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

Abstract

To provide adeno-associated virus virions with variant capsids and methods of use thereof.SOLUTION: The present disclosure provides recombinant adeno-associated virus (AAV) virions with altered capsid protein, where the recombinant AAV (rAAV) virions exhibit greater ability to cross barriers between intravitreal fluid and retinal cells, and thus greater infectivity of a retinal cell compared to wild-type AAV, and where the rAAV virions comprise a heterologous nucleic acid. The present disclosure provides methods of delivering a gene product to a retinal cell in an individual.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] Vision is mediated by cells in the retina, a thin, layered structure that covers the back of the eye. Photoreceptors on the back of the retina respond to the absorption of photons, initiating a stream of signal processing that passes through secondary and tertiary neurons in the retina, including bipolar, horizontal, and amacrine cells. Retinal pigment epithelial (RPE) cells, located beneath the photoreceptors, are essential for facilitating this photoreceptor function as they promote the regeneration of the photon-detecting molecule 11-cisretinal via the visual cycle pathway. Retinal ganglion cells (RGCs) in the inner retina receive visual signals from tertiary neurons and transmit these visual signals to the brain in the form of action potentials.

[0002] Gene mutations expressed within retinal cells, including those in photoreceptors, RPEs, bipolar cells, and transcripts in other cells, lead to disruptions in 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 co-infection with a helper virus, such as an adenovirus, to facilitate replication, while AAVs establish latent infection in the absence of a helper. The virion consists 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 non-structural rep gene encodes four regulatory proteins essential for viral replication, while cap encodes three structural proteins (VP1-3) that assemble to form a 60-mer capsid shell. This viral capsid mediates the AAV vector's ability to overcome many of the biological barriers to viral transduction (including cell surface receptor binding, endocytosis, intracellular trafficking, and nuclear unpackaging). [Overview of the project]

[0004] This disclosure provides recombinant adeno-associated virus (AAV) virions having a modified capsid protein, exhibiting a higher ability to cross the barrier between intravitreous fluid and retinal cells compared to wild-type AAV, and thus exhibiting higher infectivity to retinal cells, and also containing heterologous nucleic acids, thereby providing recombinant AAV (rAAV) virions. This disclosure also provides a method for delivering gene products to retinal cells in an individual. [Brief explanation of the drawing]

[0005] [Figure 1] A schematic diagram of the directional evolution method used for the generation of primate retinal AAV variants is shown. [Figure 2] A table showing peptide insertions and substitutions in variant AAV capsids is provided. [Figure 3A] The amino acid sequence of an example guide RNA-directed endonuclease is shown. [Figure 3B] The amino acid sequence of an example guide RNA-directed endonuclease is shown. [Figure 3C] The amino acid sequence of an example guide RNA-directed endonuclease is shown. [Figure 4] The amino acid sequence of the AAV2 capsid protein VP1 is shown. Amino acids 587 and 588 (NP) are shown in bold and underlined. [Figure 5] This shows the amino acid sequences corresponding to amino acids 570-610 of the AAV capsid protein VP1 of various AAV serotypes. [Figure 6A] This shows the amino acid sequence alignment of the AAV capsid protein loop IV (GH loop) region. Insertion sites are indicated in bold and underlined. [Figure 6B] This shows the amino acid sequence alignment of the AAV capsid protein loop IV (GH loop) region. Insertion sites are indicated in bold and underlined. [Figure 6C] This shows the amino acid sequence alignment of the AAV capsid protein loop IV (GH loop) region. Insertion sites are 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 shows 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 - J show the amino acid sequences of exemplary heterologous gene products. [Figure 7-7] K shows the amino acid sequence of an exemplary heterologous gene product. [Figure 7-8] L shows the amino acid sequence of an exemplary heterologous gene product. [Figure 7-9] M shows the amino acid sequence of an exemplary heterologous gene product. [Figure 7-10] N and O show the amino acid sequences of exemplary heterologous gene products. [Figure 7-11] P shows the amino acid sequence of an exemplary heterologous gene product. [Figure 7-12] Q and R show the amino acid sequences of exemplary heterologous gene products. [Figure 7-13] S shows the amino acid sequence of an exemplary heterologous gene product. [Figure 7-14] T shows the amino acid sequence of an exemplary heterologous gene product. [Figure 7-15] U shows the amino acid sequence of an exemplary heterologous gene product. [Figure 7-16] V shows 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 is shown. Table 1 shows the ranking of primate - derived variants and controls recovered from photoreceptors after injection of the green fluorescent protein (GFP) barcode library. [Figure 10] Table 2 is shown. Table 2 shows the ranking of primate-derived variants and controls recovered from RPE cells after GFP barcode library injection. [Figure 11] This shows GFP expression from a GFP barcode library in primate retinas. [Figure 12-1] A and B show the directional evolution of AAV in the primate retina. [Figure 12-2] C shows the directional evolution of AAV in the primate retina. [Figure 12-3] C shows the directional evolution of AAV in the primate retina. [Figure 12-4] C and D show the directional evolution of AAV in the primate retina. [Figure 12-5] E and F demonstrate the directional evolution of AAV in the primate retina. The sequence from top to bottom in F is described in sequence numbers 117-135. [Figure 13-1] A-F demonstrate the evolution of AAV in the primate retina. [Figure 13-2] G to L demonstrate the verification of evolved AAV in the primate retina. [Figure 13-3] M~O demonstrates the verification of evolved AAV in the primate retina. [Figure 13-4] P and Q demonstrate the validation of evolved AAV in the primate retina. [Modes for carrying out the invention]

[0006] The term “retinal cells” as used herein may refer to any cell type that constitutes 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. Unless otherwise specified, this term encompasses all subtypes and both naturally occurring and recombinant forms. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also known as recombinant AAV vector (or "rAAV vector"). The term "AAV" includes type 1 AAV (AAV-1), type 2 AAV (AAV-2), type 3 AAV (AAV-3), type 4 AAV (AAV-4), type 5 AAV (AAV-5), type 6 AAV (AAV-6), type 7 AAV (AAV-7), type 8 AAV (AAV-8), type 9 AAV (AAV-9), type 10 AAV (AAV-10), type 11 AAV (AAV-11), bird AAV, cattle AAV, dog AAV, horse AAV, primate AAV, non-primate AAV, and sheep AAV. See, for example, Mori et al. (2004) Virology 330:375. The term "AAV" also includes chimeric AAV. "Primate AAV" refers to AAVs isolated from primates, "non-primate AAV" refers to AAVs isolated from non-primate mammals, and "bovine AAV" refers to AAVs isolated from bovine mammals (for example, dairy cows).

[0008] As used herein, “rAAV vector” refers to an AAV vector containing 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 cells. Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV reverse terminal repeats (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0009] An "AAV virus," "AAV virus particle," or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically all capsid proteins of wild-type AAV) and a capsid-formed polynucleotide rAAV vector. If this particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, e.g., transgenes delivered to mammalian cells), the particle is typically called an "rAAV vector particle" or simply an "rAAV vector." Therefore, the production of rAAV particles necessarily involves the production of rAAV vectors, and thus the vector is contained within the rAAV particle.

[0010] "Packaging" refers to a series of intracellular events that result in the aggregation of AAV particles and the formation of a capsid.

[0011] The AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and capsid-forming proteins of adeno-associated viruses. In this specification, the AAV rep and cap are referred to as the AAV "packaging genes."

[0012] A “helper virus” for AAV refers to a virus that enables AAV (e.g., wild-type AAV) to replicate and package in mammalian cells. Various such helper viruses are known in this art, and include adenoviruses, herpesviruses, and poxviruses such as vaccinia. Adenoviruses encompass several different subgroups, but type 5 adenovirus of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositaries such as ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), which are also available from depositaries such as ATCC.

[0013] "Helper virus function(s)" means a function(s) encoded within the helper virus genome that enables the replication and packaging of AAV (in conjunction with other replication and packaging requirements described herein). As described herein, "Helper virus function(s)" can be provided in several ways, including by providing a helper virus or by providing, for example, a polynucleotide sequence encoding an essential function(s) to a producing cell along the way.

[0014] An “infectious” virus or viral particle is a virus or viral particle that contains polynucleotide components capable of being delivered to cells to which the virus species is tropic. This term does not necessarily mean any replication ability of the virus. As used herein, an “infectious” virus or viral particle is a virus or viral particle that can access target cells, can infect target cells, and can express heterologous nucleic acids within target cells. Thus, “infectivity” refers to the ability of a viral particle to access target cells, to infect target cells, and to express heterologous nucleic acids within target cells. Infectivity may refer to in vitro infectivity 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 the viral genome (vg) copy number. The ability of a viral particle to express heterologous nucleic acids within cells may be called “transduction.” The ability of viral particles to express heterologous nucleic acids within cells can be assayed using several techniques, such as evaluation of marker genes, e.g., green fluorescent protein (GFP) assays (where GFP is produced and detected / / or measured within cells infected with viral particles) (for example, if the virus contains a nucleotide sequence encoding GFP), or measurement of the produced protein (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 (describes the TCID50 infectivity titer assay) and Zolotukhin et al. (1999) Gene Ther.6:973.

[0015] A “replicable” virus (e.g., replicable AAV) refers to a phenotypic wild-type virus that is infectious and capable of replicating within infected cells (i.e., in the presence of a helper virus or helper virus function). In the case of AAV, replication ability generally requires the presence of a functional AAV packaging gene. Generally, the rAAV vectors described herein are non-replicable in mammalian cells (particularly human cells) due to the absence of one or more AAV packaging genes. Typically, such rAAV vectors lack any AAV packaging gene sequence to minimize the possibility of replicable AAV being generated by recombination between the AAV packaging gene and the incoming rAAV vector. In many embodiments, the rAAV vector preparations described herein contain, if present, small amounts of replicable AAV (rcAAV, also known as RCA) (e.g., 10 2 Less than approximately 1 rcAAV per rAAV particle, 10 4 Less than approximately 1 rcAAV per rAAV particle, 10 8 Less than approximately 1 rcAAV per rAAV particle, 10 12 This is a preparation containing less than approximately 1 rcAAV per rAAV particle, or rcAAV is absent.

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

[0017] A polynucleotide or polypeptide has a certain percentage of "sequence identity" to another polynucleotide or polypeptide, meaning that when two sequences are aligned, they will have the same percentage of bases or amino acids. Sequence similarity can be determined in several different ways. To determine sequence identity, sequences can be aligned using methods and computer programs, one example of which is 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 Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. 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. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth.Mol.Biol.70:173-187 (1997). GAP programs using the Needleman-Unsch alignment method can also be used for sequence alignment. See J.Mol.Biol.48:443-453 (1970).

[0018] The subject of interest is a BestFit program that determines sequence identity using the Smith-Waterman local homology algorithm (Advances in Applied Mathematics 2:482-489 (1981)). The gap generation penalty is generally in the range of 1 to 5, usually 2 to 4, and 3 in many embodiments. The gap extension penalty is generally in the range of approximately 0.01 to 0.20, and is often 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 from 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. Sequence identity percentage is calculated by FastDB based on the following parameters. Mismatch penalty: 1.00 Gap penalty: 1.00 Gap size penalty: 0.33, and Linkage penalty: 30.0.

[0020] A "gene" refers to a polynucleotide containing at least one open reading frame capable of encoding a specific protein after transcription and translation.

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

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

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

[0024] The term "recombinant" applied to polynucleotides means that the polynucleotide is the product of various combinations of procedures, including cloning, restriction, or ligation steps, and other procedures that result in a construct different from naturally occurring polynucleotides. A recombinant virus is a viral particle containing recombinant polynucleotides. This term includes both replicas 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 molecular interactions that contribute to the functional control of polynucleotides, including replication, duplication, transcription, splicing, translation, or degradation. This control can affect the frequency, speed, or specificity of a process and can be either reinforcing or inhibitory. Regulatory elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, can bind to RNA polymerase and initiate transcription of a coding region, usually located downstream (3′ direction) of the promoter.

[0026] "Operatively bound" or "operatably bound" refers to the parallel arrangement of gene elements that are in a relationship that allows them to act in the expected manner. For example, if a promoter helps initiate transcription of a coding sequence, the promoter is operationally bound to the coding region. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0027] An "expression vector" is a vector containing a region encoding a polypeptide of interest, used to induce protein expression in an intended target cell. Expression vectors also include regulatory elements that bind responsively to the encoding region to promote protein expression within the target. A combination of a regulatory element and one or more genes to which the regulatory element responsively binds for expression is sometimes called an "expression cassette," and numerous expression cassettes are known and available in the art, or can be readily constructed from components available in the art.

[0028] "Heterogeneous" means that the organism originates from an organism that is genotypeically different from the rest of the organism being compared. For example, a polynucleotide introduced into a plasmid or vector from a different species using genetic engineering techniques is a heterogeneous polynucleotide. A promoter that is extracted from a native coding sequence and binds to a coding sequence that does not naturally find binding is a heterogeneous promoter. Therefore, for example, an rAAV containing a heterogeneous nucleic acid encoding a heterogeneous gene product is an rAAV containing a nucleic acid not normally found in naturally occurring wild-type AAV, and the heterogeneous gene product it encodes is a gene product not normally encoded by naturally occurring wild-type AAV. As another example, a variant AAV capsid protein containing a heterogeneous peptide inserted into the GH loop of the capsid protein is a variant AAV capsid protein containing an insertion of a peptide not normally found in naturally occurring wild-type AAV.

[0029] The terms “genetic modification” and “genetic alteration” (and their grammatical variants) are used interchangeably herein and refer to the process by which a genetic element (e.g., polynucleotide) is introduced into a cell by means other than mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or an improved version of an element already present in the cell. Genetic modification can be brought about, for example, by transfection into a cell using a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contact with a polynucleotide-liposome complex. Genetic modification can also be brought about, for example, by transduction or infection of DNA or RNA with a virus or viral vector. Generally, the genetic element is introduced into a chromosome or minichromosome within the cell, but any modification that alters the phenotype and / or genotype of the cell and its progeny is also included in this term.

[0030] If, during in vitro extended culture of cells, the gene sequences are available to perform their function, the cells are said to have been “stable” modified, transduced, genetically modified, or transformed by the gene sequences. Generally, such cells are “genetically” modified (genetically modified) in the sense that the genetic modifications are introduced that are inherited by the offspring of the modified cells.

[0031] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. These terms also include modified amino acid polymers, e.g., disulfide bond formation, glycosylation, lipid addition, phosphorylation, or conjugate with labeling components. When discussed in the context of delivering gene products to mammalian subjects, polypeptides and compositions for polypeptides, such as anti-angiogenic polypeptides and neuroprotective polypeptides, refer to the respective intact polypeptide, or any fragment or genetically modified derivative thereof, that retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in gene product delivery to mammalian subjects (which may be called “transgenes” to be delivered to recipient cells) include polynucleotides encoding intact polypeptides or any fragment or genetically modified derivative having the desired biochemical function.

[0032] "Isolated" plasmids, nucleic acids, vectors, viruses, virions, host cells, or other substances refer to preparations of a substance that lack at least some of the other components that may be present together with the substance or similar substance when it is naturally occurring or first prepared. Thus, for example, an isolated substance can be prepared by using a purification technique to concentrate the substance from a raw material mixture. Concentration can be measured on an absolute scale, for example, on weight per unit volume of solution, or it can be measured against any second potential interfering substances present in the raw material mixture. Increasing the concentration in embodiments of the present invention means being even more isolated. Isolated plasmids, nucleic acids, vectors, viruses, host cells, or other substances are purified in some embodiments to, for example, a purity of about 80% to about 90%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, or higher.

[0033] As used herein, terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures a disease or any adverse effects that may result from it. As used herein, “treatment” encompasses any disease treatment in mammals, particularly humans, and includes (a) preventing the onset of a disease in subjects who are predisposed to the disease or at risk of developing the disease but have not yet been diagnosed with the disease, (b) inhibiting the disease, i.e., suppressing its onset, and (c) alleviating the disease, i.e., causing a regression of the disease.

[0034] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein and are not limited to include mammals, including humans and non-human primates (including monkeys and humans), mammals used for sport (e.g., horses, camels, etc.), mammals kept as livestock (e.g., sheep, goats, dairy cows, etc.), mammals kept as pets (e.g., dogs, cats, etc.), and rodents (e.g., mice, rats, etc.). In some cases, the individual is a human.

[0035] Before further description of the present invention, it should be understood that the invention is not limited to the specific embodiments described and is naturally subject to change. Furthermore, it should be understood that the terms used herein are merely for describing specific embodiments and are not intended to be restrictive, as the scope of the invention is limited solely by the appended claims.

[0036] Where a range of values ​​is given, it should be understood that each value within that range (up to one-tenth of the lower limit, unless otherwise explicitly indicated by the context) and the values ​​described or between that range are included in the invention. The upper and lower limits in these smaller ranges can be included independently within those smaller ranges, and these are also included in the invention and subject to any specifically excluded limitations in the described range. If the described range includes one or both of the upper and lower limits, the range excluding one or both of these included upper and lower limits is also included in the invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used when carrying out or testing the present invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference to disclose and explain methods and / or materials related to citations of those publications.

[0038] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include multiple referents unless otherwise explicitly stated by context. For example, “one rAAV virion” includes multiple such virions, and “the capsid protein” includes one or more variant capsid proteins and their equivalents known to those skilled in the art. Furthermore, it should be noted that claims may be constructed to exclude any optional element. Therefore, this description is intended to serve as a precedent for the use of exclusive terms such as “solely,” “only,” or “negative” limitations in relation to the enumeration of elements of a claim.

[0039] It should be understood that certain features of the Invention, described in the context of separate embodiments for clarity, may be provided in combination in a single embodiment. Conversely, various features of the Invention, described in the context of a single embodiment for brevity, may be provided separately or in any preferred partial combination. All combinations in embodiments relating to the Invention are expressly encompassed by the Invention, and all such combinations are disclosed herein as if every combination were individually and explicitly disclosed. In addition, all partial combinations in various embodiments and their elements are also expressly encompassed by the Invention, and all such partial combinations are disclosed herein as if every such combination were individually and explicitly disclosed herein.

[0040] The publications discussed herein are provided simply because they were disclosed prior to the filing date of this application. Nothing in this specification should be construed as an acknowledgment that the present invention has no prior rights to such publications by prior invention. Furthermore, the publication dates indicated may differ from the actual publication dates, which may need to be verified individually.

[0041] This disclosure provides recombinant adeno-associated virus (AAV) virions having a modified capsid protein, exhibiting a higher ability to cross the barrier between intravitreous fluid and retinal cells compared to wild-type AAV, and thus exhibiting higher infectivity to retinal cells, and also providing recombinant AAV (rAAV) virions containing heterologous nucleic acids. This disclosure also provides a method for delivering gene products to retinal cells in an organism. Furthermore, this disclosure also provides a method for modifying target nucleic acids present in retinal cells.

[0042] This disclosure provides recombinant adeno-associated virus (AAV) virions having a modified capsid protein, exhibiting higher infectivity to retinal cells compared to wild-type AAV, and containing heterologous nucleic acids, recombinant AAV (rAAV) virions. rAAV virions exhibit increased ability to cross the barrier between intravitreous fluid and retinal cells. rAAV virions exhibit higher infectivity to retinal cells compared to the infectivity of wild-type AAV to corresponding retinal cells. Retinal cells may be photoreceptor cells (e.g., rods, cones), retinal ganglion cells (RGCs), Müller cells (Müller glial cells), astrocytes (e.g., retinal astrocytes), bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelium (RPE) cells. This disclosure further provides methods for delivering gene products to retinal cells in an individual and methods for treating eye diseases. This disclosure provides an rAAV virion having a modified capsid protein, which exhibits at least a five-fold increase in localization to one or more of the inner granular layer, outer granular layer, photoreceptor layer, ganglion layer, and retinal pigment epithelium compared to the degree of localization to the inner granular layer, outer granular layer, photoreceptor layer, ganglion layer, or retinal pigment epithelium by an AAV virion containing the corresponding parent AAV capsid protein, and which contains heterologous nucleic acids.

[0043] Variant AAV capsid polypeptide This disclosure provides variant AAV capsid proteins. As described above, the variant AAV capsid proteins of this disclosure are modified compared to wild-type or other reference AAV capsid proteins. Modifications include insertions and swaps (e.g., replacing adjacent sequences of amino acids with different adjacent sequences of amino acids).

[0044] In some cases, the variant AAV capsid proteins of this disclosure involve the insertion of a heterologous peptide of 5 to 20 amino acids in length at an insertion site within a surface-accessible (e.g., solvent-accessible) portion of the parent AAV capsid protein, thereby conferring increased infectivity of retinal cells compared to the infectivity of retinal cells by an AAV virion containing the corresponding parent AAV capsid protein, when present in an AAV virion, particularly when the AAV virion is injected intravitreously. Thus, the variant AAV capsid proteins of this disclosure, when present in an AAV virion, confer increased ability to cross the barrier between the intravitreous fluid ("vitreous fluid") and retinal cells in the AAV virion. Such barriers include, for example, the internal limiting membrane (ILM), the extracellular matrix of the retina, the cell membrane of the retinal cells themselves, the inner granular layer, the outer granular 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. “Insertions of approximately 5 to 20 amino acids” are also referred to herein as “peptide insertions” (e.g., xenopeptide insertions). “Corresponding parent AAV capsid protein” refers to an AAV capsid protein of the same AAV serotype without xenopeptide insertions. In some cases, variant AAV capsids contain a single xenopeptide insertion 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).

[0045] Modifications in AAV capsids may also involve swapping (e.g., replacing adjacent sequences of amino acids with heterologous peptides). Therefore, substitution means inserting a heterologous peptide in place of adjacent sequences of amino acids. In some cases, the variant AAV capsid proteins of this disclosure involve replacing adjacent sequences of amino acids with heterologous peptides of 5 to 20 amino acids in a site within a surface-accessible (e.g., solvent-accessible) portion of the parent AAV capsid protein, thereby conferring increased infectivity of retinal cells compared to the infectivity of retinal cells by an AAV virion containing the corresponding parent AAV capsid protein, particularly when the AAV virion is injected intravitreously. Thus, the variant AAV capsid proteins of this disclosure, when present within an AAV virion, confer increased ability of the AAV virion to cross the barrier between intravitreous fluid ("vitreous fluid") and retinal cells. Examples of such barriers include the ILM, the extracellular matrix of the retina, the cell membrane of the retinal cells themselves, the inner granular layer, the outer granular 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 20 amino acids" is also referred to herein as a "peptide swap" (e.g., replacing a sequence of adjacent amino acids with a heterologous peptide). A "corresponding parent AAV capsid protein" refers to an AAV capsid protein of the same AAV serotype without heterologous peptides. In some cases, a variant AAV capsid contains a single heterologous peptide substitution 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).

[0046] For the purposes of the following explanation, "insertion" refers to both the insertion of a heterologous peptide that does not involve the substitution of a sequence of adjacent amino acids, and the insertion of a heterologous peptide that replaces a sequence of adjacent amino acids.

[0047] The insertion site is located within the GH loop or loop IV of the AAV capsid protein, for example, in the solvent-accessible portion of the GH loop or loop IV of the AAV capsid protein. For more information on the GH loop / loop IV of the AAV capsid protein, see, for example, van Vliet et al. (2006) Mol.Ther.14:809, Padron et al. (2005) J.Virol.79:5047, and Shen et al. (2007) Mol.Ther.15:1955. For example, the insertion site may be within amino acids 411–650 of the AAV capsid protein, as shown in Figures 6A–6C. For example, the insertion site may be within amino acids 570-611 of AAV2, 571-612 of AAV1, 560-601 of AAV5, 571-612 of AAV6, 572-613 of AAV7, 573-614 of AAV8, 571-612 of AAV9, or 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 within 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 within 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 within a different serotype of AAV. In some cases, the insertion site is between amino acids 584 and 585 of the AAV2 capsid protein, or a corresponding insertion site within a different serotype of AAV. In some cases, the insertion site is between amino acids 590 and 591 of the AAV2 capsid protein, or a corresponding insertion site within a different serotype of AAV. In some cases, the insertion site is between amino acids 584 and 585 of the AAV4 capsid protein, or a corresponding insertion site within a different serotype of AAV. In some cases, the insertion site is between amino acids 575 and 576 of the AAV5 capsid protein, or a corresponding insertion site within a different serotype of AAV.In some cases, the substitution site is between amino acids 584 and 598 of the AAV2 capsid protein, or a corresponding site within AAV of a different serotype.

[0048] In some cases, heterologous peptides of approximately 5 to 20 amino acids in length (e.g., 5-7, 7-10, 10-12, 12-15, or 15-20 amino acids) are inserted into insertion sites within the GH loop or loop IV of the capsid protein in contrast to the corresponding parent AAV capsid protein. For example, the insertion site may be between amino acids 587 and 588 of AAV2, or between amino acids 588 and 589 of AAV2, or at the corresponding position on the capsid subunit of another AAV serotype. Note that the insertion site 587 / 588 is based on the AAV2 capsid protein. Heterologous peptides of approximately 5 to 20 amino acids in length (e.g., 5-7, 7-10, 10-12, 12-15, or 15-20 amino acids) may also be inserted into corresponding sites within AAV serotypes other than AAV2 (e.g., AAV8, AAV9, etc.). Those skilled in the art can determine, by comparing the amino acid sequences of various AAV serotype capsid proteins, which insertion site corresponds to amino acids 587-588 of AAV2 in any given AAV serotype capsid protein. Figure 5 shows the sequences corresponding to amino acids 570-611 (see Figure 4) of the AAV2 capsid protein VP1 in various AAV serotypes. For example, see GenBank accession number NP_049542 for AAV1, NP_044927 for AAV4, AAD13756 for AAV5, AAB95459 for AAV6, YP_077178 for AAV7, YP_077180 for AAV8, AAS99264 for AAV9, AAT46337 for AAV10, and AAO88208 for AAVrh10. For ancestral AAV capsids, see, for example, Santiago-Ortiz et al. (2015) Gene Ther. 22:934.

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

[0050] In some cases, the subject capsid protein contains 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 with respect to the amino acid sequences shown in Figures 6A-6C, and having an insertion of a heterologous peptide of about 5 to about 20 amino acids (e.g., 5-7, 7-10, 10-12, 12-15, or 15-20 amino acids).

[0051] In some cases, the variant AAV capsid proteins of this disclosure include substitutions (or replacements) of consecutive amino acid segments (or sequences) within a surface-accessible (e.g., solvent-accessible) portion of the parent AAV capsid, thereby conferring increased infectivity of retinal cells compared to the infectivity of retinal cells by an AAV virion containing the corresponding parent AAV capsid protein, when present in an AAV virion, particularly when the AAV virion is injected into the vitreous humor. Thus, the subjective variant AAV capsid proteins including sequence substitutions confer increased ability to cross barriers between the vitreous humor and retinal cells in the AAV virion when present in an AAV virion. Such barriers include, for example, the internal limiting membrane, the extracellular matrix of the retina, and the cell membrane of the retinal cells themselves. "Substitutions of approximately 5 to 25 consecutive amino acids" are also referred to herein as "loop swaps" (i.e., heterologous peptide substitutions). In such cases, the "corresponding parental AAV capsid protein" refers to an AAV capsid protein of the same AAV serotype without a thematic loop swap. In some cases, variant AAV capsids contain heterologous peptide substitutions of adjacent 5-amino acid to adjacent 25-amino acid lengths, for example, 5-9, 9-11, 10-15, 15-20, or 20-25 amino acid lengths.

[0052] In some cases, heterologous peptides of approximately 5 to 25 amino acids in length (e.g., 5-9, 9-10, 10-15, 15-20, or 20-25 amino acids) are substituted for a significant number of consecutive amino acids in the corresponding parent AAV capsid protein. In some embodiments, the substitution begins around amino acid 588 of AAV2 or at the corresponding position in the capsid subunit of another AAV serotype and ends around amino acid 598 of AAV2 or at the corresponding position in the capsid subunit of another AAV serotype. Note that residues 588-598 are based on the AAV2 VP1 capsid protein. Heterologous peptides of approximately 5 to 25 amino acids in length may also be substituted for corresponding sites in AAV serotypes other than AAV2 (e.g., AAV8, AAV9, etc.). Those skilled in the art can determine, by comparing the amino acid sequences of various AAV serotype capsid proteins, which sites in any given AAV serotype capsid protein correspond to amino acids 588-598 of AAV2. Figure 5 shows the amino acid residues corresponding to amino acids 588-598 (see Figure 4) of the AAV2 capsid protein VP1 in various AAV serotypes. For example, see GenBank accession number NP_049542 for AAV1, NP_044927 for AAV4, AAD13756 for AAV5, AAB95459 for AAV6, YP_077178 for AAV7, YP_077180 for AAV8, AAS99264 for AAV9, AAT46337 for AAV10, and AAO88208 for AAVrh10.

[0053] In some cases, heterologous peptides of approximately 5 to 25 amino acids in length (e.g., 5-9, 9-10, 10-15, 15-20, or 20-25 amino acids) are substituted for a significant number of consecutive amino acids in the corresponding parent AAV capsid protein. In some embodiments, the substitution begins around amino acid 585 of AAV2 or at the corresponding position in the capsid subunit of another AAV serotype and ends around amino acid 598 of AAV2 or at the corresponding position in the capsid subunit of another AAV serotype. Note that residues 585-598 are based on the AAV2 VP1 capsid protein. Heterologous peptides of approximately 5 to 25 amino acids in length may also be substituted for corresponding sites in AAV serotypes other than AAV2 (e.g., AAV8, AAV9, etc.). Those skilled in the art can determine, by comparing the amino acid sequences of various AAV serotype capsid proteins, which sites in any given AAV serotype capsid protein correspond to amino acids 585-598 of AAV2. Figure 5 shows the amino acid residues corresponding to amino acids 585-598 (see Figure 4) of the AAV2 capsid protein VP1 in various AAV serotypes. For example, see GenBank accession number NP_049542 for AAV1, NP_044927 for AAV4, AAD13756 for AAV5, AAB95459 for AAV6, YP_077178 for AAV7, YP_077180 for AAV8, AAS99264 for AAV9, AAT46337 for AAV10, and AAO88208 for AAVrh10.

[0054] Insertion / replacement peptides As described above, heterologous peptides with a length of approximately 5 to 20 amino acids are inserted into the GH loop of the AAV capsid or replace a significant number of consecutive amino acids within the GH loop of the AAV capsid. For brevity, the term “inserted peptide” will be used below to describe both peptides inserted into the parent AAV capsid and peptides that replace adjacent amino acid segments within the GH loop of the AAV capsid. In some cases, the inserted peptide has a length of 5 to 20 amino acids. In some cases, the inserted peptide has a length of 7 to 15 amino acids. In some cases, the inserted peptide has a length of 9 to 15 amino acids. In some cases, the inserted peptide has a length of 9 to 12 amino acids. The inserted peptide has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some cases, the inserted peptide has a length of 7 amino acids. In some cases, the inserted peptide has a length of 8 amino acids. In some cases, the inserted peptide has a length of 9 amino acids. In some cases, the inserted peptide has a length of 10 amino acids. In some cases, the inserted peptide has a length of 11 amino acids. In some cases, the inserted peptide has a length of 12 amino acids. In some cases, the inserted peptide has a length of 13 amino acids. In some cases, the inserted peptide has a length of 14 amino acids. In some cases, the inserted peptide has a length of 15 amino acids.

[0055] In some cases, the peptide insert is the peptide of formula I:LA(L / N)(I / Q)(Q / E)(D / H)(S / V)(M / K)(R / N)A (Sequence ID 136).

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

[0057] In some cases, the peptide insert is the peptide of formula II: TX1X2X3X4X5X6X7X8GLX9 (Sequence ID 137). In the formula, 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] Examples of peptide inserts in formula II, though not limited to them, include (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) TGGGTTMNGLS (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 the peptide of formula III: TGX1X2X3X4X5X6X7GLS (Sequence ID 138). In the formula, X1 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] Examples of peptide inserts in formula III include, but are not limited to, (2) TGEVDLAGGGLS (SEQ ID NO: 7), (4) TGGHDSSLDGLS (SEQ ID NO: 9), (5) TGGGTTMNGLS (SEQ ID NO: 98), (6) TGGHGSAPDGLS (SEQ ID NO: 99), (8) TGASYLDNSGLS (SEQ ID NO: 101), (10) TGVMHSQASGLS (SEQ ID NO: 21), (11) TGGDGSPAAPGLS (SEQ ID NO: 22), and (12) TGSDMAHGTGLS (SEQ ID NO: 23).

[0061] In some cases, the peptide insert is the peptide of formula IV: X1GX2X3X4X5X6X7X8GLSPX9TX 10 X 11 (Sequence number 139). In the formula, 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, X 10 However, it is G or W, and X 11 However, it is either T or A.

[0062] Examples of the peptide insert 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). In the formula, X1 is L, S, A, or G, X2 is A, G, or P, X3 is D, T, or G, and X4 is H, R, or T.

[0064] Examples of the peptide insert 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). In the formula, 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] Examples of peptides in 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 peptide inserts described above can replace the same number of adjacent amino acids in the GH loop of the AAV capsid polypeptide. For example, in some cases, the peptide of formula VI: LQX1X2X3RX4X5X6X7X8X9VNX 10 Q (Sequence 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, X 10However, (F, G, T, or C) replaces a sequence of 5 to 20 adjacent amino acids within the GH loop of the AAV capsid polypeptide. In other words, in some cases, the “inserted peptide” replaces an endogenous peptide (e.g., a sequence of 5 to 20 adjacent amino acids) present within the GH loop of the AAV capsid polypeptide, resulting in a variant AAV capsid containing a heterologous peptide within the GH loop. In some cases, the “inserted peptide” replaces an endogenous sequence of adjacent amino acids of the same length as the inserted peptide. Therefore, for example, if the “inserted peptide” is 16 amino acids long, in some cases, a sequence of 16 endogenous adjacent amino acids is replaced by this inserted peptide.

[0068] Examples of peptides in 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 that replaces the endogenous amino acid sequence in 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 that replaces the endogenous amino acid sequence in the GH loop of the AAV capsid is (19)LQRGNRPVTTADVNTQ (SEQ ID NO: 30). In some cases, the peptide that replaces the intrinsic amino acid sequence within the GH loop of the AAV capsid is (20)LQKADRQPGVVVVNCQ (SEQ ID NO: 31).

[0069] In some cases, one peptide insert of any one of formulas I to VI further includes 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 includes Thr-Gly-[one peptide of any one of formulas I to VI]-Gly-Leu-Ser (SEQ ID NO: 142). As another example, in some cases, the peptide insert includes Leu-Ala-[one peptide of any one of formulas I to VI]-Ala (SEQ ID NO: 143). As yet another example, in some cases, the peptide insert includes Leu-Gln-[one peptide of any one of formulas I to VI]-Gln. In some cases, the peptide insert does not contain any linker amino acids.

[0070] In some embodiments, the subject rAAV virion capsid, in comparison to the corresponding parent AAV capsid protein, contains no other amino acid substitutions, insertions, or deletions other than the insertion of approximately 5 to 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) within the GH loop or loop IV. In other embodiments, the subject rAAV virion capsid includes insertions of approximately 5 to approximately 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) within the GH loop or loop IV compared to the corresponding parent AAV capsid protein, in addition to insertions, deletions, or substitutions of 1 to approximately 25 amino acids compared to the parent AAV capsid protein. For example, in some embodiments, the subject rAAV virion capsid includes insertions, deletions, or substitutions 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 compared to the parent AAV capsid protein, in addition to insertions 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) within the GH loop or loop IV compared to the corresponding parent AAV capsid protein. In a particular embodiment, the 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 peptide insertion site.

[0071] In some cases, the variant AAV capsid polypeptides of this disclosure do not contain one, two, three, or four of the amino acid substitutions: Y273F, Y444F, Y500F, and Y730F.

[0072] In some cases, the variant AAV capsid polypeptides of the present disclosure include, 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, the variant rAAV capsid polypeptide of the present disclosure is a chimeric capsid, for example, the capsid comprising a portion of the AAV capsid of a first AAV serotype and a portion of the AAV capsid of a second serotype, and including 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) within the GH loop or loop IV in comparison with the corresponding parental AAV capsid protein.

[0074] Recombinant AAV billion This disclosure provides recombinant AAV (rAAV) virions comprising i) a variant AAV capsid polypeptide of this disclosure and ii) a heterogeneous nucleic acid comprising a nucleotide sequence encoding a heterogeneous polypeptide (i.e., a non-AAV polypeptide).

[0075] In some cases, the rAAV virions of the present disclosure include 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 with respect to the amino acid sequence shown in Figure 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, 10, 11, or 12 amino acids) within the GH loop or loop IV in comparison with the corresponding parent AAV capsid protein. In some embodiments, the subject rAAV virion includes 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 with respect to the amino acid sequence shown in Figure 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, 10, 11, or 12 amino acids) between amino acids 587 and 588 in comparison with the amino acid sequence shown in Figure 4, or at the corresponding site in comparison with the corresponding parent AAV capsid protein.

[0076] In some cases, the rAAV virions of the present disclosure include 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 with respect to the amino acid sequence shown in Figure 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, 10, 11, or 12 amino acids) within the GH loop or loop IV in comparison with the corresponding parent AAV capsid protein. In some cases, the subject rAAV virion includes a capsid protein containing 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 with respect to the amino acid sequence shown in Figure 4, and an insertion of about 5 to 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 585 and 598 in comparison with the amino acid sequence shown in Figure 4, or at the corresponding site in comparison with the corresponding parent AAV capsid protein.

[0077] In some embodiments, the subject rAAV virion comprises a capsid protein containing a GH loop having 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 with respect to the amino acid sequence shown in Figure 5, and insertions 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 the amino acids shown in bold and underlined.

[0078] In some embodiments, the subject rAAV virion comprises a capsid protein containing 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 in comparison with another AAV genotype. In some cases, the corresponding insertion site is the site shown in bold text and underline in Figure 6B.

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

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

[0081] Whether a given rAAV virion increases the infectivity of retinal cells can be determined by evaluating the therapeutic effect of the therapeutic gene product encoded by the rAAV virion in retinal cells. Possible therapeutic effects include, for example, a) a decrease in the rate of visual function loss (e.g., visual field, visual acuity), b) an improvement in visual function (e.g., an improvement in visual field or visual acuity), c) a decrease in sensitivity to light (i.e., photophobia), and a decrease in nystagmus. For example, the rAAV virion of the Disclosure, comprising a) a variant capsid of the Disclosure including the peptide insert or peptide substitution described above, and b) a heterologous nucleotide sequence encoding a heterologous gene product, when administered intravitreously, produces a therapeutic effect in retinal cells that is at least 2 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 50 times, or more than 50 times greater than the therapeutic effect in retinal cells produced when a control rAAV virion comprising a) a control AAV capsid without peptide insert or peptide substitution and b) a heterologous nucleotide sequence encoding a therapeutic heterologous gene product is administered intravitreously. Tests of visual function are known in the art, and any such tests can be used to determine whether the rAAV virion of the Disclosure exhibits increased infectivity in retinal cells.

[0082] The rAAV virions of this disclosure exhibit an increased ability to cross the barrier between the intravitreous fluid and retinal cells by 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 compared to the ability of control AAV virions containing the corresponding parental AAV capsid protein (i.e., AAV capsid protein without insertion or substitution peptides).

[0083] In some cases, when the subject rAAV virion is administered via intravitreous injection, it exhibits at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased infectivity of retinal cells compared to when an AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreous injection.

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

[0085] In some embodiments, when the subject rAAV virion is administered via intravitreous injection, it exhibits at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased infectivity of photoreceptor cells compared to when an AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreous injection.

[0086] In some embodiments, the subjective rAAV virion exhibits an increase in RGC infectivity of 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 compared to the infectivity of RGC by an AAV virion containing the corresponding parental AAV capsid protein.

[0087] In some embodiments, when the subject rAAV virion is administered via intravitreal injection, it exhibits an increase in RGC infectivity of 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 compared to the infectivity of RGC by the AAV virion when the AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreal injection.

[0088] In some embodiments, the subjective rAAV virion exhibits an increase in the infectivity of RPE cells by 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 compared to the infectivity of RPE cells by an AAV virion containing the corresponding parental AAV capsid protein.

[0089] In some embodiments, when the subject rAAV virion is administered via intravitreous injection, it exhibits at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased infectivity of RPE cells by AAV virion cells compared to when an AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreous injection.

[0090] In some embodiments, the subjective rAAV virion exhibits an increase in Müller cell infectivity of 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 compared to the infectivity of Müller cells by an AAV virion containing the corresponding parental AAV capsid protein.

[0091] In some embodiments, when the subject rAAV virion is administered via intravitreous injection, it exhibits 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 increase in the infectivity of Müller cells by AAV virion cells compared to when an AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreous injection.

[0092] In some embodiments, the subjective rAAV virion exhibits an increase in bipolar cell infectivity of 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 compared to the infectivity of bipolar cells by an AAV virion containing the corresponding parental AAV capsid protein.

[0093] In some embodiments, when the subject rAAV virion is administered via intravitreous injection, it exhibits at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased bipolar cell infectivity compared to when an AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreous injection.

[0094] In some embodiments, the subjective rAAV virion exhibits an increase in amacrine cell infectivity of 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 compared to the infectivity of amacrine cells by an AAV virion containing the corresponding parental AAV capsid protein.

[0095] In some embodiments, when the subject rAAV virion is administered via intravitreous injection, it exhibits an increase in amacrine cell infectivity by 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 compared to the infectivity of amacrine cells by AAV virion cells when an AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreous injection.

[0096] In some embodiments, the subjective rAAV virion exhibits an increase in horizontal cell infectivity of 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 compared to the horizontal cell infectivity of an AAV virion containing the corresponding parental AAV capsid protein.

[0097] In some embodiments, when the subject rAAV virion is administered via intravitreous injection, it exhibits an increase in horizontal cell infectivity by 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 compared to the horizontal cell infectivity by AAV virion cells when an AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreous injection.

[0098] In some embodiments, the subjective rAAV virion exhibits an increase in the infectivity of retinal astrocytes by 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 compared to the infectivity of retinal astrocytes by an AAV virion containing the corresponding parental AAV capsid protein.

[0099] In some embodiments, when the subject rAAV virion is administered via intravitreous injection, it exhibits an increase in retinal astrocellular infectivity of 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 compared to the infectivity of retinal astrocellular cells by AAV virion when an AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreous injection.

[0100] In some cases, subjective rAAV virions exhibit at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased ability to traverse the retinal extracellular matrix (ECM) compared to AAV virions containing the corresponding parent AAV capsid protein.

[0101] In some cases, subject rAAV virions, when administered via intravitreal injection, exhibit at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased ability to traverse the extracellular matrix (ECM) of the retina when AAV virions containing the corresponding parent AAV capsid protein are administered via intravitreal injection.

[0102] In some cases, subjective rAAV virions exhibit an increased ability to cross the internal limiting membrane (ILM) by 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 compared to the ability to cross the ILM in AAV virions containing the corresponding parent AAV capsid protein.

[0103] In some cases, subjective rAAV virions, when administered via intravitreal injection, exhibit at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased ILM traversal ability compared to AAV virions containing the corresponding parent AAV capsid protein when administered via intravitreal injection.

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

[0105] In some cases, subjective rAAV virions exhibit increased localization to one or more of the inner granular layer, outer granular layer, photoreceptor layer, ganglion layer, and retinal pigment epithelium by 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 compared to the degree of localization to one or more of the inner granular layer, outer granular layer, photoreceptor layer, ganglion layer, and retinal pigment epithelium by AAV virions containing the corresponding parental AAV capsid protein.

[0106] In some cases, subjective rAAV virions, when injected intravitreously, exhibit at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased localization beyond the ILM by AAV virions containing the corresponding parental AAV capsid protein injected intravitreously. For example, in some cases, subjective rAAV virions, when injected intravitreously, exhibit at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased localization to the retinal pigment epithelium (RPE) layer by control AAV virions containing the corresponding parental AAV capsid protein injected intravitreously. As another example, in some cases, subjective rAAV virions, when injected intravitreously, exhibit at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased localization to the photoreceptor (PR) layer compared to the localization to the PR layer by control AAV virions containing the corresponding parental AAV capsid protein injected intravitreously. As another example, in some cases, subjective rAAV virions, when injected intravitreously, exhibit at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased localization to the inner granular layer compared to the degree of localization to the inner granular layer by AAV virions containing the corresponding parental AAV capsid protein injected intravitreously. As another example, in some cases, subjective rAAV virions, when injected intravitreously, exhibit at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increased localization to the outer granular layer compared to the degree of localization to the outer granular layer by control AAV virions containing the corresponding parent AAV capsid protein injected intravitreously.As another example, in some cases, subjective rAAV virions, when injected intravitreously, show increased localization to the ganglion cell layer by 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 compared to localization to the ganglion cell layer by control AAV virions containing the corresponding parent AAV capsid protein injected intravitreously.

[0107] In some embodiments, the subject rAAV virion selectively infects retinal cells, for example, the subject rAAV virion infects retinal cells with 10, 15, 20, 25, 50, or more than 50 times the specificity compared to non-retinal cells (e.g., cells outside the eye).

[0108] In some embodiments, the subject rAAV virion selectively infects photoreceptor cells, for example, the subject rAAV virion infects photoreceptor cells with 10-, 15-, 20-, 25-, 50-, or more than 50-fold specificity compared to non-photoreceptor cells present in the eye (e.g., retinal ganglion cells, Müller cells, etc.).

[0109] In some embodiments, when the subject rAAV virion is administered via intravitreous injection, it exhibits at least a 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold increase in the infectivity of photoreceptor cells by AAV virion cells compared to when an AAV virion containing the corresponding parent AAV capsid protein is administered via intravitreous injection.

[0110] gene product The rAAV virions of this disclosure comprise heterogeneous nucleic acids comprising nucleotide sequences encoding one or more gene products (one or more heterogeneous 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 this disclosure comprise heterogeneous nucleotide sequences encoding both heterogeneous nucleic acid gene products and heterogeneous polypeptide gene products. If the gene product is RNA, in some cases, the RNA gene product encodes a polypeptide. If the gene product is RNA, in some cases, the RNA gene product does not encode a polypeptide. In some cases, the rAAV virions of this disclosure comprise a single heterogeneous nucleic acid comprising nucleotide sequences encoding one heterogeneous gene product. In some cases, the rAAV virions of this disclosure comprise a single heterogeneous nucleic acid comprising nucleotide sequences encoding two heterogeneous gene products. If a single heterogeneous nucleic acid encodes two heterogeneous gene products, in some cases, the nucleotide sequences encoding the two heterogeneous gene products are ligibly conjugated to the same promoter. When a single heteronucleotide encodes two heterogeneous gene products, in some cases the nucleotide sequences encoding the two heterogeneous gene products are configurably bound to two different promoters. In some cases, the rAAV virion of this disclosure comprises a single heteronucleotide containing a nucleotide sequence encoding three heterogeneous gene products. When a single heteronucleotide encodes three heterogeneous gene products, in some cases the nucleotide sequences encoding the three heterogeneous gene products are configurably bound to the same promoter. When a single heteronucleotide encodes three heterogeneous gene products, in some cases the nucleotide sequences encoding the three heterogeneous gene products are configurably bound to two or three different promoters. In some cases, the rAAV virion of this disclosure comprises two heterogeneous nucleic acids, each containing a nucleotide sequence encoding one heterogeneous gene product.

[0111] In some cases, the gene product is RNA encoding a polypeptide. In some cases, the gene product is 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 clinical benefit. In some embodiments, the gene product is a site-specific nuclease that results in site-specific knockdown of gene function. In some embodiments, the gene product is an RNA guide endonuclease that results in modification of a target nucleic acid. In some embodiments, the gene product is a guide RNA comprising i) an RNA guide endonuclease that results in modification of a target nucleic acid, and ii) a first segment that binds to a target sequence in the target nucleic acid and a second segment that binds to the RNA guide endonuclease. In some cases, the gene product is a first guide RNA comprising i) an RNA guide endonuclease that causes modification of the target nucleic acid, ii) a first guide RNA comprising a first segment that binds to a first target sequence in the target nucleic acid and a second segment that binds to the RNA guide endonuclease, and iii) a first guide RNA comprising a first segment that binds to a second target sequence in the target nucleic acid and a second segment that binds to the RNA guide endonuclease.

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

[0113] Furthermore, interfering RNAs may also be directed at angiogenic products, such as vascular endothelial growth factor (VEGF) (e.g., Cand5, see 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 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, as well as U.S. Patent Nos. 7,947,659 and 7,919,473.

[0114] Aptamer When the gene product is an aptamer, exemplary aptamers of interest include those for VEGF. See, for example, Ng et al. (2006) Nat. Rev. Drug Discovery 5:123 and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:18902. For example, a VEGF aptamer may contain the nucleotide sequence 5′-cgcaaucagugaaugcuuauacauccg-3′ (SEQ ID NO: 3). 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] Polypeptide 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-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), neurotrophin 4 (NT4), nerve growth factor (NGF), and neuroturin (NTN)), anti-angiogenic polypeptides (e.g., soluble VEGF receptors, VEGF-binding antibodies, VEGF-binding antibody fragments (e.g., single-chain anti-VEGF antibodies), endostatins, tamstatins, angiostatins, soluble Flt polypeptides (Lai et al. (2005) Mol.Ther.12:659)), and Fc fusion proteins containing soluble Flt polypeptides (e.g., Pechan et al. (2009) Gene Examples include pigment epithelial-derived factor (PEDF), soluble Tie-2 receptor, etc., tissue metalloproteinase inhibitor 3 (TIMP-3), photoresponsive opsins such as rhodopsin, and anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl, XIAP). Suitable polypeptides include, but are not limited to, glial neurotrophic factor (GDNF), fibroblast growth factor, fibroblast growth factor 2, neurotrien (NTN), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), neurotrophin 4 (NT4), brain-derived neurotrophic factor (BDNF, for example, a polypeptide containing an amino acid sequence having at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or 100% amino acid sequence identity with respect to a contiguous sequence of approximately 200 to 247 amino acids adjacent to the amino acid sequence shown in Figure 7B (SEQ ID NO: 11)), epidermal growth factor, rhodopsin, X-linked apoptosis inhibitors, and sonic hedgehog.

[0116] Suitable photoresponsive opsins include, for example, the photoresponsive opsins 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 photo-gated ion channel polypeptides. See, for example, Gaub et al. (2014) Proc. Natl. Acad. Sci. USA 111:E5574. For example, a suitable polypeptide is the photo-gated ion channel glutamate receptor (LiGluR). Expression of LiGluR in retinal ganglion cells and ON bipolar cells in the presence of photocatalytic compounds makes the cells photoresponsive. LiGluR includes the L439C substitution. See Caporale et al. (2011) Mol Ther. 19:1212-1219, Volgraf et al. (2006) Nat Chem Biol. 2:47-52, and Gorostiza et al. (2007) Proc Natl Acad Sci USA. 104:10865-10870. Examples of photocatalytic compounds include maleimide-azobenzene-glutamate 0 (MAG0), which has a peak efficiency at 460 nm. 460 ) are examples. MAG0 460 It has the following structure:

[0118]

change

[0119] Furthermore, suitable polypeptides include retinosin (for example, polypeptides containing amino acid sequences that have at least 90%, at least 95%, at least 98%, at least 99%, or 100% amino acid sequence identity with respect to a contiguous sequence of approximately 200 to 224 amino acids adjacent to the amino acid sequence shown in Figure 7A (Sequence ID 10)). Suitable polypeptides include, for example, Retinitis Pigmentosa GTPase Regulatory Agent (RPGR) Interacting Protein 1 (see, for example, GenBank accession numbers Q96KN7, Q9EPQ2, and Q9GLM3) (e.g., a polypeptide containing 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 sequence of about 1150 to 1200 amino acids, or about 1200 to 1286 amino acids, of the amino acid sequence shown in Figure 7F (SEQ ID NO: 15)), Peripherin 2 (Prph2) (e.g., GenBank accession number NP_000313 (e.g., a polypeptide containing 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 sequence of about 300 to 346 amino acids, of the amino acid sequence shown in Figure 7D (SEQ ID NO: 13)), and Travis et al. See al. (1991) Genomics 10:733), peripherins (e.g., polypeptides containing amino acid sequences having at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or 100% amino acid sequence identity to approximately 400 to 470 adjacent amino acids of the amino acid sequence (SEQ ID NO: 14) shown in Figure 7E), retinal pigment epithelium-specific protein (RPE65) (e.g., polypeptides containing amino acid sequences having at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or 100% amino acid sequence identity to approximately 200 to 247 adjacent amino acids of the amino acid sequence (SEQ ID NO: 12) shown in Figure 7C) (e.g., GenBank AAC39660, and Morimura et al. (1998) Proc.Natl.Examples include rod-derived cone survival factor (RdCVF) (e.g., polypeptides containing amino acid sequences with at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 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., polypeptides containing amino acid sequences with at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or 100% amino acid sequence identity to the amino acid sequence shown in Figure 7G), and retinitis pigmentosa GTPase regulatory substances (RPGR) (e.g., polypeptides containing amino acid sequences with at least approximately 90%, at least approximately 95%, at least approximately 98%, at least approximately 99%, or 100% amino acid sequence identity to the amino acid sequence shown in any one of Figures 7S to 7V). For example, in some cases, a suitable RPGR polypeptide contains 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 with the amino acid sequence shown in Figure 7S. Another example is that, in some cases, a suitable RPGR polypeptide contains 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 with the amino acid sequence shown in Figure 7T. For example, in some cases, a suitable RPGR polypeptide contains 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 with the amino acid sequence shown in Figure 7U. For example, in some cases, a suitable RPGR polypeptide contains 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 with the amino acid sequence shown in Figure 7V.

[0120] Other suitable polypeptides include CHM (Colloideremia (Rab Escort Protein 1 (REP1))), a polypeptide that causes colloideremia when defective or absent (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 causes Leber congenital amaurosis and retinitis pigmentosa when defective or absent (see, e.g., den Hollander et al. (1999) Nat.Genet.23:217 and GenBank accession number CAM23328). For example, a suitable REP1 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 with respect 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 with respect to the amino acid sequence shown in Figure 7K. As another example, a suitable PDE6β6 isoform 1 polypeptide may contain amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity with respect to the amino acid sequence shown in Figure 7L. As yet another example, a suitable PDE6β6 isoform 2 polypeptide may contain amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity with respect to the amino acid sequence shown in Figure 7M. As yet another example, a suitable PDE6β6 polyisoform peptide 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 with respect to the amino acid sequence shown in Figure 7N.

[0122] Furthermore, suitable polypeptides include those that, when defective or absent, result in monochromacy, such as the cone photoreceptor cGMP gate channel subunit alpha (CNGA3) (e.g., GenBank accession number NP_001289, and see Booij et al. (2011) Ophthalmology 118:160-167), the cone photoreceptor cGMP gate cation channel beta subunit (CNGB3) (e.g., see Kohl et al. (2005) Eur J Hum Genet. 13(3):302), guanine nucleotide-binding proteins (G proteins), alpha-transduction-active 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 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 with respect to the amino acid sequence shown in Figure 7O. As another example, a suitable CNGA3 (also known as ACHM2) isoform 2 polypeptide may contain amino acids having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100% amino acid sequence identity with respect to the amino acid sequence shown in Figure 7P.

[0124] As another example, a suitable CNGB3 (also known as ACHM3) 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 with respect to the amino acid sequence shown in Figure 7Q. As yet another example, GNAT2 (also known as ACHM4) 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 with respect 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 results in site-specific knockdown of gene function, for example, this endonuclease knocks out an allele associated with retinal disease. For example, if, in the wild type, the dominant allele encodes a defective copy of a gene that is a structural protein of the retina and / or contributes to normal retinal function, the site-specific endonuclease can target the defective allele and knock it out. In some cases, the site-specific endonuclease is an RNA-guided endonuclease.

[0126] Site-specific nucleases can be used not only to knock out defective alleles but also to stimulate homologous recombination with donor DNA encoding a functional copy of the protein encoded by the defective allele. Therefore, for example, the subject rAAV virion can be used to deliver together 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 retinosuxin, functional RPE65, functional peripherin, etc.). See, for example, Li et al. (2011) Nature 475:217. In some embodiments, the subject rAAV virion comprises a heterogeneous nucleotide sequence encoding a site-specific endonuclease and a heterogeneous nucleotide sequence encoding a functional copy of the defective allele that encodes a functional retinal protein. Examples of functional retinal proteins include retinosuxin, RPE65, retinitis pigmentosa GTPase regulatory substance (RGPR) interacting protein 1, peripherin, peripherin 2, and RdCVF.

[0127] Suitable site-specific endonucleases for use include, for example, zinc finger nucleases (ZFNs), meganucleases, and effector nucleases for transcription activators (TALENs), which are non-naturally occurring and modified to target specific genes. Such site-specific nucleases can be manipulated to cut at specific locations in the genome, and then non-homologous end joining can repair the damage by inserting or deleting several nucleotides. Such site-specific nucleases (also called "indels") then release proteins from the frame, effectively knocking out the gene. See, for example, U.S. Patent Publication 2011 / 0301073. Suitable site-specific endonucleases include manipulated meganucleases and remanufactured homing endonucleases. Suitable endonucleases include I-Tevl nucleases. Suitable meganucleases include I-Sce1 (see, for example, Bellaiche et al. (1999) Genetics 152:1037) and I-Cre1 (Heath et al. (1997) Nature Structural Biology 4:468).

[0128] RNA guide endonuclease In some cases, the gene product is an RNA guide endonuclease. In some cases, the gene product is RNA containing a nucleotide sequence encoding an RNA guide endonuclease. In some cases, the gene product is a guide RNA, e.g., a single guide RNA. In some cases, the gene product is 1) a guide RNA and 2) an RNA guide endonuclease. The guide RNA may include a) a protein-binding region that binds to the RNA guide endonuclease, and b) a region that binds to a target nucleic acid. RNA guide endonucleases are also referred to herein as “genome editing nucleases.”

[0129] Examples of suitable genome editing nucleases are CRISPR / Cas endonucleases (e.g., Class 2 CRISPR / Cas endonucleases such as Type II, Type V, or Type VI CRISPR / Cas endonucleases). Suitable genome editing nucleases are CRISPR / Cas endonucleases (e.g., Class 2 CRISPR / Cas endonucleases such as Type II, Type V, or Type VI CRISPR / Cas endonucleases). In some cases, the genome targeting composition contains a Class 2 CRISPR / Cas endonuclease. In some cases, the genome targeting composition contains a Class 2 Type II CRISPR / Cas endonuclease (e.g., Cas9 protein). In some cases, the genome targeting composition contains a Class 2 Type V CRISPR / Cas endonuclease (e.g., Cpf1 protein, C2c1 protein, or C2c3 protein). In some cases, the genome-targeted composition contains a class 2 type VI CRISPR / Cas endonuclease (e.g., C2c2 protein (also known as Cas13a protein)). CasX protein is also suitable for use. CasY protein is also suitable for use.

[0130] In some cases, genome editing nucleases are fusion proteins that are fused with heterologous polypeptides (also called "fusion partners"). In some cases, genome editing nucleases are fused with amino acid sequences that result in intracellular localization (fusion partners), i.e., the fusion partners are intracellular localization sequences (e.g., one or more nuclear localization signals (NLS) targeting the nucleus, two or more NLS, three or more NLS, 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 a target site within a target nucleic acid sequence (e.g., a chromosomal or extrachromosomal sequence, e.g., an episome sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by association with the protein-binding segment of the Cas9 guide RNA (e.g., it is stabilized at the target site). In some cases, a preferred Cas9 polypeptide contains 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 with Streptococcus pyogenes Cas9 shown in Figure 3A. In some cases, the Cas9 polypeptide used in the compositions or methods of this disclosure is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, the saCas9 polypeptide contains 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 with the amino acid sequence shown in Figure 3.

[0132] In some cases, a preferred 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 in the amino acid sequence shown in Figure 3A are substituted with alanine, for example. For example, an HF Cas9 polypeptide may have 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, and may include an amino acid sequence in which amino acids N497, R661, Q695, and Q926 are substituted with alanine, for example.

[0133] In some cases, suitable Cas9 polypeptides exhibit modified PAM specificity. See, for example, 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 the Cpf1 protein. In some cases, the Cpf1 protein contains 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 with the amino acid sequence shown in Figure 3C.

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

[0136] Enzymatically inactive RNA guide endonuclease RNA guide endonucleases with reduced enzymatic activity are also suitable for use. Such RNA guide endonucleases are called “dead” RNA guide endonucleases. For example, a Cas9 polypeptide containing certain amino acid substitutions that exhibits substantially no endonuclease activity but still binds to the target nucleic acid when complexed with a guide RNA is called “dead” Cas9 or “dCas9”. In some cases, “dead” Cas9 proteins have reduced ability to cleave both the complementary and non-complementary strands of double-stranded target nucleic acids. For example, “nuclease-deficient” Cas9 lacks a functional RuvC domain (i.e., does not cleave the non-complementary strand of double-stranded target DNA) and lacks a functional HNH domain (i.e., does not cleave the complementary strand of double-stranded target DNA). As a non-limiting example, in some cases, nuclease-deficient Cas9 proteins have mutations at amino acid positions corresponding to residues D10 and H840 of SEQ ID NO: 15 (or the corresponding residues in the Cas9 homolog), causing the polypeptide to have reduced ability to cleave both the complementary and non-complementary strands of the target nucleic acid (e.g., not cleave at all). Such Cas9 proteins have reduced ability to cleave the target nucleic acid (e.g., single-stranded or double-stranded target nucleic acid) but retain the ability to bind to the target nucleic acid. Cas9 proteins that cannot cleave the target nucleic acid (e.g., due to one or more mutations in the catalytic domains of the RuvC and HNH domains) are called "nuclease-deficient Cas9," "dead Cas9," or simply "dCas9." Other residues can also be mutated to achieve the above effect (e.g., by inactivating any one other nuclease moiety). As a non-limiting example, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or A987 (or the corresponding amino acids of the Cas9 homolog) of Streptococcus pyogenes Cas9 can be modified (i.e., substituted).In some cases, two or more of the following amino acids in Streptococcus pyogenes Cas9 (D10, E762, H840, N854, N863, and D986) are substituted (or the corresponding amino acids in the Cas9 homolog). In some cases, D10 and N863 (or the corresponding amino acids in the Cas9 homolog) of Streptococcus pyogenes Cas9 are substituted with ala. Mutations other than alanine substitution are also preferred.

[0137] In some cases, the genome editing endonuclease is an RNA guide endonuclease (and corresponding guide RNA) known as a Cas9 synergistic activation medium (Cas9-SAM). The RNA guide endonuclease (e.g., Cas9) in the Cas9-SAM system is a “dead” Cas9 fused to a transcriptional activation domain (preferred transcriptional activation domains include, for example, VP64, p65, MyoD1, HSF1, RTA, and SET7 / 9) or a transcriptional repression domain (preferred transcriptional repression domains include, for example, the KRAB domain, NuE domain, NcoR domain, SID domain, and SID4X domain). The guide RNA in the Cas9-SAM system contains a loop that binds to an adapter protein fused to a transcriptional activation domain (e.g., VP64, p65, MyoD1, HSF1, RTA, or SET7 / 9) or a transcriptional repression domain (e.g., the KRAB domain, NuE domain, NcoR domain, SID domain, or SID4X domain). For example, in some cases, the guide RNA is a single guide RNA containing an MS2 RNA aptamer inserted into one or two loops of the sgRNA, dCas9 is a fusion polypeptide containing dCas9 fused to VP64, and the adapter / functional protein is a fusion polypeptide containing i) MS2, ii) p65, and iii) HSF1. See, for example, U.S. Patent Publication 2016 / 0355797.

[0138] Furthermore, chimeric polypeptides containing a) a dead RNA guide endonuclease and b) heterogeneous fusion polypeptides are also suitable for use. Examples of suitable heterogeneous 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 Nucleic acids that bind to class 2 CRISPR / Cas endonucleases (e.g., Cas9 protein, type V or VI CRISPR / Cas protein, Cpf1 protein, etc.) and target the complex to a specific location within the target nucleic acid are referred to herein as “guide RNA” or “CRISPR / Cas guide nucleic acid” or “CRISPR / Cas guide RNA”. The guard RNA imparts target specificity to the complex (RNP complex) by including a targeting segment containing a guide sequence (also referred to herein as the targeting sequence), which is a nucleotide sequence complementary to the sequence of the target nucleic acid.

[0140] In some cases, the guide RNA comprises two separate nucleic acid molecules: an "activator" and a "targeter," and is referred to herein as "dual guide RNA," "double-molecule guide RNA," "two-molecule guide RNA," or "dgRNA." In some cases, the guide RNA is a single molecule (for example, for some class 2 CRISPR / Cas proteins, the corresponding guide RNA is a single molecule, and in some cases the activator and targeter are covalently bonded to each other, for example, via intervening nucleotides), and this guide RNA is referred to as "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 harmful mutation within a defective allele (e.g., a harmful mutation within a retinal cell target nucleic acid), the RNA-guided endonuclease / guide RNA complex can be used, for example, via homologous recombination repair (HDR), together with a donor nucleic acid containing a nucleotide sequence that corrects the harmful mutation (e.g., a donor nucleic acid containing a nucleotide sequence encoding a functional copy of the protein encoded by the defective allele).

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

[0143] In some cases, the gene product is i) an RNA guide endonuclease and ii) a single 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 guide endonuclease, and ii) two separate sgRNAs, which result in the 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 may be cleaved by the Cpf1 polypeptide to produce two or more guide RNAs.

[0146] This disclosure provides a method for modifying a target nucleic acid in retinal cells within an individual if it contains a harmful mutation, comprising administering the rAAV virion of this disclosure to the individual (e.g., by intraocular, intravitreal administration), wherein the rAAV virion comprises a heterogeneous nucleic acid including i) a nucleotide sequence encoding an RNA guide endonuclease (e.g., Cas9 endonuclease), ii) a nucleotide sequence encoding an sgRNA containing a nucleotide sequence complementary to the target nucleic acid, and iii) a nucleotide sequence encoding a donor DNA template containing a nucleotide sequence that corrects the harmful mutation. Administration of rAAV virion results in correction of the harmful mutation in the target nucleic acid by HDR.

[0147] This disclosure provides a method for modifying a target nucleic acid in retinal cells within an individual if it contains a harmful mutation, comprising administering the individual rAAV virion of this disclosure (e.g., by intraocular, intravitreal administration), wherein the rAAV virion comprises a heterogeneous nucleic acid including i) a nucleotide sequence encoding an RNA guide endonuclease (e.g., Cas9 endonuclease), ii) a nucleotide sequence encoding a first sgRNA containing a nucleotide sequence complementary to a first sequence in the target nucleic acid, and iii) a nucleotide sequence encoding a second sgRNA containing a nucleotide sequence complementary to a second sequence in the target nucleic acid. Administration of rAAV virion results in the excision of the harmful mutation in the target nucleic acid by NHEJ.

[0148] control array In some cases, the nucleotide sequence encoding the gene product of interest is adsorbed to a transcriptional regulatory element. For example, in some cases, the nucleotide sequence encoding the gene product of interest is adsorbed to a constitutive promoter. In other cases, the nucleotide sequence encoding the gene product of interest is adsorbed to an inductive promoter. In some cases, the nucleotide sequence encoding the gene product of interest is adsorbed to a tissue-specific or cell-type-specific regulatory element. For example, in some cases, the nucleotide sequence encoding the gene product of interest is adsorbed to a retinal cell-specific promoter. For example, in some cases, the nucleotide sequence encoding the gene product of interest is adsorbed to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), which is, for example, a regulatory element that confers selective expression of an adsorbed gene within a photoreceptor cell. Suitable photoreceptor-specific regulatory elements include, for example, rhodopsin promoters, rhodopsin kinase promoters (Young et al. (2003) Ophthalmol.Vis.Sci.44:4076), beta-phosphodiesterase gene promoters (Nicoud et al. (2007) J.Gene Med.9:1015), retinitis pigmentosa gene promoters (Nicoud et al. (2007) see above), photoreceptor-retinoid-binding protein (IRBP) gene enhancers (Nicoud et al. (2007) see above), and IRBP gene promoters (Yokoyama et al. (1992) Exp Eye Res.55:225).

[0149] Pharmaceutical composition This disclosure provides a) a subject rAAV virion as described below, and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer comprising 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] Examples of such excipients, carriers, diluents, and buffers include any pharmaceutically active substance that can be administered without excessive toxicity. Examples of pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline solution, glycerol, and ethanol. These may also include pharmaceutically acceptable salts, such as inorganic salts (e.g., hydrochlorides, hydrobroms, phosphates, sulfates, etc.) and organic salts (e.g., acetates, propions, malons, benzoates, etc.). In addition, auxiliary substances such as wetting agents and emulsifiers, and pH buffers may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and do not need to be discussed in detail herein. Examples of pharmaceutically acceptable excipients include A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins, and Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) HCAnsel et al., eds., 7 th This is adequately explained in various publications, including Lippincott, Williams, & Wilkins (ed.), and Handbook of Pharmaceutical Excipients (2000), AH Kibbe et al., eds., 3rd ed., American Pharmaceutical Assoc.

[0151] Method and procedure for delivering gene products to retinal cells This disclosure provides a method for delivering a gene product to retinal cells in an individual, comprising administering the individual the rAAV virion described above. The gene product may be a polypeptide or 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 retinal cells may result in the treatment of retinal diseases. Retinal cells may be photoreceptor cells, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, or retinal pigment epithelial cells. In some cases, the retinal cells are photoreceptor cells, e.g., rod or cone cells.

[0152] This disclosure provides a method for modifying a target nucleic acid in retinal cells, comprising contacting the retinal cells with 1) an rAAV virion of this disclosure comprising a heterogeneous nucleic acid containing a nucleotide sequence encoding an RNA guide endonuclease that binds to a guide RNA, and 2) a guide RNA. This disclosure provides a method for modifying a target nucleic acid in retinal cells, comprising contacting the retinal cells with an rAAV virion of this disclosure comprising i) an RNA guide endonuclease that binds to a guide RNA, and ii) a heterogeneous nucleic acid containing a nucleotide sequence encoding the guide RNA. In some cases, the method comprises contacting the retinal cells with a donor DNA template. In some cases, the RNA guide endonuclease is a Cas9 polypeptide. In some cases, the guide RNA is a single guide RNA.

[0153] This disclosure provides a method for treating an eye disease (e.g., a retinal disease), comprising administering an effective amount of the subject rAAV virion described above to an individual in need of such treatment. The subject rAAV virion can be administered via intraocular injection, for example, 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 or intranasal administration.

[0154] The "therapeutic effective dose" falls within a relatively wide range that can be determined through experiments and / or clinical trials. For example, for in vivo injection, i.e., direct injection into the eye, the therapeutic effective dose is approximately 10 6 ~about 10 15 For example, around 10 billion rAAV 8 ~about 10 12 This results in rAAV virions. For example, for in vivo injection, i.e., direct injection into the eye, the therapeutically effective dose is approximately 10 6 Viral genome (vg) ~ approximately 10 15 vg's rAAV is around 10 billion, for example, about 10 8 vg~about 10 12 The effective amount of rAAV virions delivered to cells for in vitro transduction is approximately 10 8 ~about 10 13 This amounts to approximately 10 rAAV virions. For example, in in vitro transduction, the effective amount of rAAV virions delivered to cells is approximately 10 8 ~about 10 13 This is equivalent to approximately 10 vg of rAAV virions. As another example, for in vitro transduction, the effective amount of rAAV virions delivered to cells is approximately 10 vg / cell to approximately 10 4 This corresponds to approximately vg / cell rAAV virions. Other effective drug doses can also be easily established by those skilled in the art through standard tests to establish dose-response curves.

[0155] In some embodiments, two or more doses (e.g., two, three, four, or more doses) may be used to achieve a desired gene expression level. In some cases, the two or more doses are administered at various intervals, for example, once a day, once a week, twice a month, once a month, once every three months, once every six months, once a year, etc. In some cases, the multiple doses are administered at intervals of one to two months, two to four months, four to eight months, eight to twelve months, one to two years, two to five years, or five years or more.

[0156] Eye diseases that can be treated using the subject method include, but are not limited to, acute macular neuroretinopathy, Behçet'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, cystic macular edema, diabetic macular edema, multifocal choroiditis, eye injuries affecting the location or position of the posterior eyeball, eye 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 ophthalmitis, Vogt-Koyanagi-Harada syndrome, and diffuse uveitis. These include diffusion, posterior eye conditions caused or affected by ocular laser treatment, posterior eye conditions caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorders, retinal vein branch occlusion, anterior ischemic optic neuropathy, non-retinopathy diabetic retinal dysfunction, retinal detachment, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease (yellow spotted fundus), hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital non-progressive night blindness, choroideremia, Valdey-Beetle syndrome, macular telangiectasia, Leber hereditary optic nerve atrophy, retinopathy of prematurity, color vision deficiencies (including monochromacy, type 1 dichromacy, type 2 trichromacy, and type 3 dichromacy), and Vietti crystalline dystrophy.

[0157] This disclosure provides a method for treating retinal diseases. The method generally involves administering the rAAV virion of this disclosure, or a composition comprising the rAAV virion of this disclosure, to the eye of an individual in need of treatment. Non-limiting methods for evaluating the treatment of retinal diseases include measuring functional changes, e.g., changes in visual acuity (e.g., BCVA), visual field measurements (e.g., perimetry), electrophysiological responses to light and dark (e.g., ERG, VEP), color vision measurements, and / or contrast sensitivity measurements, measuring changes in anatomical structure or health status using anatomical and / or photographic scales (e.g., OCT, fundus photography, and / or autofluorescence), and measuring eye movements (e.g., nystagmus, fixation preference, and stability).

[0158] For example, a person skilled in the art would likely be able to easily determine an effective dose of rAAV virion by testing its effect on one or more parameters (e.g., visual acuity, visual field, electrophysiological response to light and dark, color vision, contrast sensitivity, anatomical structure, retinal health and vascular structure, eye motility, fixation preference, and stability). In some cases, administration of an effective dose of the rAAV virion of this disclosure results in a reduction in the rate of loss of retinal function, anatomical integrity, or retinal health, e.g., a 2x, 3x, 4x, or 5x reduction in the rate of loss, or more, and thus a reduction in the rate of disease progression, e.g., a 10x reduction in the rate of loss, or more, and thus a reduction in the rate of disease progression. In some cases, administration of an effective dose of the rAAV virion of this disclosure results in increased retinal function, improved retinal anatomy or health, and / or stabilization of eye movements, e.g., two-fold, three-fold, four-fold, or five-fold or greater improvement in retinal function, retinal anatomy or health, and / or orbital stability, e.g., ten-fold improvement in retinal function, retinal anatomy or health, and / or orbital stability.

[0159] Nucleic acids and host cells This disclosure provides isolated nucleic acids comprising a nucleotide sequence encoding the subject variant adeno-associated virus (AAV) capsid protein described above, wherein the variant AAV capsid protein comprises an insertion of approximately 5 to 20 amino acids within the GH loop or loop IV compared to the corresponding parental AAV capsid protein, or comprises a substitution of approximately 5 to 20 amino acids with a heterologous peptide within the GH loop or loop IV compared to the corresponding parental AAV capsid protein, and the variant capsid protein, when present in an AAV virion, results in increased infectivity of retinal cells compared to the infectivity of retinal cells by an AAV virion containing the corresponding parental AAV capsid protein. The isolated nucleic acid of the subject may be an AAV vector, for example, a recombinant AAV vector.

[0160] Inserted peptide The variant AAV capsid protein encoded by the subject nucleic acid contains an insertion peptide approximately 5 to 20 amino acids long, which is inserted into the GH loop of the AAV capsid. The insertion peptide has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Preferred insertion peptides are as described above. As described above, a suitable insertion peptide is one of the peptides from formulas I to VI. Insertion of the insertion peptide into the parent AAV capsid replaces, in some cases, an endogenous sequence of approximately 5 to 20 amino acids within the GH loop or loop IV. Therefore, in some cases, the variant AAV capsid protein encoded by the subject nucleic acid includes substitutions of approximately 5 to 20 amino acids by heterologous peptides within the GH loop or loop IV in comparison with the corresponding parent AAV capsid protein, and suitable heterologous peptides include any one of the peptides of formulas I to VI as described above.

[0161] Thematic recombinant AAV vectors can be used to generate thematic recombinant AAV virions, as described above. Therefore, this disclosure provides recombinant AAV vectors that, when introduced into suitable cells, can result in the production of thematic recombinant AAV virions.

[0162] The present invention further provides host cells containing a subject nucleic acid, for example, isolated (genetically modified) host cells. The subject host cells may be isolated cells, for example, cells in an in vitro culture. The subject host cells are useful for the production of subject rAAV virions, as described later. When the subject host cells are used for the production of subject rAAV virions, they are called "packaging cells". In some embodiments, the subject host cells are stably genetically modified with the subject nucleic acid. In other embodiments, the subject host cells are transiently genetically modified with the subject nucleic acid.

[0163] The subject nucleic acid is introduced into host cells stably or transiently using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, and liposome-mediated transfection. For stable transformation, the subject nucleic acid generally also contains a selection marker, such as one of several well-known selection markers (e.g., neomycin resistance).

[0164] Theme host cells are generated by introducing theme nucleic acids into various cells, such as 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, and CHO cells. Non-limiting examples of suitable host cells include, for example, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC No. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human fetal kidney (HEK) cells (ATCC No. CRL1573), and HLHepG2 cells. Furthermore, subject host cells can also be created by infecting insect cells, such as AAV-producing Sf9 cells, with baculoviruses (see, for example, U.S. Patent No. 7,271,002 and U.S. Patent Application No. 12 / 297,958).

[0165] In some embodiments, the subject gene-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, the subject host cell further comprises an rAAV vector. rAAV virions can be generated using the subject host cell. Methods for generating rAAV virions are described, for example, in U.S. Patent Publication 2005 / 0053922 and U.S. Patent Publication 2009 / 0202490.

[0166] Examples of non-limiting aspects of this disclosure The aspects (including embodiments) of the subject matter of the present invention described above may be useful individually or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the present disclosure, numbered 1 to 63, are shown below. As will be apparent to those skilled in the art, each of the individually numbered aspects may be used individually or in combination with any of the individually numbered aspects that precede or follow. This is intended to support all such combinations of aspects and is not limited to the combinations of aspects expressly shown below.

[0167] 1. Recombinant adeno-associated virus (rAAV) virion comprising: a) a variant AAV capsid protein comprising the insertion of one heterologous peptide of any one of formulas I to VI, which confers increased infectivity of retinal cells compared to the infectivity of 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 according to embodiment 1, which exhibits at least a five-fold increase in the infectivity of retinal cells compared to the infectivity of retinal cells caused by a control AAV virion containing the corresponding parent AAV capsid protein. 3. The rAAV virion according to embodiment 1, which exhibits at least a tenfold increase in the infectivity of retinal cells compared to the infectivity of retinal cells caused by an AAV virion containing the corresponding parent AAV capsid protein. 4. The rAAV virion according to embodiment 1, wherein the insertion of the heterologous peptide replaces a sequence of 5 to 20 adjacent amino acids of the parent AAV capsid protein. 5. The rAAV virion according to Embodiment 1, wherein the insertion site is between amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype.

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

[0169] 11. The rAAV virion according to embodiment 10, wherein the RNA guide endonuclease is an enzymatically inactive type II CRISPR / Cas polypeptide. 12. The rAAV virion according to embodiment 10, wherein the heterogeneous gene product is an RNA guide endonuclease and a guide RNA. 13. The rAAV virion according to any one of embodiments 1 to 12, wherein the heterologous peptide 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). 14. The heterologous peptide comprises (21) LALIQDSMRA (SEQ ID NO: 35) or (22) LANQEHVKNA (SEQ ID NO: 2), the rAAV virion according to any one of embodiments 1 to 12. 15. The heterologous peptide is a peptide of formula II:TX1X2X3X4X5X6X7X8GLX9 (Sequence ID 137), wherein 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, as described in any one of embodiments 1 to 12.

[0170] 16. The rAAV virion according to any one of embodiments 1 to 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 heterologous peptide is a peptide of formula III:TGX1X2X3X4X5X6X7GLS (Sequence ID 138), wherein X1 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, as described in any one of embodiments 1 to 12. 18. The rAAV virion according to any one of embodiments 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 is given by formula IV: X1GX2X3X4X5X6X7X8GLSPX9TX 10 X 11 Peptide (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, X 10 However, it is G or W, and X 11 an rAAV billion according to any one of embodiments 1 to 12, wherein T or A. 20. The rAAV virion according to any one of embodiments 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 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, as described in any one of embodiments 1 to 12, the rAAV virion. 22. The aforementioned heterologous peptide is given by formula VI: LQX1X2X3RX4X5X6X7X8X9VNX 10Peptide Q (Sequence ID 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, X 10 an rAAV billion according to any one of embodiments 1 to 12, wherein the rAAV billion is F, G, T, or C. 23. The rAAV virion according to any one of embodiments 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) a recombinant adeno-associated virus virion according to any one of embodiments 1 to 23, and b) a pharmaceutically acceptable excipient, comprising a pharmaceutical composition. 25. A method for delivering a gene product to retinal cells in the body, comprising administering to the individual an rAAV virion according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24.

[0172] 26. The method according to embodiment 25, wherein the gene product is a polypeptide. 27. The method according to embodiment 25, wherein the gene product is a short interfering RNA or an aptamer. 28. The method according to embodiment 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 according to embodiment 26, wherein the polypeptide is glial neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain-derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked apoptosis inhibitor, retinoskin, RPE65, retinitis pigmentosa GTPase-interacting protein 1, periferin, periferin-2, rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), or sonic hedgehog. 30. The method according to embodiment 26, wherein the polypeptide is an RNA guide endonuclease.

[0173] 31. A method for treating an eye disease, comprising administering to an individual in need of treatment an effective amount of rAAV virion described in any one of embodiments 1 to 23 or the pharmaceutical composition described in embodiment 24. 32. The method according to embodiment 31, wherein administration is by intraocular injection. 33. The method according to embodiment 31, wherein administration is by intravitreous injection or choroidal injection. 34. The method according to any one of embodiments 31 to 33, wherein the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinal detachment, Leber congenital amaurosis, diabetic retinopathy, monochromacy, or color vision deficiency. 35. An isolated nucleic acid comprising a nucleotide sequence encoding a variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein contains an insertion of approximately 5 to 20 amino acids within the capsid protein GH loop in comparison with the corresponding parent AAV capsid protein, and the presence of the variant capsid protein in the AAV virion results in increased infectivity of the AAV virion in retinal cells, the amino acid insertion being within the GH loop of the native AAV capsid, and this insertion being one of the peptides of formulas I to VI.

[0174] 36. The nucleic acid according to embodiment 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. Isolated gene-modified host cells containing the nucleic acid described in embodiment 35 or embodiment 36. 38. A variant adeno-associated virus (AAV) capsid protein containing an insertion of approximately 5 to 20 amino acids, wherein the amino acid insertion is within the GH loop of the native AAV capsid, and the insertion is one of the peptides of formulas I to VI. 39.a) a variant AAV capsid protein comprising an insertion of a heterologous peptide of formula VI, which confers increased infectivity of retinal cells compared to the infectivity of retinal cells by a control AAV virion comprising a corresponding parent AAV capsid protein, and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product. 40. The rAAV virion according to embodiment 39, which exhibits at least a five-fold increase in the infectivity of retinal cells compared to the infectivity of retinal cells by a control AAV virion containing the corresponding parent AAV capsid protein.

[0175] 41. The rAAV virion according to embodiment 39, which exhibits at least a tenfold increase in the infectivity of retinal cells compared to the infectivity of retinal cells by an AAV virion containing the corresponding parent AAV capsid protein. 42. The rAAV virion according to any one of embodiments 39 to 41, wherein the insertion of the heterologous peptide replaces a sequence of 5 to 20 adjacent amino acids of the parent AAV capsid protein. 43. The rAAV virion according to any one of embodiments 39 to 42, wherein the insertion site is between amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype. 44. The rAAV virion according to embodiment 43, wherein the insertion site is between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype, or the insertion site is between amino acids corresponding to amino acids 585 and 598 of VP1 of AAV2, or at a corresponding position in the capsid protein of another AAV serotype. 45. The rAAV virion according to any one of embodiments 39 to 44, wherein the heterogeneous gene product is an interfering RNA.

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

[0177] 51. The rAAV virion according to any one of embodiments 39 to 44, wherein the heterogeneous gene product is an RNA guide endonuclease and a guide RNA. 52. The rAAV virion according to any one of embodiments 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) a rAAV virion according to any one of embodiments 39 to 52, and b) a pharmaceutically acceptable excipient, comprising a pharmaceutical composition. 54. A method for delivering a gene product to retinal cells within an organism, comprising administering to the organism an rAAV virion according to any one of embodiments 39 to 52 or a pharmaceutical composition according to embodiment 53. 55. The method according to embodiment 54, wherein the gene product is a polypeptide.

[0178] 56. The method according to embodiment 54, wherein the gene product is a short interfering RNA or an aptamer. 57. The method according to embodiment 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 according to embodiment 57, wherein the polypeptide is glial neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain-derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked apoptosis inhibitor, retinoskin, RPE65, retinitis pigmentosa GTPase-interacting protein 1, periferin, periferin-2, rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), or sonic hedgehog. 59. The method according to embodiment 55, wherein the polypeptide is an RNA guide endonuclease. 60. A method for treating an eye disease, comprising administering to an individual in need of treatment an effective amount of recombinant adeno-associated virus (rAAV) virion described in any one of embodiments 39 to 52 or a pharmaceutical composition described in embodiment 53.

[0179] 61. The method according to embodiment 60, wherein administration is by intraocular injection. 62. The method according to embodiment 60, wherein administration is by intravitreal injection or choroidal injection. 63. The method according to any one of embodiments 60 to 62, wherein the eye disease is glaucoma, retinitis pigmentosa, macular degeneration, retinal detachment, Leber congenital amaurosis, diabetic retinopathy, monochromacy, or color vision deficiency. [Examples]

[0180] The following examples are provided to give a complete disclosure and explanation of how to prepare and use the present invention and are not intended to limit the scope of what the inventors consider to be the invention, nor are they intended to represent that the following experiments are all experiments performed, or that no other experiments have been performed. While efforts have been made to ensure accuracy in the figures used (e.g., quantities, temperatures, etc.), some degree of experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, and pressure is around atmospheric pressure. Standard abbreviations may be used. For example, bp stands for base pair (plural), kb for kilobase (plural), pl for picoliters (plural), s or sec for seconds (plural), min for minutes (plural), h or hr for hours (plural), aa for amino acids (plural), kb for kilobase (plural), bp for base pair (plural), nt for nucleotides (plural), im for intramuscular (in), ip for intraperitoneal (in), sc for subcutaneous (in), and so on.

[0181] Example 1: AAV virion containing variant AAV capsid Through a directional evolutionary approach, we derived multiple AAV capsid variants. AAV virions containing these variant AAV capsids infect the primate retina, for example, when administered via intravitreal injection. Primates possess a fovea for high-clarity vision and are an important preclinical model for human retinal diseases.

[0182] AAV Packaging AAV virions containing variant AAV capsids were identified by screening. For this screening, five libraries were used: 1) an AAV2-based 7-mer peptide display library containing a 7-mer peptide insertion at approximately amino acid 588, surrounded by a 5′LA linker and a 3′A linker; 2) an AAV4-based 7-mer peptide display library with a 7-mer peptide insertion at approximately amino acid 584, possessing a 5′TG linker and a 3′GLS linker; 3) an AAV5-based 7-mer peptide display library with a 7-mer peptide insertion at approximately amino acid 575, possessing a 5′TG linker and a 3′GLS linker; 4) a library based on an ancestral AAV sequence (Santiago-Ortiz et al., 2015) containing a 7-mer peptide display library at approximately amino acid 591, possessing a 5′TG linker and a 3′GLS linker; and 5) an AAV2-based library with semi-random mutations at the surface exposure position of approximately amino acid 588 (Koerber, Jang, & Schaffer, 2008). Viral packaging was performed so that each viral genome was enclosed within the capsid protein shell encoded by its genome, as previously described in Koerber et al. (2008) (see above) and Fowler et al. Nat Protoc 9, 2267-2284 (2014). Therefore, functional improvements through selection can be related to the genome 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 replaced with PBS by Amicon filtration. DNase-resistant viral genome titers were measured by quantitative real-time PCR using BioRad iCycler. From this library, variants capable of infecting primate retinas from the vitreous humor were identified using an in vivo repeated screening selection process (Figure 1). Approximately 250 μL of 1 × 10⁶ 1 13 ~1 × 10 14The patient was injected with a virus titer of vg / mL. Three weeks after injection, the eye was excised, and retinal punches were collected from the central and peripheral regions of the retina (Figure 1). DNA from various retinal layers was assayed to identify the capsid inserts. After each round of injection, the capsid sequence was recovered from the collected cells by PCR using primers HindIII_F1 and NotI_R1, AscI_R1, or SpeI_R1 (reverse primers are specific to the specific AAV skeleton) to maintain the isolation of groups in the library. Next, the PCR amplicons were digested and re-cloned to form the skeleton. RPE cells were isolated from retinal cells, and the tissue was frozen. Retinal cells were embedded and sectioned on a cryostat to isolate photoreceptors in the outer granular layer. Next, DNA was collected from the isolated photoreceptors or RPE, and the cap gene was PCR amplified. The recovered cap gene was used for subsequent AAV packaging.

[0183] Figure 1 illustrates the directional evolution method used for the generation of primate retinal AAV variants. A peptide display library was prepared, packaged into AAV vectors, and injected into primate eyes via intravitreal injection. AAV variants were positively selected from the vector pool using repeated selection rounds. Error-prone PCR was performed after three selection rounds, followed by additional selection rounds.

[0184] Deep sequencing of AAV libraries from selected rounds Following five selection rounds, Illumina deep sequencing was used to identify variants whose relative presentation rate within the library increased across rounds. Increased presentation rate within the viral library indicates positive selection and the ability to infect primate retinas from the vitreous humor. Regions of approximately 75–85 base pairs containing 7mer insertion or loop swap mutation sites were PCR-amplified from the isolated DNA. Primers included Illumina adapter sequences containing unique barcodes to enable multiplexing of amplicons from multiple selection rounds. PCR amplicons were purified and sequenced by 100 cycles of single-read runs on an Illumina HiSeq 2500. Custom Python code was written to translate DNA sequences into amino acid sequences and count reads containing unique 7mer insertion sequences. Read counts were normalized by the total number of reads in the run. Directional evolution dynamics were analyzed and plotted using Python and Pandas.

[0185] Deep sequencing analysis Approximately 1 x 10 per library 7 The top variant was selected from the variants. The variant with the best performance was chosen as the variant with the largest multiplication factor in the final selection round compared to the initial plasmid library (number of reads in the final round (normalized to the total number of reads in the round) / number of reads in the library (normalized to the total number of reads in the round)). To enable analysis of variants that do not appear in plasmid library sequencing, a pseudocount of 1 was added before normalization for each individual variant. See Fowler et al. (2014) above. The amino acid sequence of the peptide insertion is shown in Figure 2.

[0186] The variants generated through this approach enable a non-invasive panretinal gene therapy strategy in primate retinas using intravitreous 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, monochromacy, X-linked retinal detachment, CRB1, optogenetic therapy, nutritional and survival factors (e.g., GDNF, BDNF, FGF, RdCVF, RdCVFL, XIAP), and the expression of angiogenesis blockers such as sFLT. Such vectors can also be used for the delivery of gene editing tools such as CRISPR / Cas9 for gene modification or augmentation model creation 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: Method for constructing and sequencing a GFP barcode library Building a GFP barcode library A unique 25 bp DNA barcode (CAG-GFP-barcode-pA) was cloned from an AAV ITR construct containing a self-complementary CAG promoter driving eGFP. Individual variants were packaged separately with constructs containing different barcodes. The variants were then titer-matched and mixed in equal ratios, followed by injection into mice, dogs, and primates.

[0189] Deep sequencing of GFP barcode libraries Barcodes were directly PCR amplified from DNA or cDNA (created from mRNA using Superscript III reverse transcriptase) collected from canine or primate retinal tissue. Samples were collected from areas throughout the retina and from ONL or RPE. Primers amplified a region of approximately 50 bp around the GFP barcode and contained an Illumina adapter sequence and secondary barcodes to enable multiplexing of multiple samples. PCR amplicons were purified and sequenced by 100 cycles of single-read runs on MiSeq. Read counts were normalized by the total number of reads in the run. Barcode abundance was analyzed using custom code written in Python, and then plotted using Pandas. The variant with the best performance was selected based on the multiplier increase of the total library relative to the injected library (total % in recovered samples / total % in injected libraries). Analysis was performed on n=1 primate.

[0190] Figure 9 shows Table 1, and Figure 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 library contained individual variants packaged with GFP fused to unique DNA barcodes. Barcodes were amplified from DNA recovered from specific cell types in the retina using polymerase chain reaction (PCR). "Region" in Tables 1 and 2 refers to the region from which DNA was recovered. The multiplier of reads for each variant was calculated by dividing the number of reads for each unique barcode in 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 multiplier across multiple locations in the retina. Variants were ranked by the multiplier of their barcodes.

[0192] Figure 11 shows GFP expression from a GFP barcode library in primate retinas. GFP expression resulting from intravitreal injection of the pooled GFP barcode library (containing all test viruses) was primarily located in the outer retina and showed a greater tendency toward the outer retina than AAV24YF expression.

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

[0194] result Directional evolution of AAV in primate retinas Non-human primates, in addition to dogs, are critical preclinical models for human therapeutic development because they are most closely related to humans and their retinal anatomical structure is similar to that of humans. Specifically, primates are the only large animal models that possess a fovea. The fovea is a specialized high-clarity area in the retina that is most important for everyday activities such as reading, critical to quality of life, and lost in many cases of retinal degeneration. The species specificity observed in canine studies motivated the inventors to pursue further pathways of directional evolution in the primate retina. Nine libraries were packaged and included in primate screening: EP2, EP5, EP6, EP8, EP9, EP-ancestor, AAV2-7mer, ancestor-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 time-series selection rounds (one round of error-prone PCR performed after round 3). The AAV cap gene was PCR-amplified from the ONL and simultaneously from the RPE covering it. The EP library was discontinued in round 3 because variants from the EP library were not recovered from retinal tissue. In round 4, additional libraries (AAV4-7mer and AAV5-7mer) were added to the selection using separate skeletons isolated from other libraries by separate PCR annealing and restriction sites.

[0195] Deep sequencing allows for observations from canine screening, and the library is approximately 10 6 ~about 10 7 It contains individual variants, which are approximately 10 across 6 selection rounds. 4 ~about 10 5It became clear that the variants converged to one specific variant. This is a diversity that cannot be observed through Sanger sequencing (Figure 12A). As observed in canine screening, in each library analyzed, a small fraction of library members from the initial plasmid library were overpresented (Figure 12B). Analysis of results from high-throughput sequencing across selection rounds revealed a subset of variants for each library that showed a significant increase in appearance during the selection round (Figure 12C).

[0196] Screening of a secondary barcode GFP library in primate retinas Sixteen variants were selected from these five libraries (Figure 12C) and included in a secondary selection round against the GFP barcode 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 three weeks after injection (12D). GFP expression resulting from injection of the GFP barcode library was found primarily in photoreceptors, and also in some inner retinal cells. This represents a shift from AAV2 or 7m8, which resulted in more potent inner retinal expression (Figure 12E).

[0197] Figures 12A-12F show the directional evolution of AAV in primate retina. (A) Deep sequencing of the variant library revealed the convergence of variants across the selection rounds. (B) In each library evaluated, a small percentage of variants were overexpressed in the plasmid library. (C) The scatter plot shows the behavior of individual variants in the final selection round for each library injected into primate retina. Variants overexpressed in the original library are colored blue. Variants with the largest multiplier increase in expression rate in the final selection round are shown in magenta. Variants that were overexpressed in the original library and showed a significant increase in expression rate across the selection rounds are colored orange. (D) The primate retina map shows the distribution of samples collected for the selection rounds and the GFP barcode library. The variant color coding is the same as in Figure 2. (E) GFP expression derived from the barcode library revealed that expression shifted towards the outer retinal layer in the selected variants. (F) This shows the results of GFP barcode library injection into the outer retina of primates. The list of variants is ordered from the highest (top) to the lowest (bottom) performance vectors, and the attached value indicates the degree to which the variant competed with other vectors, expressed as total % in the AAV library / total % in the recovered library.

[0198] Verification of top-performing primate variants Quantification of vector performance in the outer retinal layer revealed that AAV2-based variants performed better than other serotype-based viruses. One vector, the loop-swap variant AAV2, approximately 588-LQRGVRIPSVLEVNGQ (SEQ ID NO: 116), performed better than other variants, but with lower viral titers (approximately 5 × 10⁻¹⁶). 11 This resulted in vg / mL).

[0199] Therefore, it is the second-ranked variant from GFP barcode screening, and has a high titer (approximately 5 × 10⁻⁶). 13 AAV2-LALIQDSMRA (SEQ ID NO: 117, named NHP#9), packaged at vg / mL, was selected for the first round of validation trials targeting ganglion cells in the inner retina and cones in the outer retina. Cone photoreceptors are involved in adult macular degeneration (AMD). Because macular degeneration is the most common cause of blindness, with 288 million people projected to be affected worldwide by 2040 in developed countries, cone photoreceptors are a primary target for retinal gene therapy. NHP#9 was packaged with an SNCG promoter that drives tdTomato expression within RGCs and a pR1.7 promoter that drives GFP expression in cones. Vectors encoding both of these constructs were packaged in equal proportions (approximately 1.5 × 10⁻¹⁶). 12 The mixture (vg / construct / eye) was injected intravitreously into cynomolgus monkeys. The variant 7m8 (Dalkara et al. (2013), see above), described earlier, was packaged with the same construct of equal titer and injected into the vitreous humor of the opposite eye. In the eye injected with NHP#9, the expression of the tdTomato reporter in RGCs was lower compared to 7m8, which efficiently infected ganglion cells throughout the retina, but its expression in foveal cones was greatly increased compared to 7m8. This indicates a shift in tropism from the inner retinal layer to photoreceptors in the outer retinal layer. qRT-PCR performed using ddCT revealed an 11.71-fold (10.37-13.22-fold) increase in GFP expression compared to 7m8. Images collected from flat-mounted retinas, along with labeled cell counts performed using Imaris software, confirmed a substantial decrease in the number of transduced ganglion cells and an increase in the number of cones targeted by NHP#9.

[0200] Next, validation tests were also conducted on the loop-swap variant, approximately 588-LQRGVRIPSVLEVNGQ (sequence number 118, named NHP#26), which was the top-ranked variant from GFP barcode screening, although only a small number of virus particles were produced. Approximately 5 × 10 10 NHP#26-scCAG-eGFP particles were intravitreously injected into one eye of cynomolgus monkeys. Although the number of injected particles was small, efficient GFP expression was observed throughout the fovea and retina (Figure 13G). In contrast to the spot-like and ring-like expression patterns observed in the fovea with 7m8, NHP#9 (Figure 13A), and other naturally occurring serotypes, fundus imaging of NHP#26 resulted in disc-like GFP expression in the center of the fovea (Figure 13G). Confocal imaging of flat-mounted retina confirmed this disc-like expression pattern around the fovea, along with a very small number of 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 revealed low GFP expression in ganglion cells, as indicated by the absence of GFP+ ganglion cell axons, while high levels of GFP expression were found in Müller cells, further cells in the inner granular layer, and foveal cones and rods throughout the retina (Figures 13J-13Q).

[0201] Figures 13A-13Q show validation of evolved AAVs in primate retinas. (A-F) Approximately 1.5 × 10⁻¹⁶ AAVs packaged in 7m8 and variant NHP#9. 12 Co-injection of 7m8 particles and approximately 1.5 pR1.7-eGFP into the primate retina was performed. 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 decreased expression in ganglion cells and increased GFP expression in cones compared to 7m8 (B, D, F). (G) 5×10 10Fundus imaging of primate retinas after injection of NHP#26-scCAG-GFP particles resulted in disc-shaped GFP expression in the center of the fovea and a punctate GFP expression pattern throughout the retina. (H) Confocal imaging of native GFP expression in the fovea, flatly mounted. (I) Confocal imaging of native GFP expression in the outer region of the vascular arcade. (J) Confocal imaging of native GFP expression in a cryostat section penetrating the fovea. (K) Native GFP expression in the lower retina outside the vascular arcade showed only slight GFP expression in ganglion cells, but high levels of expression in Müller cells and photoreceptors in the outer retinal layer. Autofluorescence was also observed in RPE. (L) Anti-GFP labeling in cryostat sections revealed GFP expression in photoreceptors (obvious from the outer segment), GFP expression in Müller cells (obvious from the process spreading to the retina), and GFP expression in cells in the inner granular layer (possibly interneurons) with a horizontal process. (M) Anti-GFP labeling in foveal sections revealed further transfection of cones, Müller glia, and interneurons. (N) Co-labeling with anti-cone arrestin and anti-GFP revealed GFP expression in rod photoreceptors and in cells within the inner granular layer in sections prepared adjacent to the optic disc. (O) Co-labeling with anti-cone arrestin and anti-GFP antibody in low-expression areas revealed GFP expression in inner granular layer cells. (P, Q) A montage of confocal images from cryostat sections collected from outside the vascular arcade showed efficient GFP expression in the inner granular layer and outer retina.

[0202] While the present invention has been described with reference to specific embodiments thereof, those skilled in the art will understand that various modifications 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 suit the purpose, spirit, and scope of the invention, such as specific situations, materials, compositions of substances, processes, and process steps. All such modifications are intended to be within the scope of the appended claims.

[0203] cross reference This application claims the benefits of U.S. Provisional Patent Application No. 62 / 527,871, filed on 30 June 2017, and U.S. Provisional Patent Application No. 62 / 535,042, filed on 20 July 2017, the entirety of which these applications are incorporated herein by reference.

[0204] Description of research funded by the federal government. This invention was made with government support under grant number 1R01EY022975-01A1, approved by the National Institutes of Health. The government has certain rights to 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 LQRGVRIPSVLEVNGQ (SEQ ID NO: 29) 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; Including, rAAV2 virions.

2. An rAAV2 virion as described in 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 parent AAV2 capsid protein.

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

5. The rAAV2 virion of claim 1, wherein the heterologous gene product is an interfering RNA, an aptamer, or a polypeptide.

6. 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. a) a recombinant adeno-associated virus virion 2 according to any one of claims 1 to 6; b) a pharmaceutically acceptable excipient; and 10. A pharmaceutical composition comprising:

8. A recombinant adeno-associated virus (rAAV) virion 2 according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 for use in a method of 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.

9. The rAAV virion 2 for use or pharmaceutical composition for use described in claim 8, wherein the gene product is a polypeptide, a short interfering RNA or an aptamer.

10. The polypeptide comprising: 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 10. The rAAV virion 2 for use or pharmaceutical composition for use according to claim 9, wherein 11. A recombinant adeno-associated virus (rAAV) virion 2 according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 for use in a method for treating an ocular disease, comprising: 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.

12. An isolated nucleic acid comprising a nucleotide sequence encoding a variant adeno-associated virus 2 (AAV2) capsid protein VP1, comprising: the variant AA2V capsid protein comprises an insertion of a heterologous peptide comprising the sequence LQRGVRIPSVLEVNGQ (SEQ ID NO: 29), and the variant capsid protein, when present in AAV virions, confers 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; Nucleic acid.

13. An isolated genetically modified host cell comprising the nucleic acid described in claim 12.

14. A variant adeno-associated virus 2 (AAV2) capsid protein VP1 comprising an insertion of a heterologous peptide comprising the sequence LQRGVRIPSVLEVNGQ (SEQ ID NO: 29), comprising: 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.

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