RU2773756C2 - Options of adeno-associated virus capsids and their use for angiogenesis inhibition - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: present invention relates to biotechnology, namely to a virion of a recombinant adeno-associated virus (hereinafter – rAAV) for the inhibition of the activity of a vascular endothelial growth factor (VEGF) in a mammalian eye cell. rAAV contains (a) an option of a capsid protein of an adeno-associated virus (hereinafter – AAV), containing a peptide insert in GH loop of the capsid protein, compared with the corresponding initial capsid protein AAV, where the peptide insert contains an amino acid sequence ISDQTKH (SEQ ID NO:14), and (b) heterologous nucleic acid containing a nucleotide sequence encoding polypeptide that inhibits VEGF activity, functionally associated with a sequence to control expression, where the nucleotide sequence is selected from a group consisting of: (i) a nucleotide sequence indicated as SEQ ID NO:69 (sc-ranibizumab-LH1), (ii) a nucleotide sequence indicated as SEQ ID NO:65 (aflibercept), (iii) a nucleotide sequence indicated as SEQ ID NO:74 (brolucizumab), (iv) nucleotides 61-1578 of SEQ ID NO:69, (v) nucleotides 79-1377 of SEQ ID NO:65, (vi) nucleotides 61-816 of SEQ ID NO:74, and (vii) a nucleotide sequence at least 95%, at least 98% or at least 99% identical to any of (i)-(vi). Pharmaceutical compositions containing an option of AAV capsid protein, a method for producing these capsid proteins and rAAV virions, and methods for using these capsid proteins and rAAV virions in research and clinical practice, for example, in delivery of a nucleic acid sequence to one or more retinal cells for the treatment of retinal disorders and diseases, are presented.

EFFECT: invention makes it possible to improve the expression of heterologous nucleic acid.

28 cl, 63 dwg, 1 tbl, 12 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to U.S. Provisional Application Nos. 62/590976 filed November 27, 2017 and 62/664726 filed April 30, 2018, the entire contents of each of which are incorporated herein by reference.

INCLUDING AS A REFERENCE A SEQUENCE LIST IN TEXT FILE FORM

[002] The sequence listing is provided with this description in the form of a text file "090400-5010-WO-seq-listing.txt", created on November 26, 2018 and having a size of 238 KB. The full content of the text file is given in the present description by reference.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[003] The invention described herein relates primarily to the field of adeno-associated virus (AAV) virions containing variant capsid proteins and the production of such variant capsids using directed evolution techniques.

BACKGROUND OF THE INVENTION

[004] Inherited retinal diseases comprise a large group of heterogeneous genetic disorders affecting approximately 1 in 3,000 people (more than 2 million people worldwide) and are a major source 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, with 1.7 million Americans currently living with wAMD-associated severe central vision loss and almost one third of adults over the age of 40 with diabetes have significant visual impairment. These diseases are usually associated with dysfunction or death of one or more types of retinal cells, in some cases due to the lack of expression or function of a key protein, such as RPE65 in LCA2, in other cases, due to mutations in genes that form toxic gene products, for example, dominant mutations affecting the folding of the rhodopsin protein, or in other cases, due to changes in retinal physiology induced by ectopic protein expression, for example, VEGF in wAMD.

[005] One way to meet this great unmet medical need is adeno-associated virus (AAV) mediated gene therapy, in which a recombinant adeno-associated virus (rAAV) is used to deliver a gene to one or more cell types in the retina, for example, to replace a missing gene, to correct dominantly defective gene or to provide a matrix for continuous protein therapy. While advances in clinical AAV-based gene therapy are increasing, it still has drawbacks with respect to viral vector properties, including, for example, targeting desired retinal cells with high efficiency. For example, many homologous primate AAV serotypes and many non-human primate serotypes have been identified and characterized, where AAV2 is the most well characterized of the AAV serotypes and the first to be adapted as a gene delivery vehicle to the eye. However, these AAVs (including AAV2) have not been reported to be effective for transducing deeper retinal cell types when delivered via vitreous injection. Accordingly, there is a need in the art for new AAV variants with superior transduction capability that can provide more efficient gene delivery to retinal cells for the treatment of ocular disease. There is a need in the art for those AAV variants that have an improved retinal transduction profile - in some cases broad, in other cases favored by specific retinal cell types - compared to wild-type AAV and AAV variants, as known herein. areas.

[006] Natural AAV is a single-stranded DNA virus containing three open reading frames, rep , cap and aap . The first gene, rep , encodes four proteins required for genome replication (Rep78, Rep68, Rep52, and Rep40), the second, cap , expresses three structural proteins (VP1-3) that assemble to form the viral capsid, and the third expresses an assembly-activating protein (AAP) required for capsid assembly. AAV depends on the presence of a helper virus, such as adenovirus or herpesvirus, for active replication. In the absence of a helper virus, AAV enters a latent state in which its genome is maintained episomally or integrated into the host chromosome at the AAVS1 locus.

[007]Methods of directed evolution in vitro andin vivo can be used to select AAV variants that provide improvement over current AAV-based gene delivery vectors. Such directed evolution methods are known in the art and are described, for example, in PCT Publication WO 2014/194132 and Kotterman & Schaffer (Nature Review Genetics, AOP, published online 20 May 2014; doi: 10.1038/nrg3742), the full contents of both of which given in the present description by reference. Directed evolution is a method of constructing a capsid that mimics natural evolution through alternating cycles of genetic diversity and selection processes, thus allowing the accumulation of advantageous mutations that increasingly improve the function of a biomolecule such as an AAV-based virion. In this way, in the genescap Wild-type AAVs are diversified to produce large genomic libraries that 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.

[008] Variants of AAV are described, for example, in US Patent numbers 9193956; 9;186;419; 8632764; 8663624; 8927514; 8628966; 8263396; 8734809; 8889641; 8632764; 8691948; 8299295; 8802440; 8445267; 8906307; 8574583; 8067015; 7588772; 7867484; 8163543; 8283151; 8999678; 7892809; 7906111; 7259151; 7629322; 7220577; 8802080; 7198951; 8318480; 8962332; 7790449; 7282199; 8906675; 8524446; 7712893; 6491907; 8637255; 7186522; 7105345; 6759237; 6984517; 6962815; 7749492; 7259151; and 6156303; U.S. Patent Application Publication Numbers 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; publications of European patent applications number 2692731 A1; 2383346B1; 2359865 B1; 2359866B1; 2359867 B1; and 2357010 B1; 1791858B1; 1668143 B1; 1660678B1; 1664314 B1; 1496944 B1; 1456383 B1; 2341068B1; 2338900 B1; 1456419 B1; 1310571 B1; 1456383 B1; 1633772 B1; and 1135468 B1; and International Publications (PCT) numbers WO 2014/124282; WO 2013/170078; WO2014/160092; WO2014/103957; WO2014/052789; WO 2013/174760; WO 2013/123503; W02011/038187; and WO 2008/124015; WO2003/054197; however, none of these references describe embodiments and/or features and/or structural compositions of the AAV variants described herein.

[009] The contents of all documents and references cited in the present specification and in the cited Patent Documents are incorporated herein by reference.

SUMMARY OF THE INVENTION

[0010] The present invention relates to variants of adeno-associated virus (AAV) capsid proteins having one or more modifications in amino acid sequence relative to the original AAV capsid protein, which, when present in the AAV virion, confer increased infectivity for one or more retinal cell types. , compared with the infectivity for retinal cells of the AAV virion containing the unmodified original AAV capsid protein. The present invention also relates to recombinant AAV virions and their pharmaceutical compositions containing a variant AAV capsid protein as described herein, to methods for producing variant rAAV capsid proteins and virions, and to methods for using these capsid proteins and rAAV virions in research and clinical practice, for example, in delivering a nucleic acid sequence to one or more retinal cells for the treatment of retinal disorders and diseases.

[0011] In some aspects of the description, the present invention relates to variants of the adeno-associated virus (AAV) capsid proteins, these variants of the AAV capsid proteins, have one or more modifications in the amino acid sequence, compared to the original AAV capsid, which, when present in the AAV virion confer increased infectivity to one or more retinal cell types (e.g., photoreceptor cell (e.g., rods; cones), retinal ganglion cell (RGC), glial cell (e.g., Muller glial cell, microglial cell), bipolar cell, amacrine cell, horizontal cells and/or retinal pigment epithelium (RPE) cells), compared with the infectivity for retinal cells of the AAV virion containing the original AAV capsid protein containing no amino acid sequence modification.

[0012] In some aspects, the present invention relates to recombinant AAV (rAAV) virions, these rAAV virions contain a capsid protein variant as described herein, wherein the rAAV virions have increased infectivity for one or more retinal cell types (e.g., photoreceptor cell (e.g., rods; cones), retinal ganglion cell (RGC), glial cell (e.g., Mueller glial cell, microglial cell), bipolar cell, amacrine cell, horizontal cell, and/or retinal pigment epithelial cell (RPE)), compared with infectivity for retinal cells of the AAV virion containing the corresponding unmodified original AAV capsid protein. In some embodiments, the rAAV virion has increased infectivity for all retinal cells, compared to an AAV virion containing the original AAV capsid protein. In other embodiments, the rAAV virion has increased infectivity for certain retinal cell types, but not others, compared to an AAV virion containing the original AAV capsid protein. In other words, the rAAV virion has an increased infectivity that is preferential for certain retinal cell types but not others, e.g., rAAV has a preferential increased infectivity 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 epithelial (RPE) cells, but does not have increased infectivity for all cell types.

[0013] In some embodiments, the rAAV virion contains a heterologous nucleic acid. In some such embodiments, the heterologous nucleic acid encodes an RNA that encodes a polypeptide. In other such embodiments, the heterologous nucleic acid sequence encodes an RNA that does not encode a polypeptide, e.g., the heterologous nucleic acid sequence encodes an RNA interference agent, a guide RNA for a nuclease, and so on.

[0014] The present invention also relates to pharmaceutical compositions containing the subject infectious rAAV virions and a pharmaceutically acceptable carrier.

[0015] The present invention also relates to the use of a rAAV virion containing a variant capsid protein as described herein in a method for delivering a heterologous nucleic acid to a target cell (such as a retinal cell) by contacting the target cell with the rAAV virion. In some embodiments, the target cell is in vivo , for example, in the eye of an individual in need of treatment for an eye disease. In other embodiments, the target cell is in vitro .

[0016] The present invention also relates to methods of treating eye disease by administering to a subject in need of such treatment an effective amount of rAAV virions containing a capsid protein variant as described herein, or a pharmaceutical composition containing an effective amount of rAAV virions.

[0017] The present invention also provides an isolated nucleic acid containing a sequence encoding an AAV capsid protein variant as described herein and a host cell containing the isolated nucleic acid. In other embodiments, the isolated nucleic acid and/or the isolated host cell contains rAAV.

[0018] In some aspects, a variant AAV capsid protein contains an insertion of about 5 amino acids to about 20 amino acids ("heterologous peptide" or "peptide insert") in the GH-loop of the capsid protein, compared to the corresponding parental AAV capsid protein, where the variant capsid protein, when present in the AAV virion, confers increased infectivity to the retinal cell compared to the infectivity to the retinal cell of an AAV virion containing the corresponding parent AAV capsid protein. In some embodiments, the peptide contains 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:30), LANQDYTKTA (SEQ ID NO:31), LAHDITKNIA (SEQ ID NO:32), LAHPDTTKNA (SEQ ID NO:32 :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 primarily consists of 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:15 :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: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 aspects, the AAV capsid protein variant comprises one or more amino acid substitutions compared to the corresponding parent AAV capsid protein, wherein the capsid protein variant, when present in the AAV virion, confers increased infectivity to a retinal cell compared to the infectivity to a retinal cell of the AAV virion containing the corresponding parent AAV capsid protein.

[0019] In related aspects, an AAV capsid protein variant comprises a peptide insert and one or more amino acid substitutions, compared to the corresponding parent AAV capsid protein, wherein the capsid protein variant, when present in the AAV virion, confers increased infectivity to retinal cells compared to infectivity for the retinal cell of the AAV virion containing the corresponding parental AAV capsid protein.

[0020] The present invention also relates to an AAV capsid protein variant comprising the heterologous LAISDQTKHA peptide (SEQ ID NO:28) and a P34A substitution relative to AAV2. In related embodiments, the present invention provides an infectious recombinant AAV comprising an AAV capsid protein variant at least 90% identical to the sequence set forth in SEQ ID NO:42 and a heterologous nucleic acid comprising a sequence encoding a VEGF inhibitor, preferably a VEGFa inhibitor, and to a pharmaceutical composition containing them. In some embodiments, the heterologous nucleic acid sequence encoding a VEGF inhibitor is selected from a sequence encoding aflibercept, ranibizumab, a single chain variant of ranibizumab (sc-ranibizumab LH or HL), brolucizumab, sc-ranibizumab fused to an IgG Fc domain (sc-ranibizumab -Fc), or brolucizumab fused to an IgG Fc domain (brolucizumab-Fc). In related embodiments, the recombinant AAV comprises a heterologous nucleic acid comprising two or more sequences each encoding a VEGFa inhibitor (eg, a first sequence encoding aflibercept and a second sequence encoding brolucizumab). In preferred embodiments, the heterologous nucleic acid sequence has a sequence of any of SEQ ID NOs: 65, 67, 69, 70, 72, 74, 76 or a sequence at least 90% identical to it. In other related embodiments, the present invention relates to a method for treating a patient with an eye disease associated with increased intraocular levels of VEGFa, comprising administering to the patient, preferably by intravitreal injection, an effective amount of an infectious recombinant AAV comprising an AAV capsid protein variant according to at least 90% identical to the sequence indicated as SEQ ID NO:42, and a heterologous nucleic acid containing a sequence encoding a VEGF inhibitor.

[0021] The present invention also relates to an AAV capsid protein variant comprising the heterologous peptide LAISDQTKHA (SEQ ID NO:28) and amino acid substitutions N312K, N449D, N551S, I698V, and L735Q, relative to AAV2.

[0022] The present invention also relates to methods for producing and/or delivering rAAV containing an AAV capsid variant as described herein. In addition, the present invention relates to kits containing rAAV containing a variant AAV capsid, as described in the present description, and for use in the methods described in the present description.

[0023] In other embodiments, the AAV virion containing the variant capsid protein in the preceding paragraphs may include any of the preceding or subsequently described embodiments. Indeed, it is to be understood that specific features of the invention, which, for the sake of clarity, are described in the context of separate embodiments, may also be presented in combination in one embodiment. Conversely, various features of the invention, which, for brevity, are described in the context of one embodiment, may also be presented individually or in any suitable subcombination. All combinations of embodiments related to the invention are specifically included in the invention and are described in the present description, just as if all combinations without exception were individually and explicitly described. In addition, all subcombinations of various embodiments and elements thereof are also specifically included in the invention and are described in the present description, just as if all subcombinations without exception were individually and expressly described in the present description.

[0024] The Summary of the Invention is intended neither to define the claims nor to limit the scope of the invention in any way.

[0025] Other features and advantages of the invention described in the present description will become apparent from the following figures, detailed description and claims.

[0026] Before reading the description of the present methods and compositions, it should be understood that this invention is not limited to the specific method or composition described, and thus may have variations. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, as the scope of the present invention is only limited by the appended claims.

[0027] The invention described in the present description is illustrated in the figures and in the description. However, while specific embodiments are illustrated in the figures, it is not intended to limit the invention to the specific embodiment or embodiments illustrated and/or described. Instead, the invention described herein is intended to include all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention. Thus, the figures are intended to be illustrative and not limiting.

[0028] When a range of values is presented, it is understood that each intermediate value, up to the tenth unit of the lower limit, unless the context clearly requires otherwise, between the upper and lower limits of this range, is also specifically described. Every smaller range between any value or intermediate value in the specified range and any other specified or intermediate value in that specified range is included in the invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either, none, or both of the limits are included in the smaller ranges is also included in the invention, subject to any specifically excluded limit in the specified range. Where the specified range includes one or both of the limits, ranges excluding either or both of these included limits are also included in the invention.

[0029] Unless otherwise indicated, all technical and scientific terms used in the present description have the same meaning as is generally accepted by a person skilled in the field to which this invention pertains. While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, some potential and preferred methods and materials have now been described. The content of all publications mentioned in the present description, is given in the present description as a reference for the disclosure and description of the methods and/or materials in connection with which the publications are cited. It is understood that the present description takes precedence over the description in any included publication to the extent that there is inconsistency.

[0030] As will become apparent to those skilled in the art upon reading this specification, each of the individual embodiments described and illustrated herein has discrete components and features that can be easily separated from those of any of several other embodiments or combined with them, without deviating from the scope or content of the present invention. Any of the methods mentioned can be carried out in the order in which the events are mentioned, or in any other order that is logically possible.

[0031] It should be noted that in the present specification and in the appended claims, non-specified and specific singular forms include plural reference objects unless the context clearly requires otherwise. Thus, for example, reference to "recombinant AAV virion" includes a plurality of such virions, and reference to "photoreceptor cell" includes reference to one or more photoreceptor cells and their equivalents known to those skilled in the art, and so on. In addition, it should be noted that claims may be drafted to exclude any optional element. Thus, this statement is intended to serve as a foregoing basis for the use of such exclusive terminology as "only", "only", etc., in connection with the enumeration of the elements of a claim, or for the use of a "negative" limitation.

[0032] The publications discussed in the present description are presented only according to their descriptions prior to the filing date of this application. Nothing in this specification should be construed as an admission that the present invention is not entitled to backdate such publication based on prior invention. In addition, the publication dates shown may differ from the actual publication dates, which may need to be independently verified.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The invention is better understood from the following detailed description when read in conjunction with the accompanying drawings. The patent or application file contains at least one color drawing. Copies of the publication of this patent or patent application with color drawing(s) will be provided by the patent office upon request and payment of the required fee. It should be noted that, in accordance with common practice, the various features of the figures are not to scale. In contrast, measurements of various features are arbitrarily scaled up or down for clarity. The following figures are included in the drawings.

[0034] Figure 1 shows embodiments of a guided evolution method. Step (a) shows the preparation of a viral capsid library including combinations of DNA mutagenesis methods and cap genes. Step (b) shows the packaging of viruses such that each viral particle is composed of a mutant capsid surrounding the cap gene encoding that capsid and purified. The capsid library is then subjected to in vitro or in vivo selective pressure. In this aspect of the directed evolution method, tissue or cellular material of interest is harvested to isolate AAV variants that have successfully infected that target, and the successful viruses are isolated. Stage (c) shows the enrichment of successful clones from stage 1 through repeated selection. Step (d) shows the enrichment of the cap genes selected in step 2, subjected to the steps of re-diversification and further selection to repetitively increase the fitness of the virus. Step (e) shows the variants identified as superior during vector selection steps 1 and 2 that can be commercially produced as recombinant AAV vectors and characterized by the level of transduction of different cell types and target tissues. Due to the nature of the AAV directed evolution process, the variants described herein have already been shown to be able to transduce retinal cells and deliver the genome (the genome encoding the cap gene variant) during the selection process.

[0035] Figure 2 is a diagram of fixation of retinal samples showing where the samples from which the viral genomes are amplified are collected within a wide area of the retina.

[0036] Figure 3 shows PCR amplification of viral genomes from ganglion cell layer (GCL), inner nuclear layer (INL), photoreceptor/outer nuclear layer (ONL), and retinal pigment epithelium layer (RPE) tissue from a representative selection run. Both the right eye (upper image) and the left eye (lower image) were injected with the library. Samples were taken from the inner retina (int), middle retina (mid), and outer/peripheral retina (ext). The bars within the red boxes represent successful amplification of the viral genomes.

[0037] Figures 4A-4D show the frequency of motifs in the sequencing analysis. Figure 4A shows the cycle 3 sequencing analysis. Figure 4B shows the cycle 4 sequencing analysis. Figure 4C shows the cycle 5 sequencing analysis. Figure 4D shows the cycle 6 sequencing analysis.

[0038] Figure 5 is a representative 3D model of AAV2 containing a random heptamer after amino acid 587.

[0039] Figures 6A-6W are an alignment of SEQ ID NOs: 1-11 from wild-type AAV showing amino acid localization between and among wild-type (naturally occurring) serotypes AAV1, AAV2, AAV3A, AAV3B, and AAV4-10.

[0040] Figure 7 shows Heidelberg Spectralis™ fundus fluorescence images of an African green monkey retina after 2×10 ¹¹ genomes of the AAV2 vector (gv) delivering a GFP transgene under the control of the CMV promoter (AAV2. CMV.GFP). Images were taken at baseline (A) and 14 days (B), 28 days (C) and 42 days (D) after injection.

[0041] Figure 8 shows Heidelberg Spectralis™ fundus fluorescence images of an African green monkey retina after 2×10 ¹¹ genomes of the new AAV variant LAISDQTKHA+P34A vector (gw) delivering the GFP transgene under control were introduced into the vitreous. CMV promoter (LAISDQTKHA+P34A.CMV.GFP). Images were taken at baseline (A) and 14 days (B), 28 days (C) and 42 days (D) after injection.

[0042] Figure 9 shows fundus fluorescence images obtained using cynomolgus retinas after intravitreal injection of a new AAV variant LAISDQTKHA+P34A delivering a GFP transgene under the control of the CAG promoter (LAISDQTKHA+P34A.CAG.EGFP). (A) Image of monkey retina after injection into the vitreous body of 2×10 ¹¹ gv vector, obtained 14 days (A1), 21 days (A2) and 28 days (A3) after injection. (B) Image of monkey retina after injection into the vitreous body of 1×10 ¹² gv vector, obtained 14 days (B1) and 21 days (B2) after injection.

[0043] Figures 10A-10E show the results of immunohistochemical analysis of monkey retina after intravitreal injection of 1×10 ¹² gb of the novel AAV variant LAISDQTKHA+P34A delivering the GFP transgene under the control of the CAG promoter analyzed three weeks after injection. All immunohistochemistry data are presented next to the corresponding fluorescent fundus image, where the red box indicates where approximately in the retina the analysis was performed. Figure 10A: Reliable transduction of the retinal pigment epithelium (RPE) and photoreceptors was observed using a GFP-specific antibody (red). Immunostaining of photoreceptor cones using anti-M/L-opsin antibody is shown in white. Figures 10B and 10C: Reliable transduction of photoreceptor rods and cones (Figure 10B) and RPE (Figure 10C) was observed by direct EGFP fluorescence (green) and by immunohistochemistry using GFP-specific antibody (red). Melanosomes in RPE appear black in the image. Figure 10D: Transduction of photoreceptor cones (identified by M/L-opsin, white) and retinal ganglion cells (RGCs) in and around the fovea was observed by direct EGFP fluorescence (green) and by immunohistochemistry using a GFP-specific antibody (red). The images in the middle panels represent a higher magnification (63X) of the area indicated by the white box in the left panel. Figure 10E: Transduction of retinal ganglion cells (RGC) and retinal ganglion cell layer was observed by direct EGFP fluorescence (right panels, green; lower right panel is a 63X magnification of the upper right panel); the upper left panel shows the highlight area with high illumination.

[0044] Figures 11A-11F show in vitro transduction of human retinal pigment epithelial (RPE) cells with a recombinant AAV virus containing a novel AAV capsid variant LAISDQTKHA+P34A and a GFP transgene under the control of the CAG promoter. Cells differentiated into RPE cells from a human embryonic stem cell line (Figures 11A and 11C) or from human fibroblast-derived induced pluripotent stem cells (FB-iPSC) (Figures 11B and 11D) were infected using the novel AAV variant LAISDQTKHA+P34A .CAG.GFP or wild-type control AAV2.CAG.GFP. Figures 11A and 11B: Immunofluorescence imaging of cell cultures 7 days post-infection at MOI 500 shows that the novel AAV capsid variant (left panels) transduces RPE cells better than wild-type AAV2 capsid (right panels). Figures 11C and 11D: Quantification of the percentage of GFP positive RPE cells in each culture by flow cytometry revealed that the novel AAV capsid variant provided a significant, dose-dependent improvement in the number of transduced cells compared to the wild-type AAV2 capsid, regardless of cell source. Figures 11E and 11F: Quantification of the amount of GFP in each culture by Western blotting reveals that the new AAV variant provides a significant improvement in transgene expression compared to the wild-type capsid, regardless of the cell source.

[0045] Figures 12A-12F show embodiments of anti-VEGF proteins. In FIG. 12A shows the design of aflibercept, which consists of the human Flt1 signal peptide, VEGFR1 domain 2, and VEGFR2 domain 3 fused to a human IgG1 Fc region. In FIG. 12B-C show designs of ranibizumab that include a flexible protein linker to convert a double-stranded antigen-binding fragment (Fab) to a single-stranded Fab (scFab). In FIG. 12B shows the light-heavy (LH) form, which consists of the human Ig kappa light chain signal peptide, variable light, constant light, variable heavy, and constant heavy 1 domains of ranibizumab linked via a flexible peptide. In FIG. 12C shows the heavy-light (HL) form, which is similar except that the signal peptide is derived from the human IgG heavy chain and the heavy and light domains are on opposite sides of the linker compared to the LH form. In FIG. 12D shows the design of brolucizumab that includes the variable light and variable heavy domains linked via a flexible peptide. In FIG. 12E shows an LH-Fc sc-ranibizumab design consisting of a scFab LH form fused to a human IgG1 Fc region. In FIG. 12F shows the brolucizumab-Fc design, which consists of brolucizumab fused to a human IgG1 Fc region. Red bars represent complementarity determining regions (CDRs). The genes were codon optimized for improved expression in human cells and synthesized in GeneArt or GenScript and inserted into the pAAV-CAG-SV40 pA vector between the CAG promoter and the SV40 polyA signal.

[0046] Figures 13A-B show the result of an ELISA for the detection of proteins that bind to VEGF. In FIG. 13A shows that VEGF binding activity was detected in media from HEK293T cells transfected using aflibercept (SEQ ID NO:65) or single-stranded (sc) ranibizumab (SEQ ID NO:67, 69 and 70) plasmids, but not from cells after sham transfection or transfection of a GFP-expressing vector. In FIG. 13B shows that VEGF binding activity was detected in media from HEK293T cells transfected using aflibercept (SEQ ID NO:65), sc-ranibizumab LH1 (SEQ ID NO:69) or brolucizumab (SEQ ID NO:74) expression plasmids, but not from cells transfected with a GFP expression vector. The signal for brolucizumab is very low, most likely due to poor recognition by the detection antibody. Error bars represent the standard deviation for wells transfected in quadruplicate.

[0047] Figures 14A-B are representative Western blots of media from HEK293T cells transfected with anti-VEGF constructs. In FIG. 14A shows aflibercept (SEQ ID NO:65) or sc-ranibizumab (SEQ ID NO:67, 69 and 70) expression plasmids. Clinical specimens of both Eylea and Aflibercept are reduced to distinct dimer halves, with an apparent molecular weight of 58 kDa (including glycosylation). Clinical lucentis recovers to separate 24 kD light and heavy chains, while sc-ranibizumab proteins do not separate and migrate at approximately 48 kD. There is a higher number of LH forms of the protein compared to the HL form of the protein, according to protein quantification obtained by ELISA. In FIG. 14B shows aflibercept (SEQ ID NO:65), sc-ranibizumab LH1 (SEQ ID NO:69), or brolucizumab (SEQ ID NO:74) plasmids. The signal for brolucizumab is low, most likely due to poor recognition by the detection antibody. The protein migrates with the correct molecular weight of 26 kDa. There was no signal in any of the negative control samples for sham transfection or GFP.

[0048] Figures 15A-B show the result of a competitive ELISA for the detection of free VEGF after incubation with media from HEK293T cells transfected with anti-VEGF constructs. In FIG. 15A shows aflibercept (SEQ ID NO:65) or sc-ranibizumab (SEQ ID NO:67, 69 and 70) expression plasmids. The inhibition curves for the four anti-VEGF proteins from the transfection samples were very similar to the clinical comparative Eilea and Lucentis proteins. Aflibercept and Eilea competed more strongly for VEGF than the sc-ranibizumab and lucentis variants. All three forms of sc-ranibizumab were nearly identical. In FIG. 15B shows aflibercept (SEQ ID NO:65), sc-ranibizumab LH1 (SEQ ID NO:69) or brolucizumab (SEQ ID NO:74) expression plasmids. No competition activity was present for the GFP negative control sample. Error bars represent the standard deviation for wells after transfection in duplicate.

[0049] Figure 16 shows the result of a cellular VEGF neutralization assay. The assay uses HEK293 cells expressing VEGF receptor/beta-galactosidase fusion proteins that produce active beta-galactosidase upon VEGF binding. Cells are incubated with mixtures of VEGF and various dilutions of media from HEK293T cells transfected with aflibercept (SEQ ID NO:65) or sc-ranibizumab (SEQ ID NO:69) expression plasmids. The inhibition curves of anti-VEGF proteins from transfection samples show that anti-VEGF proteins neutralize VEGF activity at levels equivalent to clinical reference proteins. Error bars represent the standard deviation for wells after duplicate analysis.

[0050] Figure 17A-B shows the result of a cellular VEGF neutralization assay performed using equal volumes of media from HEK293T cells transfected with anti-VEGF plasmids. In FIG. 17A shows plasmids expressing GFP, aflibercept (SEQ ID NO:65), sc-ranibizumab HL (SEQ ID NO:67), or sc-ranibizumab LH1 (SEQ ID NO:69). All of the anti-VEGF constructs evaluated neutralized VEGF. In FIG. 17B shows GFP, aflibercept (SEQ ID NO:65), sc-ranibizumab LH1 (SEQ ID NO:69) and brolucizumab (SEQ ID NO:74) expression plasmids. All of the anti-VEGF constructs evaluated neutralized VEGF. There was a slight matrix effect for the GFP control at all dilutions analyzed. Error bars represent the standard deviation for wells after transfection in duplicate.

[0051] Figure 18 shows the result of an ELISA for VEGF detection in media from RPE cells transfected with aflibercept (SEQ ID NO:65) or sc-ranibizumab (SEQ ID NO:67, 69, and 70) expression plasmids. A decrease in VEGF levels was observed through both aflibercept and sc-ranibizumab expression. The effect on VEGF levels of all three forms of sc-ranibizumab was similar. Error bars represent the standard deviation for wells transfected in quadruplicate.

[0052] Figure 19 shows the result of an ELISA for VEGF detection in media harvested from RPE cells six or ten days after transduction using the R100 capsid (having the amino acid sequence listed as SEQ ID NO:42) expressing aflibercept transgenes (SEQ ID NO:65), sc-ranibizumab (SEQ ID NOS:67 and 69) or brolucizumab (SEQ ID NOS:74). The endogenous level of VEGF, as shown in cells transduced with the GFP control vector, was 4500-8300 pg/ml. Transduction with all anti-VEGF vectors resulted in undetectable levels of VEGF in the media. Error bars represent the standard deviation for wells transfected in quadruplicate.

[0053] Figure 20 shows the result of an ELISA for the detection of proteins that bind to VEGF in media collected from RPE cells six or ten days after transduction using the R100 capsid (having the amino acid sequence listed as SEQ ID NO:42) expressing transgenes against VEGF. VEGF binding activity was detected in media from cells transduced expressing aflibercept (SEQ ID NO:65), sc-ranibizumab (SEQ ID NO:67 and 69), or brolucizumab (SEQ ID NO:74) vectors, but not from cells transduced expressing GFP vector. The signal for brolucizumab is very low, most likely due to poor recognition by the detection antibody. Error bars represent the standard deviation for wells transfected in quadruplicate.

[0054] Figures 21 are representative Western blots of media harvested from RPE cells six or ten days after transduction using the R100 capsid (having the amino acid sequence listed as SEQ ID NO:42) expressing anti-VEGF transgenes. Clinical samples of both Eilea and Aflibercept (SEQ ID NO:65) are reduced to distinct dimer halves, with an apparent molecular weight of 60 kDa (including glycosylation), as indicated by the black arrow. There were no bands with the correct mobility in the GFP negative control sample. Clinical lucentis is reduced to separate 24 kDa light and heavy chains, while sc-ranibizumab HL and LH (SEQ ID NOs: 67 and 69) do not separate and migrate with an apparent molecular weight of 58 kDa as indicated by the gray arrow. The signal for brolucizumab (SEQ ID NO:74) is low, most likely due to poor recognition by the detection antibody. The protein migrates at the correct molecular weight of 26 kDa, as indicated by the shaded arrow.

[0055] Figure 22 shows the result of a competitive ELISA for the detection of free VEGF after incubation with media harvested from RPE cells six or ten days after transduction using the R100 capsid (having the amino acid sequence listed as SEQ ID NO:42) expressing the transgenes against VEGF. All anti-VEGF constructs competed for VEGF. No competition activity was present for the GFP negative control sample. Levels of endogenous VEGF were higher for the lowest dilutions due to endogenous VEGF produced by RPE cells. Error bars represent the standard deviation for wells after duplicate analysis.

[0056] Figure 23 shows the result of a cellular VEGF neutralization assay performed with media harvested from RPE cells six days after transduction with an R100 capsid (having the amino acid sequence listed as SEQ ID NO:42) expressing anti-VEGF transgenes. All of the anti-VEGF constructs evaluated neutralized VEGF. No neutralization of VEGF was observed for media after GFP control transduction. Error bars represent the standard deviation for wells after duplicate analysis.

DEFINITIONS

[0057] Unless otherwise defined, all technical and scientific terms used in the present description have the same meaning as is generally accepted by a person skilled in the field to which this technology relates.

[0058] Adeno-associated virus is a non-pathogenic parvovirus consisting of a 4.7 kb single-stranded DNA genome. inside a shellless, icosahedral capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that function as the viral origin of replication and packaging signal. The ORF rep encodes four non-structural proteins that play roles in viral replication, transcriptional regulation, site-specific integration, and virion assembly. The ORF cap encodes three structural proteins (VP 1-3) that assemble to form the 60-mer viral capsid. Finally, the ORF, present as an alternative reading frame within the cap gene, forms the assembly activating protein (AAP), a viral protein that locates the AAV capsid proteins in the nucleolus and functions during the assembly of the capsid.

[0059] There are several natural (wild-type) serotypes and more than 100 known AAV variants, each of which differs in amino acid sequence, in particular within the hypervariable regions of the capsid proteins, and thus in the delivery properties of their genes. Neither AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.

[0060] For purposes of description herein, the term "AAV" is an abbreviation for adeno-associated virus, including, without limitation, the virus itself and its derivatives. Except where otherwise indicated, the term refers to all subtypes or serotypes and to both replication-competent and recombinant forms. The term "AAV" includes, without limitation, 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, equine AAV, primate AAV , non-primate AAV and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, and so on.

[0061] The genomic sequences of various AAV serotypes, as well as the sequences of natural terminal repeats (TR), Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank access 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 content of which is given in the present description as a reference for describing the nucleic acid sequences and amino acid sequences of AAV. See also, 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; Maurice et al. (2004) Virology 33:375-383; International patent publications WO 00/28061, WO 99/61601, WO 98/11244; and US Patent No. 6156303.

[0062] The sequences of natural cap (capsid) proteins associated with AAV serotypes are known in the art and include those described herein 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 "AAV capsid protein variant" or "AAV variant" refer to an AAV capsid protein containing an amino acid sequence comprising at least one modification or substitution (including deletion, insertion, point mutation, etc.) as compared to that occurring in nature or wild-type sequence of the AAV capsid protein, for example, as indicated in SEQ ID NO:1-12 in the present description. An AAV capsid protein variant may have about 80% or more identity with the amino acid sequence of the wild type capsid protein, e.g., 85% identity or more, 90% identity or more, or 95% identity or more with the amino acid sequence of the wild type capsid protein, for example, 98% or 99% identity with wild-type capsid protein. The AAV capsid protein variant may not be the wild-type capsid protein.

[0063] For purposes herein, "AAV virion" or "AAV viral particle" refers to a viral particle consisting of at least one AAV capsid protein and an encapsidated AAV polynucleotide.

[0064] For the purposes of the present description, the term "rAAV" is an abbreviation that refers to recombinant adeno-associated virus. "Recombinant", when applied to a polynucleotide, means that the polynucleotide is the product of various combinations of steps of cloning, restriction or ligation, and other methods, resulting in a construct different from the polynucleotide found in nature. A recombinant virus is a viral particle containing a recombinant polynucleotide. The terms respectively include replicas of the original polynucleotide construct and progeny of the original viral construct.

[0065] The term "rAAV vector" includes rAAV virions (i.e., rAAV viral particles) (eg, infectious rAAV virion), which by definition include a rAAV polynucleotide; and also includes rAAV-encoding polynucleotides (e.g., single-stranded rAAV-encoding polynucleotide (ss-rAAV); double-stranded rAAV-encoding polynucleotide (ds-rAAV), e.g., rAAV-encoding plasmids; and the like).

[0066] If the AAV virion contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene for delivery to a target cell, an RNAi agent or a CRISPR-based agent for delivery to a target cell, etc.) it is commonly referred to as "recombinant AAV virion (rAAV)" or "rAAV virus particle". Typically, a heterologous polynucleotide is flanked by at least one, and typically two, AAV inverted terminal repeat (ITR) sequences.

[0067] The term "packaging" refers to a series of intracellular events leading to the assembly and encapsulation of an AAV particle. The " rep " and " cap " genes of AAV refer to polynucleotide sequences encoding the replication and capsidation proteins of the adeno-associated virus. rep and cap AAV are referred to herein as AAV "packaging genes".

[0068] The term "helper virus" for AAV refers to a virus that allows AAV (eg, wild-type AAV) to undergo replication and packaging through a mammalian cell. Many such AAV helper viruses are known in the art, including adenoviruses, herpes viruses, and poxviruses such as vaccinia. Adenoviruses include a number of distinct subgroups, although adenovirus type 5 of subgroup C is the most commonly accepted. Numerous human, non-human mammalian and avian adenoviruses are known and available from depositories such as ATCC. Herpes family viruses include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

[0069] The term "helper virus function(s)" refers to the function(s) encoded in the helper virus genome that allow AAV replication and packaging (in combination with other replication and packaging requirements described herein). As described herein, "helper virus function" can be provided in a number of ways, including providing a helper virus or providing, for example, polynucleotide sequences encoding the desired function(s) in trans to the producer cell. For example, a plasmid or other expression vector containing nucleotide sequences encoding one or more adenoviral proteins is transfected into a producer cell along with the rAAV vector.

[0070] The term "infectious" virus or viral particle is one that contains a competently assembled viral capsid and is capable of delivering a polynucleotide component to a cell for which the virus species is tropic. The term does not necessarily include any ability to replicate the virus. Assays for infectious viral particles are described elsewhere in the present description and in the literature in this field. Viral infectivity can be expressed as the ratio of infectious viral particles to the total number of 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 (describing the TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973. See also examples.

[0071] The term "tropism", as used herein, refers to the preferential targeting of a virus (eg, AAV) to cells of a particular host species or to particular cell types within a host species. For example, a virus that can infect heart, lung, liver, and muscle cells has a broader (ie, increased) tropism than a virus that can only infect lung and muscle cells. Tropism may also include the dependence of the virus on specific types of host cell surface molecules. For example, some viruses can only infect cells with surface glycosaminoglycans, while other viruses can only infect cells with sialic acid (such relationships can be tested using various cell lines lacking specific classes of molecules as potential host cells for viral infection) . In some cases, the tropism of a virus describes the relative preferences of the virus. For example, the first virus may be able to infect all types of cells, but is much more successful in infecting cells with surface glycosaminoglycans. A second virus can be considered to have a similar (or identical) tropism to the first virus if the second virus also prefers the same characteristics (e.g., the second virus is also more successful at infecting cells with surface glycosaminoglycans), even if the absolute transduction efficiencies are not similar. . For example, the second virus may be more efficient than the first virus in infecting each given cell type tested, but if the relative preferences are similar (or identical), the second virus can still be considered to have similar (or identical) tropism to the first virus. In some embodiments, the tropism of the virion containing the AAV capsid protein variant in question is not altered relative to the native virion. In some embodiments, the tropism of the virion containing the AAV capsid protein variant in question is increased (ie, expanded) relative to the natural virion. In some embodiments, the tropism of the virion containing the AAV capsid protein variant in question is reduced relative to the natural virion.

[0072] The term "replication-competent" virus (e.g., replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and is also capable of replicating in an infected cell (i.e., in the presence of a helper virus or virus functions - assistant). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. Typically, rAAV vectors as described herein are replication incompetent in mammalian cells (especially human cells) due to the absence of one or more AAV packaging genes. Typically, such rAAV vectors lack the sequences of any AAV packaging genes to minimize the possibility that replication-competent AAVs are generated by recombination between the AAV packaging genes and the incoming rAAV vector. In many embodiments, rAAV vector preparations as described herein are preparations that, if at all, contain little replication-competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10 ² rAAV particles, less than about 1 rcAAV per 10 ⁴ rAAV particles, less than about 1 rcAAV per 10 rAAV particles, less than about 1 rcAAV per 10 ¹² rAAV particles, or no rcAAV).

[0073] The term "polynucleotide" refers to the polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogues. The polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be introduced before or after polymer assembly. The term polynucleotide, within the meaning of the invention, means interchangeably double- and single-stranded molecules. Unless otherwise indicated or necessary, any embodiment herein that contains a polynucleotide includes both the double-stranded form and each of two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0074] A polynucleotide or polypeptide has a certain percentage of "sequence identity" with another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when two sequences are compared. Sequence similarity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, available on the web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available from the Genetics Computing Group (GCG) software package of Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other alignment methods 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 gaps in the sequence. The Smith-Waterman algorithm is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70:173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be used to align sequences. See J. Mol. Biol. 48:443-453 (1970).

[0075] The term "gene" refers to a polynucleotide that performs a function of some sort in a cell. For example, a gene may contain an open reading frame capable of encoding a gene product. One example of a gene product is a protein transcribed and translated from a gene. Another example of a gene product is RNA, such as a functional RNA product, such as an aptamer, interfering RNA, ribosomal RNA (rRNA), transfer RNA (tRNA), non-coding RNA (ncRNA), guide RNA for nucleases, etc., which is transcribed but not broadcast.

[0076] The term "gene expression product" or "gene product" is a molecule resulting from the expression of a particular gene, as defined above. Gene expression products include, for example, a polypeptide, an aptamer, interfering RNA, messenger RNA (mRNA), rRNA, tRNA, non-coding RNA (ncRNA), and the like.

[0077] The term "siRNA-based agent"("smallinterfering" or "short interfering RNA" (or siRNA)) is an RNA duplex of nucleotides targeted to a gene of interest ("target gene"). "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule, forming a double-stranded RNA (dsRNA) region. miRNA is "targeted" to the gene, in the sense that the nucleotide sequence of the duplex portion of the miRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the siRNA duplex is less than 30 nucleotides in length. In some embodiments, the duplex may be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length. In some embodiments, the duplex is 19-25 nucleotides in length. In some embodiments, siRNA-mediated gene targeting is accomplished using DNA-guided RNA interference (ddRNAi), which is a gene knockout technique that uses DNA constructs to activate endogenous RNA interference (RNAi) pathways in an animal cell. Such DNA constructs are designed to express self-complementary double-stranded RNAs, usually short hairpin RNAs (shRNAs), which, after processing, result in the shutdown of the gene or target genes. Any RNA can be knocked out, including endogenous mRNAs or viral RNAs, by designing constructs for expression of a double-stranded RNA complementary to the desired target mRNA. Thus, a portion of the RNA duplex from the siRNA agent may form part of a short hairpin structure, referred to as shRNA. In addition to the duplex portion, the hairpin structure may comprise a loop portion located between the two sequences forming the duplex. Loop length may vary. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides long. The hairpin structure may also contain 3' or 5' overhangs. In some embodiments, the overhang is a 3' or 5' overhang 0, 1, 2, 3, 4, or 5 nucleotides long. Typically, the expression product (e.g., mRNA, polypeptide, etc.) of the target gene is reduced by an siRNA-based agent (e.g., siRNA, shRNA, etc.) containing specific double-stranded nucleotide sequences that are complementary to the fragment. at least 19-25 nucleotides in length (eg, sequences of 20-21 nucleotides) of the target gene transcript, including the 5'-untranslated (UT) region, ORF, or 3'-UT region. In some embodiments, small interfering RNAs are approximately 19-25 bp 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 US Patent Publication No. 2004/0023390 description of 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 synthetic minimal polyadenylation signal.

[0078] The term "antisense RNA" includes RNA that is complementary to a gene expression product. For example, an antisense RNA targeting a specific mRNA is an RNA-based agent (or may be a modified RNA) that is complementary to the mRNA, where hybridization of the antisense RNA to the mRNA alters the expression of the mRNA (e.g., by altering the stability of the RNA, altering the translation of the RNA etc.). Also included in "antisense RNA" are nucleic acids encoding antisense RNA.

[0079] When applied to "CRISPR/Cas9-based agents", the term "CRISPR" includes CRISPR-associated short palindromic repeat/CRISPR systems (Cas) involved in providing adaptive immunity to bacteria and archaea against viruses and plasmids using CRISPR RNA (crRNA) for targeted shutdown of penetrating nucleic acids. The Cas9 protein (or its functional equivalent and/or variant, i.e. a Cas9-like protein) naturally has DNA endonuclease activity that depends on the association of the protein with two natural or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). ). In some cases, two molecules are covalently linked to form a single molecule (also referred to as a single guide RNA ("sRNA")). Thus, a Cas9 or Cas9-like protein associates with a DNA-targeting RNA (where the term includes both a two-molecule guide RNA configuration and a single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and directs the protein to the nucleic acid sequence is the target.

[0080] If Cas9 or a Cas9-like protein retains its natural enzymatic function, it can cut the target DNA to form a double-strand break, which can lead to genome alteration (i.e., editing: deletions, insertion (when a donor polynucleotide is present), substitution, and etc.), thus changing the expression of the gene. Some variants of Cas9 (where variants are included in the term Cas9-like) are altered such that they have reduced DNA cutting activity (in some cases, they cut one strand instead of both strands of the target DNA, while, in other cases, they have greatly reduced to no DNA cutting activity). Cas9-like proteins with reduced DNA cutting activity (even without DNA cutting activity) can still be targeted to a target DNA to block RNA polymerase activity. Alternatively, a Cas9 or Cas9-like protein can be modified by fusing the VP64 transcriptional activation domain to the Cas9 protein and co-delivering the fusion protein with the MS2-P65-HSF1 helper protein and a single guide RNA containing the MS2 RNA aptamers in the tetraloop and stem-loop to formation in the cell of a synergistic activation mediator complex (Cas9-SAM), which activates transcription. Thus, enzymatically inactive Cas9-like proteins can be targeted to a specific location in a target DNA by DNA-targeting RNA to block or activate transcription of the target DNA. The term "CRISPR/Cas9 based agents", within the scope of the invention, includes all forms of CRISPR/Cas9 as described above or as known in the art.

[0081] Detailed information on CRISPR-based tools can be found, for example, in (a) Jinek et. al., Science. 2012 Aug 17;337(6096):816-21: "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity"; (b) Qi et al., Cell. Feb 28, 2013; 152(5): 1173-83: "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression" and (c) US Patent Application No. 13/842859 and PCT Application No. PCT/US13/32589; the full content of all of which is given in the present description by reference. Thus, the term "CRISPR-based agent", within the meaning of the invention, includes any agent (or a nucleic acid encoding such an agent) containing natural and/or synthetic sequences that can be used in a Cas9-based system (e.g., Cas9 or the like). Cas9 protein, any component of an RNA targeting DNA, such as crRNA-like RNA, tracr-like RNA, single guide RNA, etc., donor polynucleotide, etc.).

[0082] By "zinc finger nucleases" (ZFNs) is meant artificial DNA endonucleases obtained by fusing a zinc finger DNA binding domain with a DNA cleaving domain. ZFNs can be designed to target desired DNA sequences and this allows zinc finger nucleases to cleave unique target sequences. When introduced into a cell, ZFN can be used to edit target DNA in the cell (eg, in the cell's genome) by inducing the formation of double-strand breaks. For more information on the use of ZFN, see, for example, Asuri et al., Mol Ther. February 2012; 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. Nov 2011; 21(l1): 1638-40; the description of all of which, in relation to ZFN, is given in the present description by reference. The term "ZFN-based agent" includes a zinc finger nuclease and/or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[0083] The term “transcriptional activator-like effector nucleases” or “TALEN” agents refers to transcription activator-like effector nucleases (TALENs), which are artificial DNA endonucleases obtained by fusion of the effector DNA-binding domain of TAL ( transcription activator-like) with a DNA-cleaving domain. TALEN can be quickly designed to bind to virtually any desired DNA sequence, and once introduced into a cell, TALEN can be used to edit a target DNA in a cell (eg, in the cell's genome) by inducing double-strand breaks. For more information on the use of TALEN 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; the description of all of which, in relation to TALEN, is given in the present description by reference. The term "TALEN-based agent" includes TALEN and/or a polynucleotide containing a nucleotide sequence encoding TALEN.

[0084] The term "control element" or "control sequence" refers to a nucleotide sequence involved in molecular interactions contributing to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of a polynucleotide. Regulation may affect the frequency, rate, or specificity of the process, and may be reinforcing or inhibitory in nature. Control elements known in the art include, for example, sequences for regulating transcription such as promoters and enhancers. A promoter is a region of DNA capable, under certain conditions, of binding to an RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) of the promoter. Promoters may be ubiquitous, ie. active in a variety of cell types, eg CAG or CMV promoters; or tissue or cell specific, for example, the rho promoter, which is active in rods, or the opsin promoter, which is active in cones.

[0085] The term "operably linked" or "operably linked" refers to the immediate neighborhood of genetic elements, where the elements are in a relationship that allows them to function in the expected manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of the coding sequence. Intermediate residues between the promoter and the coding region may be present, provided that this functional relationship is maintained.

[0086] The term "expression vector" includes a vector containing a polynucleotide region that encodes a polypeptide of interest, and which is used to effect expression of the protein in the intended target cell. The expression vector may also contain control elements operably linked to the coding region to facilitate protein expression in the target. The combination of control elements and the gene or genes to which they are operably linked for expression is sometimes referred to as an "expression cassette", a large number of which are known and available in the art or can be easily constructed from components available in the art.

[0087] The term "heterologous" means derived from a molecule that is genotypically different from the rest of the molecule with which it is compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its natural coding sequence and operably linked to a coding sequence to which it is not naturally found is a heterologous promoter. Thus, for example, an rAAV comprising a heterologous nucleic acid sequence encoding a heterologous gene product is an rAAV comprising a polynucleotide not normally included in natural wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by natural wild-type AAV.

[0088] The terms "genetic change" and "genetic modification" (and their grammatical variants) are used interchangeably herein to refer to a process in which a genetic element (eg, polynucleotide) is introduced into a cell other than mitosis or meiosis. The element may be heterologous to the cell, or may be an additional copy or enhancement of an element already present in the cell. The genetic change can be accomplished, for example, by transfection using a recombinant plasmid or other polynucleotide by any method known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. The genetic change can also be carried out, for example, by transduction or infection using a DNA or RNA virus or a viral vector. Typically, the genetic element is introduced into a chromosome or minichromosome in a cell; but any change that changes the phenotype and/or genotype of a cell and its progeny is included in this term.

[0089] In relation to the modification of a cell, the term "genetically modified" or "transformed" or "transfected" or "transduced" with exogenous DNA (for example, with a recombinant virus) refers to a situation where such DNA is introduced into the cell. The presence of exogenous DNA results in a permanent or temporary genetic change. The transforming DNA may or may not be integrated (covalently linked) in the genome of the cell. A "clone" is a population of cells derived from a single cell or a common ancestor through mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

[0090] Within the scope of the invention, a cell is said to be "stably" altered, transduced, genetically modified, or transformed using a genetic sequence if the sequence is available to perform its function during long-term in vitro culture of the cell and/or over a long period of time. time in vivo . As a rule, such a cell is "hereditarily" modified (genetically modified) in the sense that a genetic change is introduced, which is also amenable to inheritance by the progeny of the modified cell.

[0091] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also include an amino acid polymer that has been modified; for example, by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling moiety. Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a gene product to a mammalian subject and compositions therefor, refer to the corresponding intact polypeptide, or any fragment or genetically modified derivative thereof, that retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivering a gene product to a mammalian subject (which may be referred to as "transgenes" for delivery to a recipient cell) include polynucleotides encoding an intact polypeptide or any fragment or genetically modified derivative having a desired biochemical function.

[0092] As used herein, an "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, protein or other substance refers to a preparation of a substance devoid of at least some of the other components that may also be present where a substance or similar substance occurs naturally or from where it was originally obtained. Thus, for example, an isolated substance can be obtained using a purification process to enrich it from a mixture of sources. Enrichment can be measured on an absolute basis, such as mass per volume of solution, or can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichment embodiments of this disclosure have increased recovery. The isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is, in some embodiments, purified, e.g., about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99% pure, or greater.

[0093] As used herein, the terms "treatment", "treatment", and the like, refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in relation to the complete or partial prevention of a disease or symptom thereof, and/or may be therapeutic in relation to the partial or complete cure of the disease and/or adverse effect attributed to the disease. "Treatment", within the meaning of the invention, refers to any treatment of a disease in a mammal, in particular a human, and includes: (a) preventing the onset of the disease (and/or the symptoms caused by the disease) in a subject who may be predisposed to the disease, or risk of getting the disease but not yet diagnosed as having it; (b) inhibition of the disease (and/or symptoms caused by the disease), i.e. stop its development; and (c) alleviating the disease (and/or symptoms caused by the disease), i. e. causing regression of the disease (and/or symptoms caused by the disease), i.e. alleviation of the disease and/or one or more symptoms of the disease. For example, contemplated compositions and methods may be directed to the treatment of retinal disease. Non-limiting methods for evaluating and treating retinal diseases include measuring retinal function and its changes, such as changes in visual acuity (eg, best corrected visual acuity [BCVA], ability to ambulate, navigate, detect and distinguish objects), changes in visual field (eg, static and kinetic perimetry of the visual field), clinical examination (eg, slit lamp examination of the anterior and posterior segment of the eye), measurement of the ability to respond to electrophysiological signals of all wavelengths in light and in the dark (eg, all forms of electroretinography (ERG) [full-format , multifocal and patterns], all forms of induced visual potential (VEP), electrooculography (EOG), assessment of color vision, dark adaptation and/or contrast sensitivity; measurement of changes in anatomy or health status using anatomical and/or photographic indicators, for example, optical coherence tomography (OCT) , photographing the fundus, laser scanning ophthalmoscopy with an adaptive optics system, fluorescence and / or autofluorescence; measurement of eyeball mobility and eye movement (e.g., nystagmus, fixation preference and stability), measurement of reported outcomes (patient-reported changes in visually mediated and non-visually mediated behaviors and activities, patient-reported outcomes [PRO] based on QoL questionnaires) , activities of daily living, and measures of neurological function (eg, functional magnetic resonance imaging (MRI)).

[0094] The terms "individual", "host", "subject" and "patient" are used interchangeably in the present description, and refer to a mammal, including, but not limited to, humans; non-human primates, including monkeys; mammalian sports animals (eg horses); mammalian farm animals (eg sheep, goats, etc.); mammalian domestic animals (dogs, cats, etc.); and rodents (eg mice, rats, etc.).

[0095] In some embodiments, the individual is a human previously naturally exposed to AAV and, as a result, is a carrier of anti-AAV antibodies (ie, AAV-neutralizing antibodies). In some embodiments, the subject is a human who has previously been injected with an AAV vector (and who may as a result carry anti-AAV antibodies) and who needs reintroduction of the vector to treat another condition or to additionally treat the same condition. Based on the positive results in clinical studies involving gene delivery via AAV, for example, in the liver, muscle and retina - all tissues affected by neutralizing antibodies against this carrier - there are many such therapeutic applications/target diseases.

[0096] The term "effective amount", within the scope of the invention, is an amount sufficient to obtain advantageous or desirable clinical results. An effective amount may be administered in one or more administrations. For the purposes of this disclosure, an effective amount of a compound (e.g., an infectious rAAV virion) is an amount sufficient to alleviate, mitigate, stabilize, reverse, prevent, slow, or delay the progression (and/or associated symptoms) of a particular disease state (e.g., disease retina). Accordingly, an effective amount of an infectious rAAV virion is an amount of an infectious rAAV virion that is capable of efficiently delivering a heterologous nucleic acid to a target cell (or cells) of an individual. Effective amounts can be determined preclinically by, for example, detection in a cell or tissue of a gene product (RNA, protein) encoded by a heterologous nucleic acid sequence using methods well known in the art, for example, RT-PCR, Western blot, ELISA , fluorescence or other reporter readings, etc. Effective amounts can be determined clinically, by, for example, detecting a change in the onset or progression of the disease using methods known in the art, such as fundus autofluorescence, fluorescein angiography, OCT, microperimetry, adaptive optics, and the like. and the like, as described herein and as known in the art.

[0097] The term "retinal cell" as used herein refers to any of the types of cells that make up the retina, such as, but not limited to, retinal ganglion (RG) cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, Mueller glial cells, microglial cells and retinal pigment epithelium (RPE). The term "photoreceptor cells" as used herein refers to, without limitation, rod or "rod" cells and cone or "cone" cells. The term "Müller cells" or "Müller glia" refers to the glial cells that support neurons in the vertebrate retina.

[0098] The term "directed evolution" refers to a method of constructing a capsid, in vitro and/or in vivo , mimics natural evolution through alternating cycles of genetic diversity and selection processes, thereby accumulating advantageous mutations, increasingly improving the function of the biomolecule. Guided evolution often involves an in vivo method, referred to as "biopanning", to select AAV variants from a library where the variants have a more efficient level of infectivity for the cell or tissue type of interest.

DETAILED DESCRIPTION

[0099] Adeno-associated viruses (AAVs) are a family of parvoviruses with a 4.7 kb single-stranded DNA genome contained within a non-enveloped capsid. The viral genome of natural AAV has 2 inverted terminal repeats (ITRs) - functioning as the viral origin of replication and packaging signal - flanking 2 primary open reading frames (ORFs): rep (coding for proteins that function in viral replication, transcriptional regulation, site-specific integration and assembly of the virion) and cap . The ORF cap encodes 3 structural proteins that assemble to form the 60-mer viral capsid. Many naturally occurring variants and serotypes of AAV have been identified, and none have been associated with human disease.

[00100] Recombinant AAV variants can be used as gene delivery vectors, where the marker or therapeutic gene of interest is inserted between the ITR instead of rep and cap . These vectors have been shown to transduce both dividing and non-dividing cells in vitro and in vivo and can provide stable transgene expression for several years in post-mitotic tissue. 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-associated viruses 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 immunological characteristics. Proc Natl Acad Sci USA 1966; 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 a genital site. Virology 1984; 134:52-63; Mayor HD, Melnick JL. Small deoxyribonucleic acid-containing viruses (picodnavirus group). Nature 1966; 210:331-2; Mori S, Wang L, Takeuchi T, Kanda T. Two novel adeno-associated viruses from cynomolgus monkey: pseudotyping characterization of capsid protein. Virology 2004; 330:375-83; Flotte TR. Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors. Gene Ther 2004; 11:805-10.

[00101]For recombinant AAV (referred to herein simply as "AAV") has shown promising results in an increasing number of clinical trials. However, there are disadvantages of gene delivery that may limit the usefulness of AAV, such as immune responses against the capsid, low tissue-specific transduction, inability to target specific cell types, and relatively low transfer capacity. In many situations, there is insufficient knowledge of the mechanisms for effectively extending rational design with the possibility of improving AAV. Alternatively, directed evolution has emerged as a way to generate new AAV variants that meet specific biomedical needs. Directed evolution methods use genetic diversity and selection techniques to allow for the accumulation of advantageous mutations, increasingly improving the function of the biomolecule. In this way, in the genescap Wild-type AAVs are diversified in several ways to generate large genomic libraries, which are packaged to produce viral particle libraries, and then selective pressure is applied to isolate new variants that can overcome gene delivery barriers. Importantly, it is not necessary to know the mechanistic basis underlying the gene delivery problem for directed evolution of function, which can thus accelerate the development of improved vectors.

[00102] As a rule, the options described in the present description, obtained using the library and/or libraries of AAV. Such AAV library or libraries are/are generated by mutagenesis of the cap gene, the gene encoding the structural proteins of the AAV capsid, by a number of directed evolution methods known and readily available to one of skill in the field of genetic engineering of viruses. See, for example, Bartel et al. Am. soc. Gene Cell Ther. 15th ^Annu . 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. Gene Ther. 18, 367-378 (2007); and Koerber, JT et al. Mol. Ther. 17, 2088-2095 (2009). Such methods, without limitation, are as follows: i) reduced fidelity PCR to introduce random point mutations into the open reading frame (ORF) cap AAV at a predetermined, modifiable frequency; ii) recombination or "DNA shuffling" of viruses in vitro or in vivo to generate random chimeras of AAV cap genes to generate a gene library with multiple AAV serotypes; iii) inserting random peptides into specific regions of the capsid by ligating the degenerate oligonucleotides into the ORF cap ; iv) specific insertions of peptide coding sequences into random locations in the AAV ORF cap by transposon mutagenesis; v) replacing the surface loops of AAV capsids with bioinformatically designed peptide sequence libraries based on the level of conservatism of each amino acid position among natural AAV serotypes and variants to obtain loop swap libraries; vi) random amino acid substitution at degeneracy positions between AAV serotypes to generate libraries of ancestral variants (Santiago-Ortiz et al., 2015); and a combination of such methods.

[00103]Using shuffling DNA chimeras are produced by combining their original properties in unique and often advantageous ways; however, some may be unpackable, which actually reduces the diversity of the library. Library diversity concentrations are achieved by peptide insertion methods such as, but not limited to, iii-iv) above. Library diversity is also concentrated by methods such as v) above, and such concentration is directed to a plurality of hypervariable regions lying on the surface of the exposed loops of the AAV capsid. While many methods generate capsid variants with mutations in only a small region of the capsid, these methods can be combined with additional mutagenesis methods to modify the complete capsid.

[00104] Once the AAV library or libraries are/are generated, the viruses are then packaged such that each AAV particle consists of a mutant capsid surrounding the cap gene encoding that capsid and purified. The library variants are then selectively pressured by in vitro and/or in vivo methods known and readily available to those skilled in the art of AAV. See, for example, Maheshri, N. et al. nature biotech. 24, 198-204 (2006); Dalkara, 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, without limitation, AAV variants can be selected using i) affinity columns, by eluting different fractions from which variants with altered binding properties are obtained; ii) primary cells - isolated from tissue samples or immortalized cell lines mimicking the behavior of cells in the human body - whereby AAV variants with increased potency and/or tissue specificity are obtained; iii) animal models - which mimic the clinical gene therapy environment - whereby AAV variants that successfully infect the target tissue are obtained; iv) human xenograft models that produce AAV variants that infect transplanted human cells; and/or combinations of methods for their selection.

[00105] Once the viruses have been selected, they can be isolated by known methods such as, but not limited to, adenovirus mediated replication, PCR amplification, next generation sequencing and cloning, and the like. The virus clones are then enriched through repeated cycles of selection methods and AAV DNA is isolated to isolate selected cap gene variants of interest. Such selected variants can be subjected to further modification or mutagenesis and thus serve as a new starting point for additional selection steps to step up the fitness of the AAV virus. However, in specific cases, successful capsids are obtained without additional mutations.

[00106] The AAV variants described herein are generated, at least in part, using an in vivo directed evolution method, such as the methods described above, involving the use of screening in the primate retina after intravitreal injection. Thus, the AAV capsid variants described herein contain one or more modifications in amino acid sequence that allow for more efficient transduction of primate retinal cells than the corresponding parent AAV capsid protein. As used herein, "corresponding parent AAV capsid protein" refers to an AAV capsid protein from the same wild-type AAV serotype or AAV serotype variant as the AAV capsid protein variant in question, but which does not contain one or more amino acid sequence modifications of the AAV capsid protein variant in question. .

[00107] In some embodiments, the subject AAV capsid protein variant comprises a heterologous peptide of about 5 amino acids to about 20 amino acids inserted via a covalent bond into the GH loop or loop IV of the AAV capsid protein compared to the corresponding parental AAV capsid protein. By "GH loop" or loop IV of the AAV capsid protein is meant the solvent-accessible portion, referred to in the art as the GH loop or loop IV, of the AAV capsid protein. For the GH loop/loop 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. Thus, for example, the insertion site may be within approximately amino acids 411-650 of the AAV VP1 capsid protein. For example, the insertion site may be within amino acids 571-612 of VP1 AAV1, amino acids 570-611 of VP1 AAV2, within amino acids 571-612 of VP1 AAV3A, within amino acids 571-612 of VP1 AAV3B, within amino acids 569-610 of VP1 of AAV4, within within amino acids 560-601 VP1 AAV5, within amino acids 571-612 VP1 AAV6, within amino acids 572-613 VP1 AAV7, within amino acids 573-614 VP1 AAV8, within amino acids 571-612 VP1 AAV9 or within amino acids 573-614 VP1 AAV10 VP1, or the corresponding amino acids of any variant thereof. One skilled in the art will know, from comparison of the amino acid sequences of capsid proteins from different AAV serotypes, where the insertion site "corresponding to AAV2 amino acids" can be found in a capsid protein from any given AAV serotype. See also in Figure 6 the alignment of SEQ ID NOs: 1-11 from wild-type AAV showing amino acid localization between and among the wild-type (naturally occurring) serotypes AAV1, AAV2, AAV3A, AAV3B and AAV4-10.

[00108] In specific embodiments, the insertion site is a single insertion site between two adjacent amino acids located between amino acids 570-614 VP1 from any wild-type AAV serotype or AAV variant, e.g., the insertion site is between two adjacent amino acids located within VP1 amino acids 570-610, amino acids 580-600, amino acids 570-575, amino acids 575-580, amino acids 580-585, amino acids 585-590, amino acids 590-600, or amino acids 600-614 from any AAV serotype or variant. For example, the insertion site may be between amino acids 580 and 581, amino acids 581 and 582, amino acids 583 and 584, amino acids 584 and 585, amino acids 585 and 586, amino acids 586 and 587, amino acids 587 and 588, amino acids 588 and 589, or amino acids 589 and 590. The insertion site can be between amino acids 575 and 576, amino acids 576 and 577, amino acids 577 and 578, amino acids 578 and 579, or amino acids 579 and 580. The insertion site can be between amino acids 590 and 591, amino acids 591 and 592, amino acids 592 and 593, amino acids 593 and 594, amino acids 594 and 595, amino acids 595 and 596, amino acids 596 and 597, amino acids 597 and 598, amino acids 598 and 599, or amino acids 599 and 600. For example, the insertion site may be between amino acids 587 and 588 of AAV2 , between amino acids 590 and 591 AAV1, between amino acids 588 and 589 AAV3A, between amino acids 588 and 589 AAV3B, between amino acids 584 and 585 AAV4, between 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.

[00109] In some embodiments, the peptide insert described herein is 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids , 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids or 20 amino acids. In another embodiment, the peptide insert described herein contains 1 to 4 spacer amino acids at the amino-terminus (N-terminus) and/or carboxy-terminus (C-terminus) of any of the peptide inserts described herein. . Exemplary spacer amino acids include, without limitation, leucine (L), alanine (A), glycine (G), serine (S), threonine (T), and proline (P). In specific embodiments, the peptide insert contains 2 spacer amino acids at the N-terminus and 2 spacer amino acids at the C-terminus. In other embodiments, the peptide insert contains 2 spacer amino acids at the N-terminus and 1 spacer amino acid at the C-terminus.

[00110] The peptide inserts described herein have not been previously described and/or inserted into an AAV capsid. Without intending to be bound by theory, the presence of any of the described peptide inserts may act to decrease the affinity of the capsid variant for heparin sulfate, which likely reduces binding to the extracellular matrix upstream of the primate retina. In addition, the peptide insert motifs described herein can provide improved transduction of primate retinal cells by adding a cell surface receptor binding domain.

[00111] In some preferred embodiments, the peptide insert contains an amino acid sequence according to any of the formulas below.

[00112] In some aspects, the peptide insert may be a 7-10 amino acid long peptide of Formula 1a:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ ,

where each of Y ₁ -Y ₃ , if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

X ₁ selected from Gln, Asn, His, Ile and Ala

X ₂ selected from Ala, Gln, Asp, Ser, Lys and Pro

X ₃ selected from Asp, Ile, Thr and Asn

X ₄ is selected from Thr, Ser, Tyr, Gln, Glu and Ala

X ₅ selected from Thr, Lys and Asn

X ₆ selected from Lys, Asn and Glu

X ₇ is selected from Asn, Thr, Ile, His, Asp and Ala.

[00113] In specific embodiments, the peptide insert of Formula 1a comprises an amino acid sequence selected from 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).

[00114] In other aspects, the peptide insert may be a 7-10 amino acid long peptide of formula 1b:

Y ₁ Y ₂ X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y ₃ ,

where each of Y ₁ -Y ₃ , if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

X ₁ selected from Gln, Asn, His and Ile

X ₂ selected from Ala, Gln, Asp and Ser

X ₃ selected from Asp and Ile

X ₄ selected from Thr, Tyr and Gln

X ₅ selected from Thr and Lys

X ₆ selected from Lys and Asn

X ₇ selected from Asn, Thr, Ile and His

[00115] In specific embodiments, the Formula 1b peptide insert comprises an amino acid sequence selected from QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), NQDYTKT (SEQ NO:16), HDITKNI (SEQ ID NO:16), 17) and HQDTTKN (SEQ ID NO:19).

[00116] In other aspects, the peptide insert may be a 7-10 amino acid long peptide of formula 1c

Y ₁ Y ₂ X ₁ X ₂ AspX ₃ ThrLysX ₄ Y ₃ ,

where each of Y ₁ -Y ₃ , if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

X ₁ selected from Gln, Asn, His and Ile

X ₂ selected from Ala, Gln and Ser

X ₃ selected from Thr, Tyr and Gln

X ₄ selected from Asn, Thr and His

[00117] In specific embodiments, the Formula 1c peptide insert comprises an amino acid sequence selected from QADTTKN (SEQ ID NO:13), ISDQTKH (SEQ ID NO:14), NQDYTKT (SEQ NO:16), and HQDTTKN (SEQ ID NO:16) 19).

[00118] In other aspects, the peptide insert may be a 7-10 amino acid long peptide of formula 1d:

Y ₁ Y ₂ X ₁ X ₂ AspX ₃ ThrThrX ₄ Y ₃ ,

where each of Y ₁ -Y ₃ , if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

X ₁ selected from Gln and Ile

X ₂ selected from Ala and Ser

X ₃ selected from Thr and Gln

X ₄ selected from Asn and His

[00119] In specific embodiments, the Formula 1d peptide insert contains an amino acid sequence selected from QADTTKN (SEQ ID NO:13) and ISDQTKH (SEQ ID NO:14).

[00120] In other aspects, the peptide insert may be a 7-11 amino acid long peptide of Formula Ie

Y ₁ Y ₂ X ₁ X ₂ AsnX ₃ AsnGluX ₄ Y ₃ ,

where each of Y ₁ -Y ₃ , if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

X ₁ selected from Gln and Ile

X ₂ selected from Ala and Ser

X ₃ selected from Glu and Ala

X ₄ selected from Asn and His

[00121] In other embodiments, the implementation, the peptide insert of formula 1e contains an amino acid sequence selected from ISNENEH (SEQ ID NO:21) and QANANEN (SEQ ID NO:22).

[00122] In another embodiment, the peptide insert may be a 7-11 amino acid long peptide of formula IIa:

Y ₁ Y ₂ X ₁ X ₂ DX ₃ TKX ₄ Y ₃ ,

where each of Y ₁ -Y ₃ , if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

X ₁ is selected from Q, N, A, H and I;

X ₂ is selected from Q, A, P and S;

X ₃ is selected from T, Y, S and Q; and

X ₄ is selected from T, N, A and H.

[00123] In a further embodiment of a peptide insert from the amino acid sequence of formula X ₁ X ₂ DX ₃ TKX ₄ , the peptide insert 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).

[00124] In some such embodiments, the peptide insert may be a 7-11 amino acid long peptide of Formula IIb:

Y ₁ Y ₂ X ₁ X ₂ DX ₃ TKX ₄ Y ₃ ,

[00125] where each of Y ₁ -Y ₃ , if present, is independently selected from Ala, Leu, Gly, Ser, Thr, Pro

X ₁ is selected from N, A and H;

X ₂ is selected from Q, P and S;

X ₃ is selected from T, Y and S; and

X ₄ is selected from T, N and A.

[00126] In the following embodiment of a peptide insert from the amino acid sequence of formula X ₁ X ₂ DX ₃ TKX ₄ , the peptide insert is selected from the group consisting of ASDSTKA, NQDYTKT (SEQ NO:16), HQDTTKN (SEQ ID NO:19), and HPDTTKN (SEQ ID NO:18).

[00127] In other embodiments, the peptide insert comprises an amino acid sequence selected from KDRAPST (SEQ ID NO:26), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25), and GKSKVID (SEQ ID NO:23 ).

[00128] In some embodiments, the peptide insert comprises an amino acid sequence selected from 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), TNRTSPD (SEQ ID NO:24), PNSTHGS (SEQ ID NO:25) and KDRAPST (SEQ ID NO:26).

[00129] In other preferred embodiments, the peptide insert has 1 to 3 spacer amino acids (Y ₁ -Y ₃ ) at the amino and/or carboxy terminus of an amino acid sequence selected from 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) and KDRAPST (SEQ ID NO:26). In specific such embodiments, the peptide insert 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).

[00130] In some embodiments, the subject AAV capsid protein variant does not include any other amino acid sequence modifications other than a peptide insert of about 5 amino acids to about 20 amino acids in the GH loop or loop IV. For example, in some embodiments, the subject AAV capsid protein variant comprises 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), 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), and the AAV capsid variant does not include any other amino acids other substitutions, insertions or deletions (i.e. the AAV capsid protein variant contains the indicated insert and is otherwise identical to the corresponding AAV capsid protein). In other words, the AAV capsid protein variant containing said insert is otherwise identical to the original AAV capsid protein into which the peptide is inserted. As another example, the contemplated AAV capsid protein variant contains a peptide insert having an amino acid sequence selected from 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: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:37 :38), LATNRTSPDA (SEQ ID NO:39), LAPNSTHGSA (SEQ ID NO:40), and LAKDRAPSTA (SEQ ID NO:41), where the peptide insert is located between amino acids 587 and 588 of VP1 from the AAV2 capsid, or corresponding between amino acids 588 and 589 of VP1 from AAV1, AAV3A, AAV3B, AAV6, or AAV9, between amino acids 586 and 587 of VP1 from AAV4, between amino acids 577 and 578 of VP1 from AAV5, between amino acids 589 and 590 VP1 from AAV7, between amino acids 590-591 VP1 from AAV8 or AAV10, etc. where the protein sequence of the AAV capsid variant is otherwise identical to the sequence of the corresponding parental AAV capsid protein, eg, any of SEQ ID NOs: 1-12.

[00131] In other embodiments, the AAV capsid protein variant in question, in addition to containing a peptide insert, e.g., as described herein or as known in the art, in the GH loop, contains from about 1 to about 100 amino acid substitutions or deletions eg 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 25-50, about 50-100 amino acid substitutions or deletions, compared to the original AAV capsid protein. Thus, in some embodiments, the capsid protein variant in question comprises an amino acid sequence having a sequence identity of 85% or more, 90% or more, 95% or more, or 98% or more, for example, or 99% identity with the corresponding parental capsid. AAV, for example, a wild-type capsid protein as indicated in SEQ ID NOS: 1-12.

[00132] In the following embodiment, one or more amino acid substitutions are at the position of amino acid residue(s) 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 VP1 AAV2 capsid protein, as numbered before insertion peptide, or corresponding amino acid residue(s) of another AAV capsid protein. In some such embodiments, 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 the AAV2 VP1 capsid protein, or insertion peptide (residues) of amino acids of another AAV capsid protein.

[00133] In a preferred embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert contains an amino acid sequence selected from ISDQTKH (SEQ ID NO:14), LGISDQTKHA (SEQ ID NO:29) and LAISDQTKHA (SEQ ID NO:28), and b) one or more of the following amino acid substitutions, compared to the AAV2 amino acid sequence (SEQ ID NO:2) or a corresponding substitution in another parent AAV serotype (i.e. e. other than AAV2), where the substituted amino acid(s) do not occur naturally at the respective positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E and combinations thereof. In some embodiments, 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 insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype.

[00134] In a particularly preferred embodiment, the AAV capsid variant comprises a peptide insert containing the amino acid sequence ISDQTKH (SEQ ID NO:14), or containing, primarily 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 from AAV2 or the corresponding amino acids from another AAV capsid, and additionally contains an amino acid substitution P34A at residue position 34, relative to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or the corresponding residue from another AAV capsid. An AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or greater, full-length amino acid sequence identity. the amino acid sequence specified in SEQ ID NO:2, or with the corresponding original AAV capsid. In a particularly preferred embodiment, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity to the following amino acid sequence:

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPK A LAISDQTKHA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SERPIGTRYLTRNL (SE:2Q) ID).

[00135] In another particularly preferred embodiment, the AAV capsid variant comprises a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO:14), or comprising, primarily 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 protein, or at the corresponding position in the capsid protein of another AAV serotype, and contains the amino acid substitution N312K, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or the corresponding substitution in the other parent AAV serotype, and optionally further comprising the amino acid substitutions N449D, D472N, N551S, I698V, and/or L735Q, compared to the amino acid sequence of the AAV2 capsid, or the corresponding substitutions in the different parent AAV serotype. In another particularly preferred embodiment, the AAV capsid variant comprises a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO:14), or comprising, essentially consisting of, or consisting primarily 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 or at the corresponding position in the capsid protein of another AAV serotype, and contains the amino acid substitutions N312K, N449D, D472N, N551S, I698V and L735Q, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or substitution of corresponding residues in another original AAV serotype. An AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater, amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO. :2. In a particularly preferred embodiment, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity to the following amino acid sequence:

MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRL K FKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT D TPSGTTTQSRLQFSQAGASDIR N QSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKT S VDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN LAISDQTKHA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPE V QYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRN Q (SEQ ID NO:43).

[00136] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert located between amino acids 588 and 589 of VP1 from AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 from AAV4, amino acids 577 and 578 from AAV5, amino acids 589 and 590 from AAV7, or amino acids 590-591 from AAV8 or AAV10, where the peptide insert contains an amino acid sequence selected from ISDQTKH (SEQ ID NO:14), LGISDQTKHA (SEQ ID NO:29) and LAISDQTKHA ( SEQ ID NO:28), and b) a valine to isoleucine substitution at amino acid position 709 of AAV3A or AAV3B, an alanine to isoleucine substitution at position 709 of AAV1 or AAV6, an asparagine to isoleucine substitution at amino acid position 707 of AAV4 or amino acid 709 of AAV9 , or a threonine to isoleucine substitution at amino acid position 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid position 697 of AAV5, and optionally otherwise identical mu any of SEQ ID NOs: 1 and 3-12. In preferred embodiments, the capsid protein variant comprises a) a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO:14) or comprising, primarily consisting of, or consisting primarily 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) the amino acid substitution of valine for isoleucine at amino acid position 708, compared to the amino acid sequence of AAV2, where the capsid protein variant contains 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[00137] In another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO:14), or comprising, primarily consisting of, or consisting primarily 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) the amino acid substitution of valine for isoleucine at amino acid position 708, compared to the amino acid sequence of AAV2, and is otherwise identical to the amino acid sequence of SEQ ID NO :2.

[00138] In another embodiment, the capsid protein variant comprises a) a peptide insert comprising the amino acid sequence ISDQTKH (SEQ ID NO:14), or comprising, primarily 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 is otherwise identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LGISDQTKHA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:44).

[00139] In a preferred embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert contains an amino acid sequence 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 AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) do not occur naturally at the respective 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, V719M, S721L, L735Q and combinations thereof, preferably selected from S109T, P250S, A524T, A5983E, I6983E, and /or V719M. The peptide insert site is preferably located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. In a particularly preferred embodiment, the AAV capsid variant comprises a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO:13) or comprising, essentially consisting of, or consisting primarily of the amino acid sequence LAQADTTKNA (SEQ ID NO:27) between amino acids 587 and 588 an AAV2 capsid, or at the corresponding position in the capsid protein of another AAV serotype, and contains the I698V amino acid substitution, compared to the AAV2 amino acid sequence, or the corresponding substitution in the other parent AAV serotype, where the replaced amino acid(s) do not naturally occur at the appropriate position. An AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater, amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO. :2. In some embodiments, the corresponding amino acid substitution is I699V amino acid substitution versus AAV3A, AAV3B or AAV9 capsid amino acid sequence I687V substitution versus AAV5 capsid amino acid substitution I700V substitution versus AAV7 amino acid sequence I701V substitution , compared to the amino acid sequence of AAV8 or AAV10. In a particularly preferred embodiment, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity to the following amino acid sequence:

( SEQ ID NO :45).

[00140] In other preferred embodiments, the AAV capsid variant comprises a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO:13), or comprising, primarily consisting of or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO:27), between amino acids 587 and 588 of the AAV2 capsid or at the corresponding position in the capsid protein of another AAV serotype, and contains the amino acid substitution V719M and optionally the substitution V708I, compared to the amino acid sequence of AAV2, or the corresponding substitution in the other parent AAV serotype, where the amino acid(s) replaced do not occur in nature in the corresponding position.

[00141] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert located between amino acids 588 and 589 of VP1 from AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 from AAV4, amino acids 577 and 578 from AAV5, amino acids 589 and 590 from AAV7, or amino acids 590-591 from AAV8 or AAV10, wherein the peptide insert contains an amino acid sequence selected from QADTTKN (SEQ ID NO:13) and LAQADTTKNA (SEQ ID NO:27), and b) substitution of valine for isoleucine at amino acid position 709 of AAV3A or AAV3B, substitution of alanine for isoleucine at position 709 of AAV1 or AAV6, substitution of asparagine for isoleucine at amino acid position 707 of AAV4 or amino acid 709 of AAV9, or substitution of threonine for isoleucine at position amino acid 710 from AAV7 or amino acid 711 from AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid position 697 from AAV5. In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert located between amino acids 588 and 589 of VP1 from AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 from AAV4, amino acids 577 and 578 from AAV5, amino acids 589 and 590 from AAV7, or amino acids 590-591 from AAV8 or AAV10, where the peptide insert contains an amino acid sequence selected from QADTTKN (SEQ ID NO:13) and LAQADTTKNA (SEQ ID NO:27), and b) amino acid a serine to threonine 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 preferred embodiments, the AAV capsid variant comprises a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO:13) or comprising, primarily consisting of or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO:27), between amino acids 587 and 588 AAV2 capsid, and contains a serine to threonine substitution at amino acid position 109 (S109T) or a valine to isoleucine amino acid substitution at amino acid position 708 (V708I), compared to the AAV2 amino acid sequence, where the capsid protein variant contains from 1 to 5, from 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 greater amino acid sequence identity over the entire length of the amino acid the sequence indicated in SEQ ID NO:2. In other preferred embodiments, the AAV capsid variant comprises a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO:13), or comprising, primarily consisting of or consisting of the amino acid sequence LAQADTTKNA (SEQ ID NO:27), between amino acids 587 and 588 capsid of AAV2 or at the corresponding position in the capsid protein of another AAV serotype, and contains a serine to threonine substitution at amino acid position 109 and a valine to isoleucine amino acid substitution at amino acid position 708, compared to the amino acid sequence of AAV2.

[00142] In another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO:13), or comprising, essentially 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 b) at least one amino acid substitution, wherein the amino acid sequence of the capsid variant does not contain the amino acid substitution of valine to isoleucine at amino acid position 708, compared to the amino acid sequence of AAV2, and does not contain the substitution of serine to threonine at amino acid position 109, compared to the amino acid sequence of AAV2.

[00143] In another embodiment, the capsid protein variant comprises a) a peptide insert comprising the amino acid sequence QADTTKN (SEQ ID NO:13), or comprising, essentially 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 is otherwise identical to the amino acid sequence of SEQ ID NO:2.

[00144] In another preferred embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence selected from HDITKNI (SEQ ID NO:17), IAHDITKNIA ( SEQ ID NO:60) and LAHDITKNIA (SEQ ID NO:32), and b) one or more of the following amino acid substitutions, compared to the AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in another parent AAV serotype ( i.e. other than AAV2) where the substituted amino acid(s) do not occur naturally at the respective positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y , G226E, G236V, I240T, P250S, N312K, P363L, D368H, R389S, N449D, T456K, S463Y, D472N, R484C, A524T, P535S, N551S, A593E, I698V, V708I, V719M, S721L and their combinations In some embodiments, the AAV capsid protein contains one or more amino acid substitutions selected from S109T, R389S, A593E, and/or V708I. Preferably, the peptide insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. In one preferred embodiment, the AAV capsid variant comprises a peptide insert containing the amino acid sequence HDITKNI (SEQ ID NO:17) or containing, primarily 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 contains the amino acid substitution S109T, compared to the amino acid sequence of the AAV2 capsid, or a corresponding substitution in another parental AAV serotype. The AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO: 2.

[00145] In another embodiment, the capsid variant comprises a) a peptide insert comprising the amino acid sequence HDITKNI (SEQ ID NO:17) or comprising, primarily 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 is otherwise identical to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LAHDITKNIA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:46).

[00146] In other embodiments, the capsid variant comprises a) a peptide insert comprising, primarily consisting of, or consisting primarily 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 The capsid variant does not contain the amino acid substitution of valine for isoleucine at amino acid position 708, compared to the amino acid sequence of AAV2. In other embodiments, the capsid variant comprises a) a peptide insert comprising the amino acid sequence DITKNIA (SEQ ID NO:61), or comprising, primarily consisting of, or consisting primarily 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 AAV2 amino acid sequence. In other embodiments, the variant capsid comprises a) a peptide insert comprising, essentially consisting of, or consisting of 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, where the amino acid the capsid variant sequence contains the amino acid substitution of valine for isoleucine at amino acid position 708, compared to the AAV2 amino acid sequence.

[00147] In another preferred embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert contains an amino acid sequence selected from NQDYTKT (SEQ ID NO:16) and LANQDYTKTA ( SEQ ID NO:31), and b) one or more of the following amino acid substitutions, compared to the AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in a different parental AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) do not occur naturally at the respective positions: 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 contains one or more amino acid substitutions selected from S109T, S109T+S463Y, D368H, and V708I. Preferably, the peptide insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. In one preferred embodiment, the AAV capsid variant comprises a peptide insert containing the amino acid sequence NQDYTKT (SEQ ID NO:16), or containing, primarily consisting of or consisting of the amino acid sequence LANQDYTKTA (SEQ ID NO:31), between amino acids 587 and 588 capsid of AAV2 and contains the amino acid substitution V708I, compared to the amino acid sequence of the AAV2 capsid or the corresponding substitution in another parental AAV serotype. The AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO: 2. In another embodiment, the capsid variant comprises a) a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO:16) or comprising, primarily consisting of, or consisting primarily of the amino acid sequence LANQDYTKTA (SEQ ID NO:31) between amino acids 587 and 588 the AAV2 capsid, and is otherwise identical to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LANQDYTKTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:47).

[00148] In other embodiments, the capsid variant comprises a) a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO:16), or comprising, primarily 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) an S109T amino acid substitution compared to the sequence from SEQ ID NO:2, and optionally an S463Y amino acid substitution, wherein the capsid variant has at least about 85%, at least about 90%, of at least about 95%, at least about 98% identity with the entire length of the amino acid sequence specified in SEQ ID NO:2. In related embodiments, the capsid variant comprises a) a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO:16) or comprising, primarily consisting of, or consisting primarily of the amino acid sequence LANQDYTKTA (SEQ ID NO:31) between amino acids 587 and 588 the AAV2 capsid, and b) the S109T amino acid substitution compared to the amino acid sequence of SEQ ID NO:2, and is otherwise identical to the amino acid sequence of SEQ ID NO:2.

[00149] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert located between amino acids 588 and 589 of VP1 from AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 from AAV4, amino acids 577 and 578 from AAV5, amino acids 589 and 590 from AAV7, or amino acids 590-591 from AAV8 or AAV10, wherein the peptide insert contains an amino acid sequence selected from NQDYTKT (SEQ ID NO:16) and LANQDYTKTA (SEQ ID NO:31), and b) an asparagine to lysine amino acid change 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 a serine to lysine change at position 312 of AAV3A or AAV3B, or at position 315 of AAV8 or AAV10, or an arginine to lysine substitution at position 303 of AAV4 or AAV5, or at position 314 of AAV7. In another embodiment, the capsid variant comprises a) a peptide insert comprising the amino acid sequence NQDYTKT (SEQ ID NO:16) or comprising, primarily consisting of, or consisting primarily of the amino acid sequence LANQDYTKTA (SEQ ID NO:31) between amino acids 587 and 588 an AAV2 capsid, and b) an N312K amino acid substitution, wherein the capsid protein variant contains 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions.

[00150] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence 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 AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) do not occur naturally at the respective 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 insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. In one preferred embodiment, the AAV capsid variant contains a peptide insert containing the amino acid sequence PNSTHGS (SEQ ID NO:25), or containing, primarily consisting of or consisting of the amino acid sequence LAPNSTHGSA (SEQ ID NO:40), between amino acids 587 and 588 capsid of AAV2, and contains the amino acid substitution V708I, compared to the amino acid sequence of the AAV2 capsid, or a corresponding substitution in another original AAV serotype. The AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO: 2. In another embodiment, the capsid variant comprises a) a peptide insert comprising the amino acid sequence PNSTHGS (SEQ ID NO:25), or comprising, primarily consisting of or consisting of the amino acid sequence LAPNSTHGSA (SEQ ID NO:40), between amino acids 587 and 588 capsid of AAV2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LAPNSTHGSA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSERPIGTRYLTRNL (SEQ IDNO:48).

[00151] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert contains an amino acid sequence selected from NKTTNKDA (SEQ ID NO:62) and LANKTTNKDA (SEQ ID NO:35), and b) one or more of the following amino acid substitutions, compared to the AAV2 amino acid sequence (SEQ ID NO:2) or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) not found in nature at the appropriate 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 insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. In one preferred embodiment, the AAV capsid variant comprises a peptide insert containing the amino acid sequence NKTTNKDA (SEQ ID NO:62), or containing, primarily consisting of or consisting of the amino acid sequence LANKTTNKDA (SEQ ID NO:35), between amino acids 587 and 588 capsid of AAV2 and contains the amino acid substitution N449D, compared to the amino acid sequence of the AAV2 capsid, or the corresponding substitution in another original AAV serotype. The AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO: 2. In another embodiment, the capsid variant comprises a) a peptide insert comprising the amino acid sequence NKTTNKDA (SEQ ID NO:62), or comprising, primarily consisting of or consisting of the amino acid sequence LANKTTNKDA (SEQ ID NO:35), between amino acids 587 and 588 capsid of AAV2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LANKTTNKDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:49).

[00152] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence selected from TNRTSPD (SEQ ID NO:24) and LATNRTSPDA (SEQ ID NO:39), and b) one or more of the following amino acid substitutions, compared to the AAV2 amino acid sequence (SEQ ID NO:2) or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) not found in nature at the appropriate 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, V719M, S721L, L735Q and combinations thereof. Preferably, the peptide insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. In a related embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert located between amino acids 588 and 589 of VP1 from AAV1, AAV3A, AAV3B, AAV6 or AAV9, amino acids 586 and 587 from AAV4, amino acids 577 and 578 from AAV5, amino acids 589 and 590 from AAV7, or amino acids 590-591 from AAV8 or AAV10, where the peptide insert contains an amino acid sequence selected from TNRTSPD (SEQ ID NO:24) and LATNRTSPDA (SEQ ID NO:39), and b) substitution valine to isoleucine at amino acid 709 of AAV3A or AAV3B, alanine to isoleucine at 709 of AAV1 or AAV6, asparagine to isoleucine at amino acid 707 of AAV4 or amino acid 709 of AAV9, or a threonine to isoleucine at amino acid 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a glutamine to isoleucine substitution at amino acid position 697 of AAV5. In other embodiments, the capsid protein variant comprises a) a peptide insert comprising, essentially 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 b) the amino acid substitution of valine for isoleucine at position amino acids 708, compared to the AAV2 amino acid sequence, where the capsid protein variant contains 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions. In another embodiment, the capsid protein variant comprises a) a peptide insert containing the amino acid sequence TNRTSPD (SEQ ID NO:24) between amino acids 587 and 588 of the AAV2 capsid, and AAV2 sequence. The AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO: 2.

[00153] In another embodiment, the variant capsid protein comprises a) a peptide insert comprising the amino acid sequence TNRTSPD (SEQ ID NO:24), or comprising, essentially 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 is otherwise identical to the amino acid sequence of SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LATNRTSPDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSERPIGTRYLTRNL (SEQ ID NO:50).

[00154] In another embodiment, the present invention provides an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence 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 AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) do not occur naturally at the respective positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K and combinations thereof. Preferably, the peptide insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. The AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO: 2. In some embodiments, the AAV capsid variant comprises a peptide insert located between amino acids 587 and 588 of the AAV2 capsid, containing the amino acid sequence GKSKVID (SEQ ID NO:23), or containing, primarily consisting of, or consisting of the amino acid sequence LAGKSKVIDA (SEQ ID NO: :38), and is otherwise identical to the amino acid sequence of SEQ ID NO:2. In other embodiments, the AAV capsid variant comprises a) a peptide insert comprising, essentially consisting of, or consisting of the amino acid sequence LAGKSKVIDA (SEQ ID NO:38) between amino acids 587 and 588 of the AAV2 capsid and contains at least one amino acid substitution.

[00155] In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identical to the following amino acid sequence:

LAGKSKVIDA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:51).

[00156] In another embodiment, the present invention provides an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence 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 AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) do not occur naturally at the respective 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 insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. An AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2 . In another embodiment, the variant capsid contains a peptide insert containing the amino acid sequence ASDSTKA (SEQ ID NO:15), or containing, mainly consisting of or consisting of the amino acid sequence LAASDSTKAA (SEQ ID NO:30), between amino acids 587 and 588 of the capsid AAV2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LAASDSTKAA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:52).

[00157] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence 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 AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) do not occur naturally at the respective positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K and combinations thereof. Preferably, the peptide insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. An AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO:2 . In another embodiment, the variant capsid comprises a peptide insert comprising the amino acid sequence KDRAPST (SEQ ID NO:26), or comprising, primarily 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 is otherwise identical to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LAKDRAPSTA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSERPIGTRYLTRNL (SEQ ID NO:53).

[00158] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence 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 AAV2 amino acid sequence (SEQ ID NO:2) or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) not naturally occurring at one or more of the respective 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 insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. The AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO: 2. In another embodiment, the variant capsid comprises a peptide insert comprising the amino acid sequence HQDTTKN (SEQ ID NO:19) or comprising, essentially consisting of or consisting 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 set forth in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LAHQDTTKNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:54).

[00159] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert contains an amino acid sequence 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 AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) does not naturally occur at one or more of the corresponding positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T and combinations thereof. Preferably, the peptide insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. An AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2 . In another embodiment, the variant capsid contains a peptide insert containing the amino acid sequence ISNENEH (SEQ ID NO:21), or containing, mainly consisting of or consisting of the amino acid sequence LPISNENEHA (SEQ ID NO:36), between amino acids 587 and 588 of the capsid AAV2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

( SEQ ID NO:55).

[00160] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence 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 AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) do not occur naturally at the respective positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K and combinations thereof. Preferably, the peptide insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. An AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2 . In another embodiment, the variant capsid contains a peptide insert containing the amino acid sequence QANANEN (SEQ ID NO:22), or containing, mainly consisting of or consisting of the amino acid sequence LPQANANENA (SEQ ID NO:37), between amino acids 587 and 588 of the capsid AAV2, and is otherwise identical to the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

LPQANANENA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:56).

[00161] In another embodiment, the present invention relates to an AAV capsid protein variant comprising a) a peptide insert in the GH-loop of the capsid protein, wherein the peptide insert comprises an amino acid sequence 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 AAV2 amino acid sequence (SEQ ID NO:2), or a corresponding substitution in another parent AAV serotype (i.e., other than AAV2), where the substituted amino acid(s) do not occur naturally at the respective positions: M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K and combinations thereof. Preferably, the peptide insert site is located between amino acids 587 and 588 of the AAV2 capsid, or at the appropriate position in the capsid protein of another AAV serotype. An AAV capsid variant may have at least about 85%, at least about 90%, at least about 95%, at least about 98%, or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO:2 . In another embodiment, the variant capsid comprises a peptide insert comprising the amino acid sequence HPDTTKN (SEQ ID NO:18), or comprising, essentially 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 is otherwise identical to the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the AAV capsid variant has an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98% sequence identity, or 100% identity with the following amino acid sequences:

[00162]

LAHPDTTKNA RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO:57).

[00163] In several aspects, the present invention relates to an AAV capsid protein variant comprising one or more amino acid substitutions, as compared to the corresponding parent AAV capsid protein, wherein the capsid protein variant, when present in the AAV virion, confers increased infectivity to retinal cells along compared with the infectivity for retinal cells of the AAV virion containing the corresponding parental AAV capsid protein.

[00164] In some embodiments, the AAV capsid protein variant comprises a P34A amino acid substitution compared to an AAV2 capsid amino acid sequence (SEQ ID NO:2) or a P33A amino acid substitution compared to an AAV5 capsid amino acid sequence (SEQ ID NO:6) . In some preferred embodiments, the capsid protein variant 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 greater, 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 contains the amino acid substitution P34A or P33A, compared to the amino acid sequence of the AAV2 or AAV5 capsid, respectively. In some preferred embodiments, the capsid protein variant comprises an amino acid sequence containing the P34A amino acid substitution compared to the amino acid sequence set forth in SEQ ID NO:2 and is otherwise identical to the amino acid sequence set forth in SEQ ID NO:2. In related embodiments, the capsid protein variant contains the amino acid substitution P34A, compared to the amino acid sequence of SEQ ID NO:2, where the capsid protein variant contains 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions, compared to the amino acid the AAV2 capsid protein sequence shown in SEQ ID NO:2.

[00165] In other embodiments, the AAV capsid protein variant contains an amino acid substitution at amino acid position 164, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position of another parental AAV serotype (i.e., other than AAV2) where the amino acid substituted does not occur naturally at the corresponding position. In some preferred embodiments, the capsid protein variant 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 greater amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO 2 and contains an amino acid substitution at amino acid position 164, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the rAAV virion comprises an amino acid substitution of glutamine for lysine at amino acid position 164, compared to the amino acid sequence from AAV1, AAV2, or AAV6, or at amino acid position 165, compared to the amino acid sequence from AAV7, AAV8, or AAV10; or contains a serine to lysine substitution at amino acid position 160 of AAV5, or contains an alanine to lysine substitution at amino acid position 164 of AAV9. In related embodiments, the capsid protein variant contains an amino acid substitution at amino acid position 164 (e.g., Q164K), compared to the AAV2 capsid amino acid sequence (SEQ ID NO:2), where the capsid protein variant contains 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 capsid protein variant comprises an amino acid sequence containing a Q164K amino acid substitution compared to the amino acid sequence set forth in SEQ ID NO:2, and is otherwise identical to the amino acid sequence set forth in SEQ ID NO:2. In other embodiments, the variant capsid protein contains the amino acid substitutions Q164K and V708I, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or the corresponding substitutions in a different parental AAV serotype (i.e., other than AAV2), and has both at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or greater, amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO:2.

[00166] In other embodiments, the AAV capsid protein variant contains an amino acid substitution at amino acid position 698, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position of another parental AAV serotype (i.e., non-AAV2 ), where the substituted amino acid does not occur naturally at the corresponding position. In some preferred embodiments, the capsid protein variant 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 greater, amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2 and contains an amino acid substitution at amino acid position 698, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the rAAV virion contains an isoleucine to valine amino acid substitution at amino acid position 698, compared to the amino acid sequence from AAV2, or at amino acid position 699, compared to the amino acid sequence from AAV3A, AAV3B, or AAV9, or at amino acid position 687 from AAV5, or at amino acid position 700 from AAV7, or at amino acid position 701 from AAV8 or AAV10. In related embodiments, the capsid protein variant contains an amino acid substitution at amino acid position 699 (e.g., I698V), compared to the AAV2 capsid amino acid sequence (SEQ ID NO:2), where the capsid protein variant contains 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 capsid protein variant contains an amino acid sequence containing the I698V amino acid substitution compared to the amino acid sequence set forth in SEQ ID NO:2 and is otherwise identical to the amino acid sequence set forth in SEQ ID NO:2.

[00167] In other embodiments, a variant AAV capsid protein contains an amino acid substitution at amino acid position 109, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or at the corresponding position of another parental AAV serotype (i.e., non-AAV2 ). In some preferred embodiments, the capsid protein variant 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 greater amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO 2 and contains an amino acid substitution at amino acid position 109, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the capsid protein variant comprises the amino acid substitution of serine for threonine 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 capsid protein variant contains the S109T amino acid substitution, compared to the AAV2 amino acid sequence, where the capsid protein variant contains 1 to 5, 5 to 10, or 10 to 15 amino acid substitutions. In other related embodiments, the capsid protein variant contains the S109T amino acid substitution and the A593E amino acid substitution, compared to the AAV2 amino acid sequence. In some embodiments, the variant capsid protein contains the amino acid substitutions S109T and A493V, and optionally A593E and/or V708I, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or corresponding substitutions in another parent AAV 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 greater, amino acid sequence identity throughout the length of the amino acid sequence shown in SEQ ID NO 2. In some preferred embodiments, the variant capsid protein contains the amino acid substitutions S109T, A493V, A593E, and V708I, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or corresponding substitutions in a different parental AAV 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 greater, amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO 2. In other preferred embodiments, implementation , the capsid protein variant contains the amino acid substitutions S109T and V708I, 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 greater, amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO 2, or otherwise identical with the amino acid sequence of SEQ ID NO:2.

[00168] In other embodiments, the AAV capsid protein variant contains an amino acid substitution at amino acid position 593, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or at the corresponding position of another parental AAV serotype (i.e., different from AAV2). In some preferred embodiments, the capsid protein variant 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 greater, amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2 and contains an amino acid substitution at amino acid position 593, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the capsid protein variant comprises the amino acid substitution of glycine for glutamate at amino acid position 594, compared to the amino acid sequence of AAV1, AAV3A, AAV6, or AAV9, or at amino acid position 583 of AAV5, or at amino acid position 596 of AAV8 or AAV10, or the amino acid substitution of arginine to glutamate at amino acid position 594 of AAV3B, or the amino acid substitution of aspartate to glutamate at amino acid position 592 of AAV4, or the amino acid substitution of glutamine to glutamate at position 595 of AAV7. In other embodiments, the capsid protein variant contains the A593E amino acid substitution compared to the AAV2 amino acid sequence and does not contain one or more of the following amino acid substitutions compared to the AAV2 amino acid sequence: I19V, V369A, K26R, N215D, G355S, V46A, and S196P. In related embodiments, the capsid protein variant contains the amino acid substitutions A593E and N596D, compared to the amino acid sequence of AAV2, and is 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 specified in SEQ ID NO 2. In other embodiments, the capsid variant contains the amino acid substitutions A593E and N596D compared to the amino acid sequence of AAV2 and is otherwise identical to the amino acid AAV2 sequence. In other embodiments, the capsid variant contains the amino acid substitutions A593E and V708I, compared to the AAV2 amino acid sequence, and is 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 specified in SEQ ID NO 2. In other embodiments, the capsid variant contains the amino acid substitutions A593E and V708I, compared to the AAV2 amino acid sequence, and is otherwise identical to the amino acid sequence AAV2.

[00169] In other embodiments, the AAV capsid protein variant contains an amino acid substitution at amino acid position 708, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position of another parental AAV serotype (i.e., other than AAV2) where the replaced amino acid does not occur naturally in the corresponding position. Preferably, the rAAV virion does not contain a proline-to-serine substitution at amino acid position 250, compared to AAV2, or the corresponding amino acid in the other parental AAV serotype. In some embodiments, the capsid protein variant 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 contains an amino acid substitution at amino acid position 708, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In preferred embodiments, the capsid protein variant contains a valine to isoleucine (V708I) substitution at amino acid position 708, compared to the AAV2 capsid amino acid sequence, and has at least about 85%, at least about 90%, at least about 95 %, at least about 98%, or at least about 99%, or greater, amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO 2, or otherwise identical with the amino acid sequence of SEQ ID NO:2 where the capsid protein variant does not contain the P250S amino acid substitution. In some embodiments, the capsid protein variant comprises a valine to isoleucine change at amino acid position 709 of AAV3A or AAV3B, an alanine to isoleucine change at position 709 of AAV1 or AAV6, an asparagine to isoleucine change at amino acid position 707 of AAV4 or amino acid 709 of AAV9, or a replacement of threonine with isoleucine at amino acid position 710 of AAV7 or amino acid 711 of AAV8 or AAV10, or a replacement of glutamine with isoleucine at amino acid position 697 of AAV5. In related embodiments, the capsid protein variant contains the V708I amino acid substitution, compared to the AAV2 amino acid sequence, where the capsid protein variant contains 2 to 5, 5 to 10, or 10 to 15 amino acid substitutions, and where the capsid protein variant does not contain the amino acid P250S replacement. In other embodiments, the capsid protein variant contains the V708I amino acid substitution and also contains the A593E and/or S109T amino acid substitution, compared to the AAV2 amino acid sequence. In other related embodiments, the capsid variant contains the amino acid substitutions V708I and A593E, compared to the AAV2 amino acid sequence, where the capsid protein variant is otherwise identical to the AAV2 amino acid sequence. In other related embodiments, the capsid variant contains V708I and S109T amino acid substitutions compared to the AAV2 amino acid sequence, where the capsid protein variant is otherwise identical to the AAV2 amino acid sequence. In other embodiments, the capsid protein variant contains the amino acid substitutions V708I and V719M, compared to the AAV2 amino acid sequence, and is at least about 85%, at least about 90%, at least about 95%, at least about 98% , or at least about 99%, or greater, amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO 2, or is otherwise identical with the amino acid sequence of SEQ ID NO:2. In other embodiments, the capsid protein variant contains the amino acid substitutions V708I and R733C, compared to the amino acid sequence of AAV2, and is at least about 85%, at least about 90%, at least about 95%, at least about 98 % or at least about 99%, or greater, amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO 2 or otherwise identical with the amino acid sequence of SEQ ID NO:2. In other embodiments, the capsid protein variant contains the amino acid substitutions V708I and G727D, compared to the amino acid sequence of AAV2, and is at least about 85%, at least about 90%, at least about 95%, at least about 98 % or at least about 99% or greater amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO 2, or is otherwise identical with the amino acid sequence of SEQ ID NO:2.

[00170] In other embodiments, the AAV capsid protein variant contains an amino acid substitution at amino acid position 196, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position of another parental AAV serotype (i.e., non-AAV2 ), where the substituted amino acid does not occur naturally in the corresponding position and is optionally other than proline. In some preferred embodiments, the capsid protein variant 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 greater, amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO:2 and contains an amino acid substitution at amino acid position 196, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), and is optionally different from S196P replacements. In preferred embodiments, the capsid protein variant comprises an amino acid substitution of serine for tyrosine at amino acid position 196 of AAV2 or AAV9, or at amino acid position 197 of AAV7, AAV8 or AAV10, or at amino acid position 186 of AAV5; or an alanine to tyrosine substitution at amino acid position 196 of AAV1 or AAV6; or a methionine to tyrosine substitution at amino acid position 191 of AAV4; or substitution of threonine for tyrosine at amino acid position 196 of AAV3A or AAV3B. In a related embodiment, the variant capsid protein contains an amino acid sequence containing the S196Y amino acid substitution compared to the amino acid sequence set forth in SEQ ID NO:2 and is otherwise identical to the amino acid sequence set forth in SEQ ID NO:2. In related embodiments, the capsid protein variant contains an amino acid substitution at amino acid position 196 other than the S196P substitution (e.g., contains the S196Y substitution) compared to the AAV2 capsid amino acid sequence (SEQ ID NO:2), where the capsid protein variant contains from 1 up 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.

[00171] In other embodiments, the AAV capsid protein variant contains an amino acid substitution at amino acid position 175, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2), or at the corresponding position of another parental AAV serotype (i.e., different from AAV2) where the amino acid substituted does not occur naturally at the corresponding position. In some preferred embodiments, the capsid protein variant 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 greater amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO 2 and contains an amino acid substitution at amino acid position 175, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the capsid variant comprises a Q175H amino acid substitution, compared to the AAV2 amino acid sequence as set forth in SEQ ID NO:2, or a glutamine to histidine substitution at the corresponding position of another parental AAV serotype. In related embodiments, the capsid protein variant contains an amino acid substitution at amino acid position 175 (e.g., Q175H), compared to the AAV2 capsid amino acid sequence (SEQ ID NO:2), where the capsid protein variant contains 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.

[00172]In other embodiments, the AAV capsid protein variant contains an amino acid substitution at amino acid position 64, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2) or at the corresponding position of another parental AAV serotype (i.e., other than AAV2), where the amino acid substituted does not occur naturally in the corresponding position. In some preferred embodiments, the capsid protein variant 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 greater amino acid sequence identity with the entire length of the amino acid sequence shown in SEQ ID NO 2 and contains an amino acid substitution at amino acid position 64, compared to the amino acid sequence of the AAV2 capsid (SEQ ID NO:2). In some embodiments, the rAAV virion contains an amino acid substitution P64S, compared to the AAV2 amino acid sequence as indicated in SEQ ID NO:2, or a proline to serine substitution at the corresponding position of the other parental AAV serotype. In related embodiments, the capsid protein variant contains an amino acid substitution at amino acid position 64 (e.g., P64S), compared to the AAV2 capsid amino acid sequence (SEQ ID NO:2), where the capsid protein variant contains 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.

[00173] In other embodiments, the AAV capsid protein variant comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity with a wild-type AAV capsid sequence selected from of the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 10, 11 and 12, and also contains 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) a peptide insert 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:26), HQDTTKN (SEQ ID NO:19), HPDTTKN (SEQ ID NO:18), NKTTNKD (SEQ ID NO:18), :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 ID NO:40), LAKDRAPSTA (SEQ ID NO:41), LAHQDTTKNA (SEQ ID NO:34), LAHPDTTKNA (SEQ ID NO:33), LANKTTNKDA (SEQ ID NO:33 :35), LAGKSKVIDA (SEQ ID NO:38) and LPISNENEHA (SEQ ID NO:36). In some embodiments, the AAV capsid variant contains the indicated one or more amino acid substitutions and/or peptide inserts and is otherwise identical to a sequence selected from the group consisting of SEQ ID NOs: 1-12.

[00174] In some embodiments, the AAV capsid protein variant is an ancestral capsid protein. An ancestral capsid protein is understood to be the evolutionary ancestor of a capsid protein currently found in nature, e.g. random substitution of amino acids at degeneracy positions between the AAV capsid proteins currently found in nature. One non-limiting example of an ancestral capsid is shown below, where the degeneracy positions are (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 as "X":

[00175]

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSXSXGXTNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLXRTQSTGGTAGXXELLFSQXGPXXMSXQAKNWLPGPCYRQQRVSKTLXQNNNSNFAWTGATKYHLNGRXSLVNPGVAMATHKDDEXRFFPSSGVLIFGKXGAGXNNTXLXNVMXTXEEEIKTTNPVATEXYGVVAXNLQSSNTAPXTGXVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPXXFXXAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYAKSXNVDFAVXXXGVYXEPRPIGTRYLTRNL (SEQ ID NO:58).

[00176] In some embodiments, the ancestral 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 greater, amino acid sequence identity with the entire length of the amino acid sequence specified in SEQ ID NO:58. In some embodiments, the ancestral 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 AAV2 amino acid sequence, for example, as indicated in SEQ ID NO 2. In some embodiments, the ancestral capsid protein contains an amino acid sequence having at least about 85%, at least about 90%, at least at least about 95%, at least about 98%, or at least about 99%, or greater, amino acid sequence identity with the entire length of the amino acid sequence from the ancestral sequence described in SEQ ID NO:58 or SEQ ID NO:2, and contains one or more amino acid residues selected from gr a 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, alanine (A) at 551, alanine (A) at 555, glutamic acid (E) at 557, methionine (M) at 561, serine (S) at 563, glutamine (Q) at 577, serine (S) at 583, valine (V) at 593, threonine (T) at 596, alanine (A) at 661, valine (V) at 662, threonine (T) at 664, proline (P) at 665, threonine (T) at 710, aspartic acid (D) at 717, asparagine (N) at 718, glutamic acid (E) at 719, and serine (S) at 723. In some preferred embodiments, the capsid protein variant comprises an amino acid sequence having at least approximately 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, in in some cases 100% amino acid sequence identity with the entire length of the following amino acid sequence 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, asparagine (D) at 533, glutamine (Q) at 547, alanine (A) at 551, alanine (A) at 555, glutamic acid (E) at 557, methionine ( M) at 561, serine (S) at 563, glutamine (Q) at 577, serine (S) at 583, valine (V) at 593, threonine (T) at 596, alanine (A) at 661, valine (V ) at 662, threonine (T) at 664, proline (P) at 665, threonine (T) at 710, aspartic acid (D) at 717, asparagine (N) at 718, glutamic acid (E) at 719, and serine ( S) in 723:

[00177]

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASAGSTNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSTGGTAGTRELLFSQAGPSNMSAQAKNWLPGPCYRQQRVSKTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPSSGVLIFGKQGAGANNTALENVMMTSEEEIKTTNPVATEQYGVVASNLQSSNTAPVTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYAKSTNVDFAVDNEGVYSEPRPIGTRYLTRNL (SEQ ID NO:59).

[00178] In other embodiments, the AAV capsid protein variant comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 98% identity with a wild-type AAV capsid sequence selected from the group consisting of the ancestral variant described herein as SEQ ID NO:58 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, asparagine (D) at 533, glutamine (Q) at 547, alanine (A) at 551, alanine (A) at 555, glutamic acid (E) at 557, methionine (M) at 561, serine (S) at 563, glutamine (Q) at 577, serine (S) at 583, valine (V) at 593, threonine (T) at 596, alanine (A) at 661 , valine (V) at 662, threonine (T) at 664, proline (P) at 665, threo nin (T) at 710, aspartic acid (D) at 717, asparagine (N) at 718, glutamic acid (E) at 719, and serine (S) at 723; and also contains 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) a peptide insert 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: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 ID NO:40), LAKDRAPSTA (SEQ ID NO:41), LAHQDTTKNA (SEQ ID NO:34), LAHPDTTKNA (SEQ ID NO:33) , LANKTTNKDA (SEQ ID NO:35), LAGKSKVIDA (SEQ ID NO:38), and LPISNENEHA (SEQ ID NO:36). In some embodiments, the AAV capsid variant contains the indicated one or more amino acid substitutions and/or peptide inserts and is otherwise identical to SEQ ID NO:59.

[00179] The AAV variants described herein were generated using in vivo directed evolution involving the use of primate retinal screening after intravitreal injection. In some embodiments, the capsid protein variants described herein, when present in an AAV virion, provide more efficient transduction of retinal cells, compared to transduction of retinal cells by an AAV virion containing the corresponding parental AAV or wild-type AAV capsid protein. For example, in some embodiments, the capsid protein variants described herein, when present in an AAV virion, provide more efficient transduction of primate retinal cells than AAV virions containing the corresponding native AAV capsid protein or wild-type AAV capsid protein, e.g. retinas take up more AAV virions containing the variant AAV capsid protein in question than AAV virions containing the original AAV capsid protein or wild-type AAV. In some such embodiments, for an AAV virion variant or rAAV variant is shown at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or greater than 50-fold increased retinal cell transduction compared to retinal cell transduction with a wild-type AAV virion or rAAV containing the corresponding parental AAV capsid protein. In specific such embodiments, the capsid protein variants described herein, when present in an AAV virion, provide greater transduction of primate retinal cells than AAV virions containing the corresponding parental AAV capsid protein or wild-type AAV capsid protein. In other words, the AAV virion variant transduces cell types not transduced by virions containing the corresponding parent AAV capsid protein, and thus more retinal cell types than the corresponding parent AAV virion. In some embodiments, the AAV virion variant preferentially transduces a retinal cell, for example, the rAAV virion in question infects a retinal cell with 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50 times higher specificity than another retinal or non-retinal cell, such as a cell outside the eye. In some embodiments, the transduced retinal cell is a photoreceptor cell (eg, rods; cones). In some embodiments, the retinal cell is a retinal ganglion cell (RGC). In some embodiments, the retinal cell is a retinal epithelial cell (RPE cell). In some embodiments, the retinal cell is a Mueller glial cell. In some embodiments, the retinal cell is a microglial cell. In some embodiments, the retinal cell is an amacrine cell. In some embodiments, the retinal cell is a bipolar cell. In some embodiments, the retinal cell is a horizontal cell. Increases in retinal cell transduction, eg, increased transduction efficiency, wider transduction, more preferential transduction, etc. can easily be achieved in vitro or in vivo by any number of methods known in the art for measuring gene expression. For example, it is possible to package an AAV with a genome containing an expression cassette containing a reporter gene, e.g., a fluorescent protein, under the control of a ubiquitously expressing or tissue-specific promoter, and evaluate the degree of transduction by detecting the fluorescent protein, e.g., by fluorescence microscopy. As another example, an AAV can be packaged with a genome containing a barcoded nucleic acid sequence and the degree of transduction can be assessed by detecting the nucleic acid sequence, for example by PCR. As another example, an AAV can be packaged with a genome containing an expression cassette containing a therapeutic gene for the treatment of a retinal disease, and the degree of transduction can be assessed by detecting the treatment of the retinal disease in an affected patient treated with AAV.

[00180] Ophthalmic diseases that can be treated using a rAAV vector or virion variant and/or method described 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; Betti's crystalline dystrophy; choroideremia; chorioretinal atrophy; chorioretinal degeneration; cone or rod-shaped dystrophies (autosomal dominant, autosomal recessive, and X-linked); hereditary stationary night blindness (autosomal dominant, autosomal recessive and X-linked); color vision disorders including achromatopsia (including ACHM2, ACHM3, ACHM4 and ACHM5), protanopia, deuteranopia and tritanopia; Friedreich's ataxia; Leber's congenital amaurosis (autosomal dominant and autosomal recessive), including, but not limited to, LCA1, LCA2, LCA3, LCA4, LCA6, LCA7, LCA8, LCA12, and LCA15; Leber's hereditary optic neuropathy; macular degeneration (autosomal dominant and autosomal recessive), including, but not limited to, acute macular degeneration, Best's vitelliform macular degeneration, patterned degeneration, North Carolina macular degeneration, hereditary drusen, Sorsby fundus dystrophy, malattia levantanese , and genetically defined retinopathy of prematurity; disease of ocular-retinal development; ocular albinism; optic nerve atrophy (autosomal dominant, autosomal recessive and X-linked); retinitis pigmentosa (autosomal dominant, autosomal recessive, X-linked and mitochondrially inherited traits), examples of which include RP1, RP2, RP3, RP10, RP20, RP38, RP40 and RP43; X-linked retinoschisis; Stargardt's disease; and Usher syndrome including, but not limited to, USH1B, USH1C, USH1D, USH1F, USH1G, USH2A, USH2C, USH2D, and USH3. Examples of complex genetic diseases include, but are not limited to, glaucoma (open-angle, angle-closure, hypotensive, normotensive, congenital, neovascular, pigmentary, pseudoexfoliative); age-related and other forms of macular degeneration, both exudative and non-exudative forms (autosomal dominant and autosomal recessive), such as acute macular degeneration, vitelliform macular degeneration; retinopathy of prematurity; and Vogt-Koyanagi-Harada (VKH) syndrome. Examples of acquired diseases include, but are not limited to, acute macular neuroretinopathy; anterior ischemic optic neuropathy and posterior ischemic optic neuropathy; Behcet's disease; retinal vein occlusion; choroidal neovascularization; diabetic retinopathy, including proliferative diabetic retinopathy and associated complications; diabetic uveitis; edema such as macular edema, cystoid macular edema and diabetic macular edema; violations of the epiretinal membrane; telangiectosis of the macula; multifocal choroiditis; non-retinopathic diabetic retinal dysfunction; eye tumors; atrophy of the optic nerve; retinal detachment; retinal disorders such as central retinal vein occlusion, proliferative vitreoretinopathy (PVR), retinal artery and vein occlusion, vascular occlusion, uveal retinal disease; uveal effusion; infectious and infiltrating disease of the retina; optic nerve diseases such as acquired optic atrophy. Examples of traumatic injuries include, but are not limited to, histoplasmosis; optic nerve injury; eye injury affecting the posterior segment or area of the eye; retinal injury; viral infection of the eye; viral infection of the optic nerve; a condition affecting the posterior segment of the eye that is caused or affected by laser eye therapy; conditions affecting the posterior segment of the eye that are caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; and sympathetic ophthalmia.

[00181] In another embodiment, the capsid variant described herein comprises a heterologous nucleic acid comprising 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, aptamer , polypeptide, secreted antibody, single chain antibody, V _HH domain, soluble receptor, affitelo, nottin, darpin, centurin, chaperone, site-specific nuclease providing site-specific knockdown of gene function or modified site-specific nuclease providing gene-specific activation of transcription.

[00182] The rAAV virion variant described herein contains a heterologous nucleic acid containing a nucleotide sequence encoding a gene product. In some embodiments, the gene product is an interfering RNA. In some embodiments, the gene product is a long non-coding RNA. In some embodiments, the gene product is a short non-coding RNA. In some embodiments, the gene product is an 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 a V _HH domain. In some embodiments, the gene product is a soluble receptor. In some embodiments, the gene product is an affitelo. In some embodiments, the gene product is a nottin. In some embodiments, the gene product is a darpin. In some embodiments, the gene product is a centurin. In some embodiments, the gene product is a chaperone. In some embodiments, the gene product is a site-specific nuclease that provides site-specific knockdown of gene function.

[00183] Uses of the gene product include, but are not limited to, increasing the level of a factor in a cell, increasing the level of a factor in a neighboring cell by secreting a factor, decreasing the level of a factor in a cell, or decreasing the level of a factor in a neighboring cell by secreting a factor. A gene product can be designed to supplement the level of a defective or missing gene product, reduce the level of a defective or missing gene product, introduce a new maintenance gene product, supplement the level of a maintenance gene product, decrease the level of an inhibitory gene product, or both reduce the level of an inhibitory gene product and introduce or supplementing the product level of a maintenance gene.

[00184] Gene products delivered by contemplated AAV variants can be used to alter the level of gene products or the activity of gene products directly or indirectly associated with retinal diseases and trauma. Genes whose products are directly or indirectly associated with genetic diseases include, for example, ADP-ribosylation factor-like protein 6 (ARL6) genes; BBsome-interacting protein 1 (BBIP1); BBsome protein 1 (BBS1); protein 2 BBsomes (BBS2); protein 4 BBsomes (BBS4); protein 5 BBsomes (BBS5); BBsome protein 7 (BBS7); BBsome protein 9 (BBS9); protein 10 BBsomes (BBS10); protein 12 BBsomes (BBS12); centrosome protein 290 kDa (CEP290); intraflagellar transport protein 172 (IFT172); intraflagellar transport protein 27 (IFT27); inositol polyphosphate 5-phosphatase E (INPP5E); member 13 of subfamily J of the potassium channel of internal rectification (KCNJ13); transcription factor-like leucine zipper protein-1 (LZTFL1); McKusick-Kaufman syndrome protein (MKKS); protein Meckel syndrome type 1 (MKS1); nephronophthisis protein 3 (NPHP1); serologically defined colon cancer antigen 8 (SDCCAG8); containing a three-component motif of protein 32 (TRIM32); domain 8 of the tetratricopeptide repeat (TTC8); the Batten disease protein (CLN3); accompanying Rab protein 1 (CHM); (PRDM13); (RGR; (TEAD1); aryl hydrocarbon-like receptor protein 1 (AIPL1); otx-like transcription factor for homeobox cone-rod photoreceptor photoreceptors (CRX); guanylate cyclase-activating protein 1A (GUCA1A); retinal-specific guanylate cyclase (GUCY2D); member 3 the family of membrane-associated transport proteins phosphatidylinositol (PITPNM3); prominin 1 (PROM1); peripherin (PRPH); peripherin 2 (PRPH2); synaptic membrane exocytosis-regulating protein 1 (RIMS1); semaphorin 4A (SEMA4A); human homologue of the unc119 C protein. elegans (UNC119); ATP-binding cassette transporter - retina (ABCA4); ADAM metallopeptidase domain 9 (ADAM9); transcription-activating factor 6 (ATF6); chromosome 21 open reading frame 2 (C21orf2); chromosome 8 open reading frame 37 (C8orf37) ; calcium channel; voltage-gated; subunit 4 alpha 2/delta (CACNA2D4); cadherin-related family member 1 (protocadherin 21) (CDHR1); cera-like protein mid-kinase (CERKL); alpha subunit of the cGMP-driven cation channel of photoreceptor cones (CNGA3); beta 3 subunits of the cone cyclic nucleotide-gated cation channel (CNGB3); cyclin M4 (CNNM4); a nucleotide-binding guanine protein (G-protein); polypeptide 2 with alpha transduction activity (GNAT2); member 2 of subfamily V potassium channels (KCNV2); phosphodiesterase 6C (PDE6C); phosphodiesterase 6H (PDE6H); centriole protein B of the centriole proteome 1 (POC1B); RAB28 member of the RAS oncogene family (RAB28); retinal and anterior neural fold transcription factor homeobox 2 (RAX2); 11-cis-retinol dehydrogenase 5 (RDH5); protein 1 interacting with the GTPase regulator in RP (RPGRIP1); member 5 of the tubulin-tyrosine ligase-like protein family (TTLL5); subunits alpha-1 voltage-gated calcium channel L-type (CACNA1F); GTPase regulator in retinitis pigmentosa (RPGR); (GNAT1); (PDE6B); (RHO); CABP4); GPR179); (GRK1); GRM6); LRIT3); SLC24A1); TRPM1); NYX); OPN1LW); OPN1MW); blue conopsin (OPN1SW); frataxin (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-ND] genes); (BEST1); C1QTNF5EFEMP1); ELOVL4); FSCN2); GUCA1B); HMCN1); IMPG1); RP1L1); TIMP3); DRAM2); MFN2); NR2F1); optic nerve atrophy protein 1 (OPA1); TMEM126A); TIMM8A); CA4); HK1); KLHL7); NR2E3); NRL); OR2W3); PRPF3); PRPF4); PRPF6); PRPF8); PRPF31); ROM1); retinitis pigmentosa protein 1 (RP1); RP9); SNRNP200); SPP2); TOPORS); ARL2BP); C2orf71); CLRN1); CNGA1); CNGB1); CYP4V2); DHDDS); DHX38); EMC1); EYS); FAM161A); GPR125); HGSNAT); IDH3B); IMPG2); KIAA1549); K.I.Z.); MAC); MERTC); MVK); NEK2); NEUROD1); PDE6A); PDE6G); PRCD); RBP3); RLBP1); SLC7A14); USH2A); ZNF408); ZNF513); OFD1); RP2); retinoschisin (RS1); ABHD12); CDH23); CEP250); CIB2); DFNB31); GPR98); HARS); MYO7A); PCDH15); 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).

[00185] Genes whose products induce or stimulate apoptosis are referred to herein as "pro-apoptotic genes", and the products of these genes (mRNA; protein) are referred to as "pro-apoptotic gene products". Proapoptotic targets include, for example, Bax gene products; Bid gene products; Bak gene products; Bad gene products; Bcl-2; Bcl-X1. Anti-apoptotic gene products include an X-linked inhibitor of apoptosis.

[00186] Genes whose products induce or stimulate angiogenesis are referred to herein as "pro-angiogenic genes", and the products of these genes (mRNA; protein) are referred to as "pro-angiogenic gene products". Proangiogenic targets include, for example, vascular endothelial growth factor (VEGFa, VEGFb, VEGFc, VEGFd); vascular endothelial growth factor receptor 1 (VEGFR1); vascular endothelial growth factor receptor 2 (VEGFR2); related Fms tyrosine kinase 1 (Flt1); placental growth factor (PGF); platelet-derived growth factor (PDGF); angiopoietins; sonic hedgehog. Genes whose products inhibit angiogenesis are referred to herein as "anti-angiogenic genes", and the products of these genes (mRNA; protein) are referred to as "anti-angiogenic gene products". Antiangiogenic gene products include endostatin; tumstatin; angiostatin; pigment epithelial factor (PEDF); and fusion proteins or antibodies that are specific for pro-angiogenic targets and/or their receptors, e.g., anti-VEGF sFLT1 or Eylea fusion proteins, VEGF-specific antibodies Lucentis™ and Avastin™, etc.

[00187] In some embodiments, the gene product(s) delivered by the contemplated AAV variants act to inhibit angiogenesis. In specific preferred embodiments, the gene product(s) delivered by the contemplated AAV variants act to inhibit the activity of one or more mammalian VEGF proteins selected from the group consisting of VEGFa, VEGFb, VEGFc, VEGFd, and PGF. In particularly preferred embodiments, the gene product(s) delivered by the contemplated AAV variants inhibit VEGFa activity. VEGFa has 9 alternative splicing isoforms, the most physiologically significant of which is VEGF165. VEGFa levels have been found to be increased in the vitreous of patients with wet age-related macular degeneration, diabetic macular edema, and retinal vein occlusion. Gene product(s) that inhibit VEGFa activity in the eye, and which are thus effective for treating patients with elevated vitreous VEGFa levels, include, but are not limited to, aflibercept, ranibizumab, brolucizumab, bevacizumab, and soluble fms-like tyrosine kinase 1 (sFLT1) (GenBank Access No. U01134). In some embodiments, the present invention provides an infectious recombinant AAV virion (rAAV) comprising (i) an AAV capsid protein variant as described herein and (ii) a heterologous nucleic acid comprising a plurality of sequences, each of which encodes a distinct inhibitor VEGFa.

[00188] In one preferred embodiment, the gene product is aflibercept. Aflibercept (EYLEA®) is a recombinant fusion protein containing the extracellular domains of human VEGF receptors 1 and 2 fused to a human IgG1 Fc fragment. Aflibercept acts as a soluble decoy receptor that binds VEGFa and PGF with greater affinity than natural receptors. The approved dose for intravitreal injection of aflibercept is 2.0 mg, the dosage varies according to the indication. Aflibercept is indicated for the treatment of neovascular (wet) age-related macular degeneration, macular edema after retinal vein occlusion, diabetic macular edema, and diabetic retinopathy. In a particularly preferred embodiment, the present invention relates to a novel codon-optimized nucleic acid sequence encoding aflibercept (corresponding to Figure 12A) comprising or consisting of:

[00189]

ATGGTTTCTTACTGGGACACCGGCGTGCTGCTGTGTGCCCTGCTTTCTTGTCTGCTGCTGACCGGCTCTAGCAGCGGCTCTGATACCGGCAGACCCTTCGTGGAAATGTACAGCGAGATCCCCGAGATCATCCACATGACCGAGGGCAGAGAGCTGGTCATCCCTTGCAGAGTGACAAGCCCCAACATCACCGTGACTCTGAAGAAGTTCCCTCTGGACACACTGATCCCCGACGGCAAGAGAATCATCTGGGACAGCCGGAAGGGCTTCATCATCAGCAACGCCACCTACAAAGAGATCGGCCTGCTGACCTGTGAAGCCACCGTGAATGGCCACCTGTACAAGACCAACTACCTGACACACAGACAGACCAACACCATCATCGACGTGGTGCTGAGCCCTAGCCACGGCATTGAACTGTCTGTGGGCGAGAAGCTGGTGCTGAACTGTACCGCCAGAACCGAGCTGAACGTGGGCATCGACTTCAACTGGGAGTACCCCAGCAGCAAGCACCAGCACAAGAAACTGGTCAACCGGGACCTGAAAACCCAGAGCGGCAGCGAGATGAAGAAATTCCTGAGCACCCTGACCATCGACGGCGTGACCAGAAGTGACCAGGGCCTGTACACATGTGCCGCCAGCTCTGGCCTGATGACCAAGAAAAACAGCACCTTCGTGCGGGTGCACGAGAAGGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAATAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGG TGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAATGA (SEQ ID NO:65).

[00190] In some embodiments, the present invention provides a nucleic acid sequence encoding aflibercept, 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 greater, nucleic acid identity to the entire length of the nucleic acid sequence specified in SEQ ID NO:65 or to nucleotides 79-1377 of SEQ ID NO:65 (without underlined nucleotides encoding the Flt1 signal sequence). In related embodiments, the gene product(s) delivered by the contemplated AAV variant(s) are encoded by a nucleic acid sequence consisting of or containing 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 her. In other related embodiments, the gene product(s) delivered by the contemplated AAV variants comprise an amino acid sequence having at least about at least about 90%, at least about 95%, at least about 96%, at least at least about 97%, at least about 98%, at least about 99% identity, or 100% identity with the following amino acid sequence:

[00191]

MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:66).

[00192] In other related embodiments, the AAV variant(s) comprise a heterologous nucleic acid comprising a nucleotide sequence encoding 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% identity, or 100% identity with amino acids 27-458 of SEQ ID NO:66 (corresponding to the amino acid sequence of aflibercept without the underlined signal peptide sequence).

[00193] In another preferred embodiment, the gene product is a single chain variant of ranibizumab (sc-ranibizumab). Ranibizumab (LUCENTIS®) is a fragment (Fab) of an IgG1 monoclonal antibody that binds and blocks all VEGFa isoforms. LUCENTIS® is expressed as two separate chains (light and heavy) connected via a disulfide bond between a constant light domain (CL) and a constant heavy domain 1 (CH1). The approved intravitreal dose of ranibizumab is either 0.3 or 0.5 mg in 0.05 ml, depending on the indication. Ranibizumab is approved for the treatment of age-related wet macular degeneration, macular edema after retinal vein occlusion, diabetic macular edema, and diabetic retinopathy. In a particularly preferred embodiment, the present invention relates to a novel codon-optimized nucleic acid sequence encoding a single-stranded heavy-light (HL) form of ranibizumab (sc-ranibizumab HL) corresponding to Figure 12C, wherein the nucleic acid sequence comprises or consists of:

[00194]

ATGGACTGGACCTGGTCCATCCTGTTTCTGGTGGCTGCCGCCACAGGCACATACTCTGAAGTGCAGCTGGTGGAATCTGGCGGCGGACTTGTTCAACCTGGCGGCTCTCTGAGACTGAGCTGTGCCGCCTCTGGCTACGACTTTACCCACTACGGCATGAACTGGGTCCGACAGGCCCCTGGCAAAGGCCTTGAATGGGTCGGATGGATCAACACCTACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAGATTCACCTTCAGCCTGGACACCAGCAAGAGCACCGCCTACCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTATCCCTACTACTACGGCACCAGCCACTGGTACTTCGACGTGTGGGGACAGGGCACACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCACTGGCTCCTAGCAGCAAGTCTACCTCCGGTGGAACAGCCGCTCTGGGCTGTCTGGTCAAGGACTACTTTCCCGAGCCTGTGACCGTGTCCTGGAATAGCGGAGCACTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAAAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCAAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGTGGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACACACCTCGGCGGAAGCTCTGGAAGCGGCTCTGGATCTACCGGCACAAGCTCTAGCGGAACAGGCACAAGCGCTGGCACAACCGGAACAAGCGCTTCTACATCTGGCTCTGGTTCTGGCGGAGGCGGAGGATCAGGTGGTGGTGGATCTGCTGGCGGAACAGCTACAGCTGGCGCCTCTTCTGGCAGCGACATTCAGCTGACACAGAGCCCTTCTAGCCTGAGCGCCTCTGTGGGCGACAGAGTGACCATCACATGTA GCGCCAGCCAGGACATCTCCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGGTGCTGATCTACTTTACCAGCAGCCTGCACTCCGGCGTGCCCAGCAGATTTTCTGGATCTGGCTCCGGCACCGACTTCACCCTGACAATATCTAGCCTCCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCACCGTGCCTTGGACATTTGGCCAGGGCACAAAGGTGGAAATCAAGCGGACAGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAAGTGGACAACGCCCTCCAGTCCGGCAACAGCCAAGAGTCTGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGTCCAGCACACTGACACTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAGAGCTTCAACCGGGGCGAGTGTTGA (SEQ ID NO:67).

[00195] In some embodiments, the present invention provides a nucleic acid sequence encoding sc-ranibizumab HL, 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 greater, nucleic acid identity to the entire length of the nucleic acid sequence specified in SEQ ID NO:67, or to nucleotides 58-1575 of SEQ ID NO:67 (no underlined nucleotides encoding the human IGHV7-8 signal sequence). In related embodiments, the gene product(s) delivered by the subject AAV variant(s) are encoded by a nucleic acid sequence consisting of or containing 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 her. In other related embodiments, the gene product(s) delivered by the contemplated AAV variant(s) comprise an amino acid sequence having at least about at least about 90%, at least about 95%, at least about 96% , at least about 97%, at least about 98%, at least about 99% identity, or 100% identity with the following amino acid sequence:

[00196]

MDWTWSILFLVAAATGTYSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHLGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:68).

[00197] In other related embodiments, the AAV variant(s) comprise a heterologous nucleic acid comprising a nucleotide sequence encoding 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% identity, or 100% identity with 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).

[00198] In another particularly preferred embodiment, the present invention relates to a novel codon-optimized nucleic acid sequence encoding a single-stranded light-heavy (LH) form of ranibizumab (sc-ranibizumab LH) corresponding to Figure 12B, wherein the nucleic acid sequence comprises or comprises:

[00199]

ATGGTCCTCCAGACACAGGTGTTCATCAGCCTGCTGCTGTGGATCTCTGGCGCCTACGGCGATATCCAGCTGACACAGAGCCCTAGCAGCCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACATGTAGCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGGTGCTGATCTACTTTACCAGCAGCCTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATATCTAGCCTCCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCACCGTGCCTTGGACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGACAGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTCCAGAGCGGCAATAGCCAAGAGAGCGTGACCGAGCAGGACAGCAAGGACTCTACCTACTCTCTGAGCAGCACACTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAGTGTGGCGGCAGCTCTGGAAGCGGATCTGGAAGCACAGGCACAAGCTCTAGCGGAACCGGAACAAGCGCTGGCACAACAGGCACATCTGCCAGCACAAGCGGTTCTGGTTCTGGCGGAGGCGGAGGATCTGGTGGTGGCGGATCTGCTGGCGGAACAGCTACAGCTGGTGCCTCTTCTGGATCCGAGGTGCAGCTGGTTGAATCTGGCGGTGGACTGGTTCAGCCAGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTACGATTTCACCCACTACGGCATGAACTGGGTCCGACAGG CCCCTGGCAAAGGCCTTGAATGGGTCGGATGGATCAACACCTACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAGATTCACCTTCAGCCTGGACACCTCCAAGAGCACCGCCTACCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTATCCCTACTACTACGGCACCAGCCACTGGTACTTCGACGTGTGGGGACAGGGCACACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCACTGGCTCCTAGCAGCAAGAGCACATCAGGCGGAACTGCTGCCCTGGGCTGTCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACAGCGGAGCACTGACATCTGGCGTGCACACCTTTCCAGCCGTGCTCCAAAGCAGCGGCCTGTATTCTCTGTCCAGCGTCGTGACAGTGCCTAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGTCGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACCTCTGA (SEQ ID NO:69).

[00200] In another particularly preferred embodiment, the present invention relates to a novel codon-optimized nucleic acid sequence encoding a single-stranded light-heavy (LH) form of ranibizumab (sc-ranibizumab LH) corresponding to Figure 12B, wherein the nucleic acid sequence comprises or comprises:

[00201]

ATGGTGCTCCAGACCCAGGTGTTTATTTCACTGCTGCTGTGGATTAGCGGGGCTTACGGAGACATTCAGCTGACCCAGAGTCCTTCATCTCTGAGCGCCTCCGTGGGCGACAGGGTGACCATCACATGCTCTGCCAGCCAGGATATCTCCAACTATCTGAATTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGGTGCTGATCTATTTCACCAGCTCCCTGCACAGCGGAGTGCCATCCCGCTTCTCCGGCTCTGGCAGCGGCACCGACTTTACCCTGACAATCTCTAGCCTCCAGCCAGAGGATTTCGCCACATACTATTGCCAGCAGTACAGCACCGTGCCCTGGACATTTGGCCAGGGCACCAAGGTGGAGATCAAGCGGACAGTGGCCGCCCCAAGCGTGTTCATCTTTCCCCCTAGCGACGAGCAGCTGAAGTCTGGCACCGCCAGCGTGGTGTGCCTGCTGAACAATTTCTATCCCAGAGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCCCTCCAGTCCGGCAATTCTCAGGAGAGCGTGACCGAGCAGGACTCCAAGGATTCTACATACAGCCTGTCCTCTACCCTGACACTGTCCAAGGCCGACTATGAGAAGCACAAGGTGTACGCATGCGAGGTGACCCACCAGGGCCTGAGCTCCCCAGTGACAAAGAGCTTTAACAGGGGAGAGTGTGGAGGATCTAGCGGATCCGGATCTGGAAGCACCGGCACATCCTCTAGCGGAACCGGCACAAGCGCCGGCACCACAGGCACCTCCGCCTCTACAAGCGGCAGCGGATCTGGCGGAGGAGGAGGATCCGGAGGAGGAGGATCTGCCGGCGGCACCGCCACAGCCGGCGCCTCCTCTGGCTCCGAGGTGCAGCTGGTGGAGTCTGGAGGAGGCCTGGTGCAGCCTGGAGGCTCCCTGAGGCTGTCTTGCGCAGCAAGCGGCTATGATTTCACCCACTACGGAATGAACTGGGTGCGCCAGG CACCTGGCAAGGGCCTGGAGTGGGTGGGCTGGATCAATACCTATACAGGCGAGCCAACCTACGCCGCCGACTTTAAGCGGAGATTCACATTTTCCCTGGATACCAGCAAGTCCACAGCCTACCTCCAGATGAACAGCCTGAGGGCAGAGGACACCGCCGTGTACTATTGCGCCAAGTATCCTTACTATTACGGCACAAGCCACTGGTACTTCGACGTGTGGGGACAGGGCACCCTGGTGACAGTGAGCTCCGCCAGCACCAAGGGCCCATCCGTGTTTCCTCTGGCCCCATCTAGCAAGTCTACCAGCGGAGGAACAGCCGCCCTGGGATGTCTGGTGAAGGACTACTTCCCAGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACCTCCGGCGTGCACACATTTCCCGCCGTGCTCCAGTCCTCTGGCCTGTATAGCCTGAGCTCCGTGGTGACCGTGCCTTCTAGCTCCCTGGGCACCCAGACATACATCTGTAACGTGAATCACAAGCCTTCAAATACCAAAGTCGATAAAAAAGTGGAACCAAAATCCTGTGATAAAACCCATCTGTGA (SEQ ID NO:70).

[00202] In some embodiments, the present invention provides a nucleic acid sequence encoding sc-ranibizumab LH, 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 greater, nucleic acid identity with the entire length of the nucleic acid sequence specified in SEQ ID NO:69 or SEQ ID NO:70, or with nucleotides 61-1578 of SEQ ID NO:69 or SEQ ID NO:70 (without underlined nucleotides encoding the kappa Ig signal sequence). In related embodiments, the gene product(s) delivered by the contemplated AAV variant(s) are encoded by a nucleic acid sequence consisting of or containing a nucleic acid sequence from SEQ ID NO:69 or SEQ ID NO:70, 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 her. In other related embodiments, the gene product(s) delivered by the contemplated AAV variant(s) comprise an amino acid sequence having at least about at least about 90%, at least about 95%, at least about 96% , at least about 97%, at least about 98%, at least about 99% identity, or 100% identity with the following amino acid sequence:

[00203]

MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHL (SEQ ID NO:71).

[00204] In other related embodiments, the AAV variant(s) comprise a heterologous nucleic acid comprising a nucleotide sequence encoding 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% identity, or 100% identity with 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).

[00205] In another preferred embodiment, the present invention relates to a novel nucleic acid sequence encoding a single-stranded light-heavy (LH) form of ranibizumab fused to a human IgG1 Fc region (sc-ranibizumab-Fc) corresponding to Figure 12E, wherein the nucleic acid sequence acid contains or consists of:

[00206]

ATGGTCCTCCAGACACAGGTGTTCATCAGCCTGCTGCTGTGGATCTCTGGCGCCTACGGCGATATCCAGCTGACACAGAGCCCTAGCAGCCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACATGTAGCGCCAGCCAGGACATCAGCAACTACCTGAACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGGTGCTGATCTACTTTACCAGCAGCCTGCACAGCGGCGTGCCCAGCAGATTTTCTGGCTCTGGCAGCGGCACCGACTTCACCCTGACAATATCTAGCCTCCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGTACAGCACCGTGCCTTGGACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGGACAGTGGCCGCTCCTAGCGTGTTCATCTTTCCACCTAGCGACGAGCAGCTGAAGTCTGGCACAGCCTCTGTCGTGTGCCTGCTGAACAACTTCTACCCCAGAGAAGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTCCAGAGCGGCAATAGCCAAGAGAGCGTGACCGAGCAGGACAGCAAGGACTCTACCTACTCTCTGAGCAGCACACTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAAGTGTACGCCTGCGAAGTGACCCACCAGGGCCTTTCTAGCCCTGTGACCAAGAGCTTCAACAGAGGCGAGTGTGGCGGCAGCTCTGGAAGCGGATCTGGAAGCACAGGCACAAGCTCTAGCGGAACCGGAACAAGCGCTGGCACAACAGGCACATCTGCCAGCACAAGCGGTTCTGGTTCTGGCGGAGGCGGAGGATCTGGTGGTGGCGGATCTGCTGGCGGAACAGCTACAGCTGGTGCCTCTTCTGGATCCGAGGTGCAGCTGGTTGAATCTGGCGGTGGACTGGTTCAGCCAGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTACGATTTCACCCACTACGGCATGAACTGGGTCCGACAGG CCCCTGGCAAAGGCCTTGAATGGGTCGGATGGATCAACACCTACACCGGCGAGCCAACATACGCCGCCGACTTCAAGCGGAGATTCACCTTCAGCCTGGACACCTCCAAGAGCACCGCCTACCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGCGCCAAGTATCCCTACTACTACGGCACCAGCCACTGGTACTTCGACGTGTGGGGACAGGGCACACTGGTCACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTTTTCCCACTGGCTCCTAGCAGCAAGAGCACATCAGGCGGAACTGCTGCCCTGGGCTGTCTGGTCAAGGACTACTTTCCTGAGCCTGTGACCGTGTCCTGGAACAGCGGAGCACTGACATCTGGCGTGCACACCTTTCCAGCCGTGCTCCAAAGCAGCGGCCTGTATTCTCTGTCCAGCGTCGTGACAGTGCCTAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAATGTGAACCACAAGCCTAGCAACACCAAGGTCGACAAGAAGGTGGAACCCAAGAGCTGCGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAATAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCTGACCTG CCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAATGAGCCACGCGTAACACGTGCATGCGAGAGATCTGA (SEQ ID NO:72).

[00207] In some embodiments, the present invention provides a nucleic acid sequence encoding sc-ranibizumab-Fc, 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 greater, nucleic acid identity with the entire length of the nucleic acid sequence specified in SEQ ID NO:72, or with nucleotides 61-2277 of SEQ ID NO:72 (no underlined nucleotides encoding the Ig kappa signal sequence). In related embodiments, the gene product(s) delivered by the contemplated AAV variants are encoded by a nucleic acid sequence consisting of or containing the nucleic acid sequence of SEQ ID NO:72, or a nucleic acid having at least about 90%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with her. In other related embodiments, the gene product(s) delivered by the contemplated AAV variant(s) comprise an amino acid sequence having at least about at least about 90%, at least about 95%, at least about 96% , at least about 97%, at least about 98%, at least about 99% identity, or 100% identity with the following amino acid sequence:

[00208]

MVLQTQVFISLLLWISGAYGDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGSSGSGSGSTGTSSSGTGTSAGTTGTSASTSGSGSGGGGGSGGGGSAGGTATAGASSGSEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHLDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 73).

[00209] In other related embodiments, the AAV variant(s) comprise a heterologous nucleic acid comprising a nucleotide sequence encoding 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% identity, or 100% identity with 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)

[00210] In another preferred embodiment, the gene product is brolucizumab. Brolucizumab (RTH258) is a single chain variable fragment (scFv) that binds and blocks all VEGFa isoforms. Brolucizumab is currently in phase III clinical trials evaluating 3 mg and 6 mg doses for the treatment of age-related wet macular degeneration. In a particularly preferred embodiment, the present invention relates to a novel codon-optimized nucleic acid sequence encoding brolucizumab (corresponding to Figure 12D), wherein the nucleic acid sequence comprises or consists of:

[00211]

ATGGTCCTCCAGACACAGGTGTTCATCAGCCTGCTGCTGTGGATCTCTGGCGCCTATGGCGAGATCGTGATGACACAGAGCCCCAGCACACTGTCTGCCAGCGTGGGAGACAGAGTGATCATCACATGCCAGGCCAGCGAGATCATCCACAGCTGGCTGGCTTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACCTGGCCTCTACACTGGCCAGCGGAGTGCCTAGCAGATTTTCTGGCTCTGGATCTGGCGCCGAGTTCACCCTGACAATCTCTAGCCTCCAGCCTGACGACTTCGCCACCTACTACTGCCAGAACGTGTACCTGGCCAGCACCAACGGCGCCAATTTTGGCCAGGGCACCAAGCTGACAGTGCTTGGCGGAGGCGGAGGTTCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCAGGCGGTGGTGGATCTGAAGTGCAGCTGGTGGAATCAGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGAGCTGTACCGCCTCTGGCTTCTCCCTGACCGACTACTACTACATGACCTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAGTGGGTCGGATTCATCGACCCCGACGACGATCCTTACTACGCCACATGGGCCAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCGGCGGAGATCACAATAGCGGCTGGGGACTCGATATCTGGGGCCAGGGAACACTGGTCACCGTGTCTAGTTGA (SEQ ID NO:74).

[00212] In some embodiments, the present invention provides a nucleic acid sequence encoding brolucizumab, 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 greater, nucleic acid identity to the entire length of the nucleic acid sequence specified in SEQ ID NO:74, or to nucleotides 61-816 of SEQ ID NO:74 (without underlined nucleotides encoding the Ig kappa signal sequence). In related embodiments, the gene product(s) delivered by the contemplated AAV variants are encoded by a nucleic acid sequence consisting of or containing the nucleic acid sequence of SEQ ID NO:74, or a nucleic acid having at least about 90%, at least 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 other related embodiments, the gene product(s) delivered by the contemplated AAV variants comprise an amino acid sequence having at least about at least about 90%, at least about 95%, at least about 96%, at least at least about 97%, at least about 98%, at least about 99% identity, or 100% identity with the following amino acid sequence:

[00213]

MVLQTQVFISLLLWISGAYGEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSS (SEQ ID NO:75).

[00214] In other related embodiments, the AAV variant(s) comprise a heterologous nucleic acid comprising a nucleotide sequence encoding 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% identity, or 100% identity with amino acids 21-271 of SEQ ID NO:75 (corresponding to the amino acid sequence of brolucizumab without the underlined signal peptide sequence).

[00215] In another preferred embodiment, the present invention relates to a novel nucleic acid sequence encoding brolucizumab fused to a human IgG1 Fc region (brolucizumab-Fc) corresponding to Figure 12F, wherein the nucleic acid sequence comprises or consists of:

[00216]

ATGGTCCTCCAGACACAGGTGTTCATCAGCCTGCTGCTGTGGATCTCTGGCGCCTATGGCGAGATCGTGATGACACAGAGCCCCAGCACACTGTCTGCCAGCGTGGGAGACAGAGTGATCATCACATGCCAGGCCAGCGAGATCATCCACAGCTGGCTGGCTTGGTATCAGCAGAAGCCTGGCAAGGCCCCTAAGCTGCTGATCTACCTGGCCTCTACACTGGCCAGCGGAGTGCCTAGCAGATTTTCTGGCTCTGGATCTGGCGCCGAGTTCACCCTGACAATCTCTAGCCTCCAGCCTGACGACTTCGCCACCTACTACTGCCAGAACGTGTACCTGGCCAGCACCAACGGCGCCAATTTTGGCCAGGGCACCAAGCTGACAGTGCTTGGCGGAGGCGGAGGTTCTGGTGGCGGAGGAAGTGGCGGCGGAGGATCAGGCGGTGGTGGATCTGAAGTGCAGCTGGTGGAATCAGGCGGAGGACTGGTTCAACCTGGCGGCTCTCTGAGACTGAGCTGTACCGCCTCTGGCTTCTCCCTGACCGACTACTACTACATGACCTGGGTCCGACAGGCCCCTGGCAAAGGACTTGAGTGGGTCGGATTCATCGACCCCGACGACGATCCTTACTACGCCACATGGGCCAAGGGCAGATTCACCATCAGCCGGGACAACAGCAAGAACACCCTGTACCTCCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCCGGCGGAGATCACAATAGCGGCTGGGGACTCGATATCTGGGGCCAGGGAACACTGGTCACCGTGTCTAGTGACAAGACCCACACCTGTCCTCCATGTCCTGCTCCAGAACTGCTCGGCGGACCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGATCCCGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGG AAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAATAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAAACCATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTTTACACACTGCCTCCAAGCAGGGACGAGCTGACAAAGAACCAGGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGTACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGTCTCCTGGCAAATGAGCCACGCGTAACACGTGCATGCGAGAGATCTGA (SEQ ID NO:76).

[00217] In some embodiments, the present invention provides a nucleic acid sequence encoding brolucizumab-Fc, comprising a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least at least about 97%, at least about 98%, or at least about 99%, or greater, nucleic acid identity to the entire length of the nucleic acid sequence specified in SEQ ID NO:76 or to nucleotides 61-1530 of SEQ ID NO :76 (without underlined nucleotides encoding the Ig kappa signal sequence). In related embodiments, the gene product(s) delivered by the contemplated AAV variants are encoded by a nucleic acid sequence consisting of or containing the nucleic acid sequence of SEQ ID NO:76, or a nucleic acid having at least about 90%, at least 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 other related embodiments, the gene product(s) delivered by the contemplated AAV variant(s) comprise an amino acid sequence having at least about at least about 90%, at least about 95%, at least about 96% , at least about 97%, at least about 98%, at least about 99% identity, or 100% identity with the following amino acid sequence.

[00218]

MVLQTQVFISLLLWISGAYGEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 77).

[00219] In other related embodiments, the AAV variant(s) comprise a heterologous nucleic acid comprising a nucleotide sequence encoding 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% identity, or 100% identity with amino acids 21-498 of SEQ ID NO:77 (the corresponding amino acid sequence of brolucizumab-Fc without the underlined signal peptide sequence).

[00220] In particularly preferred embodiments, the present invention provides an infectious recombinant AAV virion (rAAV) comprising an AAV capsid protein variant comprising a peptide insert in the GH-loop of the capsid protein, compared to the corresponding parental AAV capsid protein, wherein the peptide insert comprises the amino acid sequence ISDQTKH (SEQ ID NO:14) or LAISDQTKHA (SEQ ID NO:28), and a heterologous nucleic acid encoding a polypeptide that inhibits VEGF activity, preferably VEGFa, wherein the capsid protein variant confers increased infectivity to the retinal cell compared to infectivity the corresponding original AAV capsid protein for the retinal cell. In some embodiments, the insertion site is between amino acids corresponding to amino acids 587 and 588 of VP1 from AAV2 (SEQ ID NO:2), or at the corresponding position in the capsid protein of another AAV serotype. Preferably, the AAV capsid protein variant also contains the P34A amino acid substitution, relative to AAV2 capsid VP1 (SEQ ID NO:2), or a corresponding substitution in another AAV serotype.

[00221] In some embodiments, the present invention provides an rAAV virion comprising an AAV capsid protein variant comprising (i) a peptide insert between amino acids corresponding to amino acids 587 and 588 of VP1 from AAV2 (SEQ ID NO:2), or at the corresponding position in the capsid protein of another AAV capsid protein serotype, compared to the corresponding parent AAV capsid protein, and (ii) a P34A amino acid substitution, relative to AAV2 capsid VP1 (SEQ ID NO:2), or a corresponding substitution in another AAV serotype, and a heterologous nucleic acid , containing the sequence encoding aflibercept. In preferred embodiments, the nucleic acid sequence encoding aflibercept consists of or contains 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 it. In a particularly preferred embodiment, the present invention provides an rAAV virion comprising an AAV capsid protein variant having an amino acid sequence at least 90% identical, at least 95% identical, or at least 99% identical to the sequence indicated as SEQ ID NO:42, and a heterologous nucleic acid comprising the nucleic acid sequence of SEQ ID NO:65, wherein the variant capsid protein confers increased infectivity to a retinal cell compared to the infectivity of the corresponding parental AAV capsid protein to a retinal cell. 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, brolucizumab, and brolucizumab- fc. In related embodiments, the present invention relates to a pharmaceutical composition containing such rAAV. In other related embodiments, the present invention relates to a method of treating a VEGFa-associated eye disease comprising administering to a subject in need an effective amount of an rAAV virion comprising an AAV capsid protein variant having an amino acid sequence at least 90% identical to the sequence indicated as SEQ ID NO:42, and a heterologous nucleic acid comprising the nucleic acid sequence from SEQ ID NO:65 and optionally one or more additional nucleic acid sequences, each encoding a separate VEGFa inhibitor. rAAV can be administered by subretinal, suprachoroidal, topical, intracameral, or vitreous injection, but is preferably administered by vitreous injection. In some embodiments, the VEGFa-associated eye disease is selected from wet (neovascular, exudative) age-related macular degeneration; macular edema after retinal vein occlusion; retinal neovascularization due to retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative diabetic retinopathy and proliferative diabetic retinopathy), myopic macular degeneration, branch retinal vein occlusion, gemiretinal vein occlusion and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectosis; neovascular glaucoma; hemorrhages of the vitreous body; retinal and choroidal neovascularization secondary to retinal diseases including, but not limited to, uveitis, trauma, degenerative retinal disorders, retinal and/or choroid genetic disease, eye tumors, corneal and iris neovascularization. In some preferred embodiments, the VEGFa associated 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.

[00222] In some embodiments, the present invention provides an rAAV virion comprising an AAV capsid protein variant comprising (i) a peptide insert between amino acids corresponding to amino acids 587 and 588 of VP1 from AAV2 (SEQ ID NO:2), or at the corresponding position in the capsid protein of another AAV capsid protein serotype, compared to the corresponding parent AAV capsid protein, and (ii) a P34A amino acid substitution, relative to AAV2 capsid VP1 (SEQ ID NO:2), or a corresponding substitution in another AAV serotype, and a heterologous nucleic acid containing a nucleotide sequence encoding ranibizumab, sc-ranibizumab HL, sc-ranibizumab LH, or sc-ranibizumab-Fc. In preferred embodiments, the nucleic acid sequence encoding sc-ranibizumab consists of or contains the nucleic acid sequence listed in any of SEQ ID NOs: 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 her. In a particularly preferred embodiment, the present invention provides an rAAV virion comprising an AAV capsid protein variant having an amino acid sequence at least 90% identical, at least 95% identical, or at least 99% identical to the sequence indicated as SEQ ID NO:42, and a heterologous nucleic acid comprising the nucleic acid sequence of any of SEQ ID NOs: 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 separate VEGFa inhibitor, preferably selected from aflibercept, brolucizumab, and brolucizumab-Fc. In related embodiments, the present invention relates to a pharmaceutical composition containing such rAAV. In other related embodiments, the present invention relates to a method of treating an ocular disease associated with elevated levels of intraocular VEGFa comprising administering to a subject in need thereof an effective amount of an rAAV virion comprising an AAV capsid protein variant having an amino acid sequence at least 90% identical to sequence indicated as SEQ ID NO:42, and a heterologous nucleic acid sequence containing a nucleic acid sequence from any of SEQ ID NO: 67, 69, 70 and 72. Preferably, rAAV is administered by injection into the vitreous body. In some embodiments, the VEGFa-associated eye disease is selected from wet (neovascular, exudative) age-related macular degeneration; macular edema after retinal vein occlusion; retinal neovascularization due to retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative diabetic retinopathy and proliferative diabetic retinopathy), myopic macular degeneration, branch retinal vein occlusion, gemiretinal vein occlusion and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectosis; neovascular glaucoma; hemorrhages of the vitreous body; retinal and choroidal neovascularization secondary to retinal diseases including, but not limited to, uveitis, trauma, degenerative retinal disorders, retinal and/or choroid genetic disease, eye tumors, corneal and iris neovascularization. In some preferred embodiments, the VEGFa associated 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.

[00223] In some embodiments, the present invention provides an rAAV virion comprising an AAV capsid protein variant comprising (i) a peptide insert between amino acids corresponding to amino acids 587 and 588 of VP1 from AAV2 (SEQ ID NO:2), or at the corresponding position in the capsid protein of another AAV capsid protein serotype, compared to the corresponding parent AAV capsid protein, and (ii) a P34A amino acid substitution, relative to AAV2 capsid VP1 (SEQ ID NO:2), or a corresponding substitution in another AAV serotype, and a heterologous nucleic acid , containing the nucleotide sequence encoding brolucizumab or brolucizumab-Fc. In preferred embodiments, the nucleotide sequence encoding brolucizumab or brolucizumab-Fc consists of or contains the nucleic acid sequence set forth 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 her. In a particularly preferred embodiment, the present invention provides an rAAV virion comprising an AAV capsid protein variant having an amino acid sequence at least 90% identical, at least 95% identical, or at least 99% identical to the sequence indicated as SEQ ID NO:42, and a 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 a retinal cell compared to the infectivity of the corresponding parental AAV capsid protein to the cell retina. In other related embodiments, the heterologous nucleic acid further comprises one or more nucleotide sequences each encoding a distinct VEGFa inhibitor, preferably selected from aflibercept, ranibizumab, sc-ranibizumab HL, sc-ranibizumab LH, and sc-ranibizumab-Fc. In related embodiments, the present invention relates to a pharmaceutical composition containing such rAAV. In other related embodiments, the present invention relates to a method of treating an ocular disease associated with elevated levels of intraocular VEGFa comprising administering to a subject in need thereof an effective amount of an rAAV virion comprising an AAV capsid protein variant having an amino acid sequence at least 90% identical to the sequence indicated as SEQ ID NO:42, and a heterologous nucleic acid sequence containing the nucleic acid sequence from SEQ ID NO:74 or SEQ ID NO:76. Preferably, rAAV is administered by intravitreal injection. In some embodiments, the VEGFa-associated eye disease is selected from wet (neovascular, exudative) age-related macular degeneration; macular edema after retinal vein occlusion; retinal neovascularization due to retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative diabetic retinopathy and proliferative diabetic retinopathy), myopic macular degeneration, branch retinal vein occlusion, gemiretinal vein occlusion and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectosis; neovascular glaucoma; hemorrhages of the vitreous body; retinal and choroidal neovascularization secondary to retinal diseases including, but not limited to, uveitis, trauma, degenerative retinal disorders, retinal and/or choroid genetic disease, eye tumors, corneal and iris neovascularization. In some preferred embodiments, the VEGFa associated 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.

[00224] Genes whose products function as immunomodulators, eg complement factors, toll-like receptors, are referred to as "immunomodulatory genes". Exemplary immunomodulatory genes include genes for cytokines, chemokines, and fusion proteins or antibodies specific for them and/or their receptors, e.g., the anti-IL-6 fusion protein rilonacept™, the complement factor H-specific antibody lampamizumab, and the like. Genes whose products function as neuroprotective factors, such as platelet-derived growth factor receptor (PDGFR); glial neurotrophic factor (GDNF); rod-derived cone viability factor (RdCVF); fibroblast growth factor (FGF); neurturin (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neutrophin-4 (NT4); brain-derived neurotrophic factor (BDNF); epidermal growth factor. Genes whose products function as light-responsive opsins, such as opsin; rhodopsin; channel rhodopsin; halorhodopsin.

[00225] In some cases, the product of the gene 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 a retinal disease. For example, when a dominant allele encodes a defective copy of a gene that, when of wild type, encodes a retinal structural protein and/or provides normal retinal function, a site-specific endonuclease can target the defective allele and knock out the defective allele.

[00226] In addition to knocking out the defective allele, a site-specific nuclease can also be used to promote homologous recombination with donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, for example, the contemplated rAAV virion can be used to deliver a site-specific endonuclease that knocks out the defective allele, and can be used to deliver a functional copy of the defective allele resulting in repair of the defective allele, thus allowing production of a functional retinal protein (e.g., functional retinoschisin, functional RPE65, functional peripherin, etc.). See, for example, Li et al. (2011) Nature 475:217. In some embodiments, the rAAV virion described herein contains a heterologous nucleotide sequence encoding a site-specific endonuclease; and a heterologous nucleotide sequence encoding a functional copy of the defective allele, wherein the functional copy encodes a functional retinal protein. Functional retinal proteins include, for example, retinoschisin, RPE65, retinitis pigmentosa GTPase regulator-interacting protein-1 (RGPR), peripherin, peripherin-2, and the like.

[00227] Suitable site-specific endonucleases include, for example, meganucleases; zinc finger nucleases (ZFN); effector nucleases like transcription activators (TALEN); and associated with uniformly spaced clustered short palindromic repeats/CRISPR (Cas), where such site-specific endonucleases do not occur naturally and are modified to target a specific gene. Such site-specific nucleases can be designed to cut at specific locations in the genome, and then, by joining the non-homologous ends, the break can be repaired while at the same time inserting or deleting a few nucleotides. Such site-specific endonucleases (also referred to as introducing "indels") then effect the protein out of frame shift and efficient gene knockout. See, for example, US Patent Publication No. 2011/0301073.

[00228] In some embodiments of the variant rAAV vector described herein, the nucleotide sequence encoding the product of the gene of interest is operably linked to a constitutive promoter. Suitable constitutive promoters include, for example, the cytomegalovirus (CMV) promoter (Stinski et al. (1985) Journal of Virology 55(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 elongation factor 1α promoter (EF1α ) (Kim et al. (1990) Gene 91(2): 217-223), human phosphoglycerate kinase (PGK) promoter (Singer-Sam et al. (1984) Gene 32(3): 409-417, mitochondrial heavy chain promoter genome (Loderio et al. (2012) PNAS 109(17): 6513-6518), ubiquitin promoter (Wulff et al. (1990) FEBS Letters 261: 101-105). of the gene of interest is operably linked to an inducible promoter In some cases, the nucleotide sequence encoding the product of the gene of interest is operably linked to a tissue a non-specific or cell-type-specific regulatory element. For example, in some instances, the nucleotide sequence encoding the product of the gene of interest is operably linked to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), e.g., a regulatory element that selectively expresses the operably linked gene in a photoreceptor cell. Suitable photoreceptor-specific regulatory elements include, for example, the rhodopsin promoter; rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); beta-phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); enhancer of the interphotoreceptor retinol-binding protein (IRBP) gene (Nicoud et al. (2007) supra); IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225), opsin gene promoter (Tucker et al. (1994) PNAS 91:2611-2615), retinoschisin gene promoter (Park et al. (2009) Gene Therapy 16(7): 916-926), CRX homeodomain protein gene promoter (Furukawa et al. (2002) The Journal of Neuroscience 22(5): 1640-1647), guanine nucleotide-binding protein polypeptide 1 gene promoter with activity alpha transduction (GNAT1) (Lee et al. (2010) Gene Therapy 17:1390-1399), neutral retinal-specific leucine zipper (NRL) gene promoter (Akimoto et al. (2006) PNAS 103(10): 3890-3895), the human cone arrestin promoter (hCAR) (Li et al. (2002) Biochemistry and Molecular Biology 43: 1375-1383), and the PR2.1, PR1.7, PR1.5 and PR1.1 promoters (Ye et al (2016) Human Gene Therapy 27(1): 72-82)). In some instances, the nucleotide sequence encoding the gene product of interest is operably linked to a retinal pigment epithelial (RPE) cell-specific regulatory element (e.g., an RPE-specific promoter), e.g. RPE. Suitable RPE-specific regulatory elements include, for example, the RPE65 gene promoter (Meur et al. (2007) Gene Therapy 14: 292-303), the cellular retinaldehyde binding protein (CRALBP) gene promoter (Kennedy et al. (1998) Journal of Biological Chemistry 273: 5591-5598), the pigment epithelial factor (PEDF, also called serpin F1) gene promoter (Kojima et al. (2006) Molecular and Cellular Biochemistry 293(1-2): 63-69), and the vitelliform macular degeneration gene promoter ( VMD2) promoter (Esumi et al. (2004) The Journal of Biological Chemistry 279(18): 19064-19073). In some instances, the nucleotide sequence encoding the gene product of interest is operably linked to a Mueller glia-specific regulatory element (e.g., a glia-specific promoter), e.g., a regulatory element that selectively expresses the operably linked gene in a retinal glial cell. Suitable glia-specific regulatory elements include, for example, the glial fibrillar acidic protein (GFAP) promoter (Besnard et al. (1991) Journal of Biological Chemistry 266(28): 18877-18883). In some instances, the nucleotide sequence encoding the gene product of interest is operably linked to a bipolar cell-specific regulatory element (e.g., a bipolar cell-specific promoter), e.g., a regulatory element that selectively expresses the operably linked gene in the bipolar cell. Suitable bipolar cell-specific regulatory elements include, for example, the GRM6 promoter (Cronin et al. (2014) EMBO Molecular Medicine 6(9): 1175-1190).

[00229] For the purposes of the invention, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding an AAV capsid protein variant as described above. The isolated nucleic acid may be an AAV vector, eg a recombinant AAV vector.

[00230] The present invention also relates to a method of treating retinal disease comprising administering to an individual in need thereof an effective amount of an rAAV virion variant comprising a transgene of interest as described above and described herein. One skilled in the art can readily determine the effective amount of the rAAV virion in question and that the disease has been treated by testing for a change in one or more functional or anatomical parameters, e.g., visual acuity, visual field, ability to respond to electrophysiological signals in light and in the dark, color vision, contrast sensitivity, anatomy, state and vasculature of the retina, eyeball mobility, preference and stability of fixation.

[00231] Non-limiting methods for assessing retinal function and changes include visual acuity assessment (e.g., best corrected visual acuity [BCVA], ability to ambulate, navigate, detect and distinguish objects), visual field assessment (e.g., static and kinetic perimetry of the visual field ), performing a clinical examination (eg, slit lamp examination of the anterior and posterior segments of the eye), assessing the ability to respond to electrophysiological signals of all wavelengths in light and in the dark (eg, all forms of electroretinography (ERG) [full-format, multifocal, and patterns] , all forms of induced visual potential (VEP), electrooculography (EOG), assessment of color vision, dark adaptation and/or contrast sensitivity). Non-limiting methods for evaluating retinal anatomy and health and changes include optical coherence tomography (OCT), fundus photography, adaptive optics laser scanning ophthalmoscopy (AO-SLO), fluorescence and/or autofluorescence; measurement of eyeball mobility and eye movement (eg, nystagmus, fixation preference and stability), measures of recorded outcomes (patient-reported changes in visually mediated and non-visually mediated behaviors and activities, patient-reported outcomes [PRO] based on QoL questionnaires) , activities of daily living, and measures of neurological function (eg, functional magnetic resonance imaging (MRI)).

[00232] In some embodiments, an effective amount of the rAAV virion in question results in a reduction in the rate of loss of retinal function, anatomical integrity, or retinal health, such as a 2-fold, 3-fold, 4-fold, or 5-fold reduction, or more, the rate of loss, and thus the progression of the disease, for example, to reduce, 10 times or more, the rate of loss, and thus the progression of the disease. In some embodiments, an effective amount of the rAAV virion in question results in an increase in visual function, retinal function, improvement in retinal anatomy or health, and/or improvement in eyeball motility and/or improvement in neurological function, e.g. times, 3 times, 4 times, or 5 times or more retinal function, retinal anatomy or health, and/or improvement in eyeball mobility, e.g., 10 times or more improvement in retinal function, anatomy or retinal health, and/or improved eyeball mobility. As understood by one of skill in the art, the dose required to achieve the desired effect of treatment can typically range from 1 x 10 ⁸ to about 1 x 10 ¹⁵ recombinant virions, generally referred to as those of skill in the art, as from 1×10 ⁸ to approximately 1×10 ¹⁵ "vector genomes".

[00233] The rAAV virion in question can be administered by intraocular injection, for example, by intraocular injection, subretinal injection, by suprachoroidal injection, or by any convenient mode or route of administration that can deliver the rAAV virion to the eye. Other convenient modes or routes of administration include, without limitation, intravenous, intra-arterial, periocular, intracameral, subconjunctival and sub-Tenon injections, and topical and intranasal administration. When administered by injection into the vitreous, the subject rAAV virion is able to move through the vitreous and cross 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 retina and vitreous body, formed by astrocytes and the ends of the processes of Muller cells), and / or move through the layers of the retina more efficiently, compared with the ability of the AAV virion containing the corresponding original AAV capsid protein.

[00234] The capsid protein variant described herein is isolated, eg, purified. In some embodiments, the capsid protein variant described herein is incorporated into an AAV vector or a recombinant AAV virion (rAAV). In other embodiments, such AAV vector variants and/or AAV virion variants are used in an in vivo or ex vivo method for treating eye disease in the primate retina.

[00235] The present invention further relates to host cells, such as, without limitation, isolated (genetically modified) host cells containing the nucleic acid in question. The host cell of the invention described herein may be an isolated cell, such as a cell from an in vitro cell culture. Such a host cell can be used to produce the variant rAAV virion in question, as described herein. In one embodiment, such a host cell is stably genetically modified using a nucleic acid. In other embodiments, the host cell is temporarily genetically modified using a nucleic acid. Such a nucleic acid is introduced stably or transiently into a host cell using established methods including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like. For stable transformation, the nucleic acid typically further comprises a selectable marker, for example any of several well known selectable markers such as neomycin resistance and the like. Such a host cell is obtained by introducing the nucleic acid into any of a variety of cells, eg, mammalian cells, including, eg, mouse cells and primate (eg, human) cells. Exemplary mammalian cells include, but are not limited to, primary cells and cell lines, where 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. .P. Exemplary host cells include, without limitation, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. . CRL-1573), Vero cells, NIH 3T3 cells (e.g. ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g. ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), cells COS, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. A host cell can also be obtained using a baculovirus to infect insect cells, such as AAV-producing Sf9 cells (see, for example, US Patent No. 7271002; US Patent Application Ser. No. 12/297958). In some embodiments, the genetically modified host cell includes, in addition to a nucleic acid comprising a nucleotide sequence encoding an AAV capsid protein variant as described above, a nucleic acid comprising a nucleotide sequence encoding one or more AAV rep proteins. In other embodiments, the host cell further comprises a variant of the rAAV vector. The rAAV virion variant can be generated using such host cells. Methods for producing the rAAV virion are described, for example, in US Patent Publication No. 2005/0053922 and US Patent Publication No. 2009/0202490.

[00236] The present invention further provides a pharmaceutical composition comprising: a) an rAAV virion variant as described above and described 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 a human or non-human patient. Such excipients, carriers, diluents and buffers include any drug that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerin, and ethanol. Pharmaceutically acceptable salts may include, for example, inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and organic acid salts such as acetates, propionates, malonates, benzoates, and the like. In addition, adjuvants such as humectants or emulsifiers, surfactants, pH buffering agents, and the like may be present in such carriers. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described in detail in a variety of 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., 7th ^ed ., Lippincott, Williams, &Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. ^Kibbe et al., eds., 3rd ed. amer. Pharmaceutical Assoc. In some aspects of the present invention, the present invention relates to a pharmaceutical composition containing from about 1x10 ⁸ to about 1x10 ¹⁵ recombinant viruses, or from 1x10 ⁸ to about 1x10 ¹⁵ vector genomes, where each specified recombinant virus contains a genome encoding one or more gene products.

[00237] Some embodiments of the invention are illustrated in the following paragraphs 1-36:

1. An adeno-associated virus (AAV) capsid protein variant containing a peptide insert in the GH-loop of the capsid protein, compared to the corresponding parental AAV capsid protein, where the peptide insert contains the amino acid sequence ISDQTKH (SEQ ID NO:14) and where the capsid protein variant imparts increased infectivity for the retinal cell, compared with the infectivity of the corresponding original AAV capsid protein for the retinal cell.

2. The AAV capsid protein variant of claim 1, wherein the peptide insert contains the amino acid sequence Y ₁ Y ₂ ISDQTKHY ₃ , where each of Y ₁ -Y ₃ is independently selected from Ala, Leu, Gly, Ser, Thr, and Pro.

3. The AAV capsid protein variant of claim 2, wherein the peptide insert contains the amino acid sequence LAISDQTKHA (SEQ ID NO:28).

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

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

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

7. An AAV capsid protein variant according to any one of paragraphs 1-6, wherein the capsid protein comprises (i) the amino acid sequence ISDQTKH (SEQ ID NO:14) and (ii) the P34A amino acid substitution and is at least 90% identical, at least at least 95% identical or at least 99% identical to the sequence indicated as SEQ ID NO:42.

8. The AAV capsid protein variant of claim 7, wherein the capsid protein primarily consists of the amino acid sequence shown as SEQ ID NO:42.

9. A recombinant AAV virion (rAAV) containing an AAV capsid protein variant according to any one of paragraphs 1-8 and a heterologous nucleic acid containing a nucleotide sequence encoding a gene product.

10. rAAV according to item 9, where the gene product is a polypeptide.

11. The rAAV of claim 10, wherein the heterologous nucleic acid comprises a nucleotide sequence encoding a polypeptide that inhibits vascular endothelial growth factor (VEGF) activity.

12. rAAV according to item 11, wherein the nucleotide sequence encodes a fusion protein.

13. rAAV according to item 12, wherein the nucleotide sequence encodes aflibercept (eylea).

14. rAAV according to item 13, where the nucleotide sequence is at least 90% identical, at least 95% identical, or at least 99% identical with the nucleic acid sequence indicated as SEQ ID NO: 65, or with nucleotides 79-1377 of SEQ ID NO:65, and preferably encodes the amino acid sequence shown as SEQ ID NO:66 or amino acids 27-458 of SEQ ID NO:66.

15. rAAV according to item 11, where the nucleotide sequence encodes a monoclonal antibody or antigen-binding fragment.

16. The rAAV of item 15, wherein the nucleotide sequence encodes ranibizumab (Lucentis).

17. rAAV according to item 15, where the nucleotide sequence (i) contains a sequence at least 90% identical, at least 95% identical, or at least 99% identical with the sequence of SEQ ID NO:67 (sc- ranibizumab HL) or nucleotides 58-1575 of SEQ ID NO:67 and encodes the amino acid sequence shown as SEQ ID NO:68, or (ii) encodes an amino acid sequence of amino acids 20-524 of SEQ ID NO:68.

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

19. rAAV according to item 15, where the nucleotide sequence (i) has a sequence of at least 90% identical, at least 95% identical or at least 99% identical with the sequence of SEQ ID NO:72 (sc- ranibizumab-Fc) or nucleotides 61-2277 of SEQ ID NO:72 and encodes the amino acid sequence shown as SEQ ID NO:73, or (ii) encodes an amino acid sequence of amino acids 21-752 of SEQ ID NO:73.

20. rAAV according to item 15, wherein the nucleotide sequence encodes brolucizumab.

21. rAAV according to item 20, where the nucleotide sequence has a sequence that is at least 90% identical, at least 95% identical, or at least 99% identical to the nucleic acid sequence indicated as SEQ ID NO:74, or with nucleotides 61-816 of SEQ ID NO:74, and preferably encodes the amino acid sequence shown as SEQ ID NO:75 or the amino acid sequence of amino acids 21-271 of SEQ ID NO:75.

22. rAAV according to item 15, where the nucleotide sequence of (i) has a sequence of at least 90% identical, at least 95% identical, or at least 99% identical with the sequence of SEQ ID NO: 76 (brolucizumab- Fc) or nucleotides 61-1530 of SEQ ID NO:76 and encodes the amino acid sequence shown as SEQ ID NO:77, or (ii) encodes the amino acid sequence of amino acids 21-498 of SEQ ID NO:77.

23. The rAAV of claim 11 wherein the heterologous nucleic acid comprises (i) an aflibercept coding sequence, preferably wherein the aflibercept coding sequence is at least 90% identical, at least 95% identical, or at least 99% identical. % identical to the nucleic acid sequence indicated as SEQ ID NO: 65 and encodes the amino acid sequence indicated as SEQ ID NO: 66, and (ii) the sequence encoding brolucizumab, preferably, where the sequence encoding brolucizumab is at least 90 % identical, at least 95% identical, or at least 99% identical to the nucleic acid sequence indicated as SEQ ID NO: 74 or SEQ ID NO: 76, and preferably encodes the amino acid sequence indicated as SEQ ID NO: 75 or SEQ ID NO:77.

24. An rAAV according to any one of 9-23, wherein the nucleotide sequence encoding the gene product is operably linked to an expression control sequence.

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

26. A method for delivering a VEGF inhibitor to a retinal cell, choroidal cell, lens cell, ciliated cell, iris cell, optic nerve cell, and/or corneal cell in a subject, comprising administering to the subject an rAAV virion according to any one of paragraphs 11-24 or a pharmaceutical composition according to paragraph 24.

27. The method of claim 26, wherein the rAAV virion or pharmaceutical composition is administered to the vitreous body of the subject.

28. A method of treating a VEGFa associated eye disease selected from wet (neovascular, exudative) age-related macular degeneration; macular edema after retinal vein occlusion; retinal neovascularization due to retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative diabetic retinopathy and proliferative diabetic retinopathy); myopic macular degeneration; branch retinal vein occlusion, gemiretinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectosis; neovascular glaucoma; hemorrhages of the vitreous body; retinal and choroidal neovascularization secondary to retinal diseases including, but not limited to, uveitis, trauma, retinal degenerative disorders, retinal and/or choroid genetic disease, eye tumors, corneal and iris neovascularization, in a subject in need of such treatment, by administering to the subject an effective amount of an rAAV according to any one of paragraphs 11-24 or a pharmaceutical composition according to paragraph 25.

29. The method of claim 28, wherein the VEGFa-associated 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.

30. The method according to any one of paragraphs 26-29, wherein the rAAV comprises a capsid protein essentially consisting of the amino acid sequence shown as SEQ ID NO:42 and a heterologous nucleic acid containing an aflibercept-encoding sequence, preferably, wherein the aflibercept-encoding sequence , has the sequence shown in SEQ ID NO:65.

31. The method according to any one of paragraphs 26-29, where rAAV contains a capsid protein, mainly consisting of the amino acid sequence indicated as SEQ ID NO:42, and a heterologous nucleic acid containing a sequence encoding ranibizumab, sc-ranibizumab HL, sc- ranibizumab LH, or sc-ranibizumab-Fc, preferably, wherein the sequence encoding the sc-ranibizumab HL, sc-ranibizumab LH, or sc-ranibizumab-Fc is the sequence shown in any of SEQ ID NOs: 67, 69, 70 and 72.

32. The method according to any one of paragraphs 26-29, where the rAAV contains a capsid protein, mainly consisting of the amino acid sequence indicated as SEQ ID NO:42, and a heterologous nucleic acid containing a sequence encoding brolucizumab or brolucizumab-Fc, preferably, where the sequence encoding brolucizumab or brolucizumab-Fc has the sequence shown in SEQ ID NO:74 or SEQ ID NO:76.

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

34. The method of any one of items 26-33, wherein the subject is a human.

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

36. An isolated, genetically modified host cell containing the nucleic acid according to paragraph 35.

EXAMPLES

[00238] The following examples are provided so as to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are intended neither to limit the scope of what the present inventors consider their invention nor to represent that the experiments below represent all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (eg quantities, temperature, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or so.

[00239] General methods of molecular and cellular biochemistry can be found in such standard manuals as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (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), the contents of which are incorporated herein by reference. The reagents, cloning vectors, and genetic manipulation kits referred to herein are available from commercial vendors such as BioRad, Stratagene, Invitrogen, Sigma-Aldrich, and ClonTech.

[00240] Example 1

[00241] Vitreous injection and tissue collection. One male cynomolgus macaque ( macaca fascicularis ) aged 4-10 years and weighing at least 4 kg was dosed by intravitreal injection through the sclera (approximately 3 mm posterior to the limbus using a delivery method and device suitable for human use). ). The animal was anesthetized and injected with a local anesthetic. 100 μl of the library was injected into each eye.

[00242] Euthanasia was performed by a qualified veterinarian using 100 mg/kg sodium pentobarbital on day 14±3. The eyes were excised and kept at 4° C. until slicing.

[00243] Cutting fabrics. The eyes were cut along the border between the visual and blind parts of the retina using a scalpel, and the anterior segment was removed. Scalpel exit incisions were made in the retina around the fovea to allow fixation of the retina, and the vitreous humor was removed. Six retinal samples from each quadrant (anterior, posterior, nasal and temporal) were collected as shown in Figure 2 and cellular material was isolated corresponding to RPE cells, photoreceptors, bipolar cells, amacrine cells, horizontal cells and/or ganglion cells.

[00244] Directed evolution. The directed evolution method is shown in Figures 1A-1E. Briefly, a viral capsid library containing 20+ patented combinations of DNA mutagenesis methods and cap genes was obtained (FIG. 1A). The viruses are then packaged (Figure 1B) - such that each particle consists of a mutant capsid surrounding the cap gene encoding this capsid - and purified. The capsid library is subjected to selective pressure in vivo . Tissue or cellular material of interest is harvested to isolate AAV variants that successfully infect that target, and successful viruses are isolated . Successful clones enrich through repeated selection (stage 1 - figure 1D). The selected cap genes are then subjected to patented re-diversification and enriched through additional selection steps to repetitively increase virus fitness (Stage 2 - FIG. 1D). Variants identified during vector selection steps 1 and 2 show the transducibility of primate retinal cells (FIG. 1E).

[00245] Successful isolation of AAV capsid genomes: cycles 1-6. The capsids isolated from each selection cycle were used to package the library, which was injected to initiate the subsequent selection cycle. Isolation of capsid genes from tissue represents the successful internalization of a library of vectors into the tissue of interest. After cycle 4, the library was re-diversified before library packaging and injection for cycle 5. Isolation of viral genomes from RPE, PR, inner nuclear layer (INL) and ganglion cell layer (GCL) of retinal tissue from a representative selection cycle is shown in Figure 3 The bars within the boxes represent successful isolation of viral genomes.

[00246] Sequencing analysis: cycles 3-6. During cycles 3-6, sequencing of individual clones in the library was performed to determine the frequency of variants within the population. Variants were evaluated by the presence of motifs in the sequencing data. Variants were motif grouped based on the presence of a unifying variant (eg, a specific point mutation or a specific peptide insert sequence at a permanent location within the capsid) occurring in multiple sequences. Motives representing at least 5% of the sequenced population in two or more selection cycles, or at least 10% of the sequenced population in one or more selection cycles, are shown in Figure 4A (cycle 3 sequencing analysis), 4B (cycle 4 analysis sequencing), 4C (run 5 sequencing analysis) and 4D (run 6 sequencing analysis).

[00247] Several representative clones identified as conferring increased infectivity for retinal cells are listed in Table 1 below (each clone contains identified substitution(s) and/or peptide insert and is otherwise identical to SEQ ID NO:2; selection cycle, the number of sequences and frequency (in brackets) are listed for each clone):

Table 1 Amino acid sequence of the AAV VP1 capsid protein conferring increased infectivity on one or more retinal cells. The substitutions listed in column 2 are based on the wild-type AAV2 amino acid sequence, ie. in the absence of the inserted peptide. Insert Replacement Whole retina RPE Photoreceptor 588~LAISDQTKHA~ (SEQ ID NO:28) Not Cycle 4, 5, 6 Cycle 3, 5, 6 Cycle 4, 5 588~LAISDQTKHA~ (SEQ ID NO:28) +M1L+L15P +P535S Cycle 6
1 (1.61%) 588~LAISDQTKHA~ (SEQ ID NO:28) +P34A Cycle 5
1 (1.89%) 588~LAISDQTKHA~ (SEQ ID NO:28) +P34A+S731L Cycle 5
1 (1.82%) 588~LAISDQTKHA~ (SEQ ID NO:28) +N57D Cycle 4
1 (1.33%) 588~LAISDQTKHA~ (SEQ ID NO:28) +N66K Cycle 5
1 (1.82%) 588~LAISDQTKHA~ (SEQ ID NO:28) +R81Q Cycle 6
1 (1.61%) 588~LAISDQTKHA~ (SEQ ID NO:28) +Q101R Cycle 4
1 (2.27%) 588~LAISDQTKHA~ (SEQ ID NO:28) +S109T Cycle 3
1 (1.85%) 588~LAISDQTKHA~ (SEQ ID NO:28) +R144K Cycle 5
1 (1.82%) 588~LAISDQTKHA~ (SEQ ID NO:28) +R144M Cycle 5
1 (1.82%) 588~LAISDQTKHA~ (SEQ ID NO:28) +Q164K Cycle 4
1 (2.27%) 588~LAISDQTKHA~ (SEQ ID NO:28) +Q164K+V708I Cycles 3 and 4
2 (3.7%)
1 (1.33%) 588~LAISDQTKHA~ (SEQ ID NO:28) +T176P Cycle 4
1 (1.33%) 588~LAISDQTKHA~ (SEQ ID NO:28) +L188I Cycle 5
1 (2.27%) 588~LAISDQTKHA~ (SEQ ID NO:28) +S196Y Cycle 4
1 (1.33%) 588~LAISDQTKHA~ (SEQ ID NO:28) +G226E Cycle 4
1 (2.27%) 588~LAISDQTKHA~ (SEQ ID NO:28) +G236V Cycle 4
1 (1.33%) 588~LAISDQTKHA~ (SEQ ID NO:28) +I240T Cycle 3
1 (1.85%) 588~LAISDQTKHA~ (SEQ ID NO:28) +N312K Cycle 4
1 (1.33%) 588~LAISDQTKHA~ (SEQ ID NO:28) +P363L Cycle 6
1 (1.61%) 588~LAISDQTKHA~ (SEQ ID NO:28) +T456K Cycle 6
1 (1.61%) 588-LAISDQTKHA~ (SEQ ID NO:28) +I698V Cycle 5
1 (1.89%) 588-LAISDQTKHA~ (SEQ ID NO:28) +V708I Cycle 4, 5, 6 Cycle 3, 4, 5 Cycle 4, 5 588~LAISDQTKHA~ (SEQ ID NO:28) +V708I+R484C Cycle 5
1 (2.27%) 588~LAISDQTKHA~ (SEQ ID NO:28) N312K+N449D+N551S+I698V+L735Q Constructed Constructed 588~LGISDQTKHA~ (SEQ ID NO:29) Not Cycle 5
1 (1.89%) 588~LAQADTTKNA~ (SEQ ID NO:27) Not Cycle 3, 4, 5, 6 Cycle 3, 4, 5 Cycle 4, 5 588~LAQADTTKNA~ (SEQ ID NO:27) +E36D Cycle 5
1 (2.27%) 588~LAQADTTKNA~ (SEQ ID NO:27) +P250S Cycle 6
1 (1.61%) 588~LAQADTTKNA~ (SEQ ID NO:27) +A524T Cycle 3
1 (1.85%) 588~LAQADTTKNA~ (SEQ ID NO:27) +A593E Cycle 4
1 (1.33%) 588~LAQADTTKNA~ (SEQ ID NO:27) +I698V Cycles 5 and 6
1 (1.61%)
1 (1.89%) 588~LAQADTTKNA~ (SEQ ID NO:27) +V708I Cycle 3, 4, 5, 6 Cycle 3, 4, 5 588~LAQADTTKNA~ (SEQ ID NO:27) +V708I+V719M Cycles 3 and 4
1 (2.08%)
2 (4.55%) 588~LAQADTTKNA~ (SEQ ID NO:27) +V719M Cycle 4
1 (2.27%) 588~LAHQDTTKNA~ (SEQ ID NO:34) Not Cycle 5
2 (3.77%) 588~LANQDYTKTA~ (SEQ ID NO:31) Not Cycles 3, 4 and 5
5 (9.26%)
1 (1.33%)
3 (5.66%) Cycles 3, 4 and 5
2 (4.17%)
2 (4.55%)
2 (2.27%) Cycle 5
1 (1.82%) 588~LANQDYTKTA~ (SEQ ID NO:31) +S109T+S463Y Cycle 3
1 (1.85%) 588~LANQDYTKTA~ (SEQ ID NO:31) +D368H Cycle 3
1 (1.85%) 588~LANQDYTKTA~ (SEQ ID NO:31) +V708I Cycle 4
1 (1.33%) Cycle 3
1 (2.08%) 588~LAHDITKNIA~ (SEQ ID NO:32) Not Cycles 3, 4 5 and 6
1 (1.85%)
1 (1.33%)
1 (1.89%)
2 (3.23%) 588~LAHDITKNIA~ (SEQ ID NO:32) +S109T Cycle 4
1 (1.33%) 588~LAHDITKNIA~ (SEQ ID NO:32) +R389S Cycle 5
1 (1.89%) 588~LAHDITKNIA~ (SEQ ID NO:32) +A593E Cycle 3
1 (1.85%) 588~LAHDITKNIA~ (SEQ ID NO:32) +V708I Cycle 4, 5 588~IAHDITKNIA~ (SEQ ID NO: 60) +V708I Cycle 3
1 (2.08%) 588~LAPNSTHGSA~ (SEQ ID NO:40) +V708I Cycle 3
1 (1.85%) 588~LANKTTNKDA~ (SEQ ID NO:35) Not Cycle 5
1 (2.27%) 588~LANKTTNKDA~ (SEQ ID NO:35) +N449D Cycle 4
1 (2.17%) 588~LAHPDTTKNA~ (SEQ ID NO:33) Cycle 6
1 (1.61%) 588~LATNRTSPDA~ (SEQ ID NO:39) Cycle 6
1 (1.61%) 588~LPQANANENA~ (SEQ ID NO:37) Cycle 5
1 (1.89%) 588~LAASDSTKAA~ (SEQ ID NO:30) Cycles 3 and 4
1 (1.85%)
1 (1.33%) 588~LAKDRAPSTA~ (SEQ ID NO:41) Cycle 3
1 (1.85%) 588~LPISNENEHA~ (SEQ ID NO:36) Cycle 4
1 (2.17%) 588~LAGKSKVIDA~ (SEQ ID NO:38) Cycle 5
1 (1.82%) NO P34A Cycle 5
1 (1.89%) Cycles 4, 5
3 (6.82%)
1 (2.27%) Cycle 4
1 (2.17%) NO P64S Cycle 3
1 (1.85%) Cycle 4
1 (2.27%) NO S109T Cycle 4
2 (2.67%) Cycles 3, 4, 5
4 (8.33%)
1 (2.27%)
1 (2.27%) NO S109T+P8L Cycle 3
1 (2.08%) NO S109T+Q120R Cycle 3
1 (2.08%) NO S109T+A493V
+A593E+V708I Cycle 3
1 (1.85%) NO Q164K Cycles 4 and 5
2 (2.67%)
1 (1.89%) Cycle 4
1 (2.27%) NO Q175H Cycle 3
1 (1.85%) Cycle 4
1 (2.17%) NO S196Y Cycle 3
1 (1.85%) Cycle 4
1 (2.27%) NO A593E Cycles 3, 4, 5
3 (5.56%)
7 (9.33%)
1 (1.89%) Cycles 3, 4, 5
12 (25%)
7 (15.9%)
14(31.8%) Cycle 4
1 (2.17%) NO A593E+Q464R Cycle 3
1 (2.08%) NO A593E+N596D Cycle 4
1 (2.27%) NO A593E+N596D
+T491A Cycle 3
2 (4.17%) NO A593E+V708I Cycles 3, 4
2 (3.7%)
2 (2.67%) Cycles 3, 4, 5
4 (8.33%)
1 (2.27%)
1 (2.27%) NO I698V Cycle 5
1 (1.89%) Cycle 5
1 (2.27%) NO V708I Cycles 3, 4,
2 (3.7%)
5 (6.67%) Cycles 3, 4, 5
1 (2.08%)
4 (9.09%)
4 (9.09%) NO V708I+V719M Cycle 4
2 (4.55%) NO V708I+G727D Cycle 5
1 (2.27%) NO V708I+R733C Cycle 4
1 (2.17%)

[00248] A clone having the following capsid VP1 ancestral sequence has been identified as a capsid having increased infectivity for one or more retinal cells:

[00249]

MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPLGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASAGSTNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLARTQSTGGTAGTRELLFSQAGPSNMSAQAKNWLPGPCYRQQRVSKTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEDRFFPSSGVLIFGKQGAGANNTALENVMMTSEEEIKTTNPVATEQYGVVASNLQSSNTAPVTGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPANPPAVFTPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYAKSTNVDFAVDNEGVYSEPRPIGTRYLTRNL. (SEQ ID NO:59).

[00250] This ancestral capsid variant evolved from an ancestral capsid of SEQ ID NO:58 in which degeneracy 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) evolved to contain alanine (A) in 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, alanine (A) at 551, alanine (A) at 555, glutamic acid (E) at 557, methionine (M) at 561, serine (S) at 563, glutamine (Q) at 577, serine (S) at 583, valine (V) at 593, threonine (T) at 596, alanine (A) at 661, valine (V) at 662, threonine (T) at 664, proline (P) at 665, threonine (T) at 710, aspartic acid (D) at 717, asparagine (N) at 718, glutamic acid (E) at 719 and serine (S) at 723.

[00251] The AAV virion variants described herein may include appropriate parameters, features, modifications, advantages, and rational design options clearly apparent to one skilled in the art of constructing AAV viral vectors.

[00252] Example 2

[00253] Directed evolution has been used to discover new adeno-associated virus (AAV) variants with superior gene delivery to retinal cells after intravitreal injection (IVT), an administration method with significant advantages over other methods of gene delivery to the human eye (Example 1). Cell tropism following vitreous administration of a novel AAV variant containing the P34A substitution and a LAISDQTKHA peptide (SEQ ID NO:28) inserted at amino acid position 588 (LAISDQTKHA+P34A; SEQ ID NO:42) was assessed in vivo in non-human primates (NHP ) as a representative example of the ability of ISDQTKH (SEQ ID NO:14) AAV variants to transduce retinal cells.

[00254] Recombinant AAV virions containing either an AAV2 capsid or a novel capsid variant LAISDQTKHA+P34A (SEQ ID NO:42) and a genome containing a green fluorescent protein (GFP) transgene operably linked to the CMV promoter (AAV2.CMV.GFP and LAISDQTKHA+P34A.CMV.GFP, respectively) or the CAG promoter (AAV2.CAG.EGFP and LAISDQTKHA+P34A.CAG.EGFP, respectively) was generated using standard methods. African green monkeys (Figures 7, 8) or cynomolgus monkeys (Figure 9) were intravitreally injected with varying doses of vector ranging from 4×10 ¹⁰ GV to 1×10 ¹² GV per eye (see Figure legends for details), and retinal cell transduction was assessed in life by fundus fluorescence imaging using Heidelberg Spectralis™.

[00255]Delivery into the vitreous body of AAVs containing the novel LAISDQTKHA+P34A variant (SEQ ID NO:42) resulted in broader and more robust transgene expression in the NHP retina than AAV2 (Figures 7-9). The images revealed that new AAV capsid variant provides robust expression in the center of the fovea (a region rich in cones); in the parafoveal ring (in an area rich in retinal ganglion cells) and in the periphery (in an area rich in many cell types including rods, Muller glial cells, amacrine cells, bipolar cells) as early as 2 weeks after injection. In contrast, and in line with results published by other authors, wild-type AAV2 provides weaker expression, present primarily in the parafoveal ring, and can only be detected at later time points. Immunohistochemical analysis of various areas of the retina, performed 3 weeks after injection, confirmed that a variety of retinal cell types, including retinal pigment epithelial cells, photoreceptor rods and cones, and retinal ganglion cells, were successfully transduced into the retina (Figures 10A-10E).

[00256] This study illustrates superior gene delivery by an ISDQTKH-containing variant following a clinically preferred route of administration compared to clinically significant AAV2. Similar efficacy is achievable using other variants containing this peptide insert motif. Likewise, similar efficacy is achievable using the other variants described herein, identified using the same directed evolution method.

[00257] Example 3

[00258] The cell tropism of the novel AAV variant LAISDQTKHA+P34A (SEQ ID NO:42) for retinal pigment epithelial (RPE) and photoreceptor (PR) cells was assessed in vitro using RPE cells and PR cells derived from fibroblast-derived induced pluripotent human stem cells (FB-iPSC) or human embryonic stem cells (ESC).

[00259] AAV virions containing either an AAV2 capsid or a new capsid variant LAISDQTKHA+P34A (SEQ ID NO:42) and a genome containing a green fluorescent protein (EGFP) transgene operably linked to the CAG promoter (AAV2.CAG.EGFP and LAISDQTKHA+P34A.CAG.EGFP, respectively) were obtained using standard methods. Human RPE cell cultures were derived from the ESI-017 human embryonic stem cell line or human fibroblast-derived induced pluripotent stem cells (FB-iPSC) using a 45-day differentiation method. Maturation to 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 phagocytosis of the outer segments of the rods. PR cultures were generated by a multi-step ophthalmic cup formation paradigm and confirmed to contain PR by detection of recoverin and S-opsin expression after 179 days in culture.

[00260] Compared to AAV2, LAISDQTKHA+P34A (SEQ ID NO:42) provided significantly higher transduction efficiency and transgene expression in human RPE cultures seven days post-infection as determined by immunofluorescence (Figures 11A-B), flow cytometry (2.7 times magnification; Figures 11C-D) and Western blot analysis (Figures 11E-F). Similarly, robust transduction and expression was achieved using LAISDQTKHA+P34A.CAG.EGFP in human PR cultures 32 days post-infection. This study illustrates the superior ability of ISDQTKH (SEQ ID NO:14)-containing variants to deliver genes to retinal cells.

[00261] Example 4

[00262] Design and construction of vectors expressing anti-VEGF proteins. The amino acid sequence of aflibercept consists of the human Flt1 signal peptide, VEGFR1 domain 2 and VEGFR2 domain 3 fused to a human IgG1 Fc region (Figure 12A; SEQ ID NO:66). The amino acid sequences of the light and heavy chains of ranibizumab were linked via a flexible protein linker to convert the double-stranded antigen-binding fragment (Fab) to a single-stranded Fab (scFab). The light-heavy (LH) form consists of the human kappa Ig light chain signal peptide, variable light, constant light, variable heavy, and constant heavy 1 domains of ranibizumab linked via a flexible peptide (Figure 12B; SEQ ID NO:71). The heavy-light (HL) form is similar except that the signal peptide is derived from the heavy chain of human IgG and the heavy and light domains are on opposite sides of the linker compared to the LH form (Figure 12C; SEQ ID NO:68) . The brolucizumab design includes the variable light and variable heavy domains linked via a flexible peptide to a human Ig kappa light chain signal peptide for secretion from mammalian cells (Figure 12D; SEQ ID NO:75). The open reading frames were codon optimized for improved expression in human cells and synthesized in GeneArt or GenScript and provided in a standard plasmid cloning vector. DNA of interest was excised from this plasmid using the restriction enzymes PstI and BglII and inserted into the pAAV-CAG-SV40 pA plasmid between the CAG promoter and the SV40 polyA signal. The sc-ranibizumab-Fc design consists of the LH form of sc-ranibizumab fused to the human IgG1 Fc region from the aflibercept design (Figure 12E; SEQ ID NO:73). The brolucizumab-Fc design consists of brolucizumab fused to the human IgG1 Fc region from the aflibercept design (Figure 12F; SEQ ID NO:77). The sc-ranibizumab LH, brolucizumab, and Fc regions were amplified by PCR from the original cloning vectors, with extensions containing the BsmBI type IIS restriction enzyme site at the 3' end (sc-ranibizumab LH and brolucizumab) or the 5' end (Fc). The resulting PCR products were digested with restriction enzymes PstI and BsmBI (sc-ranibizumab LH and brolucizumab) or BsmBI and BglII (Fc) and inserted into the pAAV-CAG-SV40 pA plasmid between the CAG promoter and the SV40 polyA signal. Ligation reactions were used to transform E. coli and positive clones were identified by restriction digestion. Clones were then grown on a larger scale and plasmid DNA was purified using the Qiagen Endo-Free Maxiprep kit. The identity of the plasmids was verified by restriction digestion and sequencing. The pAAV-CAG-SV40 polyA plasmid contains AAV2 inverted terminal repeats (ITRs) allowing packaging of anti-VEGF sequences into AAV capsids and delivery to cells of interest in the retina via intravitreal injection.

[00263] HEK293T cells were sham-transfected or transfected with a plasmid containing either a GFP transgene or an anti-VEGF protein (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), BRO (SEQ ID NO:74)) under the control of the CAG promoter using the FuGene6 transfection reagent in two separate experiments. Media were collected 48 hours after transfection and analyzed using an aflibercept ELISA kit (Eagle Biosciences, Immunoguide IG-AA115). Anti-VEGF proteins were detected with the anti-Fc IgG HRP provided in the kit as a reagent (dummy, GFP and AFLB) or the anti-H+L IgG HRP (sc-RNBZ HL, sc-RNBZ LH1 and sc-RNBZ LH2) . Concentrations were calculated relative to clinical ilea (false, GFP and AFLB) or lucentis (sc-RNBZ HL, sc-RNBZ LH1, sc-RNBZ LH2 and BRO). In the first of two experiments, aflibercept expression was approximately 15 μg/mL and sc-ranibizumab expression ranged from 1.6 to 2.8 μg/mL, where the LH forms were expressed at similar concentrations and the HL form was expressed at approximately two times lower concentration (figure 13A). In the second experiment, aflibercept expression was approximately 50 μg/ml and sc-ranibizumab LH1 expression was approximately 4.0 μg/ml (Figure 13B). The signal for brolucizumab was very low, most likely due to poor recognition by the detection antibody. VEGF binding activity was not detected in media after mock transfection or GFP transfection.

[00264] Example 5

[00265] For direct visualization of anti-VEGF proteins and confirmation of protein size, Western blot analysis of media from cells transfected with GFP expression plasmids, aflibercept (SEQ ID NO:65), sc-ranibizumab (SEQ ID NO:67, 69 and 70) or brolucizumab (SEQ ID NO:74) (Figure 14). Media from transfected HEK293T cells were separated on Bolt 4-12% Bis-Tris Plus gel (Invitrogen, Cat# NW04122BOX) and the separated proteins were transferred to nitrocellulose using the iBlot2 device. The blot was probed with HRP-conjugated goat anti-human IgG Fc (Thermo, cat# 31413, left panel) or goat anti-human Fab IgG (Thermo, cat# 31482, right panel), using an iBind Flex device, and visualized using SuperSignal West Dura chemiluminescent substrate (Thermo cat# 34076). Images were acquired using an iBright FL1000 imaging device.

[00266] The aflibercept sample appeared similar to the Eilea clinical reference protein. No signal was present in the negative control samples. The clinical reference protein lucentis is restored to separate 24 kD light and heavy chains, while the sc-ranibizumab proteins migrate at approximately 48 kD, due to the existence of a polypeptide linker linking the light and heavy chains into a single protein. There is more of the LH form of the protein compared to the HL form of the protein, according to protein quantification obtained by ELISA (FIG. 13A). The signal for brolucizumab is low, most likely due to poor recognition by the detection antibody. The protein migrates with the correct molecular weight of 24 kDa.

[00267] Example 6

[00268] As an additional method to determine VEGF binding activity, a competitive ELISA of VEGF was performed (Figure 15). Eylea or Lucentis clinical reference proteins and media from transfected HEK293T cells were incubated with 13 pM VEGF at room temperature overnight. Samples were analyzed for free VEGF using the Quantikine VEGF ELISA kit (R&D Systems, Cat# DVE00). In the first of two experiments, the inhibition curves for the four anti-VEGF proteins from the transfection samples were very similar to clinical ilea and lucentis. Aflibercept and Eylea competed more strongly for VEGF than the sc-ranibizumab and Lucentis variants (Figure 15A). All three forms of sc-ranibizumab (SEQ ID NOS: 67, 69 and 70) were nearly identical. In the second experiment, all anti-VEGF constructs competed for VEGF (Figure 15B). No competition activity was present for the GFP negative control sample.

[00269] Example 7

[00270] To confirm that anti-VEGF proteins expressed in transfected cells block VEGF binding to its receptor and thus its function, equal concentrations of clinical reference proteins and media from transfected HEK293T cells were mixed with 20 ng/mL VEGF and plated to PathHunter KDR cells (DiscoverX 93-0996Y1). These cells express VEGF receptor/beta-galactosidase fusion proteins that form active beta-galactosidase when bound to VEGF. Cells were lysed after 22 hours and analyzed for beta-galactosidase activity. Protein inhibition curves of anti-VEGF proteins from transfection samples are equivalent to those for clinical reference proteins, showing that the expressed proteins block VEGF function in a cell-based assay (Figure 16). In two subsequent experiments, cellular VEGF neutralization assays were also performed using equal volumes of media from HEK293T cells transfected with anti-VEGF constructs. In the first of these experiments, plasmids were evaluated to express GFP, aflibercept (SEQ ID NO:65), sc-ranibizumab HL (SEQ ID NO:67) or sc-ranibizumab LH1 (SEQ ID NO:69). All of the anti-VEGF constructs evaluated neutralized VEGF (Figure 17A). The sc-ranibizumab LH form neutralized VEGF more strongly than the sc-ranibizumab HL form. In the second experiment, plasmids for the expression of GFP, aflibercept (SEQ ID NO:65), sc-ranibizumab LH1 (SEQ ID NO:69) or brolucizumab (SEQ ID NO:74) were mixed with 8 ng/ml VEGF and placed in PathHunter cells KDR (DiscoverX 93-0996Y1). Cells were lysed after 18 hours and analyzed for beta-galactosidase activity. Again, all of the anti-VEGF constructs evaluated neutralized VEGF (Figure 17B). There was a slight matrix effect for the GFP control at all dilutions analyzed.

[00271] Example 8

[00272] Retinal pigment epithelial (RPE) cells were sham transfected or transfected with a plasmid containing either a GFP transgene or an anti-VEGF transgene under the control of the CAG promoter using the FuGeneHD transfection reagent. Media were collected 48 hours after transfection. Transfection efficiency for RPE cells was unusually low, as evidenced by the number of GFP expressing cells (data not shown), and thus VEGF binding activity was not detectable in the media following these transfections. The total concentration of VEGF in the media was assessed to determine whether low concentrations of anti-VEGF agents can reduce the secretion of VEGF in RPE. Media were collected 48 hours after transfection and analyzed using the Quantikine VEGF ELISA kit (R&D Systems, cat#DVE00). A strong decrease in VEGF levels was observed after expression of aflibercept (SEQ ID NO:65) and a decrease in VEGF levels after expression of sc-ranibizumab (SEQ ID NO:67, 69 and 70) (Figure 18). The effect on VEGF levels of all three forms of sc-ranibizumab was similar. Upon delivery through more efficient AAV transduction, the anti-VEGF transgenes resulted in higher concentrations of anti-VEGF proteins, which were quantified by the aflibercept ELISA kit.

[00273] Example 9

[00274] AAV expression plasmids carrying genes for aflibercept (SEQ ID NO:65), sc-ranibizumab (SEQ ID NO:67 and 69) or brolucizumab (SEQ ID NO:74) were used to package viral DNA into a capsid variant R100 (having the amino acid sequence shown as SEQ ID NO:42). Mature RPE cells were transduced 30 days after seeding into 12-well plates at MOI 5000 of these vectors in a volume of 1.0 ml of X-Vivo10 media. The medium was changed three days after transduction, collected and changed six days after transduction, and again collected and changed ten days after transduction.

[00275] Total VEGF concentration in media was analyzed using the Quantikine VEGF ELISA kit (R&D Systems Cat#DVE00) to determine the effect of transduction using anti-VEGF vectors on the level of free detectable endogenous VEGF secreted by RPE cells into the media (Figure 19). The endogenous level of VEGF, as shown in cells transduced with the GFP control vector, was 4500 pg/ml in the sample after 6 days and 8300 pg/ml in the sample after 10 days. Transduction with all anti-VEGF vectors resulted in low to undetectable levels of VEGF in media. It is likely that the amount of VEGF secreted by the RPE cells did not change, but rather that the VEGF in the media was bound by anti-VEGF proteins so that it was not detectable by ELISA. Additional experiments, including Western blotting, can be performed to determine the total amount of bound and unbound VEGF.

[00276] Media after RPE transductions were also analyzed using the aflibercept ELISA kit (Eagle Biosciences, Immunoguide IG-AA115). Anti-VEGF proteins were detected using an anti-IgG H+L HRP antibody (ThermoFisher, Cat# 31410). Concentrations were calculated relative to clinical ilea (AFLB and GFP) or lucentis (sc-RNBZ HL, sc-RNBZ LH1 and BRO). The expression of aflibercept (SEQ ID NO:65) was approximately 1800 ng/ml in the 6 day sample and 2800 ng/ml in the 10 day sample. The expression of sc-ranibizumab ranged from 700 ng/ml for HL (SEQ ID NO:67) in the sample after 6 days to 1700 ng/ml for LH (SEQ ID NO:69) in the sample after 10 days (figure 20). The signal for brolucizumab (SEQ ID NO:74) was very low, most likely due to poor recognition by the detection antibody. VEGF binding activity was not detected in media after GFP transduction.

[00277] Example 10

[00278] To directly visualize anti-VEGF proteins and confirm protein size, Western blot analysis of media from transduced RPE cells was performed (Figure 21). Equal volumes of media for day 6 and day 10 were separated on Bolt 4-12% Bis-Tris Plus gels (Invitrogen, Cat# NW04122BOX) and the separated proteins were transferred to nitrocellulose using the iBlot2 device. The blot was probed with HRP-conjugated goat anti-human IgG Fc (Thermo, cat# 31413, left panel) or goat anti-human Fab IgG (Thermo, cat# 31482, right panel), using an iBind Flex device, and visualized using SuperSignal West Dura chemiluminescent substrate (Thermo, Cat# 34076). Images were acquired using a ChemiDoc MP imaging device.

[00279] Samples of aflibercept (SEQ ID NO:65) appeared to be similar to the Eilea clinical reference protein (black arrow). No bands were present with the correct mobility in the GFP negative control sample. Clinical lucentis is reduced to separate 24 kDa light and heavy chains, while sc-ranibizumab HL (SEQ ID NO:67) and LH (SEQ ID NO:69) do not separate and migrate at an apparent molecular weight of 58 kDa as shown gray arrow. The signal for brolucizumab (SEQ ID NO:74) is low, most likely due to poor recognition by the detection antibody. The protein migrates at the correct molecular weight of 26 kDa, as indicated by the shaded arrow. Protein levels are almost similar in samples after 6 and 10 days.

[00280] Example 11

[00281] As an additional method to determine VEGF binding activity, a competitive ELISA of VEGF was performed (Figure 22). Equal volumes of media from transduced RPE cells were incubated with 13 pM VEGF at room temperature overnight. Samples were analyzed for free VEGF using the Quantikine VEGF ELISA kit (R&D Systems, Cat# DVE00). Media from cells transduced with all anti-VEGF constructs competed for VEGF. The results were similar for samples after 6 and 10 days. No competition activity was present for the GFP negative control sample. Free VEGF levels were higher for the lowest dilutions due to endogenous VEGF produced by RPE cells.

[00282] Example 12

[00283] To compare the neutralization activity of VEGF proteins against VEGF expressed in RPE cells from viral vectors, equal volumes of media from transduced RPE cells were mixed with 8 ng/ml VEGF and placed on PathHunter KDR (DiscoverX 93-0996Y1) cells (Figure 23) . Cells were lysed after 18 hours and analyzed for beta-galactosidase activity. Media from cells transduced with all anti-VEGF constructs neutralized VEGF. There was no neutralization of VEGF observed for media after GFP control transduction.

[00284] The foregoing merely illustrates the principles of the invention. It is understood that a person skilled in the art is able to develop various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its scope and scope. In addition, all examples and conventions cited in this specification are principally intended to assist the reader in understanding the principles of the invention and the concepts presented by the inventors of the present invention for the development of the prior art, and should be construed as not limiting such specifically cited examples and terms.

[00285] In addition, all statements in the present description, listing the principles, aspects and embodiments of the invention, as well as their specific examples, are intended to include both structural and functional equivalents. Further, such equivalents are intended to include both currently known equivalents and equivalents to be developed in the future, i.e. any element designed to perform the same function, regardless of structure. The scope of the present invention, therefore, is not limited to the illustrative embodiments shown and described in the present description. Instead, the scope and content of the present invention are contained in the appended claims.

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SEQUENCE LIST

<110> 4D Molecular Therapeutics Inc.

<120> ADENO-ASSOCIATED VIRUS CAPSID VARIANTS

AND USE FOR ANGIOGENESIS INHIBITION

<130> 090400-5010 WO

<150> US 62/590976

<151> 2017-11-27

<150> US 62/664726

<151> 2018-04-30

<160> 77

<170> PatentIn version 3.5

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Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Arg Gln Ala Ala Thr

580 585 590

Ala Asp Val Asn Thr Gln Gly Val Leu Pro Gly Met Val Trp Gln Asp

595 600 605

Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr

610 615 620

Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Lys

625 630 635 640

His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Asn

645 650 655

Pro Ser Thr Thr Phe Ser Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln

660 665 670

Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Lys

675 680 685

Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr

690 695 700

Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr

705 710 715 720

Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 3

<211> 736

<212> protein

<213> Adeno-associated virus 3A

<400> 3

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Gly

130 135 140

Ala Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly

145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Arg Gly Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

435 440 445

Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser

450 455 460

Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn

485 490 495

Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn

500 505 510

Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly

530 535 540

Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln

565 570 575

Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr

580 585 590

Thr Gly Thr Val Asn His Gln Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 4

<211> 736

<212> protein

<213> Adeno-associated virus 3B

<400> 4

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly

145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

435 440 445

Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser

450 455 460

Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn

485 490 495

Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn

500 505 510

Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly

530 535 540

Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln

565 570 575

Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr

580 585 590

Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 5

<211> 734

<212> protein

<213> Adeno-associated virus 4

<400> 5

Met Thr Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Glu

1 5 10 15

Gly Val Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro Lys

20 25 30

Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro Val

50 55 60

Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Gln

65 70 75 80

Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Gln Arg Leu Gln Gly Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Leu

115 120 125

Gly Leu Val Glu Gln Ala Gly Glu Thr Ala Pro Gly Lys Lys Arg Pro

130 135 140

Leu Ile Glu Ser Pro Gln Gln Pro Asp Ser Ser Thr Gly Ile Gly Lys

145 150 155 160

Lys Gly Lys Gln Pro Ala Lys Lys Lys Leu Val Phe Glu Asp Glu Thr

165 170 175

Gly Ala Gly Asp Gly Pro Pro Glu Gly Ser Thr Ser Gly Ala Met Ser

180 185 190

Asp Asp Ser Glu Met Arg Ala Ala Ala Gly Gly Ala Ala Val Glu Gly

195 200 205

Gly Gln Gly Ala Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys

210 215 220

Asp Ser Thr Trp Ser Glu Gly His Val Thr Thr Thr Ser Thr Arg Thr

225 230 235 240

Trp Val Leu Pro Thr Tyr Asn Asn His Leu Tyr Lys Arg Leu Gly Glu

245 250 255

Ser Leu Gln Ser Asn Thr Tyr Asn Gly Phe Ser Thr Pro Trp Gly Tyr

260 265 270

Phe Asp Phe Asn Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln

275 280 285

Arg Leu Ile Asn Asn Asn Trp Gly Met Arg Pro Lys Ala Met Arg Val

290 295 300

Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Thr Ser Asn Gly Glu

305 310 315 320

Thr Thr Val Ala Asn Asn Leu Thr Ser Thr Val Gln Ile Phe Ala Asp

325 330 335

Ser Ser Tyr Glu Leu Pro Tyr Val Met Asp Ala Gly Gln Glu Gly Ser

340 345 350

Leu Pro Pro Phe Pro Asn Asp Val Phe Met Val Pro Gln Tyr Gly Tyr

355 360 365

Cys Gly Leu Val Thr Gly Asn Thr Ser Gln Gln Gln Thr Asp Arg Asn

370 375 380

Ala Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Met Leu Arg Thr Gly

385 390 395 400

Asn Asn Phe Glu Ile Thr Tyr Ser Phe Glu Lys Val Pro Phe His Ser

405 410 415

Met Tyr Ala His Ser Gln Ser Leu Asp Arg Leu Met Asn Pro Leu Ile

420 425 430

Asp Gln Tyr Leu Trp Gly Leu Gln Ser Thr Thr Thr Gly Thr Thr Leu

435 440 445

Asn Ala Gly Thr Ala Thr Thr Asn Phe Thr Lys Leu Arg Pro Thr Asn

450 455 460

Phe Ser Asn Phe Lys Lys Asn Trp Leu Pro Gly Pro Ser Ile Lys Gln

465 470 475 480

Gln Gly Phe Ser Lys Thr Ala Asn Gln Asn Tyr Lys Ile Pro Ala Thr

485 490 495

Gly Ser Asp Ser Leu Ile Lys Tyr Glu Thr His Ser Thr Leu Asp Gly

500 505 510

Arg Trp Ser Ala Leu Thr Pro Gly Pro Pro Met Ala Thr Ala Gly Pro

515 520 525

Ala Asp Ser Lys Phe Ser Asn Ser Gln Leu Ile Phe Ala Gly Pro Lys

530 535 540

Gln Asn Gly Asn Thr Ala Thr Val Pro Gly Thr Leu Ile Phe Thr Ser

545 550 555 560

Glu Glu Glu Leu Ala Ala Thr Asn Ala Thr Asp Thr Asp Met Trp Gly

565 570 575

Asn Leu Pro Gly Gly Asp Gln Ser Asn Ser Asn Leu Pro Thr Val Asp

580 585 590

Arg Leu Thr Ala Leu Gly Ala Val Pro Gly Met Val Trp Gln Asn Arg

595 600 605

Asp Ile Tyr Tyr Gln Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp

610 615 620

Gly His Phe His Pro Ser Pro Leu Ile Gly Gly Phe Gly Leu Lys His

625 630 635 640

Pro Pro Pro Gln Ile Phe Ile Lys Asn Thr Pro Val Pro Ala Asn Pro

645 650 655

Ala Thr Thr Phe Ser Ser Thr Pro Val Asn Ser Phe Ile Thr Gln Tyr

660 665 670

Ser Thr Gly Gln Val Ser Val Gln Ile Asp Trp Glu Ile Gln Lys Glu

675 680 685

Arg Ser Lys Arg Trp Asn Pro Glu Val Gln Phe Thr Ser Asn Tyr Gly

690 695 700

Gln Gln Asn Ser Leu Leu Trp Ala Pro Asp Ala Ala Gly Lys Tyr Thr

705 710 715 720

Glu Pro Arg Ala Ile Gly Thr Arg Tyr Leu Thr His His Leu

725 730

<210> 6

<211> 724

<212> protein

<213> Adeno-associated virus 5

<400> 6

Met Ser Phe Val Asp His Pro Pro Asp Trp Leu Glu Glu Val Gly Glu

1 5 10 15

Gly Leu Arg Glu Phe Leu Gly Leu Glu Ala Gly Pro Pro Lys Pro Lys

20 25 30

Pro Asn Gln Gln His Gln Asp Gln Ala Arg Gly Leu Val Leu Pro Gly

35 40 45

Tyr Asn Tyr Leu Gly Pro Gly Asn Gly Leu Asp Arg Gly Glu Pro Val

50 55 60

Asn Arg Ala Asp Glu Val Ala Arg Glu His Asp Ile Ser Tyr Asn Glu

65 70 75 80

Gln Leu Glu Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Asp

85 90 95

Ala Glu Phe Gln Glu Lys Leu Ala Asp Asp Thr Ser Phe Gly Gly Asn

100 105 110

Leu Gly Lys Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Phe

115 120 125

Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Thr Gly Lys Arg Ile

130 135 140

Asp Asp His Phe Pro Lys Arg Lys Lys Ala Arg Thr Glu Glu Asp Ser

145 150 155 160

Lys Pro Ser Thr Ser Ser Asp Ala Glu Ala Gly Pro Ser Gly Ser Gln

165 170 175

Gln Leu Gln Ile Pro Ala Gln Pro Ala Ser Ser Leu Gly Ala Asp Thr

180 185 190

Met Ser Ala Gly Gly Gly Gly Pro Leu Gly Asp Asn Asn Gln Gly Ala

195 200 205

Asp Gly Val Gly Asn Ala Ser Gly Asp Trp His Cys Asp Ser Thr Trp

210 215 220

Met Gly Asp Arg Val Val Thr Lys Ser Thr Arg Thr Trp Val Leu Pro

225 230 235 240

Ser Tyr Asn Asn His Gln Tyr Arg Glu Ile Lys Ser Gly Ser Val Asp

245 250 255

Gly Ser Asn Ala Asn Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr

260 265 270

Phe Asp Phe Asn Arg Phe His Ser His Trp Ser Pro Arg Asp Trp Gln

275 280 285

Arg Leu Ile Asn Asn Tyr Trp Gly Phe Arg Pro Arg Ser Leu Arg Val

290 295 300

Lys Ile Phe Asn Ile Gln Val Lys Glu Val Thr Val Gln Asp Ser Thr

305 310 315 320

Thr Thr Ile Ala Asn Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp

325 330 335

Asp Asp Tyr Gln Leu Pro Tyr Val Val Gly Asn Gly Thr Glu Gly Cys

340 345 350

Leu Pro Ala Phe Pro Pro Gln Val Phe Thr Leu Pro Gln Tyr Gly Tyr

355 360 365

Ala Thr Leu Asn Arg Asp Asn Thr Glu Asn Pro Thr Glu Arg Ser Ser

370 375 380

Phe Phe Cys Leu Glu Tyr Phe Pro Ser Lys Met Leu Arg Thr Gly Asn

385 390 395 400

Asn Phe Glu Phe Thr Tyr Asn Phe Glu Glu Val Pro Phe His Ser Ser

405 410 415

Phe Ala Pro Ser Gln Asn Leu Phe Lys Leu Ala Asn Pro Leu Val Asp

420 425 430

Gln Tyr Leu Tyr Arg Phe Val Ser Thr Asn Asn Thr Gly Gly Val Gln

435 440 445

Phe Asn Lys Asn Leu Ala Gly Arg Tyr Ala Asn Thr Tyr Lys Asn Trp

450 455 460

Phe Pro Gly Pro Met Gly Arg Thr Gln Gly Trp Asn Leu Gly Ser Gly

465 470 475 480

Val Asn Arg Ala Ser Val Ser Ala Phe Ala Thr Thr Asn Arg Met Glu

485 490 495

Leu Glu Gly Ala Ser Tyr Gln Val Pro Pro Gln Pro Asn Gly Met Thr

500 505 510

Asn Asn Leu Gln Gly Ser Asn Thr Tyr Ala Leu Glu Asn Thr Met Ile

515 520 525

Phe Asn Ser Gln Pro Ala Asn Pro Gly Thr Thr Ala Thr Tyr Leu Glu

530 535 540

Gly Asn Met Leu Ile Thr Ser Glu Ser Glu Thr Gln Pro Val Asn Arg

545 550 555 560

Val Ala Tyr Asn Val Gly Gly Gln Met Ala Thr Asn Asn Gln Ser Ser

565 570 575

Thr Thr Ala Pro Ala Thr Gly Thr Tyr Asn Leu Gln Glu Ile Val Pro

580 585 590

Gly Ser Val Trp Met Glu Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp

595 600 605

Ala Lys Ile Pro Glu Thr Gly Ala His Phe His Pro Ser Pro Ala Met

610 615 620

Gly Gly Phe Gly Leu Lys His Pro Pro Met Met Leu Ile Lys Asn

625 630 635 640

Thr Pro Val Pro Gly Asn Ile Thr Ser Phe Ser Asp Val Pro Val Ser

645 650 655

Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val Thr Val Glu Met Glu

660 665 670

Trp Glu Leu Lys Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln

675 680 685

Tyr Thr Asn Asn Tyr Asn Asp Pro Gln Phe Val Asp Phe Ala Pro Asp

690 695 700

Ser Thr Gly Glu Tyr Arg Thr Thr Arg Pro Ile Gly Thr Arg Tyr Leu

705 710 715 720

Thr Arg Pro Leu

<210> 7

<211> 736

<212> protein

<213> Adeno-associated virus 6

<400> 7

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Phe Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Ile Gly

145 150 155 160

Lys Thr Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Thr Pro Ala Ala Val Gly Pro Thr Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ala

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Ala Ser Thr Gly Ala Ser Asn Asp Asn His

260 265 270

Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe

275 280 285

His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn

290 295 300

Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln

305 310 315 320

Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn Asn

325 330 335

Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu Pro

340 345 350

Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala

355 360 365

Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly

370 375 380

Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro

385 390 395 400

Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe

405 410 415

Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp

420 425 430

Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg

435 440 445

Thr Gln Asn Gln Ser Gly Ser Ala Gln Asn Lys Asp Leu Leu Phe Ser

450 455 460

Arg Gly Ser Pro Ala Gly Met Ser Val Gln Pro Lys Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Lys Thr Asp Asn

485 490 495

Asn Asn Ser Asn Phe Thr Trp Thr Gly Ala Ser Lys Tyr Asn Leu Asn

500 505 510

Gly Arg Glu Ser Ile Ile Asn Pro Gly Thr Ala Met Ala Ser His Lys

515 520 525

Asp Asp Lys Asp Lys Phe Phe Pro Met Ser Gly Val Met Ile Phe Gly

530 535 540

Lys Glu Ser Ala Gly Ala Ser Asn Thr Ala Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Lys Ala Thr Asn Pro Val Ala Thr Glu Arg

565 570 575

Phe Gly Thr Val Ala Val Asn Leu Gln Ser Ser Ser Thr Asp Pro Ala

580 585 590

Thr Gly Asp Val His Val Met Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Ala Glu Phe Ser Ala Thr Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Val Gln Tyr Thr Ser Asn

690 695 700

Tyr Ala Lys Ser Ala Asn Val Asp Phe Thr Val Asp Asn Asn Gly Leu

705 710 715 720

Tyr Thr Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 8

<211> 737

<212> protein

<213> Adeno-associated virus 7

<400> 8

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Ala Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Ser Val Gly Ser Gly Thr Val Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Glu Gly Ala Asp Gly Val Gly Asn

210 215 220

Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Ser Glu Thr Ala Gly Ser Thr Asn Asp Asn

260 265 270

Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Lys Leu Arg Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Ile Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn

370 375 380

Gly Ser Gln Ser Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr Ser

405 410 415

Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ala

435 440 445

Arg Thr Gln Ser Asn Pro Gly Gly Thr Ala Gly Asn Arg Glu Leu Gln

450 455 460

Phe Tyr Gln Gly Gly Pro Ser Thr Met Ala Glu Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Phe Arg Gln Gln Arg Val Ser Lys Thr Leu Asp

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asn Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Asp Arg Phe Phe Pro Ser Ser Gly Val Leu Ile

530 535 540

Phe Gly Lys Thr Gly Ala Thr Asn Lys Thr Thr Leu Glu Asn Val Leu

545 550 555 560

Met Thr Asn Glu Glu Glu Ile Arg Pro Thr Asn Pro Val Ala Thr Glu

565 570 575

Glu Tyr Gly Ile Val Ser Ser Asn Leu Gln Ala Ala Asn Thr Ala Ala

580 585 590

Gln Thr Gln Val Val Asn Asn Gln Gly Ala Leu Pro Gly Met Val Trp

595 600 605

Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro

610 615 620

His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly

625 630 635 640

Leu Lys His Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro

645 650 655

Ala Asn Pro Pro Glu Val Phe Thr Pro Ala Lys Phe Ala Ser Phe Ile

660 665 670

Thr Gln Tyr Ser Thr Gly Glyn Val Ser Val Glu Ile Glu Trp Glu Leu

675 680 685

Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser

690 695 700

Asn Phe Glu Lys Gln Thr Gly Val Asp Phe Ala Val Asp Ser Gln Gly

705 710 715 720

Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn

725 730 735

Leu

<210> 9

<211> 738

<212> protein

<213> Adeno-associated virus 8

<400> 9

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ala Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Thr Tyr

405 410 415

Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Thr Thr Gly Gly Thr Ala Asn Thr Gln Thr Leu Gly

450 455 460

Phe Ser Gln Gly Gly Pro Asn Thr Met Ala Asn Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Thr Gly

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Ala Gly Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asn Ser Leu Ala Asn Pro Gly Ile Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Asn Gly Ile Leu Ile

530 535 540

Phe Gly Lys Gln Asn Ala Ala Arg Asp Asn Ala Asp Tyr Ser Asp Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Glu Tyr Gly Ile Val Ala Asp Asn Leu Gln Gln Gln Asn Thr Ala

580 585 590

Pro Gln Ile Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ser Lys Leu Asn Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Ser Val Asp Phe Ala Val Asn Thr Glu

705 710 715 720

Gly Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 10

<211> 736

<212> protein

<213> Adeno-associated virus 9

<400> 10

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly

145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro

180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn

260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu

405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser

435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser

450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro

465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn

485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn

500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly

530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser

565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln

580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met

625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu

725 730 735

<210> 11

<211> 738

<212> protein

<213> Adeno-associated virus 10

<400> 11

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Glu Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro

180 185 190

Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Ser Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr

405 410 415

Thr Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Ser Thr Gly Gly Thr Gln Gly Thr Gln Gln Leu Leu

450 455 460

Phe Ser Gln Ala Gly Pro Ala Asn Met Ser Ala Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met

530 535 540

Phe Gly Lys Gln Gly Ala Gly Arg Asp Asn Val Asp Tyr Ser Ser Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Ala Asn Thr Gly

580 585 590

Pro Ile Val Gly Asn Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Ser Gln Ala Lys Leu Ala Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu

705 710 715 720

Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 12

<211> 738

<212> protein

<213> Adeno-associated virus rh10

<400> 12

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro

180 185 190

Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Gly Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Ser Leu Asn Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr

405 410 415

Gln Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu

450 455 460

Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser

485 490 495

Gln Asn Asp Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met

530 535 540

Phe Gly Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala

580 585 590

Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Ser Gln Ala Lys Leu Ala Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Asp

705 710 715 720

Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 13

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 13

Gln Ala Asp Thr Thr Lys Asn

fifteen

<210> 14

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 14

Ile Ser Asp Gln Thr Lys His

fifteen

<210> 15

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 15

Ala Ser Asp Ser Thr Lys Ala

fifteen

<210> 16

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 16

Asn Gln Asp Tyr Thr Lys Thr

fifteen

<210> 17

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 17

His Asp Ile Thr Lys Asn Ile

fifteen

<210> 18

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 18

His Pro Asp Thr Thr Lys Asn

fifteen

<210> 19

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 19

His Gln Asp Thr Thr Lys Asn

fifteen

<210> 20

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 20

Asn Lys Thr Thr Asn Lys Asp

fifteen

<210> 21

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 21

Ile Ser Asn Glu Asn Glu His

fifteen

<210> 22

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 22

Gln Ala Asn Ala Asn Glu Asn

fifteen

<210> 23

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 23

Gly Lys Ser Lys Val Ile Asp

fifteen

<210> 24

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 24

Thr Asn Arg Thr Ser Pro Asp

fifteen

<210> 25

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 25

Pro Asn Ser Thr His Gly Ser

fifteen

<210> 26

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 26

Lys Asp Arg Ala Pro Ser Thr

fifteen

<210> 27

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 27

Leu Ala Gln Ala Asp Thr Thr Lys Asn Ala

1 5 10

<210> 28

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 28

Leu Ala Ile Ser Asp Gln Thr Lys His Ala

1 5 10

<210> 29

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 29

Leu Gly Ile Ser Asp Gln Thr Lys His Ala

1 5 10

<210> 30

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 30

Leu Ala Ala Ser Asp Ser Thr Lys Ala Ala

1 5 10

<210> 31

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 31

Leu Ala Asn Gln Asp Tyr Thr Lys Thr Ala

1 5 10

<210> 32

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 32

Leu Ala His Asp Ile Thr Lys Asn Ile Ala

1 5 10

<210> 33

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 33

Leu Ala His Pro Asp Thr Thr Lys Asn Ala

1 5 10

<210> 34

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 34

Leu Ala His Gln Asp Thr Thr Lys Asn Ala

1 5 10

<210> 35

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 35

Leu Ala Asn Lys Thr Thr Asn Lys Asp Ala

1 5 10

<210> 36

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 36

Leu Pro Ile Ser Asn Glu Asn Glu His Ala

1 5 10

<210> 37

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 37

Leu Pro Gln Ala Asn Ala Asn Glu Asn Ala

1 5 10

<210> 38

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 38

Leu Ala Gly Lys Ser Lys Val Ile Asp Ala

1 5 10

<210> 39

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 39

Leu Ala Thr Asn Arg Thr Ser Pro Asp Ala

1 5 10

<210> 40

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 40

Leu Ala Pro Asn Ser Thr His Gly Ser Ala

1 5 10

<210> 41

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> heterologous peptide insert

<400> 41

Leu Ala Lys Asp Arg Ala Pro Ser Thr Ala

1 5 10

<210> 42

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 42

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Ala Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Ile Ser Asp

580 585 590

Gln Thr Lys His Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 43

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 43

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Lys Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asp Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asn Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Ser Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Ile Ser Asp

580 585 590

Gln Thr Lys His Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Gln

740 745

<210> 44

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 44

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Gly Ile Ser Asp

580 585 590

Gln Thr Lys His Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 45

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 45

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Gln Ala Asp

580 585 590

Thr Thr Lys Asn Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Val Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 46

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 46

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala His Asp Ile

580 585 590

Thr Lys Asn Ile Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 47

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 47

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Asn Gln Asp

580 585 590

Tyr Thr Lys Thr Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 48

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 48

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Pro Asn Ser

580 585 590

Thr His Gly Ser Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 49

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 49

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Asn Lys Thr

580 585 590

Thr Asn Lys Asp Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 50

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 50

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Thr Asn Arg

580 585 590

Thr Ser Pro Asp Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 51

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 51

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Gly Lys Ser

580 585 590

Lys Val Ile Asp Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 52

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 52

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Ala Ser Asp

580 585 590

Ser Thr Lys Ala Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 53

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 53

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala Lys Asp Arg

580 585 590

Ala Pro Ser Thr Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 54

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 54

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala His Gln Asp

580 585 590

Thr Thr Lys Asn Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 55

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 55

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Pro Ile Ser Asn

580 585 590

Glu Asn Glu His Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 56

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 56

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Pro Gln Ala Asn

580 585 590

Ala Asn Glu Asn Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 57

<211> 745

<212> protein

<213> Artificial sequence

<220>

<223> AAV capsid variant

<400> 57

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Thr Leu Ser

1 5 10 15

Glu Gly Ile Arg Gln Trp Trp Lys Leu Lys Pro Gly Pro Pro Pro Pro

20 25 30

Lys Pro Ala Glu Arg His Lys Asp Asp Ser Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Arg Gln Leu Asp Ser Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu His Ser Pro Val Glu Pro Asp Ser Ser Ser Gly Thr Gly

145 150 155 160

Lys Ala Gly Gln Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ala Asp Ser Val Pro Asp Pro Gln Pro Leu Gly Gln Pro Pro

180 185 190

Ala Ala Pro Ser Gly Leu Gly Thr Asn Thr Met Ala Thr Gly Ser Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Thr Trp Met Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Arg Thr

435 440 445

Asn Thr Pro Ser Gly Thr Thr Thr Gln Ser Arg Leu Gln Phe Ser Gln

450 455 460

Ala Gly Ala Ser Asp Ile Arg Asp Gln Ser Arg Asn Trp Leu Pro Gly

465 470 475 480

Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Ser Ala Asp Asn Asn

485 490 495

Asn Ser Glu Tyr Ser Trp Thr Gly Ala Thr Lys Tyr His Leu Asn Gly

500 505 510

Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Asp

515 520 525

Asp Glu Glu Lys Phe Phe Pro Gln Ser Gly Val Leu Ile Phe Gly Lys

530 535 540

Gln Gly Ser Glu Lys Thr Asn Val Asp Ile Glu Lys Val Met Ile Thr

545 550 555 560

Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Tyr

565 570 575

Gly Ser Val Ser Thr Asn Leu Gln Arg Gly Asn Leu Ala His Pro Asp

580 585 590

Thr Thr Lys Asn Ala Arg Gln Ala Ala Thr Ala Asp Val Asn Thr Gln

595 600 605

Gly Val Leu Pro Gly Met Val Trp Gln Asp Arg Asp Val Tyr Leu Gln

610 615 620

Gly Pro Ile Trp Ala Lys Ile Pro His Thr Asp Gly His Phe His Pro

625 630 635 640

Ser Pro Leu Met Gly Gly Phe Gly Leu Lys His Pro Pro Gln Ile

645 650 655

Leu Ile Lys Asn Thr Pro Val Pro Ala Asn Pro Ser Thr Thr Phe Ser

660 665 670

Ala Ala Lys Phe Ala Ser Phe Ile Thr Gln Tyr Ser Thr Gly Gln Val

675 680 685

Ser Val Glu Ile Glu Trp Glu Leu Gln Lys Glu Asn Ser Lys Arg Trp

690 695 700

Asn Pro Glu Ile Gln Tyr Thr Ser Asn Tyr Asn Lys Ser Val Asn Val

705 710 715 720

Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Ser Glu Pro Arg Pro Ile

725 730 735

Gly Thr Arg Tyr Leu Thr Arg Asn Leu

740 745

<210> 58

<211> 737

<212> protein

<213> Artificial sequence

<220>

<223> Ancestral capsid protein

<220>

<221> undefined attribute

<222> (264)..(264)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (266)..(266)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (268)..(268)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (448)..(448)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (459)..(460)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (467)..(467)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (470)..(471)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (474)..(474)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (495)..(495)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (516)..(516)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (533)..(533)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (547)..(547)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (551)..(551)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (555)..(555)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (557)..(557)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (561)..(561)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (563)..(563)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (577)..(577)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (583)..(583)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (593)..(593)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (596)..(596)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (661)..(662)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (664)..(665)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (710)..(710)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (717)..(719)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> undefined attribute

<222> (723)..(723)

<223> Xaa can be any naturally occurring amino acid

<400> 58

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Glu Gly Ala Asp Gly Val Gly Asn

210 215 220

Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Ser Xaa Ser Xaa Gly Xaa Thr Asn Asp Asn

260 265 270

His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr

405 410 415

Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Xaa

435 440 445

Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Xaa Xaa Glu Leu Leu Phe

450 455 460

Ser Gln Xaa Gly Pro Xaa Xaa Met Ser Xaa Gln Ala Lys Asn Trp Leu

465 470 475 480

Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Leu Xaa Gln

485 490 495

Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His Leu

500 505 510

Asn Gly Arg Xaa Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr His

515 520 525

Lys Asp Asp Glu Xaa Arg Phe Phe Pro Ser Ser Gly Val Leu Ile Phe

530 535 540

Gly Lys Xaa Gly Ala Gly Xaa Asn Asn Thr Xaa Leu Xaa Asn Val Met

545 550 555 560

Xaa Thr Xaa Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu

565 570 575

Xaa Tyr Gly Val Val Ala Xaa Asn Leu Gln Ser Ser Asn Thr Ala Pro

580 585 590

Xaa Thr Gly Xaa Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp

595 600 605

Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro

610 615 620

His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly

625 630 635 640

Leu Lys His Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro

645 650 655

Ala Asn Pro Pro Xaa Xaa Phe Xaa Xaa Ala Lys Phe Ala Ser Phe Ile

660 665 670

Thr Gln Tyr Ser Thr Gly Glyn Val Ser Val Glu Ile Glu Trp Glu Leu

675 680 685

Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser

690 695 700

Asn Tyr Ala Lys Ser Xaa Asn Val Asp Phe Ala Val Xaa Xaa Xaa Gly

705 710 715 720

Val Tyr Xaa Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn

725 730 735

Leu

<210> 59

<211> 737

<212> protein

<213> Artificial sequence

<220>

<223> Ancestral capsid variant

<400> 59

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro

180 185 190

Pro Ala Gly Pro Ser Gly Leu Gly Ser Gly Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Glu Gly Ala Asp Gly Val Gly Asn

210 215 220

Ala Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Ser Ala Ser Ala Gly Ser Thr Asn Asp Asn

260 265 270

His Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg

275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn

290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile

305 310 315 320

Gln Val Lys Glu Val Thr Thr Asn Asp Gly Val Thr Thr Ile Ala Asn

325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Ser Asp Ser Glu Tyr Gln Leu

340 345 350

Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro

355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn

370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe

385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Thr Phe Ser Tyr Thr

405 410 415

Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu

420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ala

435 440 445

Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Arg Glu Leu Leu Phe

450 455 460

Ser Gln Ala Gly Pro Ser Asn Met Ser Ala Gln Ala Lys Asn Trp Leu

465 470 475 480

Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Lys Thr Leu Ser Gln

485 490 495

Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His Leu

500 505 510

Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr His

515 520 525

Lys Asp Asp Glu Asp Arg Phe Phe Pro Ser Ser Gly Val Leu Ile Phe

530 535 540

Gly Lys Gln Gly Ala Gly Ala Asn Asn Thr Ala Leu Glu Asn Val Met

545 550 555 560

Met Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu

565 570 575

Gln Tyr Gly Val Val Ala Ser Asn Leu Gln Ser Ser Asn Thr Ala Pro

580 585 590

Val Thr Gly Thr Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val Trp

595 600 605

Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro

610 615 620

His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly

625 630 635 640

Leu Lys His Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro

645 650 655

Ala Asn Pro Pro Ala Val Phe Thr Pro Ala Lys Phe Ala Ser Phe Ile

660 665 670

Thr Gln Tyr Ser Thr Gly Glyn Val Ser Val Glu Ile Glu Trp Glu Leu

675 680 685

Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser

690 695 700

Asn Tyr Ala Lys Ser Thr Asn Val Asp Phe Ala Val Asp Asn Glu Gly

705 710 715 720

Val Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn

725 730 735

Leu

<210> 60

<211> 10

<212> protein

<213> Artificial sequence

<220>

<223> Heterologous peptide insert

<400> 60

Ile Ala His Asp Ile Thr Lys Asn Ile Ala

1 5 10

<210> 61

<211> 7

<212> protein

<213> Artificial sequence

<220>

<223> Heterologous peptide insert

<400> 61

Asp Ile Thr Lys Asn Ile Ala

fifteen

<210> 62

<211> 8

<212> protein

<213> Artificial sequence

<220>

<223> Heterologous peptide insert

<400> 62

Asn Lys Thr Thr Asn Lys Asp Ala

fifteen

<210> 63

<211> 8

<212> protein

<213> Artificial sequence

<220>

<223> Heterologous peptide insert

<400> 63

Pro Gln Ala Asn Ala Asn Glu Asn

fifteen

<210> 64

<211> 8

<212> protein

<213> Artificial sequence

<220>

<223> Heterologous peptide insert

<400> 64

Pro Ile Ser Asn Glu Asn Glu His

fifteen

<210> 65

<211> 1377

<212> DNA

<213> Artificial sequence

<220>

<223> codon-optimized sequence,

encoding aflibercept

<400> 65

atggtttctt actgggacac cggcgtgctg ctgtgtgccc tgctttcttg tctgctgctg 60

accggctcta gcagcggctc tgataccggc agacccttcg tggaaatgta cagcgagatc 120

cccgagatca tccacatgac cgagggcaga gagctggtca tcccttgcag agtgacaagc 180

cccaacatca ccgtgactct gaagaagttc cctctggaca cactgatccc cgacggcaag 240

agaatcatct gggacagccg gaagggcttc atcatcagca acgccaccta caaagagatc 300

ggcctgctga cctgtgaagc caccgtgaat ggccacctgt acaagaccaa ctacctgaca 360

cacagacaga ccaacaccat catcgacgtg gtgctgagcc ctagccacgg cattgaactg 420

tctgtgggcg agaagctggt gctgaactgt accgccagaa ccgagctgaa cgtgggcatc 480

gacttcaact gggagtaccc cagcagcaag caccagcaca agaaactggt caaccgggac 540

ctgaaaaccc agagcggcag cgagatgaag aaattcctga gcaccctgac catcgacggc 600

gtgaccagaa gtgaccaggg cctgtacaca tgtgccgcca gctctggcct gatgaccaag 660

aaaaacagca ccttcgtgcg ggtgcacgag aaggacaaga cccacacctg tcctccatgt 720

cctgctccag aactgctcgg cggaccttcc gtgttcctgt ttcctccaaa gcctaaggac 780

accctgatga tcagcagaac ccctgaagtg acctgcgtgg tggtggatgt gtccccgag 840

gatcccgaag tgaagttcaa ttggtacgtg gacggcgtgg aagtgcacaa cgccaagacc 900

aagcctagag aggaacagta caatagcacc tacagagtgg tgtccgtgct gaccgtgctg 960

caccaggatt ggctgaacgg caaagagtac aagtgcaagg tgtccaacaa ggccctgcct 1020

gctcctatcg agaaaaccat ctccaaggcc aagggccagc ctagggaacc ccaggtttac 1080

acactgcctc caagcaggga cgagctgaca aagaaccagg tgtccctgac ctgcctggtc 1140

aagggcttct acccttccga tatcgccgtg gaatgggaga gcaatggcca gcctgagaac 1200

aactacaaga caacccctcc tgtgctggac agcgacggct cattcttcct gtacagcaag 1260

ctgacagtgg acaagagcag atggcagcag ggcaacgtgt tcagctgctc cgtgatgcac 1320

gaggccctgc acaaccacta cacccagaag tccctgagcc tgtctcctgg caaatga 1377

<210> 66

<211> 458

<212> protein

<213> Artificial sequence

<220>

<223> Aflibercept

<400> 66

Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser

1 5 10 15

Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Asp Thr Gly Arg Pro

20 25 30

Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu

35 40 45

Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr

50 55 60

Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys

65 70 75 80

Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala Thr

85 90 95

Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His

100 105 110

Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile

115 120 125

Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu

130 135 140

Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile

145 150 155 160

Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu

165 170 175

Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe

180 185 190

Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu

195 200 205

Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr

210 215 220

Phe Val Arg Val His Glu Lys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

355 360 365

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

385 390 395 400

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

405 410 415

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

420 425 430

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

435 440 445

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 67

<211> 1575

<212> DNA

<213> Artificial sequence

<220>

<223> codon-optimized sequence,

encoding single-chain heavy-light ranibizumab

<400> 67

atggactgga cctggtccat cctgtttctg gtggctgccg ccacaggcac atactctgaa 60

gtgcagctgg tggaatctgg cggcggactt gttcaacctg gcggctctct gagactgagc 120

tgtgccgcct ctggctacga ctttacccac tacggcatga actgggtccg acaggcccct 180

ggcaaaggcc ttgaatgggt cggatggatc aacacctaca ccggcgagcc aacatacgcc 240

gccgacttca agcggagatt caccttcagc ctggacacca gcaagagcac cgcctacctc 300

cagatgaaca gcctgagagc cgaggacacc gccgtgtact actgcgccaa gtatccctac 360

tactacggca ccagccactg gtacttcgac gtgtggggac agggcacact ggtcacagtg 420

tctagcgcct ctacaaaggg ccccagcgtt ttcccactgg ctcctagcag caagtctacc 480

tccggtggaa cagccgctct gggctgtctg gtcaaggact actttcccga gcctgtgacc 540

gtgtcctgga atagcggagc actgacaagc ggcgtgcaca cctttccagc cgtgctgcaa 600

agcagcggcc tgtactctct gagcagcgtc gtgacagtgc caagcagctc tctgggcacc 660

cagacctaca tctgcaatgt gaaccacaag cctagcaaca ccaaggtgga caagaaggtg 720

gaacccaaga gctgcgacaa gacacacctc ggcggaagct ctggaagcgg ctctggatct 780

accggcacaa gctctagcgg aacaggcaca agcgctggca caaccggaac aagcgcttct 840

acatctggct ctggttctgg cggaggcggga ggatcaggtg gtggtggatc tgctggcgga 900

acagctacag ctggcgcctc ttctggcagc gacattcagc tgacacagag cccttctagc 960

ctgagcgcct ctgtgggcga cagagtgacc atcacatgta gcgccagcca ggacatctcc 1020

aactacctga actggtatca gcagaagccc ggcaaggccc ctaaggtgct gatctacttt 1080

accagcagcc tgcactccgg cgtgcccagc agattttctg gatctggctc cggcaccgac 1140

ttcaccctga caatatctag cctccagcct gaggacttcg ccacctacta ctgccagcag 1200

tacagcaccg tgccttggac atttggccag ggcacaaagg tggaaatcaa gcggacagtg 1260

gccgctccta gcgtgttcat ctttccacct agcgacgagc agctgaagtc tggcacagcc 1320

tctgtcgtgt gcctgctgaa caacttctac cccagagaag ccaaggtgca gtggaaagtg 1380

gacaacgccc tccagtccgg caacagccaa gagtctgtga ccgagcagga cagcaaggac 1440

tccacctaca gcctgtccag cacactgaca ctgagcaagg ccgactacga gaagcacaaa 1500

gtgtacgcct gcgaagtgac ccaccagggc ctttctagcc ctgtgaccaa gagcttcaac 1560

cggggcgagt gttga 1575

<210> 68

<211> 524

<212> protein

<213> Artificial sequence

<220>

<223> single chain heavy-light ranibizumab

<400> 68

Met Asp Trp Thr Trp Ser Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Thr Tyr Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Asp Phe

35 40 45

Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser

85 90 95

Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser His Trp Tyr

115 120 125

Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

130 135 140

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr

145 150 155 160

Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

165 170 175

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

180 185 190

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

195 200 205

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile

210 215 220

Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val

225 230 235 240

Glu Pro Lys Ser Cys Asp Lys Thr His Leu Gly Gly Ser Ser Gly Ser

245 250 255

Gly Ser Gly Ser Thr Gly Thr Ser Ser Ser Gly Thr Gly Thr Ser Ala

260 265 270

Gly Thr Thr Gly Thr Ser Ala Ser Thr Ser Gly Ser Gly Ser Gly Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Gly Thr Ala Thr Ala

290 295 300

Gly Ala Ser Ser Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser

305 310 315 320

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser

325 330 335

Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys

340 345 350

Ala Pro Lys Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val

355 360 365

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

370 375 380

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

385 390 395 400

Tyr Ser Thr Val Pro Trp Thr Phe Gly Glyn Gly Thr Lys Val Glu Ile

405 410 415

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

420 425 430

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

435 440 445

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

450 455 460

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

465 470 475 480

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

485 490 495

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

500 505 510

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

515 520

<210> 69

<211> 1578

<212> DNA

<213> Artificial sequence

<220>

<223> codon-optimized sequence,

encoding single-chain light-heavy ranibizumab

<400> 69

atggtcctcc agacacaggt gttcatcagc ctgctgctgt ggatctctgg cgcctacggc 60

gatatccagc tgacacagag ccctagcagc ctgtctgcct ctgtgggcga cagagtgacc 120

atcacatgta gcgccagcca ggacatcagc aactacctga actggtatca gcagaagccc 180

ggcaaggccc ctaaggtgct gatctacttt accagcagcc tgcacagcgg cgtgcccagc 240

agattttctg gctctggcag cggcaccgac ttcaccctga caatatctag cctccagcct 300

gaggacttcg ccacctacta ctgccagcag tacagcaccg tgccttggac atttggccag 360

ggcaccaagg tggaaatcaa gcggacagtg gccgctccta gcgtgttcat ctttccacct 420

agcgacgagc agctgaagtc tggcacagcc tctgtcgtgt gcctgctgaa caacttctac 480

cccagagaag ccaaggtgca gtggaaggtg gacaacgccc tccagagcgg caatagccaa 540

gagagcgtga ccgagcagga cagcaaggac tctacctact ctctgagcag cacactgacc 600

ctgagcaagg ccgactacga gaagcacaaa gtgtacgcct gcgaagtgac ccaccaggggc 660

ctttctagcc ctgtgaccaa gagcttcaac agaggcgagt gtggcggcag ctctggaagc 720

ggatctggaa gcacaggcac aagctctagc ggaaccggaa caagcgctgg cacaacaggc 780

acatctgcca gcacaagcgg ttctggttct ggcggaggcg gaggatctgg tggtggcgga 840

tctgctggcg gaacagctac agctggtgcc tcttctggat ccgaggtgca gctggttgaa 900

tctggcggtg gactggttca gccaggcgga tctctgagac tgtcttgtgc cgccagcggc 960

tacgatttca cccactacgg catgaactgg gtccgacagg cccctggcaa aggccttgaa 1020

tgggtcggat ggatcaacac ctacaccggc gagccaacat acgccgccga cttcaagcgg 1080

agattcacct tcagcctgga cacctccaag agcaccgcct acctccagat gaacagcctg 1140

agagccgagg acaccgccgt gtactactgc gccaagtatc cctactacta cggcaccagc 1200

cactggtact tcgacgtgtg gggacagggc acactggtca cagtgtctag cgcctctaca 1260

aagggcccca gcgttttccc actggctcct agcagcaaga gcacatcagg cggaactgct 1320

gccctgggct gtctggtcaa ggactacttt cctgagcctg tgaccgtgtc ctggaacagc 1380

ggagcactga catctggcgt gcacaccttt ccagccgtgc tccaaagcag cggcctgtat 1440

tctctgtcca gcgtcgtgac agtgcctagc agctctctgg gcacccagac ctacatctgc 1500

aatgtgaacc acaagcctag caacaccaag gtcgacaaga aggtggaacc caagagctgc 1560

gacaagaccc acctctga 1578

<210> 70

<211> 1578

<212> DNA

<213> Artificial sequence

<220>

<223> codon optimized single strand

light-heavy ranibizumab

<400> 70

atggtgctcc agacccaggt gtttatttca ctgctgctgt ggattagcgg ggcttacgga 60

gacattcagc tgacccagag tccttcatct ctgagcgcct ccgtgggcga cagggtgacc 120

atcacatgct ctgccagcca ggatatctcc aactatctga attggtacca gcagaagccc 180

ggcaaggccc ctaaggtgct gatctatttc accagctccc tgcacagcgg agtgccatcc 240

cgcttctccg gctctggcag cggcaccgac tttaccctga caatctctag cctccagcca 300

gaggatttcg ccacatacta ttgccagcag tacagcaccg tgccctggac atttggccag 360

ggcaccaagg tggagatcaa gcggacagtg gccgccccaa gcgtgttcat ctttccccct 420

agcgacgagc agctgaagtc tggcaccgcc agcgtggtgt gcctgctgaa caatttctat 480

cccagagagg ccaaggtgca gtggaaggtg gataacgccc tccagtccgg caattctcag 540

gagagcgtga ccgagcagga ctccaaggat tctacataca gcctgtcctc taccctgaca 600

ctgtccaagg ccgactatga gaagcacaag gtgtacgcat gcgaggtgac ccaccagggc 660

ctgagctccc cagtgacaaa gagctttaac aggggagagt gtggaggatc tagcggatcc 720

ggatctggaa gcaccggcac atcctctagc ggaaccggca caagcgccgg caccacaggc 780

acctccgcct ctacaagcgg cagcggatct ggcggaggag gaggatccgg aggaggagga 840

tctgccggcg gcaccgccac agccggcgcc tcctctggct ccgaggtgca gctggtggag 900

tctggaggag gcctggtgca gcctggaggc tccctgaggc tgtcttgcgc agcaagcggc 960

tatgatttca cccactacgg aatgaactgg gtgcgccagg cacctggcaa gggcctggag 1020

tgggtgggct ggatcaatac ctatacaggc gagccaacct acgccgccga ctttaagcgg 1080

agattcacat tttccctgga taccagcaag tccacagcct acctccagat gaacagcctg 1140

agggcagagg acaccgccgt gtactattgc gccaagtatc cttactatta cggcacaagc 1200

cactggtact tcgacgtgtg gggacagggc accctggtga cagtgagctc cgccagcacc 1260

aagggcccat ccgtgtttcc tctggcccca tctagcaagt ctaccagcgg aggaacagcc 1320

gccctgggat gtctggtgaa ggactacttc ccagagcccg tgaccgtgtc ctggaattct 1380

ggcgccctga cctccggcgt gcacacattt cccgccgtgc tccagtcctc tggcctgtat 1440

agcctgagct ccgtggtgac cgtgccttct agctccctgg gcaccagac atacatctgt 1500

aacgtgaatc acaagccttc aaataccaaa gtcgataaaa aagtggaacc aaaatcctgt 1560

gataaaaccc atctgtga 1578

<210> 71

<211> 525

<212> protein

<213> Artificial sequence

<220>

<223> single chain light-heavy ranibizumab

<400> 71

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp

35 40 45

Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser

100 105 110

Thr Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Ser Ser Gly Ser

225 230 235 240

Gly Ser Gly Ser Thr Gly Thr Ser Ser Ser Gly Thr Gly Thr Ser Ala

245 250 255

Gly Thr Thr Gly Thr Ser Ala Ser Thr Ser Gly Ser Gly Ser Gly Gly

260 265 270

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Gly Thr Ala Thr Ala

275 280 285

Gly Ala Ser Ser Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

290 295 300

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

305 310 315 320

Tyr Asp Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly

325 330 335

Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro

340 345 350

Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr

355 360 365

Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

370 375 380

Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser

385 390 395 400

His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

405 410 415

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

420 425 430

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

435 440 445

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

450 455 460

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

465 470 475 480

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

485 490 495

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

500 505 510

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Leu

515 520 525

<210> 72

<211> 2277

<212> DNA

<213> Artificial sequence

<220>

<223> sequence encoding a single-stranded

light-heavy ranibizumab-Fc

<400> 72

atggtcctcc agacacaggt gttcatcagc ctgctgctgt ggatctctgg cgcctacggc 60

gatatccagc tgacacagag ccctagcagc ctgtctgcct ctgtgggcga cagagtgacc 120

atcacatgta gcgccagcca ggacatcagc aactacctga actggtatca gcagaagccc 180

ggcaaggccc ctaaggtgct gatctacttt accagcagcc tgcacagcgg cgtgcccagc 240

agattttctg gctctggcag cggcaccgac ttcaccctga caatatctag cctccagcct 300

gaggacttcg ccacctacta ctgccagcag tacagcaccg tgccttggac atttggccag 360

ggcaccaagg tggaaatcaa gcggacagtg gccgctccta gcgtgttcat ctttccacct 420

agcgacgagc agctgaagtc tggcacagcc tctgtcgtgt gcctgctgaa caacttctac 480

cccagagaag ccaaggtgca gtggaaggtg gacaacgccc tccagagcgg caatagccaa 540

gagagcgtga ccgagcagga cagcaaggac tctacctact ctctgagcag cacactgacc 600

ctgagcaagg ccgactacga gaagcacaaa gtgtacgcct gcgaagtgac ccaccaggggc 660

ctttctagcc ctgtgaccaa gagcttcaac agaggcgagt gtggcggcag ctctggaagc 720

ggatctggaa gcacaggcac aagctctagc ggaaccggaa caagcgctgg cacaacaggc 780

acatctgcca gcacaagcgg ttctggttct ggcggaggcg gaggatctgg tggtggcgga 840

tctgctggcg gaacagctac agctggtgcc tcttctggat ccgaggtgca gctggttgaa 900

tctggcggtg gactggttca gccaggcgga tctctgagac tgtcttgtgc cgccagcggc 960

tacgatttca cccactacgg catgaactgg gtccgacagg cccctggcaa aggccttgaa 1020

tgggtcggat ggatcaacac ctacaccggc gagccaacat acgccgccga cttcaagcgg 1080

agattcacct tcagcctgga cacctccaag agcaccgcct acctccagat gaacagcctg 1140

agagccgagg acaccgccgt gtactactgc gccaagtatc cctactacta cggcaccagc 1200

cactggtact tcgacgtgtg gggacagggc acactggtca cagtgtctag cgcctctaca 1260

aagggcccca gcgttttccc actggctcct agcagcaaga gcacatcagg cggaactgct 1320

gccctgggct gtctggtcaa ggactacttt cctgagcctg tgaccgtgtc ctggaacagc 1380

ggagcactga catctggcgt gcacaccttt ccagccgtgc tccaaagcag cggcctgtat 1440

tctctgtcca gcgtcgtgac agtgcctagc agctctctgg gcacccagac ctacatctgc 1500

aatgtgaacc acaagcctag caacaccaag gtcgacaaga aggtggaacc caagagctgc 1560

gacaagaccc acacctgtcc tccatgtcct gctccagaac tgctcggcgg accttccgtg 1620

ttcctgtttc ctccaaagcc taaggacacc ctgatgatca gcagaacccc tgaagtgacc 1680

tgcgtggtgg tggatgtgtc ccacgaggat cccgaagtga agttcaattg gtacgtggac 1740

ggcgtggaag tgcacaacgc caagaccaag cctagagagg aacagtacaa tagcacctac 1800

agagtggtgt ccgtgctgac cgtgctgcac caggattggc tgaacggcaa agagtacaag 1860

tgcaaggtgt ccaacaaggc cctgcctgct cctatcgaga aaaccatctc caaggccaag 1920

ggccagccta gggaacccca ggtttacaca ctgcctccaa gcagggacga gctgacaaag 1980

aaccaggtgt ccctgacctg cctggtcaag ggcttctacc cttccgatat cgccgtggaa 2040

tgggagagca atggccagcc tgagaacaac tacaagacaa cccctcctgt gctggacagc 2100

2160

aacgtgttca gctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 2220

ctgagcctgt ctcctggcaa atgagccacg cgtaacacgt gcatgcgaga gatctga 2277

<210> 73

<211> 752

<212> protein

<213> Artificial sequence

<220>

<223> single chain light-heavy ranibizumab fused with Fc

<400> 73

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp

35 40 45

Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Val Leu Ile Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser

100 105 110

Thr Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

115 120 125

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

130 135 140

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

145 150 155 160

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

165 170 175

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

180 185 190

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

195 200 205

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

210 215 220

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Ser Ser Gly Ser

225 230 235 240

Gly Ser Gly Ser Thr Gly Thr Ser Ser Ser Gly Thr Gly Thr Ser Ala

245 250 255

Gly Thr Thr Gly Thr Ser Ala Ser Thr Ser Gly Ser Gly Ser Gly Gly

260 265 270

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Gly Thr Ala Thr Ala

275 280 285

Gly Ala Ser Ser Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly

290 295 300

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

305 310 315 320

Tyr Asp Phe Thr His Tyr Gly Met Asn Trp Val Arg Gln Ala Pro Gly

325 330 335

Lys Gly Leu Glu Trp Val Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro

340 345 350

Thr Tyr Ala Ala Asp Phe Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr

355 360 365

Ser Lys Ser Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

370 375 380

Thr Ala Val Tyr Tyr Cys Ala Lys Tyr Pro Tyr Tyr Tyr Gly Thr Ser

385 390 395 400

His Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser

405 410 415

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser

420 425 430

Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

435 440 445

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

450 455 460

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Ser Gly Leu Tyr

465 470 475 480

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln

485 490 495

Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

500 505 510

Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Leu Asp Lys Thr

515 520 525

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

530 535 540

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

545 550 555 560

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

565 570 575

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

580 585 590

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

595 600 605

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

610 615 620

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

625 630 635 640

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

645 650 655

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

660 665 670

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

675 680 685

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

690 695 700

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

705 710 715 720

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

725 730 735

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

740 745 750

<210> 74

<211> 816

<212> DNA

<213> Artificial sequence

<220>

<223> codon-optimized sequence,

encoding brolucizumab

<400> 74

atggtcctcc agacacaggt gttcatcagc ctgctgctgt ggatctctgg cgcctatggc 60

gagatcgtga tgacacagag ccccagcaca ctgtctgcca gcgtgggaga cagagtgatc 120

atcacatgcc aggccagcga gatcatccac agctggctgg cttggtatca gcagaagcct 180

ggcaaggccc ctaagctgct gatctacctg gcctctacac tggccagcgg agtgcctagc 240

agattttctg gctctggatc tggcgccgag ttcaccctga caatctctag cctccagcct 300

gacgacttcg ccacctacta ctgccagaac gtgtacctgg cggcaccaa cggcgccaat 360

tttggccagg gcaccaagct gacagtgctt ggcgggaggcg gaggttctgg tggcggagga 420

agtggcggcg gaggatcagg cggtggtgga tctgaagtgc agctggtgga atcaggcgga 480

ggactggttc aacctggcgg ctctctgaga ctgagctgta ccgcctctgg cttctccctg 540

accgactact actacatgac ctgggtccga caggcccctg gcaaaggact tgagtgggtc 600

ggattcatcg accccgacga cgatccttac tacgccacat gggccaaggg cagattcacc 660

atcagccggg acaacagcaa gaacaccctg tacctccaga tgaacagcct gagagccgag 720

gacaccgccg tgtactattg tgccggcgga gatcacaata gcggctgggg actcgatatc 780

tggggccagg gaacactggt caccgtgtct agttga 816

<210> 75

<211> 271

<212> protein

<213> Artificial sequence

<220>

<223> Brolucizumab

<400> 75

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Ile Ile Thr Cys Gln Ala Ser Glu Ile

35 40 45

Ile His Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Leu Leu Ile Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Tyr

100 105 110

Leu Ala Ser Thr Asn Gly Ala Asn Phe Gly Gln Gly Thr Lys Leu Thr

115 120 125

Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

145 150 155 160

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser

165 170 175

Gly Phe Ser Leu Thr Asp Tyr Tyr Tyr Met Thr Trp Val Arg Gln Ala

180 185 190

Pro Gly Lys Gly Leu Glu Trp Val Gly Phe Ile Asp Pro Asp Asp Asp

195 200 205

Pro Tyr Tyr Ala Thr Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

225 230 235 240

Asp Thr Ala Val Tyr Tyr Cys Ala Gly Gly Asp His Asn Ser Gly Trp

245 250 255

Gly Leu Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265 270

<210> 76

<211> 1530

<212> DNA

<213> Artificial sequence

<220>

<223> Brolucizumab fused with Fc

<400> 76

atggtcctcc agacacaggt gttcatcagc ctgctgctgt ggatctctgg cgcctatggc 60

gagatcgtga tgacacagag ccccagcaca ctgtctgcca gcgtgggaga cagagtgatc 120

atcacatgcc aggccagcga gatcatccac agctggctgg cttggtatca gcagaagcct 180

ggcaaggccc ctaagctgct gatctacctg gcctctacac tggccagcgg agtgcctagc 240

agattttctg gctctggatc tggcgccgag ttcaccctga caatctctag cctccagcct 300

gacgacttcg ccacctacta ctgccagaac gtgtacctgg cggcaccaa cggcgccaat 360

tttggccagg gcaccaagct gacagtgctt ggcgggaggcg gaggttctgg tggcggagga 420

agtggcggcg gaggatcagg cggtggtgga tctgaagtgc agctggtgga atcaggcgga 480

ggactggttc aacctggcgg ctctctgaga ctgagctgta ccgcctctgg cttctccctg 540

accgactact actacatgac ctgggtccga caggcccctg gcaaaggact tgagtgggtc 600

ggattcatcg accccgacga cgatccttac tacgccacat gggccaaggg cagattcacc 660

atcagccggg acaacagcaa gaacaccctg tacctccaga tgaacagcct gagagccgag 720

gacaccgccg tgtactattg tgccggcgga gatcacaata gcggctgggg actcgatatc 780

tggggccagg gaacactggt caccgtgtct agtgacaaga cccacacctg tcctccatgt 840

cctgctccag aactgctcgg cggaccttcc gtgttcctgt ttcctccaaa gcctaaggac 900

accctgatga tcagcagaac ccctgaagtg acctgcgtgg tggtggatgt gtcccacgag 960

gatcccgaag tgaagttcaa ttggtacgtg gacggcgtgg aagtgcacaa cgccaagacc 1020

aagcctagag aggaacagta caatagcacc tacagagtgg tgtccgtgct gaccgtgctg 1080

caccaggatt ggctgaacgg caaagagtac aagtgcaagg tgtccaacaa ggccctgcct 1140

gctcctatcg agaaaaccat ctccaaggcc aagggccagc ctaggggaacc ccaggtttac 1200

acactgcctc caagcaggga cgagctgaca aagaaccagg tgtccctgac ctgcctggtc 1260

aagggcttct acccttccga tatcgccgtg gaatgggaga gcaatggcca gcctgagaac 1320

aactacaaga caacccctcc tgtgctggac agcgacggct cattcttcct gtacagcaag 1380

ctgacagtgg acaagagcag atggcagcag ggcaacgtgt tcagctgctc cgtgatgcac 1440

gaggccctgc acaaccacta cacccagaag tccctgagcc tgtctcctgg caaatgagcc 1500

acgcgtaaca cgtgcatgcg agagatctga 1530

<210> 77

<211> 498

<212> protein

<213> Artificial sequence

<220>

<223> Brolucizumab fused with Fc

<400> 77

Met Val Leu Gln Thr Gln Val Phe Ile Ser Leu Leu Leu Trp Ile Ser

1 5 10 15

Gly Ala Tyr Gly Glu Ile Val Met Thr Gln Ser Pro Ser Thr Leu Ser

20 25 30

Ala Ser Val Gly Asp Arg Val Ile Ile Thr Cys Gln Ala Ser Glu Ile

35 40 45

Ile His Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

50 55 60

Lys Leu Leu Ile Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Ala Glu Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Tyr

100 105 110

Leu Ala Ser Thr Asn Gly Ala Asn Phe Gly Gln Gly Thr Lys Leu Thr

115 120 125

Val Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly

145 150 155 160

Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Thr Ala Ser

165 170 175

Gly Phe Ser Leu Thr Asp Tyr Tyr Tyr Met Thr Trp Val Arg Gln Ala

180 185 190

Pro Gly Lys Gly Leu Glu Trp Val Gly Phe Ile Asp Pro Asp Asp Asp

195 200 205

Pro Tyr Tyr Ala Thr Trp Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp

210 215 220

Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

225 230 235 240

Asp Thr Ala Val Tyr Tyr Cys Ala Gly Gly Asp His Asn Ser Gly Trp

245 250 255

Gly Leu Asp Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp

260 265 270

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

275 280 285

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

290 295 300

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

305 310 315 320

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

325 330 335

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

340 345 350

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

355 360 365

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

370 375 380

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

385 390 395 400

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

405 410 415

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

420 425 430

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

435 440 445

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

450 455 460

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

465 470 475 480

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

485 490 495

Gly Lys

<---

Claims (35)

1. A recombinant adeno-associated virus (rAAV) virion for inhibiting vascular endothelial growth factor (VEGF) activity in a mammalian eye cell, containing (a) an adeno-associated virus (AAV) capsid protein variant containing a peptide insert in the GH-loop of the capsid protein, compared with the corresponding parent AAV capsid protein, where the peptide insert contains the amino acid sequence ISDQTKH (SEQ ID NO:14), and (b) a heterologous nucleic acid containing a nucleotide sequence encoding a polypeptide that inhibits VEGF activity operably linked to an expression control sequence, where the nucleotide sequence is selected from the group consisting of: (i) the nucleotide sequence indicated as SEQ ID NO: 69 (sc-ranibizumab-LH1), (ii) the nucleotide sequence shown as SEQ ID NO:65 (aflibercept), (iii) the nucleotide sequence shown as SEQ ID NO:74 (brolucizumab), (iv) nucleotides 61-1578 of SEQ ID NO:69, (v) nucleotides 79-1377 of SEQ ID NO:65, (vi) nucleotides 61-816 of SEQ ID NO:74, and (vii) a nucleotide sequence having at least 95%, at least 98% or at least 99% identity with any of (i)-(vi). 2. rAAV according to claim 1, wherein the peptide insert contains the amino acid sequence Y1Y2ISDQTKHY3, where each of Y1-Y3 is independently selected from Ala, Leu, Gly, Ser, Thr, and Pro. 3. rAAV according to claim 2, wherein the peptide insert contains the amino acid sequence LAISDQTKHA (SEQ ID NO:28). 4. rAAV according to any one of paragraphs. 1-3, where the insertion site is between amino acids corresponding to amino acids 587 and 588 of VP1 from AAV2 (SEQ ID NO:2), or at the corresponding position in the capsid protein of another AAV serotype. 5. rAAV according to any one of paragraphs. 1-4, wherein the capsid protein contains one or more amino acid substitutions relative to AAV2 capsid VP1 (SEQ ID NO:2) or one or more corresponding substitutions in another AAV serotype. 6. rAAV according to any one of paragraphs. 1-5, wherein the capsid protein contains an amino acid substitution P34A relative to VP1 of the AAV2 capsid (SEQ ID NO:2) or a corresponding substitution in another AAV serotype. 7. rAAV according to any one of paragraphs. 1-6, wherein the capsid protein contains (i) the amino acid sequence ISDQTKH (SEQ ID NO:14) and (ii) the amino acid substitution P34A and is at least 90% identical, at least 95% identical, or at least 99 % identical to the sequence indicated as SEQ ID NO:42. 8. rAAV according to claim 7, wherein the capsid protein mainly consists of the amino acid sequence shown as SEQ ID NO:42. 9. rAAV according to claim 8, wherein the nucleotide sequence is at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical with the entire length of the sequence indicated as SEQ ID NO :69 (sc-ranibizumab LH) or nucleotides 61-1578 of SEQ ID NO:69 and encodes the amino acid sequence shown as SEQ ID NO:71 or encodes an amino acid sequence of amino acids 21-525 of SEQ ID NO:71. 10. rAAV according to claim 9, wherein the nucleotide sequence comprises nucleotides 61-1578 of SEQ ID NO:69 and preferably contains nucleotides 1-1578 of SEQ ID NO:69. 11. rAAV according to claim 8, where the nucleotide sequence is at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical with the entire length of the sequence indicated as SEQ ID NO :65 (aflibercept), or nucleotides 79-1377 of SEQ ID NO:65 and encodes the amino acid sequence shown as SEQ ID NO:66, or encodes the amino acid sequence of amino acids 27-458 of SEQ ID NO:66. 12. rAAV according to claim 11, where the nucleotide sequence contains nucleotides 79-1377 of SEQ ID NO:65 and preferably contains nucleotides 1-1377 of SEQ ID NO:65. 13. rAAV according to claim 8, where the nucleotide sequence is at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical with the entire length of the sequence indicated as SEQ ID NO :74 (brolucizumab) or nucleotides 61-816 of SEQ ID NO:74 and encodes the amino acid sequence shown as SEQ ID NO:75 or encodes the amino acid sequence of amino acids 21-271 of SEQ ID NO:75. 14. rAAV according to claim 13, wherein the nucleotide sequence comprises nucleotides 61-816 of SEQ ID NO:74 and preferably contains nucleotides 1-816 of SEQ ID NO:74. 15. rAAV according to claim 8, where the heterologous nucleic acid contains (i) a nucleotide sequence at least 95% identical, at least 98% identical, at least 99% identical, or 100% identical with the nucleic acid sequence , indicated as SEQ ID NO: 65 and encoding the amino acid sequence indicated as SEQ ID NO: 66, or (ii) a nucleotide sequence at least 95% identical, at least 98% identical, at least 99% identical or 100% identical to the nucleic acid sequence indicated as SEQ ID NO: 74 and encoding the amino acid sequence indicated as SEQ ID NO: 75. 16. rAAV according to any one of paragraphs. 1-15, where the expression control sequence contains a ubiquitous promoter. 17. rAAV according to any one of paragraphs. 1-16, where the variant capsid protein confers increased infectivity to the retinal cell compared to the infectivity of the corresponding parental AAV capsid protein to the retinal cell. 18. Pharmaceutical composition containing rAAV according to any one of paragraphs. 1-17 and a pharmaceutically acceptable carrier. 19. A method for delivering a VEGF inhibitor to a retinal cell, choroidal cell, lens cell, ciliated cell, iris cell, optic nerve cell, and/or corneal cell in a subject, comprising administering to the subject an rAAV virion according to any one of paragraphs. 1-17 or a pharmaceutical composition according to claim 18. 20. The method of claim 19, wherein the rAAV virion or pharmaceutical composition is administered to the vitreous body of the subject. 21. A method of treating a VEGFa associated eye disease selected from wet (neovascular, exudative) age-related macular degeneration; macular edema after retinal vein occlusion; retinal neovascularization due to retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative diabetic retinopathy and proliferative diabetic retinopathy); myopic macular degeneration; branch retinal vein occlusion, gemiretinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectosis; neovascular glaucoma; hemorrhages of the vitreous body; retinal and choroidal neovascularization secondary to retinal diseases including, but not limited to, uveitis, trauma, retinal degenerative disorders, retinal and/or choroid genetic disease, eye tumors, corneal and iris neovascularization, in a subject in need of such treatment, by administering to the subject an effective amount of rAAV according to any one of paragraphs. 1-17 or a pharmaceutical composition according to claim 18. 22. The method of claim 21, wherein the VEGFa-associated 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. 23. The method of claim 21 or 22, wherein the rAAV or pharmaceutical composition is administered to the vitreous body of the subject. 24. The method according to any one of paragraphs. 19-23, where the subject is a human. 25. Virion rAAV according to any one of paragraphs. 1-17, wherein the heterologous nucleic acid further comprises a nucleotide sequence encoding an interfering RNA that inhibits angiogenesis, wherein the interfering RNA reduces the expression of a pro-angiogenic gene product selected from VEGFa, VEGFb, VEGFc, VEGFd, VEGFR1, VEGGFFR2, VEGFR3, PGF, PD, and angiopoietin. 26. The rAAV virion according to claim 25, wherein the heterologous nucleic acid further comprises a nucleotide sequence encoding an interfering RNA that inhibits angiogenesis, wherein the interfering RNA reduces VEGFc expression. 27. A pharmaceutical composition containing the rAAV virion according to claim 25 or 26 and a pharmaceutically acceptable excipient. 28. A method of treating a VEGFa associated eye disease selected from wet (neovascular, exudative) age-related macular degeneration; macular edema after retinal vein occlusion; retinal neovascularization due to retinal vein occlusion; diabetic macular edema, diabetic retinopathy (including all stages of non-proliferative diabetic retinopathy and proliferative diabetic retinopathy); myopic macular degeneration; branch retinal vein occlusion, gemiretinal vein occlusion, and central retinal vein occlusion; retinopathy of prematurity; idiopathic choroidal neovascularization; myopic macular degeneration and secondary retinal and choroidal neovascularization; retinal telangiectosis; neovascular glaucoma; hemorrhages of the vitreous body; retinal and choroidal neovascularization secondary to retinal diseases including, but not limited to, uveitis, trauma, retinal degenerative disorders, retinal and/or choroid genetic disease, eye tumors, corneal and iris neovascularization, in a subject in need of such treatment, by administering to the subject an effective amount of an rAAV according to claim 25 or 26 or a pharmaceutical composition according to claim 27.

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