein expression in the target. The control elements, and combinations of one or more genes operatively linked thereto for expression, are sometimes referred to as "expression cassettes," many of which are known and available in the art, or can be readily constructed from components available in the art.

[0081] The term "heterologous" refers to an entity with a genotype that differs from the entity being compared to. For example, a polynucleotide introduced into a plasmid or vector derived from a different species via genetic engineering is a heteropolynucleotide. A promoter that has been removed from its natural coding sequence and is operatively linked to a coding sequence not found to be linked naturally is a heteropromoter. Thus, for example, an rAAV containing a heterologous nucleic acid sequence encoding a heterologous gene product is an rAAV that includes polynucleotides not typically found in naturally occurring wild-type AAVs, and the encoded heterologous gene product is a gene product not typically encoded by naturally occurring wild-type AAVs.

[0082] The terms “genetic alteration” and “genetic modification” (and their grammatical variations) are used interchangeably herein to refer to the process in which a genetic element (e.g., a polynucleotide) is introduced into a cell other than during mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alterations can be achieved, for example, by transfecting cells with recombinant plasmids or other polynucleotides using any method known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic alterations can also be achieved, for example, by transduction or infection with a DNA or RNA virus or viral vector. Typically, genetic elements are introduced into chromosomes or mini-chromosomes within the cell; however, any alteration of the phenotype and / or genotype of the cell and its offspring is included in this terminology.

[0083] Regarding cell modification, the terms "genetic modification," "transformation," "transfection," or "transduction" (e.g., via recombinant viruses) involving exogenous DNA refer to the introduction of such DNA into the cell. The presence of exogenous DNA results in permanent or transient genetic changes. Transformed DNA may or may not integrate (covalently linked) into the cell's genome. A "clone" is a population of cells derived from a single cell or common ancestor through mitosis. A "cell line" is a clone of primary cells capable of stably growing for many generations in vitro.

[0084] As used herein, a cell is said to be “stable” altered, transduced, genetically modified, or transformed with a gene sequence if the sequence can be used to perform its function during extended cell culture in vitro and / or during extended time in vivo. Typically, such cells are “heritably altered” (genetically modified) because genetic changes are introduced that can also be inherited by the offspring of the altered cells.

[0085] The terms “polypeptide,” “peptide,” and “protein,” used interchangeably herein, refer to polymers of amino acids of any length. These terms also cover modified amino acid polymers; for example, those with disulfide bond formation, glycosylation, esterification, phosphorylation, or conjugation with a labeled component. When discussed in the context of delivering gene products to mammalian subjects, polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and combinations thereof refer to the corresponding complete polypeptide, or any fragment or genetically engineered derivative thereof, that retains the biochemical functions required for the complete protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids used for delivering gene products to mammalian subjects (which may be referred to as “transgenic” cells to be delivered to recipient cells) include polynucleotides encoding complete polypeptides or any fragment or genetically engineered derivative having the desired biochemical functions.

[0086] As used herein, "isolated" plasmids, nucleic acids, vectors, viruses, viral particles, host cells, proteins, or other substances refer to formulations lacking at least some of the other components, which may also be present in naturally occurring substances or substances originally prepared therefrom, or similar substances. Therefore, for example, the isolated substance can be prepared by enriching it from a source mixture using purification techniques. Enrichment can be measured on an absolute basis, such as the weight per volume of solution, or relative to a second potential interfering substance present in the source mixture. The increasing enrichment in embodiments of this disclosure is increasingly focused. In some embodiments, the isolated plasmids, nucleic acids, vectors, viruses, host cells, or other substances are purified, for example, to a purity of about 80% to about 90%, a purity of at least about 90%, a purity of at least about 95%, a purity of at least about 98%, or a purity of at least about 99% or more.

[0087] As used herein, the terms “treatment”, “treating”, etc., refer to achieving a desired pharmacological and / or physiological effect. Such effect may be preventative in relation to the complete or partial prevention of a disease or its symptoms, and / or therapeutic in relation to the partial or complete cure of a disease and / or side effects attributable to the disease. As used herein, “treatment” encompasses any treatment of diseases in mammals, particularly humans, and includes: (a) prevention of a disease (and / or symptoms caused by the disease) that may occur in a subject who may be susceptible to or at risk of the disease but has not yet been diagnosed with it; (b) suppression of the disease (and / or symptoms caused by the disease), i.e., prevention of its development; and (c) relief of the disease (and / or symptoms caused by the disease), i.e., causing the disease (and / or symptoms caused by the disease) to subside, i.e., improvement of the disease and / or one or more symptoms of the disease. For example, the subject composition and method may be for the treatment of retinal diseases. Non-limiting methods for assessing retinal diseases and their treatment include measuring retinal function and its changes, such as changes in visual acuity (e.g., best-corrected visual acuity [BCVA], movement, navigation, object detection, and discrimination), changes in visual fields (e.g., visual field testing of static and dynamic visual fields), clinical examination (e.g., slit-lamp examination of the anterior and posterior segments of the eye), electrophysiological responses to all light and dark wavelengths (e.g., all forms of electroretinography (ERG) [full field, multifield, and modal], all forms of visual evoked potentials (VEP), electrooculography (EOG), color vision, dark adaptation, and / or contrast sensitivity); measuring anatomical or health changes using anatomical and / or photographic measures, such as optical coherence tomography (OCT), fundus photography, adaptive optics scanning laser ophthalmoscopy, fluorescence, and / or autofluorescence; measuring ocular movements and motions (e.g., nystagmus, fixation preference, and stability), measuring reported outcomes (patient-reported changes in visual and non-visual guided behaviors and activities, patient-reported outcomes [PRO]), questionnaire-based quality of life assessments, and measurements of daily activities and neurological function (e.g., functional magnetic resonance imaging (MRI)).

[0088] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably in this document and refer to mammals, including but not limited to: humans; non-human primates, including apes; mammalian locomotion animals (such as horses); mammalian farm animals (such as sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0089] In some implementations, the individual is someone who has been previously naturally exposed to AAV and therefore carries anti-AAV antibodies (i.e., AAV neutralizing antibodies). In other implementations, the individual is someone who has previously received an AAV vector (and may therefore carry anti-AAV antibodies) and requires re-administration of the vector to treat a different condition or to further treat the same condition. Based on positive results from clinical trials delivering the AAV gene to all tissues affected by neutralizing antibodies against the vector, such as the liver, muscle, and retina, numerous such therapeutic applications / disease targets exist.

[0090] As used herein, the term "effective amount" is an amount sufficient to achieve a beneficial or desired clinical outcome. An effective amount may be administered in one or more doses. For the purposes of this disclosure, an effective amount of a compound (e.g., infectious rAAV viral particles) is an amount sufficient to alleviate, improve, stabilize, reverse, prevent, slow, or delay (and / or associated symptoms) the progression of a specific disease state (e.g., retinal disease). Thus, an effective amount of infectious rAAV viral particles is an amount of infectious rAAV viral particles capable of efficiently delivering heterologous nucleic acids to target cells (or multiple target cells) of an individual. An effective amount can be preclinically determined by detecting gene products (RNA, protein) encoded by a heterologous nucleic acid sequence in cells or tissues, for example, using techniques well known in the art such as RT-PCR, Western blotting, ELISA, fluorescence, or other reporter factor readings. As described herein and as known in the art, an effective amount can be clinically determined by detecting changes in the onset or progression of disease, for example, using methods known in the art such as fundus autofluorescence, fluorescein angiography, OCT, micrometry, adaptive optics, etc.

[0091] The term "retinal cell" in this document refers to any cell type containing the retina, such as, but not limited to, retinal ganglion (RG) cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, Müller glial cells, microglia, and retinal pigment epithelium (RPE). The term "photoreceptor cell" in this document refers to, but is not limited to, rod cells or "rods" and cone cells or "cones." The term "Müller cell" or "Müller glial" refers to the glial cells that support neurons in the vertebrate retina.

[0092] The term "directed evolution" refers to an in vitro and / or in vivo in vitro engineering methodology that mimics natural evolution through iterative rounds of genetic diversification and selection processes to accumulate beneficial mutations that gradually improve the function of biomolecules. Directed evolution often involves an in vivo approach known as "bio-panning," used to select AAV variants from a library that have a more effective level of infectivity for the cell or tissue type of interest. Detailed Implementation

[0093] Adeno-associated virus (AAV) is a family of parvoviruses with a 4.7 kb single-stranded DNA genome contained within a non-enveloped capsid. The viral genome of naturally occurring AAV has two inverted terminal repeats (ITRs) – which function as the source of viral signals for replication and packaging – flanked by two major open reading frames (ORFs): rep (encoding proteins involved in viral replication, transcriptional regulation, site-specific integration, and viral particle assembly) and cap. The cap ORF encodes three structural proteins that assemble to form a 60-mer viral capsid. Numerous naturally occurring AAV variants and serotypes have been isolated, and none are associated with human disease.

[0094] Recombinant versions of AAV can be used as gene delivery vectors, in which a marker or therapeutic gene of interest is inserted between the rep and cap ITRs. These vectors have been shown to transduce dividing and non-dividing cells in vitro and in vivo, and can lead to stable transgene expression for many years in post-mitotic tissues. See, for example, Knipe DM, Howley PM. Fields' Virology . Lippincott Williams&Wilkins, Philadelphia, PA, USA, 2007; Gao GP,Alvira MR, Wang L, Calcedo R, Johnston J, Wilson JM. Novel adeno-associatedviruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci USA 2002; 99: 11854–9; Atchison RW, Casto BC, Hammon WM. Adenovirus-Associated Defective Virus Particles. Science 1965; 149: 754–6; Hoggan MD, Blacklow NR, Rowe WP. Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunologicalcharacteristics. Proc Natl Acad Sci USA1966; 55: 1467–74; Blacklow NR,Hoggan MD, Rowe WP. Isolation of adenovirus-associated viruses from man. Proc Natl Acad Sci USA 1967; 58: 1410–5; Bantel-Schaal U, zur Hausen H.Characterization of the DNA of a defective human parvovirus isolated from agenital site. Virology 1984; 134: 52–63; Mayor HD, Melnick JL. Smalldeoxyribonucleic acid-containing viruses (picodnavirus group). Nature 1966;210: 331–2; Mori S, Wang L, Takeuchi T, Kanda T. Two novel adeno-associatedviruses from cynomolgus monkey: pseudotyping characterization of capsidprotein. Virology 2004; 330: 375–83; Flotte TR. Gene therapy progress andprospects: recombinant adeno-associated virus (rAAV) vectors. Gene Ther 2004;11: 805–10。

[0095] Recombinant AAV (hereinafter referred to as "AAV") has shown promising results in a growing number of clinical trials. However, barriers to gene delivery can limit the utility of AAV, such as anti-capsid immune responses, low transduction in certain tissues, inability to target specific cell types, and relatively low carrying capacity. In many cases, there is insufficient mechanistic knowledge to effectively endow rational designs with the ability to improve AAV. As an alternative, directed evolution has emerged as a strategy for creating novel AAV variants that meet specific biomedical needs. Directed evolution strategies utilize genetic diversification and selection processes to accumulate beneficial mutations that progressively improve the function of the biomolecule. In this process, wild-type AAV cap genes are diversified through several methods to produce large genetic libraries, which are packaged to produce viral particle libraries, and then selective pressure is applied to isolate novel variants that can overcome gene delivery barriers. Importantly, the mechanistic basis of the gene delivery problem does not need to be known by directed evolution of function, which can therefore accelerate the development of enhanced vectors.

[0096] Typically, the variants disclosed herein are generated using one and / or multiple AAV libraries. Such one or more AAV libraries are produced by mutating the cap gene, a gene encoding a structural protein of the AAV capsid, using a range of directed evolution techniques known and readily available to those skilled in the art of viral genome engineering. See, for example, Bartel et al., Am. Soc. Gene Cell Ther. 15 thAnnu. Meet. 20, S140 (2012); Bowles, D. et al., J. Virol. 77, 423-432 (2003); Gray et al., Mol. Ther. 18, 570-578 (2010); Grimm, D. et al., J. Virol. 82, 5887-5911; Koerber, JT et al., Mol. Ther. 16, 1703-1709 (2008); Li W. et al., Mol. Ther. 16, 1252-1260 (2008); Koerber, JT et al., Methods Mol. Biol. 434, 161-170 (2008); Koerber, JT et al., Hum. GeneTher. 18, 367-378 (2007); and Koerber, JT et al., Mol. Ther. 17, 2088-2095 (2009). This technique is not limited to the following: i) error-prone PCR for introducing random point mutations into the AAV cap open reading frame (ORF) at a predetermined, modifiable rate; ii) in vitro or in vivo viral recombination or “DNA shuffling” for generating random chimeras of the AAV cap gene to produce gene libraries with multiple AAV serotypes; iii) random peptide insertion at a defined site on the capsid by linking degenerate oligonucleotides in the cap ORF; iv) defining the insertion of peptide-coding sequences at random sites on the AAV cap ORF using transposon mutagenesis; v) replacing the surface loop of the AAV capsid with a bioinformatically designed peptide sequence library based on the level of conservation of each amino acid position in the native AAV serotype and variants to produce a “loop exchange” library; vi) random amino acid substitution at degenerate positions between AAV serotypes to produce a library of ancestral variants (Santiago-Ortiz et al., 2015); and combinations of such techniques.

[0097] DNA shuffling produces chimeras that combine their parental traits in a unique and often beneficial way; however, some may fail to be packaged, which can actually reduce library diversity. Library diversity concentrations are achieved through peptide insertion techniques, such as, but not limited to, those described in iii-iv above. Library diversity is also concentrated in techniques such as v above, and this concentration is directed towards multiple hypervariable regions located on the surface-exposed loops of the AAV capsid. While many techniques produce variant capsids with only a small portion of the capsid mutated, these techniques can be paired with additional mutagenesis strategies to modify the entire capsid.

[0098] Once one or more AAV libraries are generated, the virus is then packaged such that each AAV particle consists of a mutant capsid surrounding a cap gene encoding a mutant capsid and purified. The variants of the library are then subjected to in vitro and / or in vivo selective stress techniques known to and readily available to those skilled in the art of AAV. See, for example, Maheshri, N. et al., Nature Biotech. 24, 198-204 (2006); Dakara, D. et al., Sci. Transl. Med. 5, 189ra76 (2013); Lisowski, L. et al., Nature. 506, 382-286 (2013); Yang, L. et al., PNAS. 106, 3946-3951 (2009); Gao, G. et al., Mol. Ther. 13, 77-87 (2006); and Bell, P. et al., Hum. Gene. Ther. 22, 985-997 (2011). For example, but not limited to, the following AAV variants may be selected: i) affinity columns, wherein elution of different fractions produces variants with altered binding properties; ii) primary cells – isolated from tissue samples or immortalized cell lines that mimic human cell behavior – that produce AAV variants with enhanced efficiency and / or tissue specificity; iii) animal models – which mimic clinical gene therapy environments – that produce AAV variants that have been successfully infected with target tissues; iv) human xenograft models that produce AAV variants that have been infected with transplanted human cells; and / or combinations of other selection techniques.

[0099] Once viruses are selected, they can be recovered using known techniques, such as, but not limited to, adenovirus-mediated replication, PCR amplification, next-generation sequencing, and cloning. Viral clones are then enriched using repeated rounds of selection techniques, and AAV DNA is isolated to recover the cap gene of the selected variant of interest. This selected variant can undergo further modification or mutation, and thus serve as a new starting point for further selection steps to iteratively increase AAV viral fitness. However, in some cases, a successful capsid has already been generated without additional mutations. The AAV variants disclosed herein are generated at least in part using in vivo directed evolution methods (such as those described above), which involve selection using primate retina after intravitreal administration. Therefore, the AAV variant capsids disclosed herein contain one or more modifications in the amino acid sequence that confer more efficient transduction to primate retinal cells compared to the corresponding parental AAV capsid protein. As used herein, “corresponding parental AAV capsid protein” refers to an AAV capsid protein of the same wild-type or variant AAV serotype as the subject variant AAV capsid protein, but without the one or more amino acid sequence modifications of the subject variant AAV capsid protein.

[0100] In some embodiments, the subject variant AAV capsid protein comprises a heteropeptide of about 5 to about 20 amino acids covalently linked to the GH ring or ring IV of the corresponding parent AAV capsid protein. The “GH ring” or ring IV of the AAV capsid protein refers to the solvent-accessible portion of the GH ring or ring IV of the AAV capsid protein as referred to in the art. For the GH ring / ring IV of the AAV capsid, see, for example, van Vliet et al., (2006). Mol. Ther. 14:809; Padron et al., (2005) J. Virol. 79:5047; and Shen et al., (2007) Mol. Ther. 15:1955. Therefore, for example, the insertion site can be within amino acids 411-650 of the AAV VP1 capsid protein. For example, the insertion site can be within amino acids 571-612 of AAV1 VP1, amino acids 570-611 of AAV2 VP1, amino acids 571-612 of AAV3A VP1, amino acids 571-612 of AAV3B VP1, amino acids 569-610 of AAV4 VP1, amino acids 560-601 of AAV5 VP1, amino acids 571-612 of AAV6 VP1, amino acids 572-613 of AAV7 VP1, amino acids 573-614 of AAV8 VP1, amino acids 571-612 of AAV9 VP1, or amino acids 573-614 of AAV10 VP1, or the corresponding amino acids of any variant thereof. Based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, those skilled in the art will know that the insertion site “corresponding to the amino acid of AAV2” will be present in the capsid protein of any given AAV serotype. See also Figure 6 for an illustration of the alignment of wild-type AAV SEQ ID NO:1-11, which provides the amino acid positions between and across the wild-type (naturally occurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10.

[0101] In some embodiments, the insertion site is a single insertion site between two adjacent amino acids 570-614 of VP1 in any wild-type AAV serotype or AAV variant. For example, the insertion site is between two adjacent amino acids 570-610, 580-600, 570-575, 575-580, 580-585, 585-590, 590-600, or 600-614 of VP1 in any AAV serotype or variant. For example, the insertion site may be between amino acids 580 and 581, 581 and 582, 583 and 584, 584 and 585, 585 and 586, 586 and 587, 587 and 588, 588 and 589, or 589 and 590. The insertion site can be between amino acids 575 and 576, 576 and 577, 577 and 578, 578 and 579, or 579 and 580. The insertion site can also be between amino acids 590 and 591, 591 and 592, 592 and 593, 593 and 594, 594 and 595, 595 and 596, 596 and 597, 597 and 598, 598 and 599, or 599 and 600. For example, the insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 588 and 589 of AAV3A, between amino acids 588 and 589 of AAV3B, between amino acids 584 and 585 of AAV4, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.

[0102] In some embodiments, the peptide inserts disclosed herein are of length 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In another embodiment, the peptide inserts disclosed herein include 1 to 4 spacer amino acids at the N-terminus and / or C-terminus of any of the peptide inserts disclosed herein. Exemplary spacer amino acids include, but are not limited to, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In some embodiments, the peptide insert comprises 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, the peptide insert comprises 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus.

[0103] The peptide insertions disclosed herein are previously undescribed and / or inserted into the AAV capsid. Without wishing to be bound by theory, the presence of any disclosed peptide insertion could serve to reduce the affinity of the variant capsid for heparin sulfate, which may reduce binding to the extracellular matrix in front of the primate retina. Furthermore, the peptide insertion motifs disclosed herein could confer enhanced transduction to primate retinal cells by adding cell surface receptor-binding domains.

[0104] In some preferred embodiments, the insert peptide comprises an amino acid sequence of any one of the following formulas.

[0105] In some respects, the inserted peptide can be a peptide of formula 1a with a length of 7 to 10 amino acids: Y1Y2X1X2X3X4X5X6X7Y3 Each of Y1-Y3 (if it exists) is independently selected from Ala, Leu, Gly, Ser, Thr, Pro. X1 is selected from Gln, Asn, His, Ile, and Ala X2 is selected from Ala, Gln, Asp, Ser, Lys, and Pro. X3 is selected from Asp, Ile, Thr, and Asn. X4 is selected from Thr, Ser, Tyr, Gln, Glu, and Ala. X5 is selected from Thr, Lys, and Asn. X6 is selected from Lys, Asn, and Glu. X7 is selected from Asn, Thr, Ile, His, Asp, and Ala.

[0106] In some embodiments, the insert peptide of Formula 1a comprises an amino acid sequence selected from the following: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ NO:16), HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), and QANANEN (SEQ ID NO:22).

[0107] In other respects, the insert peptide can be a peptide of Formula 1b with a length of 7 to 10 amino acids: Y1Y2X1X2X3X4X5X6X7Y3 Each of Y1-Y3 (if it exists) is independently selected from Ala, Leu, Gly, Ser, Thr, Pro. X1 is selected from Gln, Asn, His, and Ile. X2 is selected from Ala, Gln, Asp, and Ser. X3 is selected from Asp and Ile X4 is selected from Thr, Tyr, and Gln. X5 is selected from Thr and Lys X6 is selected from Lys and Asn X7 is selected from Asn, Thr, Ile, and His In some embodiments, the insert peptide of Formula 1b comprises an amino acid sequence selected from the following: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), NQDYTKT (SEQ NO:16), HDITKNI (SEQ ID NO:17), and HQDTTKN (SEQ ID NO:19).

[0108] In other respects, the inserted peptide can be a peptide of formula 1c with a length of 7 to 10 amino acids: Y1Y2X1X2AspX3ThrLysX4Y3 Each of Y1-Y3 (if it exists) is independently selected from Ala, Leu, Gly, Ser, Thr, Pro. X1 is selected from Gln, Asn, His, and Ile. X2 is selected from Ala, Gln, and Ser. X3 is selected from Thr, Tyr, and Gln. X4 is selected from Asn, Thr, and His. In some embodiments, the insert peptide of Formula 1c comprises an amino acid sequence selected from the following: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), NQDYTKT (SEQ NO:16), and HQDTTKN (SEQ ID NO:19).

[0109] In other respects, the inserted peptide can be a peptide of formula 1d with a length of 7 to 10 amino acids: Y1Y2X1X2AspX3ThrThrX4Y3 Each of Y1-Y3 (if it exists) is independently selected from Ala, Leu, Gly, Ser, Thr, Pro. X1 is selected from Gln and Ile X2 is selected from Ala and Ser X3 is selected from Thr and Gln. X4 is selected from Asn and His In some embodiments, the insert peptide of Formula 1d comprises an amino acid sequence selected from the following: QADTTKN (SEQ ID NO: 13) and ISDQTKH (SEQ ID NO: 14).

[0110] In other respects, the inserted peptide can be a peptide of formula 1e with a length of 7 to 11 amino acids: Y1Y2X1X2AsnX3AsnGluX4Y3 Each of Y1-Y3 (if it exists) is independently selected from Ala, Leu, Gly, Ser, Thr, Pro. X1 is selected from Gln and Ile X2 is selected from Ala and Ser X3 is selected from Glu and Ala X4 is selected from Asn and His In other embodiments, the insert peptide of Formula 1e comprises an amino acid sequence selected from the following: ISNENEH (SEQ ID NO: 21) and QANANEN (SEQ ID NO: 22).

[0111] In yet another embodiment, the insert peptide can be a peptide of formula IIa with a length of 7 to 11 amino acids: Y1Y2X1X2DX3TKX4Y 3. Each of Y1-Y3 (if it exists) is independently selected from Ala, Leu, Gly, Ser, Thr, Pro. X1 is selected from Q, N, A, H, and I; X2 is selected from Q, A, P, and S. X3 is selected from T, Y, S, and Q; and X4 is selected from T, N, A, and H.

[0112] In another embodiment of the peptide insertion of the amino acid sequence of formula X1X2DX3TKX4, the peptide insertion is selected from the group consisting of: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA, NQDYTKT (SEQ NO:16), HQDTTKN (SEQ ID NO:19) and HPDTTKN (SEQ ID NO:18).

[0113] In some of these embodiments, the inserted peptide can be a peptide of formula IIb with a length of 7 to 11 amino acids: Y1Y2X1X2DX3TKX4Y3 Each of Y1-Y3 (if it exists) is independently selected from Ala, Leu, Gly, Ser, Thr, Pro. X1 is selected from N, A, and H; X2 is selected from Q, P, and S; X3 is selected from T, Y, and S; and X4 is selected from T, N, and A.

[0114] In another embodiment of the peptide insertion of the amino acid sequence of formula X1X2DX3TKX4, the peptide insertion is selected from the group consisting of: ASDSTKA, NQDYTKT (SEQ NO:16), HQDTTKN (SEQ ID NO:19) and HPDTTKN (SEQ ID NO:18).

[0115] In other embodiments, the insert peptide comprises an amino acid sequence selected from the following: KDRAPST (SEQ ID NO:26), TNTTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), and GKSKVID (SEQ ID NO:23).

[0116] In some embodiments, the insert peptide comprises an amino acid sequence selected from the following: ASDSTKA (SEQ ID NO:15), QANANEN (SEQ ID NO:22), QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), GKSKVID (SEQ ID NO:23), TNTTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), and KDRAPST (SEQ ID NO:26).

[0117] In other preferred embodiments, the insert peptide has 1 to 3 spacer amino acids (Y1-Y3) at the amino and / or carboxyl ends of the amino acid sequences selected from the following: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), QANANEN (SEQ ID NO:22), GKSKVID (SEQ ID NO:23), TNTTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), and KDRAPST (SEQ ID NO:26). In some of these embodiments, the insert peptide is selected from the group consisting of: LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA (SEQ ID NO:30), LANQDYTKTA (SEQ ID NO:31), LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA (SEQ ID NO:33), LAHQDTTKNA (SEQ ID NO:34), LANKTTNKDA (SEQ ID NO:35), LPISNENEHA (SEQ ID NO:36), LPQANANENA (SEQ ID NO:37), LAGKSKVIDA (SEQ ID NO:38), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ ID NO:40), and LAKDRAPSTA (SEQ ID NO:41).

[0118] In some embodiments, the subject variant AAV capsid protein does not contain any amino acid sequence modifications other than peptide insertions of about 5 to about 20 amino acids in the GH ring or ring IV. For example, in some embodiments, the subject variant AAV capsid protein includes a peptide insert comprising an amino acid sequence selected from the group consisting of: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), QANANEN (SEQ ID NO:22), GKSKVID (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST (SEQ ID NO:26), LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), QADTTKN (SEQ ID NO:10), ISDQTKHA (SEQ ID NO:20), LGISDQTKHA (SEQ ID NO:20), and LGISDQTKHA (SEQ ID NO:20). The variant AAV capsid contains no other amino acid substitutions, insertions, or deletions (i.e., the variant AAV capsid protein contains the insertions and is otherwise identical to the corresponding AAV capsid protein). In other words, the variant AAV capsid protein containing the insertions is otherwise identical to the parental AAV capsid protein with the inserted peptide.As another example, the theme variant AAV capsid protein contains peptide insertions having an amino acid sequence selected from the following: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), HPDTTKN (SEQ ID NO:18), HQDTTKN (SEQ ID NO:19), NKTTNKD (SEQ ID NO:20), ISNENEH (SEQ ID NO:21), QANANEN (SEQ ID NO:22), GKSKVID (SEQ ID NO:23), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST (SEQ ID NO:26), LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29). NO:29), LAASDSTKAA (SEQ ID NO:30), LANQDYTKTA (SEQ ID NO:31), LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA (SEQ ID NO:33), LAHQDTTKNA (SEQ ID NO:34), LANKTTNKDA (SEQ IDNO:35), LPISNENEHA (SEQ ID NO:36), LPQANANENA (SEQ ID NO:37), LAGKSKVIDA (SEQ ID NO:38), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ ID NO:40) and LAKDRAPSTA (SEQID NO:41), wherein the peptide is inserted between amino acids 587 and 588 of VP1 in the AAV2 capsid or between corresponding amino acids of VP1 in another parent AAV, for example between amino acids 588 and 589 of VP1 in AAV1, AAV3A, AAV3B, AAV6, or AAV9, between amino acids 586 and 587 of VP1 in AAV4, between amino acids 577 and 578 of VP1 in AAV5, between amino acids 589 and 590 of VP1 in AAV7, between amino acids 590 and 591 of VP1 in AAV8 or AAV10, etc., wherein the variant AAV capsid protein sequence is otherwise identical to the corresponding parent AAV capsid protein sequence, for example, any one of SEQ ID NO:1-12.

[0119] In other embodiments, in addition to including peptide insertions in the GH ring, such as those disclosed herein or known in the art, the subject variant AAV capsid protein comprises about 1 to about 100 amino acid substitutions or deletions, for example, 1 to about 5, about 2 to about 4, about 2 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 50, or about 50 to about 100 amino acid substitutions or deletions compared to the parental AAV capsid protein. Thus, in some embodiments, the subject variant capsid protein comprises 85% or more, 90% or more, 95% or more, or 98% or more, for example, 99% sequence identity with the corresponding parental AAV capsid (e.g., the wild-type capsid protein shown in SEQ ID NO: 1-12).

[0120] In a further embodiment, the one or more amino acids replace one or more amino acid residues 1, 15, 34, 57, 66, 81, 101, 109, 144, 164, 176, 188, 196, 226, 236, 240, 250, 312, 363, 368, 449, 456, 463, 472, 484, 524, 535, 551, 593, 698, 708, 719, 721 and / or 735 of the AAV2VP1 capsid protein, or one or more corresponding amino acid residues of another AAV capsid protein. In some of these embodiments, the one or more amino acid substitutions are selected from the group consisting of: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, and L735Q of another AAV capsid protein.

[0121] In a preferred embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises an amino acid sequence selected from the following: ISDQTKH (SEQ ID NO:14), LGISDQTKHA (SEQ ID NO:29), and LAISDQTKHA (SEQ ID NO:28), and b) a corresponding substitution of one or more of the following amino acid substitutions or another AAV parental serotype (i.e., other than AAV2) compared to the amino acid sequence of AAV2 (SEQ ID NO:2), wherein the substituted one or more amino acids are not naturally present at the following corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q and combinations thereof. In some embodiments, the one or more amino acid substitutions are selected from the group consisting of: M1L+L15P+P535S, P34A, P34A+S721L, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, Q164K+V708I, T176P, L188I, S196Y, G226E, G236V, I240T, N312K, N312K+N449D+D472N+N551S+I698V+L735Q, P363L, R484C+V708I, T456K, and V708I. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype.

[0122] In a particularly preferred embodiment, the variant AAV capsid comprises a peptide insert containing the amino acid sequence ISDQTKH (SEQ ID NO:14) or comprising, substantially consisting of, or consisting of, the amino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ ID NO:29) between amino acids 587 and 588 of VP1 of AAV2 or between corresponding amino acids of another AAV capsid, and further comprising a P34A amino acid substitution at residue 34 of the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding residue of another AAV capsid. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or higher, amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2 or the corresponding parental AAV capsid. In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or has 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPK AAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEVPKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLN FGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKR LNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQYLYYLSRT NTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAISDQTKHA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:42) In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO:14) or comprising, substantially comprising, or comprising the amino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ ID NO:29) at the corresponding position between amino acids 587 and 588 of the AAV2 capsid protein or in the capsid protein of another AAV serotype, and comprising an N312K amino acid substitution compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or a corresponding substitution in another AAV parent serotype, and optionally further comprising N449D, D472N, N551S, I698V and / or L735Q amino acid substitutions compared to the amino acid sequence of the AAV2 capsid or the corresponding substitution in another AAV parent serotype. In another particularly preferred embodiment, the variant AAV capsid comprises a peptide insert containing the amino acid sequence ISDQTKH (SEQ ID NO:14) or comprising, substantially consisting of, or consisting of, the amino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ ID NO:29) at the corresponding position between amino acids 587 and 588 of the AAV2 capsid or in the capsid protein of another AAV serotype, and comprising substitutions of the N312K, N449D, D472N, N551S, I698V, and L735Q amino acids compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or substitutions of the corresponding residues in another AAV parent serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or has 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPKPPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDS SSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRL K FKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQYLYYLSRT D TPSGTTTQSRLQFSQAGASDIR N QSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT S VDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAISDQTKHA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPE V QYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRN Q (SEQ ID NO:43) In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9; between amino acids 586 and 587 of AAV4; between amino acids 577 and 578 of AAV5; between amino acids 589 and 590 of AAV7; or between amino acids 590 and 591 of AAV8 or AAV10, the peptide insertion comprising an amino acid sequence selected from: ISDQTKH (SEQ ID NO:14), LGISDQTKHA (SEQ ID NO:29), and LAISDQTKHA (SEQ ID NO:29). NO:28), and b) valine to isoleucine at position 709 of amino acid AAV3A or AAV3B, alanine to isoleucine at position 709 of AAV1 or AAV6, asparagine to isoleucine at position 707 of amino acid AAV4 or 709 of amino acid AAV9, threonine to isoleucine at position 710 of amino acid AAV7 or 711 of amino acid AAV8 or AAV10, or glutamine to isoleucine at position 697 of amino acid AAV5, and optionally otherwise identical to any one of SEQ ID NO:1 and 3-12. In a preferred embodiment, the variant capsid protein comprises: a) a peptide insertion comprising the amino acid sequence ISDQTKH (SEQ ID NO:14) or comprising, substantially comprising, or comprising the amino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ ID NO:29) between amino acids 587 and 588 of the AAV2 capsid and b) a valine to isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0123] In yet another embodiment, the variant capsid protein comprises: a) a peptide insertion comprising the amino acid sequence ISDQTKH (SEQ ID NO:14) or comprising, substantially consisting of, or consisting of the amino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ ID NO:29) between amino acids 587 and 588 of the AAV2 capsid and b) a valine to isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2, and otherwise identical to the amino acid sequence SEQ ID NO:2.

[0124] In yet another embodiment, the variant capsid protein comprises: a) a peptide insertion comprising the amino acid sequence ISDQTKH (SEQ ID NO:14) or comprising, substantially consisting of, or consisting of the amino acid sequence LAISDQTKHA (SEQ ID NO:28) or LGISDQTKHA (SEQ ID NO:29) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LGISDQTKHARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:44) In a preferred embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from QADTTKN (SEQ ID NO:13) and LAQADTTKNA (SEQ ID NO:27), and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids are not naturally present at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G23 6V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V719M, S721L, L735Q, and combinations thereof, preferably selected from S109T, P250S, A524T, A593E, I698V, V708I, and / or V719M. The peptide insertion site is preferably located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. In a particularly preferred embodiment, the variant AAV capsid comprises a peptide insert containing the amino acid sequence QADTTKN (SEQ ID NO:13) or comprising, substantially consisting of, or consisting of, the amino acid sequence LAQADTTKNA (SEQ ID NO:27) at a corresponding position in the capsid protein of another AAV serotype, between amino acids 587 and 588 of the AAV2 capsid, and containing an I698V amino acid substitution compared to the amino acid sequence of AAV2, or a corresponding substitution in another AAV parent serotype, wherein one or more substituted amino acids do not naturally appear at the corresponding position. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the corresponding amino acid substitutions are I699V amino acid substitutions compared to the amino acid sequence of the AAV3A, AAV3B, or AAV9 capsid, I687V substitutions compared to the amino acid sequence of the AAV5 capsid, I700V substitutions compared to the amino acid sequence of the AAV7 capsid, and I701V substitutions compared to the amino acid sequence of the AAV8 or AAV10 capsid.In a particularly preferred embodiment, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences or has 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAQADTTKNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPE V QYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ IDNO:45) In other preferred embodiments, the variant AAV capsid comprises a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO:13) or comprising, substantially comprising, or comprising the amino acid sequence LAQADTTKNA (SEQ ID NO:27) at a corresponding position between amino acids 587 and 588 of the AAV2 capsid or in the capsid protein of another AAV serotype, and comprising a V719M amino acid substitution compared to the amino acid sequence of AAV2 and optionally a V708I substitution or a corresponding substitution in another AAV parent serotype, wherein one or more substituted amino acids do not naturally appear at the corresponding position.

[0125] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insertion located between amino acids 588 and 589 of VP1 in AAV1, AAV3A, AAV3B, AAV6, or AAV9; between amino acids 586 and 587 of AAV4; between amino acids 577 and 578 of AAV5; between amino acids 589 and 590 of AAV7; or between amino acids 590 and 591 of AAV8 or AAV10, the peptide insertion comprising an amino acid sequence selected from: QADTTKN (SEQ ID NO:13) and LAQADTTKNA (SEQ ID NO:14). NO:27), and b) valine to isoleucine at position 709 of AAV3A or AAV3B, alanine to isoleucine at position 709 of AAV1 or AAV6, asparagine to isoleucine at position 707 of AAV4 or AAV9, threonine to isoleucine at position 710 of AAV7 or threonine at position 711 of AAV8 or AAV10, or glutamine to isoleucine at position 697 of AAV5. In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insertion between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9, between amino acids 586 and 587 of AAV4, between amino acids 577 and 578 of AAV5, between amino acids 589 and 590 of AAV7, or between amino acids 590 and 591 of AAV8 or AAV10, the peptide insertion comprising an amino acid sequence selected from QADTTKN (SEQ ID NO:13) and LAQADTTKNA (SEQ ID NO:27), and b) a serine to threonine amino acid substitution at position 109 compared to the amino acid sequence of AAV1, AAV3A, AAV3B, AAV4, AAV7, AAV8, AAV9, or AAV10, or at position 108 compared to the amino acid sequence of AAV5 or AAV6.In a preferred embodiment, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence QADTTKN (SEQ ID NO:13) or comprising, substantially comprising, or comprising the amino acid sequence LAQADTTKNA (SEQ ID NO:27) between amino acids 587 and 588 of the AAV2 capsid, and comprising a serine-to-threonine substitution at amino acid 109 (S109T) or a valine-to-isoleucine substitution at amino acid 708 (V708I) compared to the AAV2 amino acid sequence, wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions, and preferably has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In other preferred embodiments, the variant AAV capsid includes a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO:13) or comprising, substantially comprising, or comprising the amino acid sequence LAQADTTKNA (SEQ ID NO:27) at a corresponding position between amino acids 587 and 588 of the AAV2 capsid or in the capsid protein of another AAV serotype, and comprising a serine-threonine substitution at amino acid 109 and a valine-isoleucine substitution at amino acid 708 compared to the amino acid sequence of AAV2.

[0126] In yet another embodiment, the variant capsid protein comprises: a) a peptide insertion comprising the amino acid sequence QADTTKN (SEQ ID NO:13) or comprising, substantially comprising, or comprising the amino acid sequence LAQADTTKNA (SEQ ID NO:27) between amino acids 587 and 588 of the AAV2 capsid and b) at least one amino acid substitution, wherein the amino acid sequence of the variant capsid does not contain a valine-to-isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2, and does not contain a serine-to-threonine substitution at amino acid 109 compared to the amino acid sequence of AAV2.

[0127] In yet another embodiment, the variant capsid protein comprises: a) a peptide insertion comprising the amino acid sequence QADTTKN (SEQ ID NO:13) or comprising, substantially consisting of, or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO:27) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence SEQ ID NO:2.

[0128] In another preferred embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from HDITKNI (SEQ ID NO:17), IAHDITKNIA (SEQ ID NO:60), and LAHDITKNIA (SEQ ID NO:32), and b) a corresponding substitution of one or more of the following amino acid substitutions or another AAV parental serotype (i.e., other than AAV2) compared to the amino acid sequence of AAV2 (SEQ ID NO:2), wherein the substituted one or more amino acids are not naturally present at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188 The AAV capsid protein comprises one or more amino acid substitutions selected from S109T, R389S, A593E, and / or V708I. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. In some embodiments, the AAV capsid protein contains one or more amino acid substitutions selected from S109T, R389S, A593E, and / or V708I. In a preferred embodiment, the variant AAV capsid comprises a peptide insert containing the amino acid sequence HDITKNI (SEQ ID NO:17) or comprising, substantially consisting of, or consisting of the amino acid sequence IAHDITKNIA (SEQ ID NO:60) or LAHDITKNIA (SEQ ID NO:32) between amino acids 587 and 588 of the AAV2 capsid, and containing an S109T amino acid substitution compared to the amino acid sequence of the AAV2 capsid or a corresponding substitution in another AAV parent serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2.

[0129] In yet another embodiment, the variant capsid comprises: a) a peptide insertion comprising the amino acid sequence HDITKNI (SEQ ID NO:17) or comprising, substantially consisting of, or consisting of the amino acid sequence IAHDITKNIA (SEQ ID NO:60) or LAHDITKNIA (SEQ ID NO:32) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence described in SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAHDITKNIARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ IDNO:46) In other embodiments, the variant capsid comprises: a) a peptide insertion comprising, substantially consisting of, or consisting of the amino acid sequence LAHDITKNIA between amino acids 587 and 588 of the AAV2 capsid, and b) at least one amino acid substitution, wherein the amino acid sequence of the variant capsid does not contain a valine to isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2. In yet another embodiment, the variant capsid comprises: a) a peptide insertion comprising the amino acid sequence DITKNIA (SEQ ID NO: 61) or comprising, substantially consisting of, or consisting of, the amino acid sequence IAHDITKNIA (SEQ ID NO: 60) or LAHDITKNIA (SEQ ID NO: 32) between amino acids 587 and 588 of the AAV2 capsid, and b) a V708I substitution compared to the amino acid sequence of AAV2. In other embodiments, the variant capsid comprises: a) a peptide insertion comprising, substantially comprising, or comprising the amino acid sequence LAHDITKNIA (SEQ ID NO:32) between amino acids 587 and 588 of the AAV2 capsid, and b) two or more amino acid substitutions, wherein the amino acid sequence of the variant capsid comprises a valine to isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2.

[0130] In another preferred embodiment, a variant AAV capsid protein is provided comprising a) an inserted peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from NQDYTKT (SEQ ID NO:16) and LANQDYTKTA (SEQ ID NO:31), and b) an amino acid sequence of AAV2 (SEQ ID NO:31). NO:2) Compared to the corresponding substitutions in one or more of the following amino acid substitutions or another AAV parental serotype (i.e., other than AAV2), wherein one or more substituted amino acids are not naturally present at the corresponding position: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q and combinations thereof. In some embodiments, the AAV capsid protein comprises one or more amino acid substitutions selected from S109T, S109T+S463Y, D368H, and V708I. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at a corresponding position in the capsid protein of another AAV serotype. In a preferred embodiment, the variant AAV capsid comprises a peptide insertion comprising the amino acid sequence NQDYTKT (SEQ ID NO:16) or comprising, substantially comprising, or comprising the amino acid sequence LANQDYTKTA (SEQ ID NO:31) between amino acids 587 and 588 of the AAV2 capsid, and comprising a V708I amino acid substitution compared to the amino acid sequence of the AAV2 capsid or a corresponding substitution in another AAV parent serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the variant capsid comprises: a) a peptide insertion comprising the amino acid sequence NQDYTKT (SEQ ID NO:16) or comprising, substantially consisting of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO:31) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence described in SEQ ID NO:2.In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences or has 100% sequence identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANQDYTKTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ IDNO:47) In other embodiments, the variant capsid comprises: a) a peptide insertion comprising the amino acid sequence NQDYTKT (SEQ ID NO: 16) or comprising, substantially comprising, or comprising the amino acid sequence LANQDYTKTA (SEQ ID NO: 31) between amino acids 587 and 588 of the AAV2 capsid and b) an S109T amino acid substitution compared to the sequence of SEQ ID NO: 2 and optionally an S463Y amino acid substitution, wherein the variant capsid has at least about 85%, at least about 90%, at least about 95%, and at least about 98% identity with the entire length of the amino acid sequence shown in SEQ ID NO: 2. In relevant embodiments, the variant capsid comprises: a) a peptide insertion comprising the amino acid sequence NQDYTKT (SEQ ID NO:16) or comprising, substantially comprising, or comprising the amino acid sequence LANQDYTKTA (SEQ ID NO:31) between amino acids 587 and 588 of the AAV2 capsid and b) an S109T amino acid substitution compared to the amino acid sequence SEQ ID NO:2, and otherwise identical to the amino acid sequence SEQ ID NO:2.

[0131] In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insertion located between amino acids 588 and 589 of VP1 of AAV1, AAV3A, AAV3B, AAV6, or AAV9; between amino acids 586 and 587 of AAV4; between amino acids 577 and 578 of AAV5; between amino acids 589 and 590 of AAV7; or between amino acids 590 and 591 of AAV8 or AAV10, the peptide insertion comprising an amino acid sequence selected from NQDYTKT (SEQ ID NO: 16) and LANQDYTKTA (SEQ ID NO: 16). ID NO: 31) and b) asparagine to lysine amino acid substitution at position 313 compared to the amino acid sequence of AAV1 or AAV6 or at position 314 compared to the amino acid sequence of AAV9, or serine to lysine substitution at position 312 of AAV3A or AAV3B or at position 315 of AAV8 or AAV10, or arginine to lysine substitution at position 303 of AAV4 or AAV5 or at position 314 of AAV7. In another embodiment, the variant capsid comprises: a) a peptide insertion comprising the amino acid sequence NQDYTKT (SEQ ID NO: 16) or comprising, substantially consisting of, or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO: 31) between amino acids 587 and 588 of the AAV2 capsid and b) N312K amino acid substitution, wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[0132] In another embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from PNSTHGS (SEQ ID NO:25) and LAPNSTHGSA (SEQ ID NO:40), and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids are not naturally present at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. In a preferred embodiment, the variant AAV capsid comprises a peptide insert containing the amino acid sequence PNSTHGS (SEQ ID NO:25) or comprising, substantially comprising, or comprising the amino acid sequence LAPNSTHGSA (SEQ ID NO:40) between amino acids 587 and 588 of the AAV2 capsid, and containing a V708I amino acid substitution compared to the amino acid sequence of the AAV2 capsid or a corresponding substitution in another AAV parent serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the variant capsid comprises: a) a peptide insertion comprising the amino acid sequence PNSTHGS (SEQ ID NO:25) or comprising, substantially consisting of, or consisting of the amino acid sequence LAPNSTHGSA (SEQ ID NO:40) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence described in SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAPNSTHGSA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQIDNO:48) In another embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from NKTTNKDA (SEQ ID NO:62) and LANKTTNKDA (SEQ ID NO:35), and b) a corresponding substitution of one or more of the following amino acid substitutions or another AAV parental serotype (i.e., other than AAV2) compared to the amino acid sequence of AAV2 (SEQ ID NO:2), wherein the substituted one or more amino acids are not naturally present at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L 188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. In a preferred embodiment, the variant AAV capsid comprises a peptide insert containing the amino acid sequence NKTTNKDA (SEQ ID NO: 62) or comprising, substantially comprising, or comprising the amino acid sequence LANKTTNKDA (SEQ ID NO: 35) between amino acids 587 and 588 of the AAV2 capsid, and containing an N449D amino acid substitution compared to the amino acid sequence of the AAV2 capsid or a corresponding substitution in another AAV parent serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO: 2. In yet another embodiment, the variant capsid comprises: a) a peptide insertion comprising the amino acid sequence NKTTNKDA (SEQ ID NO: 62) or comprising, substantially consisting of, or consisting of the amino acid sequence LANKTTNKDA (SEQ ID NO: 35) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence described in SEQ ID NO: 2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPKPPAERHKDDSRGLVLPGYKYYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LANKTTNKDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:49) In another embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from: TNTTSPD (SEQ ID NO:24) and LATNRTSPDA (SEQ ID NO:39), and b) an amino acid sequence of AAV2 (SEQ ID NO:39). NO:2) Compared to one or more of the following amino acid substitutions or the corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein one or more substituted amino acids are not naturally present at the corresponding position: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V719M, S721L, L735Q and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at a corresponding position in the capsid protein of another AAV serotype. In a related embodiment, a variant AAV capsid protein is provided comprising a) a peptide insertion located between amino acids 588 and 589 of VP1 in AAV1, AAV3A, AAV3B, AAV6, or AAV9; between amino acids 586 and 587 in AAV4; between amino acids 577 and 578 in AAV5; between amino acids 589 and 590 in AAV7; or between amino acids 590 and 591 in AAV8 or AAV10, the peptide insertion comprising an amino acid sequence selected from: TNTRSPD (SEQ ID NO: 24) and LATNRTSPDA (SEQ ID NO: 24). NO:39), and b) valine to isoleucine at position 709 of AAV3A or AAV3B, alanine to isoleucine at position 709 of AAV1 or AAV6, asparagine to isoleucine at position 707 of AAV4 or AAV9, threonine to isoleucine at position 710 of AAV7 or threonine at position 711 of AAV8 or AAV10, or glutamine to isoleucine at position 697 of AAV5.In other embodiments, the variant capsid protein comprises: a) a peptide insertion comprising, substantially comprising, or comprising the amino acid sequence LATNRTSPDA (SEQ ID NO:39) between amino acids 587 and 588 of the AAV2 capsid and b) a valine-to-isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions. In yet another embodiment, the variant capsid protein comprises: a) a peptide insertion comprising the amino acid sequence TNTTSPD (SEQ ID NO:24) between amino acids 587 and 588 of the AAV2 capsid and b) a valine-to-isoleucine amino acid substitution at amino acid 708 compared to the amino acid sequence of AAV2. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2.

[0133] In yet another embodiment, the variant capsid protein comprises: a) a peptide insertion comprising the amino acid sequence TNTRSPD (SEQ ID NO:24) or comprising, substantially consisting of, or consisting of the amino acid sequence LATNRTSPDA (SEQ ID NO:39) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPKPPAERHKDDSRGLVLPGYKYYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LATNRTSPDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:50) In another embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from GKSKVID (SEQ ID NO:23) and LAGKSKVIDA (SEQ ID NO:38), and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids are not naturally present at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the variant AAV capsid comprises a peptide insertion between amino acids 587 and 588 of the AAV2 capsid, said peptide insertion comprising the amino acid sequence GKSKVID (SEQ ID NO:23) or comprising, substantially comprising, or comprising the amino acid sequence LAGKSKVIDA (SEQ ID NO:38), and otherwise identical to the amino acid sequence SEQ ID NO:2. In other embodiments, the variant AAV capsid comprises: a) a peptide insertion comprising, substantially comprising, or comprising the amino acid sequence LAGKSKVIDA (SEQ ID NO:38) between amino acids 587 and 588 of the AAV2 capsid, and comprising at least one amino acid substitution.

[0134] In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or has 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPKPPAERHKDDSRGLVLPGYKYYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAGKSKVIDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:51) In another embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from ASDSTKA (SEQ ID NO:15) and LAASDSTKAA (SEQ ID NO:30), and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids are not naturally present at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the variant capsid comprises a peptide insert containing the amino acid sequence ASDSTKA (SEQ ID NO:15) or comprising, substantially consisting of, or consisting of the amino acid sequence LAASDSTKAA (SEQ ID NO:30) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence described in SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPKPPAERHKDDSRGLVLPGYKYYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAASDSTKAA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:52) In another embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from KDRAPST (SEQ ID NO:26) and LAKDRAPTSA (SEQ ID NO:41), and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids are not naturally present at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the variant capsid comprises a peptide insert containing the amino acid sequence KDRAPST (SEQ ID NO:26) or comprising, substantially consisting of, or consisting of the amino acid sequence LAKDRAPTSA (SEQ ID NO:41) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence described in SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPKPPAERHKDDSRGLVLPGYKYYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAKDRAPSTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:53) In another embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from HQDTTKN (SEQ ID NO:19) and LAHQDTTKNA (SEQ ID NO:34), and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids are not naturally present at one or more of the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T 176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the variant capsid comprises a peptide insert containing the amino acid sequence HQDTTKN (SEQ ID NO:19) or contains, substantially consists of, or consists of the amino acid sequence LAHQDTTKNA (SEQ ID NO:34) between amino acids 587 and 588 of the AAV2 capsid, and is otherwise identical to the amino acid sequence described in SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or has 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPKPPAERHKDDSRGLVLPGYKYYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAHQDTTKNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:54) In another embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from ISNENEH (SEQ ID NO:21) and LPISNENEHA (SEQ ID NO:36), and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids are not naturally present at one or more of the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T 176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the variant capsid comprises a peptide insert containing the amino acid sequence ISNENEH (SEQ ID NO:21) or comprising, substantially consisting of, or consisting of the amino acid sequence LPISNNENEHA (SEQ ID NO:36) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence described in SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPKPPAERHKDDSRGLVLPGYKYYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LPISNENEHA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:55) In another embodiment, a variant AAV capsid protein is provided comprising a) a peptide insertion in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from QANANEN (SEQ ID NO:22) and LPQANANENA (SEQ ID NO:37), and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids are not naturally present at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the variant capsid comprises a peptide insert containing the amino acid sequence QANANEN (SEQ ID NO:22) or comprising, substantially consisting of, or consisting of the amino acid sequence LPQANANENA (SEQ ID NO:37) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence described in SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPKPPAERHKDDSRGLVLPGYKYYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFSHSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LPQANANENA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:56) In another embodiment, a variant AAV capsid protein is provided comprising a) an insertion peptide in the GH-ring of the capsid protein, wherein the peptide insertion comprises a peptide insertion selected from HPDTTKN (SEQ ID NO:18) and LAHPDTTKNA (SEQ ID NO:33), and b) one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2 (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), wherein the substituted one or more amino acids are not naturally present at the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, and combinations thereof. Preferably, the peptide insertion site is located between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype. The variant AAV capsid may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the variant capsid comprises a peptide insertion comprising the amino acid sequence HPDTTKN (SEQ ID NO:18) or comprising, substantially consisting of, or consisting of the amino acid sequence LAHPDTTKNA (SEQ ID NO:33) between amino acids 587 and 588 of the AAV2 capsid, and otherwise identical to the amino acid sequence described in SEQ ID NO:2. In some embodiments, the variant AAV capsid has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity with the following amino acid sequences, or having 100% identity with the following amino acid sequences: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPV EHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSP RDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQ YLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAHPDTTKNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ IDNO:57). In several aspects, a variant AAV capsid protein is provided comprising one or more amino acid substitutions relative to the corresponding parental AAV capsid protein, wherein, when present in AAV viral particles, the variant AAV capsid protein confers increased infectivity to retinal cells compared to the infectivity of AAV viral particles containing the corresponding parental AAV capsid protein to retinal cells.

[0135] In some embodiments, the variant AAV capsid protein comprises a P34A amino acid substitution compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or a P33A amino acid substitution compared to the amino acid sequence of the AAV5 capsid (SEQ ID NO:6). In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:6, and comprises a P34A or P33A amino acid substitution compared to the amino acid sequence of the AAV2 or AAV5 capsid, respectively. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence comprising a P34A amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO:2. In related embodiments, the variant capsid protein comprises a P34A amino acid substitution compared to the amino acid sequence SEQ ID NO:2, wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of the AAV2 capsid protein shown in SEQ ID NO:2.

[0136] In other embodiments, the variant AAV capsid protein comprises an amino acid substitution at amino acid 164 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position in another AAV parent serotype (i.e., other than AAV2), wherein the substituted amino acid does not naturally appear at the corresponding position. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and comprises an amino acid substitution at amino acid 164 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the rAAV viral particle comprises a glutamine-to-lysine amino acid substitution at amino acid 164 compared to the amino acid sequence of AAV1, AAV2, or AAV6, or at amino acid 165 compared to the amino acid sequence of AAV7, AAV8, or AAV10; or comprises a serine-to-lysine substitution at amino acid 160 of AAV5, or an alanine-to-lysine substitution at amino acid 164 of AAV9. In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 164 (e.g., Q164K) compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of the AAV2 capsid protein shown in SEQ ID NO:2. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence comprising the Q164K amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO:2. In other embodiments, the variant capsid protein comprises Q164K and V708I amino acid substitutions compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2.

[0137] In other embodiments, the variant AAV capsid protein comprises amino acid 698, which is identical to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or an amino acid substitution at the corresponding position in another AAV parent serotype (i.e., other than AAV2), wherein the substituted amino acid does not naturally occur at the corresponding position. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and comprises an amino acid substitution at amino acid 698, which is identical to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the rAAV viral particle comprises an isoleucine to valine amino acid substitution at amino acid 698 compared to the amino acid sequence of AAV2, or at amino acid 699 compared to the amino acid sequence of AAV3A, AAV3B, or AAV9, or at amino acid 687 of AAV5, or at amino acid 700 of AAV7, or at amino acid 701 of AAV8 or AAV10. In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 699 (e.g., I698V) compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of the AAV2 capsid protein shown in SEQ ID NO:2. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence containing the I698V amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO:2.

[0138] In other embodiments, the variant AAV capsid protein comprises amino acid substitution at position 109 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position in another AAV parent serotype (i.e., other than AAV2). In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and comprises an amino acid substitution at position 109 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the variant capsid protein comprises a serine to threonine amino acid substitution at position 109 compared to the amino acid sequence of AAV1, AAV3A, AAV3B, AAV4, AAV7, AAV8, AAV9, or AAV10, or at position 108 compared to the amino acid sequence of AAV5 or AAV6. In related embodiments, the variant capsid protein comprises an S109T amino acid substitution compared to the amino acid sequence AAV2, wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions. In other related embodiments, the variant capsid protein comprises an S109T amino acid substitution and an A593E amino acid substitution compared to the amino acid sequence of AAV2. In some embodiments, the variant capsid protein comprises S109T and A493V, and optionally A593E and / or V708I amino acid substitutions or corresponding substitutions in another AAV parental serotype (i.e., other than AAV2), compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In some preferred embodiments, the variant capsid protein comprises S109T, A493V, A593E, and V708I amino acid substitutions compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or corresponding substitutions of another AAV parental serotype (i.e., other than AAV2), and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or higher amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2.In other preferred embodiments, the variant capsid protein comprises S109T and V708I amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and is otherwise consistent with the amino acid sequence SEQ ID NO:2.

[0139] In other embodiments, the variant AAV capsid protein comprises amino acid 593, which is identical to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or an amino acid substitution at the corresponding position in another AAV parent serotype (i.e., other than AAV2). In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and comprises an amino acid substitution at amino acid 593, which is identical to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the variant capsid protein comprises a glycine-to-glutamic acid substitution at amino acid 594 of AAV1, AAV3A, AAV6, or AAV9, amino acid 583 of AAV5, or amino acid 596 of AAV8 or AAV10, an arginine-to-glutamic acid substitution at amino acid 594 of AAV3B, an aspartic acid-to-glutamic acid substitution at amino acid 592 of AAV4, or a glutamine-to-glutamic acid substitution at position 595 of AAV7. In other embodiments, the variant capsid protein comprises an A593E amino acid substitution compared to the amino acid sequence of AAV2, and does not comprise one or more of the following amino acid substitutions compared to the amino acid sequence of AAV2: I19V, V369A, K26R, N215D, G355S, V46A, and S196P. In related embodiments, the variant capsid protein comprises amino acid substitutions of A593E and N596D compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In other embodiments, the variant capsid comprises amino acid substitutions of A593E and N596D compared to the amino acid sequence of AAV2, and is otherwise identical to the amino acid sequence of AAV2. In other embodiments, the variant capsid protein comprises amino acid substitutions of A593E and V708I compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity with the entire length of the amino acid sequence shown in SEQ ID NO:2. In other embodiments, the variant capsid contains A593E and V708I amino acid substitutions compared to the amino acid sequence of AAV2, and is otherwise identical to the amino acid sequence of AAV2.

[0140] In other embodiments, the variant AAV capsid protein comprises amino acid 708, which is the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or an amino acid substitution at the corresponding position in another AAV parent serotype (i.e., other than AAV2), wherein the substituted amino acid does not naturally occur at the corresponding position. Preferably, the rAAV viral particle does not contain proline-serine substitutions at amino acid 250, which is the amino acid sequence of AAV2, or at the corresponding amino acid in another AAV parent serotype. In some embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and comprises an amino acid substitution at amino acid 708, which is the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In a preferred embodiment, the variant capsid protein comprises a valine-to-isoleucine (V708I) substitution at amino acid 708 compared to the amino acid sequence of the AAV2 capsid, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and is otherwise identical to the amino acid sequence of SEQ ID NO:2, wherein the variant capsid protein does not contain the P250S amino acid substitution. In some embodiments, the variant capsid protein comprises a valine-to-isoleucine substitution at amino acid 709 of AAV3A or AAV3B, an alanine-to-isoleucine substitution at amino acid 709 of AAV1 or AAV6, an asparagine-to-isoleucine substitution at amino acid 707 of AAV4 or amino acid 709 of AAV9, a threonine-to-isoleucine substitution at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine-to-isoleucine substitution at amino acid 697 of AAV5. In related embodiments, the variant capsid protein comprises a V708I amino acid substitution compared to the amino acid sequence of AAV2, wherein the variant capsid protein comprises 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions, and wherein the variant capsid protein does not contain a P250S amino acid substitution. In other embodiments, the variant capsid protein comprises a V708I amino acid substitution and also comprises A593E and / or S109T amino acid substitutions compared to the amino acid sequence of AAV2. In other related embodiments, the variant capsid comprises V708I and A593E amino acid substitutions compared to the amino acid sequence of AAV2, wherein the variant capsid protein is otherwise identical to the amino acid sequence of AAV2.In other related embodiments, the variant capsid comprises V708I and S109T amino acid substitutions compared to the amino acid sequence of AAV2, wherein the variant capsid protein is otherwise identical to the amino acid sequence of AAV2. In other embodiments, the variant capsid protein comprises V708I and V719M amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO:2, and is otherwise identical to the amino acid sequence of SEQ ID NO:2. In other embodiments, the variant capsid protein comprises V708I and R733C amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity over the entire length of the amino acid sequence shown in SEQ ID NO:2, and is otherwise identical to the amino acid sequence of SEQ ID NO:2. In other embodiments, the variant capsid protein comprises V708I and G727D amino acid substitutions compared to the amino acid sequence of AAV2, and has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and is otherwise consistent with the amino acid sequence SEQ ID NO:2.

[0141] In other embodiments, the variant AAV capsid protein comprises an amino acid substitution at amino acid 196 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position in another AAV parental serotype (i.e., other than AAV2), wherein the substituted amino acid does not naturally appear at the corresponding position and optionally, in addition to proline. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and comprises an amino acid substitution at amino acid 196 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) and optionally, in addition to the S196P substitution. In a preferred embodiment, the variant capsid protein comprises a serine-to-tyrosine amino acid substitution at amino acid 196 of AAV2 or AAV9, or at amino acid 197 of AAV7, AAV8 or AAV10, or at amino acid 186 of AAV5; or an alanine-to-tyrosine substitution at amino acid 196 of AAV1 or AAV6; or a methionine-to-tyrosine substitution at amino acid 191 of AAV4; or a threonine-to-tyrosine substitution at amino acid 196 of AAV3A or AAV3B. In a related embodiment, the variant capsid protein comprises an amino acid sequence containing the S196Y amino acid substitution compared to the amino acid sequence shown in SEQ ID NO:2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO:2. In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 196, other than the S196P substitution (e.g., including the S196Y substitution), compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of the AAV2 capsid protein shown in SEQ ID NO:2.

[0142] In other embodiments, the variant AAV capsid protein comprises an amino acid substitution at amino acid 175 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position in another AAV parent serotype (i.e., other than AAV2), wherein the substituted amino acid does not naturally occur at the corresponding position. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and comprises an amino acid substitution at amino acid 175 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the variant capsid comprises a Q175H amino acid substitution compared to the amino acid sequence of AAV2 shown in SEQ ID NO:2 or a glutamine-to-histidine substitution at the corresponding position in another AAV parent serotype. In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 175 (e.g., Q175H) compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of the AAV2 capsid protein shown in SEQ ID NO:2.

[0143] In other embodiments, the variant AAV capsid protein comprises an amino acid substitution at position 64 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position in another AAV parent serotype (i.e., other than AAV2), wherein the substituted amino acid does not naturally occur at the corresponding position. In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2, and comprises an amino acid substitution at position 64 compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the rAAV viral particle comprises a P64S amino acid substitution compared to the amino acid sequence of AAV2 shown in SEQ ID NO:2 or a proline-serine substitution at the corresponding position in another AAV parent serotype. In related embodiments, the variant capsid protein comprises an amino acid substitution at amino acid 64 (e.g., P64S) compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), wherein the variant capsid protein comprises 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions compared to the amino acid sequence of the AAV2 capsid protein shown in SEQ ID NO:2.

[0144] In other embodiments, the variant AAV capsid protein comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity with the wild-type AAV capsid sequence selected from the group consisting of: SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, and 12, and further comprises i) one or more amino acid substitutions selected from the group consisting of: P34A, S109T+V708I, A593E+N596D, V708I+V719M, V708I+G727D, S109T+A493V+A593E+V708I, V708I+R733C, Q164K, and I698V and / or (ii) peptide insertions selected from the group consisting of: QADTTKN (SEQ ID NO: 13), ISDQTKH (SEQ ID NO: 14), ASDSTKA (SEQ ID NO: 15), and ISDQTKH (SEQ ID NO: 16), and ISDQTKH (SEQ ID NO: 16), and ISDQTKH (SEQ ID NO: 17), and ISDQTKH (SEQ ID NO: 18), and ISDQTKH (SEQ ID NO: 19 ... NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), PQANANEN (SEQ ID NO:63), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST (SEQ ID NO:26), HQDTTKN (SEQ ID NO:19), HPDTTKN (SEQ ID NO:18), NKTTNKD (SEQ ID NO:20), GKSKVID (SEQ ID NO:23), PISNENEH (SEQ ID NO:64), LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA (SEQ ID NO:30), LAHDITKNIA (SEQ ID NO:32), LPQANANENA (SEQ IDNO:37), LANQDYTKTA (SEQ ID NO:31), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ ID NO:40), LAKDRAPSTA (SEQ ID NO:41), LAHQDTTKNA (SEQ ID NO:34), LAHPDTTKNA (SEQ IDNO:33), LANKTTNKDA (SEQ ID NO:35), LAGKSKVIDA (SEQ ID NO:38) and LPISNENEHA (SEQ ID NO:36).In some embodiments, the variant AAV capsid contains one or more specified amino acid substitutions and / or peptide insertions, and otherwise conforms to sequences selected from the group consisting of: SEQ ID NO:1-12.

[0145] In some implementations, the variant AAV capsid protein is the ancestral capsid protein. An ancestral capsid protein refers to the evolutionary ancestor of capsid proteins found in nature today, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, AAV12, and AAV13, which is generated by computer simulation through random amino acid substitutions at degeneracy positions among AAV capsid proteins found in nature today. Below is a non-restrictive example of an ancestral capsid, where the degenerate positions (residues 264, 266, 268, 448, 459, 460, 467, 470, 471, 474, 495, 516, 533, 547, 551, 555, 557, 561, 563, 577, 583, 593, 596, 661, 662, 664, 665, 710, 717, 718, 719, 723) are marked with “X”: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSXSXGXTNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLXRTQSTGGTAGXXELLFSQXGPXXMSXQAKNWLPGPCYRQQRVSKTLXQNNNSNFAWTGATKYHLNGRXSLVNPGVAMATHKDDEXRFFPSSGVLIFGKXGAGXNNTXLXNVMXTXEEEIKTTNPVATEXYGVVAXNLQSSNTAPXTGXVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPXXFXXAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYAKSXNVDFAVXXXGVYXEPRPIGTRYLTRNL (SEQ ID NO:58) In some embodiments, the ancestral capsid protein comprises an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:58. In some embodiments, the ancestral capsid protein comprises an amino acid sequence that has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or more amino acid sequence identity with the entire length of the amino acid sequence of, for example, AAV2 shown in SEQ ID NO:2. In some embodiments, the ancestral capsid protein comprises an amino acid sequence that is identical to SEQ ID NO:58 or SEQ ID NO:58. The ancestral sequence disclosed in NO:2 has an amino acid sequence identity of at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or higher throughout its entire length, and contains one or more amino acid residues selected from the group consisting of: alanine (A) at 264, alanine (A) at 266, serine (S) at 268, alanine (A) at 448, threonine (T) at 459, arginine (R) at 460, alanine (A) at 467, serine (S) at 470, asparagine (N) at 471, alanine (A) at 474, serine (S) at 495, asparagine (D) at 516, and so on. Asparagine (D) at position 3, glutamine (Q) at position 547, alanine (A) at position 551, alanine (A) at position 555, glutamic acid (E) at position 557, methionine (M) at position 561, serine (S) at position 563, glutamine (Q) at position 577, serine (S) at position 583, valine (V) at position 593, threonine (T) at position 596, alanine (A) at position 661, valine (V) at position 662, threonine (T) at position 664, proline (P) at position 665, threonine (T) at position 710, aspartic acid (D) at position 717, asparagine (N) at position 718, glutamic acid (E) at position 719, and serine (S) at position 723.In some preferred embodiments, the variant capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, and in some cases 100% amino acid sequence identity with the entire length of the following amino acid sequences, and comprising one or more amino acid residues selected from the group consisting of: alanine (A) at 264, alanine (A) at 266, serine (S) at 268, alanine (A) at 448, threonine (T) at 459, arginine (R) at 460, alanine (A) at 467, serine (S) at 470, asparagine (N) at 471, alanine (A) at 474, and serine (S) at 495. Asparagine (D) at position 516, asparagine (D) at position 533, glutamine (Q) at position 547, alanine (A) at position 551, alanine (A) at position 555, glutamic acid (E) at position 557, methionine (M) at position 561, serine (S) at position 563, glutamine (Q) at position 577, serine (S) at position 583, valine (V) at position 593, threonine (T) at position 596, alanine (A) at position 661, valine (V) at position 662, threonine (T) at position 664, proline (P) at position 665, threonine (T) at position 710, aspartic acid (D) at position 717, asparagine (N) at position 718, glutamic acid (E) at position 719, and serine (S) at position 723. MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASAGSTNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSTGGTAGTRELLFSQAGPSNMSAQAKNWLPGPCYRQQRVSKTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPSSGVLIFGKQGAGANNTALENVMMTSEEEIKTTNPVATEQYGVVASNLQSSNTAPVTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYAKSTNVDFAVDNEGVYSEPRPIGTRYLTRNL (SEQ ID NO:59). In other embodiments, the variant AAV capsid protein comprises an amino acid sequence that is identical in at least 85%, at least 90%, at least 95%, or at least 98% to the wild-type AAV capsid sequence selected from the group consisting of: the ancestral variant disclosed herein as SEQ ID NO:58, comprising one or more amino acid residues selected from the group consisting of: alanine (A) at 264, alanine (A) at 266, serine (S) at 268, alanine (A) at 448, threonine (T) at 459, arginine (R) at 460, alanine (A) at 467, serine (S) at 470, asparagine (N) at 471, alanine (A) at 474, serine (S) at 495, asparagine (D) at 516, asparagine (D) at 533, glutamine (Q) at 547, and so on. Alanine (A), alanine at position 555 (A), glutamic acid at position 557 (E), methionine at position 561 (M), serine at position 563 (S), glutamine at position 577 (Q), serine at position 583 (S), valine at position 593 (V), threonine at position 596 (T), alanine at position 661 (A), valine at position 662 (V), threonine at position 664 (T), proline at position 665 (P), threonine at position 710 (T), aspartic acid at position 717 (D), asparagine at position 718 (N), glutamic acid at position 719 (E), and serine at position 723 (S);It also includes i) one or more amino acid substitutions selected from the group consisting of: P34A, S109T+V708I, A593E+N596D, V708I+V719M, V708I+G727D, S109T+A493V+A593E+V708I, V708I+R733C, Q164K and I698V and / or (ii) peptide insertions selected from the group consisting of: QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), ASDSTKA (SEQ ID NO:15), NQDYTKT (SEQ ID NO:16), HDITKNI (SEQ ID NO:17), PQANANEN (SEQ ID NO:63), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), KDRAPST (SEQ ID NO:25), QADTTKN (SEQ ID NO:16), ISDQTKH (SEQ ID NO:17), ISDQTKH (SEQ ID NO:18), ISDQTKH (SEQ ID NO:19 ... NO:26), HQDTTKN (SEQ ID NO:19), HPDTTKN (SEQ ID NO:18), NKTTNKD (SEQ ID NO:20), GKSKVID (SEQ ID NO:23), PISNENEH (SEQ ID NO:64), LAQADTTKNA (SEQ ID NO:27), LAISDQTKHA (SEQ ID NO:28), LGISDQTKHA (SEQ ID NO:29), LAASDSTKAA (SEQ ID NO:30), LAHDITKNIA (SEQ ID NO:32), LPQANANENA (SEQ ID NO:37), LANQDYTKTA (SEQ ID NO:31), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ IDNO:40), LAKDRAPSTA (SEQ ID NO:41), LAHQDTTKNA (SEQ ID NO:34), LAHPDTTKNA (SEQ IDNO:33), LANKTTNKDA (SEQ (SEQ ID NO:35), LAGKSKVIDA (SEQ ID NO:38), and LPISNENEA (SEQ ID NO:36). In some embodiments, the variant AAV capsid contains one or more specified amino acid substitutions and / or peptide insertions, and is otherwise consistent with SEQ ID NO:59.

[0146] The AAV variants disclosed herein are generated through directed in vivo evolution, including screening with primate retina after intravitreal administration. In some embodiments, the variant capsid proteins disclosed herein, when present in AAV viral particles, confer increased transduction to retinal cells compared to transduction of retinal cells by AAV viral particles containing the corresponding parental AAV capsid protein or wild-type AAV. For example, in some embodiments, when present in AAV viral particles, the variant capsid proteins disclosed herein confer more efficient transduction to primate retinal cells compared to AAV viral particles containing the corresponding parental AAV capsid protein or wild-type AAV capsid protein, such that retinal cells take up more AAV viral particles containing the subject variant AAV capsid protein than AAV viral particles containing the parental AAV capsid protein or wild-type AAV. In some such embodiments, AAV variant viral particles or variant rAAV exhibit at least a 2-fold, at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than 50-fold increase in transduction of retinal cells compared to transduction of retinal cells by wild-type AAV viral particles or rAAV containing the corresponding parental AAV capsid protein. In some such embodiments, when present in AAV viral particles, the variant capsid proteins disclosed herein confer broader transduction to primate retinal cells compared to AAV viral particles containing the corresponding parental AAV capsid protein or wild-type AAV capsid protein. In other words, variant AAV viral particles transduce cell types not transduced by viral particles containing the corresponding parental AAV capsid protein, and thus transduce more cell types in the retina than with the corresponding parental AAV viral particles. In some embodiments, AAV variant viral particles preferentially transduce retinal cells, for example, by infecting retinal cells with 2, 5, 10, 15, 20, 25, 50, or more than 50 times the specificity of other retinal or non-retinal cells, such as cells outside the eye. In some embodiments, the transduced retinal cells are photoreceptor cells (e.g., rods; cones). In some embodiments, the retinal cells are retinal ganglion cells (RGCs). In some embodiments, the retinal cells are retinal epithelial cells (RPE cells). In some embodiments, the retinal cells are Müller glial cells. In some embodiments, the retinal cells are microglia. In some embodiments, the retinal cells are amacrine cells. In some embodiments, the retinal cells are bipolar cells. In some embodiments, the retinal cells are horizontal cells. Increased retinal cell transduction, such as increased transduction efficiency, broader transduction, more preferential transduction, etc., can be readily assessed in vitro or in vivo using any number of methods in the art for measuring gene expression.For example, AAV can be packaged with a genome containing an expression cassette that includes a reporter gene, such as a fluorescent protein, under the control of a ubiquitous or tissue-specific promoter, and the transduction level can be assessed by detecting the fluorescent protein, for example, using fluorescence microscopy. As another example, AAV can be packaged with a genome containing a barcode nucleic acid sequence, and the transduction level can be assessed by detecting the nucleic acid sequence, for example, using PCR. As yet another example, AAV can be packaged with a genome containing an expression cassette that includes a therapeutic gene for treating retinal diseases, and the transduction level can be assessed by detecting the treatment of retinal diseases in afflicted patients who have received AAV.

[0147] Ocular diseases treatable using the variant rAAV vectors or viral particles and / or methods disclosed herein include, but are not limited to, monogenic diseases, complex genetic diseases, acquired diseases, and traumatic injuries. Examples of monogenic diseases include, but are not limited to, Bardet-Biedl syndrome; Batten's disease; Bietti's crystal dystrophy; achoroidal disease; choroidal-retinal atrophy; choroidal-retinal degeneration; cone or cone-rod dystrophy (autosomal dominant, autosomal recessive, and X-linked); congenital stationary night blindness (autosomal dominant, autosomal recessive, and X-linked); color vision disorders, including achromatopsia (including ACHM2, ACHM3, ACHM4, and ACHM5), red-green color blindness, and blue-green color blindness; Friedreich syndrome. Ataxia; Leber congenital amaurosis (autosomal dominant and autosomal recessive inheritance), including but not limited to LCA1, LCA2, LCA3, LCA4, LCA6, LCA7, LCA8, LCA12, and LCA15; Leber hereditary optic neuropathy; macular dystrophy (autosomal dominant and autosomal recessive inheritance), including but not limited to acute macular degeneration, optimal vitrectomyeloid macular dystrophy, pattern macular dystrophy, and North Carolina macular dystrophy. Carolina Macular Dystrophy, hereditary drusen, Sorsby's fundus dystrophy, malattia levantanese, and genetically determined retinopathy of prematurity; ocular retinal developmental disorders; ocular albinism; optic atrophy (autosomal dominant, autosomal recessive, and X-linked); retinitis pigmentosa (autosomal dominant, autosomal recessive, X-linked, and mitochondrial inherited traits), examples of which include RP1, RP2, RP3, RP10, RP20, RP38, RP40, and RP43; X-linked retinoschisis; Stargardt disease; and Usher syndrome, including but not limited to USH1B, USH1C, USH1D, USH1F, USH1G, USH2A, USH2C, USH2D, and USH3. Examples of complex genetic disorders include, but are not limited to, glaucoma (open-angle, closed-angle, hypotony, normal-tension, congenital, neovascular, pigmentary, pseudoexfoliation); age-related and other forms of macular degeneration, exudative and non-exudative forms (autosomal dominant and autosomal recessive), such as acute macular degeneration, vitrectomycosis; retinopathy of prematurity; and Vogt-Koyanagi-Harada (VKH) syndrome.Examples of acquired diseases include, but are not limited to, acute macular retinopathy; anterior and posterior ischemic optic neuropathy; Behcet's disease; branch retinal vein occlusion; choroidal neovascularization; diabetic retinopathy, including proliferative diabetic retinopathy and related complications; diabetic uveitis; edema, such as macular edema, cystoid macular edema, and diabetic macular edema; epiretinal membrane diseases; macular telangiectasia; multifocal choroiditis; non-retinal diabetic retinal dysfunction; ocular tumors; optic atrophy; retinal detachment; retinal diseases, such as central retinal vein occlusion, proliferative vitreoretinopathy (PVR), retinal artery and vein occlusion, vascular occlusion, uveitis; uveal leakage; retinal infections and infiltrative diseases; and optic nerve diseases, such as acquired optic atrophy. Examples of traumatic injury include, but are not limited to, histoplasmosis; optic nerve injury; ocular trauma affecting the posterior part or location of the eye; retinal injury; viral infection of the eye; viral infection of the optic nerve; posterior ocular conditions caused or affected by laser treatment of the eye; posterior ocular conditions caused or affected by photodynamic therapy; photocoagulation; radiation-induced retinopathy; and sympathetic ophthalmia.

[0148] In another embodiment, the variant capsid disclosed herein comprises a heterologous nucleic acid containing a nucleotide sequence encoding a gene product, such as, but not limited to, interfering RNA, long non-coding RNA, short non-coding RNA, antisense RNA, aptamers, peptides, secreted antibodies, single-chain antibodies, V... HH Domains, soluble receptors, affibody, knottin, DARPin, centurin, chaperone proteins, site-specific nucleases that provide site-specific knockdown of gene function or site-specific modified nucleases that provide gene-specific activation of transcription.

[0149] The rAAV variant viral particles disclosed herein contain heterologous nucleic acids, including nucleotide sequences encoding a gene product. In some embodiments, the gene product is interfering RNA. In some embodiments, the gene product is long non-coding RNA. In some embodiments, the gene product is short non-coding RNA. In some embodiments, the gene product is antisense RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide. In some embodiments, the gene product is a secreted antibody. In some embodiments, the gene product is a single-chain antibody. In some embodiments, the gene product is V... HHDomain. In some embodiments, the gene product is a soluble receptor. In some embodiments, the gene product is an affinity protein. In some embodiments, the gene product is a knotting agent. In some embodiments, the gene product is DARPin. In some embodiments, the gene product is centurin. In some embodiments, the gene product is a chaperone protein. In some embodiments, the gene product is a site-specific nuclease that provides site-specific knockdown of gene function.

[0150] The uses of gene products include, but are not limited to: increasing the levels of cytokines in cytokines; increasing the levels of cytokines in adjacent cytokines by secreting factors; decreasing the levels of cytokines in cytokines; or decreasing the levels of cytokines in adjacent cytokines by secreting factors. Gene products can be designed to: supplement the level of a missing gene product; reduce the level of a missing gene product; introduce new supporting gene products; supplement the level of supporting gene products; reduce the level of a hindering gene product; or both reduce the level of a hindering gene product and introduce or supplement the level of a supporting gene product.

[0151] Gene products delivered by the subject AAV variant can be used to alter the levels of gene products or gene product activity that are directly or indirectly associated with retinal diseases and trauma. Genes whose gene products are directly or indirectly associated with hereditary diseases include, for example, ADP-ribosylation factor-like 6 (ARL6); BBSome interacting protein 1 (BBIP1); BBSome protein 1 (BBS1); BBSome protein 2 (BBS2); BBSome protein 4 (BBS4); BBSome protein 5 (BBS5); BBSome protein 7 (BBS7); BBSome protein 9 (BBS9); BBSome protein 10 (BBS10); BB... Some proteins 12 (BBS12); centrosome protein 290 kDa (CEP290); intraciliary transport protein 172 (IFT172); intraciliary transport protein 27 (IFT27); inositol polyphosphate-5-phosphatase E (INPP5E); inward rectifying potassium channel subfamily J member 13 (KCNJ13); leucine zipper transcription factor-like 1 (LZTFL1); McKusick-Kaufman syndrome protein (MKKS); Meckel's protein (Mecke's protein). l) Syndrome type 1 protein (MKS1); Renal tuberculosis 3 protein (NPHP1); Serologically defined colon cancer antigen 8 (SDCCAG8); Protein 32 containing a triple motif (TRIM32); Tetratricopeptide repeat domain 8 (TTC8); Barth disease protein (CLN3); Rab escort protein 1 (CHM); (PRDM13); (RGR); (TEAD1); Aromatic hydrocarbon interaction receptor protein-like 1 (AIPL) 1); cone-rod OTX-like photoreceptor homeobox transcription factor (CRX); guanylate cyclase activator protein 1A (GUCA1A); retinal-specific guanylate cyclase (GUCY2D); phosphatidylinositol transfer membrane-associated family member 3 (PITPNM3); prominin 1 (PROM1); peripheral protein (PRPH); peripheral protein 2 (PRPH2); regulatory synaptic exocytogenin 1 (RIMS1); brachiopancreatin 4A (SEMA4A); Caenorhabditis elegans (C. elegans). Human homolog of unc119 protein (UNC119); ATP-binding cassette transporter retinaldehyde (ABCA4); ADAM metallopeptidase domain 9 (ADAM9); activated transcription factor 6 (ATF6); chromosome 21 open reading frame 2 (C21orf2); chromosome 8 open reading frame 37 (C8orf37); calcium channel; voltage-dependent; α2 / δ subunit 4 (CACNA2D4); cadherin-associated family member 1 (protocadherin 21) (CDHR1); ceramide kinase-like protein (CERKL);Cone photoreceptor cGMP-gated cation channel α subunit (CNGA3); cone cyclic nucleotide-gated cation channel β3 subunit (CNGB3); cyclin M4 (CNNM4); guanine nucleotide-binding protein (G protein); α-transduction-active polypeptide 2 (GNAT2); potassium channel subfamily V member 2 (KCNV2); phosphodiesterase 6C (PDE6C); phosphodiesterase 6H (PDE6H); centriolein 1 centrin B proteome (POC1B); RAB28 member of the RAS oncogene family (RAB28); retinal and anterior neural folding homeobox 2 transcription factor (RAX2); 11-cis-retinol dehydrogenase 5 (RDH5); RP GTPase regulator interactor 1 (RPGRIP1); Tubulin tyrosine ligase-like family member 5 (TTLL5); L-type voltage-gated calcium channel α-1 subunit (CACNA1F); GTPase regulator for retinitis pigmentosa (RPGR); (GNAT1); (PDE6B); (RHO); CABP4); GPR179; (GRK1); GRM6; LRIT3; SLC24A1; TRPM1; NYX; OPN1LW; OPN1MW; Blue cone Opsin (OPN1SW); Conaquinone (FXN); (IMPDH1); (OTX2); CRB1); DTHD1GDF6; IFT140; IQCB1; LCA5; LRAT; NMNAT1; RD3; RDH12; RPE65; SPATA7; TULP1; Mitochondrial genes (KSS, LHON, MT-ATP6, MT-TH, MT-TL1, MT-TP, MT-TS2, mitochondrial-encoded NADH dehydrogenase [MT-N] D]); (BEST1); C1QTNF5EFEMP1); ELOVL4); FSCN2); GUCA1B); HMCN1); IMPG1); RP1L1); TIMP3); DRAM2); MFN2); NR2F1); Optic atrophy 1 (OPA1); TMEM126A); TIMM8A); CA4); HK1); KLHL7); NR2E3); NRL); OR2W3); PRPF3); PRPF4); PRPF6); PRPF 8); PRPF31); ROM1); Retinitis pigmentosa protein (RP1); RP9); SNRNP200); SPP2); TOPORS); ARL2BP); C2orf71); CLRN1); CNGA1); CNGB1); CYP4V2); DHDDS); DHX38); EMC1); EYS); FAM161A); GPR125); HGSNAT); IDH3B); IMPG2); KIAA1549); KIZ); MAK);MERTK); MVK); NEK2); NEUROD1); PDE6A); PDE6G); PRCD); RBP3); RLBP1); SLC7A14); USH2A); ZNF408); ZNF513); OFD1); RP2); Retinoschisis protein (RS1); ABHD12); CDH23); CEP250); CIB2); DFNB31); GPR98); HARS); MYO7A); PC DH15); USH1C); USH1G); NDP); PGK1); CAPN5); FZD4); ITM2B); LRP5); MIR204); RB1); TSPAN12); C12orf65 ); CDH3); MFRP); OAT); PLA2G5); RBP4); RGS9); RGS9BP); ARMS2; ERCC6); FBLN5); HTRA1); TLR3); and TLR4).; Genes whose gene products induce or promote apoptosis are referred to in this paper as "pro-apoptotic genes," and the products of these genes (mRNA; protein) are referred to as "pro-apoptotic gene products." Pro-apoptotic targets include, for example, Bax gene products; Bid gene products; Bak gene products; Bad gene products; Bcl-2; and Bcl-X1. Anti-apoptotic gene products include X-linked apoptosis inhibitors.

[0152] Genes whose products induce or promote angiogenesis are referred to as "pro-angiogenic genes" in this paper, and the products of these genes (mRNA; protein) are referred to as "pro-angiogenic gene products." Pro-angiogenic targets include, for example, vascular endothelial growth factors (VEGFa, VEGFb, VEGFc, VEGFd); vascular endothelial growth factor receptor 1 (VEGFR1); vascular endothelial growth factor receptor 2 (VEGFR2); Fms-associated tyrosine kinase 1 (Flt1); placental growth factor (PGF); platelet-derived growth factor (PDGF); angiopoietin; and sonic hedgehogs. Genes whose products inhibit angiogenesis are referred to as "anti-angiogenic genes" in this paper, and the products of these genes (mRNA; protein) are referred to as "anti-angiogenic gene products." Anti-angiogenic gene products include endostatin; tumor stomatine; angiostatin; pigment epithelial-derived factor (PEDF); and fusion proteins or antibodies specific to pro-angiogenic targets and / or their receptors, such as the anti-VEGF fusion protein sFLT1 or Eylea, and the VEGF-specific antibody Lucentis. TM and Avastin TM wait.

[0153] In some embodiments, the gene product delivered by the subject AAV variant is used to inhibit angiogenesis. In some preferred embodiments, the gene product delivered by the subject AAV variant is used to inhibit the activity of one or more mammalian VEGF proteins selected from the group consisting of VEGFa, VEGFb, VEGFc, VEGFd, and PGF. In a particularly preferred embodiment, the gene product delivered by the subject AAV variant inhibits the activity of VEGFa. VEGFa has nine isoforms generated by alternative splicing, of which VEGF165 is physiologically most relevant. Elevated levels of VEGFa in the vitreous have been found in patients with wet age-related macular degeneration, diabetic macular edema, and retinal vein occlusion. Gene products that inhibit VEGFa activity in the eye and thus effectively treat patients with elevated vitreous VEGFa include, but are not limited to, aflibercept, ranibizumab, bromizumab, bevacizumab, and soluble fms-like tyrosine kinase 1 (sFLT1) (GenBank accession number U01134). In some embodiments, an infectious recombinant AAV (rAAV) viral particle is provided, comprising (i) a variant AAV capsid protein as described herein and (ii) a heterologous nucleic acid comprising a plurality of sequences, each of which encodes a different VEGFa inhibitor.

[0154] In a preferred embodiment, the gene product is aflibercept. Aflibercept (EYLEA®) is a recombinant fusion protein containing extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. Aflibercept acts as a soluble decoy receptor, binding to VEGFa and PGF with a greater affinity than the natural receptor. The approved dose of aflibercept for intravitreal injection is 2.0 mg, with doses varying depending on the indication. Aflibercept is indicated for the treatment of neovascular (wet) age-related macular degeneration, macular edema following retinal vein occlusion, diabetic macular edema, and diabetic retinopathy. In a particularly preferred embodiment, a gene encoding aflibercept (corresponding to...) is provided. Figure 12A The novel codons of the optimized nucleic acid sequence contain or consist of the following: ATGGTTTCTTACTGGGACACCGGCGTGCTGCTGTGTGCCCTGCTTTCTTGTCTGCTGCTGACCGGCTC TAGCAGCGGC

[0155] In some embodiments, a nucleic acid sequence encoding aflibercept is provided, comprising a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or higher, nucleic acid identity with the full length of the nucleic acid sequence shown in SEQ ID NO: 65 or with nucleotides 79-1377 of SEQ ID NO: 65 (excluding the underlined nucleotides encoding the Flt1 signal sequence). In related embodiments, the gene product delivered by the subject AAV variant is encoded by a nucleic acid sequence consisting of, or comprising, the nucleic acid sequence of SEQ ID NO: 65 or a nucleic acid having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with, the nucleic acid sequence of SEQ ID NO: 65. In other related embodiments, the gene product delivered by the subject AAV variant comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the following amino acid sequences: MVSYWDTGVLLCALLSCLLLTGSSSG SDTGPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSP SHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:66) In other related embodiments, the AAV variant comprises a heterologous nucleic acid comprising a nucleotide sequence encoding amino acids 27-458 of SEQ ID NO: 66 (corresponding to the amino acid sequence of aflibercept without the underlined signal peptide sequence) having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the amino acid sequence.

[0156] In another preferred embodiment, the gene product is ranibizumab in a single-chain form (sc-ranibizumab). Ranibizumab (LUCENTIS®) is a monoclonal IgG1 antibody fragment (Fab) that binds to and blocks all isotypes of VEGFa. LUCENTIS® is expressed in bacteria as two separate chains (light chain and heavy chain) linked by a disulfide bond between a constant light chain (CL) and a constant heavy chain 1 (CH1) domain. The approved dose of intravitreal ranibizumab is 0.3 or 0.5 mg in 0.05 mL, depending on the indication. Ranibizumab is approved for the treatment of wet age-related macular degeneration, macular edema following retinal vein occlusion, diabetic macular edema, and diabetic retinopathy. In a particularly preferred embodiment, a coding corresponding to... Figure 12C A novel codon-optimized nucleic acid sequence for ranibizumab in the single-chain heavy-light (HL) form (sc-ranibizumab HL), the nucleic acid sequence comprising or consisting of the following: ATGGACTGGACCTGGTCCATCCTGTTTCTGGTGGCTGCCGCCACAGGCACATACTCT In some embodiments, a nucleic acid sequence encoding sc-ranibizumab HL is provided, comprising a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or higher nucleic acid identity with the full length of the nucleic acid sequence shown in SEQ ID NO: 67 or with nucleotides 58-1575 of SEQ ID NO: 67 (excluding the underlined nucleotides encoding the human IGHV7-8 signal sequence). In related embodiments, the gene product delivered by the subject AAV variant is encoded by a nucleic acid sequence consisting of, or comprising, the nucleic acid sequence of SEQ ID NO: 67 or a nucleic acid having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with, the nucleic acid sequence of SEQ ID NO: 67. In other related embodiments, the gene product delivered by the subject AAV variant comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the following amino acid sequences: MDWTWSILFLVAAATGTYS EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHLGGSSGSGSGSTGTSSSGTGTSAG TTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSDIQLTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ YSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:68).

[0157] In other related embodiments, the AAV variant comprises a heterologous nucleic acid comprising a nucleotide sequence encoding amino acids 20-524 of SEQ ID NO: 68 (corresponding to the amino acid sequence of sc-ranibizumab HL without the underlined signal peptide sequence) having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the amino acid sequence.

[0158] In another particularly preferred embodiment, an encoding corresponding to Figure 12B A novel codon-optimized nucleic acid sequence for the single-chain light-heavy (LH) form of ranibizumab (sc-ranibizumab LH), the nucleic acid sequence comprising or consisting of the following: ATGGTCCTCCAGACACAGGTGTTCATCAGCCTGCTGCTGTGGATCTCTGGCGCCTACGGC In another particularly preferred embodiment, an encoding corresponding to Figure 12B A novel codon-optimized nucleic acid sequence for the single-chain light-heavy (LH) form of ranibizumab (sc-ranibizumab LH), the nucleic acid sequence comprising or consisting of the following: ATGGGTGCTCCAGACCCAGGTGTTTATTTCACTGCTGCTGTGGATTAGCGGGCTTACGGA

[0159] In some embodiments, a nucleic acid sequence encoding sc-ranibizumab LH is provided, comprising a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or higher nucleic acid identity with the full length of the nucleic acid sequence shown in SEQ ID NO: 69 or SEQ ID NO: 70, or with nucleotides 61-1578 (excluding the underlined nucleotides encoding the Igκ signal sequence) of SEQ ID NO: 69 or SEQ ID NO: 70. In related embodiments, the gene product delivered by the subject AAV variant is encoded by a nucleic acid sequence comprising, or comprising, a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with, or containing, the nucleic acid sequence of SEQ ID NO: 69 or SEQ ID NO: 70. In other related embodiments, the gene product delivered by the subject AAV variant comprises an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical or 100% identical to the following amino acid sequences: MVLQTQVFISLLLWISGAYGDIQLTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGG GSGGGGSAGGTATAGASSGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSH WYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL(SEQ ID NO:71) In other related embodiments, the AAV variant comprises a heterologous nucleic acid comprising a nucleotide sequence encoding amino acids 21-525 of SEQ ID NO: 71 (corresponding to the amino acid sequence of sc-ranibizumab LH without the underlined signal peptide sequence) having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the amino acid sequence.

[0160] In another preferred embodiment, an encoding corresponding to Figure 12E A novel nucleic acid sequence of ranibizumab in a single-chain light-heavy (LH) form fused to the Fc region of human IgG1 (sc-ranibizumab-Fc), the nucleic acid sequence comprising or consisting of the following: ATGGTCCTCCAGACACAGGTGTTCATCAGCCTGCTGCTGTGGATCTCTGGCGCCTACGGC

[0161] In some embodiments, a nucleic acid sequence encoding sc-ranibizumab-Fc is provided, comprising a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or higher nucleic acid identity with the full length of the nucleic acid sequence shown in SEQ ID NO: 72 or with nucleotides 61-2277 of SEQ ID NO: 72 (excluding the underlined nucleotides encoding the Igκ signal sequence). In related embodiments, the gene product delivered by the subject AAV variant is encoded by a nucleic acid sequence consisting of, or comprising, the nucleic acid sequence of SEQ ID NO: 72 or a nucleic acid having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with, the nucleic acid sequence of SEQ ID NO: 72. In other related embodiments, the gene product delivered by the subject AAV variant comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the following amino acid sequences: MVLQTQVFISLLLWISGAYG(SEQ ID NO: 73) In other related embodiments, the AAV variant comprises a heterologous nucleic acid comprising a nucleotide sequence encoding amino acids 21-752 of SEQ ID NO: 73 (corresponding to the amino acid sequence of sc-ranibizumab-Fc without the underlined signal peptide sequence) having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the amino acid sequence.

[0162] In another preferred embodiment, the gene product is bromizumab. Bromizumab (RTH258) is a single-chain variable fragment (scFv) that binds to and blocks all isotypes of VEGFa. Bromizumab is currently in a Phase III clinical trial evaluating 3 mg and 6 mg doses for the treatment of wet age-related macular degeneration. In a particularly preferred embodiment, a gene product encoding bromizumab (corresponding to...) is provided. Figure 12D A novel codon-optimized nucleic acid sequence, the nucleic acid sequence comprising or consisting of the following: ATGGTCCTCCAGACACAGGTGTTCATCAGCCTGCTGCTGTGGATCTCTGGCGCCTATGGC GAGATCGTGATGACACAGAGCCCCAGCACACTGTCTGCCAGCGTGGGAGACAGAGTGATCATCACATGCCAGGCCAGCGAGATCATCCACAGCTGGCTGGCTTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACCTGGCCTCTACACTGGCCAGCGGAGTGCCTAGCAGATTTTCT GGCTCTGGATCTGGCCGAGTTCACCCTGACAATCTCTAGCCTCCAGCCTGACGACTTCGCCACCTACTACTGCCAGAACGTGTACCTGGCCAGCACCAACGGCGCCAATTTTGGCCAGGGCACCAAGCTGACAGTGCTTGGCGGAGGCGGAGGTTCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCA GGCGGTGGTGGATCTGAAGTGCAGCTGGTGGAATCAGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGAGCTGTACCGCCTCTGGCTTCTCCCTGACCGACTACTACATGACCTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAGTGGGTCGGATTCATCGACCCCGACGACGATCCT TACTACGCCACATGGGCCAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCCGGCGGAGATCACAATAGCGGCTGGGGACTCGATATCTGGGGCCAGGGAACACTGGTCACCGTGTCTAGTTGA (SEQ ID NO:74).

[0163] In some embodiments, a nucleic acid sequence encoding bromizumab is provided, the nucleic acid sequence comprising a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or higher nucleic acid identity with the full length of the nucleic acid sequence shown in SEQ ID NO: 74 or with nucleotides 61-816 of SEQ ID NO: 74 (excluding the underlined nucleotides encoding the Igκ signal sequence). In related embodiments, the gene product delivered by the subject AAV variant is encoded by a nucleic acid sequence comprising, or comprising, the nucleic acid sequence of SEQ ID NO: 74 or a nucleic acid having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with the nucleic acid sequence of SEQ ID NO: 74. In other related embodiments, the gene product delivered by the subject AAV variant comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the following amino acid sequences: MVLQTQVFISLLLWISGAYG EIVMTQSPSTLSASSVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGG SGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSS (SEQ ID NO:75) In other related embodiments, the AAV variant comprises a heterologous nucleic acid comprising a nucleotide sequence encoding amino acids 21-271 of SEQ ID NO: 75 (corresponding to the amino acid sequence of bromoizumab without the underlined signal peptide sequence) having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the amino acid sequence.

[0164] In another preferred embodiment, an encoding corresponding to Figure 12F A novel nucleic acid sequence of bromizumab fused to the Fc region of human IgG1 (bromoizumab-Fc), said nucleic acid sequence comprising or consisting of the following: ATGGTCCTCCAGACACAGGTGTTCATCAGCCTGCTGCTGTGGATCTCTGGCGCCTATGGC

[0165] In some embodiments, a nucleic acid sequence encoding bromozumab-Fc is provided, the nucleic acid sequence comprising a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% or higher nucleic acid identity with the full length of the nucleic acid sequence shown in SEQ ID NO: 76 or with nucleotides 61-1530 of SEQ ID NO: 76 (excluding the underlined nucleotides encoding the Igκ signal sequence). In related embodiments, the gene product delivered by the subject AAV variant is encoded by a nucleic acid sequence comprising, or comprising, the nucleic acid sequence of SEQ ID NO: 76 or a nucleic acid having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with the nucleic acid sequence of SEQ ID NO: 76. In other related embodiments, the gene product delivered by the subject AAV variant comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the following amino acid sequences: MVLQTQVFISLLLWISGAYG EIVMTQSPSTLSASSVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLD IWGQGTLVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 77).

[0166] In other related embodiments, the AAV variant comprises a heterologous nucleic acid comprising a nucleotide sequence encoding amino acids 21-498 of SEQ ID NO: 77 (corresponding to the amino acid sequence of bromoizumab without the underlined signal peptide sequence) having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the amino acid sequence.

[0167] In a particularly preferred embodiment, an infectious recombinant AAV (rAAV) viral particle is provided comprising a variant AAV capsid protein and a heterologous nucleic acid encoding a polypeptide that inhibits VEGF (preferably VEGFa) activity, wherein the variant AAV capsid protein comprises a peptide insertion in the GH ring of the capsid protein relative to the corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO: 14) or LAISDQTKHA (SEQ ID NO: 28), wherein the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of the corresponding parental AAV capsid protein to retinal cells. In some embodiments, the insertion site is an amino acid between amino acids 587 and 588 of VP1 (SEQ ID NO: 2) corresponding to AAV2 or at a corresponding position in the capsid protein of another AAV serotype. Preferably, the variant AAV capsid protein further comprises a P34A amino acid substitution relative to the VP1 capsid (SEQ ID NO: 2) of AAV2, or a corresponding substitution in another AAV serotype.

[0168] In some embodiments, rAAV viral particles comprising a variant AAV capsid protein and a heterologous nucleic acid are provided, the variant AAV capsid protein comprising (i) a peptide insertion located between amino acids 587 and 588 of VP1 (SEQ ID NO: 2) corresponding to AAV2, or at a corresponding position in the capsid protein of another AAV serotype of the capsid protein relative to the corresponding parental AAV capsid protein, and (ii) a substitution of amino acid P34A relative to the VP1 capsid (SEQ ID NO: 2) of AAV2 or a corresponding substitution in another AAV serotype, the heterologous nucleic acid comprising a sequence encoding aflibercept. In a preferred embodiment, the nucleic acid sequence encoding aflibercept consists of, or comprises, the nucleic acid sequence of SEQ ID NO: 65 or a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with the nucleic acid sequence of SEQ ID NO: 65. In a particularly preferred embodiment, an rAAV viral particle is provided comprising a variant AAV capsid protein and a heterologous nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 65, wherein the variant AAV capsid protein has an amino acid sequence having at least 90%, at least 95%, or at least 99% identity with the sequence shown in SEQ ID NO: 42, wherein the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of the corresponding parental AAV capsid protein to retinal cells. In other related embodiments, the heterologous nucleic acid further comprises one or more sequences, each encoding an additional VEGFa inhibitor, preferably selected from ranibizumab, sc-ranibizumab HL, sc-ranibizumab LH, sc-ranibizumab-Fc, bromizumab, and bromizumab-Fc. In related embodiments, a pharmaceutical composition comprising such rAAV is provided. In other related embodiments, a method for treating VEGFa-related eye disease is provided, comprising administering to a subject in need an effective amount of rAAV viral particles, said rAAV viral particles comprising a variant AAV capsid protein having an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 42 and a heterologous nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 65, and optionally one or more additional nucleic acid sequences, each encoding a different VEGFa inhibitor. rAAV can be administered by subretinal, suprachoroidal, topical, intra-anterior chamber, or intravitreal injection, but is preferably administered by intravitreal injection.In some implementations, VEGFa-related eye diseases are selected from: wet (neovascular, exudative) age-related macular degeneration; macular edema following retinal vein occlusion; retinal neovascularization caused by retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative and proliferative diabetic retinopathy), myopic macular degeneration, branch retinal vein occlusion, hemilateral retinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectasia; neovascular glaucoma; vitreous hemorrhage; and retinal and choroidal neovascularization secondary to retinal diseases, including but not limited to uveitis, trauma, retinal degenerative diseases, hereditary retinal and / or choroidal diseases, ocular tumors, and corneal and iris neovascularization. In some preferred embodiments, VEGFa-related eye diseases are selected from wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; and myopic choroidal neovascularization.

[0169] In some embodiments, rAAV viral particles comprising a variant AAV capsid protein and a heterologous nucleic acid are provided, the variant AAV capsid protein comprising (i) a peptide insertion located between amino acids 587 and 588 of VP1 (SEQ ID NO: 2) of AAV2, or at a corresponding position in the capsid protein of another AAV serotype of the capsid protein relative to the corresponding parental AAV capsid protein, and (ii) a P34A amino acid substitution relative to the VP1 capsid (SEQ ID NO: 2) of AAV2 or a corresponding substitution in another AAV serotype, the heterologous nucleic acid comprising a nucleotide sequence encoding ranibizumab, sc-ranibizumab HL, sc-ranibizumab LH, or sc-ranibizumab-Fc. In a preferred embodiment, the nucleic acid sequence encoding sc-ranibizumab consists of or contains the nucleic acid sequence shown in any one of SEQ ID NO: 67, 69, 70, and 72, or has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In a particularly preferred embodiment, an rAAV viral particle is provided comprising a variant AAV capsid protein and a heterologous nucleic acid, the variant AAV capsid protein having an amino acid sequence having at least 90%, at least 95%, or at least 99% identity with the sequence shown in SEQ ID NO: 42, and the heterologous nucleic acid comprising the nucleic acid sequence of any one of SEQ ID NO: 67, 69, 70, and 72, wherein the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of the corresponding parental AAV capsid protein to retinal cells. In other related embodiments, the heterologous nucleic acid further comprises one or more additional nucleic acid sequences, each encoding a different VEGFa inhibitor, preferably selected from aflibercept, bromizumab, and bromizumab-Fc. In related embodiments, a pharmaceutical composition comprising such rAAV is provided. In other related embodiments, a method for treating an eye disease associated with elevated intraocular VEGFa is provided, the method comprising administering to a subject in need an effective amount of rAAV viral particles, the rAAV viral particles comprising a variant AAV capsid protein and a heterologous nucleic acid sequence, the variant AAV capsid protein having an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 42, and the heterologous nucleic acid sequence comprising any one of SEQ ID NO: 67, 69, 70, and 72. Preferably, rAAV is administered via intravitreal injection.In some implementations, VEGFa-related eye diseases are selected from: wet (neovascular, exudative) age-related macular degeneration; macular edema following retinal vein occlusion; retinal neovascularization caused by retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative and proliferative diabetic retinopathy), myopic macular degeneration, branch retinal vein occlusion, hemilateral retinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectasia; neovascular glaucoma; vitreous hemorrhage; and retinal and choroidal neovascularization secondary to retinal diseases, including but not limited to uveitis, trauma, retinal degenerative diseases, hereditary retinal and / or choroidal diseases, ocular tumors, and corneal and iris neovascularization. In some preferred embodiments, VEGFa-related eye diseases are selected from wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; and myopic choroidal neovascularization.

[0170] In some embodiments, rAAV viral particles comprising a variant AAV capsid protein and a heterologous nucleic acid are provided, the variant AAV capsid protein comprising (i) a peptide insertion located between amino acids 587 and 588 of VP1 (SEQ ID NO: 2) corresponding to AAV2, or at a corresponding position in the capsid protein of another AAV serotype of the capsid protein relative to the corresponding parental AAV capsid protein, and (ii) a substitution of amino acid P34A relative to the VP1 capsid (SEQ ID NO: 2) of AAV2 or a corresponding substitution in another AAV serotype, the heterologous nucleic acid comprising a nucleotide sequence encoding bromizumab or bromizumab-Fc. In a preferred embodiment, the nucleic acid sequence encoding bromizumab or bromizumab-Fc consists of or comprises the nucleic acid sequence shown in SEQ ID NO: 74 or SEQ ID NO: 76, or has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with it. In a particularly preferred embodiment, an rAAV viral particle is provided comprising a variant AAV capsid protein and a heterologous nucleic acid, the variant AAV capsid protein having an amino acid sequence having at least 90%, at least 95%, or at least 99% identity with the sequence shown in SEQ ID NO: 42, and the heterologous nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 74 or SEQ ID NO: 76, wherein the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of the corresponding parental AAV capsid protein to retinal cells. In other related embodiments, the heterologous nucleic acid further comprises one or more nucleotide sequences, each encoding a different VEGFA inhibitor, preferably selected from aflibercept, ranibizumab, sc-ranibizumab HL, sc-ranibizumab LH, and sc-ranibizumab-Fc. In related embodiments, a pharmaceutical composition comprising such rAAV is provided. In other related embodiments, a method for treating an eye disease associated with elevated intraocular VEGFa is provided, the method comprising administering an effective amount of rAAV viral particles to a subject in need, the rAAV viral particles comprising a variant AAV capsid protein and a heterologous nucleic acid sequence, the variant AAV capsid protein having an amino acid sequence having at least 90% identity with the sequence shown in SEQ ID NO: 42, and the heterologous nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 74 or SEQ ID NO: 76. Preferably, rAAV is administered via intravitreal injection.In some implementations, VEGFa-related eye diseases are selected from: wet (neovascular, exudative) age-related macular degeneration; macular edema following retinal vein occlusion; retinal neovascularization caused by retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative and proliferative diabetic retinopathy), myopic macular degeneration, branch retinal vein occlusion, hemilateral retinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectasia; neovascular glaucoma; vitreous hemorrhage; and retinal and choroidal neovascularization secondary to retinal diseases, including but not limited to uveitis, trauma, retinal degenerative diseases, hereditary retinal and / or choroidal diseases, ocular tumors, and corneal and iris neovascularization. In some preferred embodiments, VEGFa-related eye diseases are selected from wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; and myopic choroidal neovascularization.

[0171] Genes whose product acts as immunomodulators, such as complement factors and Toll-like receptors, are called "immunomodulatory genes." Exemplary immunomodulatory genes include cytokines, chemokines, and fusion proteins or antibodies specific to them and / or their receptors, such as the anti-IL-6 fusion protein Rilonacept. TM Examples of genes whose products act as neuroprotective factors include: platelet-derived growth factor receptor (PDGFR); glial-derived neurotrophic factor (GDNF); rod-derived conviability factor (RdCVF); fibroblast growth factor (FGF); neuronal rank protein (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophic factor-4 (NT4); brain-derived neurotrophic factor (BDNF); and epidermal growth factor. Genes whose products act as light-responsive opsins include opsins, rhodopsin, channel rhodopsin, and halo rhodopsin.

[0172] In some cases, the gene product of interest is a site-specific endonuclease that provides site-specific knockdown of gene function, for example, where the endonuclease knocks out an allele associated with retinal disease. For instance, when a dominant allele encodes a defective copy of a gene that, when wild-type, is a retinal structural protein and / or provides normal retinal function, a site-specific endonuclease can be targeted to and knock out the defective allele.

[0173] In addition to knocking out defective alleles, site-specific nucleases can also be used to stimulate homologous recombination with donor DNA that encodes a functional copy of a protein encoded by the defective allele. Therefore, for example, the subject rAAV viral particle can be used to deliver a site-specific endonuclease that knocks out a defective allele, and can also be used to deliver a functional copy of the defective allele, leading to the repair of the defective allele and thereby providing the production of functional retinal proteins (e.g., functional retinal schistosome protein, functional RPE65, functional peripheral proteins, etc.). See Li et al., (2011). Nature 475:217. In some embodiments, the rAAV viral particles disclosed herein comprise a heterologous nucleotide sequence encoding a site-specific endonuclease; and a heterologous nucleotide sequence encoding a functional copy of a defective allele, wherein the functional copy encodes a functional retinal protein. Functional retinal proteins include, for example, retinal schistosome protein, RPE65, retinitis pigmentosa GTPase regulator (RGPR)-interacting protein-1, peripheral proteins, peripheral proteins-2, etc.

[0174] Suitable site-specific endonucleases include, for example, broad-spectrum nucleases; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (TALENs); and periodically clustered interstitial short palindromic repeats / CRISPR-associated (Cas) nucleases, where these site-specific endonucleases are not naturally occurring and are modified to target specific genes. Such site-specific nucleases can be engineered to cut specific sites within the genome, and then non-homologous end joining can repair the break when a few nucleotides are inserted or deleted. Such site-specific endonucleases (also known as “INDELs”) then eject the protein from the frame and efficiently knock out the gene. See, for example, U.S. Patent Publication No. 2011 / 0301073.

[0175] In some embodiments of the variant rAAV vectors disclosed herein, the nucleotide sequence encoding the gene product of interest is operatively linked to a constitutive promoter. Suitable constitutive promoters include, for example, cytomegalovirus promoters (CMV) (Stinski et al., (1985)). Journal of Virology55(2): 431-441), CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG) (Miyazaki et al., (1989)). Gene 79(2): 269-277,CB SB (Jacobson et al., (2006)) Molecular Therapy 13(6): 1074-1084), Human extension factor 1α promoter (EF1α) (Kim et al., (1990) Gene 91(2): 217-223), Human phosphoglycerate kinase promoter (PGK) (Singer-Sam et al., (1984)). Gene 32(3): 409-417, Mitochondrial heavy chain promoter (Loderio et al., (2012)). PNAS 109(17): 6513-6518), ubiquitous promoter (Wulff et al., (1990) FEBS Letters 261: 101-105). In other embodiments, the nucleotide sequence encoding the gene product of interest is operatively linked to an inducible promoter. In some cases, the nucleotide sequence encoding the gene product of interest is operatively linked to a tissue-specific or cell-type-specific regulatory element. For example, in some cases, the nucleotide sequence encoding the gene product of interest is operatively linked to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), which, for example, confers selective expression of the gene operatively linked in photoreceptor cells. Suitable photoreceptor-specific regulatory elements include, for example, rhodopsin promoters; rhodopsin kinase promoters (Young et al., (2003)). Ophthalmol. Vis. Sci. 44:4076); β-phosphodiesterase gene promoter (Nicoud et al., (2007)). J. Gene Med. 9:1015); Retinitis pigmentosa gene promoter (Nicoud et al., (2007) ibid.); Interretinal retinoic acid binding protein (IRBP) gene enhancer (Nicoud et al., (2007) ibid.); IRBP gene promoter (Yokoyama et al., (1992) Exp Eye Res. 55:225), opsin gene promoter (Tucker et al., (1994) PNAS 91:2611-2615), retinal schistosome gene promoter (Park et al., (2009)). Gene Therapy 16(7): 916-926), CRX homeo domain protein gene promoter (Furukawa et al., (2002)). The Journal of Neuroscience22(5): 1640-1647), promoter of the guanine nucleotide-binding protein α transduction active polypeptide 1 (GNAT1) gene (Lee et al., (2010)). Gene Therapy 17:1390-1399, promoter of the neuroretinal-specific leucine zipper protein (NRL) gene (Akimoto et al., (2006)). PNAS 103(10): 3890-3895), Human cone repressor (hCAR) promoter (Li et al., (2002) Biochemistry and Molecular Biology 43: 1375-1383), and PR2.1, PR1.7, PR1.5 and PR1.1 promoters (Ye et al., (2016)). Human Gene Therapy 27(1): 72-82). In some cases, the nucleotide sequence encoding the product of interest is operatively linked to a retinal pigment epithelial (RPE) cell-specific regulatory element (e.g., an RPE-specific promoter), which, for example, confers selective expression of the operatively linked gene in RPE cells. Suitable RPE-specific regulatory elements include, for example, the RPE65 gene promoter (Meur et al., (2007)). Gene Therapy 14: 292-303), promoter of the cellular retinaldehyde-binding protein (CRALBP) gene (Kennedy et al., (1998)). Journal of Biological Chemistry 273: 5591-5598), Pigment epithelial-derived factor (PEDF aka serpin F1) gene promoter (Kojima et al., (2006)). Molecular and Cellular Biochemistry 293(1-2): 63-69), and the yolk-like macular dystrophy (VMD2) promoter (Esumi et al., (2004)). The Journal of Biological Chemistry 279(18): 19064-19073). In some cases, the nucleotide sequence encoding the product of interest is operatively linked to a Müller glial cell-specific regulatory element (e.g., a glial-specific promoter), such as a regulatory element in retinal glial cells that confers selective expression of the linked gene. Suitable glial-specific regulatory elements include, for example, the glial fibrillary acidic protein (GFAP) promoter (Besnard et al., (1991)). Journal of Biological Chemistry266(28): 18877-18883). In some cases, the nucleotide sequence encoding the gene product of interest is operatively linked to a bipolar cell-specific regulatory element (e.g., a bipolar-specific promoter), which, for example, confers selective expression of the linked gene in bipolar cells. Suitable bipolar-specific regulatory elements include, for example, the GRM6 promoter (Cronin et al., (2014)). EMBO Molecular Medicine 6(9):1175-1190).

[0176] For the purposes of this invention, the disclosure herein provides isolated nucleic acids comprising a nucleotide sequence encoding a variant AAV capsid protein as described above. The isolated nucleic acid may be an AAV vector, such as a recombinant AAV vector.

[0177] The disclosure herein also provides a method for treating retinal diseases, the method comprising administering an effective amount of rAAV variant viral particles, comprising the transgenes of concern as described above and disclosed herein, to an individual in need. Those skilled in the art will be able to readily determine the effective amount of the subject rAAV viral particles and treat the disease by testing changes in one or more functional or anatomical parameters, such as visual acuity, visual field, electrophysiological response to light and dark, color vision, contrast sensitivity, anatomy, retinal health and vascular system, eye mobility, fixation preference, and stability.

[0178] Non-limiting methods for assessing retinal function and its changes include: assessing visual acuity (e.g., best-corrected visual acuity [BCVA], movement, navigation, object detection, and discrimination); assessing visual field (e.g., static and dynamic visual field measurements); performing clinical examinations (e.g., slit-lamp examination of the anterior and posterior segments of the eye); and assessing electrophysiological responses to all bright and dark wavelengths (e.g., all forms of electroretinography (ERG) [all-angle, multifocal, and modal], all forms of visual evoked potentials (VEP), electrooculography (EOG), color vision, dark adaptation, and / or contrast sensitivity). Non-limiting methods for assessing anatomical and retinal health and its changes include optical coherence tomography (OCT), fundus photography, adaptive optics scanning laser ophthalmoscopy (AO-SLO), fluorescence and / or autofluorescence; measurement of ocular movement and motion (e.g., nystagmus, fixation preference, and stability); measurement of reported outcomes (patient-reported changes in visual and non-visual guided behaviors and activities, patient-reported outcomes [PRO]), questionnaire-based quality of life assessments, and measurements of daily activities and neurological function (e.g., functional magnetic resonance imaging (MRI)).

[0179] In some embodiments, an effective amount of the subject rAAV viral particles results in a reduced rate of loss of retinal function, anatomical integrity, or retinal health, e.g., a 2-, 3-, 4-, or 5-fold or greater reduction in the rate of loss and thus disease progression, e.g., a 10-fold or greater reduction in the rate of loss and thus disease progression. In some embodiments, an effective amount of the subject rAAV viral particles results in an increased improvement in visual function, retinal function, retinal anatomy or health, and / or eye mobility and / or neurological function, e.g., a 2-, 3-, 4-, or 5-fold or greater increase in the improvement in retinal function, retinal anatomy or health, and / or eye mobility, e.g., a 10-fold or greater increase in the improvement in retinal function, retinal anatomy or health, and / or eye mobility. As will be readily understood by those skilled in the art, the dose required to achieve the desired therapeutic effect is typically in the range of 1 x 10⁻⁶. 8 To approximately 1 x 10 15 Within the range of individual recombinant viral particles, commonly referred to by ordinary technicians as 1 x 10-1 8 To approximately 1 x 10 15 A "carrier genome".

[0180] The subject rAAV viral particles can be administered via intraocular injection, such as intravitreal injection, subretinal injection, suprachoroidal injection, or any other convenient mode of administration or route leading to the delivery of rAAV viral particles to the eye. Other convenient modes of administration or routes include, but are not limited to, intravenous, intra-arterial, periocular, anterior chamber, subconjunctival, and sub-tenons injection and local and intranasal administration. When administered via intravitreal injection, the subject rAAV viral particles are able to cross the vitreous body and the internal limiting membrane (also referred to herein as the internal limiting membrane, or “ILM”; a thin, transparent, cell-free membrane on the surface of the retina that forms the boundary between the retina and the vitreous body, formed by the terminal feet of astrocytes and Müller cells), and / or move more efficiently across the retinal layer than AAV viral particles containing the corresponding parental AAV capsid protein.

[0181] The variant capsid proteins disclosed herein are isolated, for example, purified. In some embodiments, the variant capsid proteins disclosed herein are contained in an AAV vector or recombinant AAV (rAAV) viral particles. In other embodiments, such AAV variant vectors and / or AAV variant viral particles are used in in vivo or in vitro methods for treating ocular diseases in the retina of primates.

[0182] The disclosure herein also provides host cells, such as, but not limited to, isolated (genetically modified) host cells containing the subject nucleic acid. The host cells disclosed herein according to the invention can be isolated cells, such as cells derived from in vitro cell cultures. As described herein, such host cells are used to generate subject rAAV variant viral particles. In one embodiment, such host cells are stably genetically modified with nucleic acids. In other embodiments, host cells are transiently genetically modified with nucleic acids. The nucleic acid is stably or transiently introduced into the host cell using established techniques, including but not limited to electroporation, calcium phosphate precipitation, liposome-mediated transfection, etc. For stable transformation, the nucleic acid typically also contains an optional marker, such as any of several well-known optional markers, such as neomycin resistance. Such host cells are generated by introducing the nucleic acid into any cell of a variety of cells, such as mammalian cells, including, for example, mouse cells and primate cells (e.g., human cells). Exemplary mammalian cells include, but are not limited to, primary cells and cell lines, wherein exemplary cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, etc. Exemplary host cells include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human embryonic kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, and so on. Baculoviruses can also be used to infect host cells to infect insect cells, such as Sf9 cells, which produce AAV (see, for example, U.S. Patent No. 7,271,002; U.S. Patent Application Serial No. 12 / 297,958). In some embodiments, as described above, in addition to a nucleic acid containing a nucleotide sequence encoding a variant AAV capsid protein, the genetically modified host cell comprises a nucleic acid containing a nucleotide sequence encoding one or more AAVrep proteins. In other embodiments, the host cell further comprises an rAAV variant vector. Such host cells can be used to generate rAAV variant viral particles.For example, methods for generating rAAV viral particles are described in U.S. Patent Publication No. 2005 / 0053922 and U.S. Patent Publication No. 2009 / 0202490.

[0183] This disclosure further provides a pharmaceutical composition comprising: a) rAAV variant viral particles as described above and disclosed herein; and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for human or non-human patients. Such excipients, carriers, diluents, and buffers include any reagent that can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. They may include pharmaceutically acceptable salts, such as inorganic acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. Furthermore, excipients such as wetting agents or emulsifiers, pH buffers, etc., may be present in such carriers. Many pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been well described in various publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy”, 20th edition; Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999); HC Ansel et al., eds., 7. th ed., Lippincott, Williams,&Wilkins; and Handbook of Pharmaceutical Excipients (2000) AH Kibbe et al., eds., 3 rd ed. Amer. Pharmaceutical Association. In some aspects of the invention, the invention provides a pharmaceutical composition comprising about 1 × 10 8 Up to approximately 1×10 15 One recombinant virus or 1×10 8 Up to approximately 1×10 15 Each of the recombinant viruses comprises a vector genome, wherein each of the recombinant viruses contains a genome encoding one or more gene products.

[0184] Some embodiments of the present invention are illustrated by way of example in the following items 1 to 36: 1. A variant adeno-associated virus (AAV) capsid protein comprising a peptide insertion in the GH ring of the capsid protein relative to a corresponding parental AAV capsid protein, wherein the peptide insertion comprises the amino acid sequence ISDQTKH (SEQ ID NO:14), and wherein the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of the corresponding parental AAV capsid protein to retinal cells.

[0185] 2. The variant AAV capsid protein according to Project 1, wherein the peptide insertion comprises the amino acid sequence Y1Y2ISDQTKHY3, wherein each of Y1-Y3 is independently selected from Ala, Leu, Gly, Ser, Thr and Pro.

[0186] 3. The variant AAV capsid protein according to Project 2, wherein the peptide insertion comprises the amino acid sequence LAISDQTKHA (SEQ ID NO:28).

[0187] 4. A variant AAV capsid protein according to any one of items 1-3, wherein the insertion site is located between amino acids 587 and 588 of VP1 (SEQ ID NO:2) corresponding to AAV2 or at the corresponding position in the capsid protein of another AAV serotype.

[0188] 5. A variant AAV capsid protein according to any one of items 1-4, wherein the capsid protein comprises one or more amino acid substitutions relative to the VP1 capsid (SEQ ID NO:2) of AAV2 or one or more corresponding substitutions in another AAV serotype.

[0189] 6. A variant AAV capsid protein according to any one of items 1-5, wherein the capsid protein comprises a P34A amino acid substitution relative to the VP1 capsid (SEQ ID NO:2) of AAV2 or a corresponding substitution in another AAV serotype.

[0190] 7. A variant AAV capsid protein according to any one of items 1-6, wherein the capsid protein comprises (i) the amino acid sequence ISDQTKH (SEQ ID NO:14) and (ii) P34A amino acid substitutions, and has at least 90%, at least 95%, or at least 99% identity with the sequence shown in SEQ ID NO:42.

[0191] 8. The variant AAV capsid protein according to Item 7, wherein the capsid protein is substantially composed of the amino acid sequence shown in SEQ ID NO:42.

[0192] 9. A recombinant AAV (rAAV) viral particle comprising a variant AAV capsid protein according to any one of items 1 to 8 and a heterologous nucleic acid, said heterologous nucleic acid comprising a nucleotide sequence encoding a gene product.

[0193] 10. The rAAV according to Item 9, wherein the gene product is a polypeptide.

[0194] 11. The rAAV according to Item 10, wherein the heteronucleotide comprises a nucleotide sequence encoding a polypeptide that inhibits the activity of vascular endothelial growth factor (VEGF).

[0195] 12. The rAAV according to Item 11, wherein the nucleotide sequence encodes a fusion protein.

[0196] 13. The rAAV according to Item 12, wherein the nucleotide sequence encodes aflibercept (Eylea).

[0197] 14. The rAAV according to item 13, wherein the nucleotide sequence has at least 90%, at least 95%, or at least 99% identity with the nucleic acid sequence shown in SEQ ID NO: 65 or with nucleotides 79-1377 of SEQ ID NO: 65, and preferably encodes the amino acid sequence shown in SEQ ID NO: 66 or amino acids 27-458 of SEQ ID NO: 66.

[0198] 15. The rAAV according to item 11, wherein the nucleotide sequence encodes a monoclonal antibody or an antigen-binding fragment thereof.

[0199] 16. The rAAV according to item 15, wherein the nucleotide sequence encodes ranibizumab (Lucentis).

[0200] 17. The rAAV according to item 15, wherein the nucleotide sequence (i) comprises a sequence having at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 67 (sc-ranibizumab HL) or nucleotides 58-1575 of SEQ ID NO: 67, and encodes the amino acid sequence shown in SEQ ID NO: 68, or (ii) encodes the amino acid sequence of amino acids 20-524 of SEQ ID NO: 68.

[0201] 18. The rAAV according to item 15, wherein the nucleotide sequence (i) has a sequence having at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 69 (sc-ranibizumab LH1) or SEQ ID NO: 70 (sc-ranibizumab LH2) or with nucleotides 61-1578 of SEQ ID NO: 69 or SEQ ID NO: 70, and encodes the amino acid sequence shown in SEQ ID NO: 71, or (ii) encodes the amino acid sequence of amino acids 21-525 of SEQ ID NO: 71.

[0202] 19. The rAAV according to item 15, wherein the nucleotide sequence (i) has at least 90%, at least 95%, or at least 99% identity with the sequence of SEQ ID NO: 72 (sc-ranibizumab-Fc) or with nucleotides 61-2277 of SEQ ID NO: 72, and encodes the amino acid sequence shown in SEQ ID NO: 73, or (ii) encodes the amino acid sequence of amino acids 21-752 of SEQ ID NO: 73.

[0203] 20. The rAAV according to Item 15, wherein the nucleotide sequence encodes bromizumab.

[0204] 21. The rAAV according to item 20, wherein the nucleotide sequence has at least 90%, at least 95%, or at least 99% identity with the nucleic acid sequence shown in SEQ ID NO: 74 or with nucleotides 61-816 of SEQ ID NO: 74, and preferably encodes the amino acid sequence shown in SEQ ID NO: 75 or the amino acid sequence of amino acids 21-271 of SEQ ID NO: 75.

[0205] 22. The rAAV according to item 15, wherein the nucleotide sequence (i) has at least 90%, at least 95%, or at least 99% identity with the sequence of SEQ ID NO: 76 (bromoizumab-Fc) or with nucleotides 61-1530 of SEQ ID NO: 76, and encodes the amino acid sequence shown in SEQ ID NO: 77, or (ii) encodes the amino acid sequence of amino acids 21-498 of SEQ ID NO: 77.

[0206] 23. The rAAV according to item 11, wherein the heterologous nucleic acid comprises (i) a sequence encoding aflibercept, preferably wherein the sequence encoding aflibercept has at least 90%, at least 95%, or at least 99% identity with the nucleic acid sequence shown in SEQ ID NO: 65, and encodes the amino acid sequence shown in SEQ ID NO: 66, and (ii) a sequence encoding bromizumab, preferably wherein the sequence encoding bromizumab has at least 90%, at least 95%, or at least 99% identity with the nucleic acid sequence shown in SEQ ID NO: 74 or SEQ ID NO: 76, and preferably encodes the amino acid sequence shown in SEQ ID NO: 75 or SEQ ID NO: 77.

[0207] 24. The rAAV according to any one of items 9-23, wherein the nucleotide sequence encoding the gene product is operatively linked to an expression control sequence.

[0208] 25. A pharmaceutical composition comprising rAAV according to any one of items 11-24 and a pharmaceutically acceptable carrier.

[0209] 26. A method for delivering a VEGF inhibitor to retinal cells, choroidal cells, lens cells, ciliary cells, iris cells, optic nerve cells, and / or corneal cells of a subject, comprising administering to the subject an rAAV viral particle according to any one of items 11-24 or a pharmaceutical composition according to claim 24.

[0210] 27. The method according to item 26, wherein the rAAV viral particles or pharmaceutical composition are administered into the vitreous body of the subject.

[0211] 28. A method for treating VEGFa-related eye disease in subjects requiring treatment, wherein the VEGFa-related eye disease is selected from wet (neovascular, exudative) age-related macular degeneration; macular edema following retinal vein occlusion; retinal neovascularization caused by retinal vein occlusion; and diabetic macular edema. Diabetic retinopathy (including all stages of non-proliferative and proliferative diabetic retinopathy); myopic macular degeneration; branch retinal vein occlusion, hemilateral retinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectasia; neovascular glaucoma; vitreous hemorrhage; retinal and choroidal neovascularization secondary to retinal diseases, including but not limited to uveitis, trauma, retinal degeneration, hereditary retinal and / or choroidal diseases, ocular tumors, corneal and iris neovascularization, wherein the method is performed by administering to the subject an effective amount of rAAV according to any one of items 11-24 or the pharmaceutical composition according to claim 25. 29. The method according to Item 28, wherein the VEGFa-related eye disease is selected from wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; and myopic choroidal neovascularization.

[0212] 30. The method according to any one of items 26 to 29, wherein the rAAV comprises a capsid protein consisting substantially of the amino acid sequence shown in SEQ ID NO: 42 and a heterologous nucleic acid comprising a sequence encoding aflibercept, preferably wherein the sequence encoding aflibercept has the sequence shown in SEQ ID NO: 65.

[0213] 31. The method according to any one of items 26 to 29, wherein the rAAV comprises a capsid protein consisting substantially of the amino acid sequence shown in SEQ ID NO: 42 and a heterologous nucleic acid comprising a sequence encoding ranibizumab, sc-ranibizumab HL, sc-ranibizumab LH or sc-ranibizumab-Fc, preferably wherein the sequence encoding sc-ranibizumab HL, sc-ranibizumab LH or sc-ranibizumab-Fc has the sequence shown in any one of SEQ ID NO: 67, 69, 70 and 72.

[0214] 32. The method according to any one of items 26 to 29, wherein the rAAV comprises a capsid protein consisting substantially of an amino acid sequence as shown in SEQ ID NO: 42 and a heterologous nucleic acid comprising a sequence encoding bromizumab or bromizumab-Fc, preferably wherein the sequence encoding bromizumab or bromizumab-Fc has the sequence shown in SEQ ID NO: 74 or SEQ ID NO: 76.

[0215] 33. The method according to any one of items 28-32, wherein the rAAV or pharmaceutical composition is administered into the vitreous body of the subject.

[0216] 34. The method according to any one of items 26-33, wherein the subject is a human being.

[0217] 35. An isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein according to any one of items 1-8.

[0218] 36. An isolated, genetically modified host cell comprising the nucleic acid described in item 35. Example

[0219] The following embodiments are provided to provide a complete disclosure and description of how to prepare and use the invention to those skilled in the art, and are not intended to limit the scope of the invention as viewed by the inventors, nor are they intended to represent all or only the experiments performed. While every effort has been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), some experimental errors and biases should still be taken into account. Unless otherwise specified, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are degrees Celsius, and pressures are atmospheric pressure or close to atmospheric pressure.

[0220] General methods of molecular and cellular biochemistry can be found in standard textbooks such as *Molecular Cloning: A Laboratory Manual*, 3rd edition (Sambrook et al., Harbor Laboratory Press, 2001); *Short Protocols in Molecular Biology*, 4th edition (Ausubel et al., eds., John Wiley & Sons, 1999); *Protein Methods* (Bollag et al., John Wiley & Sons, 1996); *Nonviral Vectors for Gene Therapy* (Wagner et al., eds., Academic Press, 1999); *Viral Vectors* (Kaplift & Loewy eds., Academic Press, 1995); *Immunology Methods Manual* (I. Lefkovits ed., Academic Press, 1997); and *Cell and Tissue Culture: Laboratory Procedures in Biotechnology* (Doyle & Griffiths, John Wiley & Sons, 1998). Their contents are incorporated herein by reference. The reagents, cloning vectors, and kits for genetic manipulation mentioned in this disclosure are available from commercial suppliers such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[0221] Example 1 Intravitreal injection and tissue harvesting. Intravitreal injection was administered to a single male cynomolgus macaca (macaca fascicularis) aged 4 to 10 years and weighing at least 4 kg via the sclera (approximately 3 mm posterior to the limbus using a procedure and delivery device suitable for human use). The animal was anesthetized and given a local anesthetic. 100 μL of the library was administered to each eye.

[0222] On day 14±3, euthanasia was performed by intravenous administration of sodium pentobarbital at a dose of 100 mg / kg by a trained veterinarian. The eye was nucleated and stored at 4°C until dissection.

[0223] Tissue dissection. The eye was opened along the serrated edge with a scalpel, and the anterior segment was removed. A decompression incision was made on the retina around the fovea to allow the retina to mount flat, and the vitreous humor was removed. Six retinal samples were collected from each quadrant (superior, inferior, nasal, and temporal), such as... Figure 2 As shown, cellular materials corresponding to RPE cells, photoreceptors, biopolar cells, amacrine cells, horizontal cells, and / or ganglion cells were isolated.

[0224] Directed evolution. Figure 1 The diagram illustrates the directed evolution process. In short, it describes the creation of a system incorporating over 20 DNA mutation techniques. cap A library of viral capsids with proprietary combinations of genes ( Figure 1 (a)). Then package the virus ( Figure 1 (b) — such that each particle is composed of surrounding elements that encode the capsid. cap The mutant capsid of the gene is composed and purified. The capsid library is placed under in vivo selection pressure. Tissue or cellular material of interest is harvested to isolate AAV variants that have been successfully infected with the target, and the successful viruses are recovered. Successful clones are enriched by repeated selection (stages I to II). Figure 1 (c) in the middle. The selected... cap The genes then undergo proprietary redistribution and are enriched through further selection steps to iteratively increase viral fitness (stage 2 – Figure 1 (d)). Variants identified during vector selection phases 1 and 2 demonstrated the ability to transduce primate retinal cells. Figure 1 (e) in the middle.

[0225] AAV capsid genome was successfully recovered. Rounds 1 through 6. The capsid recovered from each round of selection was used to package the injected library to begin the next round of selection. Recovery of the capsid gene from the tissue indicates that the library vector has been successfully internalized into the tissue of interest. After round 4, additional re-diversification of the library was incorporated before library packaging and injection in round 5. Figure 3 The image shows the recovery of viral genomes from RPE, PR, INL, and GCL retinal tissues in representative selection rounds. The bands within the box represent successful viral genome recovery.

[0226] Sequencing analysis: Rounds 3 to 6. During rounds 3 to 6, individual clones within the library are sequenced to determine the frequency of variants within the population. The presence of motifs in the sequencing data is assessed. Variants are grouped into motifs based on the presence of uniform variations occurring across multiple sequences (e.g., specific point mutations or specific peptide insertions at consistent positions within the capsid). Figure 4 A (3rd round of sequencing analysis) Figure 4 B (Round 4 sequencing analysis) Figure 4 C (5th round of sequencing analysis) and 4D (6th round of sequencing analysis) represent motifs representing at least 5% of the sequencing population in two or more rounds of selection or at least 10% of the sequencing population in one or more rounds of selection.

[0227] Table 1 below lists several representative clones that confer increased infectivity to retinal cells (each clone contains one or more substitutions and / or peptide insertions for the recognition and is otherwise identical to SEQ ID NO: 2; the selection rounds, sequence numbers, and frequencies for each clone are listed (in parentheses): Table 1. Amino acid sequence modifications of the AAV VP1 capsid protein that confer increased infectivity to one or more cells of the retina. The substitutions listed in column 2 are based on the amino acid sequence of wild-type AAV2, i.e., the peptides in which the insertions are not present.

[0228] The capsids also identified as having increased infectivity to one or more retinal cells are clones with the following ancestral VP1 capsid sequence: MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASAGSTNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSTGGTAGTRELLFSQAGPSNMSAQAKNWLPGPCYRQQRVSKTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPSSGVLIFGKQGAGANNTALENVMMTSEEEIKTTNPVATEQYGVVASNLQSSNTAPVTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYAKSTNVDFAVDNEGVYSEPRPIGTRYLTRNL. (SEQ ID NO:59) This ancestral capsid variant is derived from the ancestral capsid SEQ ID NO:58 evolved, with degenerate positions (residues 264, 266, 268, 448, 459, 460, 467, 470, 471, 474, 495, 516, 533, 547, 551, 555, 557, 561, 563, 577, 583, 593, 596, 661, 662, 664, 665, 710, 717, 718, 719, 723) evolving to include: alanine (A) at 264, alanine (A) at 266, serine (S) at 268, alanine (A) at 448, threonine (T) at 459, arginine (R) at 460, alanine (A) at 467, serine (S) at 470, asparagine (N) at 471, and alanine (A) at 474. The following are listed: serine at position 495 (S), asparagine at position 516 (D), asparagine at position 533 (D), glutamine at position 547 (Q), alanine at position 551 (A), alanine at position 555 (A), glutamic acid at position 557 (E), methionine at position 561 (M), serine at position 563 (S), glutamine at position 577 (Q), serine at position 583 (S), valine at position 593 (V), threonine at position 596 (T), alanine at position 661 (A), valine at position 662 (V), threonine at position 664 (T), proline at position 665 (P), threonine at position 710 (T), aspartic acid at position 717 (D), asparagine at position 718 (N), glutamic acid at position 719 (E), and serine at position 723 (S).

[0229] The AAV variant viral particles disclosed herein can incorporate reasonable design parameters, characteristics, modifications, advantages, and variations, which are obvious to those skilled in the art in the field of engineered AAV viral vectors. Example 2 Directed evolution was employed to discover novel adeno-associated virus (AAV) variants exhibiting superior gene delivery to retinal cells upon intravitreal (IVT) administration, a route of administration significantly superior to other methods of gene delivery to the human eye (Example 1). Cell tropism following intravitreal administration of the novel AAV variant (LAISDQTKHA+P34A; SEQ ID NO:42) containing the peptide LAISDQTKHA (SEQ ID NO:28) with a P34A substitution and insertion at amino acid 588 is a representative example of the ability of an AAV variant containing ISDQTKH (SEQ ID NO:14) to transduce retinal cells, assessed in vivo in non-human primates (NHP).

[0230] Recombinant AAV virus particles were manufactured using standard methods, comprising an AAV2 capsid or a novel variant capsid LAISDQTKHA+P34A (SEQ ID NO:42) and a genome containing a green fluorescent protein (GFP) transgene operatively linked to a CMV promoter (AAV2.CMV.GFP and LAISDQTKHA+P34A.CMV.GFP, respectively) or a CAG promoter (AAV2.CAG.EGFP and LAISDQTKHA+P34A.CAG.EGFP, respectively). African green monkeys ( Figure 7 , Figure 8 ) or crab-eating macaque ( Figure 9 Various doses of carrier are injected intravitreally, with an injection area of ​​4 × 10⁻⁶ per eye. 10 vg to 1×10 12 vg (see legend for details), and using Heidelberg Spectralis TM Assess retinal cell transduction lifespan using fundus fluorescence imaging.

[0231] Intravitreal delivery of AAV containing the novel variant LAISDQTKHA+P34A (SEQ ID NO:42) resulted in more extensive and robust transgene expression across the NHP retina than AAV2. Figures 7-9 Images show that the novel AAV variant capsid provides robust expression in the center of the fovea (a cone-rich region); as early as 2 weeks post-injection, in the parafoveal ring (a region rich in retinal ganglion cells) and in the periphery (a region rich in many cell types, including rods, Müller glial cells, amacrine cells, and bipolar cells). In contrast, and consistent with results reported by others, wild-type AAV2 provides weaker expression, primarily in the parafoveal ring and detectable only at later time points. Immunohistochemical analysis of various regions of the retina 3 weeks post-injection confirmed successful transduction of many retinal cell types throughout the retina, including retinal pigment epithelial cells, rod and cone photoreceptors, and retinal ganglion cells. Figures 10A-10E ). This study demonstrates superior gene delivery of variants containing ISDQTKH following a clinically preferred route of administration compared to clinically relevant AAV2. Similar efficacy can be achieved with other variants containing this peptide insert motif. Similarly, similar efficacy can be achieved using other variants identified using the same directed evolution methods disclosed herein.

[0232] Example 3 Cell tropism of a novel AAV variant, LAISDQTKHA+P34A (SEQ ID NO:42), for retinal pigment epithelial (RPE) and photoreceptor (PR) cells was evaluated in vitro using RPE cells and PR cells derived from fibroblast-derived human induced pluripotent stem cells (FB-iPSCs) or human embryonic stem cells (ESCs).

[0233] AAV viral particles were manufactured using standard methods, comprising an AAV2 capsid or a novel variant capsid LAISDQTKHA+P34A (SEQ ID NO:42) and a genome containing a green fluorescent protein (EGFP) transgene operatively linked to the CAG promoter (AAV2.CAG.EGFP and LAISDQTKHA+P34A.CAG.EGFP, respectively). Human RPE cell cultures were generated from human embryonic stem cell line ESI-017 or human fibroblast-derived induced pluripotent stem cells (“FB-iPSC”) using a 45-day differentiation protocol. Maturation into RPE cells was confirmed by detecting the expression of mature RPE markers including RPE65 and BEST1; synthesis of VEGF and PEDF; and the ability to phagocytose the outer segments of rods. PR cultures were generated using a multi-step eyecup formation paradigm, and PR content was confirmed by detecting the expression of recovery protein and S-opsin after 179 days of culture.

[0234] Compared to AAV2, such as by immunofluorescence ( Figures 11A to 11B ), flow cytometry (increased by 2.7 times; Figures 11C to 11D ) and Western blot analysis ( Figures 11E to 11F As determined by [the study], LAISDQTKHA+P34A (SEQ ID NO:42) provided significantly higher transduction efficiency and transgene expression in human RPE cultures at 7 days post-infection. At 32 days post-infection, robust transduction and expression were also achieved in human PR cultures using LAISDQTKHA+P34A.CAG.EGFP. This study demonstrates the superior ability of variants containing ISDQTKH (SEQ ID NO:14) to deliver genes to retinal cells.

[0235] Example 4 Design and construction of an anti-VEGF expression vector. The amino acid sequence of aflibercept consists of a human Flt1 signal peptide fused to the Fc region of human IgG1, VEGFR1 domain 2, and VEGFR2 domain 3. Figure 12A(SEQ ID NO: 66). The amino acid sequences of the light and heavy chains of ranibizumab are linked via a flexible protein linker to convert the double-chain antigen-binding fragment (Fab) into a single-chain Fab (scFab). The light-heavy (LH) form consists of a human Igκ light chain signal peptide linked by a flexible peptide, and the variable light chain, constant light chain, variable heavy chain, and constant heavy chain 1 domain of ranibizumab. Figure 12B ; SEQ ID NO: 71). The heavy-light (HL) form is similar to the LH form, except that the signal peptide is derived from the human IgG heavy chain and the heavy and light chain domains are on opposite sides of the linker. Figure 12C ;SEQ ID NO: 68). Bromoizumab is designed to include variable light chain and variable heavy chain domains linked by flexible peptides, and a human Igκ light chain signal peptide for secretion from mammalian cells (…). Figure 12D (SEQ ID NO: 75). To improve expression from human cells, the open reading frame was codon-optimized, synthesized via GeneArt or GenScript, and provided in a standard plasmid cloning vector. The DNA of interest was excised from the plasmid using restriction enzymes PstI and BglII and inserted between the CAG promoter and SV40 polyA signal in the pAAV-CAG-SV40 pA plasmid. The sc-ranibizumab-Fc design consists of an LH form of sc-ranibizumab fused to the Fc region of human IgG1 designed from aflibercept. Figure 12E ;SEQ ID NO: 73). The bromizumab-Fc design consists of bromizumab fused to the Fc region of human IgG1 designed from aflibercept. Figure 12F (SEQ ID NO: 77). The sc-ranibizumab LH, bromizumab, and Fc regions were amplified by PCR from a starting cloning vector having an extension containing the type II restriction enzyme BsmBI site at the 3' end (sc-ranibizumab LH and bromizumab) or the 5' end (Fc). The resulting PCR product was digested with restriction enzymes PstI and BsmBI (sc-ranibizumab LH and bromizumab) or BsmBI and BglII (Fc) and inserted between the CAG promoter and the SV40 polyA signal in the pAAV-CAG-SV40 pA plasmid. The ligation reaction was used to transform *E. coli*, and positive clones were identified by restriction digestion. The clones were then grown on a larger scale, and the plasmid DNA was purified using the Qiagen endonuclease-free Maxiprep kit. The identity of the plasmid was verified by restriction enzyme digestion and sequencing. The pAAV-CAG-SV40 polyA plasmid contains AAV2 inverted terminal repeats (ITRs), which allow anti-VEGF sequences to be packaged within an AAV capsid and delivered to cells of interest in the retina via intravitreal administration.

[0236] In two independent experiments, HEK293T cells were transfected or simulated using the FuGene6 transfection reagent with plasmids containing GFP or anti-VEGF transgenes controlled by the CAG promoter (sc-RNBZ HL (SEQ ID NO:67), sc-RNBZ LH1 (SEQ ID NO:69), and sc-RNBZ-LH2 (SEQ ID NO:70), AFLB (SEQ ID NO:65), and BRO (SEQ ID NO:74)). Culture medium was collected 48 hours post-transfection and assayed using an aflibercept ELISA kit (Eagle Biosciences, Immunoguide IG-AA115). Anti-VEGF protein was detected by HRP-anti-IgG Fc (simulated, GFP, and AFLB) or HRP-anti-IgG H+L (sc-RNBZ HL, sc-RNBZ LH1, and sc-RNBZ LH2) provided as reagents in the kit. Concentrations were calculated relative to clinical Eylea (simulated, GFP, and AFLB) or Lucentis (sc-RNBZ HL, sc-RNBZ LH1, sc-RNBZ LH2, and BRO). In the first of both experiments, aflibercept expression was approximately 15 μg / ml, sc-ranibizumab expression ranged from 1.6 to 2.8 μg / ml, LH form expression was at similar concentrations, and HL form expression was approximately twice as low. Figure 13A In the second experiment, aflibercept was expressed at approximately 50 μg / ml, and ranibizumab LH1 was expressed at approximately 4.0 μg / ml. Figure 13B The signal from bromizumab was very low, most likely due to poor recognition by the detection antibody. No VEGF binding activity was detected in simulated or GFP-transfected media.

[0237] Example 5 To directly visualize and confirm the size of anti-VEGF proteins, Western blot analysis was performed on the culture medium from cells transfected with expression plasmids of GFP, aflibercept (SEQ ID NO: 65), sc-ranibizumab (SEQ ID NO: 67, 69, and 70), or bromizumab (SEQ ID NO: 74) (Figure 14). Culture medium from transfected HEK293T cells was run on a Bolt 4-12% Bis-Tris Plus gel (Invitrogen Cat# NW04122BOX), and isolated proteins were transferred to nitrocellulose using an iBlot2 device. The blots were detected using an iBind Flex device with HRP-conjugated goat anti-human IgG Fc (Thermo Cat# 31413, left panel) or goat anti-human IgG Fab (Thermo Cat# 31482, right panel), and visualized using a SuperSignal West Dura chemiluminescent substrate (Thermo Cat# 34076). Images were captured using an iBright FL1000 imager.

[0238] The aflibercept sample appeared similar to the clinical comparative protein Eylea. No signal was detected in the negative control sample. The clinical comparative protein Lucentis was reduced to individual light and heavy chains of 24 kD, while the sc-ranibizumab protein ran at approximately 48 kD, attributed to the presence of a polypeptide linker that binds the light and heavy chains into a single protein. Higher amounts of the LH form of protein were present compared to the HL form, consistent with protein quantification obtained by ELISA. Figure 13A The low signal from bromizumab is most likely due to poor antibody recognition. The protein migrates at its correct molecular weight of 24 kD.

[0239] Example 6 As another method to determine VEGF binding activity, a VEGF competition ELISA was performed (Figure 15). Clinical comparison proteins Eylea or Lucentis and culture media from transfected HEK293T cells were incubated overnight at room temperature with 13 pM VEGF. Free VEGF in the samples was measured using the Quantikine VEGF ELISA kit (R&D Systems Cat# DVE00). In the first of the two experiments, the inhibition curves of the four anti-VEGF proteins from the transfected samples were very similar to those of clinical Eylea and Lucentis. Aflibercept and Eylea competed more strongly with VEGF than the sc-ranibizumab variant and Lucentis. Figure 15AAll three forms of sc-ranibizumab (SEQ ID NO: 67, 69, and 70) are nearly identical. In the second experiment, all anti-VEGF constructs competed with VEGF ( Figure 15B The GFP negative control sample showed no competitive activity.

[0240] Example 7 To confirm that the anti-VEGF protein expressed from transfected cells blocks the binding of VEGF to its receptor and thus inhibits its function, equal concentrations of the clinical comparative protein and the culture medium from transfected HEK293T cells were mixed with 20 ng / ml VEGF and placed on PathHunter KDR cells (DiscoverX 93-0996Y1). These cells express a VEGF receptor / β-galactosidase fusion protein, which produces active β-galactosidase upon VEGF binding. The cells were lysed after 22 hours, and β-galactosidase activity was measured. The inhibition curve of the anti-VEGF protein from the transfected samples was the same as that of the clinical comparative protein, demonstrating that the expressed protein blocks VEGF function in cell-based assays. Figure 16 In the two subsequent experiments, cellular VEGF neutralization assays were also performed using an equal volume of culture medium derived from HEK293T cells transfected with an anti-VEGF construct. In the first of these experiments, expression plasmids for GFP, aflibercept (SEQ ID NO: 65), sc-ranibizumab HL (SEQ ID NO: 67), or sc-ranibizumab LH1 (SEQ ID NO: 69) were evaluated. All anti-VEGF constructs assessed the neutralization of VEGF (…). Figure 17A The LH form of sc-ranibizumab neutralized VEGF more strongly than the HL form. In a second experiment, expression plasmids of GFP, aflibercept (SEQ ID NO: 65), sc-ranibizumab LH1 (SEQ ID NO: 69), or bromizumab (SEQ ID NO: 74) were mixed with 8 ng / ml VEGF and placed on PathHunter KDR cells (DiscoverX 93-0996Y1). The cells were lysed after 18 hours and β-galactosidase activity was measured. Again, all anti-VEGF constructs were evaluated for neutralized VEGF (…). Figure 17B In the measured dilution, the GFP control sample showed a slight matrix effect.

[0241] Example 8 Retinal pigment epithelial (RPE) cells were transfected or simulated using FuGeneHD transfection reagent with plasmids containing GFP or anti-VEGF transgenes under the control of the CAG promoter. Culture medium was collected 48 hours post-transfection. As evidenced by the number of GFP-expressing cells (data not shown), RPE cell transfection efficiency was extremely low; therefore, VEGF binding activity was not detectable in the culture medium from these transfections. The total concentration of VEGF in the culture medium was assessed to determine whether low concentrations of anti-VEGF agents could reduce VEGF secretion by RPE cells. Culture medium was collected 48 hours post-transfection and measured using the Quantikine VEGF ELISA kit (R&D Systems Cat# DVE00). A significant decrease in VEGF levels was observed after aflibercept (SEQ ID NO: 65) expression, and a slight decrease in VEGF levels after sc-ranibizumab (SEQ ID NO: 67, 69, and 70) expression. Figure 18 All three sc-ranibizumab forms have similar effects on VEGF levels. Once delivered via more efficient AAV transduction, the anti-VEGF transgene results in higher concentrations of anti-VEGF protein, which are quantified using an aflibercept ELISA kit.

[0242] Example 9 Viral DNA was packaged into an R100 variant capsid (with the amino acid sequence shown in SEQ ID NO: 42) using an AAV expression plasmid carrying the genes for aflibercept (SEQ ID NO: 65), ranibizumab (SEQ ID NO: 67 and 69), or bromizumab (SEQ ID NO: 74). Mature RPE cells were transduced into 12-well plates at an MOI of 5,000 in 1.0 ml X-Vivo10 medium for 30 days. The medium was changed three days after transduction, collected and replaced six days after transduction, and collected and replaced again ten days after transduction.

[0243] The total concentration of VEGF in the culture medium was determined using the Quantikine VEGF ELISA kit (R&D Systems Cat# DVE00) to determine the effect of anti-VEGF vector transduction on the level of free, detectable endogenous VEGF secreted into the culture medium by RPE cells. Figure 19Cells transduced with the GFP control vector showed endogenous VEGF levels of 4,500 pg / ml in day 6 samples and 8,300 pg / ml in day 10 samples. Transduction with all anti-VEGF vectors produced little to no detectable VEGF in the culture medium. It is likely that the amount of VEGF secreted by RPE cells is not altered, but rather that VEGF in the culture medium is bound by anti-VEGF proteins, making it undetectable by ELISA. Further experiments, including Western blotting, can be performed to determine the total amount of bound and unbound VEGF.

[0244] Culture media derived from RPE transduction were also measured using an aflibercept ELISA kit (Eagle Biosciences, Immunoguide IG-AA115). Anti-VEGF protein was detected using HRP-anti-IgG H+L antibody (ThermoFisher Cat# 31410). Concentrations were calculated relative to clinical Eylea (AFLB and GFP) or Lucentis (sc-RNBZ HL, sc-RNBZ LH1, and BRO). Aflibercept (SEQ ID NO: 65) expression was approximately 1,800 ng / ml in samples on day 6 and 2,800 ng / ml in samples on day 10. sc-ranibizumab expression ranged from 700 ng / ml in HL (SEQ ID NO: 67) samples on day 6 to 1,700 ng / ml in LH (SEQ ID NO: 69) samples on day 10. Figure 20 The signal from bromoizumab (SEQ ID NO: 74) was very low, most likely due to poor recognition by the detection antibody. No VEGF binding activity was detected in the medium derived from GFP transduction.

[0245] Example 10 To directly visualize anti-VEGF proteins and confirm their size, Western blot analysis was performed on the culture medium from transduced RPE cells. Figure 21Equal volumes of day 6 and day 10 medium were run on Bolt 4-12% Bis-Tris Plus gels (Invitrogen Cat#NW04122BOX), and the isolated proteins were transferred to nitrocellulose using an iBlot2 device. Blots were detected using an iBind Flex device with HRP-conjugated goat anti-human IgG Fc (Thermo Cat# 31413, left panel) or goat anti-human IgG Fab (Thermo Cat# 31482, right panel) and observed with SuperSignal West Dura chemiluminescent substrate (Thermo Cat# 34076). Images were captured using a ChemiDoc MP imager.

[0246] The aflibercept (SEQ ID NO: 65) sample appeared similar to the clinical comparative protein Eylea (black arrow). No band with the correct migration rate was observed in the GFP negative control sample. Clinical Lucentis was reduced to separate light and heavy chains of 24 kD, while sc-ranibizumab HL (SEQ ID NO: 67) and LH (SEQ ID NO: 69) did not separate and migrated at an apparent molecular weight of 58 kD, as indicated by the gray arrow. Brombizumab (SEQ ID NO: 74) showed a low signal, most likely due to poor recognition by the detection antibody. The protein migrated at the correct molecular weight of 26 kD, as indicated by the dotted arrow. Protein levels were quite similar in the samples from day 6 and day 10.

[0247] Example 11 As another method to determine VEGF binding activity, VEGF competitive ELISA is performed. Figure 22 An equal volume of culture medium from transduced RPE cells was incubated overnight at room temperature with 13 pM VEGF. Free VEGF levels in the samples were measured using the Quantikine VEGF ELISA kit (R&D Systems Cat# DVE00). VEGF was competitively expressed from culture medium derived from cells transduced with all anti-VEGF constructs. Results for samples on day 6 and day 10 were similar. The GFP-negative control sample showed no competitive activity. Free VEGF levels were high at the lowest dilution due to endogenous VEGF produced by RPE cells.

[0248] Example 12 To compare the VEGF neutralizing activity of anti-VEGF proteins expressed in RPE cells derived from the viral vector, an equal volume of culture medium from transduced RPE cells was mixed with 8 ng / ml VEGF and placed on PathHunter KDR cells (DiscoverX 93-0996Y1). Figure 23 The cells were lysed 18 hours later, and β-galactosidase activity was measured. VEGF was neutralized in the culture medium derived from cells transduced with all anti-VEGF constructs. No VEGF neutralization was observed in the culture medium derived from the GFP control.

[0249] The foregoing only illustrates the principles of the invention. It will be understood that those skilled in the art will be able to devise different arrangements, which, while not expressly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all instances and conditional language described herein are primarily intended to help the reader understand the principles and concepts of the invention contributed by the inventors to advance the field, and are to be interpreted as not being limited to these specifically described instances and conditions.

[0250] Furthermore, all statements herein referring to the principles, aspects, and embodiments of the invention, together with specific examples thereof, are intended to cover both their structural and functional equivalents. Additionally, such equivalents are contemplated to include both currently known equivalents and future-developed equivalents, i.e., any element developed to perform the same function regardless of structure. Therefore, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention are embodied in the appended claims.

Claims

1. A recombinant adeno-associated virus (rAAV) viral particle comprising (a) a variant adeno-associated virus (AAV) capsid protein, said variant AAV capsid protein comprising a peptide insertion in the GH ring of said capsid protein relative to a corresponding parental AAV capsid protein, said peptide insertion comprising the amino acid sequence ISDQTKH (SEQ ID NO: 14), and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide that inhibits the activity of vascular endothelial growth factor (VEGF), said nucleotide sequence being selected from: (i) The nucleotide sequence shown in SEQ ID NO: 65 (abfcecept), (ii) Nucleotides 79-1377 of SEQ ID NO: 65, and (iii) A nucleotide sequence having at least 98% identity with (i) and encoding the amino acid sequence shown in SEQ ID NO: 66, or a nucleotide sequence having at least 98% identity with (ii) and encoding amino acids 27-458 of SEQ ID NO:

66.

2. The rAAV viral particle according to claim 1, wherein the length of the peptide insertion is 10 or 11 amino acids and comprises the amino acid sequence Y1Y2ISDQTKHY3, wherein each of Y1-Y3 is independently selected from Ala, Leu, Gly, Ser, Thr and Pro, preferably wherein the inserted peptide is LAISDQTKHA (SEQ ID NO: 28).

3. The rAAV viral particle according to claim 1 or 2, wherein the insertion site is located between amino acids 587 and 588 of VP1 (SEQ ID NO: 2) corresponding to AAV2 or at the corresponding position in the capsid protein of another AAV serotype.

4. The rAAV virus particle according to any one of claims 1-3, wherein the capsid protein comprises a P34A amino acid substitution relative to the VP1 capsid (SEQ ID NO: 2) of AAV2.

5. The rAAV virus particle according to any one of claims 1-4, wherein the capsid protein comprises (i) the amino acid sequence ISDQTKH (SEQ ID NO:14) and (ii) P34A amino acid substitutions, and has at least 90%, at least 95%, or at least 99% identity with the sequence shown in SEQ ID NO:

42.

6. The rAAV virus particle according to claim 5, wherein the capsid protein consists of or contains the amino acid sequence shown in SEQ ID NO:

42.

7. The rAAV viral particle according to claim 6, wherein the nucleotide sequence is at least 99% identical to the full length of the sequence shown in SEQ ID NO: 65 (abfcep) or to nucleotides 79-1377 of SEQ ID NO: 65, and encodes the amino acid sequence shown in SEQ ID NO: 66 or the amino acid sequence encodes amino acids 27-458 of SEQ ID NO:

66.

8. The rAAV viral particle according to claim 7, wherein the nucleotide sequence is 100% identical to the full length of the sequence shown in SEQ ID NO: 65 or to nucleotides 79-1377 of SEQ ID NO: 65, and encodes the amino acid sequence shown in SEQ ID NO: 66 or the amino acid sequence encodes amino acids 27-458 of SEQ ID NO:

66.

9. The rAAV viral particle according to claim 7, wherein the nucleotide sequence comprises nucleotides 79-1377 of SEQ ID NO: 65, and preferably comprises nucleotides 1-1377 of SEQ ID NO:

65.

10. The rAAV viral particle according to any one of claims 1-9, wherein the nucleotide sequence encoding the polypeptide that inhibits vascular endothelial growth factor (VEGF) activity is operatively linked to an expression control sequence, preferably wherein the expression control sequence comprises a constitutive promoter, preferably wherein the constitutive promoter is selected from CMV, CAG and CBA promoters.

11. The rAAV viral particle according to any one of claims 1-10, wherein the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of the corresponding parental AAV capsid protein to retinal cells.

12. A pharmaceutical composition comprising rAAV viral particles according to any one of claims 1-11 and a pharmaceutically acceptable carrier.

13. The rAAV viral particles according to any one of claims 1-11 or the pharmaceutical composition according to claim 12, for the treatment of VEGFa-related eye diseases, said VEGFa-related eye diseases being selected from: wet (neovascular, exudative) age-related macular degeneration; macular edema following retinal vein occlusion; retinal neovascularization caused by retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative diabetic retinopathy and proliferative diabetic retinopathy); myopic macular degeneration; branched retinal veins Retinal vein occlusion, hemilateral retinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectasia; neovascular glaucoma; vitreous hemorrhage; retinal and choroidal neovascularization secondary to retinal diseases, including but not limited to uveitis, trauma, retinal degeneration, hereditary retinal and / or choroidal diseases, ocular tumors, corneal and iris neovascularization, preferably wherein the rAAV or pharmaceutical composition is administered via intravitreal injection.

14. The rAAV viral particles or pharmaceutical composition according to claim 13, wherein the VEGFa-related eye disease is selected from wet (neovascular, exudative) age-related macular degeneration; diabetic macular edema; macular edema after retinal vein occlusion; diabetic retinopathy; and myopic choroidal neovascularization.

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