US20200121746A1 - Adeno-associated virus virions with variant capsids and methods of use thereof - Google Patents

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

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US20200121746A1
US20200121746A1 US16/486,681 US201816486681A US2020121746A1 US 20200121746 A1 US20200121746 A1 US 20200121746A1 US 201816486681 A US201816486681 A US 201816486681A US 2020121746 A1 US2020121746 A1 US 2020121746A1
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amino acids
aav
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David V. Schaffer
Leah C. Byrne
Timothy P. Day
John G. Flannery
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University of California
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Definitions

  • Photoreceptors which lie at the back of the retina, respond to the absorption of photons, initiating a stream of signal processing that passes through second and third order neurons in the retina, including bipolar, horizontal and amacrine cells.
  • Retinal pigment epithelium (RPE) cells which lie underneath photoreceptors, promote the regeneration of the photon-detecting molecule, 11-cis retinal, via the visual cycle pathway and hence are essential for promoting this photoreceptor function.
  • Retinal ganglion cells (RGCs) in the inner retina receive visual signals from third order neurons, and communicate the visual signals in the form of action potentials to the brain.
  • Adeno-associated virus belongs to the Parvoviridae family and Dependovirus genus, whose members require co-infection with a helper virus such as adenovirus to promote replication, and AAV establishes a latent infection in the absence of a helper.
  • Virions are composed of a 25 nm icosahedral capsid encompassing 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, whereas cap encodes three structural proteins (VP1-3) that assemble into a 60-mer capsid shell.
  • This viral capsid mediates the ability of AAV vectors to overcome many of the biological barriers of viral transduction—including cell surface receptor binding, endocytosis, intracellular trafficking, and unpackaging in the nucleus.
  • 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.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • the present disclosure provides methods of delivering a gene product to a retinal cell in an individual.
  • FIG. 1 provides a schematic depiction of the directed evolution methodology used to develop primate retinal AAV variants.
  • FIG. 2 provides a table of peptide insertions and peptide replacements in variant AAV capsids.
  • FIG. 3A-3C provide amino acid sequences of exemplary guide-RNA-directed endonucleases.
  • FIG. 4 provides an amino acid sequence of AAV2 capsid protein VP1. Amino acids 587 and 588 (NP) are in bold and underlined.
  • FIG. 5 provides amino acid sequences corresponding to amino acids 570-610 of AAV capsid protein VP1 of various AAV serotypes.
  • FIG. 6A-6C provide an alignment of amino acid sequences of AAV capsid protein loop IV (GH loop) regions. Insertion sites are shown in bold and underlining.
  • FIG. 7A-7V provide amino acid sequences of exemplary heterologous gene products.
  • FIG. 8A-8B provide amino acid sequences of AAV4 capsid ( FIG. 8A ) and an ancestral AAV capsid ( FIG. 8B ).
  • FIG. 9 provides Table 1.
  • Table 1 provides a ranking of primate-derived variants and controls recovered from photoreceptors following injection of a green fluorescent protein (GFP)-Barcode library.
  • GFP green fluorescent protein
  • FIG. 10 provides Table 2.
  • Table 2 provides a ranking of primate-derived variants and controls recovered from RPE cells following injection of a GFP-Barcode library.
  • FIG. 11 depicts GFP expression of GFP-barcoded libraries in primate retina.
  • FIG. 12A-12F depict directed evolution of AAV in primate retina.
  • the sequences in FIG. 12F from top to bottom are set forth in SEQ ID NOs:117-135.
  • FIG. 13A-13Q depict validation of evolved AAV variants in primate retina.
  • retinal cell can refer herein to any of the cell types that comprise the retina, such as retinal ganglion cells; amacrine cells; horizontal cells; bipolar cells; photoreceptor cells including rods and cones; Miller glial cells; astrocytes (e.g., a retinal astrocyte); and retinal pigment epithelium.
  • AAV is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise.
  • the abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”).
  • AAV includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. See, e.g., Mori et al. (2004) Virology 330:375.
  • AAV also includes chimeric AAV.
  • Prime AAV refers to AAV isolated from a primate
  • non-primate AAV refers to AAV isolated from a non-primate mammal
  • bovine AAV refers to AAV isolated from a bovine mammal (e.g., a cow), etc.
  • rAAV vector refers to an AAV vector comprising a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a sequence of interest for the genetic transformation of a cell.
  • the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector”. Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.
  • Packaging refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.
  • AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.”
  • helper virus for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell.
  • helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • the adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used.
  • Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC.
  • Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • Helper virus function(s) refers to function(s) encoded in a helper virus genome which allow AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, “helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.
  • an “infectious” virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus.
  • an “infectious” virus or viral particle is one that can access a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell.
  • “infectivity” refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity.
  • Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles.
  • Total viral particles can be expressed as the number of viral genome (vg) copies.
  • the ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as “transduction.”
  • the ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and/or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA).
  • GFP green fluorescent protein
  • Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles.
  • Methods of determining the ratio of infectious viral particle to total viral particle are known in the art. See, e.g., Grainger et al. (2005) Mol. Ther. 11:S337 (describing a TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther. 6:973.
  • a “replication-competent” virus refers to a phenotypically wild-type virus that is infectious, and is also capable of being replicated in an infected cell (i.e. in the presence of a helper virus or helper virus functions).
  • replication competence generally requires the presence of functional AAV packaging genes.
  • rAAV vectors as described herein are replication-incompetent in mammalian cells (especially in human cells) by virtue of the lack of one or more AAV packaging genes.
  • rAAV vector preparations as described herein are those which contain few if any replication competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10 2 rAAV particles, less than about 1 rcAAV per 10 4 rAAV particles, less than about 1 rcAAV per 10 8 rAAV particles, less than about 1 rcAAV per 10 12 rAAV particles, or no rcAAV).
  • rcAAV also referred to as RCA
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide or polypeptide has a certain percent “sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over 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, Wis., USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol.
  • GCG Genetics Computing Group
  • the program has default parameters determined by the sequences inputted to be compared.
  • the sequence identity is determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wis., USA.
  • GCG Genetics Computing Group
  • FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters:
  • a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.
  • guide RNA refers to an RNA that comprises: i) an “activator” nucleotide sequence that binds to a guide RNA-directed endonuclease (e.g., a class 2 CRISPR/Cas endonuclease such as a type II, type V, or type VI CRISPR/Cas endonuclease) and activates the RNA-directed endonuclease; and ii) a “targeter” nucleotide sequence that comprises a nucleotide sequence that hybridizes with a target nucleic acid.
  • a guide RNA-directed endonuclease e.g., a class 2 CRISPR/Cas endonuclease such as a type II, type V, or type VI CRISPR/Cas endonuclease
  • the “activator” nucleotide sequence and the “targeter” nucleotide sequence can be on separate RNA molecules (e.g., a “dual-guide RNA”); or can be on the same RNA molecule (a “single-guide RNA”).
  • a “small interfering” or “short interfering RNA” or siRNA is an RNA duplex of nucleotides that is targeted to a gene interest (a “target gene”).
  • An “RNA duplex” refers to the structure formed by the complementary pairing between 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 a nucleotide sequence of the targeted gene.
  • the length of the duplex of siRNAs is less than 30 nucleotides.
  • the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length.
  • the length of the duplex is 19-25 nucleotides in length.
  • the RNA duplex portion of the siRNA can be part of a hairpin structure.
  • the hairpin structure may contain a loop portion positioned between the two sequences that form the duplex.
  • the loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length.
  • the hairpin structure can also contain 3′ or 5′ overhang portions. In some embodiments, the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.
  • microRNA refers to any type of interfering RNAs, including but not limited to, endogenous microRNAs and artificial microRNAs (e.g., synthetic miRNAs). Endogenous microRNAs are small RNAs naturally encoded in the genome which are capable of modulating the productive utilization of mRNA.
  • An artificial microRNA can be any type of RNA sequence, other than endogenous microRNA, which is capable of modulating the activity of an mRNA.
  • a microRNA sequence can be an RNA molecule composed of any one or more of these sequences.
  • MicroRNA or “miRNA” sequences have been described in publications such as Lim, et al., 2003, Genes & Development, 17, 991-1008, Lim et al., 2003, Science, 299, 1540, Lee and Ambrose, 2001, Science, 294, 862, Lau et al., 2001, Science 294, 858-861, Lagos-Quintana et al., 2002, Current Biology, 12, 735-739, Lagos-Quintana et al., 2001, Science, 294, 853-857, and Lagos-Quintana et al., 2003, RNA, 9, 175-179.
  • microRNAs include any RNA that is a fragment of a larger RNA or is a miRNA, siRNA, stRNA, sncRNA, tncRNA, snoRNA, smRNA, shRNA, snRNA, or other small non-coding RNA. See, e.g., US Patent Applications 20050272923, 20050266552, 20050142581, and 20050075492.
  • a “microRNA precursor” refers to a nucleic acid having a stem-loop structure with a microRNA sequence incorporated therein.
  • a “mature microRNA” includes a microRNA that has been cleaved from a microRNA precursor (a “pre-miRNA”), or that has been synthesized (e.g., synthesized in a laboratory by cell-free synthesis), and has a length of from about 19 nucleotides to about 27 nucleotides, e.g., a mature microRNA can have a length of 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, or 27 nt.
  • a mature microRNA can bind to a target mRNA and inhibit translation of the target mRNA.
  • Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • a recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
  • control element or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3′ direction) from the promoter.
  • “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • An “expression vector” is a vector comprising a region which encodes a polypeptide of interest, and is used for effecting the expression of the protein in an intended target cell.
  • An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target.
  • the combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.
  • Heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
  • a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter.
  • an rAAV that includes a heterologous nucleic acid encoding a heterologous gene product is an rAAV that includes a nucleic acid not normally included in a naturally-occurring, wild-type AAV
  • the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV.
  • a variant AAV capsid protein that comprises a heterologous peptide inserted into the GH loop of the capsid protein is a variant AAV capsid protein that includes an insertion of a peptide not normally included in a naturally-occurring, wild-type AAV.
  • genetic alteration and “genetic modification” (and grammatical variants thereof), are used interchangeably herein to refer to a process wherein a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis.
  • the element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell.
  • Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or contacting with a polynucleotide-liposome complex.
  • Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector.
  • the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration that changes the phenotype and/or genotype of the cell and its progeny is included in this term.
  • a cell is said to be “stably” altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro.
  • a cell is “heritably” altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.
  • polypeptide refers to polymers of amino acids of any length.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.
  • references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivery of a gene product to a mammalian subject include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.
  • an “isolated” plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from.
  • an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated.
  • An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse affect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses, camels, etc.); mammalian farm animals (e.g., sheep, goats, cows, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). In some cases, the individual is a human.
  • mammalian sport animals e.g., horses, camels, etc.
  • mammalian farm animals e.g., sheep, goats, cows, etc.
  • mammalian pets dogs, cats, etc.
  • rodents e.g., mice, rats, etc.
  • 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.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus
  • the present disclosure provides methods of delivering a gene product to a retinal cell in an individual.
  • the present disclosure also provides methods of modifying a target nucleic acid present in a retinal cell.
  • the present disclosure provides recombinant adeno-associated virus (AAV) virions with altered capsid protein, where the recombinant AAV (rAAV) virions exhibit greater infectivity of a retinal cell compared to wild-type AAV; and where the rAAV virions comprise a heterologous nucleic acid.
  • the rAAV virions exhibit increased ability to cross a barrier between intravitreal fluid and retinal cells.
  • the rAAV virions exhibit greater infectivity of a retinal cell, compared to the infectivity of a corresponding wild-type AAV for the retinal cell.
  • the retinal cell can be a photoreceptor (e.g., rods; cones), a retinal ganglion cell (RGC), a Müller cell (a Müller glial cell), an astrocyte (e.g., a retinal astrocyte), a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelium (RPE) cell.
  • the present disclosure further provides methods of delivering a gene product to a retinal cell in an individual, and methods of treating an ocular disease.
  • the present disclosure provides an rAAV virion with an altered capsid protein, where the rAAV virion exhibits at least 5-fold increased localization to one or more of the inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion cell layer, and the retinal pigment epithelium, compared to the extent of localization to the inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion cell layer, or the retinal pigment epithelium, by an AAV virion comprising the corresponding parental AAV capsid protein; and where the rAAV virions comprise a heterologous nucleic acid.
  • the present disclosure provides a variant AAV capsid protein.
  • a variant AAV capsid protein of the present disclosure is altered, compared to a wild-type or other reference AAV capsid protein. Alterations include insertions and swaps (e.g., replacements of a contiguous stretch of amino acids with a different contiguous stretch of amino acids).
  • a variant AAV capsid protein of the present disclosure comprises an insertion of a heterologous peptide of from 5 amino acids to 20 amino acids in length in an insertion site in a surface-accessible (e.g., solvent-accessible) portion of a parental AAV capsid protein, such that the variant capsid protein, when present in an AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein, particularly when the AAV virion is injected intravitreally.
  • a heterologous peptide of from 5 amino acids to 20 amino acids in length in an insertion site in a surface-accessible (e.g., solvent-accessible) portion of a parental AAV capsid protein
  • a variant AAV capsid protein of the present disclosure when present in an AAV virion, confers increased ability of the AAV virion to cross a barrier between the intravitreal fluid (“vitreous”) and a retinal cell, where such barriers include, e.g., the inner limiting membrane (ILM), the extracellular matrix of the retina, the cell membranes of the retinal cells themselves, inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion cell layer, and the retinal pigment epithelium.
  • the retinal cell is a Müller cell.
  • Other retinal cells include amacrine cells, bipolar cells, and horizontal cells.
  • an “insertion of from about 5 amino acids to about 20 amino acids” is also referred to herein as a “peptide insertion” (e.g., a heterologous peptide insertion).
  • a “corresponding parental AAV capsid protein” refers to an AAV capsid protein of the same AAV serotype, without a heterologous peptide insertion.
  • the variant AAV capsid comprises a single heterologous peptide insert of from 5 amino acids to 20 amino acids (e.g., from 5 to 7, from 7 to 10, from 10 to 12, from 12 to 15, or from 15 to 20 amino acids) in length.
  • An alteration in an AAV capsid can also be a swap, e.g., a replacement of a contiguous stretch of amino acids with a heterologous peptide.
  • a replacement is an insertion of a heterologous peptide in place of a contiguous stretch of amino acids.
  • a variant AAV capsid protein of the present disclosure comprises replacement of a contiguous stretch of amino acids with a heterologous peptide of from 5 amino acids to 20 amino acids in length in a site in a surface-accessible (e.g., solvent-accessible) portion of a parental AAV capsid protein, such that the variant capsid protein, when present in an AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein, particularly when the AAV virion is injected intravitreally.
  • a surface-accessible (e.g., solvent-accessible) portion of a parental AAV capsid protein such that the variant capsid protein, when present in an AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein, particularly when the AAV virion is
  • a variant AAV capsid protein of the present disclosure when present in an AAV virion, confers increased ability of the AAV virion to cross a barrier between the intravitreal fluid (“vitreous”) and a retinal cell, where such barriers include, e.g., ILM, the extracellular matrix of the retina, the cell membranes of the retinal cells themselves, inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion cell layer, and the retinal pigment epithelium.
  • the retinal cell is a Müller cell.
  • Other retinal cells include amacrine cells, bipolar cells, and horizontal cells.
  • a “replacement of from about 5 amino acids to about 20 amino acids” is also referred to herein as a “peptide swap” (e.g., a replacement of a contiguous stretch of amino acids with a heterologous peptide).
  • a “corresponding parental AAV capsid protein” refers to an AAV capsid protein of the same AAV serotype, without a heterologous peptide.
  • the variant AAV capsid comprises a single heterologous peptide replacement of from 5 amino acids to 20 amino acids (e.g., from 5 to 7, from 7 to 10, from 10 to 12, from 12 to 15, or from 15 to 20 amino acids) in length.
  • insertion refers to both insertion of a heterologous peptide without replacement of a contiguous stretch of amino acids, and to insertion of a heterologous peptide that replaces a contiguous stretch of amino acids.
  • the insertion site is in the GH loop, or loop IV, of the AAV capsid protein, e.g., in a solvent-accessible portion of the GH loop, or loop IV, of the AAV capsid protein.
  • the insertion site can be within amino acids 411-650 of an AAV capsid protein, as depicted in FIG. 6A-6C .
  • the insertion site can be within amino acids 570-611 of AAV2, within amino acids 571-612 of AAV1, within amino acids 560-601 of AAV5, within amino acids 571 to 612 of AAV6, within amino acids 572 to 613 of AAV7, within amino acids 573 to 614 of AAV8, within amino acids 571 to 612 of AAV9, or within amino acids 573 to 614 of AAV10, as depicted in FIG. 5 .
  • the insertion site is between amino acids 588 and 589 of an AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype.
  • the insertion site is between amino acids 587 and 588 of an AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 575 and 576 of an AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 584 and 585 of an AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 590 and 591 of an AAV2 capsid protein, or a corresponding insertion site in an AAV of a different serotype.
  • the insertion site is between amino acids 584 and 585 of an AAV4 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the insertion site is between amino acids 575 and 576 of an AAV5 capsid protein, or a corresponding insertion site in an AAV of a different serotype. In some cases, the site for replacement is between amino acids 584 and 598 of an AAV2 capsid protein, or a corresponding site in an AAV of a different serotype.
  • a heterologous peptide of from about 5 amino acids to about 20 amino acids is inserted in an insertion site in the GH loop or loop IV of the capsid protein relative to a corresponding parental AAV capsid protein.
  • the insertion site can be between amino acids 587 and 588 of AAV2, or between amino acids 588 and 589 of AAV2, or the corresponding positions of the capsid subunit of another AAV serotype. It should be noted that the insertion site 587/588 is based on an AAV2 capsid protein.
  • a heterologous peptide of 5 amino acids to about 20 amino acids (e.g., from 5 to 7, from 7 to 10, from 10 to 12, from 12 to 15, or from 15 to 20 amino acids) in length can be inserted in a corresponding site in an AAV serotype other than AAV2 (e.g., AAV8, AAV9, etc.).
  • AAV8 AAV9
  • Those skilled in the art would know, based on a comparison of the amino acid sequences of capsid proteins of various AAV serotypes, where an insertion site “corresponding to amino acids 587-588 of AAV2” would be in a capsid protein of any given AAV serotype. Sequences corresponding to amino acids 570-611 of capsid protein VP1 of AAV2 (see FIG.
  • GenBank Accession No. NP_049542 for AAV1; GenBank Accession No. NP_044927 for AAV4; GenBank Accession No. AAD13756 for AAV5; GenBank Accession No. AAB95459 for AAV6; GenBank Accession No. YP_077178 for AAV7; GenBank Accession No. YP_077180 for AAV8; GenBank Accession No. AAS99264 for AAV9; GenBank Accession No. AAT46337 for AAV10; and GenBank Accession No. AA088208 for AAVrh10. See, e.g., Santiago-Ortiz et al. (2015) Gene Ther. 22:934 for ancestral AAV capsid.
  • the insertion site can be between amino acids 587 and 588 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, between amino acids 588 and 589 of AAV10, or between amino acids 585 and 586 of AAV4.
  • the insertion sites are underlined in FIG. 5 ; the amino acid numbering is based on the numbering depicted in FIG. 5 .
  • a subject capsid protein includes a GH loop comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence set forth in FIG. 6A-6C ; and having an insertion of a heterologous peptide of from 5 to 20 amino acids (e.g., from 5 to 7, from 7 to 10, from 10 to 12, from 12 to 15, or from 15 to 20 amino acids) in length.
  • a variant AAV capsid protein of the present disclosure comprises a replacement, or substitution, of a segment, or sequence of consecutive amino acids, in a surface-accessible (e.g., solvent-accessible) portion of a parental AAV capsid, such that the variant capsid protein, when present in an AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein, particularly when the AAV virion is injected intravitreally.
  • a surface-accessible (e.g., solvent-accessible) portion of a parental AAV capsid such that the variant capsid protein, when present in an AAV virion, confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein, particularly when the AAV virion is injected intravitreally.
  • a subject variant AAV capsid protein comprising the sequence substitution when present in an AAV virion, confers increased ability of the AAV virion to cross a barrier between the vitreous and a retinal cell, where such barriers include, e.g., the inner limiting membrane, the extracellular matrix of the retina, and the cell membranes of the retinal cells themselves.
  • a “replacement of from about 5 consecutive amino acids to about 25 consecutive amino acids” is also referred to herein as a “loop swap” (i.e. a heterologous peptide substitution).
  • a “corresponding parental AAV capsid protein” in such instances refers to an AAV capsid protein of the same AAV serotype, without the subject loop swap.
  • the variant AAV capsid comprises a heterologous peptide substitution of from 5 contiguous amino acids to 25 contiguous amino acids, e.g. from 5 to 9, from 9 to 11, from 10 to 15, from 15 to 20, or from 20 to 25 amino acids in length.
  • a heterologous peptide of from about 5 amino acids to about 25 amino acids is substituted in for an equivalent number of consecutive amino acids in a corresponding parental AAV capsid protein.
  • the substitution begins at around amino acid 588 of AAV2, or the corresponding position of the capsid subunit of another AAV serotype, and ends at around amino acid 598 of AAV2 or the corresponding position of the capsid subunit of another AAV serotype. It should be noted that the residues 588-598 are based on an AAV2 VP1 capsid protein.
  • a heterologous peptide of 5 amino acids to about 25 amino acids in length can be substituted into a corresponding site in an AAV serotype other than AAV2 (e.g., AAV8, AAV9, etc.).
  • AAV8 AAV9
  • Those skilled in the art would know, based on a comparison of the amino acid sequences of capsid proteins of various AAV serotypes, where a substitution site “corresponding to amino acids 588-598 of AAV2” would be in a capsid protein of any given AAV serotype.
  • the amino acid residue corresponding to amino acids 588-598 of capsid protein VP1 of AAV2 see FIG. 4
  • FIG. 5 See, e.g., GenBank Accession No.
  • NP_049542 for AAV1; GenBank Accession No. NP_044927 for AAV4; GenBank Accession No. AAD13756 for AAV5; GenBank Accession No. AAB95459 for AAV6; GenBank Accession No. YP_077178 for AAV7; GenBank Accession No. YP_077180 for AAV8; GenBank Accession No. AAS99264 for AAV9, GenBank Accession No. AAT46337 for AAV10, and GenBank Accession No. AAO88208 for AAVrh10.
  • a heterologous peptide of from about 5 amino acids to about 25 amino acids is substituted in for an equivalent number of consecutive amino acids in a corresponding parental AAV capsid protein.
  • the substitution begins at around amino acid 585 of AAV2, or the corresponding position of the capsid subunit of another AAV serotype, and ends at around amino acid 598 of AAV2 or the corresponding position of the capsid subunit of another AAV serotype. It should be noted that the residues 585-598 are based on an AAV2 VP1 capsid protein.
  • a heterologous peptide of 5 amino acids to about 25 amino acids in length can be substituted into a corresponding site in an AAV serotype other than AAV2 (e.g., AAV8, AAV9, etc.).
  • AAV8 AAV9
  • Those skilled in the art would know, based on a comparison of the amino acid sequences of capsid proteins of various AAV serotypes, where a substitution site “corresponding to amino acids 585-598 of AAV2” would be in a capsid protein of any given AAV serotype.
  • the amino acid residue corresponding to amino acids 585-598 of capsid protein VP1 of AAV2 (see FIG. 4 ) in various AAV serotypes are shown in FIG. 5 . See, e.g., GenBank Accession No.
  • NP_049542 for AAV1; GenBank Accession No. NP_044927 for AAV4; GenBank Accession No. AAD13756 for AAV5; GenBank Accession No. AAB95459 for AAV6; GenBank Accession No. YP_077178 for AAV7; GenBank Accession No. YP_077180 for AAV8; GenBank Accession No. AAS99264 for AAV9, GenBank Accession No. AAT46337 for AAV10, and GenBank Accession No. AAO88208 for AAVrh10.
  • a heterologous peptide of from about 5 amino acids to about 20 amino acids in length is inserted into the GH loop of an AAV capsid, or replaces an equivalent number of consecutive amino acids in the GH loop of an AAV capsid.
  • the term “insertion peptide” is used below to describe both a peptide that is inserted into a parental AAV capsid and a peptide that replaces a segment of contiguous amino acids in the GH loop of an AAV capsid.
  • the insertion peptide has a length of from 5 amino acids to 20 amino acids.
  • the insertion peptide has a length of from 7 amino acids to 15 amino acids.
  • the insertion peptide has a length of from 9 amino acids to 15 amino acids. In some cases, the insertion peptide has a length of from 9 amino acids to 12 amino acids. The insertion peptide has a length of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids. In some cases, the insertion peptide has a length of 7 amino acids. In some cases, the insertion peptide has a length of 8 amino acids. In some cases, the insertion peptide has a length of 9 amino acids.
  • the insertion peptide has a length of 10 amino acids. In some cases, the insertion peptide has a length of 11 amino acids. In some cases, the insertion peptide has a length of 12 amino acids. In some cases, the insertion peptide has a length of 13 amino acids. In some cases, the insertion peptide has a length of 14 amino acids. In some cases, the insertion peptide has a length of 15 amino acids.
  • the peptide insert is, in some cases, a peptide of Formula I:
  • a peptide of Formula I comprises the following amino acid sequence: (21) LALIQDSMRA (SEQ ID NO: 35). In some cases, a peptide of Formula I comprises the following amino acid sequence: (22) LANQEHVKNA (SEQ ID NO:2).
  • the peptide insert is, in some cases, a peptide of Formula II:
  • X 1 is G, V, or S
  • X 2 is V, E, P, G, D, M, A, or S;
  • X 3 is M, V, Y, H, G, S, or D;
  • X 4 is R, D, S, G, V, Y, T, H, or M;
  • X 5 is S, L, G, T, Q, P, or A;
  • X 6 is T, A, S, M, D, Q, or H;
  • X 7 is N, G, S, L, M, P, G, or A;
  • X 8 is S, G, D, N, A, I, P, or T;
  • X 9 is S or N.
  • Peptide inserts of Formula II include, but are not limited to: (1) TGVMRSTNSGLN (SEQ ID NO: 6); (2) TGEVDLAGGGLS (SEQ ID NO: 7); (3) TSPYSGSSDGLS (SEQ ID NO: 8); (4) TGGHDSSLDGLS (SEQ ID NO: 9); (5) TGDGGTTMNGLS (SEQ ID NO: 98); (6) TGGHGSAPDGLS (SEQ ID NO: 99); (7) TGMHVTMMAGLN (SEQ ID NO: 100); (8) TGASYLDNSGLS (SEQ ID NO: 101); (9) TVVSTQAGIGLS (SEQ ID NO: 135); (10) TGVMHSQASGLS (SEQ ID NO: 21); (11) TGDGSPAAPGLS (SEQ ID NO: 22); and (12) TGSDMAHGTGLS (SEQ ID NO: 23).
  • the peptide insert is (1) TGVMRSTNSGLN (SEQ ID NO: 6). In some cases, the peptide insert is (2) TGEVDLAGGGLS (SEQ ID NO: 7). In some cases, the peptide insert is (3) TSPYSGSSDGLS (SEQ ID NO: 8). In some cases, the peptide insert is (4) TGGHDSSLDGLS (SEQ ID NO: 9). In some cases, the peptide insert is (5) TGDGGTTMNGLS (SEQ ID NO: 98). In some cases, the peptide insert is (6) TGGHGSAPDGLS (SEQ ID NO: 99). In some cases, the peptide insert is (7) TGMHVTMMAGLN (SEQ ID NO: 100).
  • 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).
  • the peptide insert is, in some cases, a peptide of Formula III:
  • X 1 is V, E, P, G, D, M, A, or S;
  • X 2 is M, V, Y, H, G, S, or D;
  • X 3 is R, D, S, G, V, Y, T, H, or M;
  • X 4 is S, L, G, T, Q, P, or A;
  • X 5 is T, A, S, M, D, Q, or H;
  • X 6 is N, G, S, L, M, P, G, or A;
  • Peptide inserts of Formula III include, but are not limited to: (2) TGEVDLAGGGLS (SEQ ID NO: 7); (4) TGGHDSSLDGLS (SEQ ID NO: 9); (5) TGDGGTTMNGLS (SEQ ID NO: 98); (6) TGGHGSAPDGLS (SEQ ID NO: 99); (8) TGASYLDNSGLS (SEQ ID NO: 101); (10) TGVMHSQASGLS (SEQ ID NO: 21); (11) TGDGSPAAPGLS (SEQ ID NO: 22); and (12) TGSDMAHGTGLS (SEQ ID NO: 23).
  • the peptide insert is, in some cases, a peptide of Formula IV:
  • X 1 is T or N
  • X 2 is L, S, A, or G
  • X 3 is D or V
  • X 4 is A, G, or P
  • X 5 is T or D
  • X 6 is R or Y
  • X 7 is D, T, or G
  • X 8 is H, R, or T
  • X 9 is V or A
  • X 10 is G or W
  • X 11 is T or A.
  • Peptide inserts of Formula IV include, but are not limited to: (13) TGLDATRDHGLSPVTGT (SEQ ID NO: 24); (14) TGSDGTRDHGLSPVTWT (SEQ ID NO: 25); (15) NGAVADYTRGLSPATGT (SEQ ID NO: 26); and (16) TGGDPTRGTGLSPVTGA (SEQ ID NO: 27).
  • the peptide insert is (13) TGLDATRDHGLSPVTGT (SEQ ID NO: 24).
  • the peptide insert is (14) TGSDGTRDHGLSPVTWT (SEQ ID NO: 25).
  • the peptide insert is (15) NGAVADYTRGLSPATGT (SEQ ID NO: 26).
  • the peptide insert is (16) TGGDPTRGTGLSPVTGA (SEQ ID NO: 27).
  • the peptide insert is, in some cases, a peptide of Formula V:
  • X 1 is L, S, A, or G
  • X 2 is A, G, or P
  • X 3 is D, T, or G
  • X 4 is H, R, or T.
  • Peptide inserts of Formula V include, but are not limited to: (13) TGLDATRDHGLSPVTGT (SEQ ID NO: 24); (14) TGSDGTRDHGLSPVTWT (SEQ ID NO: 25); and (16) TGGDPTRGTGLSPVTGA (SEQ ID NO: 27).
  • the peptide insert is, in some cases, a peptide of Formula VI:
  • X 1 is K or R
  • X 2 is N, G, or A
  • X 3 is A, V, N, or D;
  • X 5 is A, P, or V
  • X 7 is T or V
  • X 8 is E, L, A, or V;
  • X 9 is S, E, D, or V
  • X 10 is F, G, T, or C.
  • Peptides of Formula VI include, but are not limited to: (17) LQKNARPASTESVNFQ (SEQ ID NO: 28); (18) LQRGVRIPSVLEVNGQ (SEQ ID NO: 29); (19) LQRGNRPVTTADVNTQ (SEQ ID NO: 30); and (20) LQKADRQPGVVVVNCQ (SEQ ID NO: 31).
  • the peptide insert is (17) LQKNARPASTESVNFQ (SEQ ID NO: 28).
  • the peptide insert is (18) LQRGVRIPSVLEVNGQ (SEQ ID NO: 29).
  • the peptide insert is (19) LQRGNRPVTTADVNTQ (SEQ ID NO: 30).
  • the peptide insert is (20) LQKADRQPGVVVVNCQ (SEQ ID NO: 31).
  • peptide inserts can replace an equal number of contiguous amino acids in the GH loop of an AAV capsid polypeptide.
  • a peptide of Formula VI a peptide of Formula VI:
  • X 1 is K or R
  • X 2 is N, G, or A
  • X 3 is A, V, N, or D;
  • X 4 is P, I, or Q
  • X 5 is A, P, or V
  • X 6 is S, T, or G
  • X 7 is T or V
  • X 8 is E, L, A, or V;
  • X 9 is S, E, D, or V
  • X 10 is F, G, T, or C
  • an “insert peptide” replaces an endogenous peptide (e.g., a contiguous stretch of from 5 amino acids to 20 amino acids) present in in the GH loop of an AAV capsid polypeptide, resulting in a variant AAV capsid comprising a heterologous peptide in the GH loop.
  • the “insert peptide” replaces an endogenous contiguous stretch of amino acids of the same length as the insert peptide.
  • an endogenous contiguous stretch of 16 amino acids is replaced by the insert peptide.
  • Peptides of Formula VI include, but are not limited to: (17) LQKNARPASTESVNFQ (SEQ ID NO: 28); (18) LQRGVRIPSVLEVNGQ (SEQ ID NO: 29); (19) LQRGNRPVTTADVNTQ (SEQ ID NO:30); and (20) LQKADRQPGVVVVNCQ (SEQ ID NO: 31).
  • the peptide that replaces an endogenous amino acid sequence in the GH loop of an AAV capsid is (17) LQKNARPASTESVNFQ (SEQ ID NO: 28).
  • the peptide insert is (18) LQRGVRIPSVLEVNGQ (SEQ ID NO: 29).
  • the peptide that replaces an endogenous amino acid sequence in the GH loop of an AAV capsid is (19) LQRGNRPVTTADVNTQ (SEQ ID NO: 30). In some cases, the peptide that replaces an endogenous amino acid sequence in the GH loop of an AAV capsid is (20) LQKADRQPGVVVVNCQ (SEQ ID NO: 31).
  • a peptide insert of any one of Formulas I-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.
  • a peptide insert comprises: Thr-Gly-[peptide of any one of Formulas I-VI]-Gly-Leu-Ser (SEQ ID NO: 142).
  • a peptide insert comprises: Leu-Ala-[peptide of any one of Formulas I-VI]-Ala (SEQ ID NO: 143).
  • a peptide insert comprises: Leu-Gln-[peptide of any one of Formulas I-VI]-Gln.
  • a peptide insert does not include any linker amino acids.
  • a subject rAAV virion capsid does not include any other amino acid substitutions, insertions, or deletions, other than an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) in the GH loop or loop IV relative to a corresponding parental AAV capsid protein.
  • a subject rAAV virion capsid includes from 1 to about 25 amino acid insertions, deletions, or substitutions, compared to the parental AAV capsid protein, in addition to an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) in the GH loop or loop IV relative to a corresponding parental AAV capsid protein.
  • a subject rAAV virion capsid includes from 1 to about 5, from about 5 to about 10, from about 10 to about 15, from about 15 to about 20, or from about 20 to about 25 amino acid insertions, deletions, or substitutions, compared to the parental AAV capsid protein, in addition to an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) in the GH loop or loop IV relative to a corresponding parental AAV capsid protein.
  • the deletion of one or more amino acids compared to the parental AAV capsid protein occurs at the site of peptide insertion.
  • a variant AAV capsid polypeptide of the present disclosure does not include one, two, three, or four, of the following amino acid substitutions: Y273F, Y444F, Y500F, and Y730F.
  • a variant AAV capsid polypeptide of the present disclosure comprises, in addition to an insertion peptide as described above, one, two, three, or four, of the following amino acid substitutions: Y273F, Y444F, Y500F, and Y730F.
  • a variant AAV capsid polypeptide of the present disclosure is a chimeric capsid, e.g., the capsid comprises a portion of an AAV capsid of a first AAV serotype and a portion of an AAV capsid of a second serotype; and comprises an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) in the GH loop or loop IV relative to a corresponding parental AAV capsid protein.
  • the capsid comprises a portion of an AAV capsid of a first AAV serotype and a portion of an AAV capsid of a second serotype; and comprises an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g.,
  • the present disclosure provides a recombinant AAV (rAAV) virion comprising: i) a variant AAV capsid polypeptide of the present disclosure; and ii) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous polypeptide (i.e., a non-AAV polypeptide).
  • rAAV recombinant AAV
  • an rAAV virion of the present disclosure comprises a capsid protein comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to the amino acid sequence provided in FIG. 4 ; and an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) in the GH loop or loop IV relative to a corresponding parental AAV capsid protein.
  • a subject rAAV virion comprises a capsid protein comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to the amino acid sequence provided in FIG. 4 ; and an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) between amino acids 587 and 588 relative to the amino acid sequence depicted in FIG. 4 , or at a corresponding site relative to a corresponding parental AAV capsid protein.
  • amino acids e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids
  • an rAAV virion of the present disclosure comprises a capsid protein comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to the amino acid sequence provided in FIG. 4 ; and an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) in the GH loop or loop IV relative to a corresponding parental AAV capsid protein.
  • a subject rAAV virion comprises a capsid protein comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to the amino acid sequence provided in FIG. 4 ; and an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) between amino acids 585 and 598 relative to the amino acid sequence depicted in FIG. 4 , or at a corresponding site relative to a corresponding parental AAV capsid protein.
  • amino acids e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids
  • a subject rAAV virion comprises a capsid protein that includes a GH loop comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence set forth in FIG. 5 , and comprising an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) between the bolded and underlined amino acids.
  • a GH loop comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to an amino acid sequence set forth in FIG. 5 , and comprising an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
  • a subject rAAV virion comprises a capsid protein comprising an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to any one of the amino acid sequences provided in FIG. 6A-6C ; and an insertion of from about 5 amino acids to about 20 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids; e.g., 9 amino acids, 10 amino acids, 11 amino acids, or 12 amino acids) between amino acids 587 and 588 of AAV2, or at a corresponding site relative to another AAV genotype.
  • the corresponding insertion site is a site as indicated by bold text and underlining in FIG. 6B .
  • An rAAV virion of the present disclosure 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 a retinal cell, compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • Whether a given rAAV virion exhibits increased infectivity of a retinal cell can be determined by detecting expression in a retinal cell of a heterologous gene product encoded by the rAAV virion, following intravitreal administration of the rAAV virion.
  • an rAAV virion of the present disclosure that comprises: a) a variant capsid of the present disclosure comprising a peptide insert or a peptide replacement, as described above; and b) a heterologous nucleotide sequence encoding a heterologous gene product, when administered intravitreally, results in a level of the heterologous gene product in a retinal cell, that is at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, greater than the level of the gene product in the retinal cell that results when a control rAAV virion that comprises: a) a control AAV capsid that does not comprises the peptide insert or the peptide replacement; and b) heterologous nucleotide sequence encoding the heterologous gene product is administered intravitreally.
  • Whether a given rAAV virion exhibits increased infectivity of a retinal cell can be determined by assessing a therapeutic effect of a therapeutic gene product encoded by the rAAV virion in a retinal cell.
  • Therapeutic effects can include, e.g., a) a decrease in the rate of loss of visual function, 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; a decrease in nystagmus; etc.
  • an rAAV virion of the present disclosure that comprises: a) a variant capsid of the present disclosure comprising a peptide insert or a peptide replacement, as described above; and b) a heterologous nucleotide sequence encoding a heterologous therapeutic gene product, when administered intravitreally, results in a therapeutic effect of the therapeutic gene product in a retinal cell, that is at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold, greater than the therapeutic effect in the retinal cell that results when a control rAAV virion that comprises: a) a control AAV capsid that does not comprises the peptide insert or the peptide replacement; and b) heterologous nucleotide sequence encoding the heterologous therapeutic gene product is administered intravitreally.
  • Tests for visual function are known in the art; and any such test can be used to determine whether an
  • An rAAV virion of the present disclosure 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 ability to cross a barrier between the intravitreal fluid and a retinal cell, compared to the ability of a control rAAV virion comprising the corresponding parental AAV capsid protein (i.e., the AAV capsid protein without the insert peptide or replacement peptide).
  • a subject rAAV virion 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 a retinal cell, when administered via intravitreal injection, compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • a subject rAAV virion 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 a photoreceptor (rod or cone) cell, compared to the infectivity of the photoreceptor cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion 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 a photoreceptor (rod or cone) cell, when administered via intravitreal injection, compared to the infectivity of the photoreceptor cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • a subject rAAV virion 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 an RGC, compared to the infectivity of the RGC by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion 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 an RGC, when administered via intravitreal injection, compared to the infectivity of the RGC by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • a subject rAAV virion 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 an RPE cell, compared to the infectivity of the RPE cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion 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 an RPE cell, when administered via intravitreal injection, compared to the infectivity of the RPE cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • a subject rAAV virion 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 a Müller cell, compared to the infectivity of the Müller cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion 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 a Müller cell, when administered via intravitreal injection, compared to the infectivity of the Müller cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • a subject rAAV virion 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 a bipolar cell, compared to the infectivity of the bipolar cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion 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 a bipolar cell, when administered via intravitreal injection, compared to the infectivity of the bipolar cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • a subject rAAV virion 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 an amacrine cell, compared to the infectivity of the amacrine cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion 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 an amacrine cell, when administered via intravitreal injection, compared to the infectivity of the amacrine cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • a subject rAAV virion 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 a horizontal cell, compared to the infectivity of the horizontal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion 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 a horizontal cell, when administered via intravitreal injection, compared to the infectivity of the horizontal cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • a subject rAAV virion 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 a retinal astrocyte, compared to the infectivity of the retinal astrocyte by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion 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 a retinal astrocyte, when administered via intravitreal injection, compared to the infectivity of the retinal astrocyte by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • a subject rAAV virion 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 ability to cross extracellular matrix (ECM) of the retina, compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ECM of the retina.
  • ECM extracellular matrix
  • a subject rAAV virion 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 ability, when administered via intravitreal injection, to cross extracellular matrix (ECM) of the retina, compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ECM of the retina when administered via intravitreal injection.
  • ECM extracellular matrix
  • a subject rAAV virion 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 ability to cross the internal limiting membrane (ILM), compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ILM.
  • ILM internal limiting membrane
  • a subject rAAV virion 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 ability, when administered via intravitreal injection, to cross the ILM, compared to the ability of an AAV virion comprising the corresponding parental AAV capsid protein to cross the ILM when administered via intravitreal injection.
  • a subject rAAV virion can cross the ILM, and can also traverse cell layers, including Müller cells, amacrine cells, etc., to reach the photoreceptor cells and or RPE cells.
  • a subject rAAV virion when administered via intravitreal injection, can cross the ILM, and can also traverse cell layers, including Müller cells, amacrine cells, etc., to reach the photoreceptor cells and or RPE cells.
  • a subject rAAV virion 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 localization to one or more of the inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion cell layer, and the retinal pigment epithelium, compared to the extent of localization to the inner nuclear layer, the outer nuclear layer, the photoreceptor layer, the ganglion cell layer, or the retinal pigment epithelium, by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion when injected intravitreally, 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 localization past the ILM, compared to the extent of localization past the ILM by an intravitreally injected control AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion when injected intravitreally, 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 localization to the retinal pigment epithelium (RPE), compared to the extent of localization to the RPE layer by an intravitreally injected control AAV virion comprising the corresponding parental AAV capsid protein.
  • RPE retinal pigment epithelium
  • a subject rAAV virion when injected intravitreally, 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 localization to the photoreceptor (PR) layer, compared to the extent of localization to the PR layer by an intravitreally injected control AAV virion comprising the corresponding parental AAV capsid protein.
  • PR photoreceptor
  • a subject rAAV virion when injected intravitreally, 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 localization to the inner nuclear layer, compared to the extent of localization to the inner nuclear layer by an intravitreally injected control AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion when injected intravitreally, 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 localization to the outer nuclear layer, compared to the extent of localization to the outer nuclear layer by an intravitreally injected control AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion when injected intravitreally, 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 localization to the ganglion cell layer, compared to the extent of localization to the ganglion cell layer by an intravitreally injected control AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject rAAV virion selectively infects a retinal cell, e.g., a subject rAAV virion infects a retinal cell with 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold, specificity than a non-retinal cell, e.g., a cell outside the eye.
  • a subject rAAV virion selectively infects a retinal cell, e.g., a subject rAAV virion infects a photoreceptor cell with 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold, specificity than a non-retinal cell, e.g., a cell outside the eye.
  • a retinal cell e.g., a subject rAAV virion infects a photoreceptor cell with 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold, specificity than a non-retinal cell, e.g., a cell outside the eye.
  • a subject rAAV virion selectively infects a photoreceptor cell, e.g., a subject rAAV virion infects a photoreceptor cell with 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold, specificity than a non-photoreceptor cell present in the eye, e.g., a retinal ganglion cell, a Miller cell, etc.
  • a subject rAAV virion exhibits 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 a photoreceptor cell, when administered via intravitreal injection, compared to the infectivity of the photoreceptor cell by an AAV virion comprising the corresponding parental AAV capsid protein, when administered via intravitreal injection.
  • An rAAV virion of the present disclosure comprises a heterologous nucleic acid comprising a nucleotide sequence encoding one or more gene products (one or more heterologous gene products).
  • the gene product is a polypeptide.
  • the gene product is an RNA.
  • an rAAV virion of the present disclosure comprises a heterologous nucleotide sequence encoding both a heterologous nucleic acid gene product and a heterologous polypeptide gene product.
  • the gene product is an RNA
  • the RNA gene product encodes a polypeptide.
  • the gene product is an RNA
  • the RNA gene product does not encode a polypeptide.
  • an rAAV virion of the present disclosure comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding a single heterologous gene product. In some cases, an rAAV virion of the present disclosure comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding two heterologous gene products. Where the single heterologous nucleic acid encodes two heterologous gene products, in some cases, nucleotide sequences encoding the two heterologous gene products are operably linked to the same promoter.
  • nucleotide sequences encoding the two heterologous gene products are operably linked to two different promoters.
  • an rAAV virion of the present disclosure comprises a single heterologous nucleic acid comprising a nucleotide sequence encoding three heterologous gene products.
  • nucleotide sequences encoding the three heterologous gene products are operably linked to the same promoter.
  • nucleotide sequences encoding the three heterologous gene products are operably linked to two or three different promoters.
  • an rAAV virion of the present disclosure comprises two heterologous nucleic acids, each comprising a nucleotide sequence encoding a heterologous gene product.
  • the gene product is a polypeptide-encoding RNA. In some cases, the gene product is an interfering RNA. In some cases, the gene product is an aptamer. In some cases, the gene product is a polypeptide. In some cases, the gene product is a therapeutic polypeptide, e.g., a polypeptide that provides clinical benefit. In some embodiments, the gene product is a site-specific nuclease that provide for site-specific knock-down of gene function. In some embodiments, the gene product is an RNA-guided endonuclease that provides for modification of a target nucleic acid.
  • the gene products are: i) an RNA-guided endonuclease that provides for modification of a target nucleic acid; and ii) a guide RNA that comprises a first segment that binds to a target sequence in a target nucleic acid and a second segment that binds to the RNA-guided endonuclease.
  • the gene products are: i) an RNA-guided endonuclease that provides for modification of a target nucleic acid; ii) a first guide RNA that comprises a first segment that binds to a first target sequence in a target nucleic acid and a second segment that binds to the RNA-guided endonuclease; and iii) a first guide RNA that comprises a first segment that binds to a second target sequence in the target nucleic acid and a second segment that binds to the RNA-guided endonuclease.
  • RNAi interfering RNA
  • suitable RNAi include RNAi that decrease the level of an apoptotic or angiogenic factor in a cell.
  • an RNAi can be an shRNA or siRNA that reduces the level of a gene product that induces or promotes apoptosis in a cell.
  • Genes whose gene products induce or promote apoptosis are referred to herein as “pro-apoptotic genes” and the products of those genes (mRNA; protein) are referred to as “pro-apoptotic gene products.”
  • Pro-apoptotic gene products include, e.g., Bax, Bid, Bak, and Bad gene products. See, e.g., U.S. Pat. No. 7,846,730.
  • Interfering RNAs could also be against an angiogenic product, for example vascular endothelial growth factor (VEGF) (e.g., Cand5; see, e.g., U.S. Patent Publication No. 2011/0143400; U.S. Patent Publication No. 2008/0188437; and Reich et al. (2003) Mol. Vis. 9:210); VEGF receptor-1 (VEGFR1) (e.g., Sirna-027; see, e.g., Kaiser et al. (2010) Am. J. Ophthalmol. 150:33; and Shen et al. (2006) Gene Ther. 13:225); or VEGF receptor-2 (VEGFR2) (Kou et al. (2005) Biochem. 44:15064). See also, U.S. Pat. Nos. 6,649,596, 6,399,586, 5,661,135, 5,639,872, and 5,639,736; and 7,947,659 and 7,919,473.
  • exemplary aptamers of interest include an aptamer against VEGF. See, e.g., Ng et al. (2006) Nat. Rev. Drug Discovery 5:123; and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:18902.
  • a VEGF aptamer can comprise the nucleotide sequence 5′-cgcaaucagugaaugcuuauacauccg-3′ (SEQ ID NO:3).
  • PDGF platelet-derived growth factor
  • the polypeptide is a polypeptide that enhances function of a retinal cell, e.g., the function of a rod or cone photoreceptor cell, a retinal ganglion cell, a Miller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell.
  • polypeptides include neuroprotective polypeptides (e.g., glial cell derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), neurotrophin-4 (NT4), nerve growth factor (NGF), and neurturin (NTN)); anti-angiogenic polypeptides (e.g., a soluble VEGF receptor; a VEGF-binding antibody; a VEGF-binding antibody fragment (e.g., a single chain anti-VEGF antibody); endostatin; tumstatin; angiostatin; a soluble Flt polypeptide (Lai et al. (2005) Mol. Ther.
  • neuroprotective polypeptides e.g., glial cell derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), neurotrophin-4 (NT4), nerve growth factor (NGF), and neurturin (NTN)
  • anti-angiogenic polypeptides e.g., a
  • an Fc fusion protein comprising a soluble Flt polypeptide (see, e.g., Pechan et al. (2009) Gene Ther. 16:10); pigment epithelium-derived factor (PEDF); a soluble Tie-2 receptor; etc.); tissue inhibitor of metalloproteinases-3 (TIMP-3); a light-responsive opsin, e.g., a rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl; XIAP); and the like.
  • PDF pigment epithelium-derived factor
  • TMP-3 tissue inhibitor of metalloproteinases-3
  • a light-responsive opsin e.g., a rhodopsin
  • anti-apoptotic polypeptides e.g., Bcl-2, Bcl-Xl; XIAP
  • Suitable polypeptides include, but are not limited to, glial derived neurotrophic factor (GDNF); fibroblast growth factor; fibroblast growth factor 2; neurturin (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain derived neurotrophic factor (BDNF; e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 200 amino acids to 247 amino acids of the amino acid sequence depicted in FIG. 7B (SEQ ID NO: 11)); epidermal growth factor; rhodopsin; X-linked inhibitor of apoptosis; and Sonic hedgehog.
  • GDNF glial derived neurotrophic factor
  • NTN ciliary neurotrophic factor
  • NTF nerve growth factor
  • NT4 neurotrophin-4
  • BDNF brain
  • Suitable light-responsive opsins include, e.g., a light-responsive opsin as 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. See, Thyagarajan et al. (2010) J Neurosci.
  • Suitable polypeptides include light-gated ion channel polypeptides. See, e.g., Gaub et al. (2014) Proc. Natl. Acad. Sci. USA 111:E5574.
  • a suitable polypeptide is a light-gated ionotropic glutamate receptor (LiGluR). Expression of LiGluR in retinal ganglion cells and ON-bipolar cells, in the presence of a photoisomerizable compound, renders the cells responsive to light.
  • LiGluR comprises a 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.
  • Photoisomerizable compounds include, e.g., maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0 460 ).
  • MAG0 460 has the following structure:
  • Suitable polypeptides also include retinoschisin (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 200 amino acids to 224 amino acids of the amino acid sequence depicted in FIG. 7A (SEQ ID NO: 10).
  • Suitable polypeptides include, e.g., retinitis pigmentosa GTPase regulator (RPGR)-interacting protein-1 (see, e.g., GenBank Accession Nos.
  • Q96KN7, Q9EPQ2, and Q9GLM3 e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 1150 amino acids to about 1200 amino acids, or from about 1200 amino acids to 1286 amino acids, of the amino acid sequence depicted in FIG. 7F (SEQ ID NO:15); peripherin-2 (Prph2) (see, e.g., GenBank Accession No.
  • NP_000313 e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 300 amino acids to 346 amino acids of the amino acid sequence depicted in FIG. 7D (SEQ ID NO:13); and Travis et al. (1991) Genomics 10:733); peripherin (e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 400 amino acids to about 470 amino acids of the amino acid sequence depicted in FIG.
  • peripherin e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of
  • a retinal pigment epithelium-specific protein e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 200 amino acids to 247 amino acids of the amino acid sequence depicted in FIG. 7C (SEQ ID NO:12)) (see, e.g., GenBank AAC39660; and Morimura et al. (1998) Proc. Natl. Acad. Sci.
  • RPE65 retinal pigment epithelium-specific protein
  • rod-derived cone viability factor e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in any one of FIGS. 7H, 7I, and 7J
  • Rab escort protein 1 REP1
  • FIG. 7 a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG.
  • retinitis pigmentosa GTPase regulator e.g., a polypeptide comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in one of FIG. 7S-7V ); and the like.
  • RPGR retinitis pigmentosa GTPase regulator
  • a suitable RPGR polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 7S .
  • a suitable RPGR polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 7T .
  • a suitable RPGR polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 7U .
  • a suitable RPGR polypeptide comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 7V .
  • Suitable polypeptides also include: CHM (choroideremia (Rab escort protein 1 (REP1))), a polypeptide that, when defective or missing, causes choroideremia (see, e.g., Donnelly et al. (1994) Hum. Mol. Genet. 3:1017; and van Bokhoven et al. (1994) Hum. Mol. Genet. 3:1041); and Crumbs homolog 1 (CRB1), a polypeptide that, when defective or missing, causes Leber congenital amaurosis and retinitis pigmentosa (see, e.g., den Hollander et al. (1999) Nat. Genet. 23:217; and GenBank Accession No. CAM23328).
  • CHM choroideremia (Rab escort protein 1 (REP1))
  • REP1 choroideremia
  • CRB1 Crumbs homolog 1
  • a suitable REP1 polypeptide can comprise an amino acid having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set depicted in FIG. 7G .
  • Suitable polypeptides include Rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit alpha (PDE6 ⁇ ), Rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta isoform 1 (PDE6 ⁇ isoform 1), Rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta isoform 2 (PDE6 ⁇ isoform 2), Rod cGMP-specific 3′,5′-cyclic phosphodiesterase subunit beta isoform 3 (PDE6 ⁇ isoform 3).
  • a suitable PDE6a polypeptide can comprise an amino acid having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set depicted in FIG. 7K .
  • a suitable PDE6 ⁇ 6 isoform 1 polypeptide can comprise an amino acid having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set depicted in FIG. 7L .
  • a suitable PDE6 ⁇ 6 isoform 2 polypeptide can comprise an amino acid having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set depicted in FIG. 7M .
  • a suitable PDE6 ⁇ 6 isoform 3 polypeptide can comprise an amino acid having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set depicted in FIG. 7N .
  • Suitable polypeptides also include polypeptides that, when defective or missing, lead to achromotopsia, where such polypeptides include, e.g., cone photoreceptor cGMP-gated channel subunit alpha (CNGA3) (see, e.g., GenBank Accession No. NP_001289; and Booij et al. (2011) Ophthalmology 118:160-167); cone photoreceptor cGMP-gated cation channel beta-subunit (CNGB3) (see, e.g., Kohl et al. (2005) Eur J Hum Genet.
  • CNGA3 cone photoreceptor cGMP-gated channel subunit alpha
  • CNGB3 cone photoreceptor cGMP-gated cation channel beta-subunit
  • G protein guanine nucleotide binding protein
  • GNAT2 alpha transducing activity polypeptide 2
  • ACHM5 alpha transducing activity polypeptide 5
  • polypeptides that, when defective or lacking, lead to various forms of color blindness e.g., L-opsin, M-opsin, and S-opsin.
  • a suitable CNGA3 also known as ACHM2 isoform 1 polypeptide can comprise an amino acid having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set depicted in FIG. 7O .
  • a suitable CNGA3 also known as ACHM2 isoform 2 polypeptide can comprise an amino acid having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set depicted in FIG. 7P .
  • a suitable CNGB3 (also known as ACHM3) polypeptide can comprise an amino acid having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set depicted in FIG. 7Q .
  • GNAT2 also known as ACHM4
  • GNAT2 can comprise an amino acid having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence set depicted in FIG. 7R .
  • a gene product of interest is a site-specific endonuclease that provide for site-specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a retinal disease.
  • a site-specific endonuclease can be targeted to the defective allele and knock out the defective allele.
  • a site-specific endonuclease is an RNA-guided endonuclease.
  • a site-specific nuclease can also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele.
  • a subject rAAV virion can be used to deliver both a site-specific endonuclease that knocks out a defective allele, and can be used to deliver a functional copy of the defective allele, resulting in repair of the defective allele, thereby providing for production of a functional retinal protein (e.g., functional retinoschisin, functional RPE65, functional peripherin, etc.). See, e.g., Li et al. (2011) Nature 475:217.
  • a subject rAAV virion comprises a heterologous nucleotide sequence that encodes a site-specific endonuclease; and a heterologous nucleotide sequence that encodes a functional copy of a defective allele, where the functional copy encodes a functional retinal protein.
  • Functional retinal proteins include, e.g., retinoschisin, RPE65, retinitis pigmentosa GTPase regulator (RGPR)-interacting protein-1, peripherin, peripherin-2, RdCVF, and the like.
  • Site-specific endonucleases that are suitable for use include, e.g., zinc finger nucleases (ZFNs); meganucleases; and transcription activator-like effector nucleases (TALENs), where such site-specific endonucleases are non-naturally occurring and are modified to target a specific gene.
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • site-specific endonucleases can be engineered to cut specific locations within a genome, and non-homologous end joining can then repair the break while inserting or deleting several nucleotides.
  • site-specific endonucleases also referred to as “INDELs” then throw the protein out of frame and effectively knock out the gene. See, e.g., U.S. Patent Publication No. 2011/0301073.
  • Suitable site-specific endonucleases include engineered meganucleases and re-engineered homing endonucleases.
  • Suitable endonucleases include an I-Tevl nuclease.
  • Suitable meganucleases include I-Scel (see, e.g., Bellaiche et al. (1999) Genetics 152:1037); and I-Cre1 (see, e.g., Heath et al. (1997) Nature Structural Biology 4:468).
  • the gene product is an RNA-guided endonuclease.
  • the gene product is an RNA comprising a nucleotide sequence encoding an RNA-guided endonuclease.
  • the gene product is a guide RNA, e.g., a single-guide RNA.
  • the gene products are: 1) a guide RNA; and 2) an RNA-guided endonuclease.
  • the guide RNA can comprise: a) a protein-binding region that binds to the RNA-guided endonuclease; and b) a region that binds to a target nucleic acid.
  • An RNA-guided endonuclease is also referred to herein as a “genome editing nuclease.”
  • Suitable genome editing nucleases are CRISPR/Cas endonucleases (e.g., class 2 CRISPR/Cas endonucleases such as a type II, type V, or type VI CRISPR/Cas endonucleases).
  • a suitable genome editing nuclease is a CRISPR/Cas endonuclease (e.g., a class 2 CRISPR/Cas endonuclease such as a type II, type V, or type VI CRISPR/Cas endonuclease).
  • a genome targeting composition includes a class 2 CRISPR/Cas endonuclease.
  • a genome targeting composition includes a class 2 type II CRISPR/Cas endonuclease (e.g., a Cas9 protein).
  • a genome targeting composition includes a class 2 type V CRISPR/Cas endonuclease (e.g., a Cpf1 protein, a C2c1 protein, or a C2c3 protein).
  • a genome targeting composition includes a class 2 type VI CRISPR/Cas endonuclease (e.g., a C2c2 protein; also referred to as a “Cas13a” protein).
  • a CasX protein is also suitable for use is a CasY protein.
  • a genome editing nuclease is a fusion protein that is fused to a heterologous polypeptide (also referred to as a “fusion partner”).
  • a genome editing nuclease is fused to an amino acid sequence (a fusion partner) that provides for subcellular localization, i.e., the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.).
  • a fusion partner e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.
  • the genome-editing endonuclease is a Type II CRISPR/Cas endonuclease.
  • the genome-editing endonuclease is a Cas9 polypeptide.
  • the Cas9 protein is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by virtue of its association with the protein-binding segment of the Cas9 guide RNA.
  • a target nucleic acid sequence e.g., a chromosomal sequence or an extrachromosomal sequence, e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.
  • a Cas9 polypeptide comprises an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more than 99%, amino acid sequence identity to the Streptococcus pyogenes Cas9 depicted in FIG. 3A .
  • the Cas9 polypeptide used in a composition or method of the present disclosure is a Staphylococcus aureus Cas9 (saCas9) polypeptide.
  • the saCas9 polypeptide comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the saCas9 amino acid sequence depicted in FIG. 3B .
  • a suitable Cas9 polypeptide is a high-fidelity (HF) Cas9 polypeptide.
  • HF high-fidelity
  • an HF Cas9 polypeptide can comprise an amino acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the amino acid sequence depicted in FIG. 3A , where amino acids N497, R661, Q695, and Q926 are substituted, e.g., with alanine.
  • a suitable Cas9 polypeptide exhibits altered PAM specificity. See, e.g., Kleinstiver et al. (2015) Nature 523:481.
  • the genome-editing endonuclease is a type V CRISPR/Cas endonuclease.
  • a type V CRISPR/Cas endonuclease is a Cpf1 protein.
  • a Cpf1 protein comprises an amino acid sequence having at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 90%, or 100%, amino acid sequence identity to the Cpf1 amino acid sequence depicted in FIG. 3C .
  • the genome-editing endonuclease is a CasX or a CasY polypeptide.
  • CasX and CasY polypeptides are described in Burstein et al. (2017) Nature 542:237.
  • RNA-guided endonuclease with reduced enzymatic activity.
  • a “dead” RNA-guided endonuclease for example, a Cas9 polypeptide that comprises certain amino acid substitutions such that it exhibits substantially no endonuclease activity, but such that it still binds to a target nucleic acid when complexed with a guide RNA, is referred to as a “dead” Cas9 or “dCas9.”
  • a “dead” Cas9 protein has a reduced ability to cleave both the complementary and the non-complementary strands of a double stranded target nucleic acid.
  • a “nuclease defective” Cas9 lacks a functioning RuvC domain (i.e., does not cleave the non-complementary strand of a double stranded target DNA) and lacks a functioning HNH domain (i.e., does not cleave the complementary strand of a double stranded target DNA).
  • the nuclease defective Cas9 protein harbors mutations at amino acid positions corresponding to residues D10 and H840 (e.g., D10A and H840A) of SEQ ID NO: 15 (or the corresponding residues of a homolog of Cas9) such that the polypeptide has a reduced ability to cleave (e.g., does not cleave) both the complementary and the non-complementary strands of a target nucleic acid.
  • Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single stranded or double stranded target nucleic acid) but retains the ability to bind a target nucleic acid.
  • a Cas9 protein that cannot cleave target nucleic acid e.g., due to one or more mutations, e.g., in the catalytic domains of the RuvC and HNH domains
  • Other residues can be mutated to achieve the above effects (i.e. inactivate one or the other nuclease portions).
  • residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 of Streptococcus pyogenes Cas9 can be altered (i.e., substituted).
  • two or more of D10, E762, H840, N854, N863, and D986 of Streptococcus pyogenes Cas9 are substituted.
  • D10 and N863 of Streptococcus pyogenes Cas9 are substituted with Ala.
  • mutations other than alanine substitutions are suitable.
  • the genome-editing endonuclease is an RNA-guided endonuclease (and it corresponding guide RNA) known as Cas9-synergistic activation mediator (Cas9-SAM).
  • the RNA-guided endonuclease (e.g., Cas9) of the Cas9-SAM system is a “dead” Cas9 fused to a transcriptional activation domain (wherein suitable transcriptional activation domains include, e.g., VP64, p65, MyoD1, HSF1, RTA, and SET7/9) or a transcriptional repressor domain (where suitable transcriptional repressor domains include, e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, and a SID4X domain).
  • the guide RNA of the Cas9-SAM system comprises a loop that binds an adapter protein fused to a transcriptional activator domain (e.g., VP64, p65, MyoD1, HSF1, RTA, or SET7/9) or a transcriptional repressor domain (e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, or a SID4X domain).
  • a transcriptional activator domain e.g., VP64, p65, MyoD1, HSF1, RTA, or SET7/9
  • a transcriptional repressor domain e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, or a SID4X domain.
  • the guide RNA is a single-guide RNA comprising an MS2 RNA aptamer inserted into one or two loops of the sgRNA;
  • the dCas9 is a fusion polypeptide comprising dCas9 fused to VP64;
  • the adaptor/functional protein is a fusion polypeptide comprising: i) MS2; ii) p65; and iii) HSF1. See, e.g., U.S. Patent Publication No. 2016/0355797.
  • a chimeric polypeptide comprising: a) a dead RNA-guided endonuclease; and b) a heterologous fusion polypeptide.
  • suitable heterologous fusion polypeptides include a polypeptide having, e.g., methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, or nucleic acid binding activity.
  • a nucleic acid that binds to a class 2 CRISPR/Cas endonuclease e.g., a Cas9 protein; a type V or type VI CRISPR/Cas protein; a Cpf1 protein; etc.
  • a guide RNA or “CRISPR/Cas guide nucleic acid” or “CRISPR/Cas guide RNA.”
  • a guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid.
  • a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to herein as a “dual guide RNA”, a “double-molecule guide RNA”, a “two-molecule guide RNA”, or a “dgRNA.”
  • the guide RNA is one molecule (e.g., for some class 2 CRISPR/Cas proteins, the corresponding guide RNA is a single molecule; and in some cases, an activator and targeter are covalently linked to one another, e.g., via intervening nucleotides), and the guide RNA is referred to as a “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.”
  • the gene product can modify a target nucleic acid.
  • a target nucleic acid comprises a deleterious mutation in a defective allele (e.g., a deleterious mutation in a retinal cell target nucleic acid)
  • the RNA-guided endonuclease/guide RNA complex together with a donor nucleic acid comprising a nucleotide sequence that corrects the deleterious mutation (e.g., a donor nucleic acid comprising a nucleotide sequence that encodes a functional copy of the protein encoded by the defective allele)
  • HDR homology-directed repair
  • the gene products are an RNA-guided endonuclease and 2 separate sgRNAs, where the 2 separate sgRNAs provide for deletion of a target nucleic acid via non-homologous end joining (NHEJ).
  • NHEJ non-homologous end joining
  • the gene products are: i) an RNA-guided endonuclease; and ii) one guide RNA.
  • the guide RNA is a single-molecule (or “single guide”) guide RNA (an “sgRNA”). In some cases, the guide RNA is a dual-molecule (or “dual-guide”) guide RNA (“dgRNA”).
  • the gene products are: i) an RNA-guided endonuclease; and ii) 2 separate sgRNAs, where the 2 separate sgRNAs provide for deletion of a target nucleic acid via non-homologous end joining (NHEJ).
  • the guide RNAs are sgRNAs.
  • the guide RNAs are dgRNAs.
  • the gene products are: i) a Cpf1 polypeptide; and ii) a guide RNA precursor; in these cases, the precursor can be cleaved by the Cpf1 polypeptide to generate 2 or more guide RNAs.
  • the present disclosure provides a method of modifying a target nucleic acid in a retinal cell in an individual, where the target nucleic acid comprises a deleterious mutation, the method comprising administering to the individual (e.g., by intraocular; intravitreal; etc.
  • an rAAV virion of the present disclosure where the rAAV virion comprises a heterologous nucleic acid comprising: i) a nucleotide sequence encoding an RNA-guided endonuclease (e.g., a Cas9 endonuclease); ii) a nucleotide sequence encoding a sgRNA that comprises a nucleotide sequence that is complementary to the target nucleic acid; and iii) a nucleotide sequence encoding a donor DNA template that comprises a nucleotide sequence that corrects the deleterious mutation.
  • Administration of the rAAV virion results in correction of the deleterious mutation in the target nucleic acid by HDR.
  • the present disclosure provides a method of modifying a target nucleic acid in a retinal cell in an individual, where the target nucleic acid comprises a deleterious mutation, the method comprising administering to the individual (e.g., by intraocular; intravitreal; etc.
  • an rAAV virion of the present disclosure where the rAAV virion comprises a heterologous nucleic acid comprising: i) a nucleotide sequence encoding an RNA-guided endonuclease (e.g., a Cas9 endonuclease); ii) a nucleotide sequence encoding a first sgRNA that comprises a nucleotide sequence that is complementary to a first sequence in the target nucleic acid; and iii) a nucleotide sequence encoding a second sgRNA that comprises a nucleotide sequence that is complementary to a second sequence in the target nucleic acid.
  • Administration of the rAAV virion results in excision of the deleterious mutation in the target nucleic acid by NHEJ.
  • a nucleotide sequence encoding a gene product of interest is operably linked to a transcriptional control element.
  • a nucleotide sequence encoding a gene product of interest is operably linked to a constitutive promoter.
  • a nucleotide sequence encoding a gene product of interest is operably linked to an inducible promoter.
  • a nucleotide sequence encoding a gene product of interest is operably linked to a tissue-specific or cell type-specific regulatory element.
  • a nucleotide sequence encoding a gene product of interest is operably linked to a retinal cell-specific promoter.
  • a nucleotide sequence encoding a gene product of interest is operably linked to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked gene in a photoreceptor cell.
  • a photoreceptor-specific regulatory element e.g., a photoreceptor-specific promoter
  • Suitable photoreceptor-specific regulatory elements include, e.g., a rhodopsin promoter; a rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med.
  • a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra); an interphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra); an IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225).
  • IRBP interphotoreceptor retinoid-binding protein
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a) a subject rAAV virion, as described above; and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer.
  • the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a human.
  • excipients include any pharmaceutical agent that can be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the present disclosure provides a method of delivering a gene product to a retinal cell in an individual, the method comprising administering to the individual a subject rAAV virion as described above.
  • the gene product can be a polypeptide or an interfering RNA (e.g., an shRNA, an siRNA, and the like), an aptamer, or a site-specific endonuclease (e.g., an RNA-guided endonuclease), as described above.
  • Delivering a gene product to a retinal cell can provide for treatment of a retinal disease.
  • the retinal cell can be a photoreceptor, a retinal ganglion cell, a Müller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigmented epithelial cell.
  • the retinal cell is a photoreceptor cell, e.g., a rod or cone cell.
  • the present disclosure provides a method modifying a target nucleic acid in a retinal cell, the method comprising contacting the retinal cell with: 1) an rAAV virion of the present disclosure, wherein the rAAV virion comprises a heterologous nucleic acid comprising a nucleotide sequence encoding an RNA-guided endonuclease that binds a guide RNA; and 2) the guide RNA.
  • the present disclosure provides a method modifying a target nucleic acid in a retinal cell, the method comprising contacting the retinal cell with an rAAV virion of the present disclosure, wherein the rAAV virion comprises a heterologous nucleic acid comprising a nucleotide sequence encoding: i) an RNA-guided endonuclease that binds a guide RNA; and ii) the guide RNA.
  • the method comprises contacting the retinal cell with a donor DNA template.
  • the RNA-guided endonuclease is a Cas9 polypeptide.
  • the guide RNA is a single-guide RNA.
  • the present disclosure provides a method of treating an ocular disease (e.g., a retinal disease), the method comprising administering to an individual in need thereof an effective amount of a subject rAAV virion as described above.
  • a subject rAAV virion can be administered via intraocular injection, e.g. by intravitreal injection, by subretinal injection, by suprachoroidal injection, or by any other convenient mode or route of administration.
  • Other convenient modes or routes of administration include, e.g., intravenous, intranasal, etc.
  • a “therapeutically effective amount” will fall in a relatively broad range that can be determined through experimentation and/or clinical trials.
  • a therapeutically effective dose will be on the order of from about 10 6 to about 10 15 of the rAAV virions, e.g., from about 10 8 to 10 12 rAAV virions.
  • a therapeutically effective dose will be on the order of from about 10 6 viral genomes (vg) to about 10 15 vg of the rAAV virions, e.g., from about 10 8 vg to 10 12 vg.
  • an effective amount of rAAV virions to be delivered to cells will be on the order of from about 10 8 to about 10 13 of the rAAV virions.
  • an effective amount of rAAV virions to be delivered to cells will be on the order of from about 10 8 to about 10 13 vg of the rAAV virions.
  • an effective amount of rAAV virions to be delivered to cells will be on the order of from about 10 vg/cell to about 10 4 vg/cell.
  • Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.
  • more than one administration may be employed to achieve the desired level of gene expression.
  • the more than one administration is administered at various intervals, e.g., daily, weekly, twice monthly, monthly, every 3 months, every 6 months, yearly, etc.
  • multiple administrations are administered over a period of time of from 1 month to 2 months, from 2 months to 4 months, from 4 months to 8 months, from 8 months to 12 months, from 1 year to 2 years, from 2 years to 5 years, or more than 5 years.
  • Ocular diseases that can be treated using a subject method include, but are not limited to, acute macular neuroretinopathy; Behcet's disease; choroidal neovascularization; diabetic uveitis; histoplasmosis; macular degeneration, such as acute macular degeneration, non-exudative age related macular degeneration and exudative age related macular degeneration; edema, such as macular edema, cystoid macular edema and diabetic macular edema; multifocal choroiditis; ocular trauma which affects a posterior ocular site or location; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal arterial occlusive disease, retinal detachment, uveitic retinal disease; sympathetic opthalmia; Vogt Koyanagi-Ha
  • the present disclosure provides methods of treating retinal disease.
  • the methods generally involve administering an rAAV virion of the present disclosure, or a composition comprising an rAAV virion of the present disclosure, to an eye of an individual in need thereof.
  • Non-limiting methods for assessing treatment of retinal diseases include measuring functional changes, e.g. changes in visual acuity (e.g. BCVA), visual field (e.g. visual field perimetry), electrophysiological responsiveness to light and dark (e.g. ERG, VEP), color vision, and/or contrast sensitivity; measuring changes in anatomy or health using anatomical and/or photographic measures, e.g. OCT, fundus photography, and/or autofluorescence; and measuring ocular motility (e.g. nystagmus, fixation preference, and stability).
  • functional changes e.g. changes in visual acuity (e.g. BCVA), visual field (e.g. visual field perimetry), electrophysiological responsiveness to light and dark (e.g.
  • an effective amount of rAAV virions by testing for an effect on one or more parameters, e.g. visual acuity, visual field, electrophysiological responsiveness to light and dark, color vision, contrast sensitivity, anatomy, retinal health and vasculature, ocular motility, fixation preference, and stability.
  • administering an effective amount of an rAAV virion of the present disclosure results in a decrease in the rate of loss of retinal function, anatomical integrity, or retinal health, e.g. a 2-fold, 3-fold, 4-fold, or 5-fold or more decrease in the rate of loss and hence progression of disease, e.g.
  • administering an effective amount of an rAAV virion of the present disclosure results in a gain in retinal function, an improvement in retinal anatomy or health, and/or a stabilization in ocular motility, e.g. a 2-fold, 3-fold, 4-fold or 5-fold improvement or more in retinal function, retinal anatomy or health, and/or stability of the orbital, e.g. a 10-fold improvement or more in retinal function, retinal anatomy or health, and/or stability of the orbital.
  • the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence that encodes a subject variant adeno-associated virus (AAV) capsid protein as described above, where the variant AAV capsid protein comprises an insertion of from about 5 amino acids to about 20 amino acids in the GH loop or loop IV relative to a corresponding parental AAV capsid protein, or where the variant AAV capsid protein comprises a replacement of from about 5 amino acids to about 20 amino acids in the GH loop or loop IV relative to a corresponding parental AAV capsid protein with a heterologous peptide of from about 5 amino acids to about 20 amino acids; and where the variant capsid protein, when present in an AAV virion, provides for increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • a subject isolated nucleic acid can be an AAV vector, e.g., a recombinant AAV vector.
  • a variant AAV capsid protein encoded by a subject nucleic acid has an insertion peptide of from about 5 amino acids to about 20 amino acids in length is inserted into the GH loop of an AAV capsid.
  • the insertion peptide has a length of 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids.
  • Suitable insertion peptides are as described above.
  • Suitable insertion peptides include a peptide of any one of Formulas I-VI, as described above.
  • a variant AAV capsid protein encoded by a subject nucleic acid comprises a replacement of from about 5 amino acids to about 20 amino acids in the GH loop or loop IV relative to a corresponding parental AAV capsid protein with a heterologous peptide of from about 5 amino acids to about 20 amino acids, where suitable heterologous peptides include a peptide of any one of Formulas I-VI, as described above.
  • a subject recombinant AAV vector can be used to generate a subject recombinant AAV virion, as described above.
  • the present disclosure provides a recombinant AAV vector that, when introduced into a suitable cell, can provide for production of a subject recombinant AAV virion.
  • the present invention further provides host cells, e.g., isolated (genetically modified) host cells, comprising a subject nucleic acid.
  • a subject host cell can be an isolated cell, e.g., a cell in in vitro culture.
  • a subject host cell is useful for producing a subject rAAV virion, as described below. Where a subject host cell is used to produce a subject rAAV virion, it is referred to as a “packaging cell.”
  • a subject host cell is stably genetically modified with a subject nucleic acid.
  • a subject host cell is transiently genetically modified with a subject nucleic acid.
  • a subject nucleic acid is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like.
  • a subject nucleic acid will generally further include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, and the like.
  • a subject host cell is generated by introducing a subject nucleic acid into any of a variety of cells, e.g., mammalian cells, including, e.g., murine cells, and primate cells (e.g., human cells).
  • Suitable mammalian cells include, but are not limited to, primary cells and cell lines, where suitable cell lines include, but are not limited to, 293 cells, 293T cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO cells, and the like.
  • suitable host cells include, e.g., HeLa cells (e.g., American Type Culture Collection (ATCC) No.
  • ATCC American Type Culture Collection
  • CCL-2 CHO cells
  • CHO cells e.g., ATCC Nos. CRL9618, CCL61, CRL9096
  • 293 cells e.g., ATCC No. CRL-1573
  • Vero cells e.g., ATCC No. CRL-1573
  • Vero cells e.g., ATCC No. CRL-1658
  • Huh-7 cells BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.
  • HEK human embryonic kidney
  • a subject host cell can also be made using a baculovirus to infect insect cells such as Sf9 cells, which produce AAV (see, e.g., U.S. Pat. No. 7,271,002; U.S. patent application Ser. No. 12/297,958)
  • a subject genetically modified host cell includes, in addition to a nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, as described above, a nucleic acid that comprises a nucleotide sequence encoding one or more AAV rep proteins.
  • a subject host cell further comprises an rAAV vector.
  • An rAAV virion can be generated using a subject host cell. Methods of generating an rAAV virion are described in, e.g., U.S. Patent Publication No. 2005/0053922 and U.S. Patent Publication No. 2009/0202490.
  • a recombinant adeno-associated virus (rAAV) virion comprising: a) a variant AAV capsid protein, wherein the variant AAV capsid protein comprises an insertion of a heterologous peptide of any one of Formulas I-VI, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by a control AAV virion comprising the corresponding parental AAV capsid protein; and b) a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product.
  • Aspect 2 The rAAV virion of aspect 1, wherein the rAAV virion exhibits at least 5-fold increased infectivity of a retinal cell compared to the infectivity of the retinal cell by a control AAV virion comprising the corresponding parental AAV capsid protein.
  • Aspect 3 The rAAV virion of aspect 1, wherein the rAAV virion exhibits at least 10-fold increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • Aspect 4 The rAAV virion of aspect 1, wherein the insertion of the heterologous peptide replaces a contiguous stretch of from 5 amino acids to 20 amino acids of the parental AAV capsid protein.
  • Aspect 5 The rAAV virion of aspect 1, wherein the insertion site is between amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype.
  • Aspect 6 The rAAV virion of aspect 4, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype; or wherein the insertion site is located between amino acids corresponding to amino acids 585 and 598 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype.
  • Aspect 7 The rAAV virion of any one of aspects 1-6, wherein gene product is an interfering RNA or an aptamer.
  • Aspect 8 The rAAV virion of any one of aspects 1-6, wherein the gene product is a polypeptide.
  • Aspect 9 The rAAV virion of aspect 8, wherein the polypeptide is a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances function of a retinal cell.
  • Aspect 10 The rAAV virion of aspect 8, wherein the polypeptide is an RNA-guided endonuclease selected from a type II CRISPR/Cas polypeptide, a type V CRISPR/Cas polypeptide, and a type VI CRISPR/Cas polypeptide.
  • the polypeptide is 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.
  • Aspect 11 The rAAV virion of aspect 10, wherein the RNA-guided endonuclease is an enzymatically inactive type II CRISPR/Cas polypeptide.
  • Aspect 12 The rAAV virion of aspect 10, wherein the gene product is an RNA-guided endonuclease and a guide RNA.
  • Aspect 13 The rAAV virion of any one of aspects 1-12, 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).
  • Aspect 14 The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide comprises (21) LALIQDSMRA (SEQ ID NO: 35) or (22) LANQEHVKNA (SEQ ID NO: 2).
  • Aspect 15 The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide is a peptide of Formula II: TX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 GLX 9 (SEQ ID NO: 137), where:
  • X 1 is G, V, or S
  • X 2 is V, E, P, G, D, M, A, or S;
  • X 3 is M, V, Y, H, G, S, or D;
  • X 4 is R, D, S, G, V, Y, T, H, or M;
  • X 5 is S, L, G, T, Q, P, or A;
  • X 6 is T, A, S, M, D, Q, or H;
  • X 7 is N, G, S, L, M, P, G, or A;
  • X 8 is S, G, D, N, A, I, P, or T;
  • X 9 is S or N.
  • Aspect 16 The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide comprises: (1) TGVMRSTNSGLN (SEQ ID NO: 6); (2) TGEVDLAGGGLS (SEQ ID No: 7); (3) TSPYSGSSDGLS (SEQ ID NO: 8); (4) TGGHDSSLDGLS (SEQ ID NO: 9); (5) TGDGGTTMNGLS (SEQ ID NO: 98); (6) TGGHGSAPDGLS (SEQ ID NO: 99); (7) TGMHVTMMAGLN (SEQ ID NO: 100); (8) TGASYLDNSGLS (SEQ ID NO: 101); (9) TVVSTQAGIGLS (SEQ ID NO: 135); (10) TGVMHSQASGLS (SEQ ID NO: 21); (11) TGDGSPAAPGLS (SEQ ID NO: 22); or (12) TGSDMAHGTGLS (SEQ ID NO: 23)
  • Aspect 17 The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide is a peptide of Formula III: TGX 1 X 2 X 3 X 4 X 5 X 6 X 7 GLS (SEQ ID NO: 138), where:
  • X 1 is V, E, P, G, D, M, A, or S;
  • X 2 is M, V, Y, H, G, S, or D;
  • X 3 is R, D, S, G, V, Y, T, H, or M;
  • X 4 is S, L, G, T, Q, P, or A;
  • X 5 is T, A, S, M, D, Q, or H;
  • X 6 is N, G, S, L, M, P, G, or A;
  • X 7 is S, G, D, N, A, I, P, or T.
  • Aspect 18 The rAAV virion of any one of aspects 1-12, 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).
  • 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) TGASYLDNS
  • Aspect 19 The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide is a peptide of Formula IV: X 1 GX 2 X 3 X 4 X 5 X 6 X 7 X 8 GLSPX 9 TX 10 X 11 (SEQ ID NO: 139), where
  • X 1 is T or N
  • X 2 is L, S, A, or G
  • X 3 is D or V
  • X 4 is A, G, or P
  • X 5 is T or D
  • X 6 is R or Y
  • X 7 is D, T, or G
  • X 8 is H, R, or T
  • X 9 is V or A
  • X 10 is G or W
  • X 11 is T or A.
  • Aspect 20 The rAAV virion of any one of aspects 1-12, 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).
  • Aspect 21 The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide is a peptide of Formula V: TGX 1 DX 2 TRX 3 X 4 GLSPVTGT (SEQ ID NO: 140), where
  • X 1 is L, S, A, or G
  • X 2 is A, G, or P
  • X 3 is D, T, or G
  • X 4 is H, R, or T
  • Aspect 22 The rAAV virion of any one of aspects 1-12, wherein the heterologous peptide is a peptide of Formula VI: LQX 1 X 2 X 3 RX 4 X 5 X 6 X 7 X 8 X 9 VNX 10 Q (SEQ ID NO: 141), where
  • X 1 is K or R
  • X 2 is N, G, or A
  • X 3 is A, V, N, or D;
  • X 4 is P, I, or Q
  • X 5 is A, P, or V
  • X 6 is S, T, or G
  • X 7 is T or V
  • X 8 is E, L, A, or V;
  • X 9 is S, E, D, or V
  • X 10 is F, G, T, or C.
  • Aspect 23 The rAAV virion of any one of aspects 1-12, 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).
  • a pharmaceutical composition comprising:
  • a method of delivering a gene product to a retinal cell in an individual comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according any one of aspects 1-23 or the composition of aspect 24.
  • rAAV adeno-associated virus
  • Aspect 26 The method of aspect 25, wherein the gene product is a polypeptide.
  • Aspect 27 The method of aspect 25, wherein the gene product is a short interfering RNA or an aptamer.
  • Aspect 28 The method of aspect 26, wherein the polypeptide is a neuroprotective factor, an anti-angiogenic polypeptide, an anti-apoptotic factor, or a polypeptide that enhances function of a retinal cell.
  • Aspect 29 The method of aspect 26, wherein the polypeptide is glial derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis, retinoschisin, RPE65, retinitis pigmentosa GTPase-interacting protein-1, peripherin, peripherin-2, a rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), or Sonic hedgehog.
  • the polypeptide is glial derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis, retinoschisin, RPE65, retin
  • Aspect 30 The method of aspect 26, wherein the polypeptide is an RNA-guided endonuclease.
  • a method of treating an ocular disease comprising administering to an individual in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion according to any one of aspects 1-23 or the composition of aspect 24.
  • rAAV recombinant adeno-associated virus
  • Aspect 32 The method of aspect 31, wherein said administering is by intraocular injection.
  • Aspect 33 The method of aspect 31, wherein said administering is by intravitreal injection or by suprachoroidal injection.
  • Aspect 34 The method of any one of aspects 31-33, wherein the ocular disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's Congenital Amaurosis, diabetic retinopathy, achromotopsia, or color blindness.
  • Aspect 35 An isolated nucleic acid comprising a nucleotide sequence that encodes a variant adeno-associated virus (AAV) capsid protein, wherein the variant AAV capsid protein comprises an insertion of from about 5 amino acids to about 20 amino acids in the capsid protein GH loop relative to a corresponding parental AAV capsid protein, and wherein the variant capsid protein, when present in an AAV virion, provides for increased infectivity of the AAV virion of a retinal cell, and wherein the amino acid insertion is in the GH loop of a native AAV capsid, wherein the insertion is a peptide of any one of Formulas I-VI.
  • AAV adeno-associated virus
  • Aspect 36 The isolated nucleic acid of aspect 35, wherein the insertion site is between amino acids 587 and 588 of AAV2, between amino acids 585 and 598 of AAV2, between amino acids 590 and 591 of AAV1, between amino acids 575 and 576 of AAV5, between amino acids 590 and 591 of AAV6, between amino acids 589 and 590 of AAV7, between amino acids 590 and 591 of AAV8, between amino acids 588 and 589 of AAV9, or between amino acids 588 and 589 of AAV10.
  • Aspect 37 An isolated, genetically modified host cell comprising the nucleic acid of aspect 35 or aspect 36.
  • a variant adeno-associated virus (AAV) capsid protein wherein the variant AAV capsid protein comprises an insertion of from about 5 amino acids to about 20 amino acids wherein the amino acid insertion is in the GH loop of a native AAV capsid, wherein the insertion is a peptide of any one of Formulas I-VI.
  • AAV adeno-associated virus
  • a recombinant adeno-associated virus (rAAV) virion comprising:
  • variant AAV capsid protein comprises an insertion of a heterologous peptide of Formula VI, and wherein the variant capsid protein confers increased infectivity of a retinal cell compared to the infectivity of the retinal cell by a control AAV virion comprising the corresponding parental AAV capsid protein;
  • a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product.
  • Aspect 40 The rAAV virion of aspect 39, wherein the rAAV virion exhibits at least 5-fold increased infectivity of a retinal cell compared to the infectivity of the retinal cell by a control AAV virion comprising the corresponding parental AAV capsid protein.
  • Aspect 41 The rAAV virion of aspect 39, wherein the rAAV virion exhibits at least 10-fold increased infectivity of a retinal cell compared to the infectivity of the retinal cell by an AAV virion comprising the corresponding parental AAV capsid protein.
  • Aspect 42 The rAAV virion of any one of aspects 39-41, wherein the insertion of the heterologous peptide replaces a contiguous stretch of from 5 amino acids to 20 amino acids of the parental AAV capsid protein.
  • Aspect 43 The rAAV virion of any one of aspects 39-42, wherein the insertion site is between amino acids corresponding to amino acids 570 and 611 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype.
  • Aspect 44 The rAAV virion of aspect 43, wherein the insertion site is located between amino acids corresponding to amino acids 587 and 588 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype; or wherein the insertion site is located between amino acids corresponding to amino acids 585 and 598 of VP1 of AAV2, or the corresponding position in the capsid protein of another AAV serotype.
  • Aspect 45 The rAAV virion of any one of aspects 39-44, wherein gene product is an interfering RNA.
  • Aspect 46 The rAAV virion of any one of aspects 39-44, wherein gene product is an aptamer.
  • Aspect 47 The rAAV virion of any one of aspects 39-44, wherein the gene product is a polypeptide.
  • Aspect 48 The rAAV virion of aspect 47, wherein the polypeptide is a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances function of a retinal cell.
  • Aspect 49 The rAAV virion of aspect 47, wherein the polypeptide is an RNA-guided endonuclease selected from a type II CRISPR/Cas polypeptide, a type V CRISPR/Cas polypeptide, and a type VI CRISPR/Cas polypeptide.
  • the polypeptide is 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.
  • Aspect 50 The rAAV virion of aspect 49, wherein the RNA-guided endonuclease is an enzymatically inactive type II CRISPR/Cas polypeptide.
  • Aspect 51 The rAAV virion of one of aspects 39-44, wherein the gene product is an RNA-guided endonuclease and a guide RNA.
  • Aspect 52 The rAAV virion of any one of aspects 39-51, 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).
  • a pharmaceutical composition comprising:
  • a method of delivering a gene product to a retinal cell in an individual comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according any one of aspects 39-52 or the composition of aspect 53.
  • rAAV adeno-associated virus
  • Aspect 55 The method of aspect 54, wherein the gene product is a polypeptide.
  • Aspect 56 The method of aspect 54, wherein the gene product is a short interfering RNA or an aptamer.
  • Aspect 57 The method of aspect 55, wherein the polypeptide is a neuroprotective factor, an anti-angiogenic polypeptide, an anti-apoptotic factor, or a polypeptide that enhances function of a retinal cell.
  • Aspect 58 The method of aspect 57, wherein the polypeptide is glial derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis, retinoschisin, RPE65, retinitis pigmentosa GTPase-interacting protein-1, peripherin, peripherin-2, a rhodopsin, RdCVF, retinitis pigmentosa GTPase regulator (RPGR), or Sonic hedgehog.
  • the polypeptide is glial derived neurotrophic factor, fibroblast growth factor 2, neurturin, ciliary neurotrophic factor, nerve growth factor, brain derived neurotrophic factor, epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis, retinoschisin, RPE65,
  • Aspect 59 The method of aspect 55, wherein the polypeptide is an RNA-guided endonuclease.
  • a method of treating an ocular disease comprising administering to an individual in need thereof an effective amount of a recombinant adeno-associated virus (rAAV) virion according to any one of aspects 39-52 or the composition of aspect 53.
  • rAAV recombinant adeno-associated virus
  • Aspect 61 The method of aspect 60, wherein said administering is by intraocular injection.
  • Aspect 62 The method of aspect 60, wherein said administering is by intravitreal injection or by suprachoroidal injection.
  • Aspect 63 The method of any one of aspects 60-62, wherein the ocular disease is glaucoma, retinitis pigmentosa, macular degeneration, retinoschisis, Leber's Congenital Amaurosis, diabetic retinopathy, achromotopsia, or color blindness.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.
  • Example 1 AAV Virions Comprising Variant AAV Capsids
  • a number of variants of AAV capsids were derived through a directed evolution approach; AAV virions comprising the variant AAV capsids infect the primate retina, e.g., when administered via intravitreal injection. Primates are an important preclinical model for human retinal disease, with a fovea for high acuity vision, similar to humans.
  • AAV virions comprising variant AAV capsids were identified by screening. Five libraries were used for this screen: 1) a 7mer peptide display library based on AAV2, containing a 7mer peptide insertion at amino acid ⁇ 588, and surrounded by a 5′ LA linker and a 3′A linker; 2) a 7mer peptide display library based on AAV4, with a 7mer peptide insertion at amino acid ⁇ 584, with a 5′TG linker and a 3′GLS linker; 3) a 7mer peptide display library based on AAV5 with a 7mer peptide insertion at amino acid ⁇ 575 with 5′TG linker and a 3′GLS linker; 4) a library based on an ancestral AAV sequence (Santiago-Ortiz et al., 2015) and containing a 7mer peptide display library at position amino acid ⁇ 591 with a 5′TG linker and a 3′GLS linker; and 5)
  • Virus was packaged such that each viral genome was encapsidated within the capsid protein shell that that genome encoded, as previously described Koerber et al. (2008) supra; Fowler et al. Nat Protoc 9, 2267-2284 (2014). Therefore functional improvements identified through selection can be linked to the genome sequence contained within the viral capsid.
  • AAV vectors were produced by triple transient transfection of HEK293T cells, purified via iodixanol density centrifugation, and buffer exchanged into PBS by Amicon filtration. DNase-resistant viral genomic titers were measured by quantitative real time PCR using a BioRad iCycler.
  • FIG. 1 An iterative in vivo screening selection process was used to identify variants with the ability to infect the primate retina from the vitreous ( FIG. 1 ).
  • Primate eyes were injected in each round with ⁇ 250 ⁇ L of 1 ⁇ 10 ⁇ circumflex over ( ) ⁇ 13 (1E13)-1 ⁇ 10 ⁇ circumflex over ( ) ⁇ 14 (1E14) vg/mL titer virus.
  • Three weeks after injection eyes were enucleated, and retinal punches were taken from central and peripheral regions of the retina ( FIG. 1 ). DNA from various retinal layers was assayed, and the capsid inserts were identified.
  • capsid sequences were recovered by PCR from harvested cells using primers HindIII_F1 and NotI_R1, AscI_R1, or SpeI_R1, with reverse primers being specific to unique AAV backbones, in order to maintain separation of groups of libraries. PCR amplicons were then digested, and recloned into the backbone. RPE cells were separated from retinal tissue, and tissue was frozen. Retinal tissue was embedded and sectioned on a cryostat to isolate photoreceptors in the outer nuclear layer. DNA was then collected from the isolated photoreceptors or RPE, and cap genes were PCR amplified. Recovered cap genes were used for subsequent AAV packaging.
  • FIG. 1 Illustration of the directed evolution methodology used to develop primate retinal AAV variants.
  • Peptide display libraries were created, packaged into AAV vectors, and injected into the primate eye via intravitreal injections. Iterative round of selection were used to positively select AAV variants from the pool of vectors. Three rounds of selection were followed by a round of error prone PCR, followed by additional selection rounds.
  • Illumina deep sequencing was used to identify variants that increased over the rounds in relative representation in the library of AAV variants.
  • An increase of representation in the viral library indicates positive selection and ability to infect the primate retina from the vitreous.
  • a ⁇ 75-85 base pair region containing the 7mer insertion or Loop Swap mutation site was PCR amplified from harvested DNA.
  • Primers included Illumina adapter sequences containing unique barcodes to allow for multiplexing of amplicons from multiple rounds of selection. PCR amplicons were purified and sequenced with a 100-cycle single-read run on an Illumina HiSeq 2500.
  • Custom Python code was written to translate DNA sequences into amino acid sequences, and to identify and count reads containing unique 7mer insert sequences. Read counts were normalized by the total number of reads in the run. Python and Pandas were used to analyze dynamics of directed evolution and create plots.
  • AAV vectors can be used for gene augmentation therapies for retinal degenerative diseases including retinitis pigmentosa, Leber Congenital Amaurosis, Rod-cone dystrophy, cone dystrophy, achromatopsia, X-linked retinoschisis, CRB1, optogenetic therapies, expression of trophic and survival factors such as GDNF, BDNF, FGF, RdCVF, RdCVFL, XIAP, and expression of blockers of neovascularization such as sFLT.
  • the vectors can also be used to deliver gene editing tools such as CRISPR/Cas9 for gene correction or the creation of additional models of retinal disease.
  • DNA barcodes were cloned behind an AAV ITR construct containing a self-complementary CAG promoter driving eGFP (CAG-GFP-Barcode-pA). Individual variants were packaged separately with constructs containing different barcodes. Variants were then titer matched and mixed in equal ratios before injection into mice, dogs, and primates.
  • FIG. 9 provides Table 1;
  • FIG. 10 provides Table 2.
  • Table 1 provides a ranking of primate-derived variants and controls recovered from photoreceptors following injection of a GFP-Barcode library.
  • Table 2 provides a ranking of primate-derived variants and controls recovered from RPE cells following injection of a GFP-Barcode library.
  • the library contained individual variants packaged with GFP fused to a unique DNA barcode. Polymerase chain reaction (PCR) was used to amplify barcodes from DNA recovered from specific cell types in the retina. “Region” in Tables 1 and 2 indicates the region from which the DNA was recovered.
  • the fold increase of reads of each of the variants was calculated by dividing 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 indicates the average of the fold increase across multiple locations in the retina. Variants were ranked by fold increase of the barcode.
  • FIG. 11 GFP expression of GFP-barcoded libraries in primate retina.
  • GFP expression resulting from intravitreal injection of pooled, GFP-barcoded library (which contains all the tested viruses) was located primarily in the outer retina, with a tropism that was directed more toward the outer retina than expression of AAV24YF.
  • Cynomolgous monkeys between 4-10 years old were used for all studies, and intravitreal injections were made.
  • the monkey used for fluorophore expression received daily subcutaneous injections of cyclosporine at a dose of 6 mg/kg for immune suppression, and adjusted based on blood trough levels to within a 150-200 ng/ml target range.
  • Confocal scanning laser ophthalmoscopic images (Spectralis HRA, Heidelberg Engineering) were obtained from the two retinas at 3 weeks after injection, with autofluorescence settings, which leads to effective tdTomato and GFP visualization.
  • the monkey was euthanized, both retinas were lightly fixed in 4% paraformaldehyde, and tissue was examined by confocal microscopy.
  • euthanasia was achieved by administering an IV overdose of sodium pentobarbital (75 mg kg ⁇ 1), as recommended by the Panel on Euthanasia of the American Veterinary Medical Association.
  • Pieces of primate retina were then prepared in 30% sucrose, embedded in OCT media, flash frozen, and sectioned at 20 m for confocal microscopy imaging of native fluorophore expression.
  • Antibodies for labeling were: anti-GFP (A11122, Thermo, 1:250) anti-vimentin (Dako, 1:1000), peanut agglutinin (PNA) (Molecular Probes, 1:200), and anti-cone arrestin (7G6, 1:50). The procedures were conducted according to the ARVO Statement for the Use of Animals and the guidelines of the Office of Laboratory Animal Care at the University of Rochester.
  • the nonhuman primate is a critical preclinical model for human therapeutic development, as it is most closely related to, and has a retinal anatomy similar to that of humans.
  • primates are the only large animal model that possesses a fovea, the specialized high acuity area of the retina that is most important for daily activities such as reading, is critical to quality of life, and is lost in numerous retinal degenerations.
  • the species specificity observed in the canine study motivated us to pursue an additional course of directed evolution in primate retina.
  • Nine libraries were packaged and included in the primate screen: EP2, EP5, EP6, EP8, EP9, EP-Ancestral, AAV2-7mer, Ancestral-7mer (Santiago-Ortiz et al.
  • Deep sequencing revealed that, similar to observations from the canine screen, libraries contained ⁇ 1E+6- ⁇ 1E+7 individual variants, which converged to ⁇ 1E+4 ⁇ ⁇ 1E+5 variants over 6 rounds of selection, a diversity not possible to observe through Sanger sequencing ( FIG. 12A ).
  • FIG. 12B shows that in each of the libraries analyzed, a small portion of library members were over-represented in the initial plasmid library.
  • FIG. 12C Analysis of results from high throughput sequencing over the rounds of selection revealed, for each of the libraries, a subset of variants that increased significantly in their representation during rounds of selection.
  • FIG. 12C Sixteen variants, from these 5 libraries ( FIG. 12C ), were selected to be included in a secondary round of selection with GFP-barcoded libraries, along with AAV2, AAV2-4YF+TV, AAV4 and AAV5 as controls.
  • This new library was injected in both eyes of a primate, and 3 weeks after injection, biopsies were collected from locations across the retina ( FIG. 12D ).
  • GFP expression resulting from injection of the GFP-barcode libraries was primarily found in photoreceptors, as well as some inner retinal cells, a tropism that is shifted from AAV2 or 7m8, which yielded stronger inner retinal expression ( FIG. 12E ).
  • FIG. 12A-12F Directed Evolution of AAV in Primate Retina.
  • A Deep sequencing of variant libraries revealed convergence of variants over rounds of selection.
  • B In each of the libraries evaluated, a small proportion of variants are overrepresented in the plasmid library.
  • C Scatterplots illustrate the behavior of individual variants at the final round of selection for each of the libraries injected in primate retinas. Variants overrepresented in the original library are colored blue. Variants that had the greatest fold increase in representation in the final round of selection are shown in magenta. Variants that were overrepresented in the original library and increased significantly in representation over rounds of selection are colored orange.
  • D A map of the primate retina shows the distribution of samples that were collected for rounds of selection and the GFP-barcode library. Color coding of variants is the same as in FIG. 2 .
  • AAV2-LALIQDSMRA (SEQ ID NO: 117; designated NHP #9), the second ranking variant from the GFP-barcode screen, which packaged at high titers ( ⁇ 5E+13 vg/mL), was therefore selected for a first round of validation studies focusing on ganglion cells of the inner retina and cones of the outer retina. Cone photoreceptors are involved in adult macular degeneration (AMD), the most common cause of blindness in developed countries that are predicted to affect 288 million people worldwide by the year 2040, and are therefore a primary target for retinal gene therapy. NHP #9 was packaged with an SNCG promoter driving tdTomato in RGCs and the pR1.7 promoter driving expression of GFP in cones.
  • Vectors encoding both these constructs were mixed in equal ratios ( ⁇ 1.5E+12 vg/construct/eye, and injected intravitreally in a cynomolgous monkey.
  • a previously described variant, 7m8 (Dalkara et al. (2013) supra) packaged with equal titers of the same constructs was injected into the vitreous of the contralateral eye.
  • Expression of tdTomato reporter in RGC's was lower in NHP #9-injected eyes compared to 7m8, which infected ganglion cells across the expanse of the retina efficiently; however, expression in foveal cones was greatly increased relative to 7m8, indicating a shift in tropism away from the inner retina towards photoreceptors in the outer retina.
  • qRT-PCR performed using the ddCT method, revealed an 11.71 (10.37-13.22) fold increase of GFP expression in foveal cones relative to 7m8.
  • Counting of labeled cells performed with Imaris software on images collected from flatmounted retinas, also confirmed a substantial decrease in numbers of transduced ganglion cells and an increase in the number of cones targeted with NHP #9.
  • Loopswap variant ⁇ 588-LQRGVRIPSVLEVNGQ (SEQ ID NO: 118; designated NHP #26) was also tested for validation, although low numbers of viral particles were produced.
  • ⁇ 5E+10 particles of NHP #26-scCAG-eGFP were injected intravitreally into one eye of a cynomolgous monkey. Although the number of particles injected was low, efficient expression of GFP was observed in the fovea and across the retina ( FIG. 13G ). In contrast to the foveal-spot-and-ring pattern of expression that was observed with 7m8, NHP #9 ( FIG.
  • FIG. 13A fundus imaging of NHP #26 resulted in a disc of GFP expression centered on the foveola ( FIG. 13G ).
  • FIG. 13A-13Q Validation of evolved AAV variants in primate retina.
  • A-F Co-injection of ⁇ 1.5E+12 particles of SCNG-tdTomato and ⁇ 1.5E+12 pR1.7-eGFP packaged in 7m8 and variant NHP #9 in primate retina.
  • Intravitreal injection of 7m8 (A,C,E) resulted in robust tdTomato expression in ganglion cells and expression of GFP in foveal cones.
  • injection of equal number of particles of NHP #9 resulted in reduced ganglion cell expression, and increased GFP expression in cones relative to 7m8 (B,D,F).
  • G Fundus imaging in a primate eye following injection of 5E+10 particles of NHP #26-scCAG-GFP resulted in a disc of GFP expression centered on the fovea, and a punctate pattern of GFP expression across the retina.
  • H Confocal imaging of native GFP expression in the flatmounted fovea.
  • I Confocal imaging of native GFP expression in the area outside of the vascular arcade.
  • J Confocal imaging of native GFP expression in a cryostat section through the fovea.
  • K Native GFP expression in inferior retina, outside the vascular arcade, shows little GFP expression in ganglion cells, but high levels of expression in Müller cells and in photoreceptors in outer retina. Autofluorescence was also observed in RPE.
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