Variant AAV capsid for intravitreal delivery

Abstract

Variant adeno-associated virus (AAV) capsid proteins and recombinant AAV virions having one or more variant AAV capsid proteins are provided. Also provided are compositions and methods of using the recombinant AAV virions, such as for treating or preventing a disease or disorder.

Description

[0001] This application is a divisional application of a patent application with a filing date of April 24, 2020, application number 202080031274.0, and name “Variant AAV capsid for intravitreal delivery”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 839,548, filed on April 26, 2019, and U.S. Provisional Application No. 62 / 923,924, filed on October 21, 2019, the entire contents of each of which are incorporated herein by reference. Sequence listings submitted as ASCII text files

[0004] The entire contents submitted below in an ASCII text file are incorporated herein by reference: Computer Readable Form (CRF) of the Sequence Listing (File Name: 627002001140SEQLIST.TXT, Record Date: April 21, 2020, Size: 49KB). Background Art

[0005] A promising method for treating and preventing genetic and other diseases and disorders is to deliver therapeutic agents with gene therapy vectors such as virions. Illustrative examples of virions suitable for gene therapy include, but are not limited to, retroviral particles, slow virus particles, adenoviral particles, herpes virus particles, alpha virus particles, and adeno-associated virus (AAV) ions. AAV is a 4.7kb single-stranded DNA virus. Recombinant virions (rAAV virions) based on AAV have excellent clinical safety because wild-type AAV is non-pathogenic and has no causal relationship with any known disease. In addition, AAV provides the ability to carry out very efficient gene delivery and sustained transgenic expression in many tissues including eyes, muscles, lungs, and brain.

[0006] Certain challenges remain in designing viral particles for gene therapy, including optimizing viral cell tropism, and particularly in ocular gene therapy, optimizing delivery to the retina. Thus, there is a need to optimize viral particles for gene expression in selected mammalian cells. The present invention addresses this need by providing modified AAV capsid proteins that facilitate delivery of recombinant AAV viral particles to desired cells and tissues. Summary of the invention

[0007] Provided herein are recombinant adeno-associated virus (AAV) virions comprising: (a) a variant AAV capsid protein comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the amino acid residue numbering corresponds to AAV5 VP1 capsid protein; and (b) a polynucleotide sequence encoding a therapeutic gene product.

[0008] In some embodiments, the parent AAV capsid protein is an AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein. In some embodiments, the parent AAV capsid protein is an AAV2.5T capsid protein. In some embodiments, the parent AAV capsid protein is an AAV2.5T VP1 capsid protein.

[0009] In some embodiments, the modified sequence comprises LAHKFKSGDA (SEQ ID NO:3). In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the variant AAV capsid protein comprises a capsid sequence shown in SEQ ID NO:6 or SEQ ID NO:7.

[0010] In some embodiments, the rAAV virion is a variant AAV5 or a variant AAV2 and AAV5 hybrid virion. In some embodiments, the rAAV virion is a variant AAV2.5T virion.

[0011] In some embodiments, the recombinant AAV viral particles are capable of transducing cells of the retina when injected intravitreally into a mammal. In some embodiments, the recombinant AAV viral particles are capable of transducing one or more of the following when injected intravitreally into a mammal: photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, and retinal pigment epithelial cells. In some embodiments, the recombinant AAV viral particles are capable of transducing retinal pigment epithelial cells when injected intravitreally into a mammal.

[0012] In some embodiments, the therapeutic gene product is a siRNA, miRNA or protein. In some embodiments, the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product. In some embodiments, the therapeutic gene product is an opsin.

[0013] In some embodiments, the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs. In some embodiments, the one or more AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs or variants thereof. In some embodiments, the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs.

[0014] In some embodiments, the recombinant AAV virion has altered cellular tropism compared to AAV2.5T.

[0015] In some embodiments, the recombinant AAV virions disclosed herein are methods for treating retinal diseases or conditions in subjects in need thereof, wherein the method comprises administering a pharmaceutical composition comprising recombinant AAV virions to a subject by intravitreal injection. In some embodiments, the recombinant AAV virions disclosed herein are used to prepare a medicament for treating a retinal disease or condition in a subject. In some embodiments, the disease or condition is age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, or diabetic retinal edema.

[0016] Also provided herein are pharmaceutical compositions comprising the recombinant AAV virions disclosed herein.

[0017] Also provided herein is a method for producing rAAV virions, comprising: (a) culturing a host cell under conditions that produce rAAV virions, wherein the host cell comprises: (i) a polynucleotide encoding a variant AAV capsid protein comprising a modified sequence, wherein the modified sequence comprises one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1); (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide expression cassette comprising a sequence encoding a therapeutic gene product flanked by at least one AAV ITR; and (iv) AAV helper functions; and (b) recovering the rAAV virions produced by the host cell.

[0018] Also provided herein is a method of providing a therapeutic gene product to the retina of a subject, comprising administering to the subject by intravitreal injection a recombinant AAV virion disclosed herein or a pharmaceutical composition disclosed herein.

[0019] In some embodiments, the subject has been diagnosed with or is suspected of having one or more conditions selected from the group consisting of acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior part of the eye, ocular tumor, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia, Vogt's Koyanagi-Harada (VKH) syndrome, uveal ectasia, posterior eye conditions caused or affected by ocular laser therapy, posterior eye conditions caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorders, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinopathy, ischemic retinopathy, non-retinopathic diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, choroideremia, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, and color vision impairment.

[0020] Also provided herein are methods of treating a retinal disease or disorder in a subject in need thereof, comprising administering to the subject by intravitreal injection a recombinant AAV virion disclosed herein or a pharmaceutical composition disclosed herein.

[0021] In some embodiments, the disease or condition is acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior part of the eye, ocular tumor, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia, Vogt's Koyanagi-Harada (VKH) syndrome, uveal ectasia, posterior eye conditions caused or affected by ocular laser therapy, posterior eye conditions caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinopathy, ischemic retinopathy, non-retinopathic diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, choroideremia, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, or color vision impairment.

[0022] Also provided herein are variant AAV capsid proteins comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the amino acid residue numbering corresponds to AAV5 VP1 capsid protein.

[0023] In some embodiments, the parent AAV capsid protein is an AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein. In some embodiments, the parent AAV capsid protein is an AAV2.5T capsid protein. In some embodiments, the parent AAV capsid protein is an AAV2.5T VP1 capsid protein.

[0024] In some embodiments, the modified AAV capsid protein comprises LAHKFKSGDA (SEQ ID NO: 3) at amino acid residues 570-579 relative to the parent AAV capsid protein. In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the variant AAV capsid protein comprises a capsid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7.

[0025] Also provided herein are nucleic acids comprising a nucleic acid sequence encoding a variant AAV capsid protein disclosed herein.

[0026] Also provided herein is an expression vector comprising a nucleic acid disclosed herein, wherein the nucleic acid sequence encoding the variant AAV capsid protein is operably linked to a promoter sequence. In some embodiments, the expression vector further comprises a nucleic acid encoding a rep protein.

[0027] Also provided herein are cells comprising expression vectors disclosed herein. In some embodiments, the cells further comprise a nucleic acid encoding a therapeutic gene product. In some embodiments, the therapeutic gene product is a siRNA, miRNA, or protein. In some embodiments, the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product. In some embodiments, the therapeutic agent is an opsin.

[0028] In some embodiments, the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs. In some embodiments, the one or more AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs or variants thereof. In some embodiments, the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs.

[0029] All references cited herein, including patent applications and publications, are each incorporated by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1A A schematic diagram of the retina and the locations of the concentric sample punch holes is shown. Punch 1 contains the macula (small circular indentation); Punch 2 contains the fovea; the remaining tissue contains the peripheral retina, including the blood arcade.

[0031] Figure 1B-1E The relative abundance of capsid variants at different stages of the selection process is shown. Figure 1B The percentages of the top 100 variants in the original library are shown; Figure 1C The percentages of the top 100 variants identified in the second screening round in parafoveal, macular, or peripheral retinal areas are shown. Figure 1D Shown are the percentages of the top 50 evolved variants that successfully transduced both parafoveal and RPE cells in the second round library derived from the parafovea. Figure 1EThe percentage of the top 50 evolved variants that successfully transduced macular+peripheral cells in the second round library derived from the parafovea is shown.

[0032] Figure 2 Dot blot results of heparin column fractions from AAV2.5T and variant capsid AAV2.5T.LSV1 are shown. The NaCl concentrations of eluents E1 to E10 gradually increased, 0.1 M (E1), 0.2 M (E2), 0.3 M (E3), 0.4 M (E4), 0.5 M (E5), 0.6 M (E6), 0.7 M (E7), 0.8 M (E8), 0.9 M (E9), and 1.0 M (E10), respectively.

[0033] Figure 3 Shown are live images of GFP expression in 6 mm porcine retinal explants at day 7 or day 14 after infection with AAV2.5T.LSV1 virions encoding GFP at 2E+10 vg (ie, 2E10 vg) or 4E+10 vg (ie, 4E10 vg).

[0034] Figures 4A-4C The results of intravitreal administration of AAV2.5T.LSV1 virions encoding GFP on day 14 ( Figure 4A ), Day 21( Figure 4B ) and Day 28 ( Figure 4C ) OTC autofluorescence image of the African green monkey retina showing GFP expression in the fovea and blood arches.

[0035] Figure 4D-4E Provided are images of the left side of an African green monkey ( Figure 4D ) and right side ( Figure 4E ) Heidelberg spectrum image of the retina showing GFP expression in the fovea and hemorrhagic arches.

[0036] Figure 5A-5B Fluorescent images of African green monkey flat-mounted retinas following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP.

[0037] Figure 5C-5D Provided are flat-mounted fluorescent images of the retina of an African green monkey following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP, left eye ( Figure 5C ) and right eye ( Figure 5D ).

[0038] Figure 5E Provided in Figure 5C-5DGFP intensity profile plotted against macula-to-ciliary distance for the eye shown in Figure 1. Retinal area positions were calculated based on mean distance relative to the center of the fovea.

[0039] Figures 6A-6C Provided are foveal-to-ciliary fluorescence images of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig. 6A GFP expression is shown. Figure 6B DAPI staining of cell nuclei is shown. Figure 6C yes Fig. 6A and Figure 6B Composite image of .

[0040] Figure 7 Provided are fluorescent images of GFP expressed in the fovea of ​​African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP.

[0041] Figure 8 Provided are fluorescent images of GFP expressed in the fovea of ​​African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP.

[0042] Figures 9A-9D Provided are fluorescent images of the fovea of ​​African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig. 9A Shows Figures 9B-9D Composite image of . Fig. 9B DAPI staining of cell nuclei is shown. Fig. 9C GFP expression is shown. Fig.9D Rhodopsin antibody labeling of rod photoreceptors is shown.

[0043] Figures 10A-10D Provided are fluorescent images of the fovea of ​​African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig. 10A Shows Figures 10B-10D Composite image of . Fig. 10B DAPI staining of cell nuclei is shown. Fig. 10C GFP expression is shown. Fig. 10D Glutamine synthetase antibody labeling of Müller cells is shown.

[0044] Fig.11 Provided are fluorescent images of GFP expressed in the retina of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP.

[0045] Figures 12A-12CProvided are fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig. 12A yes Fig. 12B and Fig. 12C Composite image of . Fig. 12B DAPI staining of cell nuclei is shown. Figure 12 shows GFP expression.

[0046] Figures 13A-13D Provided are fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.13A Shows Figures 13B-13D Composite image of . Fig. 13B DAPI staining of cell nuclei is shown. Fig. 13C GFP expression is shown. Fig.13D Glutamine synthetase antibody labeling of Müller cells is shown.

[0047] Figures 14A-14D Provided are fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.14A Shows Figures 14B-14D Composite image of . Fig. 14B DAPI staining of cell nuclei is shown. Fig. 14C GFP expression is shown. Fig.14D Glutamine synthetase antibody labeling of Müller cells is shown.

[0048] Figures 15A-15D Provided are fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.15A Shows Figures 15B-15D Composite image of . Fig. 15B DAPI staining of cell nuclei is shown. Fig. 15C GFP expression is shown. Fig.15D Glutamine synthetase antibody labeling of Müller cells is shown.

[0049] Figures 16A-16E Provided are fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.16A Shows Figures 16B-16E Composite image of . Fig. 16B DAPI staining of cell nuclei is shown. Fig. 16C GFP expression is shown. Fig.16D PCKα antibody labeling of bipolar cells is shown. Fig.16E TUJ-1 antibody labeling of retinal ganglion axon terminal pathways is shown.

[0050] Figures 17A-17D Provided are fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.17A Shows Figures 17B-17D Composite image of . Fig. 17B DAPI staining of cell nuclei is shown. Fig. 17C GFP expression is shown. Fig.17D PCKα antibody labeling of bipolar cells is shown.

[0051] Figures 18A-18E Provided are fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.18A Shows Figures 18B-18E Composite image of . Fig.18B DAPI staining of cell nuclei is shown. Fig.18C GFP expression is shown. Fig.18D Cone arrestin antibody labeling of cone photoreceptors is shown. Fig.18E Rhodopsin antibody labeling of rod photoreceptors is shown.

[0052] Figures 19A-19E Provided are fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.19A Shows Figures 19B-19E Composite image of . Fig.19B DAPI staining of cell nuclei is shown. Fig.19C GFP expression is shown. Fig.19D Cone arrestin antibody labeling of cone photoreceptors is shown. Fig.19E Rhodopsin antibody labeling of rod photoreceptors is shown.

[0053] Figures 20A-20E Provided are fluorescent images of the retina of an African green monkey eye following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig. 20A Shows Figures 20B-20E Composite image of . Fig. 20B DAPI staining of cell nuclei is shown. Fig. 20C GFP expression is shown. Fig.20D Cone arrestin antibody labeling of cone photoreceptors is shown. Fig.20E Rhodopsin antibody labeling of rod photoreceptors is shown.

[0054] Figures 21A-21E Provided are fluorescent images of the retinal pigment epithelium (RPE) of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.21A Shows Figures 21B-21EComposite image of . Fig. 21B DAPI staining of cell nuclei is shown. Fig. 21C Native GFP expression without antibody labeling is shown. Fig.21D RPE65 antibody labeling of cones is shown. Fig.21E GFP expression at 647 nm is shown.

[0055] Figures 22A-22E Provided are fluorescent images of the retinal pigment epithelium (RPE) of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.22A Shows Figures 22B-22E Composite image of . Fig. 22B DAPI staining of cell nuclei is shown. Fig. 22C Native GFP expression without antibody labeling is shown. Fig.22D RPE65 antibody labeling of cones is shown. Fig.22E GFP expression at 647 nm is shown.

[0056] Figures 23A-23E Provided are planar fluorescence images of the retinal pigment epithelium (RPE) of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.23A Shows Figures 23B-23E Composite image of . Fig. 23B DAPI staining of cell nuclei is shown. Fig.23C Native GFP expression without antibody labeling is shown. Fig.23D RPE65 antibody labeling of cones is shown. Fig.23E GFP expression at 647 nm is shown.

[0057] Figures 24A-24E Provided are planar fluorescence images of the retinal pigment epithelium (RPE) of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.24A Shows Figures 24B-24E Composite image of . Fig. 24B DAPI staining of cell nuclei is shown. Fig.24C Native GFP expression without antibody labeling is shown. Fig.24D RPE65 antibody labeling of cones is shown. Fig.24E GFP expression at 647 nm is shown.

[0058] Figures 25A-25E Provided are planar fluorescence images of the retinal pigment epithelium (RPE) of African green monkey eyes following intravitreal administration of AAV2.5T.LSV1 virions encoding GFP. Fig.25A Shows Figures 25B-25E Composite image of . Fig.25B DAPI staining of cell nuclei is shown. Fig.25C Native GFP expression without antibody labeling is shown. Fig.25D RPE65 antibody labeling of cones is shown. Fig.25E GFP expression at 647 nm is shown.

[0059] Figures 26A-26C GFP-positive bipolar cells in various regions of the retina of African green monkey eyes after intravitreal administration of AAV2.5T.LSV1 virions encoding GFP ( Fig.26A ), cones ( Fig.26B ) and RPE cells ( Fig.26C )’s average percentage.

[0060] Figures 27A-27B Neutralizing antibody (nAB) curves against AAV2.5T.LSV1 and AAV2 are provided. Pooled human IgG antibodies (Gammagard IVIG) were combined with AAV2.5T.LSV1-CMV-GFP or AAV2-CMV-GFP. 293T cells were transduced with the mixture and incubated for 3 days before measuring GFP expression. Fig.27A Shown are IVIG neutralizing antibody curves against AAV2.5T.LSV1-CMV-GFP (N=3 for each dilution). Fig.27B IVIG neutralizing antibody curves against AAV2-CMV-GFP are shown (N=2 or 3 for each dilution). DETAILED DESCRIPTION

[0061] The present disclosure provides variant AAV capsid proteins and recombinant adeno-associated virus (AAV) virions having one or more variant AAV capsid proteins. In some embodiments, the recombinant AAV virions show one or more of the following properties compared to the corresponding viral vectors containing their natural, wild-type and / or parent capsid proteins rather than the modified capsid proteins disclosed herein: 1) increased infectivity of retinal cells; 2) altered tropism; 3) increased binding to heparin and / or heparan sulfate proteoglycans and / or inner limiting membrane (ILM); and 4) increased ability to infect and / or deliver therapeutic gene products across the ILM when administered intravitreally. Also provided are compositions and methods for promoting the expression of therapeutic gene products in individual cells (e.g., retinal cells), for example, to treat or prevent a disease or condition using any composition disclosed herein.

[0062] I. Definitions

[0063] Unless otherwise defined, all technical terms used in this application have the same meanings as commonly understood by those of ordinary skill in the art.

[0064] The terms used in this application are only used for the purpose of describing a particular situation and are not intended to be restrictive. As used in this application, unless the context clearly indicates otherwise, the singular forms "a / an" and "the" are also intended to include plural forms. In addition, to the extent that the terms "including / includes", "having / has", "with" or their variants are used in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term "comprising". The term "comprising" as used in this application is synonymous with "including" or "containing", and is inclusive or open.

[0065] Any reference to "or" in this application is intended to include "and / or" unless stated otherwise.

[0066] References herein to "about" a value or parameter include (and describe) variations with respect to that value or parameter itself. For example, description referring to "about X" includes description of "X".

[0067] In some embodiments of the invention, "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0068] As used in this application, the terms "treat / treating / treatment", "ameliorate / ameliorating" and other grammatical equivalents refer to alleviating, reducing or ameliorating an ocular disease or condition or a symptom of an ocular disease or condition, preventing additional symptoms of an ocular disease or condition, ameliorating the underlying metabolic causes of symptoms, inhibiting an ocular disease or condition, such as preventing the development of an ocular disease or condition, alleviating an ocular disease or condition, causing regression of an ocular disease or condition, or stopping symptoms of an ocular disease or condition. The term also includes achieving a therapeutic benefit. The term "therapeutic benefit" refers to eradicating or ameliorating the ocular disease or condition being treated. In addition, a therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with an ocular disease or condition, such that an improvement is observed in a patient, subject or individual, although in some embodiments, the patient, subject or individual still suffers from the ocular disease or condition.

[0069] In some embodiments, the methods of the invention provide a prophylactic benefit; for example, administering a pharmaceutical composition to a patient, subject, or individual at risk for developing an ocular disease or condition, or to a patient, subject, or individual reporting one or more physiological symptoms of an ocular disease or condition, even if a diagnosis of the disease or condition has not yet been made.

[0070] The terms "administer", "administering", "administration", and the like, as used in this application, may refer to methods used to enable delivery of a therapeutic or pharmaceutical composition to a desired site of biological action. These methods include intravitreal or subretinal injection into the eye.

[0071] The term "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount", as used in this application, can refer to a sufficient amount of at least one pharmaceutical composition or compound administered that will alleviate to some extent one or more symptoms of the eye disease or condition being treated. An "effective amount", "therapeutically effective amount" or "pharmaceutically effective amount" of a pharmaceutical composition can be administered to a subject in need as a unit dose (as described in further detail elsewhere herein).

[0072] The term "pharmaceutically acceptable", as used herein, may refer to a substance, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compounds disclosed herein and is relatively non-toxic (i.e., when the substance is administered to an individual, it neither causes undesirable biological effects nor interacts in a deleterious manner with any component of the composition in which it is contained).

[0073] The term "pharmaceutical composition", as used in this application, may refer to a biologically active compound, which is optionally mixed with at least one pharmaceutically acceptable chemical component (such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickener, excipient, etc.).

[0074] As used in this application, "AAV" vector or "rAAV vector" refers to an adeno-associated virus (AAV) vector or a recombinant AAV (rAAV) vector that contains a polynucleotide sequence of non-AAV origin (i.e., a polynucleotide heterologous to AAV, such as a nucleic acid sequence encoding a therapeutic transgene (e.g., aflibercept)) for transduction into target cells or target tissues. Typically, the heterologous polynucleotide is flanked by at least one, typically two, AAV inverted terminal repeats (ITRs). The term rAAV vector includes both rAAV vector particles and rAAV vector plasmids. rAAV vectors can be single-stranded (ssAAV) or self-complementary (scAAV).

[0075] "AAV virus" or "AAV virus particle" or "rAAV vector particle" or "rAAV particle" or "recombinant AAV virus particle" refers to a virus particle comprising at least one AAV capsid protein and a polynucleotide rAAV vector. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a target cell or target tissue), it is usually referred to as a "rAAV vector particle" or simply "rAAV vector".

[0076] The term "packaging" as used in this application may refer to the series of intracellular events that may lead to the assembly and encapsidation of rAAV particles.

[0077] 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 as AAV "packaging genes" in this application.

[0078] The terms "polypeptide" or "protein" are used interchangeably to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also includes amino acid polymers modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component or toxin. Also included in the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art.

[0079] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after polymer assembly. As used herein, the term polynucleotide may refer to a double-stranded or single-stranded molecule.

[0080] As used herein, "recombinant" can refer to a biological molecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or part of a polynucleotide in which the gene is found in nature, (3) is operably linked to a polynucleotide to which it is not linked in nature, or (4) does not occur in nature. The term "recombinant" can be used to refer to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNA encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant if, for example, it is synthesized by mRNA synthesized by a recombinant gene present in the cell.

[0081] The term "anti-VEGF gene product" includes any therapeutic agent, including a protein, polypeptide, peptide, fusion protein, multimeric protein, antibody, human monoclonal antibody, antibody fragment, aptamer, kinase inhibitor, receptor or receptor fragment, or nucleic acid molecule, which can reduce, interfere with, disrupt, block and / or inhibit the activity or function of endogenous VEGF and / or endogenous VEGF receptor (VEGFR), or the VEGF-VEGFR interaction or pathway in vivo. The anti-VEGF gene product can be any known therapeutic gene product that can reduce new blood vessel growth or formation and / or edema or swelling when delivered to cells, tissues or subjects in vivo, such as ranibizumab, brocitumab or bevacizumab. In some embodiments, the anti-VEGF gene product can be naturally occurring, non-naturally occurring or synthetic. In some embodiments, the anti-VEGF gene product can be derived from a naturally occurring molecule that is subsequently modified or mutated to confer anti-VEGF activity. In some embodiments, the anti-VEGF gene product is a fusion protein or a chimeric protein. In such proteins, functional domains or polypeptides are artificially fused to parts or polypeptides to prepare fusion proteins or chimeric proteins that can sequester VEGF in vivo or act as VEGFR decoys. In some embodiments, the anti-VEGF gene product is a fusion protein or chimeric protein that prevents endogenous VEGFR from interacting with its ligand.

[0082] As used herein, "VEGF" may refer to any isoform of VEGF or a VEGF family member, unless otherwise required, including but not limited to VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, placental growth factor (PGF), or any combination thereof, or any functional fragment or variant. As used herein, "VEGF receptor" or "VEGFR" or "VEGF-R" may be used to refer to any VEGF receptor, including but not limited to VEGFR-1 (or Flt-1), VEGFR-2 (or Flk-1 / KDR), and VEGFR-3 (or Flt-4). VEGFR may be a membrane-bound or soluble form, or a functional fragment or truncation of the receptor.

[0083] As used herein, "sFlt-1 protein" refers to a polypeptide sequence or a functional fragment thereof having at least 90% or more homology to a naturally occurring human sFLT-1 sequence, such that the sFlt-1 protein or polypeptide binds to VEGF and / or VEGF receptors, unless otherwise specifically stated. Homology refers to the % conservation of residues compared between two sequences. Naturally occurring human sFLT-1 protein may include any suitable variant of sFLT-1, including but not limited to functional fragments, sequences containing insertions, deletions, substitutions, pseudofragments, pseudogenes, splice variants, or artificially optimized sequences.

[0084] "Operably linked" or "coupled" may refer to the juxtaposition of genetic elements in which the elements are in a relationship that allows them to operate in an intended manner. For example, a promoter may be operably linked to a coding region if the promoter helps to initiate transcription of the coding sequence. Intervening residues may be present between the promoter and the coding region as long as their functional relationship is maintained.

[0085] As used herein, "expression vector" includes vectors, e.g., plasmids, minicircles, viral vectors, liposomes, etc., as described above or known in the art, comprising a polynucleotide encoding a target gene product, and used to achieve expression of the gene product in the intended target cell. The expression vector may also include control elements operatively linked to the coding region to promote expression of the gene product in the target site. Combinations of control elements, e.g., combinations of promoters, enhancers, UTRs, miRNA targeting sequences, etc., and one or more genes operably linked for expression are sometimes referred to as "expression cassettes". Many such control elements are well known and available in the art, or can be readily constructed from components available in the art.

[0086] A "gene product" is a molecule produced by the expression of a particular gene. Gene products include, for example, polypeptides, aptamers, interfering RNAs, mRNAs, etc. In specific embodiments, a "gene product" is a polypeptide, peptide, protein, or interfering RNA including small interfering RNA (siRNA), miRNA, or small hairpin RNA (shRNA). In specific embodiments, a gene product is a therapeutic gene product, e.g., a therapeutic protein.

[0087] The term "heterologous" may refer to an entity that is genotyped differently from the rest of the entity to which it is compared. For example, a polynucleotide introduced into a plasmid or vector from a different species by genetic engineering techniques may be a heterologous polynucleotide. A promoter removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally found linked may be a heterologous promoter.

[0088] Unless otherwise indicated, the methods, compositions and kits described herein can be prepared using conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, immunochemistry and ophthalmology, which are within the skill of those skilled in the art. Such conventional techniques include observing and analyzing the retina or vision of a subject, cloning and propagation of recombinant viruses, preparations of pharmaceutical compositions, and biochemical purification and immunochemical methods. The specific description of suitable techniques can be referenced to the embodiments herein. However, of course, equivalent conventional procedures can also be used. Such routine techniques and descriptions can be found in standard laboratory manuals, such as Green et al., ed., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999); Weiner et al., ed., Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler, ed., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006); and Sambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (all from Cold Spring Harbor Laboratory Press); Stryer, L., Biochemistry (4th Ed.) WH Freeman, NY (1995); Gait, "Oligonucleotide Synthesis: A Practical Approach" IRL Press, London (1984); Nelson and Cox, Lehninger, Principles of Biochemistry, 3rd Ed., WH Freeman Pub., New York (2000); and Berg et al., Biochemistry, 5th Ed., WH Freeman Pub., New York (2002), all of which are incorporated herein by reference in their entirety for all purposes.

[0089] As used herein, "variant AAV capsid protein" refers to an AAV capsid protein, wherein the AAV capsid protein comprises at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to the corresponding parent AAV, wherein the AAV capsid protein does not correspond to the amino acid sequence present in the naturally occurring AAV capsid protein. In some embodiments, the variant AAV capsid protein confers increased binding to heparin and / or heparan sulfate proteoglycans compared to the binding of AAV virions comprising the corresponding parent AAV capsid protein. In certain embodiments, the variant AAV capsid protein confers the following: a) increased infectivity of retinal cells compared to the infectivity of AAV virions comprising the corresponding parent AAV capsid protein to retinal cells; b) altered cellular tropism compared to the tropism of AAV virions comprising the corresponding parent AAV capsid protein; and / or c) increased ability to bind to and / or pass through the ILM compared to AAV virions comprising the corresponding parent AAV capsid protein.

[0090] As used herein, a "modified sequence" refers to a sequence that contains one or more substitutions, insertions and / or deletions compared to the corresponding sequence of a parent AAV or parent AAV capsid protein.

[0091] AAV's "helper virus" refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. Various such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses, and poxviruses such as vaccinia. Adenoviruses encompass many different subgroups, but the most commonly used is adenovirus type 5 in subgroup C. Many adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as ATCC. Viruses of the herpes family include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV) as well as cytomegalovirus (CMV) and pseudorabies virus (PRV); the viruses can also be obtained from depositories such as ATCC.

[0092] "One or more helper functions" refers to one or more functions encoded in the helper virus genome that allow AAV replication and packaging (in combination with other requirements for replication and packaging described herein). As described herein, "helper functions" can be provided in a variety of ways, including by providing a helper virus to the production cell in trans or providing, for example, a polynucleotide sequence encoding one or more necessary functions. For example, a plasmid or other expression vector comprising a nucleotide sequence encoding one or more adenoviral proteins is transfected into the production cell together with the rAAV vector.

[0093] "Infectious" viral particles or virions are viruses or viral particles that include a suitably assembled viral capsid and are capable of delivering a polynucleotide component to cells for which the viral species has a tropism. The term does not necessarily imply that the virus has any ability to replicate. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as a ratio of infectious viral particles to total viral particles. Methods for determining the ratio of infectious viral particles to total viral particles are well known in the art. See, e.g., Grainger et al., (2005) Mol. Ther. 11: S337 (describing a TCID50 infectivity titer assay); and Zolotukhin et al., (1999) Gene Ther. 6: 973.

[0094] II. Variant AAV Capsid Proteins

[0095] Variant adeno-associated virus (AAV) capsid proteins are provided herein. In some embodiments, the variant AAV capsid proteins disclosed herein comprise a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the amino acid residue numbering corresponds to the AAV5 VP1 capsid protein. In some embodiments, the variant AAV capsid protein comprises a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, wherein the modified sequence comprises X1X2HKFKSGDX3 (SEQ ID NO: 2), and wherein the amino acid residue numbering corresponds to the AAV5 VP1 capsid protein, wherein X 1-3 Can be independently any amino acid. In some embodiments, each X 1-3 is independently selected from A, L, G, S and T. In some embodiments, each X 1-3Independently selected from A, L, G, S and T. In some embodiments, X1 is L. In some embodiments, X2 is A. In some embodiments, X3 is A. In some embodiments, the variant AAV capsid protein comprises a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, wherein the modified sequence comprises LAHKFKSGDA (SEQ ID NO: 3), a sequence having at least 80% or at least 90% homology to SEQ ID NO: 3; a sequence having at least 80% or at least 90% identity to SEQ ID NO: 3; or having four or more, five or more, six or more, seven or more, eight or more or nine or more consecutive amino acids within SEQ ID NO: 3, and wherein the amino acid residues encode a capsid protein corresponding to AAV5 VP1. In some embodiments, the modified sequence comprises LAHKFKSGDA (SEQ ID NO: 3).

[0096] Although reference is made herein to amino acid modifications (including specific amino acid substitutions and insertions) of capsid proteins using amino acid numbering corresponding to the AAV5 VP1 capsid protein, it is understood that any of these amino acid modifications can also be introduced into AAV capsid proteins of other serotypes, for example, at positions corresponding to those of AAV5 VP1. AAV protein sequences have significant homology and similar amino acid numbering, and a skilled artisan can readily determine amino acid residues in other AAV serotypes that correspond to those specifically described herein for AAV5 VP1.

[0097] In some embodiments, the parent AAV capsid protein is a wild-type AAV capsid protein, such as AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV capsid protein. In some embodiments, the parent AAV capsid protein is an AAV5 capsid protein. In some embodiments, the parent AAV capsid protein is a VP1, VP2, or VP3 capsid protein. In some embodiments, the parent AAV capsid protein is an AAV5 VP1 capsid protein.

[0098] In some embodiments, the parent AAV capsid protein is a variant AAV capsid protein. In some embodiments, the parent AAV capsid protein is a variant of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV capsid protein. In some embodiments, the parent AAV capsid protein is a variant of AAV5 VP1 capsid protein. In some embodiments, the parent AAV capsid protein is a variant of AAV5 VP1 capsid protein having at least 90%, at least 95%, at least 98% or at least 99% homology to SEQ ID NO: 4. In some embodiments, the parent AAV capsid protein is a variant of AAV5 VP1 capsid protein having at least 90%, at least 95%, at least 98% or at least 99% sequence identity to SEQ ID NO: 4. In some embodiments, the parent AAV capsid protein is a hybrid capsid protein. In some embodiments, the parent AAV capsid protein is a hybrid of an AAV2 capsid protein and an AAV5 capsid protein. In some embodiments, the parent AAV capsid protein is an AAV2.5T capsid protein, or a variant thereof having at least 90%, at least 95%, at least 98% or at least 99% homology with the AAV2.5T capsid protein. In some embodiments, the parent AAV capsid protein is an AAV 2.5T capsid protein, or a variant thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the AAV2.5T capsid protein. In some embodiments, the parent AAV capsid protein is an AAV 2.5T capsid protein. In some embodiments, the parent AAV capsid protein is a VP1, VP2 or VP3 capsid protein. In some embodiments, the parent AAV capsid protein is AAV2.5T VP1 capsid protein, or a variant thereof having at least 90%, at least 95%, at least 98% or at least 99% homology with AAV2.5T VP1 capsid protein (SEQ ID NO: 5). In some embodiments, the parent AAV capsid protein is AAV2.5T VP1 capsid protein, or a variant thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity with AAV2.5T VP1 capsid protein (SEQ ID NO: 5).

[0099] In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the variant AAV capsid protein comprises a capsid sequence having at least 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the variant AAV capsid protein comprises a capsid protein having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% homology to the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the variant AAV capsid protein comprises a capsid protein having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the variant AAV capsid protein comprises SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the variant AAV capsid protein comprises SEQ ID NO:7.

[0100] In a specific embodiment, the variant AAV capsid protein, when present in the recombinant AAV virion, confers increased infectivity to the retinal cells, compared to the infectivity of the recombinant AAV virion comprising the corresponding parent AAV capsid protein to the retinal cells. In a specific embodiment, the variant AAV capsid protein, when present in the recombinant AAV virion, increases the infectivity of the retinal cells when administered by intravitreal injection, compared to the infectivity of the recombinant AAV virion comprising the corresponding parent AAV capsid protein to the retinal cells when administered by intravitreal injection. In some embodiments, the retinal cells are one or more of photoreceptors (e.g., rods; cones), retinal ganglion cells (RGCs), retinal pigment epithelial (RPE) cells, Müller cells, amacrine cells, bipolar cells, and horizontal cells. In a specific embodiment, the variant AAV capsid protein, when present in the recombinant AAV virion, confers an altered tropism to the recombinant AAV virion, compared to the tropism of the recombinant AAV virion comprising the corresponding parent AAV capsid protein. In certain embodiments, the variant AAV capsid protein when present in recombinant AAV virions confers increased binding to heparin and / or heparan sulfate, and / or increased ability to bind to and cross the inner limiting membrane (ILM) following intravitreal injection, compared to recombinant AAV virions comprising the corresponding parental AAV capsid protein.

[0101] III. Polynucleotides and Cells

[0102] Also provided herein are polynucleotides encoding one or more variant AAV capsid proteins described herein. In a specific embodiment, the polynucleotide is an expression vector. In some embodiments, the expression vector comprises a polynucleotide sequence encoding a modified capsid protein described herein operably connected to a promoter sequence, for example, a promoter sequence driving the polynucleotide to be expressed in a cell. In certain embodiments, the cell is a host cell. The host cell can be used to produce virions comprising modified capsid proteins. Exemplary host cells include mammalian cells (e.g., HEK293 cells), insect cells (e.g., SF9 cells), microorganisms, and yeast. In some embodiments, the cell comprises an AAV rep gene, such as an AAV rep gene encoding AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, cattle AAV, canine AAV, horse AAV, primate AAV, non-primate AAV, or sheep AAV rep proteins or variants thereof. In some embodiments, the AAV rep gene is stably maintained in the cell.

[0103] Also provided herein are cells comprising polynucleotides or vectors encoding modified capsid proteins described herein. In specific embodiments, the polynucleotides are expression vectors, and the expression vectors comprise polynucleotides encoding variant capsid proteins described herein operably linked to promoter sequences, for example, promoter sequences driving polynucleotides to be expressed in cells. In some embodiments, the polynucleotides or vectors are stably maintained in cells. In some embodiments, the cells further comprise an AAV rep gene. In some embodiments, the AAV rep gene is stably maintained in cells. In certain embodiments, the polynucleotides or vectors further comprise sequences encoding rep proteins, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or sheep AAV rep proteins or variants thereof.

[0104] In some embodiments, the cell further comprises a polynucleotide expression cassette comprising a sequence encoding a gene product, e.g., a therapeutic gene product, such as a therapeutic gene product as described herein. In certain embodiments, the polynucleotide expression cassette is flanked by one or more AAV inverted terminal repeats (ITRs). In certain embodiments, the polynucleotide expression cassette is flanked by AAV ITRs at the 5' and 3' ends. In some embodiments, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or sheep AAV ITRs or variants thereof. In some embodiments, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs. In some embodiments, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs comprising one or more nucleotide insertions, deletions, and / or substitutions.

[0105] IV. Virus particles and methods of producing the same

[0106] Also provided herein are viral particles (eg, recombinant AAV viral particles) comprising a variant AAV capsid protein described herein.

[0107] In some embodiments, the recombinant AAV virion further comprises a polynucleotide expression cassette comprising a sequence encoding a gene product, such as a therapeutic gene product, such as a therapeutic gene product described herein. In certain embodiments, the polynucleotide expression cassette is flanked by one or more AAV inverted terminal repeats (ITRs), such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or sheep AAV ITRs or variants thereof. In certain embodiments, the polynucleotide expression cassette is flanked by AAV ITRs at the 5' and 3' ends. In some embodiments, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs or variants thereof. In some embodiments, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs comprising one or more nucleotide insertions, deletions, and / or substitutions. In some embodiments, one or more ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs.

[0108] In certain embodiments, the gene product inhibits neovascularization in the retina of a subject, for example, choroidal neovascularization (CNV). Many cytokines have been found to play an important role in the regulation of CNV generation, including but not limited to vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), platelet-derived growth factor (PDGF), hypoxia-inducible factor (HIF), angiopoietin (Ang) and other cytokines, mitogen-activated protein kinase (MAPK). In some embodiments, the gene product is an inhibitor of one or more of VEGF, VEGFR, PDGF, HIF, Ang and MAPK.

[0109] In certain embodiments, the gene product inhibits neovascularization in the retina of a subject, for example, choroidal neovascularization (CNV). Many cytokines have been found to play an important role in the regulation of CNV generation, including but not limited to vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), platelet-derived growth factor (PDGF), hypoxia-inducible factor (HIF), angiopoietin (Ang) and other cytokines, mitogen-activated protein kinase (MAPK). In some embodiments, the gene product is an inhibitor of one or more of VEGF, VEGFR, PDGF, HIF, Ang and MAPK.

[0110] In some embodiments, the gene product is an interfering RNA, an aptamer or a protein. In some embodiments, the therapeutic gene product is an interfering RNA, an aptamer or a protein. In some embodiments, the gene product (or therapeutic gene product) is an siRNA, a miRNA or a protein. In some embodiments, the gene product (or therapeutic gene product) is a site-specific nuclease, which provides a site-specific knockdown of a gene function. In some embodiments, the gene product (or therapeutic gene product) is an interfering RNA. In some embodiments, the gene product (or therapeutic gene product) is an siRNA or a miRNA.

[0111] In some embodiments, the gene product (or therapeutic gene product) is an anti-VEGF gene product. In some embodiments, the gene product (or therapeutic gene product) is an anti-VEGF interfering RNA. In some embodiments, the gene product (or therapeutic gene product) is an anti-VEGF siRNA or miRNA.

[0112] In some embodiments, the gene product (or therapeutic gene product) is an aptamer. Exemplary aptamers of interest include aptamers for vascular endothelial growth factor (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. Also suitable for use are PDGF-specific aptamers, e.g., E10030; see, e.g., Ni and Hui (2009) Ophthalmologica 223:401; and Akiyama et al., (2006) J. Cell Physiol. 207:407.

[0113] In some embodiments, the gene product (or therapeutic gene product) is a protein. The protein is typically a protein that enhances retinal cell function, such as a rod or cone cell, a retinal ganglion cell, a Müller cell, a bipolar cell, amacrine cell, a horizontal cell, or a retinal pigment epithelial cell. Exemplary proteins include neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF and NTN); anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptor; VEGF binding antibody; VEGF binding antibody fragment (e.g., single-chain anti-VEGF antibody); endostatin; tumoristatin; angiostatin; soluble Flt protein (Lai et al., (2005) Mol. Ther. 12:659); Fc fusion protein comprising soluble Flt protein (see, e.g., Pechan et al., (2009) Gene Ther. 16:10); pigment epithelium-derived factor (PEDF); soluble Tie-2 receptor, etc.); tissue inhibitor of metalloproteinase-3 (TIMP-3); visual protein, e.g., rhodopsin; anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-X1), etc. Suitable polypeptides include, but are not limited to, glial-derived neurotrophic factor (GDNF); fibroblast growth factor 2; neurotrophic factor (NTN); ciliary neurotrophic factor (CNTF); nerve growth factor (NGF); neurotrophin-4 (NT4); brain-derived neurotrophic factor (BDNF); epidermal growth factor; rhodopsin; X-linked inhibitor of apoptosis; and sonic hedgehog.

[0114] Suitable opsins include, for example, light-responsive opsins as described in U.S. Patent Publication No. 2007 / 0261127 (e.g., ChR2; Chop2); U.S. Patent Publication No. 2001 / 0086421; U.S. Patent Publication No. 2010 / 0015095; and Diester et al., (2011) Nat. Neurosci. 14:387.

[0115] Suitable proteins also include retinoic acid. Suitable polypeptides include, for example, retinitis pigmentosa GTPase regulator (RGPR)-interacting protein-1 (see, e.g., GenBank accession numbers Q96KN7, Q9EPQ2, and Q9GLM3); peripherin-2 (Prph2) (see, e.g., GenBank accession number NP_000313; and Travis et al., (1991) Genomics 10:733); peripherin; retinal pigment epithelium-specific protein (RPE65) (see, e.g., GenBank AAC39660; and Morimura et al., (1998) Proc. Natl. Acad. Sci. USA 95:3088), etc.

[0116] Suitable proteins also include: CHM (choroideremia (Rab escort protein 1)), a polypeptide that, when defective or absent, causes choroideremia (see, e.g., Donnelly et al., (1994) Hum. Mol. Genet. 3:1017; and van Bokhoven et al., (1994) Hum. Mol. Genet. 3:1041); and Crumbs homolog 1 (CRB 1), a polypeptide that, when defective or absent, causes Leber congenital amaurosis and retinitis pigmentosa (see, e.g., den Hollander et al., (1999) Nat. Genet. 23:217; and GenBank Accession No. CAM23328).

[0117] Suitable proteins also include proteins that, when defective or absent, cause achromatopsia, wherein such polypeptides include, for example, 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 channel subunit beta (CNGB3) (see, e.g., Kohl et al., (2005) Eur J Hum Genet. 13(3): 302); guanine nucleotide binding protein (G protein); alpha transduction active polypeptide 2 (GNAT2) (ACHM4); and ACHM5; and polypeptides that, when defective or absent, cause various forms of color blindness (e.g., L-opsin, M-opsin, and S-opsin). See Mancuso et al., (2009) Nature 461(7265): 784-787.

[0118] In some embodiments, the gene product (or therapeutic gene product) is an anti-VEGF protein. In some embodiments, the anti-VEGF protein is an sFlt-1 protein or a fragment thereof or an anti-VEGF antibody or a fragment thereof. In some embodiments, the anti-VEGF protein is an anti-VEGF antibody or a fragment thereof, such as aflibercept, ranibizumab and bevacizumab or a fragment thereof. In some embodiments, the anti-VEGF protein is an sFlt-1 protein.

[0119] sFLT-1, a soluble truncated form of the VEGF receptor FLT-1, is the only known endogenous VEGF-specific inhibitor. In nature, it is produced by alternative mRNA splicing and lacks a membrane-proximal immunoglobulin-like domain, a transmembrane region, and an intracellular tyrosine kinase domain. Structurally, both FLT-1 and sFLT-1 proteins can contain multiple functional domains. In some variants, FLT and sFLT proteins typically share 6 interconnected domains; 3 domains are involved in the dimerization of the protein, and 3 domains are involved in the binding of ligands, such as VEGF. sFLT-1 is not limited to the cell membrane. Unbound sFLT-1 can diffuse freely in the extracellular space or solution.

[0120] The interaction between sFLT-1 and VEGF receptor is specific and can compete with 100-fold excess of unlabeled VEGF. In some cases, the angiostatic activity of sFLT-1 may be caused by inhibiting VEGF by two mechanisms: i) chelating VEGF, which binds with high affinity, and ii) forming inactive heterodimers with transmembrane isomers of VEGF receptors FLTt-1 and FLK-1 / KDR. As is well known in the art, in vitro binding assays show that sFLT-1 binds VEGF with high affinity and can also inhibit VEGF-driven human umbilical vein endothelial cell proliferation. In animal models of cancer, sFLT-1 inhibits tumor growth. In some cases, sFLT-1 may act in a substoichiometric or dominant negative manner because excess VEGF in the extracellular space may be prevented from binding and subsequently activating VEGF receptors. These properties of sFLT-1 have been described in Kendall and Thomas, 1993; Proc Natl Acad Sci. 90: 10705-10709, the entire contents of which are incorporated herein by reference. Functional fragments of sFLT-1 can be used to replace the full-length protein. More specifically, the VEGF binding domain (domain 2), or domain 2 of sFLT-1 plus domain 3 from sFLT1, KDR or other family members, can be used to bind and inactivate VEGF. Such functional fragments are described in Wiesmann et al., 1997; Cell, 91: 695-704, the entire contents of which are incorporated herein by reference.

[0121] In some embodiments, the sFlt-1 protein is a naturally occurring protein sFlt-1, such as described in U.S. Pat. No. 5,861,484 and disclosed in SEQ ID NO: 109 of US2013 / 0323302. An exemplary sFlt-1 amino acid sequence is disclosed herein as SEQ ID NO: 13. It also includes, but is not limited to, functional fragments thereof, including the sequence of sFlt-1 domain 2 or those shown in SEQ ID NO: 121 of U.S. Patent Application Publication No. 2013 / 0323302, and related constructs, such as the VEGF binding fusion protein disclosed in U.S. Pat. No. 7,635,474. An exemplary sFlt-1 functional fragment is disclosed herein as SEQ ID NO: 14. In some embodiments, the sFLT-1 protein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 100% homologous to SEQ ID NO: 13 or SEQ ID NO: 14. In some embodiments, the sFLT-1 protein has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 100% sequence identity to SEQ ID NO: 13 or SEQ ID NO: 14. The anti-VEGF protein may also include any sFLT-1 protein, variant, or fragment thereof described in U.S. Patent Application Publication No. 2013 / 0323302.

[0122] In some embodiments, the sFLT-1 protein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 100% homologous to the naturally occurring human sFLT-1 protein sequence. In some embodiments, the sFLT-1 protein is at most about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 100% homologous to the naturally occurring human sFLT-1 protein sequence. In some embodiments, the sFLT-1 protein has at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% sequence identity to the naturally occurring human sFLT-1 protein sequence. In some embodiments, the sFLT-1 protein has at most about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% sequence identity to the naturally occurring human sFLT-1 protein sequence. In some embodiments, the sFLT-1 protein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% spatially homologous to a naturally occurring human sFLT-1 protein conformation. In some cases, the sFLT-1 protein is at most about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or 100% spatially homologous to a naturally occurring human sFLT-1 protein conformation.

[0123] In some embodiments, the anti-VEGF protein is an anti-VEGF antibody. In some embodiments, the anti-VEGF protein is ranibizumab (trademark (sold by Genentech, San Francisco, CA, see FIG1 of U.S. Patent No. 7,060,269 for the heavy and light chain variable region sequences of ranibizumab); bevacizumab (sold by the trademark (sold by Genentech, San Francisco, CA, see FIG1 of U.S. Pat. No. 6,054,297 for the heavy and light chain variable region sequences of bevacizumab); aflibercept (sold by the trademark In some embodiments, bevacizumab comprises the following heavy chain and light chain variable domain sequences, respectively: EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTL (SEQ ID NO: 8) and; DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTV (SEQ ID NO: 9). In certain embodiments, ranibizumab comprises the following heavy and light chain variable domain sequences, respectively: EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDDTAVYYCAKYPYYYGTSHWYFDVWGQGTL (SEQ ID NO: 10); and

[0124] DIQLTQSPSSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKV

[0125] LIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTV (SEQ ID NO: 11). In certain embodiments, aflibercept comprises the following amino acid sequence: MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMT KKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:12). In certain embodiments, aflibercept comprises the following amino acid sequence: EIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSS (SEQ ID NO: 15).

[0126] These sequences can be expressed from DNA encoding these sequences using the genetic code, which is a standard technique understood by those skilled in the art. Those skilled in the art will appreciate that due to the degeneracy of the genetic code, anti-VEGF protein sequences can be easily expressed from many different DNA sequences.

[0127] In some cases, the gene product (or therapeutic gene product) is a site-specific endonuclease that provides for site-specific knockdown of gene function, e.g., where the endonuclease knocks out an allele associated with a retinal disease. For example, where a dominant allele encodes a defective copy of a gene that, when wild-type, is a retinal structural protein and / or provides normal retinal function, the site-specific endonuclease can be targeted to the defective allele and knock out the defective allele.

[0128] In addition to knocking out the defective allele, site-specific nucleases can also be used to stimulate homologous recombination with donor DNA encoding a functional copy of the protein encoded by the defective allele. Thus, for example, viral particles can be used to deliver site-specific endonucleases that knock out the defective allele, and can also be used to deliver functional copies of the defective allele, so that the defective allele is repaired, thereby providing the production of functional retinal proteins (e.g., functional retinoschisis proteins, functional RPE65, functional peripherin, etc.). See, for example, Li et al., (2011) Nature 475:217. In some embodiments, the viral vector includes: a heterologous nucleotide sequence encoding a site-specific endonuclease; and a heterologous nucleotide sequence encoding a functional copy of the defective allele, wherein the functional copy encodes a functional retinal protein. Functional retinal proteins include, for example, retinoschisis proteins, RPE65, retinitis pigmentosa GTPase regulator (RGPR) interacting protein-1, peripherin, peripherin-2, etc.

[0129] Site-specific endonucleases suitable for use include, for example, zinc finger nucleases (ZFNs); and transcription activator-like effector nucleases (TALENs), wherein the site-specific endonucleases are non-naturally occurring and modified to target specific genes. The site-specific nucleases can be engineered to cut specific positions in the genome, and non-homologous end ligation can then repair the break while inserting or deleting multiple nucleotides. The site-specific endonuclease (also referred to as "INDEL") then throws the protein out of the frame and effectively knocks out the gene. See, for example, U.S. Patent Publication No. 2011 / 0301073.

[0130] In some embodiments, the sequence encoding the gene product is operably connected to a promoter. In some embodiments, the sequence encoding the gene product is operably connected to a constitutive promoter. In other embodiments, the nucleotide sequence encoding the target gene product is operably connected to an inducible promoter. In some instances, the sequence encoding the gene product is operably connected to a tissue-specific or cell-specific regulatory element. For example, in some instances, the sequence encoding the gene product is operably connected to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), for example, a regulatory element that selectively expresses a gene operably connected in a photoreceptor cell is conferred. Suitable photoreceptor-specific regulatory elements include, for example: the rhodopsin promoter; the rhodopsin kinase promoter (Young et al., (2003) Ophthalmol. Vis. Sci. 44:4076); the beta phosphodiesterase gene promoter (Nicoud et al., (2007) J. Gene Med. 9:1015); the retinitis pigmentosa gene promoter (Nicoud et al., (2007) supra); the inner photoreceptor retinoid binding protein (IRBP) gene enhancer (Nicoud et al., (2007) supra); the IRBP gene promoter (Yokoyama et al., (1992) Exp Eye Res. 55:225).

[0131] In certain embodiments, the recombinant AAV virion is capable of binding to heparin and / or heparan sulfate proteoglycans (HSPGs), e.g., with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than a recombinant AAV virion comprising a corresponding parent AAV capsid protein. In certain embodiments, the recombinant AAV virion is capable of binding to heparin and / or heparan sulfate proteoglycans (HSPGs), e.g., with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than an AAV5 virion. In certain embodiments, the recombinant AAV virions are capable of binding to heparin and / or heparan sulfate proteoglycan (HSPG), e.g., with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than AAV2.5T virions.

[0132] In certain embodiments, the recombinant AAV virion is capable of binding to the inner limiting membrane (ILM), e.g., with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than a recombinant AAV virion comprising a corresponding parental AAV capsid protein. In certain embodiments, the recombinant AAV virion is capable of binding to the inner limiting membrane (ILM), e.g., with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than an AAV5 virion. In certain embodiments, the recombinant AAV virion is capable of binding to the inner limiting membrane (ILM), e.g., with at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater affinity than an AAV2.5T virion.

[0133] In a specific embodiment, the infectivity of the recombinant AAV virions to retinal cells is increased compared to the infectivity of the recombinant AAV virions comprising the corresponding parent AAV capsid protein to retinal cells. In a specific embodiment, the recombinant AAV virions are capable of passing through the ILM. In a specific embodiment, the infectivity of the recombinant AAV virions to retinal cells is increased compared to the infectivity of the recombinant AAV virions comprising the corresponding parent AAV capsid protein to retinal cells, and are capable of passing through the ILM. In a specific embodiment, the infectivity of the recombinant AAV virions to retinal cells is increased compared to the infectivity of the AAV5 virions to retinal cells. In a specific embodiment, the infectivity of the recombinant AAV virions to retinal cells is increased compared to the infectivity of the AAV2.5T virions to retinal cells. In a specific embodiment, the recombinant AAV virions are capable of transducing retinal cells when injected intravitreally into a mammal. In a specific embodiment, the recombinant AAV virions are capable of passing through the ILM. In a specific embodiment, when administered by intravitreal injection, the recombinant AAV virions are increased in infectivity to retinal cells when administered by intravitreal injection, compared to the infectivity of recombinant AAV virions containing the corresponding parent AAV capsid protein to retinal cells. In a specific embodiment, the infectivity of recombinant AAV virions to retinal cells is increased when administered by intravitreal injection, compared to the infectivity of AAV5 virions to retinal cells when administered by intravitreal injection. In a specific embodiment, the infectivity of recombinant AAV virions to retinal cells is increased when administered by intravitreal injection, compared to the infectivity of AAV2.5T virions to retinal cells when administered by intravitreal injection. In some embodiments, retinal cells are photoreceptor cells (e.g., rods; cones), retinal ganglion cells (RGCs), retinal pigment epithelial (RPE) cells, Müller cells, amacrine cells, bipolar cells, and horizontal cells. One or more. In some embodiments, retinal cells are Müller cells, cones, rods, bipolar cells, and / or RPE cells. In some embodiments, retinal cells are Müller cells. In some embodiments, the retinal cell is a cone cell. In some embodiments, the retinal cell is a bipolar cell. In some embodiments, the retinal cell is a RPE cell.

[0134] In certain embodiments, recombinant AAV virions comprising variant AAV capsid proteins described herein are capable of delivering gene products (e.g., therapeutic gene products) to the retina when injected intravitreally, compared to the infectivity of recombinant AAV virions comprising the corresponding parental AAV capsid proteins to retinal cells, e.g., the resulting expression is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher. In certain embodiments, recombinant AAV virions comprising variant AAV capsid proteins described herein are capable of delivering gene products (e.g., therapeutic gene products) to the retina when injected intravitreally, e.g., the resulting expression is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher, compared to AAV5 and / or AAV2.5T vectors.

[0135] Many methods for producing recombinant AAV virus particles are known in the art, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpes virus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71 (11): 8780-8789) and baculovirus-AAV hybrids. The rAAV production culture used to produce rAAV virus particles requires: 1) a suitable host cell, 2) a suitable helper virus function, 3) AAV rep and cap genes; 4) a nucleic acid (such as a sequence encoding a gene product) flanked by at least one AAV ITR sequence (e.g., an ultra-large rAAV vector genome); and 5) a suitable culture medium and culture medium components that support rAAV production. In some embodiments, the suitable host cell is a primate host cell. In some embodiments, the suitable host cell is a human cell line, such as HeLa, A549, 293 or Perc.6 cells.

[0136] In some embodiments, suitable helper functions are provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpes virus (HSV), baculovirus, or a plasmid construct that provides helper functions. In some embodiments, AAV helper functions are provided by adenovirus or HSV. In some embodiments, AAV helper functions are provided by baculovirus, and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cell).

[0137] In some embodiments, the AAV virions described herein are produced in insect cells. In some embodiments, the AAV virions described herein are produced in fall armyworm (Sf9) cells. In some embodiments, the AAV virions described herein are produced in human cell lines. In some embodiments, the AAV virions described herein are produced in HeLa, A549, 293 or Perc.6 cells. In some embodiments, the AAV virions described herein are produced in HEK-293 cells.

[0138] In some embodiments, the AAV rep gene can be from any AAV serotype. Generally, but not necessarily, the AAV rep gene is of the same serotype as the IIR of the AAV vector genome, as long as the rep protein encoded by the rep gene can replicate and package the rAAV genome.

[0139] Suitable culture media known in the art can be used to produce rAAV vectors. These culture media include, but are not limited to, culture media produced by Hyclone Laboratories and JRH, including modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), custom formulations, such as those described in U.S. Patent No. 6,566,118, and Sf-900II SFM culture media, such as those described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom culture medium formulations for producing recombinant AAV vectors.

[0140] A method for producing rAAV virions is a triple transfection method. In short, a plasmid containing a rep gene and a capsid gene, such as a polynucleotide encoding a variant AAV capsid protein disclosed herein, can be transfected (e.g., using a calcium phosphate method) into a cell line (e.g., HEK-293 cells) together with an auxiliary adenovirus plasmid, and virions can be collected and optionally purified. Thus, in some embodiments, rAAV virions are produced by triple transfection of a vector genome, a nucleic acid encoding AAV rep and cap (such as a polynucleotide encoding a variant capsid protein disclosed herein) and a nucleic acid encoding an AAV auxiliary function into a host cell, wherein the nucleic acid transfection enters the host cell to produce a host cell capable of producing rAAV virions.

[0141] In some embodiments, rAAV virions can be produced by a production cell line method, such as in Martin et al., (2013) Human Gene Therapy Methods 24: 253-269; U.S. Patent Publication No. US2004 / 0224411; and Liu, XL et al., (1999) Gene Ther.6: 293-299 described in exemplary production cell line methods. In brief, cell lines (e.g., HeLa, 293, A549 or Perc.6 cells) can be stably transfected with plasmids and vector genomes comprising rep genes, capsid genes (e.g., polynucleotides encoding variant capsid proteins disclosed herein). Cell lines can be screened to select leading clones for rAAV production, which can then be expanded to production bioreactors and infected with helper viruses (e.g., adenovirus or HSV) to start rAAV production. Virions can then be harvested, adenoviruses can be inactivated (e.g., by heating) and / or removed, and rAAV virions can be purified. Thus, in some embodiments, rAAV virions are produced by a producer cell line comprising one or more of a nucleic acid encoding a rAAV genome, a nucleic acid encoding an AAV rep, a polynucleotide encoding a variant capsid protein disclosed herein, and a nucleic acid encoding an AAV helper function.

[0142] In some embodiments, nucleic acids encoding AAV rep genes, AAV cap genes and / or rAAV genomes are stably maintained in production cell lines. In some embodiments, nucleic acids encoding AAV rep genes, AAV cap genes and / or rAAV genomes are introduced into one or more plasmids in a cell line to produce a production cell line. In some embodiments, AAV rep genes, AAV cap genes and rAAV genes are introduced into the same plasmid in a cell. In other embodiments, AAV rep genes, AAV cap genes and rAAV genes are introduced into two or more different plasmids in a cell. In some embodiments, a cell line stably transfected with a plasmid maintains a plasmid (e.g., 5, 10, 20, 30, 40, 50 or more than 50 passages of a cell line) in multiple passages of a cell line. For example, a plasmid can be replicated as the cell replicates, or a plasmid can be integrated into the cell genome. A variety of sequences that can replicate autonomously in cells (e.g., human cells) using plasmids have been identified (see, e.g., Krysan, PJ et al., (1989) Mol. Cell Biol. 9: 1026-1033). In some embodiments, the plasmid may contain a selective marker (e.g., an antibiotic resistance marker) that allows selection of cells that maintain the plasmid. Selective markers commonly used in mammalian cells include, but are not limited to, blasticidin, G418, hygromycin B, bleomycin, puromycin and derivatives thereof. Methods for introducing nucleic acids into cells are well known in the art, including but not limited to viral transduction, cationic transfection (e.g., using cationic polymers such as DEAE-dextran or cationic lipids such as liposomes), calcium phosphate transfection, microinjection, particle bombardment, electroporation, and nanoparticle transfection (see, e.g., Kim, TK and Eberwine, JH (2010) Anal. Bioanal. Chem. 397: 3173-3178 for more details).

[0143] In some embodiments, nucleic acids encoding AAV rep genes, AAV cap genes and / or rAAV genomes are stably integrated into the genome of a production cell line. In some embodiments, nucleic acids encoding AAV rep genes, AAV cap genes and / or rAAV genomes are introduced into one or more plasmids in a cell line to produce a production cell line. In some embodiments, AAV rep genes, AAV cap genes and rAAV genomes are introduced into the same plasmid in a cell. In other embodiments, AAV rep genes, AAV cap genes and rAAV genomes are introduced into two or more different plasmids in a cell. In some embodiments, plasmids may include selective markers (e.g., antibiotic resistance markers) that allow selection of cells that maintain plasmids. Methods for stably integrating nucleic acids into a variety of host cell lines are known. For example, repeated selection (e.g., by using selective markers) can be used to select cells that have integrated nucleic acids containing selective markers (and AAV cap genes, AAV rep genes and / or rAAV genomes). In other embodiments, nucleic acids can be integrated into a cell line in a site-specific manner to produce a production cell line. Several site-specific recombination systems are known in the art, such as FLP / FRT (see, e.g., O'Gorman, S. et al., (1991) Science 251:1351-1355), Cre / loxP (see, e.g., Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. 85:5166-5170), and phi C31-att (see, e.g., Groth, AC et al., (2000) Proc. Natl. Acad. Sci. 97:5995-6000).

[0144] In some embodiments, the production cell line is from a primate cell line (e.g., a non-human primate cell line, such as a Vero or FRhL-2 cell line). In some embodiments, the production cell line is from a human cell line. In some embodiments, the production cell line is from HeLa, 293, A549 or For example, before nucleic acid encoding AAV rep gene, AAV cap gene and / or rAAV genome is introduced and / or stably maintained / integrated into the cell line to generate the production cell line, the cell line is HeLa, 293, A549 or (Crucell) cell line or its derivatives.

[0145] In some embodiments, the production cell line is adapted for growth in suspension. It is well known that anchorage-dependent cells are generally unable to grow in suspension without a matrix (such as microcarrier beads). Adapting the cell line to growth in suspension can include, for example, culturing the cell line in a rotary culture with a stirring paddle, using a culture medium without calcium and magnesium ions to prevent agglomeration (and optionally a defoaming agent), using a culture vessel coated with a silicon compound, and selecting cells in the culture (rather than in large clumps or on the side walls of the vessel) at each passage. For further description, see, for example, the ATCC FAQ document (available at www[dot]atcc[dot]org / Global / FAQs / 9 / 1 / Adapting%20a%20monolayer%20cell%20line%20to%20suspension-40[dot]aspx) and the references cited therein.

[0146] In some aspects, a method for producing rAAV virions as disclosed herein is provided, comprising (a) culturing a host cell under conditions that produce rAAV virions, wherein the host cell comprises (i) a polynucleotide encoding a variant AAV capsid protein disclosed herein; (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide expression cassette comprising a sequence encoding a gene product (e.g., a therapeutic gene product flanked by at least one AAV ITR); and (iv) AAV helper functions; and (b) recovering the rAAV virions produced by the host cell. In some embodiments, the at least one AAV ITR is selected from the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV serotype ITR, etc. In some embodiments, the method further comprises purifying the rAAV virions.

[0147] Suitable rAAV production medium of the present invention can be supplemented with serum or serum-derived recombinant proteins at a level of 0.5%-20% (v / v or w / v). Alternatively, as is known in the art, rAAV virions can be produced under serum-free conditions, which can also be referred to as a medium without animal-derived products. It will be appreciated by those of ordinary skill in the art that commercial or custom culture media designed to support the production of rAAV virions can also be supplemented with one or more cell culture components known in the art, including but not limited to glucose, vitamins, amino acids and / or growth factors, to increase the titer of rAAV in the production culture.

[0148] rAAV production cultures can be grown under a variety of conditions (over a wide range of temperatures, for varying lengths of time, etc.) appropriate to the particular host cells being used. As is known in the art, rAAV production cultures include attachment-dependent cultures that can be cultured in suitable attachment-dependent containers, such as roller bottles, hollow fiber filters, microcarriers, and packed bed or fluidized bed bioreactors. rAAV production cultures can also include suspension-adapted host cells, such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems, such as Wave bag systems.

[0149] V. Composition

[0150] Also provided herein are compositions comprising the recombinant AAV virus particles disclosed herein, the virus particles comprising the variant AAV capsid protein described herein. In some embodiments, the composition is a pharmaceutical composition comprising the recombinant AAV virus particles disclosed herein and one or more pharmaceutically acceptable diluents, carriers or excipients.

[0151] The recombinant AAV virions can be combined with pharmaceutically acceptable carriers, diluents, and agents that can be used to prepare generally safe, nontoxic, and desirable formulations, and include excipients acceptable to primates. Such excipients can be solid, liquid, semisolid, or, in the case of aerosol compositions, gas. Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be incorporated into the formulation. Solutions or suspensions for formulations may include: sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antimicrobial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; detergents such as Tween 20 to prevent polymerization; and compounds for adjusting tonicity such as sodium chloride or dextrose. The pH may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. In a particular embodiment, the pharmaceutical composition is sterile. For example, in the case of in vivo contact with ocular cells, the recombinant AAV virions may be appropriately processed for delivery to the eye.

[0152] The pharmaceutical composition may further include a sterile aqueous solution or dispersion and a sterile powder for the temporary preparation of a sterile injectable solution or dispersion. For intravenous administration, a suitable carrier includes physiological saline, antibacterial water or phosphate buffered saline (PBS). In some cases, the composition is sterile and should have a fluidity that is easy to inject. In some embodiments, the composition is stable under manufacturing and storage conditions, and resists the contamination of microorganisms such as bacteria and fungi for preservation. The carrier can be, for example, a solvent or dispersion medium containing, for example, water, ethanol, a polyol (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.) and a suitable mixture thereof. For example, by using coatings such as lecithin, by maintaining the required particle size in the case of a dispersion and by using a surfactant, appropriate fluidity can be maintained. By various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc., it is possible to achieve the effect of preventing microorganisms. In some embodiments, the pharmaceutical composition includes one or more isotonic agents, such as sugar, polyols such as mannitol, sorbitol, sodium chloride. In some embodiments, the pharmaceutical compositions contain agents that delay absorption, such as aluminum monostearate and gelatin.

[0153] Sterile solutions can be prepared by incorporating the desired amount of recombinant AAV virions with one or a combination of the ingredients listed above into an appropriate solvent, followed by filtration sterilization, as required. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a base dispersion medium and other ingredients required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, which produces a powder of the active ingredient plus any additional desired ingredients from its previously sterile filtered solution.

[0154] In some embodiments, the pharmaceutical composition is prepared with a carrier that protects the viral vector from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers, for example, as described in U.S. Patent No. 4,522,811.

[0155] It may be advantageous to formulate oral, ophthalmic or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0156] Any concentration of recombinant AAV virions suitable for effectively transducing mammalian cells can be prepared. For example, the concentration of recombinant AAV virions can be prepared to be 10 8 vector genomes or more, for example, about 5×10 per ml 8 vector genomes; approximately 10 per ml 9 vector genomes; approximately 5×10 per ml 9 vector genomes, approximately 10 per ml 10 vector genomes, about 5×10 per ml 10 vector genomes; approximately 10 per ml 11 vector genomes; approximately 5×10 per ml 11 vector genomes; approximately 10 per ml 12 vector genomes; approximately 5×10 per ml 12 vector genomes; approximately 10 per ml 13 vector genomes; approximately 1.5 × 10 per ml 13 vector genomes; approximately 3×10 per ml 13 vector genomes; approximately 5×10 per ml 13 vector genomes; approximately 7.5 × 10 per ml 13 vector genomes; approximately 9×10 per ml 13 vector genomes; approximately 1×10 per ml 14 vector genomes, about 5×10 per ml 14 In some embodiments, the amount of the vector may be no more than about 1×10 15 The viral particles are prepared from each vector genome.

[0157] The recombinant AAV virus particles can be formulated into any suitable unit dose, including but not limited to about 1×10 8 vector genomes or more, for example, about 1×10 9 pcs, about 1×10 10 pcs, about 1×10 11 pcs, about 1×10 12 or about 1×10 13 vector genomes or more, in some cases about 1 × 10 14 In some embodiments, the recombinant AAV virus particles can be formulated to have no more than about 4×10 15 Any suitable unit dose of vector genomes. In some cases, the unit dose is up to about 5×10 15 vector genomes, for example, about 1×10 14 vector genomes or less, for example, about 1×10 13 pcs, about 1×1012 pcs, about 1×10 11 pcs, about 1×10 10 or about 1×10 9 vector genomes or fewer, in some cases 1 × 10 8 In some embodiments, the recombinant AAV virus particles can be formulated to have no less than about 1×10 8 Any suitable unit dose of vector genomes. In some cases, the unit dose is about 1×10 10 to about 1×10 11 In some cases, a unit dose is about 1×10 10 to about 3×10 12 In some cases, a unit dose is about 1×10 9 to about 3×10 13 In some cases, a unit dose is about 1×10 8 to about 3×10 14 vector genome.

[0158] In some cases, the unit dose of a pharmaceutical composition can be measured using the multiplicity of infection (MOI). MOI refers to the ratio or number of viral genomes to cells to which the nucleic acid can be delivered. In some cases, the MOI can be about 1×10 6 In some cases, the MOI may be about 1×10 5 About 1×10 7 In some cases, the MOI may be about 1×10 4 About 1×10 8 In some cases, the recombinant viral particles of the present disclosure are at least about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1×10 18In some cases, the recombinant AAV virus particles of the present disclosure are about 1×10 8 About 3×10 14 In some cases, the recombinant viral particles of the present disclosure are at most about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1×10 18 Any one of MOI.

[0159] In some aspects, the amount of the pharmaceutical composition comprises about 1 x 10 8 to about 1×10 15 recombinant AAV virus particles, about 1×10 9 to about 1×10 14 recombinant AAV virus particles, about 1×10 10 to about 1×10 13 recombinant AAV virus particles or about 1×10 11 About 3×10 12 recombinant AAV virus particles.

[0160] The pharmaceutical compositions may be included in a container, pack, or dispenser, such as a syringe, eg, a prefilled syringe, together with instructions for administration.

[0161] In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable salt. Various pharmaceutically acceptable salts are well known in the art and are described in, for example, "Remington's Pharmaceutical Sciences", 17th edition, Alfonso R.Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and its latest version), "Encyclopaedia of Pharmaceutical Technology", 3rd edition, James Swarbrick (ed.), Informa Healthcare USA (Inc.), NY, USA, 2007 and J.Pharm.Sci.66:2 (1977). In addition, for a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0162] Pharmaceutically acceptable base addition salts are formed with metals or amines such as alkali metals and alkaline earth metals or organic amines. Metals used as cations include sodium, potassium, magnesium, calcium, etc. Amines include N-N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine and procaine (see, for example, Berge et al., "Pharmaceutical Salts," J.Pharma Sci., 1977, 66, 119). The base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce a salt in a conventional manner. The free acid form can be regenerated by contacting the salt form with the acid and separating the free acid in a conventional manner. The free acid form is slightly different from its respective salt form in certain physical properties such as solubility in polar solvents, but for the purposes of the present invention, the salt is equivalent to its respective free acid.

[0163] The pharmaceutical composition can be formulated for administration to a mammal, such as a primate, such as a human. In some embodiments, the pharmaceutical composition comprises an aqueous carrier, such as a non-toxic, inert, pharmaceutically acceptable aqueous carrier. In some embodiments, the pH of the aqueous carrier is about 3 to about 8. In some embodiments, the pH of the aqueous carrier is about 6 to about 8.

[0164] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of the recombinant AAV virus particles disclosed herein mixed with a pharmaceutically acceptable carrier and / or excipient, such as saline, phosphate buffered saline, phosphate and amino acids, polymers, polyols, sugars, buffers, preservatives and other proteins. Exemplary amino acids, polymers and sugars, etc. are octylphenoxy polyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitol fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinyl pyrrolidone, polyethylene and ethylene glycol. In some embodiments, the pharmaceutical composition is stable at 4°C for at least 6 months.

[0165] In some embodiments, the pharmaceutical compositions provided herein include a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, tris buffer, glycine buffer, sterile water, and other buffers known to those of ordinary skill in the art, such as those described in Good et al., (1966) Biochemistry 5:467.

[0166] Also provided herein are compositions comprising recombinant AAV virus particles disclosed herein, the recombinant AAV virus particles comprising variant AAV capsid proteins described herein for use in the methods described herein. In some embodiments, the composition is a recombinant AAV virus particle comprising a recombinant AAV virus particle disclosed herein and one or more pharmaceutically acceptable diluents, carriers or excipients.

[0167] Also provided herein are compositions comprising polynucleotides described herein encoding variant AAV capsid proteins described herein. In some embodiments, the compositions are used in methods disclosed herein, such as methods for preparing recombinant AAV virus particles comprising variant AAV capsid proteins.

[0168] VI. Methods and Uses

[0169] As disclosed in the accompanying examples, the modified capsid proteins described herein confer enhanced or altered cell tropism to recombinant AAV virions comprising the variant AAV capsid proteins. For example, certain variant AAV capsid proteins described herein are associated with increased retinal infectivity or increased expression of gene products in the retina, increased binding to ILM, increased retinal infectivity after intravitreal injection, and / or increased expression of gene products in the retina after intravitreal injection.

[0170] Examples of retinal cell components that can be transfected by the viral particles described herein may include, but are not limited to, astrocytes, Müller cells, RGCs, RGC axons, photoreceptors, bipolar cells, amacrine cells, horizontal cells, and combinations thereof. In some examples, cell markers are used to measure transduction of nucleic acids encoding gene products to retinal cell components. Cell markers may include, but are not limited to, GFAP, vimentin, Fox, β-tubulin, rhodopsin, PKCa, parvalbumin, calcium-binding proteins, and combinations thereof. Table 1 below describes exemplary cell markers and corresponding retinal cell components.

[0171] Table 1: Summary of cell markers and cell types

[0172] Markers Cell Type GFAP Astrocytes Vimentin Müller cells Fox RGC β-Tubulin RGC axons Rhodopsin (Rho) Rod photoreceptors PKCa Bipolar cells Parvalbumin Amacrine cells Calcium Binding Protein Horizontal cells

[0173] In some examples, the ability of rAAV virions to transduce gene products can be measured by the expression of a reporter protein. In some embodiments, the reporter protein can include GFP, YFP, red cherry, β-galactosidase, or other reporter proteins known in the art.

[0174] In some examples, the expression of a reporter protein over time can be determined by capturing images of the eye at different time points. For example, the expression of GFP can be associated with an increase in fluorescence. The amount of fluorescence at a given time point can be used to quantify the expression of a gene product.

[0175] In some embodiments, immunofluorescence can be used to determine the localization of the transduced gene in the retinal cell compartments described herein. Retinal cell compartments can be labeled by using antibodies specific for cell markers disclosed herein. In some instances, the antibodies are coupled to fluorophores. In some instances, a secondary antibody can be used to detect the binding of the primary antibody. The transduced retina can be cryosectioned prior to incubation with antibodies against target cell markers, thereby allowing imaging of the bound antibodies. The cryosections can then be labeled with antibodies to detect transduction of the transgene, the presence of the target cell marker, and the overlap between the two.

[0176] Therefore, provided herein is a method for enhancing or changing the tropism of recombinant AAV virus particles, the method comprising introducing a variant AAV capsid protein disclosed herein into a recombinant AAV virus particle. Also provided herein is a method for enhancing or changing the tropism of recombinant AAV virus particles, the method comprising replacing amino acid residues 570-579 of a parent AAV capsid with an amino acid sequence comprising HKFKSGD (SEQ ID NO: 1) to produce a variant AAV capsid protein, and introducing the variant AAV capsid protein into a recombinant AAV virus particle, wherein the amino acid residue numbering corresponds to the AAV5 VP1 capsid protein. In some embodiments, the method comprises replacing amino acid residues 570-579 with an amino acid sequence comprising X1X2HKFKSGDX3 (SEQ ID NO: 2), wherein X 1-3 Can be independently any amino acid. In some embodiments, each X 1-3 In some embodiments, X1 is L. In some embodiments, X2 is A. In some embodiments, X3 is A. In some embodiments, the method comprises replacing amino acid residues 570-579 with an amino acid sequence comprising LAHKFKSGDA (SEQ ID NO: 3); having at least 80% or at least 90% homology to SEQ ID NO: 3; having at least 80% or at least 90% sequence identity to SEQ ID NO: 3; or having four or more, five or more, six or more, seven or more, eight or more, nine or more consecutive amino acids in SEQ ID NO: 3. In some embodiments, the amino acid sequence comprises LAHKFKSGDA (SEQ ID NO: 3).

[0177] In some embodiments, the parent AAV capsid protein is a wild-type AAV capsid protein, such as AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV capsid protein. In some embodiments, the parent AAV capsid protein is an AAV5 capsid protein. In some embodiments, the parent AAV capsid protein is a VP1, VP2, or VP3 capsid protein. In some embodiments, the parent AAV capsid protein is an AAV5 VP1 capsid protein.

[0178] In some embodiments, the parent AAV capsid protein is a variant AAV capsid protein. In some embodiments, the parent AAV capsid protein is a variant of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV or ovine AAV capsid protein. In some embodiments, the parent AAV capsid protein is a variant of AAV5 VP1 capsid protein. In some embodiments, the parent AAV capsid protein is a variant of AAV5 VP1 capsid protein having at least 90%, at least 95%, at least 98% or at least 99% homology with SEQ ID NO: 4. In some embodiments, the parent AAV capsid protein is a variant of AAV5 VP1 capsid protein having at least 90%, at least 95%, at least 98% or at least 99% sequence identity with SEQ ID NO: 4. In some embodiments, the parent AAV capsid protein is a hybrid. In some embodiments, the parent AAV capsid protein is a hybrid of AAV2 and AAV5. In some embodiments, the parent AAV capsid protein is AAV 2.5T, or a variant thereof having at least 90%, at least 95%, at least 98% or at least 99% homology with the AAV2.5T capsid protein. In some embodiments, the parent AAV capsid protein is AAV 2.5T, or a variant thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the AAV2.5T capsid protein. In some embodiments, the parent AAV capsid protein is AAV 2.5T. In some embodiments, the parent AAV capsid protein is VP1, VP2 or VP3 capsid protein. In some embodiments, the parent AAV capsid protein is AAV2.5T VP1 capsid protein, or a variant thereof having at least 90%, at least 95%, at least 98% or at least 99% homology with AAV2.5T VP1 capsid protein (SEQ ID NO: 5). In some embodiments, the parent AAV capsid protein is AAV2.5T VP1 capsid protein, or a variant thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity with AAV2.5T VP1 capsid protein (SEQ ID NO: 5).

[0179] The recombinant AAV virions described herein comprising the variant AAV capsid proteins described herein can be used to deliver gene products to cells (e.g., mammalian cells). For example, it can be used to study, for example, to determine the effects of gene products on cell viability and / or function. As another example, it can be used in medicine, for example, to treat a disease, for example, by delivering a therapeutic gene product to a cell or tissue. Therefore, in some embodiments, a method for expressing a gene product in a cell is provided herein, the method comprising contacting a cell with a composition comprising a recombinant AAV virion, the AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide expression cassette comprising a sequence encoding a gene product. In some embodiments, contact occurs in vitro. In some embodiments, contact occurs in vivo, that is, the composition is administered to a subject. In a specific embodiment, the composition is administered parenterally, for example, intravenously, orally or by injection. In some embodiments, the composition is administered to the eye by injection, for example, to the retina, subretina or vitreous. In some embodiments, the composition is administered by retinal injection, subretinal injection or intravitreal injection. In some embodiments, the composition is administered by intravitreal injection.

[0180] In a specific embodiment where mammalian cells are to be contacted in vitro with recombinant AAV virions comprising variant AAV capsid proteins disclosed herein, the cells can be from any mammalian species, such as rodents (e.g., mice, rats, gerbils, squirrels), rabbits, cats, dogs, goats, sheep, pigs, horses, cattle, primates, humans. The cells can be from established cell lines, e.g., WERI cells, 661W cells, or the cells can be primary cells.

[0181] In certain embodiments, to promote expression of the gene product, the cell is contacted with the recombinant AAV virion for about 30 minutes to about 24 hours or longer, for example, any of about 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 12 hours, 16 hours, 18 hours, 20 hours, or 24 hours, etc. The cell can be contacted with the recombinant AAV virion one or more times, for example, once, twice, three times, or more than three times, and after each contact event, the cell is allowed to be given a certain amount of time with the recombinant AAV virion, for example, about 16-24 hours. Contacting the cell can occur in any culture medium and under any culture conditions that promote cell survival. For example, cells can be suspended in any suitable convenient nutrient medium, such as Iscove's modified DMEM or RPMI 1640, supplemented with fetal bovine serum or heat-inactivated goat serum (about 5-10%), L-glutamine, thiols, particularly 2-mercaptoethanol, and antibiotics, such as penicillin and streptomycin. The culture may contain growth factors to which the cells respond. As defined herein, growth factors are molecules that are capable of promoting the survival, growth and / or differentiation of cells in culture or in intact tissues through specific actions on transmembrane receptors. Growth factors include polypeptide and non-polypeptide factors.

[0182] In certain embodiments, an effective amount of recombinant AAV virions comprising variant AAV capsid proteins are provided to produce expression of gene products in cells. In certain embodiments, the effective amount can be determined empirically, for example, by detecting the presence or level of the gene product, by detecting the effect on cell viability or function, etc. In certain embodiments, an effective amount of recombinant AAV virions promotes the expression of gene products in cells to be equal or higher than the same amount of reference viral vectors (such as recombinant AAV virions comprising parental AAV capsid proteins). In certain embodiments, expression is enhanced by about 2 times or more relative to expression of a reference viral vector; for example, about 3 times, 4 times, or 5 times or more, in some cases, about 10 times, 20 times, or 50 times or more, for example, 100 times. In certain embodiments, the enhanced expression occurs in a specific cell type, for example, any ocular cell described herein.

[0183] For example, in the case of in vivo contact of a cell with a composition, the composition comprises a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide expression cassette comprising a sequence encoding a gene product, and the subject can be any mammal, for example, a rodent (e.g., mouse, rat, gerbil), rabbit, cat, dog, goat, sheep, pig, horse, cattle, or primate (e.g., human or non-human primate). The methods and compositions of the present disclosure can be used to treat any condition that can be at least partially addressed by cell gene therapy. Therefore, the compositions and methods of the present disclosure can be used to treat individuals in need of cell therapy. Cells include, but are not limited to, blood, eyes, liver, kidney, heart, muscle, stomach, intestine, pancreas, and skin.

[0184] Also provided herein are methods for delivering gene products to the retina, comprising administering by intravitreal injection a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide expression cassette comprising a sequence encoding a gene product. In some embodiments, provided herein are methods for delivering gene products across the ILM, comprising administering by intravitreal injection a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide expression cassette comprising a sequence encoding a gene product. In certain embodiments, the disclosure provides methods for providing a gene product to the retina of a subject, comprising administering to a subject by intravitreal injection a pharmaceutical composition comprising a recombinant AAV virion as described herein, wherein the recombinant AAV virion comprises a variant AAV capsid provided or disclosed herein and a polynucleotide sequence encoding a gene product.

[0185] In certain embodiments, the subject has been diagnosed with or is suspected of having one or more diseases or conditions selected from the group consisting of 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 affecting the posterior part or location of the eye; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia; Vogt's disease; Koyanagi-Harada (VKH) syndrome; uveal ectasia; posterior eye conditions caused or affected by ocular laser therapy; posterior eye conditions caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; diabetic retinopathy; ischemic retinopathy; non-retinopathic diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (macular); hereditary macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroidergic hemorrhage; Bardet-Biedl syndrome; macular telangiectasia; Leber hereditary optic neuropathy; retinopathy of prematurity; and color vision disorders, including achromatopsia, hyperopia, deep-metropia, and trisopia.

[0186] In certain embodiments, the subject is at risk for developing one or more diseases or conditions selected from the group consisting of 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 affecting the posterior part or location of the eye; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia; Vogt's disease; Koyanagi-Harada (VKH) syndrome; uveal ectasia; posterior eye conditions caused or affected by ocular laser therapy; posterior eye conditions caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; diabetic retinopathy; ischemic retinopathy; non-retinopathic diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (macular); hereditary macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroidergic hemorrhage; Bardet-Biedl syndrome; macular telangiectasia; Leber hereditary optic neuropathy; retinopathy of prematurity; and color vision disorders, including achromatopsia, hyperopia, deep-metropia, and trisopia.

[0187] Also provided herein is a method for treating a retinal disease or condition in a subject in need thereof, comprising administering a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide expression cassette comprising a sequence encoding a therapeutic gene product by intravitreal injection. Also provided herein is a method for prophylactic treatment of a disease or condition in the retina of a subject in need thereof, comprising administering a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide expression cassette comprising a sequence encoding a therapeutic gene product by intravitreal injection. Also provided herein is a method for treating a determined or confirmed disease or condition in the retina of a subject in need thereof, comprising administering a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide expression cassette comprising a sequence encoding a therapeutic gene product by intravitreal injection. In some embodiments, the method comprises administering a pharmaceutical composition comprising a recombinant AAV virion comprising a variant AAV capsid protein disclosed herein and a polynucleotide expression cassette comprising a sequence encoding a therapeutic gene product.

[0188] In some embodiments, the disease or condition is selected from the group consisting of 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 affecting the posterior part or location of the eye; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia; Vogt's disease; Koyanagi-Harada (VKH) syndrome; uveal ectasia; posterior eye conditions caused or affected by ocular laser therapy; posterior eye conditions caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; diabetic retinopathy; ischemic retinopathy; non-retinopathic diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (macular); hereditary macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroidergic hemorrhage; Bardet-Biedl syndrome; macular telangiectasia; Leber hereditary optic neuropathy; retinopathy of prematurity; and color vision disorders, including achromatopsia, hyperopia, deep-metropia, and trisopia.

[0189] In certain embodiments, the subject is at risk for developing one or more diseases or conditions selected from the group consisting of 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 affecting the posterior part or location of the eye; ocular tumors; retinal disorders, such as central retinal vein occlusion, diabetic retinopathy (including proliferative diabetic retinopathy), proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia; Vogt's disease; Koyanagi-Harada (VKH) syndrome; uveal ectasia; posterior eye conditions caused or affected by ocular laser therapy; posterior eye conditions caused or affected by photodynamic therapy; photocoagulation, radiation retinopathy; epiretinal membrane disorders; branch retinal vein occlusion; anterior ischemic optic neuropathy; diabetic retinopathy; ischemic retinopathy; non-retinopathic diabetic retinal dysfunction; retinoschisis; retinitis pigmentosa; glaucoma; Usher syndrome, cone-rod dystrophy; Stargardt disease (macular); hereditary macular degeneration; chorioretinal degeneration; Leber congenital amaurosis; congenital stationary night blindness; choroidergic hemorrhage; Bardet-Biedl syndrome; macular telangiectasia; Leber hereditary optic neuropathy; retinopathy of prematurity; and color vision disorders, including achromatopsia, hyperopia, deep-metropia, and trisopia.

[0190] In certain embodiments, the gene product inhibits neovascularization in the retina of a subject, for example, choroidal neovascularization (CNV). Many cytokines have been found to play an important role in the regulation of CNV generation, including but not limited to vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), platelet-derived growth factor (PDGF), hypoxia-inducible factor (HIF), angiopoietin (Ang) and other cytokines, and mitogen-activated protein kinase (MAPK). In some embodiments, the gene product is an inhibitor of one or more of VEGF, VEGFR, PDGF, HIF, Ang and MAPK.

[0191] In some embodiments, the gene product is a gene product disclosed herein or a therapeutic gene product. In some embodiments, the gene product is an interfering RNA, an aptamer or a protein. In some embodiments, the therapeutic gene product is an interfering RNA, an aptamer or a protein. In some embodiments, the gene product (or therapeutic gene product) is an siRNA, a miRNA or a protein. In some embodiments, the gene product (or therapeutic gene product) is a site-specific nuclease that provides a site-specific knockdown of a gene function. In some embodiments, the gene product (or therapeutic gene product) is an interfering RNA. In some embodiments, the gene product (or therapeutic gene product) is an siRNA or a miRNA.

[0192] In some embodiments, the methods produce a therapeutic benefit, e.g., preventing development of a disease or condition, arresting progression of a disease or condition, reversing progression of a disease or condition, etc. In some embodiments, the methods include the step of detecting that a therapeutic benefit has been achieved.

[0193] In some examples, expression of the gene product can be observed two months or less than two months after administration, for example, about 4, 3 or 2 weeks or less than 2 weeks after administration, for example, 1 week after administration of the present composition, for example, by measuring the content of the gene product, by measuring the therapeutic effectiveness, etc. to detect. It is also expected that the expression of the gene product is maintained over time. Therefore, in some examples, expression of the gene product can be observed 2 months or more than 2 months, for example, about 4, 6, 8 or 10 months or more than 10 months, in some cases about 1 year or more than 1 year, for example, 2, 3, 4 or 5 years, and in some cases more than 5 years after administration of the present composition, for example, as detected by measuring the content of the gene product, by measuring the therapeutic effectiveness, etc.

[0194] In certain embodiments, about 1×10 8 vector genomes or more, in some cases about 1 × 10 9 , 1×10 10 , 1×10 11 , 1×10 12 or 1×10 13 vector genomes or more, and in some cases, about 1×10 14 In some cases, the amount of delivered vector genomes is up to about 1×10 15 vector genomes, e.g., 1 × 10 14 vector genomes or less, for example, about 1×10 13 , 1×10 12 , 1×10 11 , 1×10 10or 1×10 9 vector genomes or less, in some cases 1×10 8 vector genomes, and sometimes not less than 1×10 8 In some cases, the amount of vector genome delivered is about 1×10 10 to about 1×10 11 In some cases, the amount of vector genome delivered is about 1×10 10 About 3×10 12 In some cases, the amount of vector genome delivered is about 1×10 9 About 3×10 13 In some cases, the amount of vector genome delivered is about 1×10 8 About 3×10 14 vector genome.

[0195] In some cases, the unit dose of a pharmaceutical composition can be measured using the multiplicity of infection (MOI). MOI refers to the ratio or number of viral genomes to cells to which the nucleic acid can be delivered. In some cases, the MOI can be about 1×10 6 In some cases, the MOI may be about 1×10 5 About 1×10 7 In some cases, the MOI may be about 1×10 4 About 1×10 8 In some cases, the recombinant viral particles of the present disclosure are at least about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1×10 18 In some cases, the recombinant AAV virus particles of the present disclosure are about 1×10 8 About 3×10 14In some cases, the recombinant viral particles of the present disclosure are at most about 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 , 1×10 16 , 1×10 17 and 1×10 18 Any one of MOI.

[0196] In some aspects, the amount of the pharmaceutical composition comprises about 1 x 10 8 to about 1×10 15 recombinant AAV virus particles, about 1×10 9 to about 1×10 14 recombinant AAV virus particles, about 1×10 10 to about 1×10 13 recombinant AAV virus particles or about 1×10 11 About 3×10 12 recombinant AAV virus particles.

[0197] In some embodiments, more than one administration (eg, two, three, four, or more administrations) may be employed to achieve desired gene expression levels at various time intervals (eg, daily, weekly, monthly, yearly, etc.).

[0198] In some aspects, recombinant AAV virions are not detected in tear, blood, saliva, or urine samples of human subjects at about 7, about 14, about 21, or about 30 days after administration of the pharmaceutical composition. In some aspects, the presence or absence of recombinant AAV virions is detected by qPCR or ELBA.

[0199] In some aspects, following the methods of treatment described herein, the subject's best corrected visual acuity (BCVA) improves by about 1, 2, 3, 4, 5 lines or more.

[0200] In some aspects, after the administering step, neovascularization as assessed by fluorescein angiography (FA) is reduced.

[0201] In some cases, retinal thickness can be measured to check the therapeutic effect. In some cases, within about 12 months after treatment with the pharmaceutical composition of the present disclosure, the central retinal thickness of the human subject increases by no more than about 50 microns, about 100 microns or about 250 microns. In some cases, within about 3 months, about 6 months, about 9 months or about 12 months after treatment with the pharmaceutical composition of the present invention, the central retinal thickness of the human subject decreases by at least about 50 microns, about 100 microns, about 200 microns, about 250 microns, about 300 microns, about 400 microns, about 500 microns or about 600 microns. The central retinal thickness at one or more time points can be compared with the baseline measurement obtained when applying the pharmaceutical composition of the present invention for about 1, 3, 7 or 10 days or in about 1, 3, 7 or 10 days of applying the pharmaceutical composition of the present invention, to measure the reduction of the central retinal thickness of the human subject.

[0202] From the foregoing it will be appreciated that although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited by the appended claims.

[0203] VII. Exemplary Embodiments

[0204] The provided implementations include:

[0205] 1. A recombinant adeno-associated virus (AAV) virion comprising:

[0206] (a) a variant AAV capsid protein comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the amino acid residue numbering corresponds to AAV5 VP1 capsid protein; and

[0207] (b) A polynucleotide sequence encoding a therapeutic gene product.

[0208] 2. The recombinant AAV virus particle according to embodiment 1, wherein the parent AAV capsid protein is an AAV5 capsid protein or a hybrid capsid protein of AAV5 and AAV2.

[0209] 3. The recombinant AAV virus particle according to embodiment 1 or embodiment 2, wherein the parent AAV capsid protein is AAV2.5T capsid protein.

[0210] 4. The recombinant AAV virus particle of any one of embodiments 1-3, wherein the parent AAV capsid protein is AAV2.5T VP1 capsid protein.

[0211] 5. A recombinant AAV virus particle according to any one of embodiments 1-4, wherein the modified sequence comprises LAHKFKSGDA (SEQ ID NO: 3).

[0212] 6. A recombinant AAV virus particle according to any one of embodiments 1-5, wherein the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5.

[0213] 7. A recombinant AAV virus particle according to any one of embodiments 1-6, wherein the variant AAV capsid protein comprises the capsid sequence shown in SEQ ID NO:6 or SEQ ID NO:7.

[0214] 8. A recombinant AAV virus particle according to any one of embodiments 1-7, wherein the rAAV virus particle is a variant AAV5 or a variant AAV2 and AAV5 hybrid virus particle.

[0215] 9. A recombinant AAV virus particle according to any one of embodiments 1-8, wherein the rAAV virus particle is a variant AAV2.5T virus particle.

[0216] 10. The recombinant AAV virus particle of any one of embodiments 1-9, wherein the recombinant AAV virus particle is capable of transducing retinal cells when injected intravitreally into a mammal.

[0217] 11. A recombinant AAV virus particle according to embodiment 10, wherein the recombinant AAV virus particle is capable of transducing one or more of the following when injected intravitreally into a mammal: photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells and retinal pigment epithelial cells.

[0218] 12. The recombinant AAV virus particle of embodiment 10 or embodiment 11, wherein the recombinant AAV virus particle is capable of transducing retinal pigment epithelial cells when injected intravitreally into a mammal.

[0219] 13. The recombinant AAV virus particle of any one of embodiments 1-12, wherein the therapeutic gene product is siRNA, miRNA or protein.

[0220] 14. A recombinant AAV virus particle according to any one of embodiments 1-13, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product.

[0221] 15. The recombinant AAV virus particle of any one of embodiments 1-13, wherein the therapeutic gene product is opsin.

[0222] 16. The recombinant AAV virion of any one of embodiments 1-15, wherein the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs.

[0223] 17. The recombinant AAV virion of embodiment 16, wherein the one or more AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs or variants thereof.

[0224] 18. The recombinant AAV virion of embodiment 16 or embodiment 17, wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs.

[0225] 19. A recombinant AAV virus particle according to any one of embodiments 1-18, wherein the recombinant AAV virus particle has altered cell tropism compared to AAV2.5T.

[0226] 20. A pharmaceutical composition comprising the recombinant AAV virus particles of any one of embodiments 1-19.

[0227] 21. A method for producing rAAV virus particles, comprising:

[0228] (a) culturing a host cell under conditions whereby rAAV virions are produced, wherein the host cell comprises:

[0229] (i) a polynucleotide encoding a variant AAV capsid protein comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1);

[0230] (ii) a polynucleotide encoding a rep protein;

[0231] (iii) a polynucleotide expression cassette comprising a sequence encoding a therapeutic gene product flanked by at least one AAV ITR; and

[0232] (iv) AAV helper functions; and

[0233] (b) recovering the rAAV virus particles produced by the host cells.

[0234] 22. A method for providing a therapeutic gene product to the retina of a subject, comprising administering to the subject by intravitreal injection the recombinant AAV virus particle of any one of embodiments 1-19 or the pharmaceutical composition of embodiment 20.

[0235] 23. The method of embodiment 22, wherein the subject has been diagnosed with or is suspected of having one or more conditions selected from the group consisting of acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior part of the eye, ocular tumors, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia, Vogt Koyanagi-Harada (VKH) syndrome, uveal diffusion, posterior ocular conditions caused or affected by ocular laser treatment , posterior ocular conditions caused by or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorders, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinopathy, ischemic retinopathy, non-retinopathic diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, choroideremia, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, and color vision impairment.

[0236] 24. A method of treating a retinal disease or condition in a subject in need thereof, comprising administering to the subject by intravitreal injection the recombinant AAV viral particle of any one of embodiments 1-19 or the pharmaceutical composition of embodiment 20.

[0237] 25. The method of embodiment 24, wherein the disease or condition is acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior part of the eye, ocular tumors, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia, Vogt's cataract, retinal detachment ... Koyanagi-Harada (VKH) syndrome, uveal ectasia, posterior eye conditions caused or affected by ocular laser therapy, posterior eye conditions caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinopathy, ischemic retinopathy, non-retinopathic diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, choroideremia, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, or color vision impairment.

[0238] 26. A recombinant AAV virus particle according to any one of embodiments 1-19, which is used in a method for treating a retinal disease or condition in a subject in need thereof, wherein the method comprises administering a pharmaceutical composition comprising the recombinant AAV virus particle to the subject by intravitreal injection.

[0239] 27. A recombinant AAV viral particle according to any one of embodiments 1-19, which is used to prepare a medicament for treating a retinal disease or condition in a subject.

[0240] 28. The recombinant AAV virion of embodiment 26 or embodiment 27, wherein the disease or condition is age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, or diabetic retinal edema.

[0241] 29. A variant AAV capsid protein comprising a modified sequence, wherein the modified sequence comprises one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the amino acid residue numbering corresponds to AAV5 VP1 capsid protein.

[0242] 30. A variant AAV capsid protein according to embodiment 29, wherein the parent AAV capsid protein is an AAV5 capsid protein or a hybrid capsid protein of AAV5 and AAV2.

[0243] 31. A variant AAV capsid protein according to embodiment 29 or embodiment 30, wherein the parent AAV capsid protein is AAV2.5T capsid protein.

[0244] 32. The variant AAV capsid protein of any one of embodiments 29-31, wherein the parent AAV capsid protein is AAV2.5T VP1 capsid protein.

[0245] 33. A variant AAV capsid protein according to any one of embodiments 29-32, wherein the modified AAV capsid protein comprises LAHKFKSGDA (SEQ ID NO: 3) at amino acid residues 570-579 relative to the parent AAV capsid protein.

[0246] 34. A variant AAV capsid protein according to any one of embodiments 29-33, which comprises a capsid sequence having at least 85% homology to the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5.

[0247] 35. The variant AAV capsid protein of any one of embodiments 29-34, comprising the capsid sequence shown in SEQ ID NO:6 or SEQ ID NO:7.

[0248] 36. A nucleic acid comprising a nucleic acid sequence encoding the variant AAV capsid protein of any one of embodiments 29-35.

[0249] 37. An expression vector comprising the nucleic acid described in embodiment 36, wherein the nucleic acid sequence encoding the variant AAV capsid protein is operably linked to a promoter sequence.

[0250] 38. The expression vector of embodiment 37, further comprising a nucleic acid encoding a rep protein.

[0251] 39. A cell comprising the expression vector of embodiment 37 or embodiment 38.

[0252] 40. The cell of embodiment 39, further comprising a nucleic acid encoding a therapeutic gene product.

[0253] 41. The cell of embodiment 40, wherein the therapeutic gene product is siRNA, miRNA or protein.

[0254] 42. A cell according to embodiment 40 or embodiment 41, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product.

[0255] 43. The cell of embodiment 40 or embodiment 41, wherein the therapeutic agent is an opsin.

[0256] 44. The cell of any one of embodiments 40-43, wherein the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs.

[0257] 45. The cell of embodiment 44, wherein the one or more AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs or variants thereof.

[0258] 46. ​​The cell of embodiment 44 or embodiment 45, wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs.

[0259] Example

[0260] Example 1: Construction of AAV2.5T mutant library

[0261] AAV2.5T is a hybrid capsid containing regions from AAV2 and AAV5, described in U.S. Pat. No. 9,441,244, the entire disclosure of which is incorporated herein. AAV2.5T is able to transduce the retina when delivered subretinal, but not when injected intravitreally. AAV2.5T transduction can be blocked by the inner limiting membrane (ILM) which is rich in heparan sulfate proteoglycans (HSPGs).

[0262] The surface-exposed domains of AAV2.5T are identical to those of AAV5, except for a single A to T substitution in aa582 of AAV2.5T (aa581 of AAV5), a mutation that appears to increase mammalian cell infectivity without affecting canonical sialic acid receptor binding of AAV5. AAV5 and AAV2.5T have negligible binding to heparan sulfate, whereas AAV2 has a high affinity for heparan sulfate.

[0263] To develop a new AAV variant with enhanced ability to cross the ILM and transduce retinal cells when delivered by intravitreal injection, an AAV mutant library with loop substitutions and insertions was generated from a region in the AAV2.5T capsid that is analogous to the HSPG-binding region of AAV2, as determined by amino acid alignment.

[0264] Two mutation libraries were generated by synthesizing DNA fragments with random 7-amino acid loops. Since there are 20 possible amino acids at each of the 7 amino acid positions, the theoretical diversity of these fragment libraries is approximately 1x10 9 These mutant loops are flanked by amino acids LA at the 5' end and A at the 3' end.

[0265] Using these fragment libraries, loop insertion libraries (LILs) were generated by inserting a 10 amino acid loop at position 577 of the AAV2.5T capsid sequence in the receptor binding region.

[0266] A second library was generated by replacing sequences in capsid surface exposed regions with loop variant sequences between amino acids 571 and 580 (numbered from the first amino acid of VP1). This library is referred to as the loop replacement library (LSL).

[0267] These AAV variant capsids were cloned into plasmids to make a single virus packaging cell library (by transiently transfecting phiC31 integrase at a ratio of 1:50 (plasmid:phiC31), as described in WO2017112868A1). The resulting packaging cell library produces mutant AAV virions consisting of a vector genome comprising a mutant Cap gene packaged within a capsid protein encoded by the genome, with low levels of cross-packaging. To determine the diversity of the cell library, gDNA was isolated from each library and NGS was performed. The diversity was approximately 725,000 unique variants in LIL and approximately 558,000 unique variants in LSL.

[0268] Example 2: AAV library screening in NHP eyes

[0269] To investigate the ability of mutant AAV capsids to cross the ILM, a library of variant AAV virions was screened in vivo for their ability to cross the inner limiting membrane (ILM) and infect retinal cells in non-human primate (NHP) eyes.

[0270] The LIL and LSL mutant AAV capsid libraries were injected intravitreally into the eyes of three African green monkeys (Chlorocebus sabaeus) at a dose of 6x10 9 and 9x10 9 vg. Four to six weeks after injection, the animals were killed and the retinal tissue explants of each animal were isolated. The vitreous and retina with choroidal subtissue were separated, the eyeball was cut off along the corneal limbus, and the front of the eye was discarded. The vitreous was then removed, and the retina was attached to the eye cup (eyecup). The eye cup was transferred to a CO2-independent culture medium. The retina and RPE / choroid were cut into retinal flat-mounts. The entire flat-mount (photoreceptor layer downward) was placed on a polycarbonate insert membrane of a 10cm Trans-well culture plate, which contained 10mL of CO2-independent culture medium. When the retina gently settled on the insert, the culture medium was removed so that the retina was exposed to the atmosphere.

[0271] The retinas were transduced with human Ad5 virus (diluted in Neurobasal-A medium without antibiotics or antimycotics) at a multiplicity of infection (MOI) of 1000 by adding Ad5 solution on top of the tissue for 2 hours. Co-infection with Ad5 virus was used to increase the expression of Cap RNA of mutant AAV virions present in retinal tissue, thereby increasing the amount of mutant capsid RNA in infected cells for subsequent detection.

[0272] After the initial incubation, Neurobasal-A+ (B27 supplement, 1X GlutaMAX, 1X antibiotic-antimycotic) medium was added to the bottom of the wells so that it was in contact with the bottom of the insert but not covering the tissue (~10 ml), and the tissue explants were incubated at 37°C with CO2 for 2 days. After incubation, the inserts were removed from the culture plate and placed on a clean, flat surface such as Figure 1A 3 punches were prepared as shown in . In brief, a 5 mm punch of the neural retina with RPE / choroid was obtained from the macula, and then a second concentric punch was prepared using a 10 mm punch to separate the retina and RPE / choroid area of ​​the macula periphery in the blood arch, which was referred to as the parafovea. The remaining peripheral retina and RPE / choroid were collected subsequently.

[0273] RNA was then extracted from each tissue punch and converted to cDNA by RT-PCR and one round of PCR. The resulting PCR products were then cloned back into plasmids and sequenced to identify the sequence of the mutant AAV capsid that was able to successfully cross the ILM and infect the retinal cells of the monkey eye.

[0274] For the second and third rounds of screening, LIL and LSL variants extracted from each individual retinal location (punch) were mixed together to create a variant library for each location. These library mixtures were then used to generate cell and AAV capsid libraries, which were injected separately into the eyes of African green monkeys (AGM) and collected using the same retinal punch strategy as described above. In addition, in one case, the retinal pigment epithelium was isolated and variants were recovered from this tissue.

[0275] Using NGS analysis, AAV2.5T.LSV1 was identified. AAV2.5T.LSV1 is a loop substitution variant derived from the LSL library with the following amino acid loop sequence: LAHKFKSGDA (SEQ ID NO:3). AAV2.5T.LSV1 was the 48th most abundant variant in the macula in round 2 of screening. In round 3 of screening, AAV2.5T.LSV1 was the second most abundant variant found in the parafovea and the most abundant variant found in the RPE region. The relative abundance of the top variants in each round of screening is shown in Figure 2. Figure 1B-1E as shown in .

[0276] Example 3: Production of AAV2.5T.LSV1 in HEK293 cells

[0277] AAV2.5T.LSV1 was produced in HEK293 cells using a triple transfection method for further characterization. The titer of the resulting AAV2.5T.LSV1 was higher than 1 x 10 13 vg / ml, indicating that AAV2.5T.LSV1 has good packaging ability.

[0278] Example 4: Heparin Binding Affinity of AAV2.5T.LSV1

[0279] The ability of AAV2.5T.LSV1 to bind to HSPG was assessed by performing a heparin binding assay using a pre-packed GE heparin column. Heparan binding of AAV2.5T.LSV1 was compared to AAV2.5T and two other variants generated in the above screen. The vector was loaded onto the column, then washed and finally eluted with increasing concentrations of NaCl (100 mM-1 M). The loaded, flow-through, washed and eluted fractions were collected and analyzed by dot blot using the B1 antibody.

[0280] like Figure 2 As shown in , AAV2.5T did not bind to heparin. In contrast, AAV2.5T.LSV1 had the ability to bind to heparin.

[0281] Example 5: Expression and tropism in porcine retinal explants

[0282] Ex vivo porcine retinal explants were maintained in trans-wells as described in PCT / US2017 / 030636, the entire disclosure of which is incorporated herein, and transduced with a GFP reporter gene at 2×10 10 vg or 4x10 10 vg was packaged into AAV2.5T.LSV1 capsid. Live fluorescence images were captured at 1 and 2 weeks after transduction. Figure 3 Live images of porcine retinal explants are shown. These images suggest that AAV2.5T.LSV1 is able to effectively transduce at least some cells of the porcine retina in culture.

[0283] Example 6: Expression after intravitreal administration to non-human primate retina

[0284] In vivo studies were performed in African green monkeys (Chlorocebus sabaeus) using 5x10 11 African green monkeys were intravitreally injected with AAV2.5T.LSV1 carrying a GFP expression cassette. Expression in the eye was monitored weekly by fundus fluorescence and Heidelberg spectral imaging. Figures 4A-4E As shown in . Intravitreal delivery of AAV2.5T.LSV1-GFP results in GFP expression within the macula and blood arches. The blood arch region of the retina has the thickest ILM, which is thought to prevent most AAV transduction of retinal cells when injected intravitreally.

[0285] Example 7: Evaluation of expression after intravitreal administration

[0286] The retinal tropism of AAV2.5T.LSV1 was evaluated in nonhuman primates following IVT injection.

[0287] African green monkeys (Chlorocebus sabaeus) were injected intravitreally (IVT) with 5×10 11 vg / eye of AAV2.5T.LSV1 carrying a GFP expression cassette under the control of a ubiquitous promoter to assess the tropism of AAV2.5T.LSV1.

[0288] Monkeys were killed and eyes were enucleated on day 35 after IVT injection. After enucleation, eyes were fixed in 4% paraformaldehyde for 24 h and then frozen overnight with 30% sucrose / Sorensen phosphate buffer. Eyes were dissected into quadrants and the retina, including the RPE and choroid, was removed from the sclera. Retinal quadrants were flat-mounted on slides and coverslipped with 30% sucrose / Sorensen phosphate buffer. 5X tile scans of native GFP were acquired using a Zeiss Axio.Z1 and stitched using Zeiss Zen Blue software. The resulting live fluorescence images of AAV2.5T.LSV1-GFP are shown in Figures 5A-5D as shown in .

[0289] GFP intensity distribution is from Figure 5C-5D The retinal fovea was obtained from a 5X scan of the flat-mount retinal section shown in Figure 1. The measurements were made from the edge of the macula closest to the optic nerve to the ciliary body. Approximately 20,000 measurement points were plotted over a total length of 20 mm ( Figure 5E ).

[0290] Retinal flat sheets were embedded in OCT mounting medium and frozen on isopentane. Cryosections were collected at a thickness of 10 μm through the fovea, mid-periphery and periphery, and slides were stained for immunofluorescence labeling (IFL). Planar images of RPE were made by peeling the RPE from the choroid in a portion of the flat-mounted retina, then inverting the slices, placing the RPE face down on the slide, and then performing IFL staining. IFL was performed after 0.1% Triton-x permeabilization for 30 min, and then serum proteins were blocked for 1 hour with 5% normal donkey serum / 6% bovine serum albumin in Sorensen phosphate buffer. Overnight at 4 ° C, IFL was performed using the staining and antibodies shown in Table 2.

[0291] Table 2: Staining and Antibodies

[0292]

[0293]

[0294] The tissue sections were imaged and the resulting images were Figure 6A-25E A summary of the cell type tropism of AAV2.5T.LSV1 virions is shown in Table 3.

[0295] Table 3: Tropism of Virus Particles

[0296]

[0297] The percentage of GFP-positive cells was quantified by manual cell counting of colocalized cells with cell-specific markers. The number of GFP-positive bipolar cells, cones, and RPE cells in each retinal region was calculated as follows: Figures 26A-26C The numbers of GFP-positive cells varied depending on their location in the retina.

[0298] Table 4: Percentage of GFP positive cells

[0299] Cell type (location) % of total Cones (fovea) 14-16％ Photoreceptors (middle and peripheral retina) 25-55％ Bipolar cells (middle and peripheral retina) 25-50％ RPE cells (middle and peripheral retina) 2.5-4％

[0300] Example 8 Determination of neutralizing antibody curve

[0301] Neutralizing antibody (nAB) curves for AAV2.5T.LSV1 and AAV2.5T viruses were determined by in vitro assays. Briefly, serial dilutions of mixed human IgG antibodies (Gammaguard IVIG) were combined with AAV2.5T.LSV1 or AAV2.5T vectors expressing GFP and used to transduce 293T cells. GFP expression was then measured and the inhibition of GFP expression was used to calculate the IC 50 .

[0302] Example 9: Comparison of neutralizing antibody curves of AAV2.5T.LSV1 and AAV2

[0303] Neutralizing antibody (nAB) curves for AAV2.5T.LSV1 and AAV2 viruses were determined by in vitro assays. Briefly, 3-fold serial dilutions of human IgG antibodies (Gammaguard IVIG) were prepared and combined with AAV2.5T.LSV1 or AAV2 vectors expressing GFP (AAV2.5T.LSV1-CMV-GFP or AAV2-CMV-GFP, respectively). The mixture was then used to transduce 293T cells. The cells were incubated for 3 days and GFP expression was subsequently measured. The inhibition of GFP expression was used to calculate the IC 50 IC 50 Reported as the reciprocal dilution of the pooled human IgG antibody sample at which GFP expression was reduced by 50% (e.g., IC 50 =100 indicates that IVIG diluted 1:100 reduced GFP expression by 50%). 50 The values ​​indicate that the antibodies present in the mixed human IgG antibodies increased the inhibition of AAV transduction.

[0304] The inhibition curves of AAV2.5T.LSV1 and AAV2 are shown in Figures 27A-27B The IC calculated for AAV2 50 The IC calculated for AAV2.5T.LSV1 is 103.8. 50 It is 9.3.

[0305] sequence

[0306]

[0307]

[0308]

[0309]

Claims

1. A recombinant adeno-associated virus (AAV) virion comprising: (a) a variant AAV capsid protein comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the amino acid residue numbering corresponds to AAV5 VP1 capsid protein; and (b) A polynucleotide sequence encoding a therapeutic gene product.

2. The recombinant AAV virus particle according to claim 1, wherein the parent AAV capsid protein is an AAV5 capsid protein or a hybrid capsid protein of AAV5 and AAV2.

3. The recombinant AAV virus particle according to claim 1 or claim 2, wherein the parent AAV capsid protein is AAV2.5T capsid protein.

4. The recombinant AAV virus particle according to any one of claims 1 to 3, wherein the parent AAV capsid protein is AAV2.5T VP1 capsid protein.

5. The recombinant AAV virion of any one of claims 1-4, wherein the modified sequence comprises LAHKFKSGDA (SEQ ID NO: 3).

6. The recombinant AAV virus particle of any one of claims 1-5, wherein the variant AAV capsid protein comprises a capsid sequence having at least 85% homology to the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:

5.

7. The recombinant AAV virus particle of any one of claims 1-6, wherein the variant AAV capsid protein comprises the capsid sequence shown in SEQ ID NO:6 or SEQ ID NO:

7.

8. The recombinant AAV virion according to any one of claims 1-7, wherein the rAAV virion is a variant AAV5 or a variant AAV2 and AAV5 hybrid virion.

9. The recombinant AAV virion of any one of claims 1-8, wherein the rAAV virion is a variant AAV2.5T virion.

10. The recombinant AAV virion of any one of claims 1-9, wherein the recombinant AAV virion is capable of transducing cells of the retina when injected intravitreally into a mammal.

11. The recombinant AAV virion of claim 10, wherein the recombinant AAV virion is capable of transducing one or more of the following when injected intravitreally into a mammal: photoreceptors, retinal ganglion cells, Müller cells, bipolar cells, amacrine cells, horizontal cells, and retinal pigment epithelial cells.

12. The recombinant AAV virion of claim 10 or claim 11, wherein the recombinant AAV virion is capable of transducing retinal pigment epithelial cells when injected intravitreally into a mammal.

13. The recombinant AAV virion of any one of claims 1-12, wherein the therapeutic gene product is siRNA, miRNA or protein.

14. The recombinant AAV virion of any one of claims 1-13, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product.

15. The recombinant AAV virion of any one of claims 1-13, wherein the therapeutic gene product is an opsin.

16. The recombinant AAV virion of any one of claims 1-15, wherein the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs.

17. The recombinant AAV virion of claim 16, wherein the one or more AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs or variants thereof.

18. The recombinant AAV virion of claim 16 or claim 17, wherein the one or more AAVI ITRs are AAV2 ITRs or AAV5 ITRs.

19. The recombinant AAV virion of any one of claims 1-18, wherein the recombinant AAV virion has altered cell tropism compared to AAV2.5T.

20. A pharmaceutical composition comprising the recombinant AAV virus particle of any one of claims 1-19.

21. A method for producing rAAV virus particles, comprising: (a) culturing a host cell under conditions whereby rAAV virions are produced, wherein the host cell comprises: (i) a polynucleotide encoding a variant AAV capsid protein comprising a modified sequence comprising one or more amino acid substitutions within amino acid residues 570-579 relative to a parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1); (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide expression cassette comprising a sequence encoding a therapeutic gene product flanked by at least one AAVITR; and (iv) AAV helper functions; and (b) recovering the rAAV virus particles produced by the host cells.

22. A method of providing a therapeutic gene product to the retina of a subject, comprising administering to the subject by intravitreal injection the recombinant AAV viral particle of any one of claims 1-19 or the pharmaceutical composition of claim 20.

23. The method of claim 22, wherein the subject has been diagnosed with or is suspected of having one or more conditions selected from the group consisting of acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior part of the eye, ocular tumors, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia, Vogt Koyanagi-Harada (VKH) syndrome, uveal diffusion, posterior ocular conditions caused or affected by ocular laser treatment , posterior ocular conditions caused by or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorders, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinopathy, ischemic retinopathy, non-retinopathic diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, choroideremia, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, and color vision impairment.

24. A method of treating a retinal disease or disorder in a subject in need thereof, comprising administering to the subject by intravitreal injection the recombinant AAV virion of any one of claims 1-19 or the pharmaceutical composition of claim 20.

25. The method of claim 24, wherein the disease or condition is acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, macular degeneration, edema, multifocal choroiditis, ocular trauma affecting the posterior part of the eye, ocular tumors, central retinal vein occlusion, diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal artery occlusive disease, retinal detachment, uveitic retinopathy, sympathetic ophthalmia, Vogt's cataract, retinal detachment ... Koyanagi-Harada (VKH) syndrome, uveal ectasia, posterior eye conditions caused or affected by ocular laser therapy, posterior eye conditions caused or affected by photodynamic therapy, photocoagulation, radiation retinopathy, epiretinal membrane disorder, branch retinal vein occlusion, anterior ischemic optic neuropathy, diabetic retinopathy, ischemic retinopathy, non-retinopathic diabetic retinal dysfunction, retinoschisis, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod dystrophy, Stargardt disease, hereditary macular degeneration, chorioretinal degeneration, Leber congenital amaurosis, congenital stationary night blindness, choroideremia, Bardet-Biedl syndrome, macular telangiectasia, Leber hereditary optic neuropathy, retinopathy of prematurity, or color vision impairment.

26. A recombinant AAV virion according to any one of claims 1-19, for use in a method of treating a retinal disease or condition in a subject in need thereof, wherein the method comprises administering a pharmaceutical composition comprising the recombinant AAV virion to the subject by intravitreal injection.

27. The recombinant AAV virion of any one of claims 1-19, for use in the preparation of a medicament for treating a retinal disease or disorder in a subject.

28. The recombinant AAV virion of claim 26 or claim 27, wherein the disease or condition is age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, or diabetic retinal edema.

29. A variant AAV capsid protein comprising a modified sequence, wherein the modified sequence comprises one or more amino acid substitutions within amino acid residues 570-579 relative to the parent AAV capsid protein, wherein the modified sequence comprises HKFKSGD (SEQ ID NO: 1), and wherein the amino acid residue numbering corresponds to AAV5 VP1 capsid protein.

30. The variant AAV capsid protein of claim 29, wherein the parent AAV capsid protein is an AAV5 capsid protein or an AAV5 and AAV2 hybrid capsid protein.

31. The variant AAV capsid protein of claim 29 or claim 30, wherein the parent AAV capsid protein is an AAV2.5T capsid protein.

32. The variant AAV capsid protein of any one of claims 29-31, wherein the parent AAV capsid protein is AAV2.5T VP1 capsid protein.

33. The variant AAV capsid protein of any one of claims 29-32, wherein the modified AAV capsid protein comprises LAHKFKSGDA (SEQ ID NO: 3) at amino acid residues 570-579 relative to the parent AAV capsid protein.

34. The variant AAV capsid protein of any one of claims 29-33, comprising a capsid sequence having at least 85% homology to the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:

5.

35. The variant AAV capsid protein of any one of claims 29-34, comprising a capsid sequence as shown in SEQ ID NO:6 or SEQ ID NO:

7.

36. A nucleic acid comprising a nucleic acid sequence encoding the variant AAV capsid protein of any one of claims 29-35.

37. An expression vector comprising the nucleic acid of claim 36, wherein the nucleic acid sequence encoding the variant AAV capsid protein is operably linked to a promoter sequence.

38. The expression vector of claim 37, further comprising a nucleic acid encoding a rep protein.

39. A cell comprising the expression vector of claim 37 or claim 38.

40. The cell of claim 39, further comprising a nucleic acid encoding a therapeutic gene product.

41. The cell of claim 40, wherein the therapeutic gene product is a siRNA, miRNA or protein.

42. The cell of claim 40 or claim 41, wherein the therapeutic gene product is an anti-vascular endothelial growth factor (anti-VEGF) gene product.

43. The cell of claim 40 or claim 41, wherein the therapeutic agent is an opsin.

44. The cell of any one of claims 40-43, wherein the polynucleotide encoding the therapeutic gene product is flanked by one or more AAV ITRs.

45. The cell of claim 44, wherein the one or more AAV ITRs are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, or ovine AAV ITRs or variants thereof.

46. ​​The cell of claim 44 or claim 45, wherein the one or more AAV ITRs are AAV2 ITRs or AAV5 ITRs.

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