Adeno-associated virus virions with variant capsids and methods of use thereof

Abstract

The present disclosure provides recombinant adeno-associated virus (AAV) virions having modified capsid proteins that exhibit a higher ability to cross the barrier between the vitreous fluid and retinal cells compared to wild-type AAV, thereby exhibiting a higher infectivity of retinal cells, and that contain heterologous nucleic acid. The present disclosure also provides methods for delivering gene products to retinal cells in an individual.

Description

[Background technology]

[0001] Vision is mediated by cells in the retina, a thin layer of cells that lines the back of the eye. Photoreceptors at the back of the retina respond to the absorption of photons by initiating a signal processing flow that passes through second- and third-order neurons within the retina, including bipolar, horizontal, and amacrine cells. The retinal pigment epithelium (RPE) cells beneath the photoreceptors are essential for facilitating this photoreceptor function by promoting the regeneration of the photon-detecting molecule 11-cis retinal through the visual cycle pathway. Retinal ganglion cells (RGCs) within the inner retina receive visual signals from third-order neurons and transmit these signals to the brain in the form of action potentials.

[0002] Mutations in genes expressed in retinal cells, including transcripts in photoreceptors, RPE, bipolar cells, and other cells, lead to the breakdown of visual signal processing and retinal degeneration. Many of the mutations underlying retinal degenerative diseases result in the death of photoreceptor and RPE cells.

[0003] Adeno-associated viruses (AAVs) belong to the genus Dependovirus in the family Parvoviridae. Members of this family and genus require coinfection with a helper virus, such as adenovirus, to facilitate replication; in the absence of a helper, AAVs establish latent infection. Virions consist of a 25-nm icosahedral capsid, which contains a 4.7-kb single-stranded DNA genome with two open reading frames: rep and cap. The nonstructural rep gene encodes four regulatory proteins essential for viral replication, while cap encodes three structural proteins (VP1–VP3) that assemble to form the 60-mer capsid shell. This viral capsid mediates the ability of AAV vectors to overcome many of the biological barriers to viral transduction, including cell surface receptor binding, endocytosis, intracellular trafficking, and nuclear unpackaging. Summary of the Invention

[0004] The present disclosure provides recombinant adeno-associated virus (AAV) virions having modified capsid proteins that exhibit a higher ability to cross the barrier between the vitreous fluid and retinal cells compared to wild-type AAV, thereby exhibiting a higher infectivity of retinal cells, and that contain heterologous nucleic acid. The present disclosure also provides methods for delivering gene products to retinal cells in an individual. [Brief explanation of the drawings]

[0005] [Figure 1] FIG. 1 shows a schematic diagram of the directed evolution method used to generate primate retinal AAV variants. [Figure 2] 1 shows a table of peptide insertions and substitutions in variant AAV capsids. [Figure 3A] 1 shows the amino acid sequence of an exemplary guide RNA-directed endonuclease. [Figure 3B] 1 shows the amino acid sequence of an exemplary guide RNA-directed endonuclease. [Figure 3C] 1 shows the amino acid sequence of an exemplary guide RNA-directed endonuclease. [Figure 4] The amino acid sequence of AAV2 capsid protein VP1 is shown, with amino acids 587 and 588 (NP) shown in bold and underlined. [Figure 5] The amino acid sequences corresponding to amino acids 570 to 610 of the AAV capsid protein VP1 of various AAV serotypes are shown. [Figure 6A] Figure 1 shows an alignment of the amino acid sequences of the AAV capsid protein loop IV (GH loop) region, with the insertion site indicated in bold and underlined. [Figure 6B] Figure 1 shows an alignment of the amino acid sequences of the AAV capsid protein loop IV (GH loop) region, with the insertion site indicated in bold and underlined. [Figure 6C] Figure 1 shows an alignment of the amino acid sequences of the AAV capsid protein loop IV (GH loop) region, with the insertion site indicated in bold and underlined. [Figure 7-1]A and B show the amino acid sequences of exemplary heterologous gene products. [Figure 7-2] C and D show the amino acid sequences of exemplary heterologous gene products. [Figure 7-3] E shows the amino acid sequence of an exemplary heterologous gene product. [Figure 7-4] F indicates the amino acid sequence of an exemplary heterologous gene product. [Figure 7-5] G shows the amino acid sequence of an exemplary heterologous gene product. [Figure 7-6] H to J show the amino acid sequences of exemplary heterologous gene products. [Figure 7-7] K indicates the amino acid sequence of an exemplary heterologous gene product. [Figure 7-8] L indicates the amino acid sequence of an exemplary heterologous gene product. [Figure 7-9] M indicates the amino acid sequence of an exemplary heterologous gene product. [Figure 7-10] N and O indicate the amino acid sequences of exemplary heterologous gene products. [Figure 7-11] P indicates the amino acid sequence of an exemplary heterologous gene product. [Figure 7-12] Q and R indicate the amino acid sequences of exemplary heterologous gene products. [Figure 7-13] S indicates the amino acid sequence of an exemplary heterologous gene product. [Figure 7-14] T indicates the amino acid sequence of an exemplary heterologous gene product. [Figure 7-15] U indicates the amino acid sequence of an exemplary heterologous gene product. [Figure 7-16] V indicates the amino acid sequence of an exemplary heterologous gene product. [Figure 8] A shows the amino acid sequence of the AAV4 capsid, and B shows the amino acid sequence of the ancestral AAV capsid. [Figure 9] Table 1 shows the ranking of primate-derived variants and controls recovered from photoreceptors after injection of a green fluorescent protein (GFP) barcoded library. [Figure 10] Table 2 is shown. Table 2 shows the ranking of primate-derived variants and controls recovered from RPE cells after GFP barcoded library injection. [Figure 11] GFP expression of a GFP barcode library in the primate retina is shown. [Figure 12-1] A and B show directed evolution of AAV in the primate retina. [Figure 12-2] C shows directed evolution of AAV in the primate retina. [Figure 12-3] C shows directed evolution of AAV in the primate retina. [Figure 12-4] C and D show directed evolution of AAV in the primate retina. [Figure 12-5] E and F show directed evolution of AAV in the primate retina. The sequences from top to bottom in F are set forth in SEQ ID NOs: 117 to 135. [Figure 13-1] A-F show validation of evolved AAVs in the primate retina. [Figure 13-2] G–L show validation of evolved AAVs in the primate retina. [Figure 13-3] MO shows validation of evolved AAVs in the primate retina. [Figure 13-4] P and Q show validation of evolved AAV in the primate retina. DETAILED DESCRIPTION OF THE INVENTION

[0006] The term "retinal cells," as used herein, can refer 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.

[0007] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term encompasses all subtypes and both naturally occurring and recombinant forms, unless otherwise required. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as recombinant AAV vector (or "rAAV vector"). 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), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. See, e.g., Mori et al. (2004) Virology 330:375. The term "AAV" also includes chimeric AAV. "Primate AAV" refers to AAV isolated from primates, "non-primate AAV" refers to AAV isolated from non-primate mammals, "bovine AAV" refers to AAV isolated from bovine mammals (e.g., dairy cows), etc.

[0008] As used herein, "rAAV vector" refers to an AAV vector that contains a polynucleotide sequence that is not derived from AAV (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for genetic transformation of a cell. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0009] "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically from all capsid proteins of wild-type AAV) and an encapsidated polynucleotide rAAV vector. When the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, e.g., a transgene to be delivered to a mammalian cell), the particle is typically referred to as an "rAAV vector particle" or simply an "rAAV vector." Thus, production of rAAV particles necessarily includes production of rAAV vectors, and thus, the vector is contained within the rAAV particle.

[0010] "Packaging" refers to the series of intracellular events that lead to assembly and encapsidation of the AAV particle.

[0011] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0012] A "helper virus" for AAV refers to a virus that enables AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. Various such helper viruses are known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. Adenoviruses encompass several different subgroups, with subgroup C type 5 adenovirus being the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the American Type Culture Collection (ATCC). Examples of viruses from the herpes family include herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), which are also available from depositories such as the American Type Culture Collection (ATCC).

[0013] "Helper virus function(s)" refers to the function(s) encoded within the helper virus genome that enable replication and packaging of AAV (along with other replication and packaging requirements described herein). As described herein, "helper virus functions" can be provided in multiple ways, including by providing a helper virus or, for example, by providing a polynucleotide sequence encoding the essential function(s) en route to the producer cell.

[0014] An "infectious" virus or viral particle is a virus or viral particle that contains polynucleotide components that can be delivered to cells for which the viral species is tropic. This term does not necessarily imply any replicative capacity of the virus. As used herein, an "infectious" virus or viral particle is a virus or viral particle that can access, infect, and express heterologous nucleic acid in target cells. Thus, "infectivity" refers to the ability of a viral particle to access, infect, and express heterologous nucleic acid in target cells. Infectivity can refer to in vitro or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as viral genome (vg) copy number. The ability of a viral particle to express heterologous nucleic acid in a cell can be referred to as "transduction." The ability of viral particles to express heterologous nucleic acids in cells can be assayed using several techniques, including evaluation of marker genes, such as green fluorescent protein (GFP) assays (e.g., if the virus contains a nucleotide sequence encoding GFP) (GFP is produced in cells infected with viral particles and detected and / or measured), or measurement of produced proteins (e.g., by enzyme-linked immunosorbent assay (ELISA)). Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Methods for determining the ratio of infectious viral particles to total viral particles are known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11:S337 (describing the TCID50 infectious titer assay) and Zolotukhin et al. (1999) Gene Ther. 6:973.

[0015] A "replication-competent" virus (e.g., replication-competent AAV) refers to a phenotypically wild-type virus that is infectious and also capable of replicating in infected cells (i.e., in the presence of a helper virus or helper virus functions). In the case of AAV, replication ability generally requires the presence of functional AAV packaging genes. Generally, the rAAV vectors described herein are replication-incompetent in mammalian cells (particularly human cells) due to the lack of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequences to minimize the possibility of generating replication-competent AAV by recombination between the AAV packaging genes and the incoming rAAV vector. In many embodiments, the rAAV vector preparations described herein contain little, if any, replication-competent AAV (rcAAV, also known as RCA) (e.g., 10 2 Approximately 1 rcAAV per rAAV particle, less than 10 4 Approximately 1 rcAAV per rAAV particle, less than 10 8 Approximately 1 rcAAV per rAAV particle, less than 10 12 less than about 1 rcAAV per rAAV particle, or no rcAAV present).

[0016] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and can be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. As used herein, polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms that are known or predicted to form a double-stranded form.

[0017] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that when aligned, the percentage of bases or amino acids in the two sequences are the same when compared. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, including BLAST, available on the World Wide Web at ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package from 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 & Co., San Diego, California, USA). Of particular interest are alignment programs that allow gaps in sequences. The Smith-Waterman algorithm is one type of algorithm that allows gaps in sequence alignments. See Meth. Mol. Biol. 70:173-187 (1997). The GAP program, which uses the Needleman and Wunsch alignment method, can also be used to align sequences. See J. Mol. Biol. 48:443-453 (1970).

[0018] Of interest is the BestFit program, which determines sequence identity using the Smith-Waterman local homology algorithm (Advances in Applied Mathematics 2:482-489 (1981)). The gap creation penalty generally ranges from 1 to 5, usually 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. Sequence identity is preferably determined using the default parameters determined by the program. This program is also available from the Genetics Computing Group (GCG) package, Madison, Wisconsin, USA.

[0019] Another program of interest 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. Percent sequence identity is calculated by FastDB based on the following parameters: Mismatch penalty: 1.00, Gap penalty: 1.00, Gap size penalty: 0.33, and Consolidation penalty: 30.0.

[0020] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.

[0021] As used herein, the term "guide RNA" refers to an RNA that comprises i) an "activator" nucleotide sequence that binds to a guide RNA-directed endonuclease (e.g., a Class 2 CRISPR / Cas endonuclease, e.g., a Type II, Type V, or Type VI CRISPR / Cas endonuclease), and ii) a "targeter" nucleotide sequence that comprises a nucleotide sequence that hybridizes to a target nucleic acid. The "activator" nucleotide sequence and the "targeter" nucleotide sequence can be on separate RNA molecules (e.g., a "dual guide RNA") or can be on the same RNA molecule (a "single guide RNA").

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

[0023] As used herein, the term "microRNA" refers to any type of interfering RNA, including, but not limited to, endogenous microRNAs and artificial microRNAs (e.g., synthetic miRNAs). Endogenous microRNAs are small RNAs naturally encoded in the genome that can regulate the productive utilization of mRNA. Artificial microRNAs can be any type of RNA sequence, other than endogenous microRNAs, that can regulate the activity of mRNA. A microRNA sequence can be an RNA molecule composed of any one or more of these sequences. MicroRNAs (or "miRNAs") have been 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 microRNAs include any RNA that is a fragment of a larger RNA, or any RNA that is a miRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, or other small non-coding RNA. See, e.g., U.S. Patent Applications Nos. 20050272923, 20050266552, 20050142581, and 20050075492. A "microRNA precursor" (or "pre-miRNA") refers to a nucleic acid having a stem-loop structure with a microRNA sequence embedded within it."Mature microRNAs" (or "mature miRNAs") include microRNAs cleaved from microRNA precursors ("pre-miRNAs") or synthesized (e.g., synthesized in the laboratory by cell-free synthesis) and have a length of about 19 nucleotides to about 27 nucleotides; for example, mature microRNAs have a length of 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, or 27 nt. Mature microRNAs can bind to target mRNAs and inhibit translation of the target mRNAs.

[0024] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction, or ligation steps, and other procedures that result in a construct that differs from polynucleotides found in nature. A recombinant virus is a viral particle that includes a recombinant polynucleotide. The term includes copies of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0025] A "regulatory element" or "regulatory sequence" is a nucleotide sequence involved in a molecular interaction that contributes to the functional control of a polynucleotide, including its replication, duplication, transcription, splicing, translation, or degradation. This control can affect the frequency, speed, or specificity of the process and can be either reinforcing or inhibitory in nature. Regulatory elements known in the art include, for example, transcription control sequences such as promoters and enhancers. A promoter is a DNA region capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region usually located downstream (3' direction) of the promoter.

[0026] "Operably linked" or "operably linked" refers to the juxtaposition of genetic elements in a relationship permitting them to act in their expected manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. Intervening residues can be present between the promoter and coding region so long as this functional relationship is maintained.

[0027] An "expression vector" is a vector containing a region encoding a polypeptide of interest and is used to effect expression of the protein in an intended target cell. An expression vector also contains regulatory elements operably linked to the coding region to facilitate protein expression in the target. The combination of regulatory elements and one or more genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," and many expression cassettes are known and available in the art or can be readily constructed from components available in the art.

[0028] "Heterologous" means that it is derived from a genotypically different entity from the rest of the entity being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence that is not found in nature is a heterologous promoter. Thus, for example, an rAAV containing a heterologous nucleic acid encoding a heterologous gene product is an rAAV containing a nucleic acid that is not normally contained in naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by naturally occurring wild-type AAV. As another example, a variant AAV capsid protein containing a heterologous peptide inserted into the GH loop of the capsid protein is a variant AAV capsid protein containing an insertion of a peptide that is not normally contained in naturally occurring wild-type AAV.

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

[0030] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a gene sequence if the gene sequence is available to perform its function during extended in vitro culture of the cell. Generally, such cells are "genetically" altered (genetically modified) in that a genetic alteration has been introduced that is inherited by the altered cell's progeny.

[0031] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. These terms also include modified amino acid polymers, e.g., disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. When discussed in the context of delivering gene products to a mammalian subject, polypeptides and compositions for polypeptides, such as antiangiogenic polypeptides, neuroprotective polypeptides, etc., refer to the respective intact polypeptides or any fragments or genetically engineered derivatives thereof that retain the desired biochemical function of the intact protein. Similarly, reference to nucleic acids encoding antiangiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivering gene products to a mammalian subject (which may be referred to as "transgenes" to be delivered to recipient cells) includes polynucleotides encoding the intact polypeptides or any fragments or genetically engineered derivatives having the desired biochemical function.

[0032] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance that lacks at least some of the other components that may be present when the substance or similar substance naturally occurs or is initially prepared. Thus, for example, an isolated substance can be prepared by using a purification technique to enrich the substance from a source mixture. Enrichment can be measured on an absolute basis, e.g., weight per volume of solution, or can be measured relative to a second, potentially interfering substance present in the source mixture. Increasingly enriched embodiments of the invention are even more isolated. In some embodiments, an isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is purified to, for example, about 80% to about 90% purity, at least about 90% purity, at least about 95% purity, at least about 98% purity, or at least about 99% purity, or greater.

[0033] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease or adverse effects that may result from the disease. As used herein, "treatment" encompasses any disease treatment in mammals, particularly humans, and includes (a) preventing the onset of a disease in a subject who may be predisposed to or at risk for the disease but who has not yet been diagnosed with the disease; (b) inhibiting a disease, i.e., arresting its onset; and (c) palliating a disease, i.e., causing regression of a disease.

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

[0035] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0036] When a range of values ​​is stated, it is understood that each intervening value (to the nearest tenth of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of the range), and that stated or intervening value, is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. When the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the 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 incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0038] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a rAAV virion" includes a plurality of such virions, a reference to "the capsid protein" includes reference to one or more variant capsid proteins and equivalents thereof known to those skilled in the art, and so on. It should further be noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as a prelude to the use of exclusive terminology such as "solely," "only," or the use of a "negative" limitation in connection with the recitation of claim elements.

[0039] It will be understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the invention are expressly embraced by the invention, and all such combinations are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are expressly embraced by the invention, and all such subcombinations are disclosed herein as if each and every such combination were individually and explicitly disclosed herein.

[0040] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0041] The present disclosure provides recombinant adeno-associated virus (AAV) virions with modified capsid proteins that exhibit a higher ability to cross the barrier between the vitreous fluid and retinal cells compared to wild-type AAV, thereby exhibiting a higher infectivity of retinal cells, and that contain heterologous nucleic acids. The present disclosure also provides methods for delivering gene products to retinal cells in an individual. The present disclosure also provides methods for modifying target nucleic acids present in retinal cells.

[0042] The present disclosure provides recombinant adeno-associated virus (AAV) virions (rAAV) with modified capsid proteins that exhibit higher infectivity of retinal cells compared to wild-type AAV and contain heterologous nucleic acids. The rAAV virions exhibit increased ability to cross the barrier between the vitreous fluid and retinal cells. The rAAV virions exhibit higher infectivity of retinal cells compared to the infectivity of corresponding wild-type AAV for retinal cells. The retinal cells may 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 epithelial (RPE) cells. The present disclosure further provides methods for delivering gene products to retinal cells in an individual and methods for treating ocular diseases. The present disclosure provides rAAV virions having modified capsid proteins that exhibit at least a five-fold increase in localization to one or more of the inner nuclear layer, outer nuclear layer, photoreceptor layer, ganglion cell layer, or retinal pigment epithelium compared to the degree of localization to the inner nuclear layer, outer nuclear layer, photoreceptor layer, ganglion layer, or retinal pigment epithelium by AAV virions containing the corresponding parent AAV capsid proteins, and that contain heterologous nucleic acid.

[0043] Variant AAV capsid polypeptides The present disclosure provides variant AAV capsid proteins. As described above, the variant AAV capsid proteins of the present disclosure are modified relative to a wild-type or other reference AAV capsid protein. Modifications include insertions and swaps (e.g., replacing a contiguous stretch of amino acids with a different contiguous stretch of amino acids).

[0044] In some cases, the variant AAV capsid proteins of the present disclosure comprise an insertion of a heterologous peptide between 5 and 20 amino acids in length at an insertion site within a surface-accessible (e.g., solvent-accessible) portion of a parent AAV capsid protein, such that the variant capsid protein, when present within an AAV virion, confers increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV virions comprising the corresponding parent AAV capsid protein, particularly when the AAV virion is injected intravitreally. Thus, the variant AAV capsid proteins of the present disclosure, when present within an AAV virion, confers an increased ability of the AAV virion to cross barriers between the vitreous fluid ("vitreous humor") and retinal cells. Such barriers include, for example, the inner limiting membrane (ILM), the extracellular matrix of the retina, the cell membranes of the retinal cells themselves, the inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion cell layer, and the retinal pigment epithelium. In some cases, the retinal cells are Müller cells. Other retinal cells include amacrine cells, bipolar cells, and horizontal cells. An "insertion of about 5 amino acids to about 20 amino acids" is also referred to herein as a "peptide insertion" (e.g., a heterologous peptide insertion). A "corresponding parent AAV capsid protein" refers to an AAV capsid protein of the same AAV serotype without the heterologous peptide insertion. In some cases, the variant AAV capsid contains a single heterologous peptide insertion of 5 amino acids to 20 amino acids (e.g., 5-7, 7-10, 10-12, 12-15, or 15-20 amino acids) in length.

[0045] The modification in the AAV capsid can also be a swap (e.g., replacing a contiguous stretch of amino acids with a heterologous peptide). Thus, the substitution is the insertion of a heterologous peptide in place of a contiguous stretch of amino acids. In some cases, the variant AAV capsid proteins of the present disclosure comprise a replacement of a contiguous stretch of amino acids at a site within a surface-accessible (e.g., solvent-accessible) portion of a parent AAV capsid protein with a heterologous peptide of 5 to 20 amino acids in length, such that when present in AAV virions, the variant capsid protein confers increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV virions containing the corresponding parent AAV capsid protein, particularly when the AAV virions are injected intravitreally. Thus, when present in AAV virions, the variant AAV capsid proteins of the present disclosure confers increased ability of AAV virions to cross the barrier between the intravitreal fluid ("vitreous humor") and retinal cells. Such barriers include, for example, the ILM, the extracellular matrix of the retina, the cell membranes of the retinal cells themselves, the inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion cell layer, and the retinal pigment epithelium. In some cases, the retinal cells are Müller cells. Other retinal cells include amacrine cells, bipolar cells, and horizontal cells. A "substitution of about 5 to about 20 amino acids" is also referred to herein as a "peptide swap" (e.g., replacing a contiguous stretch of amino acids with a heterologous peptide). A "corresponding parent AAV capsid protein" refers to an AAV capsid protein of the same AAV serotype without the heterologous peptide. In some cases, the variant AAV capsid contains a single heterologous peptide replacement 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) in length.

[0046] For purposes of the following description, "insertion" refers both to an insertion of a heterologous peptide that does not replace a contiguous stretch of amino acids and to an insertion of a heterologous peptide that replaces a contiguous stretch of amino acids.

[0047] The insertion site can be within the GH loop or loop IV of the AAV capsid protein, e.g., within a solvent-accessible portion of the GH loop or loop IV of the AAV capsid protein. For a description of the GH loop / loop IV of the AAV capsid protein, see, e.g., 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 within amino acids 411-650 of the AAV capsid protein, as shown in Figures 6A-6C. For example, the insertion site can be within amino acids 570-611 of AAV2, within amino acids 571-612 of AAV1, within amino acids 560-601 of AAV5, within amino acids 571-612 of AAV6, within amino acids 572-613 of AAV7, within amino acids 573-614 of AAV8, within amino acids 571-612 of AAV9, or within amino acids 573-614 of AAV10, as shown in Figure 5. In some cases, the insertion site is between amino acids 588 and 589 of the AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 587 and 588 of the AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 575 and 576 of the AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 584 and 585 of the AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 590 and 591 of the AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 584 and 585 of the AAV4 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 575 and 576 of the AAV5 capsid protein, or a corresponding insertion site in an AAV of a different serotype.In some cases, the site of substitution is between amino acids 584 and 598 of the AAV2 capsid protein, or the corresponding site in an AAV of a different serotype.

[0048] In some cases, a heterologous peptide of about 5 amino acids to about 20 amino acids (e.g., 5-7, 7-10, 10-12, 12-15, or 15-20 amino acids) in length is inserted into an insertion site within the GH loop or loop IV of the capsid protein relative to the corresponding parent AAV capsid protein. For example, the insertion site can be between amino acids 587 and 588 of AAV2, or between amino acids 588 and 589 of AAV2, or a corresponding position in the capsid subunit of another AAV serotype. Note that insertion site 587 / 588 is based on the AAV2 capsid protein. A heterologous peptide of about 5 amino acids to about 20 amino acids (e.g., 5-7, 7-10, 10-12, 12-15, or 15-20 amino acids) in length may also be inserted into a corresponding site in an AAV serotype other than AAV2 (e.g., AAV8, AAV9, etc.). Based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, one skilled in the art will know where in the capsid protein of any given AAV serotype there is an insertion site corresponding to amino acids 587-588 of AAV2. The sequences corresponding to amino acids 570-611 of the AAV2 capsid protein VP1 (see Figure 4) in various AAV serotypes are shown in Figure 5. See, e.g., GenBank accession number NP_049542 for AAV1, GenBank accession number NP_044927 for AAV4, 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, GenBank accession number AAT46337 for AAV10, and GenBank accession number AAO88208 for AAVrh10. For ancestral AAV capsids, see, e.g., Santiago-Ortiz et al. (2015) Gene Ther. 22:934.

[0049] For example, the insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, between amino acids 588 and 589 of AAV10, or between amino acids 585 and 586 of AAV4. The insertion sites are underlined in Figure 5. The amino acid numbering is based on the numbering shown in Figure 5.

[0050] In some cases, a subject capsid protein comprises a GH loop 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 set forth in Figures 6A-6C, and having an insertion of a heterologous peptide between about 5 amino acids and about 20 amino acids (e.g., 5-7, 7-10, 10-12, 12-15, or 15-20 amino acids) in length.

[0051] In some cases, the variant AAV capsid proteins of the present disclosure comprise a substitution (or substitutions) of a segment (or sequence) of consecutive amino acids within a surface-accessible (e.g., solvent-accessible) portion of a parent AAV capsid, such that the variant capsid protein, when present in an AAV virion, confers increased infectivity of retinal cells compared to the infectivity of retinal cells by an AAV virion comprising the corresponding parent AAV capsid protein, particularly when the AAV virion is injected intravitreally. Thus, when present in an AAV virion, a subject variant AAV capsid protein comprising a sequence substitution confers an increased ability of the AAV virion to cross barriers between the vitreous humor and retinal cells. Such barriers include, for example, the inner limiting membrane, the extracellular matrix of the retina, and the cell membrane of the retinal cell itself. A "substitution of between about 5 consecutive amino acids and about 25 consecutive amino acids" is also referred to herein as a "loop swap" (i.e., a heterologous peptide substitution). In such cases, the "corresponding parent AAV capsid protein" refers to an AAV capsid protein of the same AAV serotype without the subject loop swap. In some cases, the variant AAV capsid contains heterologous peptide substitutions ranging from 5 contiguous amino acids to 25 contiguous amino acids in length, e.g., 5-9, 9-11, 10-15, 15-20, or 20-25 amino acids in length.

[0052] In some cases, a heterologous peptide of about 5 amino acids to about 25 amino acids (e.g., 5-9, 9-10, 10-15, 15-20, or 20-25 amino acids) in length is substituted for a substantial number of consecutive amino acids in the corresponding parent AAV capsid protein. In some embodiments, the substitution begins at about amino acid 588 of AAV2, or at a corresponding position in the capsid subunit of another AAV serotype, and ends at about amino acid 598 of AAV2, or at a corresponding position in the capsid subunit of another AAV serotype. Note that residues 588-598 are based on the AAV2 VP1 capsid protein. A heterologous peptide of about 5 amino acids to about 25 amino acids in length may also be substituted into the corresponding site in an AAV serotype other than AAV2 (e.g., AAV8, AAV9, etc.). Those skilled in the art will know, based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, which substitution sites in the capsid protein of any given AAV serotype correspond to amino acids 588-598 of AAV2. The amino acid residues in various AAV serotypes corresponding to amino acids 588-598 of the capsid protein VP1 of AAV2 (see FIG. 4) are shown in FIG. See, e.g., GenBank accession number NP_049542 for AAV1, GenBank accession number NP_044927 for AAV4, 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, GenBank accession number AAT46337 for AAV10, and GenBank accession number AAO88208 for AAVrh10.

[0053] In some cases, a heterologous peptide of about 5 amino acids to about 25 amino acids (e.g., 5-9, 9-10, 10-15, 15-20, or 20-25 amino acids) in length is substituted for a corresponding number of consecutive amino acids in the corresponding parent AAV capsid protein. In some embodiments, the substitution begins at about amino acid 585 of AAV2, or a corresponding position in the capsid subunit of another AAV serotype, and ends at about amino acid 598 of AAV2, or a corresponding position in the capsid subunit of another AAV serotype. Note that residues 585-598 are based on the AAV2 VP1 capsid protein. A heterologous peptide of about 5 amino acids to about 25 amino acids in length may also be substituted into the corresponding site in an AAV serotype other than AAV2 (e.g., AAV8, AAV9, etc.). Those skilled in the art will know, based on a comparison of the amino acid sequences of the capsid proteins of various AAV serotypes, which substitution sites in the capsid protein of any given AAV serotype correspond to amino acids 585-598 of AAV2. The amino acid residues in various AAV serotypes corresponding to amino acids 585-598 of the capsid protein VP1 of AAV2 (see FIG. 4) are shown in FIG. See, e.g., GenBank accession number NP_049542 for AAV1, GenBank accession number NP_044927 for AAV4, 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, GenBank accession number AAT46337 for AAV10, and GenBank accession number AAO88208 for AAVrh10.

[0054] Insertion / Replacement Peptides As described above, heterologous peptides of about 5 to about 20 amino acids in length are inserted into the GH loop of an AAV capsid or replace a substantial number of consecutive amino acids in the GH loop of an AAV capsid. For brevity, the term "inserted peptide" will be used hereinafter to describe both peptides inserted into a parent AAV capsid and peptides that replace a segment of adjacent amino acids in the GH loop of an AAV capsid. In some cases, the inserted peptide is 5 to 20 amino acids in length. In some cases, the inserted peptide is 7 to 15 amino acids in length. In some cases, the inserted peptide is 9 to 15 amino acids in length. In some cases, the inserted peptide is 9 to 12 amino acids in length. The inserted peptide is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In some cases, the inserted peptide is 7 amino acids in length. 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.

[0055] In some cases, the peptide insert is a peptide of Formula I: LA(L / N)(I / Q)(Q / E)(D / H)(S / V)(M / K)(R / N)A (SEQ ID NO: 136).

[0056] In some cases, the peptide of Formula I comprises the amino acid sequence of (21) LALIQDSMRA (SEQ ID NO: 35). In some cases, the peptide of Formula I comprises the amino acid sequence of (22) LANQEHVKNA (SEQ ID NO: 2).

[0057] In some cases, the peptide insert is a peptide of Formula II: TX1X2X3X4X5X6X7X8GLX9 (SEQ ID NO: 137), where X1 is G, V, or S, X2 is V, E, P, G, D, M, A, or S, X3 is M, V, Y, H, G, S, or D, X4 is R, D, S, G, V, Y, T, H, or M, X5 is S, L, G, T, Q, P, or A, X6 is T, A, S, M, D, Q, or H, X7 is N, G, S, L, M, P, G, or A, X8 is S, G, D, N, A, I, P, or T, and X9 is S or N.

[0058] Peptide inserts of Formula II include, but are not limited to, (1) TGVMRSTNSGLN (SEQ ID NO: 6), (2) TGEVDLAGGGLS (SEQ ID NO: 7), (3) TSPYSGSSDGLS (SEQ ID NO: 8), (4) TGGHDSSLDGLS (SEQ ID NO: 9), (5) TGDGGTTMNGLS (SEQ ID NO: 98), (6) TGGHGSAPDGLS (SEQ ID NO: 99), (7) TGMHVTMMAGLN (SEQ ID NO: 100), (8) TGASYLDNSGLS (SEQ ID NO: 101), (9) TVVSTQAGIGLS (SEQ ID NO: 135), (10) TGVMHSQASGLS (SEQ ID NO: 21), (11) TGDGSPAAPGLS (SEQ ID NO: 22), and (12) TGSDMAHGTGLS (SEQ ID NO: 23). In some cases, the peptide insert is (1) TGVMRSTNSGLN (SEQ ID NO: 6). In some cases, the peptide insert is (2) TGEVDLAGGGLS (SEQ ID NO:7). In some cases, the peptide insert is (3) TSPYSGSSDGLS (SEQ ID NO:8). In some cases, the peptide insert is (4) TGGHDSSLDGLS (SEQ ID NO:9). In some cases, the peptide insert is (5) TGDGGTTMNGLS (SEQ ID NO:98). In some cases, the peptide insert is (6) TGGHGSAPDGLS (SEQ ID NO:99). In some cases, the peptide insert is (7) TGMHVTMMAGLN (SEQ ID NO:100). In some cases, the peptide insert is (8) TGASYLDNSGLS (SEQ ID NO:101). In some cases, the peptide insert is (9) TVVSTQAGIGLS (SEQ ID NO:20). In some cases, the peptide insert is (10) TGVMHSQASGLS (SEQ ID NO:21). In some cases, the peptide insert is (11)TGDGSPAAPGLS (SEQ ID NO: 22). In some cases, the peptide insert is (12)TGSDMAHGTGLS (SEQ ID NO: 23).

[0059] In some cases, the peptide insert is a peptide of Formula III: TGX1X2X3X4X5X6X7GLS (SEQ ID NO: 138), where Xi is V, E, P, G, D, M, A, or S, X2 is M, V, Y, H, G, S, or D, X3 is R, D, S, G, V, Y, T, H, or M, X4 is S, L, G, T, Q, P, or A, X5 is T, A, S, M, D, Q, or H, X6 is N, G, S, L, M, P, G, or A, and X7 is S, G, D, N, A, I, P, or T.

[0060] Peptide inserts of Formula III include, but are not limited to, (2) TGEVDLAGGGLS (SEQ ID NO:7), (4) TGGHDSSLDGLS (SEQ ID NO:9), (5) TGDGGTTMNGLS (SEQ ID NO:98), (6) TGGHGSAPDGLS (SEQ ID NO:99), (8) TGASYLDNSGLS (SEQ ID NO:101), (10) TGVMHSQASGLS (SEQ ID NO:21), (11) TGDGSPAAPGLS (SEQ ID NO:22), and (12) TGSDMAHGTGLS (SEQ ID NO:23).

[0061] In some cases, the peptide insert is a peptide of Formula IV: X1GX2X3X4X5X6X7X8GLSPX9TX 10 X 11 (SEQ ID NO: 139), wherein X1 is T or N, X2 is L, S, A, or G, X3 is D or V, X4 is A, G, or P, X5 is T or D, X6 is R or Y, X7 is D, T, or G, X8 is H, R, or T, X9 is V or A, and X 10 is G or W and X 11 is T or A.

[0062] Peptide inserts of Formula IV include, but are not limited to, (13)TGLDATRDHGLSPVTGT (SEQ ID NO: 24), (14)TGSDGTRDHGLSPVTWT (SEQ ID NO: 25), (15)NGAVADYTRGLSPATGT (SEQ ID NO: 26), and (16)TGGDPTRGTGLSPVTGA (SEQ ID NO: 27). In some cases, the peptide insert is (13)TGLDATRDHGLSPVTGT (SEQ ID NO: 24). In some cases, the peptide insert is (14)TGSDGTRDHGLSPVTWT (SEQ ID NO: 25). In some cases, the peptide insert is (15)NGAVADYTRGLSPATGT (SEQ ID NO: 26). In some cases, the peptide insert is (16)TGGDPTRGTGLSPVTGA (SEQ ID NO: 27).

[0063] In some cases, the peptide insert is a peptide of Formula V: TGX1DX2TRX3X4GLSPVTGT (SEQ ID NO: 140), where X1 is L, S, A, or G, X2 is A, G, or P, X8 is D, T, or G, and X4 is H, R, or T.

[0064] Peptide inserts of Formula V include, but are not limited to, (13) TGLDATRDHGLSPVTGT (SEQ ID NO: 24), (14) TGSDGTRDHGLSPVTWT (SEQ ID NO: 25), and (16) TGGDPTRGTGLSPVTGA (SEQ ID NO: 27).

[0065] In some cases, the peptide insert is a peptide of formula VI: LQX1X2X3RX4X5X6X7X8X9VNX 10 Q (SEQ ID NO: 141), wherein X1 is K or R, X2 is N, G, or A, X3 is A, V, N, or D, X4 is P, I, or Q, X5 is A, P, or V, X6 is S, T, or G, X7 is T or V, X8 is E, L, A, or V, X9 is S, E, D, or V, and X 10 is F, G, T, or C.

[0066] Peptides of Formula VI include, but are not limited to, (17)LQKNARPASTESVNFQ (SEQ ID NO:28), (18)LQRGVRIPSVLEVNGQ (SEQ ID NO:29), (19)LQRGNRPVTTADVNTQ (SEQ ID NO:30), and (20)LQKADRQPGVVVVNCQ (SEQ ID NO:31). In some cases, the peptide insert is (17)LQKNARPASTESVNFQ (SEQ ID NO:28). In some cases, the peptide insert is (18)LQRGVRIPSVLEVNGQ (SEQ ID NO:29). In some cases, the peptide insert is (19)LQRGNRPVTTADVNTQ (SEQ ID NO:30). In some cases, the peptide insert is (20)LQKADRQPGVVVVNCQ (SEQ ID NO:31).

[0067] Any of the above-described peptide insertions can replace the same number of adjacent amino acids in the GH loop of an AAV capsid polypeptide. For example, in some cases, the peptide of Formula VI: LQX1X2X3RX4X5X6X7X8X9VNX 10 Q (SEQ ID NO: 141) (wherein Xi is K or R, X2 is N, G, or A, X3 is A, V, N, or D, X4 is P, I, or Q, X5 is A, P, or V, X6 is S, T, or G, X7 is T or V, X8 is E, L, A, or V, X9 is S, E, D, or V, and X 10A heterologous peptide (wherein R is F, G, T, or C) replaces a contiguous stretch of 5 to 20 amino acids in the GH loop of an AAV capsid polypeptide. In other words, in some cases, the "insertion peptide" replaces an endogenous peptide (e.g., a contiguous stretch of 5 to 20 amino acids) present in the GH loop of an AAV capsid polypeptide, resulting in a variant AAV capsid containing the heterologous peptide in the GH loop. In some cases, the "insertion peptide" replaces an endogenous contiguous stretch of amino acids of the same length as the insertion peptide. Thus, for example, when the "insertion peptide" has a length of 16 amino acids, in some cases, an endogenous contiguous stretch of 16 amino acids is replaced with the insertion peptide.

[0068] Peptides of Formula VI include, but are not limited to, (17)LQKNARPASTESVNFQ (SEQ ID NO:28), (18)LQRGVRIPSVLEVNGQ (SEQ ID NO:29), (19)LQRGNRPVTTADVNTQ (SEQ ID NO:30), and (20)LQKADRQPGVVVVNCQ (SEQ ID NO:31). In some cases, the peptide replacing the endogenous amino acid sequence within the GH loop of the AAV capsid is (17)LQKNARPASTESVNFQ (SEQ ID NO:28). In some cases, the peptide insert is (18)LQRGVRIPSVLEVNGQ (SEQ ID NO:29). In some cases, the peptide replacing the endogenous amino acid sequence within the GH loop of the AAV capsid is (19)LQRGNRPVTTADVNTQ (SEQ ID NO:30). In some cases, the peptide that replaces the endogenous amino acid sequence within the GH loop of the AAV capsid is (20)LQKADRQPGVVVVNCQ (SEQ ID NO: 31).

[0069] In some cases, the peptide insert of any one of Formulas I-VI further comprises one or two linker amino acids at the N-terminus of the peptide and / or one or more amino acids at the C-terminus of the peptide. For example, in some cases, the peptide insert comprises Thr-Gly-[peptide of any one of Formulas I-VI]-Gly-Leu-Ser (SEQ ID NO: 142). As another example, in some cases, the peptide insert comprises Leu-Ala-[peptide of any one of Formulas I-VI]-Ala (SEQ ID NO: 143). As another example, in some cases, the peptide insert comprises Leu-Gln-[peptide of any one of Formulas I-VI]-Gln. In some cases, the peptide insert does not comprise any linker amino acids.

[0070] In some embodiments, a subject rAAV virion capsid does not contain any other amino acid substitutions, insertions, or deletions other than the insertion of about 5 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) within the GH loop or loop IV relative to the corresponding parent AAV capsid protein. In other embodiments, a subject rAAV virion capsid comprises an insertion of from 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) within the GH loop or loop IV relative to the corresponding parent AAV capsid protein, as well as an insertion, deletion, or substitution of from 1 to about 25 amino acids relative to the parent AAV capsid protein. For example, in some embodiments, a subject rAAV virion capsid comprises an insertion of about 5 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) within the GH loop or loop IV relative to the corresponding parent AAV capsid protein, as well as an insertion, deletion, or substitution of 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 acids relative to the parent AAV capsid protein. In certain embodiments, a deletion of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids) compared to the parent AAV capsid protein occurs at the site of the peptide insertion.

[0071] In some cases, a variant AAV capsid polypeptide of the disclosure does not include one, two, three, or four of the following amino acid substitutions: Y273F, Y444F, Y500F, and Y730F.

[0072] In some cases, variant AAV capsid polypeptides of the present disclosure comprise, in addition to the inserted peptide described above, one, two, three, or four of the following amino acid substitutions: Y273F, Y444F, Y500F, and Y730F.

[0073] In some cases, a variant rAAV capsid polypeptide of the present disclosure is a chimeric capsid, e.g., the capsid comprises a portion of an AAV capsid of a first AAV serotype and a portion of an AAV capsid of a second serotype, and comprises an insertion of about 5 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) within the GH loop or loop IV relative to the corresponding parent AAV capsid protein.

[0074] Recombinant AAV virions The present disclosure provides a recombinant AAV (rAAV) virion comprising i) a variant AAV capsid polypeptide of the present disclosure and ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous polypeptide (i.e., a non-AAV polypeptide).

[0075] In some cases, a rAAV virion of the present disclosure comprises 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 an insertion of about 5 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) within the GH loop or loop IV relative to the corresponding parent AAV capsid protein. In some embodiments, a subject rAAV virion comprises 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 an insertion of between about 5 amino acids and 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 acids 587 and 588 relative to the amino acid sequence shown in FIG. 4, or at a corresponding site relative to the corresponding parent AAV capsid protein.

[0076] In some cases, a rAAV virion of the present disclosure comprises 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 an insertion of about 5 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) within the GH loop or loop IV relative to the corresponding parent AAV capsid protein. In some cases, a subject rAAV virion comprises 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 an insertion of between about 5 amino acids and 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 acids 585 and 598 relative to the amino acid sequence shown in FIG. 4, or at a corresponding site relative to the corresponding parent AAV capsid protein.

[0077] In some embodiments, a subject rAAV virion comprises a capsid protein that includes a GH loop 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 set forth in FIG. 5, and an insertion of between about 5 amino acids and 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 the amino acids shown in bold and underlined.

[0078] In some embodiments, a subject rAAV virion comprises 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 any one of the amino acid sequences shown in Figures 6A-6C, and an insertion of about 5 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, 10, 11, or 12 amino acids) between amino acids 587 and 588, or at a corresponding site relative to another AAV genotype. In some cases, the corresponding insertion site is shown in bold text and underlined in Figure 6B.

[0079] The rAAV virions of the present disclosure exhibit at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of retinal cells compared to the infectivity of retinal cells by AAV virions containing the corresponding parental AAV capsid proteins.

[0080] Whether a given rAAV virion exhibits increased infectivity of retinal cells can be determined by detecting the expression of a heterologous gene product encoded by the rAAV virion in retinal cells after intravitreal administration of the rAAV virion. For example, when administered intravitreally, a rAAV virion of the present disclosure comprising a) a variant capsid of the present disclosure comprising the above-described peptide insertion or peptide substitution, and b) a heterologous nucleotide sequence encoding the heterologous gene product, results in a heterologous gene product level in retinal cells that is at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold higher than the gene product level in retinal cells produced by intravitreal administration of a control rAAV virion comprising a) a control AAV capsid without the peptide insertion or peptide substitution, and b) a heterologous nucleotide sequence encoding the heterologous gene product.

[0081] Whether a given rAAV virion exhibits increased infectivity of retinal cells can be determined by assessing the therapeutic effect of the therapeutic gene product encoded by the rAAV virion on retinal cells, which may include, for example, a) a reduction in the rate of loss of visual function (e.g., visual field, visual acuity), b) an improvement in visual function (e.g., improved visual field or visual acuity), c) a reduction in sensitivity to light (i.e., photophobia), a reduction in nystagmus, etc. For example, a rAAV virion of the present disclosure comprising a) a variant capsid of the present disclosure that includes a peptide insertion or peptide substitution as described above, and b) a heterologous nucleotide sequence encoding a heterologous gene product, when administered intravitreally, produces a therapeutic effect in retinal cells that is at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold greater than the therapeutic effect in retinal cells produced by a control rAAV virion that includes a) a control AAV capsid that does not include the peptide insertion or peptide substitution, and b) a heterologous nucleotide sequence that encodes a therapeutic heterologous gene product, when administered intravitreally. Tests of visual function are known in the art, and any such test can be used to determine whether a rAAV virion of the present disclosure exhibits increased infectivity of retinal cells.

[0082] The rAAV virions of the present disclosure exhibit at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or greater than a 50-fold increase in ability to cross the barrier between the vitreous fluid and retinal cells compared to the ability of control AAV virions containing the corresponding parent AAV capsid protein (i.e., an AAV capsid protein without an inserted or replaced peptide).

[0083] In some cases, the subject rAAV virions, when administered via intravitreal injection, exhibit at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of retinal cells compared to the infectivity of retinal cells by AAV virions when AAV virions comprising the corresponding parent AAV capsid proteins are administered via intravitreal injection.

[0084] In some embodiments, a subject rAAV virion exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of photoreceptor cells (rod or cone) compared to the infectivity of photoreceptor cells by an AAV virion comprising the corresponding parental AAV capsid protein.

[0085] In some embodiments, a subject rAAV virion, when administered via intravitreal injection, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or greater than a 50-fold increase in infectivity of photoreceptor cells (rod or cone) compared to the infectivity of photoreceptor cells by an AAV virion when an AAV virion comprising the corresponding parent AAV capsid protein is administered via intravitreal injection.

[0086] In some embodiments, a subject rAAV virion exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of RGCs compared to the infectivity of RGCs by an AAV virion comprising the corresponding parental AAV capsid protein.

[0087] In some embodiments, a subject rAAV virion, when administered via intravitreal injection, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of RGCs compared to the infectivity of RGCs by an AAV virion comprising the corresponding parental AAV capsid protein when administered via intravitreal injection.

[0088] In some embodiments, the subject rAAV virions exhibit at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or greater than a 50-fold increase in infectivity of RPE cells compared to the infectivity of RPE cells by AAV virions comprising the corresponding parent AAV capsid proteins.

[0089] In some embodiments, the subject rAAV virions, when administered via intravitreal injection, exhibit at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of RPE cells compared to the infectivity of RPE cells by AAV virions when AAV virions comprising the corresponding parent AAV capsid proteins are administered via intravitreal injection.

[0090] In some embodiments, a subject rAAV virion exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of Müller cells compared to the infectivity of Müller cells by an AAV virion comprising the corresponding parent AAV capsid protein.

[0091] In some embodiments, a subject rAAV virion, when administered via intravitreal injection, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of Müller cells compared to the infectivity of Müller cells by the AAV virion when an AAV virion comprising the corresponding parent AAV capsid protein is administered via intravitreal injection.

[0092] In some embodiments, a subject rAAV virion exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of bipolar cells compared to the infectivity of bipolar cells by an AAV virion comprising the corresponding parental AAV capsid protein.

[0093] In some embodiments, a subject rAAV virion, when administered via intravitreal injection, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in infectivity of bipolar cells compared to the infectivity of bipolar cells by AAV virions when an AAV virion comprising the corresponding parent AAV capsid protein is administered via intravitreal injection.

[0094] In some embodiments, a subject rAAV virion exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or greater than a 50-fold increase in infectivity of amacrine cells compared to the infectivity of amacrine cells by an AAV virion comprising the corresponding parental AAV capsid protein.

[0095] In some embodiments, a subject rAAV virion, when administered via intravitreal injection, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or greater than a 50-fold increase in infectivity of amacrine cells compared to the infectivity of amacrine cells by the AAV virion when an AAV virion comprising the corresponding parent AAV capsid protein is administered via intravitreal injection.

[0096] In some embodiments, a subject rAAV virion exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in horizontal cell infectivity compared to the horizontal cell infectivity by an AAV virion comprising the corresponding parental AAV capsid protein.

[0097] In some embodiments, a subject rAAV virion, when administered via intravitreal injection, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in horizontal cell infectivity compared to the horizontal cell infectivity of AAV virions when an AAV virion comprising the corresponding parent AAV capsid protein is administered via intravitreal injection.

[0098] In some embodiments, a subject rAAV virion exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or greater than a 50-fold increase in infectivity of retinal astrocytes compared to the infectivity of retinal astrocytes by an AAV virion comprising the corresponding parental AAV capsid protein.

[0099] In some embodiments, a subject rAAV virion, when administered via intravitreal injection, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or greater than a 50-fold increase in infectivity of retinal astrocytes compared to the infectivity of retinal astrocytes by an AAV virion when an AAV virion comprising the corresponding parental AAV capsid protein is administered via intravitreal injection.

[0100] In some cases, the subject rAAV virions exhibit at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or greater than a 50-fold increase in the ability to traverse the retinal extracellular matrix (ECM) compared to the ability of AAV virions comprising the corresponding parental AAV capsid proteins to traverse the ECM.

[0101] In some cases, the subject rAAV virions, when administered via intravitreal injection, exhibit at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in the ability to cross the retinal extracellular matrix (ECM) compared to the ability of AAV virions comprising the corresponding parent AAV capsid proteins to cross the ECM when administered via intravitreal injection.

[0102] In some cases, a subject rAAV virion exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in ability to cross the inner limiting membrane (ILM) compared to the ability of an AAV virion containing the corresponding parental AAV capsid protein to cross the ILM.

[0103] In some cases, a subject rAAV virion, when administered via intravitreal injection, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in ability to cross the ILM compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ILM when administered via intravitreal injection.

[0104] A subject rAAV virion can cross the ILM and also pass through cell layers, including Müller cells, amacrine cells, etc., to reach photoreceptor cells and / or RPE cells. For example, a subject rAAV virion, when administered via intravitreal injection, can cross the ILM and also pass through cell layers, including Müller cells, amacrine cells, etc., to reach photoreceptor cells and / or RPE cells.

[0105] In some cases, a subject rAAV virion exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in localization to one or more of the inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion cell layer, and the retinal pigment epithelium compared to the degree of localization to the inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion layer, or the retinal pigment epithelium by an AAV virion comprising the corresponding parent AAV capsid protein.

[0106] In some cases, a subject rAAV virion, when injected intravitreally, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in localization across the ILM compared to the degree of localization across the ILM by an AAV virion comprising the corresponding parental AAV capsid protein injected intravitreally. For example, in some cases, a subject rAAV virion, when injected intravitreally, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in localization to the retinal pigment epithelium (RPE) layer compared to the degree of localization to the RPE layer by a control AAV virion comprising the corresponding parental AAV capsid protein injected intravitreally. As another example, in some cases, a subject rAAV virion, when injected intravitreally, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in localization to the photoreceptor (PR) layer compared to localization to the PR layer by a control AAV virion comprising the corresponding parental AAV capsid protein injected intravitreally. As another example, in some cases, a subject rAAV virion, when injected intravitreally, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in localization to the inner nuclear layer compared to the degree of localization to the inner nuclear layer by an AAV virion comprising the corresponding parental AAV capsid protein injected intravitreally. As another example, in some cases, a subject rAAV virion, when injected intravitreally, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in localization to the outer nuclear layer compared to the degree of localization to the outer nuclear layer by a control AAV virion containing the corresponding parental AAV capsid protein injected intravitreally.As another example, in some cases, a subject rAAV virion, when injected intravitreally, exhibits at least a 5-fold, at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or more than a 50-fold increase in localization to the ganglion cell layer compared to localization to the ganglion cell layer by a control AAV virion containing the corresponding parental AAV capsid protein injected intravitreally.

[0107] In some embodiments, a subject rAAV virion selectively infects retinal cells, e.g., a subject rAAV virion infects retinal cells with 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or greater than 50-fold specificity over non-retinal cells (e.g., cells other than the eye). For example, in some embodiments, a subject rAAV virion selectively infects retinal cells, e.g., a subject rAAV virion infects photoreceptor cells with 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or greater than 50-fold specificity over non-retinal cells (e.g., cells other than the eye).

[0108] In some embodiments, a subject rAAV virion selectively infects photoreceptor cells, e.g., a subject rAAV virion infects photoreceptor cells with 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or greater than 50-fold specificity over non-photoreceptor cells present in the eye (e.g., retinal ganglion cells, Muller cells, etc.).

[0109] In some embodiments, the subject rAAV virions, when administered via intravitreal injection, exhibit at least a 10-fold, at least a 15-fold, at least a 20-fold, at least a 25-fold, at least a 50-fold, or greater than a 50-fold increase in infectivity of photoreceptor cells compared to the infectivity of photoreceptor cells by AAV virions when AAV virions comprising the corresponding parent AAV capsid proteins are administered via intravitreal injection.

[0110] gene product The rAAV virions of the present disclosure comprise a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products (one or more heterologous gene products). In some cases, the gene product is a polypeptide. In some cases, the gene product is RNA. In some cases, the rAAV virions of the present disclosure comprise a heterologous nucleotide sequence encoding both a heterologous nucleic acid gene product and a heterologous polypeptide gene product. When the gene product is RNA, in some cases, the RNA gene product encodes a polypeptide. When the gene product is RNA, in some cases, the RNA gene product does not encode a polypeptide. In some cases, the rAAV virions of the present disclosure comprise a single heterologous nucleic acid comprising a nucleotide sequence encoding one heterologous gene product. In some cases, the rAAV virions of the present disclosure comprise a single heterologous nucleic acid comprising nucleotide sequences encoding two heterologous gene products. When a single heterologous nucleic acid encodes two heterologous gene products, in some cases, the nucleotide sequences encoding the two heterologous gene products are operably linked to the same promoter. When a single heterologous nucleic acid encodes two heterologous gene products, in some cases, the nucleotide sequences encoding the two heterologous gene products are operably linked to two different promoters. In some cases, the rAAV virion of the present disclosure comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding three heterologous gene products. When a single heterologous nucleic acid encodes three heterologous gene products, in some cases, the nucleotide sequences encoding the three heterologous gene products are operably linked to the same promoter. When a single heterologous nucleic acid encodes three heterologous gene products, in some cases, the nucleotide sequences encoding the three heterologous gene products are operably linked to two or three different promoters. In some cases, the rAAV virion of the present disclosure comprises two heterologous nucleic acids, each comprising a nucleotide sequence encoding one heterologous gene product.

[0111] In some cases, the gene product is an RNA encoding a polypeptide. In some cases, the gene product is an interfering RNA. In some cases, the gene product is an aptamer. In some cases, the gene product is a polypeptide. In some cases, the gene product is a therapeutic polypeptide, e.g., a polypeptide that provides a clinical benefit. In some embodiments, the gene product is a site-specific nuclease that provides site-specific knockdown of gene function. In some embodiments, the gene product is an RNA-guided endonuclease that provides modification of a target nucleic acid. In some cases, the gene product is i) an RNA-guided endonuclease that provides modification of the target nucleic acid, and ii) a guide RNA comprising: i) an RNA-guided endonuclease that provides modification of the target nucleic acid; and ii) a guide RNA that includes a first segment that binds to a target sequence in the target nucleic acid and a second segment that binds to the RNA-guided endonuclease. In some cases, the gene product is i) an RNA-guided endonuclease that effects modification of a target nucleic acid; ii) a first guide RNA that comprises a first segment that binds to a first target sequence within the target nucleic acid and a second segment that binds to the RNA-guided endonuclease; and iii) a second guide RNA that comprises a first segment that binds to a second target sequence within the target nucleic acid and a second segment that binds to the RNA-guided endonuclease.

[0112] Interfering RNA When the gene product is an interfering RNA (RNAi), suitable RNAi includes RNAi that reduces the level of apoptotic or angiogenic factors in cells. For example, RNAi can be shRNA or siRNA that reduces the level of a gene product that induces or promotes apoptosis in cells. Genes whose gene products induce or promote apoptosis are referred to herein as "pro-apoptotic genes," and the products (mRNA, proteins) of such genes are referred to as "pro-apoptotic gene products." Examples of pro-apoptotic gene products include Bax, Bid, Bak, and Bad gene products. See, for example, U.S. Patent No. 7,846,730.

[0113] Interfering RNAs can also be directed against angiogenesis 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. Patent Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and 5,639,736, and U.S. Patent Nos. 7,947,659 and 7,919,473.

[0114] Aptamers When the gene product is an aptamer, exemplary aptamers of interest include aptamers for VEGF.See, for example, Ng et al.(2006) Nat.Rev.DrugDiscovery 5:123 and Lee et al.(2005) Proc.Natl.Acad.Sci.USA 102:18902.For example, a VEGF aptamer can comprise the nucleotide sequence 5'-cgcaaucagugaaugcuuauacauccg-3' (SEQ ID NO: 3).In addition, platelet-derived growth factor (PDGF)-specific aptamers (e.g., E10030) are also suitable for use.See, for example, Ni and Hui(2009) Ophthalmologica 223:401 and Akiyama et al.(2006) J.Cell Physiol.207:407.

[0115] Polypeptides When the gene product is a polypeptide, in some cases, the polypeptide is a polypeptide that enhances the function of retinal cells, such as rod or cone photoreceptor cells, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. Exemplary polypeptides include neuroprotective polypeptides (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 receptors, VEGF-binding antibodies, VEGF-binding antibody fragments (e.g., single-chain anti-VEGF antibodies), endostatin, tumstatin, angiostatin, soluble Flt polypeptides (Lai et al. (2005) Mol. Ther. 12:659), Fc fusion proteins comprising soluble Flt polypeptides (e.g., Pechan et al. (2009) Gene. Ther. 16:10), pigment epithelium-derived factor (PEDF), soluble Tie-2 receptor, etc.), tissue inhibitor of metalloproteinase 3 (TIMP-3), light-responsive opsins, e.g., rhodopsin, anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl, XIAP), etc. Suitable polypeptides include, but are not limited to, glial-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 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 contiguous stretch of about 200 to 247 amino acids of the amino acid sequence shown in Figure 7B (SEQ ID NO: 11)), epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis, and sonic hedgehog.

[0116] 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 Diester et al. (2011) Nat. Neurosci. 14:387. 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) See J. Ophthalmic Vis.Res.9:374, Tomita et al. (2014) Mol.Ther.22:1434.

[0117] Suitable polypeptides include light-gated ion channel polypeptides. See, e.g., Gaub et al. (2014) Proc. Natl. Acad. Sci. USA 111:E5574. For example, a suitable polypeptide is the light-gated ionotropic glutamate receptor (LiGluR). Expression of LiGluR in retinal ganglion cells and ON bipolar cells in the presence of a photocatabolic compound renders the cells light-responsive. LiGluR contains an L439C substitution. See, e.g., 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. Examples of photocatabolic compounds include maleimide-azobenzene-glutamate 0 (MAG0), which has a peak efficiency at 460 nm. 460 ) are listed. MAG0 460 has the following structure:

[0118]

change

[0119] Suitable polypeptides also include 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 contiguous stretch of about 200 to 224 amino acids of the amino acid sequence shown in Figure 7A (SEQ ID NO: 10)). Suitable polypeptides include, for example, retinitis pigmentosa GTPase regulator (RPGR)-interacting protein 1 (see, e.g., GenBank Accession Nos. 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 contiguous stretch of about 1150 amino acids to about 1200 amino acids, or about 1200 amino acids to 1286 amino acids, of the amino acid sequence shown in Figure 7F (SEQ ID NO: 15)), peripherin 2 (Prph2) (e.g., GenBank Accession No. 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 contiguous stretch of about 300 amino acids to 346 amino acids of the amino acid sequence shown in Figure 7D (SEQ ID NO: 13)), and Travis et al. 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 contiguous stretch of about 400 to about 470 amino acids of the amino acid sequence shown in Figure 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 contiguous stretch of about 200 to 247 amino acids of the amino acid sequence shown in Figure 7C (SEQ ID NO: 12)) (see, e.g., GenBank AAC39660, and Morimura et al. (1998) Proc. Natl.Acad. Sci. USA 95:3088), rod-derived cone survival 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 Figures 7H, 7I, and 7J), Rab escort protein 1 (REP1) (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 Figure 7G), and 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 any one of Figures 7S-7V). 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 Figure 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 Figure 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 Figure 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 Figure 7V.

[0120] Suitable polypeptides also include CHM (choroideremia (Rab escort protein 1 (REP1))), a polypeptide that, when defective or absent, causes choroideremia (see, e.g., Donnelly et al. (1994) Hum. Mol. Genet. 3:1017 and van Bokhoven et al. (1994) Hum. Mol. Genet. 3:1041), and Crumbs homolog 1 (CRB1), a polypeptide that, when defective or absent, causes Leber congenital amaurosis and retinitis pigmentosa (see, e.g., den Hollander et al. (1999) Nat. Genet. 23:217 and GenBank Accession No. CAM23328). For example, a suitable REP1 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 Figure 7G.

[0121] 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 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 Figure 7K. As another example, a suitable PDE6β6 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 Figure 7L. As another example, a suitable PDE6β6 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 Figure 7M. As another example, a suitable PDE6β6 polyisoform peptide 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 Figure 7N.

[0122] Suitable polypeptides also include polypeptides that, when defective or absent, result in achromatopsia, such as cone photoreceptor cGMP-gated channel subunit alpha (CNGA3) (see, e.g., GenBank Accession No. NP_001289, and Booij et al. (2011) Ophthalmology 118:160-167), cone photoreceptor cGMP-gated cation channel beta subunit (CNGB3) (see, e.g., Kohl et al. (2005) Eur J Hum Genet. 13(3):302), guanine nucleotide-binding protein (G protein), alpha transduction-activating polypeptide 2 (GNAT2) (ACHM4), and polypeptides that, when defective or absent, result in various forms of color vision deficiency (e.g., L-opsin, M-opsin, and S-opsin). See Mancuso et al. (2009) Nature 461(7265):784-787.

[0123] For example, a suitable CNGA3 (also known as ACHM2) 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 Figure 7O. As another example, a suitable CNGA3 (also known as ACHM2) 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 Figure 7P.

[0124] 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 Figure 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 Figure 7R.

[0125] site-specific endonucleases In some cases, the gene product of interest is a site-specific endonuclease that causes site-specific knockdown of gene function, for example, this endonuclease knocks out the allele associated with retinal disease.For example, when the dominant allele is wild-type, if it encodes a defective copy of the gene that is a structural protein of the retina and / or that causes normal retinal function, the site-specific endonuclease can be targeted to the defective allele and knock out the defective allele.In some cases, the site-specific endonuclease is an RNA-guided endonuclease.

[0126] In addition to knocking out defective alleles, site-specific nucleases can also be used to stimulate homologous recombination with 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 both a site-specific endonuclease that knocks out a defective allele and a functional copy of the defective allele, thereby resulting in the production of 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 the defective allele that 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, etc.

[0127] Suitable site-specific endonucleases for use include, for example, zinc finger nucleases (ZFNs), meganucleases, and transcription activator effector nucleases (TALENs). Such site-specific endonucleases are non-naturally occurring and modified to target specific genes. Such site-specific nucleases can be engineered to cut specific locations within the genome, and then non-homologous end joining can repair the break, inserting or deleting several nucleotides. Such site-specific nucleases (also called "indels") then release the protein from the frame, effectively knocking out the gene. See, for example, U.S. Patent Publication No. 2011 / 0301073. Suitable site-specific endonucleases include engineered meganucleases and re-engineered homing endonucleases. Suitable endonucleases include I-Tevl nuclease. Suitable meganucleases include I-Sce1 (see, e.g., Bellaiche et al. (1999) Genetics 152:1037) and I-Cre1 (Heath et al. (1997) Nature Structural Biology 4:468).

[0128] RNA-guided endonucleases 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 the 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 a target nucleic acid. The RNA-guided endonuclease is also referred to herein as a "genome-editing nuclease."

[0129] An example of a suitable genome editing nuclease is a CRISPR / Cas endonuclease (e.g., a class 2 CRISPR / Cas endonuclease, such as a type II, type V, or type VI CRISPR / Cas endonuclease). A suitable genome editing nuclease is a CRISPR / Cas endonuclease (e.g., a class 2 CRISPR / Cas endonuclease, such as a type II, type V, or type VI CRISPR / Cas endonuclease). In some cases, the genome targeting composition comprises a class 2 CRISPR / Cas endonuclease. In some cases, the genome targeting composition comprises a class 2 type II CRISPR / Cas endonuclease (e.g., a Cas9 protein). In some cases, the genome targeting composition comprises a class 2 type V CRISPR / Cas endonuclease (e.g., a Cpf1 protein, a C2c1 protein, or a C2c3 protein). In some cases, the genome-targeting composition comprises a Class 2 Type VI CRISPR / Cas endonuclease (e.g., a C2c2 protein (also called a Cas13a protein)). A CasX protein is also suitable for use. A CasY protein is also suitable for use.

[0130] In some cases, the genome editing nuclease is a fusion protein fused with a heterologous polypeptide (also referred to as a "fusion partner"). In some cases, the genome editing nuclease is fused with an amino acid sequence (fusion partner) that confers intracellular localization, i.e., the fusion partner is an intracellular localization sequence (e.g., one or more nuclear localization signals (NLSs) that target the nucleus, two or more NLSs, three or more NLSs, etc.).

[0131] 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 to (e.g., stabilized at) a target site within a target nucleic acid sequence (e.g., a chromosomal or extrachromosomal sequence, e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by association with a protein-binding segment of the Cas9 guide RNA. In some cases, a suitable 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 Streptococcus pyogenes Cas9 shown in Figure 3A. In some cases, the Cas9 polypeptide used in the compositions or methods of the present disclosure is a Staphylococcus aureus Cas9 (saCas9) polypeptide. 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 shown in Figure 3.

[0132] 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 Figure 3A are substituted, e.g., with alanine. For example, an HF Cas9 polypeptide can include 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 Figure 3A, in which amino acids N497, R661, Q695, and Q926 are substituted, e.g., with alanine.

[0133] In some cases, suitable Cas9 polypeptides exhibit altered PAM specificity. See, e.g., Kleinstiver et al. (2015) Nature 523:481.

[0134] 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 Cpfl protein. In some cases, the Cpfl 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 shown in Figure 3C.

[0135] In some cases, the genome editing endonuclease is a CasX or CasY polypeptide. CasX and CasY polypeptides are described in Burstein et al. (2017) Nature 542:237.

[0136] Enzymatically inactive RNA-guided endonucleases RNA-guided endonucleases with reduced enzymatic activity are also suitable for use. Such RNA-guided endonucleases are referred to as "dead" RNA-guided endonucleases. For example, a Cas9 polypeptide containing certain amino acid substitutions that substantially eliminate endonuclease activity but still bind to target nucleic acids when complexed with a guide RNA is referred to as a "dead" Cas9 or "dCas9." In some cases, a "dead" Cas9 protein has a reduced ability to cleave both the complementary and non-complementary strands of a double-stranded target nucleic acid. For example, a "nuclease-defective" Cas9 lacks a functional RuvC domain (i.e., does not cleave the non-complementary strand of a double-stranded target DNA) and a functional HNH domain (i.e., does not cleave the complementary strand of a double-stranded target DNA). As a non-limiting example, in some cases, a nuclease-deficient Cas9 protein has mutations at amino acid positions corresponding to residues D10 and H840 of SEQ ID NO: 15 (or the corresponding residues of a Cas9 homolog), resulting in a reduced ability of the polypeptide to cleave both complementary and non-complementary strands of a target nucleic acid (e.g., no cleavage). Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single-stranded or double-stranded target nucleic acid), but retains the ability to bind to the target nucleic acid. A Cas9 protein that is unable to cleave a target nucleic acid (e.g., due to one or more mutations in the catalytic domain, e.g., the RuvC and HNH domains) is referred to as a "nuclease-deficient Cas9," "dead Cas9," or simply "dCas9." Other residues can also be mutated to achieve the above effects (e.g., to inactivate any one of the other nuclease moieties). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 of Streptococcus pyogenes Cas9 (or the corresponding amino acids of a Cas9 homolog) can be modified (i.e., substituted).In some cases, two or more of D10, E762, H840, N854, N863, and D986 of Streptococcus pyogenes Cas9 (or the corresponding amino acids of a Cas9 homolog) are substituted. In some cases, D10 and N863 of Streptococcus pyogenes Cas9 (or the corresponding amino acids of a Cas9 homolog) are substituted with Ala. Mutations other than alanine substitutions are also suitable.

[0137] In some cases, the genome editing endonuclease is an RNA-guided endonuclease (and corresponding guide RNA) known as Cas9 synergistic activation mediator (Cas9-SAM). The RNA-guided endonuclease (e.g., Cas9) in the Cas9-SAM system is a "dead" Cas9 fused to a transcription activation domain (e.g., VP64, p65, MyoD1, HSF1, RTA, and SET7 / 9) or a transcription repression domain (e.g., KRAB, NuE, NcoR, SID, and SID4X). The guide RNA in the Cas9-SAM system contains a loop that binds to an adaptor protein fused to a transcription activation domain (e.g., VP64, p65, MyoD1, HSF1, RTA, or SET7 / 9) or a transcription repression domain (e.g., KRAB, NuE, NcoR, SID, or SID4X). For example, in some cases, the guide RNA is a single guide RNA comprising an MS2 RNA aptamer inserted into one or two loops of the sgRNA, the dCas9 is a fusion polypeptide comprising dCas9 fused to VP64, and the adaptor / functional protein is a fusion polypeptide comprising i) MS2, ii) p65, and iii) HSF1. See, e.g., U.S. Patent Publication No. 2016 / 0355797.

[0138] Also suitable for use are chimeric polypeptides comprising a) a dead RNA-guided endonuclease and b) a heterologous fusion polypeptide. Examples of suitable heterologous fusion polypeptides include, for example, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, or nucleic acid binding activity.

[0139] guide RNA A nucleic acid that binds to a Class 2 CRISPR / Cas endonuclease (e.g., a Cas9 protein, a Type V or Type VI CRISPR / Cas protein, a Cpf1 protein, etc.) and targets the complex to a specific location within a target nucleic acid is referred to herein as a "guide RNA" or "CRISPR / Cas guide nucleic acid" or "CRISPR / Cas guide RNA." The guard RNA provides target specificity to the complex (RNP complex) by including a targeting segment that includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence complementary to the sequence of the target nucleic acid.

[0140] In some cases, a guide RNA comprises two separate nucleic acid molecules: an "activator" and a "targeter," and is referred to herein as a "dual guide RNA," "dual-molecule guide RNA," "two-molecule guide RNA," or "dgRNA." In some cases, the guide RNA is one molecule (e.g., for some Class 2 CRISPR / Cas proteins, the corresponding guide RNA is a single molecule; in some cases, the activator and targeter are covalently linked to each other, e.g., via an intervening nucleotide), and this guide RNA is referred to as a "single guide RNA," "single-molecule guide RNA," "one-molecule guide RNA," or simply "sgRNA."

[0141] When 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, when the target nucleic acid contains a deleterious mutation in a defective allele (e.g., a deleterious mutation in a retinal cell target nucleic acid), the RNA-guided endonuclease / guide RNA complex can be used together with a donor nucleic acid containing a nucleotide sequence that corrects the deleterious mutation (e.g., a donor nucleic acid containing a nucleotide sequence encoding a functional copy of the protein encoded by the defective allele) to correct the deleterious mutation, for example, via homology-directed repair (HDR).

[0142] In some cases, the gene product is an RNA-guided endonuclease and two separate sgRNAs that result in deletion of the target nucleic acid via non-homologous end joining (NHEJ).

[0143] In some cases, the gene product is i) an RNA-guided endonuclease and ii) one guide RNA. In some cases, the guide RNA is a single-molecule (or "single-guide") guide RNA ("sgRNA"). In some cases, the guide RNA is a dual-molecule (or "dual-guide") guide RNA ("dgRNA").

[0144] In some cases, the gene product is i) an RNA-guided endonuclease and ii) two separate sgRNAs, which result in deletion of the target nucleic acid via non-homologous end joining (NHEJ). In some cases, the guide RNA is an sgRNA. In some cases, the guide RNA is a dgRNA.

[0145] In some cases, the gene product is i) a Cpf1 polypeptide and ii) a guide RNA precursor, in which case the precursor can be cleaved by the Cpf1 polypeptide to generate two or more guide RNAs.

[0146] The present disclosure provides a method for modifying a target nucleic acid in a retinal cell in an individual, when the target nucleic acid contains a deleterious mutation, the method comprising administering to the individual (e.g., by intraocular, intravitreal, etc.) an rAAV virion of the present disclosure, wherein the rAAV virion contains a heterologous nucleic acid comprising: i) a nucleotide sequence encoding an RNA-guided endonuclease (e.g., 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 DNA template comprising a nucleotide sequence that corrects the deleterious mutation. Administration of the rAAV virion results in correction of the deleterious mutation in the target nucleic acid by HDR.

[0147] The present disclosure provides a method for modifying a target nucleic acid in a retinal cell in an individual, if the target nucleic acid contains a deleterious mutation, comprising administering to the individual (e.g., by intraocular, intravitreal, etc.) an rAAV virion of the present disclosure, wherein the rAAV virion contains a heterologous nucleic acid comprising: i) a nucleotide sequence encoding an RNA-guided endonuclease (e.g., 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. Administration of the rAAV virion results in excision of the deleterious mutation in the target nucleic acid by NHEJ.

[0148] Control arrays In some cases, the nucleotide sequence encoding the gene product of interest is operably linked to a transcriptional regulatory 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 control 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 control element (e.g., a photoreceptor-specific promoter), which is a control element that confers selective expression of the operably linked gene in, for example, photoreceptor cells. Suitable photoreceptor-specific control 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).

[0149] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions comprising a) a subject rAAV virion, as described below, 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.

[0150] Such excipients, carriers, diluents, and buffers include any pharmaceutical agent that may 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 may also be included, such as inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate, etc.) and organic acid salts (e.g., acetate, propionate, malonate, benzoate, etc.). In addition, auxiliary substances, such as wetting and emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients are described, for example, in 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., 3rd ed. American Pharmaceutical Assoc.

[0151] Methods for delivering gene products to retinal cells and methods of treatment The present disclosure provides a method for delivering a gene product to a retinal cell in an individual, comprising administering the above-described rAAV virion to the individual. The gene product can be a polypeptide or an interfering RNA (e.g., shRNA, siRNA, etc.), an aptamer, or a site-specific endonuclease (e.g., RNA-guided endonuclease), as described above. Delivering the gene product to a retinal cell can result in the treatment of a retinal disease. The retinal cell can be a photoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell. In some cases, the retinal cell is a photoreceptor cell, such as a rod or a cone cell.

[0152] The present disclosure provides a method for modifying a target nucleic acid in a retinal cell, the method comprising contacting the retinal cell 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 the guide RNA, and 2) a guide RNA. The present disclosure provides a method for modifying a target nucleic acid in a retinal cell, the method comprising contacting the retinal cell with an rAAV virion of the present disclosure comprising a heterologous nucleic acid comprising a nucleotide sequence encoding i) an RNA-guided endonuclease that binds to the guide RNA, and ii) a guide RNA. In some cases, the method comprises contacting the retinal cell with a donor DNA template. In some cases, the RNA-guided endonuclease is a Cas9 polypeptide. In some cases, the guide RNA is a single-guide RNA.

[0153] The present disclosure provides methods for treating ocular diseases (e.g., retinal diseases), comprising administering to an individual in need of such treatment an effective amount of a subject rAAV virion, as described above. The subject rAAV virions can be administered via intraocular injection, e.g., intravitreal injection, subretinal injection, suprachoroidal injection, or any other convenient mode or route of administration. Other convenient modes or routes of administration include, for example, intravenous, intranasal, etc.

[0154] A "therapeutically effective amount" falls 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, a therapeutically effective dose is about 10 6 ~about 10 15 Approximately 10 rAAV virions, for example, 8 ~about 10 12 For example, for in vivo injection, i.e., direct injection into the eye, a therapeutically effective dose is about 10 6 Viral genome (vg) ~ approx. 10 15 Approximately 10 vg of rAAV virions, e.g., approximately 10 8 vg~about 10 12 For in vitro transduction, the effective amount of rAAV virions delivered to cells is approximately 10 8 ~about 10 13 For example, for in vitro transduction, the effective amount of rAAV virions delivered to cells is approximately 10 8 ~about 10 13 As another example, for in vitro transduction, an effective amount of rAAV virions delivered to cells is about 10 vg / cell to about 10 4 Other effective doses can be readily established by one of ordinary skill in the art through routine testing to establish dose-response curves.

[0155] In some embodiments, two or more doses (e.g., 2, 3, 4, or more doses) may be used to achieve a desired gene expression level. In some cases, two or more doses are administered at various intervals, such as once daily, once weekly, twice monthly, once monthly, once every three months, once every six months, once every year, etc. In some cases, multiple doses are administered at intervals of 1 to 2 months, 2 to 4 months, 4 to 8 months, 8 to 12 months, 1 to 2 years, 2 to 5 years, or 5 years or more.

[0156] Ocular diseases that can be treated using the subject methods include, but are not limited to, acute macular neuroretinopathy, Behcet's syndrome, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, e.g., acute macular degeneration, non-exudative age-related macular degeneration, and exudative age-related macular degeneration, edema, e.g., macular edema, cystoid macular edema, diabetic macular edema, multifocal choroiditis, ocular injuries affecting the posterior ocular site or location, ocular tumors, retinal disorders, e.g., central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitis, sympathetic ophthalmia, Vogt-Koyanagi-Harada syndrome, diffuse uveitis, and the like. diffusion), posterior ocular conditions caused or affected by ocular laser procedures, posterior ocular conditions caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease (fundus flavicolor), hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital nonprogressive night blindness, choroideremia, Bardet-Bietle syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, color vision disorders (including monochromacy, protanopia, deuteranopia, and tritanopia), and Bietti crystalline dystrophy.

[0157] The present disclosure provides methods for treating retinal diseases. The methods generally involve administering a rAAV virion of the present disclosure, or a composition comprising a rAAV virion of the present disclosure, to the eye of an individual in need of such administration. Non-limiting methods for evaluating treatment of retinal diseases include measuring functional changes, such as measuring changes in visual acuity (e.g., BCVA), measuring visual field (e.g., perimetry), measuring electrophysiological responsiveness to light and dark (e.g., ERG, VEP), measuring color vision and / or measuring contrast sensitivity, measuring changes in anatomical structure or health using anatomical and / or photographic measures (e.g., OCT, fundus photography, and / or autofluorescence), and measuring ocular motility (e.g., nystagmus, fixation preference, and stability).

[0158] For example, one of skill in the art could readily determine an effective amount of an rAAV virion by examining its effect on one or more parameters (e.g., visual acuity, visual field, electrophysiological responsiveness to light and dark, color vision, contrast sensitivity, anatomy, retinal health and vasculature, eye motility, fixation preference, and stability). In some cases, administration of an effective amount of an rAAV virion of the present disclosure results in a reduction in the rate of loss of retinal function, anatomical integrity, or retinal health, e.g., a two-fold, three-fold, four-fold, or five-fold or more reduction in the rate of loss, thereby reducing the rate of disease progression, e.g., a ten-fold or more reduction in the rate of loss, thereby reducing the rate of disease progression. In some cases, administration of an effective amount of an rAAV virion of the present disclosure results in increased retinal function, improved retinal anatomy or health, and / or stabilization of eye motility, e.g., a 2-fold, 3-fold, 4-fold, or 5-fold or more improvement in retinal function, retinal anatomy or health, and / or orbital stability, e.g., a 10-fold improvement in retinal function, retinal anatomy or health, and / or orbital stability.

[0159] Nucleic Acids and Host Cells The present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a subject variant adeno-associated virus (AAV) capsid protein described above, wherein the variant AAV capsid protein comprises an insertion of about 5 to about 20 amino acids within the GH loop or loop IV relative to the corresponding parent AAV capsid protein, or the variant AAV capsid protein comprises a replacement of about 5 to about 20 amino acids within the GH loop or loop IV relative to the corresponding parent AAV capsid protein with a heterologous peptide of about 5 to about 20 amino acids, wherein the variant capsid protein, when present in an AAV virion, confers increased infectivity of retinal cells compared to the infectivity of retinal cells by AAV virions comprising the corresponding parent AAV capsid protein. The subject isolated nucleic acid can be an AAV vector, e.g., a recombinant AAV vector.

[0160] Insertion peptide The variant AAV capsid proteins encoded by the subject nucleic acids have an inserted peptide of about 5 to about 20 amino acids in length, which is inserted into the GH loop of the AAV capsid. The inserted peptide has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Suitable inserted peptides are as described above. Suitable inserted peptides include the peptides of any one of Formulas I-VI, as described above. Insertion of the inserted peptide into the parent AAV capsid, in some cases, replaces an endogenous stretch of about 5 to about 20 amino acids in the GH loop or loop IV. Thus, in some cases, the variant AAV capsid proteins encoded by the subject nucleic acids comprise a substitution of about 5 to about 20 amino acids within the GH loop or loop IV relative to the corresponding parent AAV capsid protein with a heterologous peptide of about 5 to about 20 amino acids, suitable heterologous peptides including any one of the peptides of Formulas I-VI, as described above.

[0161] A subject recombinant AAV vector can be used to generate a subject recombinant AAV virion, as described above. Accordingly, the present disclosure provides recombinant AAV vectors that, when introduced into suitable cells, can result in the production of a subject recombinant AAV virion.

[0162] The present invention further provides host cells, e.g., isolated (genetically modified) host cells, comprising a subject nucleic acid. A subject host cell can be an isolated cell, e.g., a cell in in vitro culture. A subject host cell is useful for producing a subject rAAV virion, as described below. When a subject host cell is used to produce a subject rAAV virion, the subject host cell is referred to as a "packaging cell." In some embodiments, a subject host cell is stably genetically modified with a subject nucleic acid. In other embodiments, a subject host cell is transiently genetically modified with a subject nucleic acid.

[0163] The subject nucleic acids are stably or transiently introduced into host cells using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, etc. For stable transformation, the subject nucleic acids generally further include a selectable marker, e.g., any of several well-known selectable markers (e.g., neomycin resistance, etc.).

[0164] A subject host cell is generated by introducing a subject nucleic acid into any of a variety of cells, e.g., mammalian cells (e.g., mouse cells, and primate cells (e.g., 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, 293T cells, COS cells, HeLa cells, Vero cells, 3T3 fibroblasts, C3H10T1 / 2 fibroblasts, CHO cells, and the like. Non-limiting examples of suitable host cells include HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), and HLHepG2 cells. The subject host cells can also be generated by using baculovirus to infect insect cells, such as Sf9 cells, that produce AAV (see, e.g., U.S. Patent No. 7,271,002; U.S. Patent Application No. 12 / 297,958).

[0165] In some embodiments, a subject genetically modified host cell comprises a nucleic acid comprising a nucleotide sequence encoding one or more AAV rep proteins in addition to a nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein. In other embodiments, a subject host cell further comprises a 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.

[0166] Examples of Non-Limiting Embodiments of the Disclosure The above-described aspects (including embodiments) of the inventive subject matter may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the present disclosure are set forth below, numbered 1 through 63. As will be apparent to one of ordinary skill in the art, each individually numbered aspect may be used or combined with any of the preceding or succeeding individually numbered aspects. This is intended to support all such combinations of aspects, and is not limited to the combinations of aspects explicitly set forth below.

[0167] 1. A recombinant adeno-associated virus (rAAV) virion comprising: a) a variant AAV capsid protein comprising an insertion of a heterologous peptide of any one of Formulas I-VI, wherein the variant AAV capsid protein confers increased infectivity of retinal cells compared to the infectivity of the retinal cells by a control AAV virion comprising the corresponding parent AAV capsid protein; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product. 2. The rAAV virion of aspect 1, which exhibits at least a 5-fold increase in infectivity of retinal cells compared to the infectivity of said retinal cells by a control AAV virion comprising the corresponding parental AAV capsid protein. 3. The rAAV virion of aspect 1, which exhibits at least a 10-fold increase in infectivity of retinal cells compared to the infectivity of said retinal cells by an AAV virion comprising the corresponding parental AAV capsid protein. 4. The rAAV virion of aspect 1, wherein the insertion of the heterologous peptide replaces a contiguous stretch of 5 to 20 amino acids of the parent AAV capsid protein. 5. The rAAV virion of embodiment 1, wherein the insertion site is between amino acids corresponding to amino acid 570 and amino acid 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype.

[0168] 6. The rAAV virion of aspect 4, wherein the insertion site is between amino acids corresponding to amino acid 587 and amino acid 588 of VP1 of AAV2, or a corresponding position in the capsid protein of another AAV serotype, or wherein the insertion site is between amino acids corresponding to amino acid 585 and amino acid 598 of VP1 of AAV2, or a corresponding position in the capsid protein of another AAV serotype. 7. The rAAV virion of any one of aspects 1 to 6, wherein the heterologous gene product is an interfering RNA or an aptamer. 8. The rAAV virion of any one of aspects 1 to 6, wherein the heterologous gene product is a polypeptide. 9. The rAAV virion of aspect 8, wherein the polypeptide is a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances the function of a retinal cell. 10. The rAAV virion of aspect 8, wherein the polypeptide is an RNA-guided endonuclease selected from a Type II CRISPR / Cas polypeptide, a Type V CRISPR / Cas polypeptide, or a Type VI CRISPR / Cas polypeptide.

[0169] 11. The rAAV virion of aspect 10, wherein the RNA-guided endonuclease is an enzymatically inactive Type II CRISPR / Cas polypeptide. 12. The rAAV virion of aspect 10, wherein the heterologous gene product is an RNA-guided endonuclease and a guide RNA. 13. The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide is a peptide of formula I:L-A(L / N)(I / Q)(Q / E)(D / H)(S / V)(M / K)(R / N)A (SEQ ID NO: 136). 14. The rAAV virion of any one of aspects 1 to 12, wherein the heterologous peptide comprises (21) LALIQDSMRA (SEQ ID NO: 35) or (22) LANQEHVKNA (SEQ ID NO: 2). 15. The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide is a peptide of formula II: TX1X2X3X4X5X6X7X8GLX9 (SEQ ID NO: 137), wherein Xi is G, V, or S; X2 is V, E, P, G, D, M, A, or S; X3 is M, V, Y, H, G, S, or D; X4 is R, D, S, G, V, Y, T, H, or M; X5 is S, L, G, T, Q, P, or A; X6 is T, A, S, M, D, Q, or H; X7 is N, G, S, L, M, P, G, or A; X8 is S, G, D, N, A, I, P, or T; and X9 is S or N.

[0170] 16. The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide comprises (1) TGVMRSTNSGLN (SEQ ID NO: 6), (2) TGEVDLAGGGLS (SEQ ID NO: 7), (3) TSPYSGSSDGLS (SEQ ID NO: 8), (4) TGGHDSSLDGLS (SEQ ID NO: 9), (5) TGDGGTTMNGLS (SEQ ID NO: 98), (6) TGGHGSAPDGLS (SEQ ID NO: 99), (7) TGMHVTMMAGLN (SEQ ID NO: 100), (8) TGASYLDNSGLS (SEQ ID NO: 101), (9) TVVSTQAGIGLS (SEQ ID NO: 20), (10) TGVMHSQASGLS (SEQ ID NO: 21), (11) TGDGSPAAPGLS (SEQ ID NO: 22), or (12) TGSDMAHGTGLS (SEQ ID NO: 23). 17. The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide is a peptide of formula III: TGX1X2X3X4X5X6X7GLS (SEQ ID NO: 138), wherein Xi is V, E, P, G, D, M, A, or S; X2 is M, V, Y, H, G, S, or D; X3 is R, D, S, G, V, Y, T, H, or M; X4 is S, L, G, T, Q, P, or A; X5 is T, A, S, M, D, Q, or H; X6 is N, G, S, L, M, P, G, or A; and X7 is S, G, D, N, A, I, P, or T. 18. The rAAV virion of any one of aspects 1 to 12, wherein the heterologous peptide comprises (2) TGEVDLAGGGLS (SEQ ID NO: 7), (4) TGGHDSSLDGLS (SEQ ID NO: 9), (5) TGDGGTTMNGLS (SEQ ID NO: 98), (6) TGGHGSAPDGLS (SEQ ID NO: 99), (8) TGASYLDNSGLS (SEQ ID NO: 101), (10) TGVMHSQASGLS (SEQ ID NO: 21), (11) TGDGSPAAPGLS (SEQ ID NO: 22), or (12) TGSDMAHGTGLS (SEQ ID NO: 23). 19. The heterologous peptide has the formula IV: X1GX2X3X4X5X6X7X8GLSPX9TX 10 X 11 A peptide of (SEQ ID NO: 139), wherein X1 is T or N, X2 is L, S, A, or G, X3 is D or V, X4 is A, G, or P, X5 is T or D, X6 is R or Y, X7 is D, T, or G, X8 is H, R, or T, X9 is V or A, and X 10 is G or W and X 11 is T or A. 20. The rAAV virion of any one of aspects 1 to 12, wherein the heterologous peptide comprises (13) TGLDATRDHGLSPVTGT (SEQ ID NO: 24), (14) TGSDGTRDHGLSPVTWT (SEQ ID NO: 25), (15) NGAVADYTRGLSPATGT (SEQ ID NO: 26), or (16) TGGDPTRGTGLSPVTGA (SEQ ID NO: 27).

[0171] 21. The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide is a peptide of formula V:TGX1DX2TRX3X4GLSPVTGT (SEQ ID NO: 140), wherein X1 is L, S, A, or G, X2 is A, G, or P, X8 is D, T, or G, and X4 is H, R, or T. 22. The heterologous peptide has the formula VI: LQX1X2X3RX4X5X6X7X8X9VNX 10Q (SEQ ID NO: 141), wherein X1 is K or R, X2 is N, G, or A, X3 is A, V, N, or D, X4 is P, I, or Q, X5 is A, P, or V, X6 is S, T, or G, X7 is T or V, X8 is E, L, A, or V, X9 is S, E, D, or V, and X 10 is F, G, T, or C. 23. The rAAV virion of any one of aspects 1 to 12, wherein the heterologous peptide comprises (17) LQKNARPASTESVNFQ (SEQ ID NO: 28), (18) LQRGVRIPSVLEVNGQ (SEQ ID NO: 29), (19) LQRGNRPVTTADVNTQ (SEQ ID NO: 30), or (20) LQKADRQPGVVVVNCQ (SEQ ID NO: 31). 24. A pharmaceutical composition comprising a) a recombinant adeno-associated virus virion according to any one of aspects 1 to 23, and b) a pharmaceutically acceptable excipient. 25. A method for delivering a gene product to a retinal cell in the body, comprising administering to the individual an rAAV virion described in any one of claims 1 to 23 or a pharmaceutical composition described in claim 24.

[0172] 26. The method of aspect 25, wherein the gene product is a polypeptide. 27. The method of aspect 25, wherein the gene product is a short interfering RNA or an aptamer. 28. The method of aspect 26, 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. 29. The method of aspect 26, wherein the polypeptide is glial-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, retinoschisin, RPE65, retinitis pigmentosa GTPase-interacting protein 1, peripherin, peripherin-2, rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), or sonic hedgehog. 30. The method of aspect 26, wherein the polypeptide is an RNA-guided endonuclease.

[0173] 31. A method for treating an eye disease, comprising administering to an individual in need thereof an effective amount of a rAAV virion according to any one of aspects 1 to 23 or a pharmaceutical composition according to aspect 24. 32. The method of aspect 31, wherein administering is by intraocular injection. 33. The method of aspect 31, wherein the administration is by intravitreal or suprachoroidal injection. 34. The method of any one of aspects 31-33, wherein the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's congenital amaurosis, diabetic retinopathy, achromatopsia, or color blindness. 35. An isolated nucleic acid comprising a nucleotide sequence encoding a variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises an insertion of about 5 amino acids to about 20 amino acids in the capsid protein GH loop relative to a corresponding parent AAV capsid protein, wherein the variant capsid protein, when present in an AAV virion, confers increased infectivity of the AAV virion in retinal cells, wherein the amino acid insertion is in the GH loop of a native AAV capsid, and the insertion is a peptide of any one of Formulas I-VI.

[0174] 36. The nucleic acid of aspect 35, wherein the insertion site is between amino acid 587 and amino acid 588 of AAV2, between amino acid 585 and amino acid 598 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. 37. An isolated genetically modified host cell comprising a nucleic acid according to embodiment 35 or embodiment 36. 38. A variant adeno-associated virus (AAV) capsid protein comprising an insertion of about 5 amino acids to about 20 amino acids, wherein the amino acid insertion is within the GH loop of a native AAV capsid, and the insertion is a peptide of any one of Formulas I-VI. 39. A rAAV virion comprising: a) a variant AAV capsid protein that includes an insertion of a heterologous peptide of formula VI and confers increased infectivity of retinal cells compared to the infectivity of said retinal cells by a control AAV virion containing the corresponding parent AAV capsid protein; and b) a heterologous nucleic acid that includes a nucleotide sequence encoding a heterologous gene product. 40. The rAAV virion of aspect 39, which exhibits at least a five-fold increase in infectivity of retinal cells compared to the infectivity of said retinal cells by a control AAV virion comprising the corresponding parental AAV capsid protein.

[0175] 41. The rAAV virion of aspect 39, which exhibits at least a 10-fold increase in infectivity of retinal cells compared to the infectivity of said retinal cells by an AAV virion comprising the corresponding parental AAV capsid protein. 42. The rAAV virion of any one of aspects 39-41, wherein the insertion of the heterologous peptide replaces a contiguous stretch of 5 to 20 amino acids of the parent AAV capsid protein. 43. The rAAV virion of any one of aspects 39 to 42, wherein the insertion site is between amino acids corresponding to amino acid 570 and amino acid 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype. 44. The rAAV virion of aspect 43, wherein the insertion site is between amino acids corresponding to amino acid 587 and amino acid 588 of VP1 of AAV2, or at a corresponding position in a capsid protein of another AAV serotype, or wherein the insertion site is between amino acids corresponding to amino acid 585 and amino acid 598 of VP1 of AAV2, or at a corresponding position in said capsid protein of another AAV serotype. 45. The rAAV virion of any one of aspects 39 to 44, wherein the heterologous gene product is an interfering RNA.

[0176] 46. ​​The rAAV virion of any one of aspects 39 to 44, wherein the heterologous gene product is an aptamer. 47. The rAAV virion of any one of aspects 39 to 44, wherein the heterologous gene product is a polypeptide. 48. The rAAV virion of aspect 47, wherein the polypeptide is a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances the function of a retinal cell. 49. The rAAV virion of aspect 47, wherein the polypeptide is an RNA-guided endonuclease selected from a Type II CRISPR / Cas polypeptide, a Type V CRISPR / Cas polypeptide, or a Type VI CRISPR / Cas polypeptide. 50. The rAAV virion of aspect 49, wherein the RNA-guided endonuclease is an enzymatically inactive type II CRISPR / Cas polypeptide.

[0177] 51. The rAAV virion of any one of aspects 39 to 44, wherein the heterologous gene product is an RNA-guided endonuclease and a guide RNA. 52. The rAAV virion of any one of aspects 39 to 51, wherein the heterologous peptide comprises (17) LQKNARPASTESVNFQ (SEQ ID NO: 28), (18) LQRGVRIPSVLEVNGQ (SEQ ID NO: 29), (19) LQRGNRPVTTADVNTQ (SEQ ID NO: 30), or (20) LQKADRQPGVVVVNCQ (SEQ ID NO: 31). 53. A pharmaceutical composition comprising: a) an rAAV virion according to any one of aspects 39 to 52; and b) a pharmaceutically acceptable excipient. 54. A method for delivering a gene product to a retinal cell in an individual, the method comprising administering to the individual an rAAV virion according to any one of aspects 39 to 52 or a pharmaceutical composition according to aspect 53. 55. The method of aspect 54, wherein the gene product is a polypeptide.

[0178] 56. The method of aspect 54, wherein the gene product is a short interfering RNA or an aptamer. 57. The method of aspect 55, 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. 58. The method of aspect 57, wherein the polypeptide is glial-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, retinoschisin, RPE65, retinitis pigmentosa GTPase-interacting protein 1, peripherin, peripherin-2, rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), or sonic hedgehog. 59. The method of aspect 55, wherein the polypeptide is an RNA-guided endonuclease. 60. A method for treating an eye disease, comprising administering to an individual in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion according to any one of aspects 39 to 52 or a pharmaceutical composition according to aspect 53.

[0179] 61. The method of aspect 60, wherein the administration is by intraocular injection. 62. The method of aspect 60, wherein the administration is by intravitreal or suprachoroidal injection. 63. The method of any one of aspects 60-62, wherein the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's congenital amaurosis, diabetic retinopathy, achromatopsia, or color blindness. [Example]

[0180] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below are all experiments performed, or that experiments in addition to those below have not been performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is near or near atmospheric. Standard abbreviations may be used. For example, bp is base pair(s), kb is kilobase(s), pl is picoliter(s), s or sec is second(s), min is minute(s), h or hr is hour(s), aa is amino acid(s), kb is kilobase(s), bp is base pair(s), nt is nucleotide(s), im is intramuscular, ip is intraperitoneal, sc is subcutaneous, etc.

[0181] Example 1: AAV virions containing variant AAV capsids Through a directed evolution approach, multiple AAV capsid variants have been derived. AAV virions containing the variant AAV capsids infect the primate retina when administered, for example, via intravitreal injection. Primates have a fovea for high-acuity vision and are an important preclinical model for human retinal diseases.

[0182] AAV packaging AAV virions containing variant AAV capsids were identified by screening using five libraries: 1) an AAV2-based 7mer peptide display library containing a 7mer peptide insert at approximately amino acid 588 and surrounded by a 5' LA linker and a 3' A linker; 2) an AAV4-based 7mer peptide display library with a 5' TG linker and a 3' GLS linker and a 7mer peptide insert at approximately amino acid 584; 3) an AAV5-based 7mer peptide display library with a 5' TG linker and a 3' GLS linker and a 7mer peptide insert at approximately amino acid 575; 4) a library based on an ancestral AAV sequence with a 5' TG linker and a 3' GLS linker containing a 7mer peptide display library at approximately amino acid 591 (Santiago-Ortiz et al., 2015); and 5) an AAV2-based library with semi-random mutations at a surface-exposed position at approximately amino acid 588 (Koerber, Jang, & Schaffer, 2008). Virus packaging was performed as previously described by Koerber et al. (2008) (see above) and Fowler et al. Nat Protoc 9, 2267-2284 (2014), with each viral genome encased within the capsid protein shell encoded by that genome. Therefore, functional improvements through selection can be linked to the genomic sequence contained within the viral capsid. Briefly, AAV vectors were produced by triple transient transfection of HEK293T cells, purified via iodixanol density centrifugation, and buffer exchanged into PBS by Amicon filtration. DNase-resistant viral genome titers were measured by quantitative real-time PCR using a BioRad iCycler. From this library, variants capable of infecting the vitreous into the primate retina were identified using an in vivo iterative screening-selection process (Figure 1). Approximately 250 μL of 1 × 10 vectors were injected into primate eyes in each round. 13 ~1×10 14Virus was injected at a titer of 10 ...

[0183] Figure 1 shows the directed evolution method used to generate primate retinal AAV variants. A peptide display library was generated, packaged into AAV vectors, and injected into primate eyes via intravitreal injection. Iterative selection rounds were used to positively select AAV variants from a pool of vectors. Three selection rounds were followed by error-prone PCR, followed by an additional selection round.

[0184] Deep sequencing of AAV libraries from selection rounds Following five selection rounds, Illumina deep sequencing was used to identify AAV variants whose relative display rate within the library increased over the rounds. Increased display rate within the viral library indicates positive selection and the ability to infect the primate retina from the vitreous. Approximately 75-85 base pair regions containing 7-mer insertion or loop swap mutation sites were PCR amplified from harvested DNA. Primers included Illumina adapter sequences containing unique barcodes, allowing multiplexing of amplicons from multiple selection rounds. PCR amplicons were purified and sequenced using a 100-cycle single-read run on an Illumina HiSeq 2500. Custom Python code was written to translate DNA sequences into amino acid sequences and count reads containing unique 7-mer insertion sequences. Read counts were normalized by the total number of reads in the run. The dynamics of directed evolution were analyzed and plotted using Python and Pandas.

[0185] Deep sequencing analysis Approximately 1 x 10 per library 7 The top variants were selected from the 100 variants. The best-performing variant was chosen as the variant with the largest fold increase in the final selection round relative to the initial plasmid library (number of reads in the final round (normalized to total number of reads in the round) / number of reads in the library (normalized to total number of reads in the round)). To allow for analysis of variants not represented in the sequencing of the plasmid library, a pseudocount of 1 was added before normalization for each individual variant. See Fowler et al. (2014) supra. The amino acid sequences of the peptide inserts are shown in Figure 2.

[0186] The variants generated through this approach enable non-invasive pan-retinal gene therapy strategies in the primate retina using intravitreal injection. Such AAV vectors can be used for gene augmentation therapy for retinal degenerative diseases, including retinitis pigmentosa, Leber congenital amaurosis, rod-cone dystrophy, cone dystrophy, achromatopsia, X-linked retinoschisis, CRB1, optogenetic therapy, expression of trophic and survival factors (e.g., GDNF, BDNF, FGF, RdCVF, RdCVFL, XIAP), and angiogenesis blockers such as sFLT. Such vectors can also be used to deliver gene editing tools such as CRISPR / Cas9 for gene correction or additional model generation in retinal diseases.

[0187] References Dalkara, D., Byrne, LC, Klimczak, RR, Visel, M., Yin, L., Merigan, WH, et al. (2013). In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Science Translational Medicine,5(189),189ra76.http: / / doi.org / 10.1126 / scitranslmed.3005708 Dalkara, D., Goureau, O., Marazova, K., & Sahel, J.-A. (2016). Let there be light: gene and cell therapy for blindness. Human Gene Therapy, hum.2015.147. http: / / doi.org / 10.1089 / hum.2015.147 Dalkara,D.,Kolstad,K.D.,Caporale,N.,Visel,M.,Klimczak,R.R.,Schaffer,D.V.,& Flannery,J.G.(2009). Inner limiting membrane barriers to AAV-mediated retinal transduction from the vitreous. Molecular Therapy :the Journal of the American Society of Gene Therapy,17(12),2096-2102.http: / / doi.org / 10.1038 / mt.2009.181 Koerber,J.T.,Jang,J.-H.,& Schaffer,D.V.(2008). DNA Shuffling of Adeno-associated Virus Yields Functionally Diverse Viral Progeny. Molecular Therapy :the Journal of the American Society of Gene Therapy,16(10),1703-1709.http: / / doi.org / 10.1038 / mt.2008.167 Maguire,A.M.,Simonelli,F.,Pierce,E.A.,Pugh,E.N.,Jr.,Mingozzi,F.,Bennicelli,J.,et al.(2008). Safety and Efficacy of Gene Transfer for Leber’s Congenital Amaurosis. N Engl J Med,358(21),2240-2248.http: / / doi.org / 10.1056 / NEJMoa0802315 Nakazawa,T.,Matsubara,A.,Noda,K.,Hisatomi,T.,She,H.,Skondra,D.,et al.(2006). Characterization of cytokine responses to retinal detachment in rats. Molecular Vision,12,867-878. Nakazawa,T.,Takeda,M.,Lewis,G.P.,Cho,K.-S.,Jiao,J.,Wilhelmsson,U.,et al.(2007). Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin. Investigative Ophthalmology & Visual Science,48(6),2760-2768.http: / / doi.org / 10.1167 / iovs.06-1398 Petrs-Silva,H.,Dinculescu,A.,Li,Q.,Min,S.-H.,Chiodo,V.,Pang,J.J.,et al.(2009). High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors. Molecular Therapy :the Journal of the American Society of Gene Therapy,17(3),463-471.http: / / doi.org / 10.1038 / mt.2008.269 Santiago-Ortiz, J., Ojala, DS, Westesson, O., Weinstein, JR, Wong, SY, Steinsapir, A., et al. (2015). AAV ancestral reconstruction library enables selection of broadly infectious viral variants. Gene Therapy, 22(12), 934-946. http: / / doi.org / 10.1038 / gt.2015.74

[0188] Example 2: Methods for constructing and sequencing GFP barcode libraries Construction of GFP barcode library A unique 25-bp DNA barcode was cloned after an AAV ITR construct containing a self-complementary CAG promoter driving eGFP (CAG-GFP-barcode-pA). Individual variants were packaged separately with constructs containing different barcodes. The variants were then titer-matched and mixed in equal ratios before being injected into mice, dogs, and primates.

[0189] Deep sequencing of GFP barcoded libraries Barcodes were PCR-amplified directly from DNA or cDNA (generated from mRNA using Superscript III reverse transcriptase) collected from canine or primate retinal tissue. Samples were collected from sections spanning the entire retina, and from the ONL or RPE. Primers amplified a ∼50-bp region surrounding the GFP barcode and contained an Illumina adapter sequence and a secondary barcode to allow multiplexing of multiple samples. PCR amplicons were purified and sequenced using a 100-cycle single-read run on a MiSeq. Read counts were normalized by the total number of reads in the run. Barcode abundance analysis was performed using custom code written in Python, and plots were then generated using Pandas. The best-performing variants were selected based on the fold increase in total library relative to the injected library (% total in recovered sample / % total in injected library). Analysis was performed on n=1 primate.

[0190] FIG. 9 shows Table 1, and FIG. 10 shows Table 2.

[0191] Table 1 shows the ranking of primate-derived variants and controls recovered from photoreceptors after injection of a GFP barcode library. Table 2 shows the ranking of primate-derived variants and controls recovered from RPE cells after injection of a GFP barcode library. The libraries contained individual variants packaged with GFP fused to unique DNA barcodes. Polymerase chain reaction (PCR) was used to amplify barcodes from DNA recovered from specific cell types within the retina. The "region" in Tables 1 and 2 indicates the region from which DNA was recovered. The fold increase in reads for each variant was calculated by dividing the number of reads for each unique barcode within the recovered cells (corresponding to each unique variant) by the number of reads for each variant in the injected library. This table shows the average fold increase across multiple locations within the retina. Variants were ranked by the fold increase in barcode.

[0192] Figure 11 shows GFP expression of the GFP-barcoded library in the primate retina. GFP expression resulting from intravitreal injection of the pooled GFP-barcoded library (containing all tested viruses) was primarily located in the outer retina, with a tropism toward the outer retina greater than that of AAV24YF.

[0193] Example 3 Primate testing Cynomolgus monkeys aged 4–10 years were used in all studies and underwent intravitreal injections. For immunosuppression, monkeys used for fluorophore expression received daily subcutaneous cyclosporine injections at a dose of 6 mg / kg, adjusted based on blood trough levels within a target range of 150–200 ng / ml. Three weeks after injection, confocal scanning laser microscope images (Spectralis HRA, Heidelberg Engineering) were acquired from both retinas using autofluorescence settings, resulting in effective visualization of tdTomato and GFP. For histological diagnosis, monkeys were euthanized, and both retinas were fixed in 4% paraformaldehyde and examined by confocal microscopy. At the conclusion of the experiment, euthanasia was achieved by administering an intravenous overdose of sodium pentobarbital (75 mg kg-1), as recommended by the American Veterinary Medical Association's Panel on Euthanasia. Next, small sections of primate retina were prepared in 30% sucrose, embedded in OCT medium, flash-frozen, and sectioned at 20 μm for confocal microscopy imaging of native fluorophore expression. The antibodies used for labeling were anti-GFP (A11122, Thermo, 1:250), anti-vimentin (Dako, 1:1000), peanut agglutinin (PNA) (Molecular Probes, 1:200), and anti-cone arrestin (7G6, 1:50). Procedures were performed in accordance with the ARVO Statement for the Use of Animals and the guidelines of the Office of Laboratory Animal Care at the University of Rochester.

[0194] result Directed evolution of AAVs in the primate retina Non-human primates, in addition to dogs, are critical preclinical models for human therapeutic development because they are most closely related to humans and have retinal anatomy similar to that of humans. Specifically, primates are the only large animal model that possesses a fovea, a specialized high-acuity area in the retina that is essential for daily activities such as reading, critical to quality of life, and lost in many retinal degenerations. The species-specificity observed in dog studies motivated the inventors to pursue further avenues of directed evolution in the primate retina. Nine libraries were packaged and included in primate screening: EP2, EP5, EP6, EP8, EP9, EP-ancestral, AAV2-7mer, ancestral-7mer (Santiago-Ortiz et al. Gene Ther 22, 934-946 (2015)), and loop swap (Koerber et al. Mol Ther 17, 2088-2095 (2009)). Libraries were injected, harvested, and repackaged for five sequential selection rounds (one round of error-prone PCR after round 3). The AAV cap gene was PCR amplified from the ONL and simultaneously from the overlying RPE. The EP library was discontinued in round 3 because no variants from the EP library were recovered from retinal tissue. In round 4, additional libraries (AAV4-7mer and AAV5-7mer) were added to the selection using separate backbones isolated from the other libraries by separate PCR annealing and restriction sites.

[0195] Deep sequencing reduced the library to approximately 10 6 ~about 10 7 These contain approximately 10 individual variants over six selection rounds. 4 ~about 10 5This revealed a convergence of variants within the initial plasmid library, a diversity that would be impossible to observe through Sanger sequencing (Figure 12A). As observed in the canine screen, a small proportion of library members within the initial plasmid library were over-represented in each analyzed library (Figure 12B). Analysis of the results from high-throughput sequencing across selection rounds revealed a subset of variants for each library whose occurrence significantly increased during the selection rounds (Figure 12C).

[0196] Screening of a secondary barcoded GFP library in the primate retina Sixteen variants were selected from these five libraries (Figure 12C) and included in a secondary round of selection against the GFP-barcoded library, along with AAV2, AAV2-4YF+TV, AAV4, and AAV5 as controls. This new library was injected into both eyes of primates, and biopsies were collected from various locations throughout the retina 3 weeks after injection (Figure 12D). GFP expression resulting from injection of the GFP-barcoded library was found primarily in photoreceptors, but also in some inner retinal cells. This is a shift in tropism from AAV2 or 7m8, which resulted in stronger inner retinal expression (Figure 12E).

[0197] Figures 12A-12F show directed evolution of AAV in the primate retina. (A) Deep sequencing of variant libraries revealed convergence of variants across selection rounds. (B) In each library evaluated, a small proportion of variants were over-represented in the plasmid library. (C) Scatter plots show the behavior of individual variants in the final selection round of each library injected into the primate retina. Variants over-represented in the original library are colored blue. Variants with the largest fold increase in display rate in the final selection round are colored magenta. Variants over-represented in the original library and that showed a significant increase in display rate across selection rounds are colored orange. (D) A map of the primate retina shows the distribution of samples collected for selection rounds and GFP-barcoded libraries. Variant color coding is the same as in Figure 2. (E) GFP expression derived from the barcoded library revealed a shift in expression toward outer retinal tropism for selected variants. (F) Results of GFP-barcoded library injections into the outer retina of primates. Variants are listed in order from best (top) to worst (bottom) performing vectors, and the values ​​indicate the degree to which the variant outcompeted other vectors, expressed as % of total in AAV library / % of total in recovered library.

[0198] Validation of top-performing primate variants Quantification of vector performance in the outer retina revealed that AAV2-based variants outperformed other serotype-based viruses. One vector, a loop-swap variant AAV2, approximately 588-LQRGVRIPSVLEVNGQ (SEQ ID NO: 116), outperformed other variants but produced lower viral titers (approximately 5 x 10 11 vg / mL).

[0199] Therefore, it was the second-ranked variant from the GFP barcode screening, which showed high titers (approximately 5 × 10 13 AAV2-LALIQDSMRA (SEQ ID NO: 117, designated NHP#9), packaged at 1000 ng / mL (vg / mL), was selected for the first round of validation studies, targeting ganglion cells in the inner retina and cones in the outer retina. Cone photoreceptors are involved in adult macular degeneration (AMD). Macular degeneration is the most common cause of blindness in developed countries, projected to affect 288 million people worldwide by 2040, making cone photoreceptors a primary target for retinal gene therapy. NHP#9 was packaged with the SNCG promoter, which drives tdTomato in RGCs, and the pR1.7 promoter, which drives GFP expression in cones. Vectors encoding both constructs were combined in equal ratios (approximately 1.5 x 10 12 The constructs were mixed at a ratio of 1:1 (vg / construct / eye) and injected intravitreally into cynomolgus monkeys. A previously described variant, 7m8 (Dalkara et al. (2013) supra), was packaged with the same construct at equal titers and injected into the vitreous of the contralateral eye. In NHP#9-injected eyes, tdTomato reporter expression in RGCs was lower than that of 7m8, which efficiently infected ganglion cells throughout the retina. However, expression in foveal cones was significantly increased compared to 7m8, indicating a shift in tropism from the inner retina to photoreceptors in the outer retina. qRT-PCR performed using the ddCT method revealed an 11.71-fold (10.37-13.22)-fold increase in GFP expression compared to 7m8. Labeled cell counts performed using Imaris software on images collected from flat-mounted retinas also confirmed a substantial reduction in the number of transduced ganglion cells and an increase in the number of cones targeted in NHP#9.

[0200] Next, the top-ranking variant from the GFP barcode screen, a loop-swap variant, approximately 588-LQRGVRIPSVLEVNGQ (SEQ ID NO: 118, designated NHP#26), was also tested for validation, although fewer viral particles were produced (approximately 5 x 10 10 NHP#26-scCAG-eGFP particles were intravitreally injected into one eye of a cynomolgus monkey. Despite the small number of particles injected, efficient GFP expression was observed within the fovea and throughout the retina (Figure 13G). In contrast to the spotted and ring-like expression patterns in the fovea observed with 7m8, NHP#9 (Figure 13A), and other naturally occurring serotypes, fundus imaging of NHP#26 revealed a disc-like GFP expression pattern in the center of the fovea (Figure 13G). Confocal imaging of flat-mounted retinas confirmed this disc-like expression pattern around the fovea, along with very few GFP-positive ganglion cell axons (Figure 13H). Punctate areas of GFP expression were often strongest around retinal blood vessels (Figure 13I) and were located throughout the retina. Imaging of cryostat sections prepared from the retina confirmed that while GFP expression was low in ganglion cells, as indicated by the absence of GFP+ ganglion cell axons, high levels of GFP expression were found in Müller cells, additional cells in the inner nuclear layer, and foveal cones and rods throughout the retina (Figures 13J-13Q).

[0201] Figures 13A-13Q show validation of evolved AAVs in the primate retina. (A-F) Approximately 1.5 x 10 7m8 and variant NHP#9 packaged AAVs. 12 We co-injected 5x10 SCNG-tdTomato particles and approximately 1.5 pR1.7-eGFP into the primate retina. Intravitreal injection of 7m8 (A, C, E) resulted in robust tdTomato expression in ganglion cells and GFP expression in foveal cones. In contrast, injection of an equal number of NHP#9 particles resulted in reduced ganglion cell expression and increased GFP expression in cones compared to 7m8 (B, D, F). (G) 5x10 10Fundus imaging of primate retinas after injection of NHP#26-scCAG-GFP particles resulted in disc-shaped GFP expression in the center of the foveola and a punctate pattern of GFP expression throughout the retina. (H) Confocal imaging of native GFP expression in the flat-mounted fovea. (I) Confocal imaging of native GFP expression in the outer segment of the vascular arcade. (J) Confocal imaging of native GFP expression in a cryostat section through the fovea. (K) Native GFP expression in the inferior retina outside the vascular arcade shows only faint GFP expression in ganglion cells, but high levels of expression in Müller cells and photoreceptors in the outer retina. Autofluorescence was also observed in the RPE. (L) Anti-GFP labeling in cryostat sections revealed GFP expression in photoreceptors (evident from the outer segments), Müller cells (evident from processes extending across the retina), and cells in the inner nuclear layer with horizontal processes (potentially interneurons). (M) Anti-GFP labeling in a foveal section reveals additional transfected cones, Müller glia, and interneurons. (N) Co-labeling with anti-cone arrestin and anti-GFP reveals GFP expression in rod photoreceptors and cells in the inner nuclear layer in a section made adjacent to the optic nerve head. (O) Co-labeling with anti-cone arrestin and anti-GFP antibodies in a low-expression area reveals GFP expression in inner nuclear layer cells. (P, Q) Montage of confocal images from cryostat sections collected outside the vascular arcade shows efficient GFP expression in the inner nuclear layer and outer retina.

[0202] While the present invention has been described with reference to specific embodiments thereof, those skilled in the art will recognize that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step(s) to the objective, spirit, and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

[0203] cross reference This application claims the benefit of U.S. Provisional Patent Application Nos. 62 / 527,871, filed June 30, 2017, and 62 / 535,042, filed July 20, 2017, which are incorporated herein by reference in their entireties.

[0204] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant No. 1R01EY022975-01A1 awarded by the National Institutes of Health. The government has certain rights in this invention.

Claims

1. A recombinant adeno-associated virus (rAAV) 2 virion, comprising: a) a variant AAV2 capsid protein VP1, comprising an insertion of a heterologous peptide comprising the sequence LALIQDSMRA (SEQ ID NO: 35) or the sequence LANQEHVKNA (SEQ ID NO: 2) at an insertion site between amino acids 570 and 611 of AAV2 capsid protein VP1, wherein the variant AAV2 capsid protein VP1 confers increased infectivity of retinal cells compared to the infectivity of said retinal cells by control AAV2 virions comprising the corresponding parent AAV2 capsid protein; b) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product; Contains, rAAV2 virions.

2. 2. The rAAV2 virion of claim 1, which exhibits at least a 5-fold increase in infectivity of retinal cells compared to the infectivity of retinal cells by a control AAV2 virion containing the corresponding parental AAV2 capsid protein.

3. 2. The rAAV2 virion of claim 1, wherein the insertion of the heterologous peptide replaces a contiguous stretch of 5 to 20 amino acids of the parent AAV2 capsid protein.

4. 2. The rAAV2 virion of claim 1, wherein the insertion site is between amino acids 587 and 588 of VP1 of AAV2, or wherein the insertion site is between amino acids 585 and 598 of VP1 of AAV2.

5. The rAAV2 virion of any one of claims 1 to 4, wherein the heterologous gene product is an interfering RNA, an aptamer, or a polypeptide.

6. 2. The rAAV2 virion of claim 1, wherein the heterologous gene product is: a) an RNA-guided endonuclease selected from a type II CRISPR / Cas polypeptide, a type V CRISPR / Cas polypeptide, and a type VI CRISPR / Cas polypeptide; b) an enzymatically inactive type II CRISPR / Cas polypeptide; or c) an RNA-guided endonuclease and a guide RNA.

7. The rAAV2 virion of any one of claims 1 to 6, wherein the heterologous peptide comprises the sequence LALIQDSMRA (SEQ ID NO: 35).

8. The rAAV2 virion of any one of claims 1 to 6, wherein the heterologous peptide comprises the sequence LANQEHVKNA (SEQ ID NO: 2).

9. a) a recombinant adeno-associated virus virion 2 according to any one of claims 1 to 8; b) a pharmaceutically acceptable excipient; and 10. A pharmaceutical composition comprising:

10. 10. A recombinant adeno-associated virus (rAAV) virion 2 according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9 for use in a method for delivering a gene product to a retinal cell in an individual, comprising: The method includes administering to the individual the rAAV virion 2 or the pharmaceutical composition. rAAV virion 2 for use or pharmaceutical composition for use.

11. 11. The rAAV virion 2 for use or pharmaceutical composition for use according to claim 10, wherein the gene product is a polypeptide, a small interfering RNA or an aptamer.

12. The polypeptide is i) neuroprotective factors, anti-angiogenic polypeptides, anti-apoptotic factors, or polypeptides that enhance the function of retinal cells; ii) glial-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, retinoschisin, RPE65, retinitis pigmentosa GTPase-interacting protein 1, peripherin, peripherin-2, rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), sonic hedgehog, or iii) RNA-guided endonucleases 12. The rAAV virion 2 for use or pharmaceutical composition for use according to claim 11, wherein

13. 10. A method for treating an eye disease comprising administering to said patient a recombinant adeno-associated virus (rAAV) virion 2 according to any one of claims 1 to 8 or a pharmaceutical composition according to claim 9. The method comprises administering to an individual in need thereof an effective amount of the rAAV virion 2 or the pharmaceutical composition. rAAV virion 2 for use or pharmaceutical composition for use.

14. 1. An isolated nucleic acid comprising a nucleotide sequence encoding a variant adeno-associated virus 2 (AAV2) capsid protein VP1, the variant AA2V capsid protein comprises an insertion of a heterologous peptide comprising the sequence LALIQDSMRA (SEQ ID NO: 35) or the sequence LANQEHVKNA (SEQ ID NO: 2), and the variant capsid protein, when present in an AAV virion, results in increased infectivity of the AAV virion in retinal cells; the heterologous peptide is inserted into an insertion site between amino acids 570 and 611 of AAV2 capsid protein VP1, between amino acids 587 and 588 of AAV2 capsid protein VP1, or between amino acids 585 and 598 of AAV2 capsid protein VP1; Nucleic acid.

15. 15. The isolated nucleic acid of claim 14, wherein the heterologous peptide comprises the sequence LALIQDSMRA (SEQ ID NO: 35).

16. 15. The isolated nucleic acid of claim 14, wherein the heterologous peptide comprises the sequence LANQEHVKNA (SEQ ID NO: 2).

17. 17. An isolated genetically modified host cell comprising a nucleic acid according to claim 15 or 16.

18. 1. A variant adeno-associated virus 2 (AAV2) capsid protein VP1 comprising an insertion of a heterologous peptide comprising the sequence LALIQDSMRA (SEQ ID NO: 35) or the sequence LANQEHVKNA (SEQ ID NO: 2), wherein the variant AAV2 capsid protein VP1, when present in AAV virions, results in increased infectivity of the AAV virions in retinal cells; the heterologous peptide is inserted into an insertion site between amino acids 570 and 611 of AAV2 capsid protein VP1, between amino acids 587 and 588 of AAV2 capsid protein VP1, or between amino acids 585 and 598 of AAV2 capsid protein VP1; Variant AAV2 capsid protein VP1.

19. 19. The variant AAV2 capsid protein VP1 of claim 18, wherein the heterologous peptide comprises the sequence LALIQDSMRA (SEQ ID NO: 35).

20. 19. The variant AAV2 capsid protein VP1 of claim 18, wherein the heterologous peptide comprises the sequence LANQEHVKNA (SEQ ID NO: 2).

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