Adeno-associated virus virions having a mutant capsid and methods of using the same

Abstract

To provide adeno-associated virus virions having mutated capsid and use methods thereof.SOLUTION: Provided is an adeno-associated virus (AAV) virion having a modified capsid protein where the AAV virion exhibits high infectivity to retinal cells as compared to a wild type AAV, and usable for delivery of a gene product to a retinal cell of an individual and for the treatment of ocular disease.SELECTED DRAWING: Figure 1

Description

Background Art

[0001] Photoreceptors are neurons that first receive and process visual information in the retina and convert visible electromagnetic radiation into a hyperpolarization response via light information transmission. As a result, such cells are lost in the overwhelming majority of hereditary retinal diseases, and this loss occurs directly (such as dominant mutations that affect the folding of rhodopsin protein) or indirectly (such as recessive mutations that affect the retinal recycling pathway in retinal pigment epithelium (RPE)).

[0002] Adeno-associated virus (AAV) belongs to the Parvoviridae family and the Dependovirus genus. To promote the replication of its members, it is necessary to coinfect with a helper virus such as adenovirus. In the absence of a helper, AAV establishes a latent infection. The virion is composed of a 25-nm icosahedral capsid containing a 4.9-kb single-stranded DNA genome with two open reading frames, rep and cap. The non-structural gene rep encodes four regulatory proteins essential for virus replication, while cap encodes three structural proteins (VP1-3) that associate with the 60-mer capsid shell. This virus capsid mediates the ability of AAV vectors to overcome many of the biological barriers to virus transduction, including cell surface receptor binding, endocytosis, intracellular trafficking, and unpackaging in the nucleus.

Summary of the Invention

[0003] The present disclosure provides recombinant adeno-associated virus (AAV) virions having a modified capsid protein, wherein the recombinant AAV (rAAV) virions exhibit high infectivity for retinal cells as compared to wild-type AAV, and the rAAV virions contain a heterologous nucleic acid. The present disclosure further provides a method for delivering a gene product to retinal cells of an individual and a method for treating an eye disease. The present disclosure provides rAAV virions, wherein the localization shown by the rAAV virions to one or more of the inner granular layer, outer granular layer, photoreceptor layer, ganglion cell layer, and retinal pigment epithelium is at least 5-fold increased compared to the degree of localization by AAV virions containing the corresponding parental AAV capsid protein to the inner granular layer, outer granular layer, photoreceptor layer, ganglion cell layer, or retinal pigment epithelium.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0005] Definition The term "retinal cell" can refer herein to any cell type that makes up the retina (such as retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells (including rods and cones), Müller glial cells, astrocytes (e.g., retinal astrocytes), and retinal pigment epithelium, etc.).

[0006] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. This term encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus and also to recombinant AAV vectors (or "rAAV vectors"). The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV isolated from primates, "non-primate AAV" refers to AAV isolated from non-primate mammals, and "bovine AAV" refers to AAV isolated from bovine mammals (e.g., female cows). such as, etc.

[0007] As used herein, an "rAAV vector" refers to an AAV vector that contains a polynucleotide sequence that is not of AAV origin (i.e., a polynucleotide that is heterologous to AAV), typically a target sequence for cell gene transformation. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV terminal inverted repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0008] "AAV virus", "AAV virus particle", or "rAAV vector particle" refers to a virus particle composed of at least one AAV capsid protein (typically, consisting of all of the capsid proteins of wild-type AAV), and a polynucleotide rAAV vector that forms the capsid. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to mammalian cells), the particle is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, the production of rAAV particles necessarily involves the production of rAAV vectors, and thus, the vector is encapsulated within the rAAV particles.

[0009] "Packaging" refers to a series of intracellular events in which the construction and capsid formation of AAV particles occur.

[0010] The "rep" gene and "cap" gene of AAV refer to polynucleotide sequences that encode the replication protein and capsid-forming protein of adeno-associated virus. Rep and cap of AAV are referred to herein as AAV "packaging genes".

[0011] A "helper virus" for AAV refers to a virus that enables the replication and packaging of AAV (e.g., wild-type AAV) by mammalian cells. Various such helper viruses for AAV are known in the art, and such helper viruses include adenoviruses, herpesviruses, and poxviruses (such as vaccinia). Adenoviruses include many different subgroups, but adenovirus type 5 of subgroup C is most commonly used. Many adenoviruses of human, non-human mammalian, and avian origin are known and are available from depository institutions such as the ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), and these are also available from depository institutions such as the ATCC.

[0012] The "helper virus function(s)" refers to a function(s) encoded by the helper virus genome that enables (in conjunction with other requirements for replication and packaging described herein) the replication and packaging of AAV. The "helper virus function" as used herein can be provided in a number of ways, including by providing a helper virus or, for example, by trans-providing a polynucleotide sequence encoding the necessary function(s) to the producer cell.

[0013] An "infectious" virus or viral particle is one that contains a polynucleotide element with the ability to deliver to the cells targeted by that virus species. This term does not necessarily imply that the virus has any replication ability. An "infectious" virus or viral particle as used herein is one that can access a target cell, infect the target cell, and express a heterologous nucleic acid in the target cell. Thus, "infectivity" is the ability of a viral particle to access a target cell, infect the target cell, and Refers to the ability to express a heterologous nucleic acid in a cell. Infectivity can refer to in vitro infectivity or in vivo infectivity. Assays for counting the number of infectious virus particles are described elsewhere in this disclosure and in the art. The infectivity of a virus can be expressed as the ratio of infectious virus particles to total virus particles. Total virus particles can be expressed as the copy number of viral genomes (vg). The ability of a virus particle to express a heterologous nucleic acid in a cell can be referred to as "transduction". The ability of a virus particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including the evaluation of marker genes (such as the green fluorescent protein (GFP) assay (e.g., the virus contains a nucleotide sequence encoding GFP), in which GFP is produced, detected, and / or measured in cells infected with the virus particle), or the measurement of the produced protein (e.g., by enzyme-linked immunosorbent assay (ELISA)). The infectivity of a virus can be expressed as the ratio of infectious virus particles to total virus particles. Methods for determining the ratio of infectious virus particles to total virus particles are known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11:S337 (describing the TCID50 infectivity titer assay), and Zolotukhin et al. (1999) Gene Ther. 6:973.

[0014] A virus with "replication ability" (e.g., AAV with replication ability) is infectious and, in addition, has the ability to replicate in infected cells (i.e., in the presence of a helper virus or helper virus function), referring to a phenotypically wild-type virus. In the case of AAV, for it to have replication ability, generally, the presence of functional AAV packaging genes is required. Generally, the rAAV vectors described herein lack one or more AAV packaging genes and thus do not have replication ability in mammalian cells (especially human cells). Typically, such rAAV vectors lack any AAV packaging gene sequences to minimize the possibility of generating AAV with replication ability by recombination between the AAV packaging genes and the new rAAV vector. In many embodiments, the rAAV vector preparations described herein contain little, if any, AAV with replication ability (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 8 rAAV particles, less than about 1 rcAAV per 10 12 rAAV particles, or no rcAAV is present).

[0015] The term "polynucleotide" refers to nucleotides in polymeric form of any length, including deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can include modified nucleotides such as methylated nucleotides and nucleotide analogs, and non-nucleotide elements may be interspersed. Modifications to the nucleotide structure, if any, can be made before or after the polymer is constructed. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide in the embodiments of the invention described herein encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to form the double-stranded form.

[0016] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, which means that when the two sequences are compared by alignment, that percentage of bases or amino acids are identical. Sequence similarity can be determined in many different ways. To determine sequence identity, sequence alignments can be taken using methods and computer programs, including BLAST available via the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, which is available in the Genetics Computing Group (GCG) package provided by Oxford Molecular Group, Inc., a wholly-owned subsidiary of Madison, Wisconsin, USA. Other alignment techniques 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 & It is described in Co., San Diego, California, USA. An alignment program that allows gaps in the sequence is particularly targeted. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program that uses the alignment method of Needleman and Wunsch can also be used for sequence alignment. See J. Mol. Biol. 48: 443-453 (1970).

[0017] The BestFit program (Advances in Applied Mathematics 2: 482-489 (1981)), which uses the Smith Waterman local homology algorithm to determine sequence identity, is targeted. The gap generation penalty generally ranges from 1 to 5, usually ranges from 2 to 4, and in many embodiments is 3. The gap extension penalty generally ranges from about 0.01 to 0.20 and in many cases is 0.10. The program has default parameters determined by the input sequences to be compared. Preferably, sequence identity is determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package provided by Madison, Wisconsin, USA.

[0018] Another targeted program is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. The percent sequence identity is calculated by FastDB based on the following parameters.

[0019] Mismatch penalty: 1.00, Gap penalty: 1.00, Gap size penalty: 0.33, and Linkage penalty: 30.0.

[0020] "Gene" refers to a polynucleotide that contains at least one open reading frame and has the ability to encode a specific protein after transcription and translation.

[0021] As used herein, the term "guide RNA" refers to an RNA that includes i) an "activator" nucleotide sequence that binds to a guide RNA-dependent endonuclease (e.g., a class 2 CRISPR / Cas endonuclease such as a type II, V, or VI CRISPR / Cas endonuclease) and activates the RNA-dependent endonuclease, and ii) a "targeter" nucleotide sequence that hybridizes to a target nucleic acid. The "activator" nucleotide sequence and the "targeter" nucleotide sequence can be present in separate RNA molecules (e.g., "dual guide RNA") or in the same RNA molecule (single guide RNA).

[0022] "Small interfering RNA" or "short interfering RNA" or siRNA is a nucleotide of an RNA duplex targeted to a gene of interest (the "target gene"). An "RNA duplex" refers to a structure formed by complementary pairing between two regions of one RNA molecule. siRNA is "targeted" to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the siRNA duplex has a length of less than 30 nucleotides. In some embodiments, the duplex can have a length of 29 nucleotides, 28 nucleotides, 27 nucleotides, 26 nucleotides, 25 nucleotides, 24 nucleotides, 23 nucleotides, 22 nucleotides, 21 nucleotides, 20 nucleotides, 19 nucleotides, 18 nucleotides, 17 nucleotides, 16 nucleotides, 15 nucleotides, 14 nucleotides, 13 nucleotides, 12 nucleotides, 11 nucleotides, or 10 nucleotides. In some embodiments, the duplex has a length of 19-25 nucleotides. The RNA duplex portion of the siRNA can be part of a hairpin structure. The hairpin structure can include, in addition to the duplex portion, a loop portion located between the two sequences that form the duplex. The length of the loop can vary. In some embodiments, the loop has a length of 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, or 13 nucleotides. The hairpin structure can also include an overhang portion at the 3' or 5'. In some embodiments, the overhang is a 3' or 5' overhang and has a length of 0 nucleotides, 1 nucleotide, 2 nucleotides, 3 nucleotides, 4 nucleotides, or 5 nucleotides.

[0023] As used herein, the term "microRNA" refers to any type of interfering RNA, including but not limited to endogenous microRNA and artificial microRNA (e.g., synthetic miRNA). Endogenous microRNA is a small molecule RNA that is naturally encoded in the genome and has the ability to regulate the productive utilization of mRNA. Artificial microRNA can be any type of RNA sequence other than endogenous microRNA, and is an RNA sequence that has the ability to regulate the activity of mRNA. A microRNA sequence can be an RNA molecule composed of any one or more of such sequences. MicroRNA (or "miRNA") sequences are described in publications such as Lim, et al., 2003, Genes & Development, 17, 991-1008, Lim et al., 2003, Science, 299, 1540, Lee and Ambrose, 2001, Science, 294, 862, Lau et al., 2001, Science 294, 858-861, Lagos-Quintana et al., 2002, Current Biology, 12, 735-739, Lagos-Quintana et al., 2001, Science, 294, 853-857, and Lagos-Quintana et al., 2003, RNA, 9, 175-179. Examples of microRNA include any RNA that is a fragment of a larger RNA, or any RNA that is miRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA or other non-coding small molecule RNA. See, for example, U.S. Patent Application Nos. 20050272923, 20050266552, 20050142581, and 20050075492. "MicroRNA precursor" (or "pre-miRNA") refers to a nucleic acid having a stem-loop structure with a microRNA sequence incorporated therein. "Mature microRNA" (or "mature miRNA") includes microRNA cleaved from a microRNA precursor ("pre-miRNA"), or synthetic micro RNA (e.g., those synthesized in the laboratory by a cell-free system) is included, and the length of such microRNA is about 19 to about 27 nucleotides. For example, the length of mature microRNA can be 19nt, 20nt, 21nt, 22nt, 23nt, 24nt, 25nt, 26nt, or 27nt. Mature microRNA can bind to target mRNA and inhibit the translation of that target mRNA.

[0024] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction, or nucleic acid ligation steps, and other procedures by which a construct different from the polynucleotide found in nature is obtained. A recombinant virus is a viral particle containing a recombinant polynucleotide. These terms include replicas of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0025] A "control element" or "control sequence" is a nucleotide sequence that is involved in the molecular interactions contributing to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. Such regulation can affect the frequency, rate, or specificity of the process and can be either enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences (such as promoters and enhancers). A promoter is a DNA region that has the ability to bind RNA polymerase under certain conditions and initiate transcription of a coding region usually located downstream (in the 3' direction) of that promoter.

[0026] "Operatively linked" or "operably linked" refers to the juxtaposition of genetic elements such that the elements are in a relationship that enables them to function in a predicted manner. For example, a promoter is operatively linked to a coding region if the promoter serves to initiate transcription of the coding sequence. Intervening residues may be present between the promoter and the coding region as long as this functional relationship is maintained.

[0027] An "expression vector" is a vector that contains a region encoding a polypeptide of interest and is used to effect protein expression in a target cell of interest. To facilitate protein expression in the target, the expression vector also contains control elements operatively linked to the coding region. A combination of a control element and one or more genes to which the control element is operably linked for expression may be referred to as an "expression cassette", many of which are known and available in the art or can be readily constructed from elements available in the art.

[0028] "Heterologous" means that it is derived from an entity that has a different genotype from that of the other part of the entity to which it is being 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 that is removed from its native coding sequence and operatively linked to a coding sequence in which it is not naturally found in a linked state is a heterologous promoter. Thus, for example, an rAAV containing a heterologous nucleic acid encoding a heterologous gene product is an rAAV that contains a nucleic acid not normally found in wild-type AAV of natural origin, and the encoded heterologous gene product is a gene product not normally encoded by wild-type AAV of natural origin. As another example, a mutant AAV capsid protein containing a heterologous peptide inserted into the GH loop of the capsid protein is a mutant AAV capsid protein that contains a peptide insertion fragment not normally found in wild-type AAV of natural origin.

[0029] "Genetic alteration" and "gen The terms "genetic modification" (as well as grammatical variants thereof) are used interchangeably herein to refer to a process by which genetic elements (e.g., polynucleotides) are introduced into a cell by other than mitosis or meiosis. Such elements may be heterologous to the cell or may be additional copies or improved versions of elements already present in the cell. Genetic modification may occur, for example, by introducing a recombinant plasmid or other polynucleotide into the cell via any process known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic modification may also occur, for example, by transduction or infection with a DNA or RNA virus or viral vector. Genetic elements are typically introduced into a chromosome or minichromosome of the cell, although any modification that alters the phenotype and / or genotype of the cell and its progeny is included in the term.

[0030] A cell is said to be "stably" modified, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during long-term culture of the cell in vitro. Generally, such cells are "genetically" modified (genetically altered) in that the modified cell contains an inheritable genetic modification.

[0031] The terms "polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. These terms also encompass modified amino acid polymers, such modifications being, for example, disulfide bond formation, glycosylation, lipid addition, phosphorylation, or complexation with a labeling element. Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when described in the context of delivery of gene products to mammalian subjects and in their compositions, refer to the respective intact polypeptides, or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein. Similarly, reference to a nucleic acid encoding an anti-angiogenic polypeptide, a nucleic acid encoding a neuroprotective polypeptide, and other such nucleic acids for use in the delivery of gene products to mammalian subjects (which may be referred to as "transgenes" to be delivered to recipient cells) includes polynucleotides encoding intact polypeptides or any fragment or genetically engineered derivative thereof having the desired biochemical function.

[0032] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of that substance that is made so as to lack at least some of the other components that may be present in the place where that substance or a similar substance occurs naturally, or in the place of its first preparation origin. Thus, for example, an isolated substance can be prepared using purification techniques to concentrate it from a mixture of sources. Concentration can be measured based on an absolute criterion such as weight per volume of solution, or can be measured in relation to a second substance that is present in the mixture of sources and that has the potential to interfere. As the concentration of embodiments of the present invention progresses, isolation further progresses. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance, in some of the purified embodiments, has, for example, a purity of about 80% to about 90%, a purity of at least about 90%, a purity of at least about 95%, a purity of at least about 98%, or a purity of at least about 99% or more.

[0033] As used herein, the terms "treatment", "treating", and the like refer to obtaining the desired pharmacological and / or physiological effects. Such effects can be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or can be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. As used herein, "t reatment" encompasses any treatment of a disease in a mammal, specifically a human, and includes (a) prevention of the onset of a disease in a subject who is prone to or at risk of having the disease but has not yet been diagnosed as having the disease, (b) suppression of the disease, i.e., arresting its onset, and (c) alleviation of the disease, i.e., inducing regression of the disease.

[0034] The terms "individual", "host", "subject", and "patient" are used interchangeably herein and refer to a mammal, including but not limited to humans and non-human primates (including monkeys and humans), mammalian sport animals (e.g., horses, camels, etc.), mammalian livestock (e.g., sheep, goats, cows, etc.), mammalian pets (dogs, cats, etc.), and rodents (e.g., mice, rats, etc.). In some cases, the individual is a human.

[0035] Prior to further description of the present invention, it is to be understood that the invention is not limited to the specific embodiments described, and accordingly, such embodiments may naturally vary. The technical terms used herein are for the purpose of describing only specific embodiments, and it is also to be understood that no limitation by the technical terms used herein is intended because the scope of the present invention is limited only by the appended claims.

[0036] When a range of values is given, unless otherwise clearly indicated in the context, each intervening value between the upper and lower limits of the range, to the unit of one-tenth of the lower limit, and any other recited value or intervening value within the recited range, is understood to be included in the present invention. The upper and lower limits of such a smaller range may be independently included in that smaller range, and such upper and lower limits are also included in the present invention and are the subject of any limit values specifically excluded in the recited range. If the recited range includes one or both of the limit values, the range excluding either or both of such included limit values is also included in the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are hereby incorporated by reference for the purpose of disclosing and describing the methods and / or materials associated with the publication.

[0038] It should be noted that, unless otherwise clearly indicated in the context, the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents. Thus, for example, references to "AAV capsid" include plural such capsids, references to "AAV virion" include one or more AAV virions, and references to equivalent forms thereof known to those skilled in the art, and the like. It should further be noted that the claims may be drafted to exclude any optional elements. Thus, this description is intended to serve as a basis for the use of exclusive terms such as "solely", "only", and the like, in connection with the recitation of claim elements, or the use of "negative" limitations.

[0039] Certain features of the invention are described in connection with separate embodiments for the sake of clarity, but it is understood that they may also be provided in combination in a single embodiment. Conversely, the various features of the invention are described in connection with a single embodiment for the sake of brevity, but may be provided separately or in any suitable sub-combination. All combinations of embodiments of the invention are expressly encompassed by the invention and are disclosed herein as if each and every combination were individually and explicitly disclosed. Further, all various embodiments and sub-combinations of their elements are also expressly encompassed by the invention and are disclosed herein as if each and every such sub-combination were individually and explicitly disclosed herein. That is, the invention is disclosed herein as if each and every such sub-combination were individually and explicitly disclosed herein.

[0040] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing in this description should be construed as an admission that the invention does not have the right to antedate such publications on the grounds of prior invention. Further, the dates of the publications provided may be different from the actual publication dates, which may need to be independently confirmed.

[0041] The present disclosure provides a recombinant adeno-associated virus (AAV) virion having a modified capsid protein, wherein the recombinant AAV (rAAV) virion exhibits a high infectivity to retinal cells as compared to wild-type AAV, and the rAAV virion contains a heterologous nucleic acid. The rAAV virion exhibits a high infectivity to retinal cells as compared to the infectivity of the corresponding wild-type AAV to retinal cells. Retinal cells can be photoreceptors (e.g., rods, cones), retinal ganglion cells (RGCs), Müller cells (Müller glial cells), astrocytes (e.g., retinal astrocytes), bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelium (RPE) cells. The present disclosure further provides a method for delivering a gene product to retinal cells of an individual and a method for treating an eye disease. The present disclosure provides an rAAV virion having a modified capsid protein, wherein the localization exhibited by the rAAV virion to one or more of the inner granular layer, outer granular layer, photoreceptor layer, ganglion cell layer, and retinal pigment epithelium is at least 5-fold increased compared to the degree of localization by an AAV virion containing the corresponding parental AAV capsid protein to the inner granular layer, outer granular layer, photoreceptor layer, ganglion cell layer, or retinal pigment epithelium, and the rAAV virion contains a heterologous nucleic acid.

[0042] Mutant AAV capsid polypeptide The present disclosure provides mutant AAV capsid proteins. The mutant AAV capsid proteins of the present disclosure contain a heterologous peptide insertion fragment having a length of 5 amino acids to 20 amino acids at an insertion site in the surface-exposed (e.g., solvent-exposed) portion of the parental AAV capsid protein. As a result, when present in an AAV virion, the mutant capsid protein increases the infectivity for retinal cells as compared to the infectivity for retinal cells by the AAV virion containing the corresponding parental AAV capsid protein. In other cases, the retinal cells are Müller cells. Other retinal cells include amacrine cells, bipolar cells, and horizontal cells. The “insertion fragment of about 5 amino acids to about 20 amino acids” is also referred to herein as a peptide insertion fragment (e.g., a heterologous peptide insertion fragment). The “corresponding parental AAV capsid protein” refers to an AAV capsid protein of the same AAV serotype that does not contain the peptide insertion fragment. In many cases, the mutant AAV capsid contains a single heterologous peptide insertion fragment having a length of 5 amino acids to 20 amino acids (e.g., 5 to 7, 7 to 10, 10 to 12, 12 to 15, or 15 to 20 amino acids).

[0043] The insertion site is located in the GH loop or loop IV of the AAV capsid protein, for example, in the solvent-exposed portion of the GH loop or loop IV of the AAV capsid protein. For the GH loop / loop IV of 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. For example, the insertion site can be between amino acids 411 and 650 of the AAV capsid protein shown in FIGS. 6A-6C. For example, the insertion site is between amino acids 570 and 611 of AAV2, between amino acids 571 and 612 of AAV1, between amino acids 560 and 601 of AAV5, between amino acids 571 and 612 of AAV6, between amino acids 572 and 613 of AAV7, between amino acids 573 and 614 of AAV8, between amino acids 571 and 612 of AAV9, or between amino acids 571 and 612 of AAV10 shown in FIG. 5 It can be between amino acids 573 and 614. In some cases, the insertion site is between amino acid 588 and amino acid 589 of the capsid protein of AAV2, or the corresponding insertion site in AAV of a different serotype. In some cases, the insertion site is between amino acid 587 and amino acid 588 of the capsid protein of AAV2, or the corresponding insertion site in AAV of a different serotype.

[0044] In some cases, a heterologous peptide having a length of about 5 amino acids to about 20 amino acids (e.g., 5 to 7, 7 to 10, 10 to 12, 12 to 15, or 15 to 20 amino acids) is inserted into the insertion site of the GH loop or loop IV of the capsid protein, as compared to the corresponding parental AAV capsid protein. For example, the insertion site can be between amino acid 587 and amino acid 588 of AAV2 or at the corresponding position of the capsid subunit of another AAV serotype. It should be noted that the insertion site 587 / 588 is based on the capsid protein of AAV2. A heterologous peptide having a length of about 5 amino acids to about 20 amino acids (e.g., 5 to 7, 7 to 10, 10 to 12, 12 to 15, or 15 to 20 amino acids) can be inserted into the corresponding site of an AAV serotype other than AAV2 (e.g., AAV8, AAV9, etc.). Based on the comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, those skilled in the art will be able to determine where the insertion site corresponding to "amino acids 587 to 588 of AAV2" is located in the capsid protein of any given AAV serotype. The sequences corresponding to amino acids 570 to 611 of the capsid protein VP1 of AAV2 in various AAV serotypes are shown in FIG. 5. For example, refer to GenBank accession number NP_049542 for AAV1, GenBank accession number AAD13756 for AAV5, GenBank accession number AAB95459 for AAV6, GenBank accession number YP_077178 for AAV7, GenBank accession number YP_077180 for AAV8, GenBank accession number AAS99264 for AAV9, and GenBank accession number AAT46337 for AAV10.

[0045] For example, the insertion site can be between amino acid 587 and amino acid 588 of AAV2, between amino acid 590 and amino acid 591 of AAV1, between amino acid 575 and amino acid 576 of AAV5, between amino acid 590 and amino acid 591 of AAV6, between amino acid 589 and amino acid 590 of AAV7, between amino acid 590 and amino acid 591 of AAV8, between amino acid 588 and amino acid 589 of AAV9, or between amino acid 588 and amino acid 589 of AAV10. The insertion site is indicated by underlining in FIG. 5, and the amino acid numbering is based on the numbering shown in FIG. 5.

[0046] In some embodiments, the target capsid protein comprises an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequences shown in FIGS. 6A - 6C, and comprises a GH loop having a heterologous peptide insertion fragment of a length of 5 to 20 amino acids (e.g., 5 to 7, 7 to 10, 10 to 12, 12 to 15, or 15 to 20 amino acids).

[0047] Inserted peptide As described above, a heterologous peptide having a length of about 5 to about 20 amino acids is inserted into the GH loop of the AAV capsid. In some cases, the inserted peptide has a length of 5 to 20 amino acids. In some cases, the inserted peptide has a length of 7 to 15 amino acids. In some cases, the inserted peptide has a length of 9 to 15 amino acids. In some cases, the inserted peptide has a length of 9 to 12 amino acids. The inserted peptide can be 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino It has a length of an amino acid, 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 some cases, the inserted peptide has a length of 7 amino acids. In some cases, the inserted peptide has a length of 8 amino acids. In some cases, the inserted peptide has a length of 9 amino acids. In some cases, the inserted peptide has a length of 10 amino acids. In some cases, the inserted peptide has a length of 11 amino acids. In some cases, the inserted peptide has a length of 12 amino acids. In some cases, the inserted peptide has a length of 13 amino acids. In some cases, the inserted peptide has a length of 14 amino acids. In some cases, the inserted peptide has a length of 15 amino acids.

[0048] The peptide insertion fragment is, in some cases, a peptide of formula I: X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, Ile, Pro, or Gln; X2 is Ala, Pro, Ser, Asp, Gly, Thr, or Val; X3 is Lys, His, Thr, Ile, Pro, Val, Arg, Ala, Asp, Glu, Asn, Gln, or Tyr; X4, if present, is Gln, Asp, Ser, Gly, Thr, Ile, Asn, Glu, Lys, or Arg; X5 is Asp, Ser, Gln, Val, Thr, Gly, Ala, Asn, Lys, or Tyr; X6 is Thr, Ala, Gln, Ser, Glu, Pro, or Ile; X7 is Thr, Ser, Asn, Pro, Leu, Gln, Lys, Ala, or Cys; X8 is Lys, Ser, Arg, Thr, Ala, Glu, Ile, or Asn; X9 is Asn, Pro, Ser, Lys, His, Ile, Thr, or Ala; and X 10 is Ala, Phe, Asp, Thr, Val, or Met.

[0049] The peptide insertion fragments of formula I include, but are not limited to, (1) LAKDATKNA (SEQ ID NO: 47), (2) PAHQDTTKNA (SEQ ID NO: 48), (3) LAHQDTTKNA (SEQ ID NO: 49), (4) LATTSQNKPA (SEQ ID NO: 50), (5) LAISDQTKHA (SEQ ID NO: 51), (6) IARGVAPSSA (SEQ ID NO: 52), (7) LAPDSTTRSA (SEQ ID NO: 53), (8) LAKGTELKPA (SEQ ID NO: 54), (9) LAIIDATKNA (SEQ ID NO: 55), (10) LAVDGAQRSA (SEQ ID NO: 56), (11) PAPQDTTKKA (SEQ ID NO: 57), (12) LPHQDTTKNA (SEQ ID NO: 58), (13) LAKDATKTIA (SEQ ID NO: 59), (14) LAKQQSASTA (SEQ ID NO: 60), (15) LAKSDQSKPA (SEQ ID NO: 61), (16) LSHQDTTKNA (SEQ ID NO: 62), (17) LAANQPSKPA (SEQ ID NO: 63), (18) LAVSDSTKAA (SEQ ID NO: 64), (19) LAAQGTAKKPA (SEQ ID NO: 65), (20) LAPDQTTRNA (SEQ ID NO: 66), (21) LAASDSTKAA (SEQ ID NO: 67), (22) LAPQDTTKNA (SEQ ID NO: 68), (23) LAKADETRPA (SEQ ID NO: 69), (24) LAHQDTAKNA (SEQ ID NO: 70), (25) LAHQDTKKNA (SEQ ID NO: 71), (26) LAHQDTTKHA (SEQ ID NO: 72), (27) LAHQDTTKKA (SEQ ID NO: 73), (28) LAHQDTTRNA (SEQ ID NO: 74), (29) LAHQDTTNA (SEQ ID NO: 75), (30) LAHQGTTKNA (SEQ ID NO: 76), (31) LAHQVTTKNA (SEQ ID NO: 77), (32) LAISDQSKPA (SEQ ID NO: 78), (33) LADATKTA (SEQ ID NO: 79), (34) LAKDTTKNA (SEQ ID NO: 80), (35) LAKSDQSRPA (SEQ ID NO: 81), (36) LAPQDTKKNA (SEQ ID NO: 82), (37) LATSDSTKAA (SEQ ID NO: 83), (38) LAVDGSQRSA (SEQ ID NO: 84), (39) LPISDQTKHA (SEQ ID NO: 85), (40) LPKDATKTIA (SEQ ID NO: 86), (41) LPPQDTTKNA (SEQ ID NO: 87), (42) PAPQDTTKNA (SEQ ID NO: 88), (43) QAHQDTTKNA (SEQ ID NO: 89). (44) LAHETSPRPA (SEQ ID NO: 90), (45) LAKSTSTAPA (SEQ ID NO: 91), (46) LADQDTTKNA (SEQ ID NO: 92), (47) LAESDQSKPA (SEQ ID NO: 93), (48) LAHKDTTKNA (SEQ ID NO: 94), (49) LAHKTQQKM (SEQ ID NO: 95), (50) LAHQDTTENA (SEQ ID NO: 96), (51) LAHQDTTINA (SEQ ID NO: 97), (52) LAHQDTTKKT (SEQ ID NO: 98), (53) LAHQDTTKND (SEQ ID NO: 99), (54) LAHQDTTKNT (SEQ ID NO: 100), (55) LAHQDTTKNV (SEQ ID NO: 101), (56) LAHQDTTKTM (SEQ ID NO: 102), (57) LAHQNTTKNA (SEQ ID NO: 103), (58) LAHRDTTKNA (SEQ ID NO: 104), (59) LAISDQTNHA (SEQ ID NO: 105), (60) LAKQKSASTA (SEQ ID NO: 106), (61) LAKSDQCKPA (SEQ ID NO: 107), (62) LAKSDQSKPD (SEQ ID NO: 108), (63) LAKSDQSNPA (SEQ ID NO: 109), (64) LAKSYQSKPA (SEQ ID NO: 110), (65) LANQDTTKNA (SEQ ID NO: 111), (66) LAPQNTTKNA (SEQ ID NO: 112), (67) LAPSSIQKPA (SEQ ID NO: 113), (68) LAQQDTTKNA (SEQ ID NO: 114), (69) LAYQDTTKNA (SEQ ID NO: 115), (70) LDHQDTTKNA (SEQ ID NO: 116), (71) LDHQDTTKSA (SEQ ID NO: 117), (72) LGHQDTTKNA (SEQ ID NO: 118), (73) LPHQDTTKND (SEQ ID NO: 119), (74) LPHQDTTKNT (SEQ ID NO: 120), (75) LPHQDTTNNA (SEQ ID NO: 121), (76) LTHQDTTKNA (SEQ ID NO: 122), (77) LTKDATKTIA (SEQ ID NO: 123), (78) LTPQDTTKNA (SEQ ID NO: 124), and (79) LVHQDTTKNA (SEQ ID NO: 125) are included.

[0050] The peptide insertion fragment, in some cases, has the formula II: X1X2X3X4X5X6X7X8X9X 10A peptide, wherein X1 is Leu, Ile, or Pro; X2 is Ala, Pro, or Ser; X3 is Lys, His, Thr, Ile, Pro, Val, Arg, or Ala; X4, if present, is Gln, Asp, Ser, Gly, Thr, Ile, or Asn; X5 is Asp, Ser, Gln, Val, Thr, Gly, or Ala; X6 is Thr, Ala, Gln, Ser, Glu, or Pro; X7 is Thr, Ser, Asn, Pro, Leu, Gln, Lys, or Ala; X8 is Lys, Ser, Arg, or Thr; X9 is Asn, Pro, Ser, Lys, His, Ile, Thr, or Ala; X 10 is Ala.

[0051] The peptide insertion fragments of formula II include, but are not limited to, (1) LAKDATKNA (SEQ ID NO: 47), (2) PAHQDTTKNA (SEQ ID NO: 48), (3) LAHQDTTKNA (SEQ ID NO: 49), (4) LATTSQNKPA (SEQ ID NO: 50), (5) LAISDQTKHA (SEQ ID NO: 51), (6) IARGVAPSSA (SEQ ID NO: 52), (7) LAPDSTTRSA (SEQ ID NO: 53), (8) LAKGTELKPA (SEQ ID NO: 54), (9) LAIIDATKNA (SEQ ID NO: 55), (10) LAVDGAQRSA (SEQ ID NO: 56), (11) PAPQDTTKKA (SEQ ID NO: 57), (12) LPHQDTTKNA (SEQ ID NO: 58), (13) LAKDATKTIA (SEQ ID NO: 59), (14) LAKQQSASTA (SEQ ID NO: 60), (15) LAKSDQSKPA (SEQ ID NO: 61), (16) LSHQDTTKNA (SEQ ID NO: 62), (17) LAANQPSKPA (SEQ ID NO: 63), (18) LAVSDSTKAA (SEQ ID NO: 64), (19) LAAQGTAKKPA (SEQ ID NO: 65), (20) LAPDQTTRNA (SEQ ID NO: 66), (21) LAASDSTKAA (SEQ ID NO: 67), (22) LAPQDTTKNA (SEQ ID NO: 68), (23) LAKADETRPA (SEQ ID NO: 69), (24) LAHQDTAKNA (SEQ ID NO: 70), (25) LAHQDTKKNA (SEQ ID NO: 71), (26) LAHQDTTKHA (SEQ ID NO: 72), (27) LAHQDTTKKA (SEQ ID NO: 73), (28 ) LAHQDTTRNA (SEQ ID NO: 74), (29) LAHQDTTNA (SEQ ID NO: 75), (30) LAHQGTTKNA (SEQ ID NO: 76), (31) LAHQVTTKNA (SEQ ID NO: 77), (32) LAISDQSKPA (SEQ ID NO: 78), (33) LADATKTA (SEQ ID NO: 79), (34) LAKDTTKNA (SEQ ID NO: 80), (35) LAKSDQSRPA (SEQ ID NO: 81), (36) LAPQDTKKNA (SEQ ID NO: 82), (37) LATSDSTKAA (SEQ ID NO: 83), (38) LAVDGSQRSA (SEQ ID NO: 84), (39) LPISDQTKHA (SEQ ID NO: 85), (40) LPKDATKTIA (SEQ ID NO: 86), (41) LPPQDTTKNA (SEQ ID NO: 87), and (42) PAPQDTTKNA (SEQ ID NO: 88) are included.

[0052] The peptides of formula II include, but are not limited to, (1) LAKDATKNA (SEQ ID NO: 47), (2) PAHQDTTKNA (SEQ ID NO: 48), (3) LAHQDTTKNA (SEQ ID NO: 49), (4) LATTSQNKPA (SEQ ID NO: 50), (5) LAISDQTKHA (SEQ ID NO: 51), (6) IARGVAPSSA (SEQ ID NO: 52), (7) LAPDSTTRSA (SEQ ID NO: 53), (8) LAKGTELKPA (SEQ ID NO: 54), (9) LAIIDATKNA (SEQ ID NO: 55), (10) LAVDGAQRSA (SEQ ID NO: 56), (11) PAPQDTTKKA (SEQ ID NO: 57), (12) LPHQDTTKNA (SEQ ID NO: 58), (13) LAKDATKTIA (SEQ ID NO: 59), (14) LAKQQSASTA (SEQ ID NO: 60), (15) LAKSDQSKPA (SEQ ID NO: 61), (16) LSHQDTTKNA (SEQ ID NO: 62), (17) LAANQPSKPA (SEQ ID NO: 63), and (18) LAVSDSTKAA (SEQ ID NO: 64). In some cases, the peptide insertion fragment is (1) LAKDATKNA (SEQ ID NO: 47). In some cases, the peptide insertion fragment is (2) PAHQDTTKNA (SEQ ID NO: 48). In some cases, the peptide insertion fragment is (3) LAHQDTTKNA (SEQ ID NO: 49). In some cases, the peptide insertion fragment is (4) LATTSQNKPA (SEQ ID NO: 50). In some cases, the peptide insertion fragment is (5) LAISDQTKHA (SEQ ID NO: 51). In some cases, the peptide insertion fragment is (6) IARGVAPSSA (SEQ ID NO: 52). In some cases, the peptide insertion fragment is (7) LAPDSTTRSA (SEQ ID NO: 53). In some cases, the peptide insertion fragment is (8) LAKGTELKPA (SEQ ID NO: 54). In some cases, the peptide insertion fragment is (9) LAIIDATKNA (SEQ ID NO: 55). In some cases, the peptide insertion fragment is (10) LAVDGAQRSA (SEQ ID NO: 56). In some cases, the peptide insertion fragment is (11) PAPQDTTKKA (SEQ ID NO: 57). In some cases, the peptide insertion fragment is (12) LPHQDTTKNA (SEQ ID NO: 58). In some cases, the peptide insertion fragment is (13) LAKDATKTIA (SEQ ID NO: 59).In some cases, the peptide insertion fragment is (14)LAKQQSASTA (SEQ ID NO: 60). In some cases, the peptide insertion fragment is (15)LAKSDQSKPA (SEQ ID NO: 61). In some cases, the peptide insertion fragment is (16)LSHQDTTKNA (SEQ ID NO: 62). In some cases, the peptide insertion fragment is (17)LAANQPSKPA (SEQ ID NO: 63). In some cases, the peptide insertion fragment is (18)LAVSDSTKAA (SEQ ID NO: 64).

[0053] The peptide insertion fragment is, in some cases, a peptide of formula III: X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, Ile, or Pro; X2 is Ala, Pro, or Ser; X3 is Lys, His, Thr, Ile, Pro, Val, Arg, or Ala; X4, if present, is Gln, Asp, Ser, Gly, Thr, Ile, or Asn; X5 is Asp, Ser, Gln, Val, Thr, Gly, or Ala; X6 is Thr, Ala, Gln, Ser, Glu, or Pro; X7 is Thr, Ser, Asn, Pro, Leu, Gln, Lys, or Ala; X8 is Lys, Ser, Arg , or Thr; X9 is Asn, Pro, Ser, Lys, His, Ile, Thr, or Ala; and X 10 is Ala, Thr, Asp, Val, or Met.

[0054] The peptide insertion fragments of formula III include, but are not limited to, (1) LAKDATKNA (SEQ ID NO: 47), (2) PAHQDTTKNA (SEQ ID NO: 48), (3) LAHQDTTKNA (SEQ ID NO: 49), (6) IARGVAPSSA (SEQ ID NO: 52), (7) LAPDSTTRSA (SEQ ID NO: 53), (8) LAKGTELKPA (SEQ ID NO: 54), (9) LAIIDATKNA (SEQ ID NO: 55), (10) LAVDGAQRSA (SEQ ID NO: 56), (11) PAPQDTTKKA (SEQ ID NO: 57), (12) LPHQDTTKNA (SEQ ID NO: 58), (13) LAKDATKTIA (SEQ ID NO: 59), (14) LAKQQSASTA (SEQ ID NO: 60), (16) LSHQDTTKNA (SEQ ID NO: 62), (17) LAANQPSKPA (SEQ ID NO: 63), (18) LAVSDSTKAA (SEQ ID NO: 64), (19) LAAQGTAKPA (SEQ ID NO: 65), (20) LAPDQTTRNA (SEQ ID NO: 66), (24) LAHQDTAKNA (SEQ ID NO: 70), (25) LAHQDTKKNA (SEQ ID NO: 71), (26) LAHQDTTKHA (SEQ ID NO: 72), (27) LAHQDTTKKA (SEQ ID NO: 73), (28) LAHQDTTRNA (SEQ ID NO: 74), (29) LAHQDTTTNA (SEQ ID NO: 75), (30) LAHQGTTKNA (SEQ ID NO: 76), (21) LAASDSTKAA (SEQ ID NO: 67), (22) LAPQDTTKNA (SEQ ID NO: 68), (31) LAHQVTTKNA (SEQ ID NO: 77), (33) LAKDATKTA (SEQ ID NO: 79), (34) LAKDTTKNA (SEQ ID NO: 80), (36) LAPQDTKKNA (SEQ ID NO: 82), (37) LATSDSTKAA (SEQ ID NO: 83), (38) LAVDGSQRSA (SEQ ID NO: 84), (41) LPPQDTTKNA (SEQ ID NO: 87), (42) PAPQDTTKNA (SEQ ID NO: 88), (52) LAHQDTTKKT (SEQ ID NO: 98), (53) LAHQDTTKND (SEQ ID NO: 99), (54) LAHQDTTKNT (SEQ ID NO: 100), (55) LAHQDTTKNV (SEQ ID NO: 101), (56) LAHQDTTKTM (SEQ ID NO: 102), (73) LPHQDTTKND (SEQ ID NO: 119), and (74) LPHQDTTKNT (SEQ ID NO: 120).

[0055] The peptide insertion fragment is, in some cases, a peptide of formula IV: X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, X2 is Ala, X3 is Lys, His, Thr, Ile, Pro, or Val, X4 is Gln, Asp, Ser, or Gly if present, X5 is Asp, Ser, or Gln, X6 is Thr, Ala, Gln, or Ser, X7 is Thr or Ser, X8 is Lys, Ser, or Arg, X9 is Asn, Pro, or Ser, and X 10 is Ala.

[0056] The peptide insertion fragments of formula IV include, but are not limited to, (1) LAKDATKNA (SEQ ID NO: 47), (3) LAHQDTTKNA (SEQ ID NO: 49), (7) LAPDSTTRSA (SEQ ID NO: 53), (15) LAKSDQSKPA (SEQ ID NO: 61), (20) LAPDQTTRNA (SEQ ID NO: 66), (22) LAPQDTTKNA (SEQ ID NO: 68), (28) LAHQDTTRNA (SEQ ID NO: 74), (32) LAISDQSKPA (SEQ ID NO: 78), (34) LAKDTTKNA (SEQ ID NO: 80), and (35) LAKSDQSRPA (SEQ ID NO: 81).

[0057] The peptide insertion fragment is, in some cases, a peptide of formula V: X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, X2 is Ala, X3 is Lys or His, X4 is Gln, Asp, Ser, or Gly if present, X5 is Asp, Ser, or Gln, X6 is Thr, Ala, Gln, or Ser, X7 is Thr or Ser, X8 is Lys, Ser, or Arg, X9 is Asn, Pro, or Ser, and X 10 is Ala.

[0058] The peptide insertion fragments of formula V include, but are not limited to, (1) LAKDATKNA (SEQ ID NO: 47), (15) LAKSDQSKPA (SEQ ID NO: 51), (34) LAKDTTKNA (SEQ ID NO: 80), and (35) LAKSDQSRPA (SEQ ID NO: 81).

[0059] The peptide insertion fragment is, in some cases, a peptide of formula VI: X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, X2 is Ala, X3 is Asn, Lys, Thr, Gln, Ser, Ile, or Leu, X4 is Ser, Ala, Thr, Glu, Gln, Gly, Lys, or Pro, X5 is Asp, Pro, Glu, Thr, Asn, or Arg, X6 is Ile, His, Thr, Gln, Asn, Tyr, Asp, or Glu, X7 is X7 is Gln, Thr, Asn, Ala, or Lys, X8 is Lys, Thr, Arg, or Asp, X9 is Pro, Asn, Thr, Arg, Lys, or Ser, and X 10 is Ala.

[0060] The peptides of formula VI include, but are not limited to, (80) LAKANQNTPA (SEQ ID NO: 126), (81) LATTPITKPA (SEQ ID NO: 127), (82) LATTPIAKPA (SEQ ID NO: 128), (83) LAIEDHTKSA (SEQ ID NO: 129), (84) LAQSEHQRPA (SEQ ID NO: 130), (85) LAKSPNKDNA (SEQ ID NO: 131), (86) LANQDYTKTA (SEQ ID NO: 132), (87) LANSTDQTRA (SEQ ID NO: 133), (88) LALGETTRPA (SEQ ID NO: 134), (89) LANSTEQTRA (SEQ ID NO: 135), (90) LAQADTTKNA (SEQ ID NO: 136), (91) LASKDITKTA (SEQ ID NO: 137), and (92) LASPRHNKKC (SEQ ID NO: 138).

[0061] In some cases, the peptide insertion fragment is a peptide of formula VII: LAHQDTTKX1X2X3 (SEQ ID NO: 148), wherein X1 is Lys, Thr, Asn, or His, X2 is Ala, Thr, Val, Ile, Met, or Asp, and X3 is Ala if present. Peptides of formula VII include, but are not limited to, (26) LAHQDTTKHA (SEQ ID NO: 72), (27) LAHQDTTKKA (SEQ ID NO: 73), (52) LAHQDTTKKT (SEQ ID NO: 98), (53) LAHQDTTKND (SEQ ID NO: 99), (54) LAHQDTTKNT (SEQ ID NO: 100), (55) LAHQDTTKNV (SEQ ID NO: 101), (56) LAHQDTTKTM (SEQ ID NO: 102), and (93) LAHQDTTKTIA (SEQ ID NO: 139).

[0062] In some cases, the peptide insertion fragment is a peptide of formula VIII: LAX1QX2TX3X4X5X6 (SEQ ID NO: 149), wherein X1 is Ala, Pro, Asp, or His, X2 is Gly or Asp, X3 is Ala, Thr, or Lys, X4 is Asn, Glu, Lys, Arg, or Thr, X5 is Leu, Asn, Lys, or Thr, and X6 is Ala, Thr, Asp, Val, or Met if present. Peptides of formula VIII include, but are not limited to, (94) LAAQGTANL (SEQ ID NO: 140), (22) LAPQDTTKNA (SEQ ID NO: 68), (46) LADQDTTKNA (SEQ ID NO: 92), (24) LAHQDTAKNA (SEQ ID NO: 70), (25) LAHQDTKKNA (SEQ ID NO: 71), (26) LAHQDTTKHA (SEQ ID NO: 72), (27) LAHQDTTKKA (SEQ ID NO: 73), (28) LAHQDTTRNA (SEQ ID NO: 74), (29) LAHQDTTTNA (SEQ ID NO: 75), (50) LAHQDTTENA (SEQ ID NO: 96), (51) LAHQDTTINA (SEQ ID NO: 97), (52) LAHQDTTKKT (SEQ ID NO: 98), (53) LAHQDTTKND (SEQ ID NO: 99), (54) LAHQDTTKNT (SEQ ID NO: 100), (55) LAHQDTTKNV (SEQ ID NO: 101), and (56) LAHQDTTKTM (SEQ ID NO: 102) are included.

[0063] In some cases, the peptide insertion fragment is a peptide of formula IX: X1AX2X3DX4TKX5A (SEQ ID NO: 150), wherein X1 is Val or Leu, X2 is Ile, Val, His, or Asp, X3 is Glu, Ser, Lys, or Gln, X4 is His, Ser, or Thr, and X5 is Ser, Ala, Asn, His, or Lys. Peptides of formula IX include, but are not limited to, (95) VAIEDHTKSA (SEQ ID NO: 141), (18) LAVSDSTKAA (SEQ ID NO: 64), (46) LADQDTTKNA (SEQ ID NO: 92), (48) LAHKDTTKNA (SEQ ID NO: 94), (26) LAHQDTTKHA (SEQ ID NO: 72), and (27) LAHQDTTKKA (SEQ ID NO: 73).

[0064] In some cases, the peptide insertion fragment is a peptide of formula X: X1X2X3AX4QX5TX6KNA (SEQ ID NO: 151), wherein X1 is Leu if present, X2 is Ala if present, X3 is Lys, Leu, or Pro, X4 is Asn, His, Pro, or Tyr, X5 is Asn, Gly, Val, or Asp, and X6 is Pro or Thr. Peptides of formula X include, but are not limited to, (96) LAKANQNTPKNA (SEQ ID NO: 142), (57) LAHQNTTKNA (SEQ ID NO: 103), (66) LAPQNTTKNA (SEQ ID NO: 112), (69) LAYQDTTKNA (SEQ ID NO: 115), (30) LAHQGTTKNA (SEQ ID NO: 76), (31) LAHQVTTKNA (SEQ ID NO: 77), and (42) PAPQDTTKNA (SEQ ID NO: 88).

[0065] In some cases, the peptide insertion fragment is LAHQDTTKKX (SEQ ID NO: 143), wherein X is any amino acid. In some cases, the peptide insertion fragment is LAHQDTTKKX (SEQ ID NO: 143), wherein X is Ala, Thr, Asp, Val, or Met. In some cases, the peptide insertion fragment is (27)LAHQDTTKKA (SEQ ID NO: 73). In some cases, the peptide insertion fragment is (52)LAHQDTTKKT (SEQ ID NO: 98). In some cases, the peptide insertion fragment is LAHQDTTKKD (SEQ ID NO: 144). In some cases, the peptide insertion fragment is LAHQDTTKKV (SEQ ID NO: 145). In some cases, the peptide insertion fragment is LAHQDTTKKM (SEQ ID NO: 146).

[0066] In some cases, the peptide insertion fragment is not (88)LALGETTRPA (SEQ ID NO: 134). In some cases, the peptide insertion fragment is not LGETTRP (SEQ ID NO: 147).

[0067] Suitable peptide insertion fragments include, but are not limited to, (1)LAKDATKNA (SEQ ID NO: 47), (2)PAHQDTTKNA (SEQ ID NO: 48), (3)LAHQDTTKNA (SEQ ID NO: 49), (4)LATTSQNKPA (SEQ ID NO: 50), (5)LAISDQTKHA (SEQ ID NO: 51), (6)IARGVAPSSA (SEQ ID NO: 52), (7)LAPDSTTRSA (SEQ ID NO: 53), (8)LAKGTELKPA (SEQ ID NO: 54), (9)LAIIDATKNA (SEQ ID NO: 55), (10)LAVDGAQRSA (SEQ ID NO: 56), (11)PAPQDTTKKA (SEQ ID NO: 57), (12)LPHQDTTKNA (SEQ ID NO: 58), (13)LAKDATKTIA (SEQ ID NO: 59), (14)LAKQQSASTA (SEQ ID NO: 60), (15)LAKSDQSKPA (SEQ ID NO: 61), (16)LSHQDTTKNA (SEQ ID NO: 62), (17)LAANQPSKPA (SEQ ID NO (17) LAVSDSTKAA (SEQ ID NO: 63), (18) LAAQGTAKKPA (SEQ ID NO: 64), (19) LAAQGTAKKPA (SEQ ID NO: 65), (20) LAPDQTTRNA (SEQ ID NO: 66), (21) LAASDSTKAA (SEQ ID NO: 67), (22) LAPQDTTKNA (SEQ ID NO: 68), (23) LAKADETRPA (SEQ ID NO: 69), (24) LAHQDTAKNA (SEQ ID NO: 70), (25) LAHQDTKKNA (SEQ ID NO: 71), (26) LAHQDTTKHA (SEQ ID NO: 72), (27) LAHQDTTKKA (SEQ ID NO: 73), (28) LAHQDTTRNA (SEQ ID NO: 74), (29) LAHQDTTNA (SEQ ID NO: 75), (30) LAHQGTTKNA (SEQ ID NO: 76), (31) LAHQVTTKNA (SEQ ID NO: 77), (32) LAISDQSKPA (SEQ ID NO: 78), (33) LADATKTA (SEQ ID NO: 79), (34) LAKDTTKNA (SEQ ID NO: 80), (35) LAKSDQSRPA (SEQ ID NO: 81), (36) LAPQDTKKNA (SEQ ID NO: 82), (37) LATSDSTKAA (SEQ ID NO: 83), (38) LAVDGSQRSA (SEQ ID NO: 84), (39) LPISDQTKHA (SEQ ID NO: 85), (40) LPKDATKTIA (SEQ ID NO: 86), (41) LPPQDTTKNA (SEQ ID NO: 87), (42) PAPQDTTKNA (SEQ ID NO: 88), (43) QAHQDTTKNA (SEQ ID NO: 89), (44) LAHETSPRPA (SEQ ID NO: 90), (45) LAKSTSTAPA (SEQ ID NO: 91), (46) LADQDTTKNA (SEQ ID NO: 92), (47) LAESDQSKPA (SEQ ID NO: 93), (48) LAHKDTTKNA (SEQ ID NO: 94), (49) LAHKTQQKM (SEQ ID NO: 95), (50) LAHQDTTENA (SEQ ID NO: 96), (51) LAHQDTTINA (SEQ ID NO: 97), (52) LAHQDTTKKT (SEQ ID NO: 98), (53) LAHQDTTKND (SEQ ID NO: 99), (54) LAHQDTTKNT (SEQ ID NO: 100), (55) LAHQDTTKNV (SEQ ID NO: 101), (56) LAHQDTTKTM (SEQ ID NO: 102), (57) LAHQNTTKNA (SEQ ID NO: 103), (58) LAHRDTTKNA (SEQ ID NO: 104), (59) LAISDQTNHA (SEQ ID NO: 105), (60) LAKQKSASTA (SEQ ID NO: 106)(61)LAKSDQCKPA (SEQ ID NO: 107), (62)LAKSDQSKPD (SEQ ID NO: 108), (63)LAKSDQSNPA (SEQ ID NO: 109), (64)LAKSYQSKPA (SEQ ID NO: 110), (65)LANQDTTKNA (SEQ ID NO: 111), (66)LAPQNTTKNA (SEQ ID NO: 112), (67)LAPSSIQKPA (SEQ ID NO: 113), (68)LAQQDTTKNA (SEQ ID NO: 114), (69)LAYQDTTKNA (SEQ ID NO: 115), (70)LDHQDTTKNA (SEQ ID NO: 116), (71)LDHQDTTKSA (SEQ ID NO: 117), (72)LGHQDTTKNA (SEQ ID NO: 118), (73)LPHQDTTKND (SEQ ID NO: 119), (74)LPHQDTTKNT (SEQ ID NO: 120), (75)LPHQDTTNNA (SEQ ID NO: 121), (76)LTHQDTTKNA (SEQ ID NO: 122), (77)LTKDATKTIA (SEQ ID NO: 123), (78)LTPQDTTKNA (SEQ ID NO: 124), (79)LVHQDTTKNA (SEQ ID NO: 125), (80)LAKANQNTPA (SEQ ID NO: 126), (81)LATTPITKPA (SEQ ID NO: 127), (82)LATTPIAKPA (SEQ ID NO: 128), (83)LAIEDHTKSA (SEQ ID NO: 129), (84)LAQSEHQRPA (SEQ ID NO: 130), (85)LAKSPNKDNA (SEQ ID NO: 131), (86)LANQDYTKTA (SEQ ID NO: 132), (87)LANSTDQTRA (SEQ ID NO: 133), (88)LALGETTRPA (SEQ ID NO: 134), (89)LANSTEQTRA (SEQ ID NO: 135), (90)LAQADTTKNA (SEQ ID NO: 136), (91)LASKDITKTA (SEQ ID NO: 137), (92)LASPRHNKKC (SEQ ID NO: 138), (93)LAHQDTTKTIA (SEQ ID NO: 139), (94)LAAQGTANL (SEQ ID NO: 140), (95)VAIEDHTKSA (SEQ ID NO: 141), and (96)LAKANQNTPKNA (SEQ ID NO: 142) are included.,

[0068] In some cases, the peptide insertion fragment is (11) PAPQDTTKKA (SEQ ID NO: 57). In some cases, the peptide insertion fragment is (7) LAPDSTTRSA (SEQ ID NO: is number 53).

[0069] In some embodiments, the subject rAAV virion capsid contains no other amino acid substitutions, amino acid insertions, or amino acid deletions except that there is an insertion fragment of about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids)) in the GH loop or loop IV as compared to the corresponding parental AAV capsid protein. In other embodiments, the subject rAAV virion capsid contains an insertion fragment of about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids)) in the GH loop or loop IV as compared to the corresponding parental AAV capsid protein, and in addition contains 1 to about 25 amino acid insertions, amino acid deletions, or amino acid substitutions as compared to the parental AAV capsid protein. For example, in some embodiments, the subject rAAV virion capsid contains an insertion fragment of about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids)) in the GH loop or loop IV as compared to the corresponding parental AAV capsid protein, and in addition contains 1 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, or about 20 to about 25 amino acid insertions, amino acid deletions, or amino acid substitutions as compared to the parental AAV capsid protein.

[0070] In some cases, the subject rAAV virion capsid does not contain 1, 2, 3, or 4 of the amino acid substitutions Y273F, Y444F, Y500F, and Y730F.

[0071] In some cases, the target mutant capsid polypeptide, in addition to the above-inserted peptide, contains one, two, three, or four of the amino acid substitutions Y273F, Y444F, Y500F, and Y730F.

[0072] In some cases, the target rAAV virion capsid is a chimeric capsid. For example, the capsid contains a portion of an AAV capsid of a first AAV serotype and a portion of an AAV capsid of a second serotype, and contains an insertion fragment of about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids)) in the GH loop or loop IV compared to the corresponding parental AAV capsid protein.

[0073] In some embodiments, the target rAAV virion contains a capsid protein comprising 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% amino acid sequence identity to the amino acid sequence shown in FIG. 4, and contains an insertion fragment of about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids)) in the GH loop or loop IV compared to the corresponding parental AAV capsid protein. In some embodiments, the target rAAV virion has at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 9 It contains a capsid protein comprising an amino acid sequence having 9% amino acid sequence identity, and contains an insertion fragment of about 5 amino acids to about 20 amino acids (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (for example, 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids)) between amino acid 587 and amino acid 588 when compared with the amino acid sequence shown in Figure 4, or at the corresponding site when compared with the corresponding parental AAV capsid protein.

[0074] In some embodiments, the subject rAAV virion contains a capsid protein comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 5, and contains an insertion fragment of about 5 amino acids to about 20 amino acids (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (for example, 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids)) between the bold underlined amino acids and contains a GH loop.

[0075] In some embodiments, the subject rAAV virions comprise 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% amino acid sequence identity to any one of the amino acid sequences shown in FIGS. 6A - 6C, and contain an insertion fragment of about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids (e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids)) between amino acid 587 and amino acid 588 of AAV2, or at the corresponding site when compared to another AAV genotype. Optionally, the corresponding insertion site is the site shown by the underlined characters in bold in FIG. 6B.

[0076] The subject rAAV virions exhibit 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 more than 50 - fold increased infectivity for retinal cells compared to the infectivity for retinal cells by AAV virions containing the corresponding parental AAV capsid protein.

[0077] Optionally, the subject rAAV virions exhibit 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 more than 50 - fold increased infectivity for retinal cells compared to the infectivity for retinal cells by AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0078] In some embodiments, the subject rAAV virions exhibit 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 more than 50 - fold increased infectivity for photoreceptor (rod or cone) cells compared to the infectivity for photoreceptor cells by AAV virions containing the corresponding parental AAV capsid protein.

[0079] In some embodiments, when the subject rAAV virions are administered via intravitreal injection, they exhibit 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 more than 50-fold increased infectivity for photoreceptor (rod or cone) cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0080] In some embodiments, the subject rAAV virions exhibit 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 more than 50-fold increased infectivity for RGCs as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein.

[0081] In some embodiments, when the subject rAAV virions are administered via intravitreal injection, they exhibit 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 more than 50-fold increased infectivity for RGCs as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0082] In some embodiments, the subject rAAV virions exhibit 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 more than 50-fold increased infectivity for RPE cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein.

[0083] In some embodiments, when the subject rAAV virions are administered via intravitreal injection, they exhibit an infectivity 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 more than 50-fold increased with respect to RPE cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0084] In some embodiments, the subject rAAV virions exhibit an infectivity 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 more than 50-fold increased with respect to Müller cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein.

[0085] In some embodiments, when the subject rAAV virions are administered via intravitreal injection, they exhibit an infectivity 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 more than 50-fold increased with respect to Müller cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0086] In some embodiments, the subject rAAV virions exhibit an infectivity 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 more than 50-fold increased with respect to bipolar cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein.

[0087] In some embodiments, when the subject rAAV virions are administered via intravitreal injection, they exhibit 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 more than 50-fold increased infectivity for bipolar cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0088] In some embodiments, the subject rAAV virions exhibit 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 more than 50-fold increased infectivity for amacrine cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein.

[0089] In some embodiments, when the subject rAAV virions are administered via intravitreal injection, they exhibit 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 more than 50-fold increased infectivity for amacrine cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0090] In some embodiments, the subject rAAV virions exhibit 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 more than 50-fold increased infectivity for horizontal cells as compared to the infectivity of AAV virions containing the corresponding parental AAV capsid protein.

[0091] In some embodiments, when the subject rAAV virions are administered via intravitreal injection, they exhibit 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 more than 50-fold increased infectivity for horizontal cells as compared to the infectivity of the AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0092] In some embodiments, the subject rAAV virions exhibit 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 more than 50-fold increased infectivity for retinal astrocytes as compared to the infectivity of the AAV virions containing the corresponding parental AAV capsid protein.

[0093] In some embodiments, when the subject rAAV virions are administered via intravitreal injection, they exhibit 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 more than 50-fold increased infectivity for retinal astrocytes as compared to the infectivity of the AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0094] In some cases, the subject rAAV virions exhibit 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 more than 50-fold increased ability to pass through the inner limiting membrane (ILM) as compared to the ability of the AAV virions containing the corresponding parental AAV capsid protein to pass through the ILM.

[0095] In some cases, when the subject rAAV virions are administered via intravitreal injection, they exhibit an increased ability to pass through the ILM that is 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 more than 50-fold greater compared to the ability of the AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0096] The subject rAAV virions can pass through the ILM and can also reach photoreceptor cells and / or RPE cells by passing through cell layers including Müller cells, amacrine cells, etc. For example, when the subject rAAV virions are administered via intravitreal injection, they can pass through the ILM and can also reach photoreceptor cells and / or RPE cells by passing through cell layers including Müller cells, amacrine cells, etc.

[0097] In some cases, when the subject rAAV virions are injected into the vitreous, they show at least 5-fold, at least 10-fold, at least 1 Exhibit an increased ILM penetration localization that is 5-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold. For example, in some cases, when the subject rAAV virions are injected intravitreally, they exhibit an RPE localization that is 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 more than 50-fold increased compared to the degree of localization to the retinal pigment epithelium (RPE) layer by control AAV virions containing the corresponding parental AAV capsid protein when injected intravitreally. As another example, in some cases, when the subject rAAV virions are injected intravitreally, they exhibit a PR layer localization that is 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 more than 50-fold increased compared to the degree of localization to the photoreceptor (PR) layer by control AAV virions containing the corresponding parental AAV capsid protein when injected intravitreally. As another example, in some cases, when the subject rAAV virions are injected intravitreally, they exhibit an inner granule layer localization that is 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 more than 50-fold increased compared to the degree of localization to the inner granule layer by control AAV virions containing the corresponding parental AAV capsid protein when injected intravitreally. As another example, in some cases, when the subject rAAV virions are injected intravitreally, they exhibit an outer granule layer localization that is 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 more than 50-fold increased compared to the degree of localization to the outer granule layer by control AAV virions containing the corresponding parental AAV capsid protein when injected intravitreally. As another example, in some cases, when the subject rAAV virions are injected intravitreally, they exhibit a ganglion cell layer localization that is 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 more than 50-fold increased compared to the degree of localization to the ganglion cell layer by control AAV virions containing the corresponding parental AAV capsid protein when injected intravitreally.

[0098] In some embodiments, the subject rAAV virions selectively infect retinal cells. For example, the subject rAAV virions infect retinal cells with a specificity that is 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold greater compared to non-retinal cells (e.g., cells outside the eye). For example, in some embodiments, the subject rAAV virions selectively infect retinal cells. For example, the subject rAAV virions infect retinal cells with a specificity that is 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold greater compared to non-retinal cells (e.g., cells outside the eye).

[0099] In some embodiments, the subject rAAV virions selectively infect photoreceptor cells. For example, the subject rAAV virions infect photoreceptor cells with a specificity that is 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold greater compared to non-photoreceptor cells present in the eye (e.g., retinal ganglion cells, Müller cells, etc.).

[0100] In some embodiments, when the subject rAAV virions are administered via intravitreal injection, they exhibit an infectivity to photoreceptor cells that is at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased compared to the infectivity to photoreceptor cells by AAV virions containing the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0101] Gene product The subject rAAV virions contain a heterologous nucleic acid comprising a nucleotide sequence encoding a gene product (heterologous gene product). Optionally, the gene product is a polypeptide. Optionally, the gene product is an RNA. When the gene product is an RNA, optionally, the RNA gene product encodes a polypeptide. Optionally, the rAAV virions of the present disclosure contain a single heterologous nucleic acid comprising a nucleotide sequence encoding a single heterologous gene product It contains an acid. In some cases, the rAAV virions of the present disclosure contain a single heterologous nucleic acid encoding nucleotide sequences for two heterologous gene products. In some cases, the rAAV virions of the present disclosure contain two heterologous nucleic acids, each containing a nucleotide sequence encoding a heterologous gene product.

[0102] In some embodiments, the gene product is an interfering 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 site-specific nuclease that site-specifically knocks down gene function. In some embodiments, the gene product is an RNA-guided endonuclease that modifies a target nucleic acid.

[0103] Interfering RNA When the gene product is an interfering RNA (RNAi), suitable RNAi includes RNAi that reduces the levels of apoptotic factors or angiogenic factors in cells. For example, the RNAi can be shRNA or siRNA that reduces the level of a gene product that induces or promotes apoptosis in cells. The gene whose gene product induces or promotes apoptosis is referred to herein as an "apoptosis-promoting gene", and the product (mRNA, protein) of such a gene is referred to as an "apoptosis-promoting gene product". Apoptosis-promoting gene products include, for example, the Bax gene product, the Bid gene product, the Bak gene product, and the Bad gene product. See, for example, U.S. Patent No. 7,846,730.

[0104] Interfering RNAs can also target angiogenic products, such as vascular endothelial growth factor (VEGF) (e.g., Cand5, see, e.g., U.S. Patent Publication No. 2011 / 0143400, U.S. Patent Publication No. 2008 / 0188437, and Reich et al. (2003) Mol. Vis. 9:210), VEGF receptor-1 (VEGFR1) (e.g., Sirna-027, see, e.g., Kaiser et al. (2010) Am. J. Ophthalmol. 150:33, and Shen et al. (2006) Gene Ther. 13:225), or VEGF receptor-2 (VEGFR2) (Kou et al. (2005) Biochem. 44:15064). See also U.S. Pat. Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and 5,639,736, as well as U.S. Pat. Nos. 7,947,659 and 7,919,473.

[0105] Aptamer When the gene product is an aptamer, examples of the aptamer of interest include aptamers that target VEGF. See, e.g., Ng et al. (2006) Nat. Rev. Drug Discovery 5:123, and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:18902. For example, a VEGF aptamer can include the nucleotide sequence 5'-cgcaaucagugaaugcuuauacauccg-3' (SEQ ID NO: / / ). Platelet-derived growth factor (PDGF)-specific aptamers (e.g., E10030) are also suitable for use, see, e.g., Ni and Hui (2009) Ophthalmologica 223:401, and Akiyama et al. (2006) J. Cell Physiol. 207:407).

[0106] Polypeptide When the gene product is a polypeptide, the polypeptide is generally a polypeptide that enhances the function of retinal cells, for example, a polypeptide that enhances the function of rod photoreceptor cells or cone photoreceptor cells, retina ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. Examples of polypeptides include neuroprotective polypeptides (e.g., glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), neurotrophin-4 (NT4), nerve growth factor (NGF), and neurturin (NTN)), anti-angiogenic polypeptides (e.g., soluble VEGF receptor, VEGF-binding antibody, VEGF-binding antibody fragment (e.g., single-chain anti-VEGF antibody), endostatin, tumstatin, angiostatin, soluble Flt polypeptide (Lai et al. (2005) Mol. Ther. 12:659), Fc fusion protein containing soluble Flt polypeptide (see, e.g., Pechan et al. (2009) Gene Ther. 16:10), pigment epithelium-derived factor (PEDF), soluble Tie-2 receptor, etc.), tissue metalloprotease inhibitor-3 (TIMP-3), light-responsive opsin (e.g., rhodopsin), anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl, XIAP), and the like. Suitable polypeptides include, but are not limited to, glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor, fibroblast growth factor 2, neurturin (NTN), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), neurotrophin-4 (NT4), brain-derived neurotrophic factor (BDNF, e.g., a polypeptide having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a continuous region of about 200 to 247 amino acids of the amino acid sequence shown in FIG. 7B (SEQ ID NO: 11)), epidermal growth factor, rhodopsin, X-linked apoptosis inhibitor, and sonic hedgehog.

[0107] Suitable light-responsive opsins include, for example, those described in U.S. Patent Publication No. 2007 / 0261127 (e.g., Channelrhodopsin-2, ChR2, Chop2), U.S. Patent Publication No. 2001 / 0086421, U.S. Patent Publication No. 2010 / 0015095, U.S. Patent Publication No. 2016 / 0002302, U.S. Patent Publication No. 2013 / 0347137, U.S. Patent Publication No. 2013 / 0019325, and light-responsive opsins described in Diester et al. (2011) Nat. Neurosci. 14:387. See Thyagarajan et al. (2010) J Neurosci. 30(26):8745-8758, Lagali et al. (2008) Nat Neurosci. 11(6):667-675, Doroudchi et al. (2011) Mol Ther. 19(7):1220-1229, Henriksen et al. (2014) J. Ophthalmic Vis.Res. 9:374, Tomita et al. (2014) Mol. Ther. 22:1434.

[0108] Suitable polypeptides include light-gated ion channel polypeptides. See, for example, Gaub et al. (2014) Proc. Natl. Acad. Sci. USA 111:E5574. For example, a suitable polypeptide is a light-gated ion channel type glutamate receptor (LiGluR). When LiGluR is expressed in retinal ganglion cells and on-type bipolar cells in the presence of a photo-isomerizable compound, the cells become light-responsive. LiGluR includes an L439C substitution. See Caporale et al. (2011) Mol Ther. 19:1212-1219, Volgraf et al. (2006) Nat Chem Biol. 2:47-52, and Gorostiza et al. (2007) Proc Natl Acad Sci USA. 104:10865-10870. Photo-isomerizable compounds include, for example, maleimide-azobenzene-glutamate 0 (MAG0 460 ) that has a maximum efficiency at 460 nm. MAG0 460 has the following structure.

[0109]

Chem.

[0110] Suitable polypeptides include, for example, retinoschisin (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a continuous stretch of about 200 to 224 amino acids of the amino acid sequence shown in FIG. 7A (SEQ ID NO: 10)). Suitable polypeptides include, for example, retinitis pigmentosa GTPase regulator (RPGR) interacting protein-1 (see, e.g., GenBank accession numbers Q96KN7, Q9EPQ2, and Q9GLM3) (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a continuous stretch of about 1150 to about 1200 amino acids or about 1200 to 1286 amino acids of the amino acid sequence shown in FIG. 7F (SEQ ID NO: 15)), peripherin-2 (Prph2) (see, e.g., GenBank accession number NP_000313 (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a continuous stretch of about 300 to 346 amino acids of the amino acid sequence shown in FIG. 7D (SEQ ID NO: 13)), and Travis et al. (1991) Genomics 10:733), peripherin (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a continuous stretch of about 400 to about 470 amino acids of the amino acid sequence shown in FIG. 7E (SEQ ID NO: 14)), retinal pigment epithelium-specific protein (RPE65) (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to a continuous stretch of about 200 to 247 amino acids of the amino acid sequence shown in FIG. 7C (SEQ ID NO: 12)) (e.g., GenBank AAC39660, and Morimura et al. (1998) Proc. Natl. Acad.(see Sci.USA 95:3088), rod-derived cone viability factor (RdCVF) (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in any one of FIGS. 7H, 7I, and 7J), total choroidal atrophy (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7G), retinitis pigmentosa GTPase regulator (RPGR) (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in one of FIGS. 7S-7V), and the like are included. For example, in some cases, a suitable RPGR polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7S. As another example, in some cases, a suitable RPGR polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown. in FIG. 7T. For example, in some cases, a suitable RPGR polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7U. For example, in some cases, a suitable RPGR polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7V.

[0111] Suitable polypeptides include CHM (complete choroid atrophy (Rab escort protein 1 (REP1))) (a polypeptide whose deficiency or deletion results in complete choroid atrophy) (see, for example, Donnelly et al. (1994) Hum. Mol. Genet. 3:1017, and van Bokhoven et al. (1994) Hum. Mol. Genet. 3:1041), as well as Crumbs homolog 1 (CRB1) (a polypeptide whose deficiency or deletion results in Leber congenital amaurosis and retinitis pigmentosa) (see, for example, den Hollander et al. (1999) Nat. Genet. 23:217, and GenBank accession number CAM23328). For example, a suitable REP1 polypeptide can include amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7G.

[0112] Suitable polypeptides include rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit alpha (PDE6α), rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta isoform 1 (PDE6β isoform 1), rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta isoform 2 (PDE6β isoform 2), and rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta isoform 3 (PDE6β isoform 3). For example, a suitable PDE6α polypeptide can include amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7K. As another example, a suitable PDE6β isoform 1 polypeptide can include amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7L. As another example, a suitable PDE6β isoform 2 polypeptide can include amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7M. As another example, a suitable PDE6β isoform 3 polypeptide can include amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7N.

[0113] Suitable polypeptides also include polypeptides whose deletion or deficiency results in color vision abnormalities. Such polypeptides include, for example, cone photoreceptor cGMP-gated channel subunit alpha (CNGA3) (see, for example, GenBank accession number NP_001289 and Booij et al. (2011) Ophthalmology 118:160-167), cone photoreceptor cGMP-gated cation channel beta subunit (CNGB3) (see, for example, Kohl et al. (2005) Eur J Hum Genet. 13(3):302), guanine nucleotide-binding protein (G protein), alpha transducin activity polypeptide 2 (GNAT2) (ACHM4), and ACHM5, as well as polypeptides whose deletion or deficiency results in various forms of color vision abnormalities (e.g., L-opsin, M-opsin, and S-opsin). See Mancuso et al. (2009) Nature 461(7265):784-787. are included.

[0114] For example, a suitable CNGA3 (also known as ACHM2) isoform 1 polypeptide may contain amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7O. As another example, a suitable CNGA3 (also known as ACHM2) isoform 2 polypeptide may contain amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7P.

[0115] As another example, a suitable CNGB3 (also known as ACHM3) polypeptide can comprise amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7Q. As another example, GNAT2 (also known as ACHM4) can comprise amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 7R.

[0116] Site-specific endonuclease In some cases, the gene product of interest is a site-specific endonuclease that site-specifically knocks down gene function. For example, the endonuclease knocks out an allele associated with a retinal disease. For example, when a gene that is a structural protein of the retina and / or provides normal retinal function when wild-type has a defective copy encoded by a dominant allele, the site-specific endonuclease can target the defective allele and knock out the defective allele. In some cases, the site-specific endonuclease is an RNA-guided endonuclease.

[0117] In addition to knocking out defective alleles, site-specific nucleases can also be used to promote homologous recombination using donor DNA encoding a functional copy of the protein encoded by the defective allele. Thus, for example, a subject rAAV virion can be used to deliver a site-specific endonuclease that knocks out a defective allele and can also be used to repair the defective allele by delivering a functional copy of the defective allele, thereby producing a functional retinal protein (e.g., functional retinoschisin, functional RPE65, functional peripherin, etc.). See, e.g., Li et al. (2011) Nature 475:217. In some embodiments, a subject rAAV virion comprises a heterologous nucleotide sequence encoding a site-specific endonuclease and a heterologous nucleotide sequence encoding a functional copy of a defective allele, wherein the functional copy encodes a functional retinal protein. Functional retinal proteins include, for example, retinoschisin, RPE65, retinitis pigmentosa GTPase regulator (RGPR)-interacting protein-1, peripherin, peripherin-2, RdCVF, and the like.

[0118] Suitable site-specific endonucleases for use include, for example, zinc finger nucleases (ZFNs), meganucleases, and transcription activator-like effector nucleases (TALENs), such site-specific endonucleases being of non-natural origin and engineered to target specific genes. Such site-specific nucleases can be engineered to cleave at specific positions within the genome, after which the cleavage is repaired by non-homologous end joining, and at the same time, insertions or deletions of several nucleotides can occur. At that time, such site-specific endonucleases (''INDEL'' By (also referred to as), the protein is out of frame and the gene is efficiently knocked out. See, for example, U.S. Patent Publication No. 2011 / 0301073. Suitable site-specific endonucleases include engineered meganucleases re-engineered homing endonucleases. Suitable endonucleases include I-Tevl nuclease. Suitable meganucleases include I-Sce1 (see, for example, Bellaiche et al. (1999) Genetics 152:1037), and I-Cre1 (see, for example, Heath et al. (1997) Nature Sructural Biology 4:468).

[0119] RNA-guided endonuclease In some cases, the gene product is an RNA-guided endonuclease. In some cases, the gene product is an RNA comprising a nucleotide sequence encoding an RNA-guided endonuclease. In some cases, the gene product is a guide RNA, for example, a single guide RNA. In some cases, the gene product is 1) a guide RNA, and 2) an RNA-guided endonuclease. The guide RNA may comprise a) a protein-binding region that binds to the RNA-guided endonuclease, and b) a region that binds to the target nucleic acid. The RNA-guided endonuclease is also referred to herein as a "genome editing nuclease".

[0120] Examples of suitable genome editing nucleases are CRISPR / Cas endonucleases (e.g., class 2 CRISPR / Cas endonucleases such as type II, type V, or type VI CRISPR / Cas endonucleases). Thus, the genome targeting composition can include a CRISPR / Cas endonuclease (e.g., class 2 CRISPR / Cas endonucleases such as type II, type V, or type VI CRISPR / Cas endonucleases). In some cases, the genome targeting composition includes a class 2 CRISPR / Cas endonuclease. In some cases, the genome targeting composition includes a class 2 type II CRISPR / Cas endonuclease (e.g., Cas9 protein). In some cases, the genome targeting composition includes a class 2 type V CRISPR / Cas endonuclease (e.g., Cpf1 protein, C2c1 protein, or C2c3 protein). In some cases, the genome targeting composition includes a class 2 type VI CRISPR / Cas endonuclease (e.g., C2c2 protein).

[0121] In some cases, the genome editing nuclease is a fusion protein fused to a heterologous polypeptide (also referred to as a “fusion partner”). In some cases, the genome editing nuclease is fused to an amino acid sequence (fusion partner) that results in intracellular localization, i.e., the fusion partner is an intracellular localization sequence (e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.).

[0122] In some cases, the genome editing endonuclease is a type II CRISPR / Cas endonuclease. In some cases, the genome editing endonuclease is a Cas9 polypeptide. The Cas9 protein is guided (e.g., stabilized at) a target site within a target nucleic acid sequence (e.g., a chromosomal sequence or an extrachromosomal sequence (e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.)) by binding to the protein-binding segment of the Cas9 guide RNA. In some cases, the Cas9 polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99% amino acid sequence identity to the Cas9 of Streptococcus pyogenes shown in FIG. 8A. In some cases, the Cas9 polypeptide used in the compositions or methods of the present disclosure is the Cas9 polypeptide of Staphylococcus aureus (saCas9). In some cases, the saCas9 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence of saCas9 shown in FIG. 8B.

[0123] In some cases, a suitable Cas9 polypeptide is a high-fidelity (HF) Cas9 polypeptide. See Kleinstiver et al. (2016) Nature 529:490. For example, amino acids N497, R661, Q695, and Q926 of the amino acid sequence shown in FIG. 8A are substituted with, for example, alanine. For example, the HF Cas9 polypeptide may comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence shown in FIG. 8A, and amino acids N497, R661, Q695, and Q926 are substituted with, for example, alanine.

[0124] In some cases, the appropriate Cas9 polypeptide exhibits modified PAM specificity. See, for example, Kleinstiver et al. (2015) Nature 523:481.

[0125] In some cases, the genome editing endonuclease is a type V CRISPR / Cas endonuclease. In some cases, the type V CRISPR / Cas endonuclease is a Cpf1 protein. In some cases, the Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100% amino acid sequence identity to the amino acid sequence of Cpf1 shown in FIG. 8C.

[0126] A nucleic acid that binds to a class 2 CRISPR / Cas endonuclease (e.g., a Cas9 protein, a type V or VI CRISPR / Cas protein, a Cpf1 protein, etc.) and targets the complex to a specific location within a target nucleic acid is herein referred to as a "guide RNA" or "CRISPR / Cas guide nucleic acid" or "CRISPR / Cas guide RNA". The guide RNA confers target specificity to the complex (RNP complex) by including a target-directed segment, which includes a guide sequence (also referred to herein as a target-directed sequence), and the guide sequence is a nucleotide sequence complementary to the sequence of the target nucleic acid.

[0127] In some cases, the guide RNA comprises two separate nucleic acid molecules, an "activator" and a "targeter", referred to herein as "dual guide RNA", "bimolecular guide RNA", "two-molecule guide RNA", or "dgRNA". In some cases, the guide RNA is a single molecule (e.g., for some Class 2 CRISPR / Cas proteins, the corresponding guide RNA is a single molecule, and in some cases, the activator and the targeter are covalently linked to each other, e.g., via intervening nucleotides), and the guide RNA is referred to as "single guide RNA", "single-molecule guide RNA", "one-molecule guide RNA", or simply "sgRNA".

[0128] If the gene product is an RNA-guided endonuclease or both an RNA-guided endonuclease and a guide RNA, the gene product can modify the target nucleic acid. In some cases, for example, if the target nucleic acid contains a mutation that is harmful to a defective allele (e.g., a harmful mutation in the target nucleic acid of retinal cells), the RNA-guided endonuclease / guide RNA complex can be used, together with a donor nucleic acid containing a nucleotide sequence that corrects the harmful mutation (e.g., a donor nucleic acid containing a nucleotide sequence encoding a functional copy of a protein encoded by the defective allele), for the correction of the harmful mutation (e.g., via homologous recombination repair (HDR)).

[0129] In some cases, the gene product is an RNA-guided endonuclease and two separate sgRNAs, and the two separate sgRNAs cause deletion of the target nucleic acid via non-homologous end joining (NHEJ).

[0130] The present disclosure provides a method for modifying a target nucleic acid in a retinal cell of an individual, the target nucleic acid comprising a deleterious mutation, the method comprising administering to the individual an rAAV virion of the present disclosure (e.g., by intravitreal administration, subretinal administration, etc.), the rAAV virion comprising a heterologous nucleic acid comprising: i) a nucleotide sequence encoding an RNA-guided endonuclease (e.g., a Cas9 endonuclease); ii) a nucleotide sequence encoding an sgRNA comprising a nucleotide sequence complementary to the target nucleic acid; and iii) a nucleotide sequence encoding a donor template DNA comprising a nucleotide sequence for correcting the deleterious mutation. When the rAAV virion is administered, the deleterious mutation in the target nucleic acid is corrected by HDR.

[0131] The present disclosure provides a method for modifying a target nucleic acid in a retinal cell of an individual, the target nucleic acid comprising a deleterious mutation, the method comprising administering to the individual an rAAV virion of the present disclosure (e.g., by intravitreal administration, subretinal administration, etc.), the rAAV virion comprising a heterologous nucleic acid comprising: i) a nucleotide sequence encoding an RNA-guided endonuclease (e.g., a Cas9 endonuclease); ii) a nucleotide sequence encoding a first sgRNA comprising a nucleotide sequence complementary to a first sequence in the target nucleic acid; and iii) a nucleotide sequence encoding a second sgRNA comprising a nucleotide sequence complementary to a second sequence in the target nucleic acid. When the rAAV virion is administered, the deleterious mutation in the target nucleic acid is removed by NHEJ.

[0132] Regulatory sequence In some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a transcriptional control element. For example, in some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a constitutive promoter. In other cases, the nucleotide sequence encoding the gene product of interest is operably linked to an inducible promoter. In some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a tissue-specific or cell-type specific regulatory element. For example, in some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a retinal cell-specific promoter. For example, in some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter) such that the gene is operably linked to a regulatory element that selectively expresses the gene in photoreceptor cells. Suitable photoreceptor-specific regulatory elements include, for example, the rhodopsin promoter, the rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076), the beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015), the retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra), the interphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra), and the IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225).

[0133] Pharmaceutical composition The present disclosure provides a pharmaceutical composition comprising a) the subject rAAV virions described above and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in humans.

[0134] Such excipients, carriers, diluents, and buffers include any pharmaceutical substance that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts can be included therein, and such pharmaceutically acceptable salts are, for example, mineral acid salts (such as hydrochloride, hydrobromide, phosphate, sulfate, and the like), as well as organic acid salts (such as acetate, propionate, malonate, benzoate, and the like). Further, such media may contain auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like. Pharmaceutically acceptable excipients are well known in the art and need not be described in detail herein. Pharmaceutically acceptable excipients are fully described in various publications, such as, 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) H. C. Ansel et al., eds., 7 th ed., Lippincott, Williams, & Wilkins, and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3 rd ed. Amer. Pharmaceutical Assoc are included.

[0135] Methods for Delivering Gene Products to Retinal Cells, and Therapeutic Methods The present disclosure provides a method for delivering a gene product to retinal cells of an individual, the method comprising administering the subject rAAV virions to the individual. The gene product can be the polypeptide or interfering RNA (e.g., shRNA, siRNA, and the like), aptamer, or site-specific endonuclease (e.g., RNA-guided endonuclease) described above. By delivering the gene product to retinal cells, retinal diseases can be treated. The retinal cells can be photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. In some cases, the retinal cells are photoreceptor cells (e.g., rod cells or cone cells).

[0136] The present disclosure provides a method for modifying a target nucleic acid in retinal cells, the method comprising contacting the retinal cells with 1) an rAAV virion of the present disclosure comprising a heterologous nucleic acid comprising a nucleotide sequence encoding an RNA-guided endonuclease that binds to a guide RNA, and 2) the guide RNA. The present disclosure provides a method for modifying a target nucleic acid in retinal cells, the method comprising contacting the retinal cells with an rAAV virion of the present disclosure, the rAAV virion comprising a heterologous nucleic acid comprising i) an RNA-guided endonuclease that binds to a guide RNA and ii) a nucleotide sequence encoding the guide RNA. In some cases, the method comprises contacting the retinal cells with a donor template DNA. In some cases, the RNA-guided endonuclease is a Cas9 polypeptide. In some cases, the guide RNA is a single guide RNA.

[0137] The present disclosure provides a method for treating an eye disease (e.g., a retinal disease), the method comprising administering to an individual in need thereof an effective amount of the subject rAAV virions. The subject rAA V virions can be administered by intravitreal injection, subretinal injection, or any other convenient mode of administration or route of administration. Other convenient modes of administration or routes of administration include, for example, intravenous, intranasal, and the like.

[0138] "A therapeutically effective amount" will fall within a relatively broad range that can be determined through experimentation and / or clinical trials. For example, for in vivo injection, i.e., direct injection into the eye, the therapeutically effective dose will be on the order of about 10 6 ~ about 10 15 rAAV virions on the order of, for example, about 10 8 ~ 10 12 rAAV virions on the order of. For in vitro transduction, the effective amount of rAAV virions to be delivered to cells will be on the order of about 10 8 ~ about 10 13 rAAV virions on the order of. Other effective dosages can be readily established by one of ordinary skill in the art through routine testing to establish a dose-response curve.

[0139] In some embodiments, multiple administrations (e.g., 2, 3, 4, or more administrations) may be used to achieve a desired level of gene expression. In some cases, the multiple administrations are performed at various intervals (e.g., daily, weekly, twice a month, monthly, every three months, every six months, annually, etc.). In some cases, the multiple administrations are performed over a period of 1 month to 2 months, 2 months to 4 months, 4 months to 8 months, 8 months to 12 months, 1 year to 2 years, 2 years to 5 years, or more than 5 years.

[0140] Eye diseases that can be treated using the subject methods include, but are not limited to, acute macular neuroretinopathy, Behçet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration (such as acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration), edema (such as macular edema, cystoid macular edema, and diabetic macular edema), multifocal choroiditis, ocular trauma affecting the posterior eye or posterior eye region, eye tumors, retinal disorders (such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal arterial occlusive diseases, retinal detachment, uveoretinal diseases, sympathetic ophthalmia, etc.), Vogt-Koyanagi-Harada (VKH) syndrome, choroidal exudation, posterior eye conditions caused by or affected by ocular laser treatment, posterior eye conditions caused by or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorders, branch retinal vein occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinal detachment syndrome, retinitis pigmentosa, glaucoma, Ascher syndrome, cone-rod dystrophy, Stargardt disease (foveal fundus), hereditary macular degeneration, choroidoretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, total choroidal atrophy, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, color vision disorders (including color vision abnormalities, type 1 dichromacy, type 2 dichromacy, and type 3 dichromacy), and Bietti crystalline dystrophy.

[0141] Nucleic acids and host cells The present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding the subject mutant adeno-associated virus (AAV) capsid protein, wherein the mutant AAV capsid protein comprises an insertion fragment of about 5 amino acids to about 20 amino acids in the GH loop or loop IV compared to the corresponding parental AAV capsid protein, and the mutant capsid protein, when present in an AAV virion, increases the infectivity for retinal cells compared to the infectivity for retinal cells by an AAV virion comprising the corresponding parental AAV capsid protein. The subject isolated nucleic acid can be an AAV vector, for example, a recombinant AAV vector.

[0142] Inserted peptide The mutant AAV capsid protein encoded by the target nucleic acid has an insertion peptide with a length of about 5 amino acids to about 20 amino acids inserted into the GH loop of the AAV capsid. The insertion peptide has a length of 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. Appropriate insertion peptides are those described above. Appropriate insertion peptides include any one of the peptides of Formula I-X described above.

[0143] The target recombinant AAV vector can be used to generate the above-described target recombinant AAV virions. Accordingly, the present disclosure provides a recombinant AAV vector that can produce target recombinant AAV virions when introduced into a suitable cell.

[0144] The present invention further provides a host cell, such as an isolated (genetically modified) host cell containing the target nucleic acid. The target host cell can be an isolated cell, for example, a cell cultured in vitro. The target host cell is useful for the production of the following target rAAV virions. When the target host cell is used for the production of the target rAAV virions, the target host cell is referred to as a "packaging cell". In some embodiments, the target host cell is stably genetically modified with the target nucleic acid. In other embodiments, the target host cell is transiently genetically modified with the target nucleic acid.

[0145] The target nucleic acid is introduced into the host cell stably or transiently using established techniques, including but not limited to electroporation, calcium phosphate precipitation, liposome-mediated gene transfer, and the like. To perform stable transformation, the target nucleic acid generally further includes a selectable marker, which can be, for example, any of several well-known selectable markers such as neomycin resistance and the like.

[0146] The target host cell is generated by introducing the target nucleic acid into any of a variety of cells, and such cells into which the target nucleic acid is introduced include, for example, mammalian cells (including, for example, mouse cells and primate cells (such as human cells)). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, and suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, and the like. Examples of suitable host cells include, but are not limited to, for example, HeLa cells (such as American Type Culture Collection (ATCC) number CCL-2), CHO cells (such as ATCC numbers CRL9618, CCL61, CRL9096), 293 cells (such as ATCC number CRL-1573), Vero cells, NIH3T3 cells (such as ATCC number CRL-1658), Huh-7 cells, BHK cells (such as ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RAT1 cells, mouse L cells (ATCC number CCLI.3), human embryonic kidney (HEK) cells (ATCC number CRL1573), HLHepG2 cells, and the like. The target host cell can also be prepared using a baculovirus for infection of insect cells such as Sf9 cells (which produce AAV) (see, for example, U.S. Patent No. 7,271,002, U.S. Patent Application No. 12 / 297,958).

[0147] In some embodiments, the subject genetically modified host cell comprises a nucleic acid comprising a nucleotide sequence encoding the above-described mutant AAV capsid protein, and in addition, a nucleic acid comprising a nucleotide sequence encoding one or more AAV rep proteins. In other embodiments, the subject host cell further comprises an rAAV vector. rAAV virions can be produced using the subject host cells. Methods for producing rAAV virions are described, for example, in U.S. Patent Publication Nos. 2005 / 0053922 and 2009 / 0202490 as described

[0148] Examples of non-limiting aspects of the present disclosure The aspects of the invention described above, including embodiments, may be advantageous alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the present disclosure are provided below numbered 1-34. As will be apparent to those skilled in the art upon reading the present disclosure, the individually numbered aspects can each be used or combined with any of the preceding or following individually numbered aspects. This is intended to facilitate all such combinations of aspects and is not limited to the combinations of aspects explicitly provided below.

[0149] 1. A recombinant adeno-associated virus (rAAV) virion comprising: a) a mutant AAV capsid protein that includes an insertion fragment of a heterologous peptide having a length of about 5 amino acids to about 20 amino acids in the capsid protein GH loop, and that increases the infectivity of the rAAV virion for retinal cells as compared to the infectivity of a control AAV virion comprising the corresponding parental AAV capsid protein for retinal cells; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product. 2. The rAAV virion according to aspect 1, which exhibits at least a 5-fold increased infectivity for the retinal cells as compared to the infectivity of a control AAV virion comprising the corresponding parental AAV capsid protein for the retinal cells. 3. The rAAV virion according to embodiment 1, which exhibits an infectivity at least 10-fold increased with respect to retinal cells as compared to the infectivity of the AAV virion containing the corresponding parental AAV capsid protein to the retinal cells. 4. The localization shown by the rAAV virion to one or more of the inner granular layer, outer granular layer, photoreceptor layer, ganglion cell layer, and retinal pigment epithelium is at least 5-fold increased as compared to the degree of localization by the AAV virion containing the corresponding parental AAV capsid protein to the inner granular layer, outer granular layer, photoreceptor layer, ganglion cell layer, or retinal pigment epithelium. The rAAV virion according to any one of embodiments 1 to 3. 5. The rAAV virion according to any one of embodiments 1 to 4, wherein the insertion site is between the amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2 or at the corresponding position of the capsid protein of another AAV serotype.

[0150] 6. The rAAV virion according to any one of embodiments 1 to 5, wherein the insertion site is located between the amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2 or at the corresponding position of the capsid protein of another AAV serotype. 7. The rAAV virion according to any one of embodiments 1 to 6, wherein the gene product is an interfering RNA or an aptamer. 8. The rAAV virion according to any one of embodiments 1 to 6, wherein the gene product is a polypeptide. 9. The rAAV virion according to embodiment 8, wherein the polypeptide is a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances the function of retinal cells. 10. The rAAV virion according to embodiment 8, wherein the polypeptide is an RNA-guided endonuclease.

[0151] 11. The rAAV virion according to embodiment 10, wherein the RNA-guided endonuclease is a Cas9 polypeptide. 12. The gene product is the rAAV virion according to embodiment 10, which is an RNA-guided endonuclease and a guide RNA. 13. The heterologous peptide inserted into the GH loop is any one of the formulas I to X The rAAV virion according to any one of embodiments 1 to 12. 14. The heterologous peptide is a peptide of formula I: X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, Ile, Pro, or Gln; X2 is Ala, Pro, Ser, Asp, Gly, Thr, or Val; X3 is Lys, His, Thr, Ile, Pro, Val, Arg, Ala, Asp, Glu, Asn, Gln, or Tyr; X4, if present, is Gln, Asp, Ser, Gly, Thr, Ile, Asn, Glu, Lys, or Arg; X5 is Asp, Ser, Gln, Val, Thr, Gly, Ala, Asn, Lys, or Tyr; X6 is Thr, Ala, Gln, Ser, Glu, Pro, or Ile; X7 is Thr, Ser, Asn, Pro, Leu, Gln, Lys, Ala, or Cys; X8 is Lys, Ser, Arg, Thr, Ala, Glu, Ile, or Asn; X9 is Asn, Pro, Ser, Lys, His, Ile, Thr, or Ala; and X 10 is Ala, Phe, Asp, Thr, Val, or Met. The rAAV virion according to any one of embodiments 1 to 12. 15. The rAAV virion according to embodiment 14, wherein the heterologous peptide comprises one of the following amino acid sequences. (1) LAKDATKNA (SEQ ID NO: 47), (2) PAHQDTTKNA (SEQ ID NO: 48), (3) LAHQDTTKNA (SEQ ID NO: 49), (4) LATTSQNKPA (SEQ ID NO: 50), (5) LAISDQTKHA (SEQ ID NO: 51), (6) IARGVAPSSA (SEQ ID NO: 52), (7) LAPDSTTRSA (SEQ ID NO: 53), (8) LAKGTELKPA (SEQ ID NO: 54), (9) LAIIDATKNA (SEQ ID NO: 55), (10) LAVDGAQRSA (SEQ ID NO: 56), (11) PAPQDTTKKA (SEQ ID NO: 57), (12) LPHQDTTKNA (SEQ ID NO: 58), (13) LAKDATKTIA (SEQ ID NO: 59), (14) LAKQQSASTA (SEQ ID NO: 60), (15) LAKSDQSKPA (SEQ ID NO: 61), (16) LSHQDTTKNA (SEQ ID NO: 62), (17) LAANQPSKPA (SEQ ID NO: 63), (18) LAVSDSTKAA (SEQ ID NO: 64), (19) LAAQGTAKKPA (SEQ ID NO: 65), (20) LAPDQTTRNA (SEQ ID NO: 66), (21) LAASDSTKAA (SEQ ID NO: 67), (22) LAPQDTTKNA (SEQ ID NO: 68), (23) LAKADETRPA (SEQ ID NO: 69), (24) LAHQDTAKNA (SEQ ID NO: 70), (25) LAHQDTKKNA (SEQ ID NO: 71), (26) LAHQDTTKHA (SEQ ID NO: 72), (27) LAHQDTTKKA (SEQ ID NO: 73), (28) LAHQDTTRNA (SEQ ID NO: 74), (29) LAHQDTTNA (SEQ ID NO: 75), (30) LAHQGTTKNA (SEQ ID NO: 76), (31) LAHQVTTKNA (SEQ ID NO: 77), (32) LAISDQSKPA (SEQ ID NO: 78), (33) LADATKTA (SEQ ID NO: 79), (34) LAKDTTKNA (SEQ ID NO: 80), (35) LAKSDQSRPA (SEQ ID NO: 81), (36) LAPQDTKKNA (SEQ ID NO: 82), (37) LATSDSTKAA (SEQ ID NO: 83), (38) LAVDGSQRSA (SEQ ID NO: 84), (39) LPISDQTKHA (SEQ ID NO: 85), (40) LPKDATKTIA (SEQ ID NO: 86), (41) LPPQDTTKNA (SEQ ID NO: 87), (42) PAPQDTTKNA (SEQ ID NO: 88), (43) QAHQDTTKNA (SEQ ID NO: 89), (44) LAHETSPRPA (SEQ ID NO: 90)(45)LAKSTSTAPA (SEQ ID NO: 91), (46)LADQDTTKNA (SEQ ID NO: 92), (47)LAESDQSKPA (SEQ ID NO: 93), (48)LAHKDTTKNA (SEQ ID NO: 94), (49)LAHKTQQKM (SEQ ID NO: 95), (50)LAHQDTTENA (SEQ ID NO: 96), (51)LAHQDTTINA (SEQ ID NO: 97), (52)LAHQDTTKKT (SEQ ID NO: 98), (53)LAHQDTTKND (SEQ ID NO: 99), (54)LAHQDTTKNT (SEQ ID NO: 100), (55)LAHQDTTKNV (SEQ ID NO: 101), (56)LAHQDTTKTM (SEQ ID NO: 102), (57)LAHQNTTKNA (SEQ ID NO: 103), (58)LAHRDTTKNA (SEQ ID NO: 104), (59)LAISDQTNHA (SEQ ID NO: 105), (60)LAKQKS, ASTA (SEQ ID NO: 106), (61)LAKSDQCKPA (SEQ ID NO: 107), (62)LAKSDQSKPD (SEQ ID NO: 108), (63)LAKSDQSNPA (SEQ ID NO: 109), (64)LAKSYQSKPA (SEQ ID NO: 110), (65)LANQDTTKNA (SEQ ID NO: 111), (66)LAPQNTTKNA (SEQ ID NO: 112), (67)LAPSSIQKPA (SEQ ID NO: 113), (68)LAQQDTTKNA (SEQ ID NO: 114), (69)LAYQDTTKNA (SEQ ID NO: 115), (70)LDHQDTTKNA (SEQ ID NO: 116), (71)LDHQDTTKSA (SEQ ID NO: 117), (72)LGHQDTTKNA (SEQ ID NO: 118), (73)LPHQDTTKND (SEQ ID NO: 119), (74)LPHQDTTKNT (SEQ ID NO: 120), (75)LPHQDTTNNA (SEQ ID NO: 121), (76)LTHQDTTKNA (SEQ ID NO: 122), (77)LTKDATKTIA (SEQ ID NO: 123), (78)LTPQDTTKNA (SEQ ID NO: 124), and (79)LVHQDTTKNA (SEQ ID NO: 125).

[0152] 16. The heterologous peptide has the formula II: X1X2X3X4X5X6X7X8X9X 10A peptide, wherein X1 is Leu, Ile, or Pro; X2 is Ala, Pro, or Ser; X3 is Lys, His, Thr, Ile, Pro, Val, Arg, or Ala; X4, if present, is Gln, Asp, Ser, Gly, Thr, Ile, or Asn; X5 is Asp, Ser, Gln, Val, Thr, Gly, or Ala; X6 is Thr, Ala, Gln, Ser, Glu, or Pro; X7 is Thr, Ser, Asn, Pro, Leu, Gln, Lys, or Ala; X8 is Lys, Ser, Arg, or Thr; X9 is Asn, Pro, Ser, Lys, His, Ile, Thr, or Ala; X 10 An rAAV virion according to any one of aspects 1 to 12, wherein X is Ala. 17. The rAAV virion according to aspect 16, wherein the peptide comprises one of the following amino acid sequences. (1) LAKDATKNA (SEQ ID NO: 47), (2) PAHQDTTKNA (SEQ ID NO: 48), (3) LAHQDTTKNA (SEQ ID NO: 49), (4) LATTSQNKPA (SEQ ID NO: 50), (5) LAISDQTKHA (SEQ ID NO: 51), (6) IARGVAPSSA (SEQ ID NO: 52), (7) LAPDSTTRSA (SEQ ID NO: 53), (8) LAKGTELKPA (SEQ ID NO: 54), (9) LAIIDATKNA (SEQ ID NO: 55), (10) LAVDGAQRSA (SEQ ID NO: 56), (11) PAPQDTTKKA (SEQ ID NO: 57), (12) LPHQDTTKNA (SEQ ID NO: 58), (13) LAKDATKTIA (SEQ ID NO: 59), (14) LAKQQSASTA (SEQ ID NO: 60), (15) LAKSDQSKPA (SEQ ID NO: 61), (16) LSHQDTTKNA (SEQ ID NO: 62), (17) LAANQPSKPA (SEQ ID NO: 63), (18) LAVSDSTKAA (SEQ ID NO: 64), (19) LAAQGTAKKPA (SEQ ID NO: 65), (20) LAPDQTTRNA (SEQ ID NO: 66), (21) LAASDSTKAA (SEQ ID NO: 67), (22) LAPQDTTKNA (SEQ ID NO: 68), (23) LAKADETRPA (SEQ ID NO: 69), (24) LAHQDTAKNA (SEQ ID NO: 70), (25) LAHQDTKKNA (SEQ ID NO: 71), (26) LAHQDTTKHA (SEQ ID NO: 72), (27) LAHQDTTKKA (SEQ ID NO: 73), (28) LAHQDTTRNA (SEQ ID NO: 74), (29) LAHQDTTNA (SEQ ID NO: 75), (30) LAHQGTTKNA (SEQ ID NO: 76), (31) LAHQVTTKNA (SEQ ID NO: 77), (32) LAISDQSKPA (SEQ ID NO: 78), (33) LADATKTA (SEQ ID NO: 79), (34) LAKDTTKNA (SEQ ID NO: 80), (35) LAKSDQSRPA (SEQ ID NO: 81), (36) LAPQDTKKNA (SEQ ID NO: 82), (37) LATSDSTKAA (SEQ ID NO: 83), (38) LAVDGSQRSA (SEQ ID NO: 84), (39) LPISDQTKHA (SEQ ID NO: 85), (40) LPKDATKTIA (SEQ ID NO: 86), (41) LPPQDTTKNA (SEQ ID NO: 87), and (42) PAPQDTTKNA (SEQ ID NO: 88). 18. The rAAV virion according to embodiment 16, wherein the peptide comprises one of the following amino acid sequences. (1) LAKDATKNA (SEQ ID NO: 47), (2) PAHQDTTKNA (SEQ ID NO: 48), (3) LAHQDTTKNA (SEQ ID NO: 49), (4) LATTSQNKPA (SEQ ID NO: 50), (5) LAISDQTKHA (SEQ ID NO: 51), (6) IARGVAPSSA (SEQ ID NO: 52), (7) LAPDSTTRSA (SEQ ID NO: 53), (8) LAKGTELKPA (SEQ ID NO: 54), (9) LAIIDATKNA (SEQ ID NO: 55), (10) LAVDGAQRSA (SEQ ID NO: 56), (11) PAPQDTTKKA (SEQ ID NO: 57), (12) LPHQDTTKNA (SEQ ID NO: 58), (13) LAKDATKTIA (SEQ ID NO: 59), (14) LAKQQSASTA (SEQ ID NO: 60), (15) LAKSDQSKPA (SEQ ID NO: 61), (16) LSHQDTTKNA (SEQ ID NO: 62), (17) LAANQPSKPA (SEQ ID NO: 63), and (18) LAVSDSTKAA (SEQ ID NO: 64). 19. A pharmaceutical composition comprising: a) a recombinant adeno-associated virus virion according to any one of embodiments 1 to 18; and b) a pharmaceutically acceptable excipient. 20. A method for delivering a gene product to retinal cells of an individual, the method comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according to any one of embodiments 1 to 18.

[0153] 21. The method according to embodiment 20, wherein the gene product is a polypeptide. 22. The method according to embodiment 20, wherein the gene product is a small interfering RNA or an aptamer. 23. The method according to embodiment 21, wherein the polypeptide is a neuroprotective factor, an anti-angiogenic polypeptide, an anti-apoptotic factor, or a polypeptide that enhances the function of retinal cells. 24. The method according to embodiment 21, wherein the polypeptide is glial cell line-derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain-derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis, retinoxysine, RPE65, retinitis pigmentosa GTPase-interacting protein-1, peripherin, peripherin-2, rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), or sonic hedgehog. 25. The method according to embodiment 21, wherein the polypeptide is an RNA-guided endonuclease.

[0154] 26. A method for treating an eye disease, comprising administering to an individual in need of treatment an effective amount of the recombinant adeno-associated virus (rAAV) virion according to any one of embodiments 1 to 18. 27. The method according to embodiment 26, wherein the administration is by intraocular injection. 28. The method according to embodiment 26, wherein the administration is by intravitreal injection. 29. The method according to embodiment 26, wherein the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinal detachment, Leber congenital cataract, diabetic retinopathy, achromotopsia, or color blindness. 30. An isolated nucleic acid comprising a nucleotide sequence encoding a mutant adeno-associated virus (AAV) capsid protein, wherein the mutant AAV capsid protein comprises an insertion fragment of about 5 amino acids to about 20 amino acids in the capsid protein GH loop as compared to the corresponding parental AAV capsid protein, and when the mutant capsid protein is present in an AAV virion, it increases the infectivity of the AAV virion for retinal cells, the amino acid insertion fragment is present in the GH loop of the native AAV capsid, and the insertion fragment is a peptide of any one of formulas I to X.

[0155] 31. The insertion site is between amino acid 587 and amino acid 588 of AAV2, AAV1 The nucleic acid according to embodiment 30, which is between amino acid 590 and amino acid 591 of, between amino acid 575 and amino acid 576 of AAV5, between amino acid 590 and amino acid 591 of AAV6, between amino acid 589 and amino acid 590 of AAV7, between amino acid 590 and amino acid 591 of AAV8, between amino acid 588 and amino acid 589 of AAV9, or between amino acid 588 and amino acid 589 of AAV10. 32. An isolated genetically modified host cell comprising the nucleic acid according to embodiment 30 or embodiment 31. 33. A mutant adeno-associated virus (AAV) capsid protein comprising an insertion fragment of about 5 amino acids to about 20 amino acids, wherein the amino acid insertion fragment is present in the GH loop of the native AAV capsid, and the insertion fragment is a peptide of any one of formulas I to X. 34. In any of embodiments 1 to 33, the heterologous peptide inserted into the GH loop can be one of formulas I to X. Formula I is X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, Ile, Pro, or Gln; X2 is Ala, Pro, Ser, Asp, Gly, Thr, or Val; X3 is Lys, His, Thr, Ile, Pro, Val, Arg, Ala, Asp, Glu, Asn, Gln, or Tyr; X4, if present, is Gln, Asp, Ser, Gly, Thr, Ile, Asn, Glu, Lys, or Arg; X5 is Asp, Ser, Gln, Val, Thr, Gly, Ala, Asn, Lys, or Tyr; X6 is Thr, Ala, Gln, Ser, Glu, Pro, or Ile; X7 is Thr, Ser, Asn, Pro, Leu, Gln, Lys, Ala, or Cys; X8 is Lys, Ser, Arg, Thr, Ala, Glu, Ile, or Asn; X9 is Asn, Pro, Ser, Lys, His, Ile, Thr, or Ala; and X 10 is Ala, Phe, Asp, Thr, Val, or Met. Formula II is X1X2X3X4X5X6X7X8X9X10 wherein X1 is Leu, Ile, or Pro; X2 is Ala, Pro, or Ser; X3 is Lys, His, Thr, Ile, Pro, Val, Arg, or Ala; X4, if present, is Gln, Asp, Ser, Gly, Thr, Ile, or Asn; X5 is Asp, Ser, Gln, Val, Thr, Gly, or Ala; X6 is Thr, Ala, Gln, Ser, Glu, or Pro; X7 is Thr, Ser, Asn, Pro, Leu, Gln, Lys, or Ala; X8 is Lys, Ser, Arg, or Thr; X9 is Asn, Pro, Ser, Lys, His, Ile, Thr, or Ala; X 10 is Ala, Formula III is X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, Ile, or Pro; X2 is Ala, Pro, or Ser; X3 is Lys, His, Thr, Ile, Pro, Val, Arg, or Ala; X4, if present, is Gln, Asp, Ser, Gly, Thr, Ile, or Asn; X5 is Asp, Ser, Gln, Val, Thr, Gly, or Ala; X6 is Thr, Ala, Gln, Ser, Glu, or Pro; X7 is Thr, Ser, Asn, Pro, Leu, Gln, Lys, or Ala; X8 is Lys, Ser, Arg, or Thr; X9 is Asn, Pro, Ser, Lys, His, Ile, Thr, or Ala; X 10 is Ala, Thr, Asp, Val, or Met, Formula IV is X1X2X3X4X5X6X7X8X9X 10wherein X1 is Leu, X2 is Ala, X3 is Lys, His, Thr, Ile, Pro, or Val, X4 is Gln, Asp, Ser, or Gly if present, X5 is Asp, Ser, or Gln, X6 is Thr, Ala, Gln, or Ser, X7 is Thr or Ser, X8 is Lys, Ser, or Arg, X9 is Asn, Pro, or Ser, X 10 is A la, and Formula V is X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, X2 is Ala, X3 is Lys or His, X4 is Gln, Asp, Ser, or Gly if present, X5 is Asp, Ser, or Gln, X6 is Thr, Ala, Gln, or Ser, X7 is Thr or Ser, X8 is Lys, Ser, or Arg, X9 is Asn, Pro, or Ser, X 10 is Ala, and Formula VI is X1X2X3X4X5X6X7X8X9X 10 wherein X1 is Leu, X2 is Ala, X3 is Asn, Lys, Thr, Gln, Ser, Ile, or Leu, X4 is Ser, Ala, Thr, Glu, Gln, Gly, Lys, or Pro, X5 is Asp, Pro, Glu, Thr, Asn, or Arg, X6 is Ile, His, Thr, Gln, Asn, Tyr, Asp, or Glu, X7 is X7 is Gln, Thr, Asn, Ala, or Lys, X8 is Lys, Thr, Arg, or Asp, X9 is Pro, Asn, Thr, Arg, Lys, or Ser, X 10 is Ala, and Formula VII is LAHQDTTKX1X2X3 (SEQ ID NO: 148) wherein X1 is Lys, Thr, Asn, or His, X2 is Ala, Thr, Val, Ile, Met, or Asp, X3 is Ala if present, Formula VIII is LAX1QX2TX3X4X5X6 (SEQ ID NO: 149), wherein X1 is Ala, Pro, Asp, or His; X2 is Gly or Asp; X3 is Ala, Thr, or Lys; X4 is Asn, Glu, Lys, Arg, or Thr; X5 is Leu, Asn, Lys, or Thr; and X6, if present, is Ala, Thr, Asp, Val, or Met. Formula IX is X1AX2X3DX4TKX5A (SEQ ID NO: 150), wherein X1 is Val or Leu; X2 is Ile, Val, His, or Asp; X3 is Glu, Ser, Lys, or Gln; X4 is His, Ser, or Thr; and X5 is Ser, Ala, Asn, His, or Lys. Formula X is X1X2X3AX4QX5TX6KNA (SEQ ID NO: 151), wherein X1, if present, is Leu; X2, if present, is Ala; X3 is Lys, Leu, or Pro; X4 is Asn, His, Pro, or Tyr; X5 is Asn, Gly, Val, or Asp; and X6 is Pro or Thr.

Example

[0156] The following examples are presented to fully disclose and describe to those skilled in the art the methods of carrying out and using the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are the following experiments intended to represent all or the only experiments conducted. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise noted, parts are by weight, molecular weights are weight-average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations can be used, for example, bp (base pair(s)), kb (kilobase(s)), pl (picoliter(s)), s or sec (second(s)), min (minute(s)), h or hr (hour(s)), aa (amino acid(s)), kb (kilobase(s)), bp (base pair(s)), nt (nucleotide(s)), i.m. (intramuscular), i.p. (intraperitoneal), s.c. (subcutaneous), and the like can be used.

[0157] Example 1: Generation and Characterization of AAV Virions Having AAV Capsid Variants In the eyes of large animals, widespread retinal infection by AAV is blocked by a powerful and complex barrier, but by using an in vivo iterative screening method, we created an AAV with a capsid variant that can overcome this barrier. Dogs are an important preclinical model for retinal degenerative diseases, their eye size and structure are similar to humans, and many types of retinal diseases occur naturally in various dog breeds. By using this screening method, we identified 96 AAV variants with the ability to cause widespread retinal infection in the dog retina. By using deep sequencing, we quantified the ability of 18 of these variants from the pool of screened AAV variants in the dog retina. Infectivity was quantified based on the levels of viral DNA and mRNA in retinal cells after intravitreal injection. These variants can be used for a variety of gene delivery strategies in the eyes of large animals and humans.

[0158] A peptide display library was created that contains random 21-nucleotide insertion fragments (flanked by a 6-nucleotide linker on the 5' side and a 3-nucleotide linker on the 3' side) at surface-exposed positions on the AAV capsid. Viral packaging was caused such that each viral genome was encapsulated within the capsid protein shell encoded by that genome. Thus, functional improvements identified through selection can be linked to the genomic sequences contained within the viral capsid. From this library, variants with the ability to infect from the vitreous into the dog retina were identified using an in vivo iterative screening and selection process (Figure 1). Approximately 250 μL of virus containing 10 13 ~10 14 virions per milliliter (vg / mL) of viral genome was injected into the eyes of dogs for each round. Three weeks after injection, the eyeballs were enucleated and retinal punch samples were taken from the central and peripheral regions of the retina. RPE cells were isolated from the retinal tissue and the tissue was frozen. Thereafter, DNA was harvested from the retinal cells and the cap gene was amplified by polymerase chain reaction (PCR) from the isolated samples. The cap gene was used for the next AAV packaging.

[0159] Figure 1 shows the directed evolution method used for the development of canine retinal AAV variants. A peptide display library was created and, after packaging into AAV vectors, injected into the eyes of dogs via intravitreal injection. By performing iterative rounds of selection, AAV variants were positively selected from the pool of vectors. After three rounds of selection, one round of error-prone PCR was performed, followed by additional rounds of selection.

[0160] After five rounds of selection, using Illumina deep sequencing, the relative amounts of variants in the library of AAV variants were shown, and variants whose relative amounts increased with each round were identified. The increase in what was shown within the virus library indicates that positive selection has occurred and that there is the ability to infect the canine retina from the vitreous. For in vivo screening, the top 96 variants were selected from a library of approximately 10 7 variants. These top 96 variants are shown in Table 1.

[0161] Table 1 Peptide number Sequence number LAKDATKNA 1 47 PAHQDTTKNA 2 48 LAHQDTTKNA 3 49 LATTSQNKPA 4 50 LAISDQTKHA 5 51 IARGVAPSSA 6 52 LAPDSTTRSA 7 53 LAKGTELKPA 8 54 LAIIDATKNA 9 55 LAVDGAQRSA 10 56 PAPQDTTKKA 11 57 LPHQDTTKNA 12 58 LAKDATKTIA 13 59 LAKQQSASTA 14 60 LAKSDQSKPA 15 61 LSHQDTTKNA 16 62 LAANQPSKPA 17 63 LAVSDSTKAA 18 64 LAAQGTAKPA 19 65 LAPDQTTRNA 20 66 LAASDSTKAA 21 67 LAPQDTTKNA 22 68 LAKADETRPA 23 69 LAHQDTAKNA 24 70 LAHQDTKKNA 25 71 LAHQDTTKHA 26 72 LAHQDTTKKA 27 73 LAHQDTTRNA 28 74 LAHQDTTTNA 29 75 LAHQGTTKNA 30 76 LAHQVTTKNA 31 77 LAISDQSKPA 32 78 LAKDATKTA 33 79 LAKDTTKNA 34 80 LAKSDQSRPA 35 81 LAPQDTKKNA 36 82 LATSDSTKAA 37 83 LAVDGSQRSA 38 84 LPISDQTKHA 39 85 LPKDATKTIA 40 86 LPPQDTTKNA 41 87 PAPQDTTKNA 42 88 QAHQDTTKNA 43 89 LAHETSPRPA 44 90 LAKSTSTAPA 45 91 LADQDTTKNA 46 92 LAESDQSKPA 47 93 LAHKDTTKNA 48 94 LAHKTQQKM 49 95 LAHQDTTENA 50 96 LAHQDTTINA 51 97 LAHQDTTKKT 52 98 LAHQDTTKND 53 99 LAHQDTTKNT 54 100 LAHQDTTKNV 55 101 LAHQDTTKTM 56 102 LAHQNTTKNA 57 103 LAHRDTTKNA 58 104 LAISDQTNHA 59 105 LAKQKSASTA 60 106 LAKSDQCKPA 61 107 LAKSDQSKPD 62 108 LAKSDQSNPA 63 109 LAKSYQSKPA 64 110 LANQDTTKNA 65 111 LAPQNTTKNA 66 112 LAPSSIQKPA 67 113 LAQQDTTKNA 68 114 LAYQDTTKNA 69 115 LDHQDTTKNA 70 116 LDHQDTTKSA 71 117 LGHQDTTKNA 72 118 LPHQDTTKND 73 119 LPHQDTTKNT 74 120 LPHQDTTNNA 75 121 LTHQDTTKNA 76 122 LTKDATKTIA 77 123 LTPQDTTKNA 78 124 LVHQDTTKNA 79 125 LAKANQNTPA 80 126 LATTPITKPA 81 127 LATTPIAKPA 82 128 LAIEDHTKSA 83 129 LAQSEHQRPA 84 130 LAKSPNKDNA 85 131 LANQDYTKTA 86 132 LANSTDQTRA 87 133 LALGETTRPA 88 134 LANSTEQTRA 89 135 LAQADTTKNA 90 136 LASKDITKTA 91 137 LASPRHNKKC 92 138 LAHQDTTKTIA 93 139 LAAQGTANL 94 140 VAIEDHTKSA 95 141 LAKANQNTPKNA 96 142

[0162] Using high-throughput sequencing, the infectivity to dog retina was further quantified for the top 18 mutants among the 96 mutants shown in Table 1. Table 2 shows the top 18 mutants selected for further quantification.

[0163] Table 2 LAKDATKNA (SEQ ID NO: 47) LAPDSTTRSA (SEQ ID NO: 53) LAKDATKTIA (SEQ ID NO: 59) PAHQDTTKNA (SEQ ID NO: 48) LAKGTELKPA (SEQ ID NO: 54) LAKQQSASTA (SEQ ID NO: 60) LAHQDTTKNA (SEQ ID NO: 49) LAIIDATKNA (SEQ ID NO: 55) LAKSDQSKPA (SEQ ID NO: 61) LATTSQNKPA (SEQ ID NO: 50) LAVDGAQRSA (SEQ ID NO: 56) LSHQDTTKNA (SEQ ID NO: 62) LAISDQTKHA (SEQ ID NO: 51) PAPQDTTKKA (SEQ ID NO: 57) LAANQPSKPA (SEQ ID NO: 63) IARGVAPSSA (SEQ ID NO: 52), LPHQDTTKNA (SEQ ID NO: 58), LAVSDSTKAA (SEQ ID NO: 64)

[0164] Eighteen mutants were packaged with the ubiquitous CAG promoter that induces GFP expression. The cDNA of GFP was fused to a unique 25-base pair barcode identifier. Each of the 18 mutants was packaged with a unique GFP barcode. The packaged mutants were mixed in equal ratios and injected into the retina. At that time, injection of an AAV2-based control vector (negative control corresponding to the parental serotype of natural origin) was also carried out. After injection, DNA and mRNA were collected from photoreceptor and RPE cells. By quantifying the levels of DNA and mRNA, the ability of the dog-derived vector to deliver DNA to the retina and cause transgene expression was determined (Figure 2).

[0165] Figure 2 shows the deep sequencing of mutants containing the GFP-barcode construct. Infection of the dog retina by dog-derived mutants was quantified by deep sequencing of tagged GFP cDNA and mRNA.

[0166] Using frozen retinal sections, confocal microscopy was used to image the expression of a library containing 18 members. Expression of GFP indicated that retinal cells within the retina and photoreceptors outside the retina were targeted by the library containing 18 members (Figure 3).

[0167] Figure 3 shows that a dog-derived AAV mutant library containing 18 members infects cells in the ganglion cell layer, inner granular layer, photoreceptor layer, and RPE layer.

[0168] Among the top 18 mutants tested, DNA and mRNA were recovered at the highest levels in two mutants. The mutant with the highest level of DNA recovery had an insertion sequence (SEQ ID NO: 57) of about 588-PAPQDTTKKA. The mutant with the highest level of mRNA expression had an insertion sequence (SEQ ID NO: 53) of about 588-LAPDSTTRSA.

[0169] Although the present invention has been described with respect to its specific embodiments, it should be understood by those skilled in the art that various changes can be made and equivalent forms can be substituted without departing from the true spirit and scope of the present invention. Furthermore, many modifications can be made to adapt a particular situation, material, composition of matter, process, or one or more process steps to the objectives, spirit, and scope of the present invention. All such modifications are intended to be included within the scope of the claims appended hereto.

[0170] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 368,929, filed Jul. 29, 2016, which is incorporated herein by reference in its entirety.

[0171] Description of Research Sponsored by the Federal Government This invention was made with government support under Contract / Award Nos. EY022975, EY018241, and EY06855 awarded by the National Institutes of Health. The government has certain rights in the invention.

Claims

1. A recombinant adeno-associated virus (rAAV) virion, comprising: a) an AAV capsid protein VP1 containing an inserted fragment of a heterologous peptide in the GH loop of the capsid protein, compared to the corresponding parental AAV capsid protein, wherein the heterologous peptide comprises the amino acid sequence KSDQSQP, VDGAQRS, TTSQNKP, or ANQPSKP, a 2-amino acid-long linker at the N-terminus of the heterologous peptide, and a 1-amino acid-long linker at the C-terminus of the heterologous peptide; b) a heterologous nucleic acid containing a nucleotide sequence encoding a heterologous gene product The rAAV virion.

2. The rAAV virion exhibits at least a 5-fold increased infectivity against retinal cells compared to the infectivity of a control AAV virion containing the corresponding parental AAV capsid protein VP1 against retinal cells, and / or The localization shown by the rAAV virion to one or more of the inner granule layer, outer granule layer, photoreceptor layer, ganglion cell layer, and retinal pigment epithelium is at least 5-fold increased compared to the degree of localization by an AAV virion containing the corresponding parental AAV capsid protein VP1 to the inner granule layer, outer granule layer, photoreceptor layer, ganglion cell layer, or retinal pigment epithelium. The rAAV virion according to Claim 1.

3. The rAAV virion according to Claim 1 or 2, wherein the inserted fragment exists between amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2.

4. The rAAV virion according to Claim 3, wherein the inserted fragment exists between amino acids 587 and 588 of VP1.

5. The heterologous gene product is a) an interfering RNA or an aptamer; b) a polypeptide, optionally a neuroprotective polypeptide, an anti-angiogenic polypeptide, an anti-apoptotic polypeptide, or a polypeptide that enhances the function of retinal cells, or an RNA-guided endonuclease; or c) an RNA-guided endonuclease and a guide RNA. The rAAV virion according to any one of Claims 1 to 4. **Claim 6**: The rAAV virion according to any one of claims 1 to 4, wherein the heterologous gene product is glial cell line-derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain-derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked apoptosis inhibitor, retinoschisin, RPE65, retinitis pigmentosa GTPase-interacting protein-1, peripherin, peripherin-2, rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), or sonic hedgehog. **Claim 7**: The rAAV virion according to any one of claims 1 to 6, wherein the heterologous peptide has a length of about 12 to 20 amino acids. **Claim 8**: a) The rAAV virion according to any one of claims 1 to 7, and b) a pharmaceutically acceptable excipient comprising a pharmaceutical composition. **Claim 9**: The recombinant adeno-associated virus (rAAV) virion according to any one of claims 1 to 7 for use in a method of delivering a gene product to retinal cells of an individual. **Claim 10**: The recombinant adeno-associated virus (rAAV) virion according to any one of claims 1 to 7 for use in a method of treating an eye disease. **Claim 11**: The rAAV virion according to claim 10, wherein the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinal detachment, Leber congenital amaurosis, diabetic retinopathy, achromotopsia, or color blindness. **Claim 12**: An isolated nucleic acid comprising a nucleotide sequence encoding an adeno-associated virus (AAV) capsid protein VP1 containing an insertion fragment of a heterologous peptide in the GH loop of the capsid protein, compared to the corresponding parental AAV capsid protein, wherein the heterologous peptide comprises the amino acid sequences KSDQSQP, VDGAQRS, TTSQNKP, or ANQPSKP, a 2-amino acid long linker at the N-terminus of the heterologous peptide, and a 1-amino acid long linker at the C-terminus of the heterologous peptide. Isolated nucleic acid. **Claim 13**: The isolated nucleic acid according to claim 12, wherein the insertion fragment is located between the amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2. **Claim 14**: The isolated nucleic acid according to claim 13, wherein the insertion fragment is located between amino acids 587 and 588 of VP1.

15. The isolated nucleic acid according to any one of claims 12 to 14, wherein the heterologous peptide has a length of about 12 to 20 amino acids.

16. An isolated genetically modified host cell comprising the isolated nucleic acid according to any one of claims 12 to 15.

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