Modified AAV capsid proteins and uses thereof

Abstract

The present invention relates to a modified adeno-associated virus capsid protein and uses thereof, the capsid protein comprising approximately 5-14 amino acid substitutions compared to the parent AAV capsid protein, and the AAV comprising the modified capsid protein has enhanced infectivity for target tissues or target cells (e.g., retinal cells) compared to the AAV comprising the unmodified parent AAV capsid protein.

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, specifically to modified AAV capsid proteins and uses thereof. [Background technology]

[0002] Vectors used in gene therapy can be divided into viral vectors and non-viral vectors. The most commonly used viral vectors include adenovirus vectors, lentivirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, etc.

[0003] Adeno-associated viruses (AAV) belong to the Parvoviridae family and the Dependovirus genus. Virions consist of a 25 nm icosahedral capsid containing a 4.7 kb single-stranded DNA genome with two open reading frames, Rep and Cap. The nonstructural Rep gene encodes four regulatory proteins required for viral replication, while Cap encodes three structural proteins (VP1-3) assembled into a 60-subunit capsid. This viral capsid mediates the ability of AAV vectors to overcome many biological barriers to viral transduction, including cell surface receptor binding, endocytosis, intracellular trafficking, and deencapsulation in the nucleus.

[0004] AAV has been widely used in many fields, such as gene transduction, gene therapy, vaccination and oncolytic therapy, and has many advantages, such as low pathogenicity, a wide range of infected tissues, a wide range of host cells (can infect and express in proliferating and non-proliferating cells), low immunogenicity, long-term expression of foreign genes in vivo, and no integration of host cell genome, and is therefore widely used in experimental and clinical studies. In 2017, the US FDA approved the first AAV-mediated gene therapy for the treatment of a rare genetic eye disease. In patients who received a single subretinal injection of rAAV vector-delivered therapeutic agent, expression of the therapeutic agent was consistently observed for more than four years.

[0005] However, currently, the application of AAV still faces several challenges, such as improving the tissue and cell targeting and transduction efficiency of vectors, as well as improving the packaging efficiency of certain vectors to accommodate industrial production. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention includes delivering a viral vector (e.g., an AAV viral vector) containing a gene of interest to a desired cell or tissue. Specifically, the present invention includes a modified capsid protein that contains one or more modifications (e.g., substitutions, insertions, or mutations) in the amino acid sequence compared to the parent AAV capsid protein, and when present in an AAV viral vector, provides enhanced infectivity to a target tissue or cell (e.g., the retina, muscle, or joint cavity) compared to an AAV viral vector containing the unmodified parent AAV capsid protein. [Means for solving the problem]

[0007] In one aspect of the present invention, a modified adeno-associated virus (AAV) capsid protein is provided, which comprises a polypeptide substitution of about 5 to 14 amino acids compared to a parent AAV capsid protein, and wherein an AAV virus comprising said modified capsid protein has improved retinal cell infectivity compared to an AAV virus comprising the corresponding parent AAV capsid protein. In some specific embodiments, the polypeptide comprises an amino acid sequence selected from RGNRQ (SEQ ID NO:1), QQNTARGNRQ (SEQ ID NO:2), RGNRQAAQQNTA (SEQ ID NO:3), RGNRQQNTA (SEQ ID NO:4), RGNRQQQNTA (SEQ ID NO:5), SGNTQ (SEQ ID NO:6), RGNQQNTARQ (SEQ ID NO:7), RGNQQPRPTSRQ (SEQ ID NO:8), RGNRQAAQQPTPTS (SEQ ID NO:9) or RGNRQQQPTPTS (SEQ ID NO:19), and the substitution is at amino acid positions 588 to 592 of the parent AAV8 or the corresponding position in another serotype capsid protein.

[0008] In some specific embodiments, the capsid protein polypeptide comprises an amino acid sequence selected from QQNTARGNRQ (SEQ ID NO:2), RGNRQQNTA (SEQ ID NO:4), SGNTQ (SEQ ID NO:6), RGNQQNTARQ (SEQ ID NO:7) or RGNRQQQPTPTS (SEQ ID NO:19).

[0009] In some specific embodiments, the capsid protein further comprises a mutation to amino acids 262-272 of the parent AAV8 capsid protein, or the corresponding position of another serotype capsid protein, and the mutated amino acid sequence is SQSGASNDNH (SEQ ID NO: 10). In one preferred embodiment, the capsid protein comprises a polypeptide substitution at amino acids 588-592 of the parent AAV8 capsid protein, or the corresponding position of another serotype capsid protein, and a mutation to amino acids 262-272 of the parent AAV8 capsid protein, or the corresponding position of another serotype capsid protein, and the polypeptide is RGNRQ (SEQ ID NO: 1) and the mutated amino acid sequence is SQSGASNDNH (SEQ ID NO: 10).

[0010] In some specific embodiments, the AAV of the present invention may be derived from any AAV serotype, for example, the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, or AAVrh10. In one preferred embodiment, the AAV of the present invention is selected from AAV8.

[0011] In some specific embodiments, the capsid protein further comprises a mutation to the amino acids at positions D80 and / or V125 of the parent AAV8 capsid protein. In one preferred embodiment, the mutation is D80N and / or V125A. In another preferred embodiment, the mutation is D80Q and / or V125G. In one specific preferred embodiment, the capsid protein comprises a polypeptide substitution at amino acids 588-592 of the parent AAV8 and a mutation to the amino acids at positions D80 and V125 of the parent AAV8 capsid protein, wherein the polypeptide is RGNQQNTARQ (SEQ ID NO: 7) and the mutation is D80N and V125A. In another specific preferred embodiment, the capsid protein comprises a polypeptide substitution at amino acids 588 to 592 of the parent AAV8 and mutations to amino acids at positions D80 and V125 of the parent AAV8 capsid protein, wherein the polypeptide is RGNQQNTARQ (SEQ ID NO:7) and the mutations are D80Q and V125G.

[0012] In another aspect of the invention, there is provided another modified adeno-associated virus (AAV) capsid protein comprising a polypeptide insertion after amino acid 589 of the parent AAV8 or the corresponding position in another serotype capsid protein, wherein an AAV virus comprising said modified capsid protein has improved retinal cell infectivity compared to an AAV virus comprising the corresponding parent AAV capsid protein.

[0013] In some specific embodiments, the polypeptide comprises an amino acid sequence selected from RGDLTTPQQ (SEQ ID NO:20), RGDLNTPQQ (SEQ ID NO:21), or RGDVSSPQQ (SEQ ID NO:22). The AAV may be from any AAV serotype, for example, the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, or AAVrh10. In one preferred embodiment, the AAV serotype is AAV8.

[0014] Another aspect of the present invention is a method for producing a i. the modified capsid protein according to the invention; ii. a heterologous nucleic acid comprising an encoded gene product.

[0015] In certain specific embodiments, the gene product is a VEGF antagonist or a TNF-α antagonist.

[0016] In certain specific embodiments, the VEGF antagonist is selected from aflibercept, conbercept, ranibizumab, and brolucizumab, preferably aflibercept.

[0017] In certain particular embodiments, the TNF-α antagonist is selected from etanercept, infliximab, adalimumab, pecelizumab or golimumab, preferably etanercept.

[0018] Another aspect of the present invention is a method for producing a a) the recombinant adeno-associated virus according to the invention; b) a pharma- ceutically acceptable excipient.

[0019] Another aspect of the present invention provides a recombinant adeno-associated virus according to the present invention for use in medicine.

[0020] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the present invention is administered by vitreous, retinal, choroidal injection, intravenous, subcutaneous, intramuscular or intraarticular injection. In a preferred embodiment, the recombinant adeno-associated virus or pharmaceutical composition according to the present invention is administered by suprachoroidal injection. In another preferred embodiment, the recombinant adeno-associated virus or pharmaceutical composition according to the present invention is administered by intramuscular or intraarticular injection. In another aspect of the invention there is provided a recombinant adeno-associated virus or a pharmaceutical composition according to the invention for use in therapy.

[0021] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the invention is administered by vitreous, retinal or choroidal injection. In a preferred embodiment, the recombinant adeno-associated virus or pharmaceutical composition according to the invention is administered by suprachoroidal injection.

[0022] In another aspect of the invention, there is provided a use of said recombinant adeno-associated virus in the preparation of a medicament for preventing or treating an eye disease.

[0023] In another aspect of the present invention, there is provided a method for preventing or treating an eye disease, comprising administering an effective amount of a recombinant adeno-associated virus or a pharmaceutical composition according to the present invention to an individual in need thereof.

[0024] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the present invention is administered by vitreous, retinal or choroidal injection. In a preferred embodiment, the recombinant adeno-associated virus or pharmaceutical composition according to the present invention is administered by suprachoroidal injection.

[0025] In some particular embodiments, the ocular disease according to the present invention is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascular ocular disease, non-infectious uveitis, or glaucoma. In some particular embodiments, the ocular disease according to the present invention is selected from age-related macular degeneration, macular edema, diabetic macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, macular edema due to branch retinal vein occlusion, diabetic retinal edema, diabetic retinopathy, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoidal choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia, or retinopathy of prematurity.

[0026] In another aspect of the invention there is provided a recombinant adeno-associated virus or a pharmaceutical composition according to the invention for use in a method for the treatment of an eye disease.

[0027] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the present invention is administered by vitreous, retinal or choroidal injection. In a preferred embodiment, the recombinant adeno-associated virus or pharmaceutical composition according to the present invention is administered by suprachoroidal injection.

[0028] In some particular embodiments, the ocular disease according to the present invention is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascular ocular disease, non-infectious uveitis, or glaucoma. In some particular embodiments, the ocular disease according to the present invention is selected from age-related macular degeneration, macular edema, diabetic macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, macular edema due to branch retinal vein occlusion, diabetic retinal edema, diabetic retinal disease, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoidal choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia, or retinopathy of prematurity.

[0029] In another aspect of the invention there is provided the use of said recombinant adeno-associated virus in the preparation of a medicament for the prevention or treatment of arthritic diseases or related conditions.

[0030] In another aspect of the present invention, there is provided a method for preventing or treating an arthritic disease or related condition, comprising administering to an individual in need thereof an effective amount of a recombinant adeno-associated virus or pharmaceutical composition according to the present invention.

[0031] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the invention is administered by intravenous, subcutaneous, intramuscular or intraarticular injection.

[0032] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the invention is administered by intramuscular or intraarticular injection.

[0033] In some specific embodiments, the arthritic disease or related condition according to the present invention is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, gout, pseudogout, spondylitis, Crohn's disease, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease.

[0034] In another aspect of the invention there is provided a recombinant adeno-associated virus or a pharmaceutical composition according to the invention for use in a method for the treatment of arthritic diseases or related conditions.

[0035] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the invention is administered by intravenous, subcutaneous, intramuscular or intraarticular injection.

[0036] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the invention is administered by intramuscular or intraarticular injection.

[0037] In some specific embodiments, the arthritic disease or related condition according to the present invention is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, gout, pseudogout, spondylitis, Crohn's disease, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease. [Brief description of the drawings]

[0038] [Figure 1A] 1 shows an alignment of amino acid sequences of the GH loop subregion of the AAV capsid protein. [Figure 1B] 1 shows an alignment of amino acid sequences of the GH loop subregion of the AAV capsid protein. [Diagram 2] Schematic diagram of AAV8 capsid protein mutant plasmid construction. [Diagram 3] 1 shows EGFP mRNA expression levels in mice intravitreally injected with AAV8 mutant viruses. [Figure 4] 1 shows EGFP fluorescent signals in whole-mount retinas of mice intravitreally injected with AAV8 mutant viruses. [Diagram 5] 1 shows EGFP fluorescent signals in whole-mount retinas from rats intravitreally injected with AAV8 mutant viruses. [Figure 6] 1 shows EGFP fluorescent signals and DAPI signals from rabbits injected with AAV8 mutant viruses into the suprachoroidal space. [Figure 7] 1 shows EGFP fluorescent signals of AAV8 mutant viruses cultured in ARPE19 cells. [Figure 8A] The number of EGFP-positive cells following infection of ARPE19 cells with AAV8 mutant viruses by flow cytometry is shown. [Figure 8B] The number of EGFP-positive cells following infection of ARPE19 cells with AAV8 mutant viruses by flow cytometry is shown. [Figure 9] The relative content of mCherry genomic DNA in rabbits injected with AAV8 mutant viruses into the suprachoroidal space is shown. [Figure 10] Luciferase fluorescence intensity of mice injected intramuscularly with AAV8 mutant viruses is shown. [Figure 11] Clinical scores of mice injected intra-articularly / intramuscularly with AAV8 mutant viruses are shown. [Figure 12] Footpad thickness of mice injected intra-articularly / intramuscularly with AAV8 mutant viruses is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The present invention relates to a capsid protein that has improved infectivity for a target tissue or target cell (e.g., the retina, muscle or joint cavity) compared to an AAV viral vector containing an unmodified parent AAV capsid protein, a rAAV that contains this capsid protein and expresses a gene of interest (e.g., a VEGF antagonist, a TNF-α antagonist), pharmaceutical compositions thereof, and uses thereof. Capsid Protein

[0040] The present invention provides modified AAV capsid proteins that contain one or more amino acid modifications compared to the corresponding wild-type or parent AAV. The modifications include substitutions, deletions, or insertions of one or more amino acids compared to the parent or AAV capsid proteins. The modifications provide improved infectivity of target tissues or cells (e.g., retina, muscle, or joint cavity) compared to AAV viral vectors that contain unmodified parent AAV capsid proteins.

[0041] In certain embodiments, the modified AAV capsid protein comprises a heterologous polypeptide substitution within a capsid protein serotype, such as VP1, belonging to AAV type 1, AAV type 2, AAV type 3, AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAVrhlO, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, bovine AAV, AAV2.7m8, AAVShH10, AAV2.5T, AAV2.5T / 7m8, AAV9 / 7m8, and AAV5 / 7m8. In some embodiments, the capsid mutant may be a chimeric capsid mutant. The sequence of the chimeric capsid mutant may comprise a portion of two or more AAV capsid serotypes or variants thereof. In some embodiments, the chimeric capsid comprises portions of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different capsid protein serotypes.

[0042] In certain embodiments, the modified AAV capsid protein comprises a heterologous polypeptide substitution of about 5-14 amino acids. In some particular embodiments, the modified capsid protein of the invention comprises a heterologous polypeptide substitution of about 5-14 amino acids at amino acid positions 588-592 of the parent AAV8, or at the corresponding positions of a capsid protein of another serotype. The amino acid sequences of AAV1-AAV9, etc., are described in patents US8962330B, US10041090B, etc. In certain embodiments, the modified capsid protein of the invention comprises a heterologous polypeptide substitution of about 5-14 amino acids at amino acid positions 588-592 of the parent AAV8. The amino acids at positions 588-592 of the parent / wild type AAV8 capsid protein are "QQNTA". Thus, a particular embodiment of the present invention is a modified capsid protein in which the amino acids "QQNTA" at positions 588-592 of the parent / wild type AAV8 capsid protein are replaced with a heterologous polypeptide of 5-14 amino acids. For example, as shown in FIG. 1A, the substitution site corresponding to amino acids 588-592 of AAV8 is at the corresponding position in other capsid proteins, e.g., at amino acids 586-590 of AAV1, amino acids 585-589 of AAV2, amino acids 586-590 of AAV3, amino acids 584-588 of AAV4, amino acids 575-579 of AAV5, amino acids 586-590 of AAV6, amino acids 587-591 of AAV7, amino acids 586-590 of AAV9, and amino acids 588-592 of AAV10.

[0043] The length of said substituted heterologous polypeptide is 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids or 11 amino acids. In some particular embodiments, the polypeptide according to the invention comprises an amino acid sequence selected from RGNRQ (SEQ ID NO: 1), QQNTARGNRQ (SEQ ID NO: 2), RGNRQAAQQNTA (SEQ ID NO: 3), RGNRQQNTA (SEQ ID NO: 4), RGNRQQQNTA (SEQ ID NO: 5), SGNTQ (SEQ ID NO: 6), RGNQQNTARQ (SEQ ID NO: 7), RGNQQPRPTSRQ (SEQ ID NO: 8), RGNRQAAQQPTPTS (SEQ ID NO: 9) or RGNRQQQPTPTS (SEQ ID NO: 19). In some preferred embodiments, the polypeptides described herein comprise an amino acid sequence selected from QQNTARGNRQ (SEQ ID NO:2), RGNRQQNTA (SEQ ID NO:4), SGNTQ (SEQ ID NO:6) or RGNQQNTARQ (SEQ ID NO:7), or RGNRQQQPTPTS (SEQ ID NO:19).

[0044] In certain embodiments, the modified AAV capsid protein further comprises a mutation compared to amino acids 262-272 of the parental AAV8 capsid protein or the corresponding positions of another serotype capsid protein, wherein amino acids 262-272 of the parental / wild-type AAV8 capsid protein are "SNGTSGGATND". Thus, certain embodiments of the invention provide modified capsid proteins mutated at amino acids 262-272 of the parental / wild-type AAV8 capsid protein, "SNGTSGGATND". The mutation site corresponding to amino acids 262-272 of AAV8 is at the corresponding position of other capsid proteins, e.g., amino acids 261-271 of AAV1, amino acids 261-271 of AAV2, amino acids 261-271 of AAV3, amino acids 255-265 of AAV4, amino acids 251-261 of AAV5, amino acids 261-271 of AAV6, amino acids 262-272 of AAV7, amino acids 261-271 of AAV9, and amino acids 262-272 of AAV10. The mutation site corresponding to amino acids 262-272 of AAV8 is at the corresponding position of other capsid proteins, e.g., as shown in FIG. 1B. In some specific embodiments, the mutated amino acid sequence according to the invention is SQSGASNDNH (SEQ ID NO: 10).

[0045] In some specific embodiments, the modified capsid protein described herein comprises a polypeptide substitution at amino acid positions 588-592 of the parent AAV8 or the corresponding positions of another serotype capsid protein, and a mutation to positions 262-272 of the parent AAV8 capsid protein or the corresponding positions of another serotype capsid protein, wherein the polypeptide is RGNRQ (SEQ ID NO: 1) and the mutated amino acid sequence is SQSGASNDNH (SEQ ID NO: 10). In some preferred embodiments, the parent capsid protein serotype described herein is AAV8.

[0046] In some further embodiments, the modified AAV capsid protein of the invention further comprises a mutation to the amino acids at positions D80 and / or V125 of the parent AAV8 capsid protein. In some specific embodiments, the mutation is D80N and / or V125A. In some specific embodiments, the mutation is D80Q and / or V125G. In some preferred embodiments, the capsid protein comprises a polypeptide substitution at amino acids 588-592 of the parent AAV8 and a mutation to the amino acids at positions D80 and V125 of the parent AAV8 capsid protein, wherein the polypeptide is RGNQQNTARQ (SEQ ID NO: 7) and the mutation is D80N and V125A. In some other preferred embodiments, the capsid protein comprises a polypeptide substitution at amino acids 588-592 of the parent AAV8 and mutations to amino acids at positions D80 and V125 of the parent AAV8 capsid protein, wherein the polypeptide is RGNQQNTARQ (SEQ ID NO:7) and the mutations are D80Q and V125G.

[0047] In some further embodiments, the modified AAV capsid protein of the invention comprises a polypeptide insertion after amino acid 589 of the parent AAV8 or the corresponding position of another serotype capsid protein. The insertion site is after amino acid 589 of AAV8 or the corresponding position of another capsid protein, e.g., after amino acid 589 of AAV1, amino acid 586 of AAV2, amino acid 587 of AAV3, amino acid 585 of AAV4, amino acid 576 of AAV5, amino acid 587 of AAV6, amino acid 588 of AAV7, amino acid 587 of AAV9, amino acid 589 of AAV10. In some specific embodiments, the inserted polypeptide comprises the amino acid sequence RGDLTTPQQ (SEQ ID NO:20), RGDLNTPQQ (SEQ ID NO:21), or RGDVSSPQQ (SEQ ID NO:22). In one preferred embodiment, the modified AAV capsid protein described in the present invention comprises a polypeptide insertion after amino acid 589 of the parent AAV8, wherein the inserted polypeptide comprises the amino acid sequence RGDLTTPQQ (SEQ ID NO: 20), RGDLNTPQQ (SEQ ID NO: 21) or RGDVSSPQQ (SEQ ID NO: 22).

[0048] In some embodiments, the modified capsid protein according to the present invention comprises a sequence having at least 85%, 90%, 95%, 98%, 99% or 100% homology to the amino acid sequence of the capsid protein having the above amino acid substitutions, mutations and / or insertions.

[0049] In some embodiments, the AAV comprising the modified capsid protein exhibits at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or even greater increase in infectivity of retinal cells compared to AAV viruses comprising the parent / wild-type capsid protein, including RPE-19 cells (a human retinal pigment epithelial cell line), retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, cone cells, rod cells, Muller glia cells, and retinal pigment epithelial cells.

[0050] In some specific embodiments, the AAV comprising the modified capsid protein exhibits at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or even greater increase in infectivity of RPE cells compared to an AAV virus comprising the corresponding parent / wild-type capsid protein.

[0051] In some specific embodiments, the AAV comprising the modified capsid protein exhibits at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or even greater increase in infectivity of retinal cells following suprachoroidal injection compared to an AAV virus comprising the corresponding parent / wild-type capsid protein.

[0052] In some specific embodiments, the AAV comprising the modified capsid protein exhibits at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or even greater increase in infectivity for retinal cells following intravitreal injection compared to an AAV virus comprising the corresponding parent / wild-type capsid protein. Recombinant adeno-associated virus (rAAV)

[0053] The gene delivery vector of the present invention is a recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV described in the present invention is a single-stranded AAV (ssAAV). Here, ssAAV refers to a rAAV that has the coding sequence of a gene of interest and the complementary sequence on separate strands, and is packaged into separate viral capsids.

[0054] In some embodiments, a recombinant adeno-associated virus (rAAV) according to the invention comprises i) a modified capsid protein according to the invention; and ii) a heterologous nucleic acid comprising an encoding gene product.

[0055] In some embodiments, a rAAV described in the present invention comprises a modified capsid protein described in the present invention such that the recombinant AAV (rAAV) described in the present invention has improved specific targeting or infectivity of a cell or tissue (e.g., eye tissue or cells, muscle tissue or joint cavity, etc.) relative to a rAAV comprising a parent or wild-type capsid protein.

[0056] In some embodiments, the rAAV according to the present invention comprises a heterologous nucleic acid encoding a gene product and a polynucleotide expression cassette of a regulatory element. The term "regulatory element" refers to a segment of a nucleic acid sequence that regulates the expression of an operably linked gene product. Thus, an expression regulatory element may include a promoter, an enhancer, an internal ribosome entry site (IRES), a transcription terminator, a start codon before a protein-coding gene, an intron splicing signal, and a terminator codon. The term may also include a nucleic acid sequence design that removes potentially undesirable start codons from said sequence, either in or out of frame. It may also include a nucleic acid sequence design that removes potentially undesirable splice sites. It may also include sequences that direct polyadenylation or the addition of polyA, and the like.

[0057] In the polynucleotide expression cassette of the invention, the heterologous nucleic acid comprises a nucleotide sequence encoding a gene product of a gene of interest, e.g., a therapeutic gene product. In some embodiments, the gene product is an interfering RNA. In some embodiments, the gene product is an aptamer. In some embodiments, the gene product is a polypeptide. In some embodiments, the gene product is a site-specific nuclease that provides site-specific knockdown of gene function. In some preferred embodiments, the gene product is a polypeptide, a protein, a fusion protein, an antibody, or the like.

[0058] In some embodiments, the gene product described herein is a VEGF antagonist, a TNF-α antagonist, a PD-1 / PD-L1 inhibitor, an Ang-2 inhibitor, a plasma kallikrein inhibitor, endostatin, an oncostatin, angiostatin, pigment epithelium-derived factor (PEDF), a soluble Tie-2 receptor, an Ang-2 antagonist, CD tissue inhibitor of metalloproteinase-3 (TIMP-3), a light-responsive opsin (e.g., rhodopsin), an anti-apoptotic polypeptide (e.g., Bcl-2, Bcl-Xl), a glial-derived neurotrophic factor (GDNF), a fibroblast growth factor 2, a neurturin (NTN), a fibroblast growth factor 2 (GFAP ... neurturin), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), neurotrophin-4 (NT4), brain-derived neurotrophic factor (BDNF), mutants having at least 80%, at least 85%, at least 90%, or at least 95% sequence identity to any of these proteins or polypeptides, and functional variants or fragments comprising at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of these proteins or polypeptides or variants thereof.

[0059] In one embodiment, the gene product according to the present invention is selected from VEGF antagonists. In a particular embodiment, the VEGF antagonist is any domain selected from extracellular domains 1 to 7 of VEGF receptor 1 (VEGFR-1, or Flt) or receptor 2 (VEGFR-2, or Flk), such as VEGFR-1 extracellular domain 2 (Flt d2) and / or VEGFR-2 extracellular domain 3 or 4.

[0060] In certain embodiments, the VEGF antagonist comprises at least one selected from the group consisting of: a) A fusion protein comprising the extracellular domain 2 of VEGFR-1 and the extracellular domain 3 of VEGFR-2 In one example, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 11 (Fltd2 Flkd3). b) A fusion protein comprising extracellular domain 2 of VEGFR-1, extracellular domain 3 and domain 4 of VEGFR-2. In one example, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 12 (Fltd2 Flkd3,4). c) having an antibody heavy chain variable region sequence set forth in SEQ ID NO:13 and an antibody light chain variable region sequence set forth in SEQ ID NO:14. d) having an antibody heavy chain variable region sequence set forth in SEQ ID NO:15 and an antibody light chain variable region sequence set forth in SEQ ID NO:16.

[0061] wherein SEQ ID NO: 12 is the sequence shown below. GRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGI DFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEV SERDTGNYTVILTNPISKEKQSHVVSLVVYVPGPGDKTHTCPLCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKATPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0062] SEQ ID NO: 13 is the sequence shown below. EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSS

[0063] SEQ ID NO: 14 is the sequence shown below. DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQ

[0064] SEQ ID NO: 15 is the sequence shown below. EVQLVESGGGLVQPGGSLRLSCTASGFSLTDYYYMTWVRQAPGKGLEWVGFIDPDDDPYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGGDHNSGWGLDIWGQGTLVTVSS SEQ ID NO: 16 is the sequence shown below. MEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLG

[0065] In a particular embodiment, the VEGF antagonist has the amino acid sequence set forth in SEQ ID NO:11. SDTGRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPS HGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 11)

[0066] In a particular embodiment, the nucleotide sequence encoding the VEGF antagonist has the sequence set forth in SEQ ID NO:23.

[0067] In some embodiments, the protein or polypeptide is selected from a TNF-α antagonist. In some specific embodiments, the VEGF antagonist is selected from etanercept, infliximab, adalimumab, pecelizumab, or golimumab.

[0068] In a particular embodiment, the TNF-α antagonist has a heavy chain variable region sequence set forth in SEQ ID NO:17 and a light chain variable region sequence set forth in SEQ ID NO:18.

[0069] SEQ ID NO: 17 is the sequence shown below. EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVSS

[0070] SEQ ID NO: 18 is the sequence shown below. DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIK

[0071] In a particular embodiment, the TNF-α antagonist has the amino acid sequence set forth in SEQ ID NO:24. LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDTVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAP LRKCRPGFGVARPGTETSDVVCKPCAPGTFSDTTSSTDICRPHQICNVVAIPGDASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 24)

[0072] In a particular embodiment, the nucleotide sequence encoding the TNF-α antagonist has the sequence set forth in SEQ ID NO:25.

[0073] In some embodiments, the VEGF antagonist or TNF-α antagonist has an amino acid sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence set forth above.

[0074] In some embodiments, the VEGF antagonist or TNF-α antagonist has a nucleotide sequence that is at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the above nucleotide sequence.

[0075] In some embodiments, the protein or polypeptide is selected from those having one or more different antigen binding sites. For example, in some embodiments, the protein or polypeptide binds to both VEGF and Ang-2. In certain embodiments, the protein or polypeptide is a bispecific antibody that binds to both VEGF and Ang-2, such as Faricimab bispecific antibody.

[0076] In some embodiments of the present invention, the coding sequence may be designed to better express the gene of interest using codon optimization. The present invention can optimize nucleotide codons encoding a polypeptide, fusion protein, antibody, or functional fragments thereof to improve the protein expression level of the gene of interest in a target cell, tissue, or species. The coding sequence is the part of the mRNA sequence that codes for the amino acids used in transcription and translation. In the transcription and translation process, every third nucleotide codon of the 61 nucleotide codons is transcribed and translated into one of the 20 amino acids. However, the frequency of tRNAs that code for the same amino acid varies depending on the cell type and animal species. If the gene sequence contains codons that are not frequently expressed, the ribosomal transcription machinery may be reduced and effective transcription may be inhibited. Therefore, one skilled in the art can improve the expression level of the gene of interest by "codon optimization". Briefly, the nucleotide sequence encoding the product is modified with synonymous codon sequences, e.g., by replacing codons expressed at low frequencies with codons expressed more frequently in the target cell, thereby increasing the expression level of the target substance in a particular cell, tissue, or substance, e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%. Coding sequences for codon optimization can be designed in a variety of ways. This optimization can be performed using methods provided online, published methods, or companies that provide codon optimization services. Codon optimization methods are described, for example, in International Patent Publication WO2015 / 012924, which is incorporated herein by reference. The entire length of the open reading frame (ORF) of the product is modified accordingly. However, in some embodiments, only a fragment of the ORF can be modified. One of these methods is used to produce a codon-optimized coding nucleotide that encodes a polypeptide.

[0077] In some embodiments, the polynucleotide expression cassette of the present invention has one or more expression regulatory elements. For example, the regulatory element comprises a constitutive promoter region, and the nucleotide sequence encoding a gene product is operably linked to the constitutive promoter, which is widespread and active in cells, tissues, or species, and promotes expression of a gene of interest in a particular cell or tissue in vivo or in vitro. In some embodiments, examples of promoters include chicken β-actin promoter (CBA), cytomegalovirus promoter (CMV), CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG), elongation factor 1 alpha promoter (EF1α), human phosphoglycerate kinase promoter (PGK), MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, opsin promoter, mitochondrial heavy chain promoter, ubiquitin promoter, and the like.

[0078] In some embodiments, the regulatory element comprises an inducible promoter region, and the nucleotide sequence encoding the gene product is operably linked to the inducible promoter. In some embodiments, the inducible promoter is a photoreceptor-specific promoter. Suitable photoreceptor-specific promoters include, for example, rhodopsin promoter, rhodopsin kinase promoter, beta phosphodiesterase gene promoter, retinitis pigmentosa gene promoter, interphotoreceptor retinoic acid binding protein (IRBP) gene enhancer, IRBP gene promoter, opsin gene promoter, retinoschisin gene promoter, CRX homeodomain protein gene promoter, guanine nucleotide-binding protein alpha transduction activity polypeptide 1 (GNAT1) gene promoter, neural retina-specific leucine zipper protein (NRL) gene promoter, human cone arrestin (hCAR) promoter, and PR2.1, PR1.7, PR1.5, and PR1.1 promoters, RPE-specific promoters (e.g., RPE65 gene promoter), cellular retinaldehyde-binding protein (CRALBP) gene promoter, pigment epithelium-derived factor (PEDF aka serpin F1 gene promoter, vitelliform macular dystrophy (VMD2) promoter, Muller glial cell-specific promoters (e.g., glial fibrillary acidic protein (GFAP) promoter), bipolar-specific promoters (e.g., GRM6 promoter), and the like.

[0079] The expression control sequence in the polynucleotide expression cassette according to the present invention can also include a polyadenylation signal. The polyadenylation signal is also called a polyadenylation site, a polyadenylic acid tail, a Poly(A) site, a Poly(A) signal, or a Poly(A) tail. A polyadenylation region refers to a covalent linkage between polyadenylic acid and a messenger RNA (mRNA) molecule. During protein biosynthesis, it is part of the process of generating mature mRNA ready for translation. In eukaryotes, polyadenylation is a mechanism that blocks the 3' end of an mRNA molecule. Polyadenylation signals protect mRNA from attack by exonucleases and are important for transcription termination, output of mRNA from the cell nucleus, and translation. Polyadenylation signals contain multiple consecutive adenosine monophosphates, typically including AAUAAA repeats. Some exemplary polyadenylation signals include simian vacuolar virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or beta globin.

[0080] The polynucleotide expression cassette according to the present invention may also comprise an inverted terminal repeat (ITR). A functional adenovirus inverted terminal repeat (ITR) refers to an ITR sequence for, for example, integrating, replicating, and packaging an AAV viral particle. Typically, the length of an ITR sequence is about 145 bp. Preferably, substantially the entire sequence encoding the ITRs, and modifications made to these ITR sequences by a person skilled in the art by conventional technical means in the art, are used in the molecule. An example of the use of an ITR molecule in the present application is a "cis" plasmid that contains a gene of interest, with the gene of interest sequence and associated regulatory elements flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the heterologous nucleotides encoding the gene of interest are flanked by the AAV ITRs (e.g., in the orientation 5'-ITR-gene of interest-ITR-3'). In some embodiments, the AAV ITRs are selected from the group consisting of AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITV, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9 ITV, AAV10 ITR, AAV11 ITR, and AAV12 ITR.

[0081] The polynucleotide expression cassette according to the present invention further comprises an RNA output signal downstream of the coding sequence and upstream of the polyadenylation site. The RNA output signal is a cis-acting post-transcriptional regulatory element that can enhance the output of RNA from the cell nucleus. Exemplary RNA output sequences include, but are not limited to, sequences from the Hepatitis B virus post-transcriptional regulatory element (HPRE) and the Guinea pig hepatitis virus post-transcriptional element (WPRE).

[0082] Those skilled in the art will appreciate that the polynucleotide expression cassettes described herein may optionally contain other elements, including, but not limited to, restriction sites that facilitate cloning, regulatory elements operably linked to individual elements, or regulatory elements that regulate gene expression. Examples include bacterial sequences in plasmid vectors, attp sites, attB sites, promoter linkers, polyadenylation sequence linkers, and the like in phage integrase vectors.

[0083] In some embodiments, a polynucleotide expression cassette as described herein comprises a combination of two or more of the elements described above and a gene of interest, generated by one of skill in the art, wherein the gene of interest coding sequence and associated regulatory elements in the polynucleotide expression cassette are flanked by 5' and 3' AAV ITR sequences.

[0084] For example, a polynucleotide expression cassette of the invention may include, in 5' to 3' order, an N-terminal AAV ITR, an enhancer / promoter / intron, a Kozak sequence, a coding sequence for a gene product, a WPRE or HPRE RNA output signal sequence, a polyadenylation signal sequence, and a C-terminal AAV ITR.

[0085] In a particular embodiment, the recombinant adeno-associated virus according to the invention comprises a polynucleotide expression cassette encoding a gene product, the polynucleotide expression cassette comprising, in order from the 5' end to the 3' end, the following: (a) 5' AAV ITR (b) CMV enhancer (c) CBA promoter (d) Chicken β-actin intron (e) the coding sequence of a gene product (f) Rabbit β-globin polyadenylation signal sequence (g) 3' AAV ITR

[0086] The coding sequence for the gene product is a nucleic acid sequence that encodes the gene product (eg, the VEGF antagonist or TNF-α antagonist).

[0087] Recombinant viral vectors containing modified capsid proteins according to the invention can be produced using standard methods, for example, comprising culturing host cells containing an artificial genome and recovering recombinant AAV encapsulating the artificial genome from the cell culture, the artificial genome comprising a cis polynucleotide expression cassette flanked by AAV ITRs, the cis polynucleotide expression cassette comprising a coding sequence for a gene product operably linked to expression control elements that control expression of the gene product in human cells, a trans expression cassette lacking AAV ITRs, the trans expression cassette encoding AAV rep and capsid proteins, the AAV rep and capsid proteins operably linked to expression control elements that drive expression of the AAV rep and capsid proteins in the host cells in culture, and an adenovirus helper having sufficient functionality to allow replication and packaging of the artificial genome via the AAV capsid proteins.

[0088] "Host cell" refers to any cell that contains or can contain a substance of interest. Typically, the host cell is a mammalian cell. In some embodiments, the host cell is a photoreceptor cell, a retinal pigment epithelial cell, a keratinocyte, a corneal cell, and / or a tumor cell. Pharmaceutical Compositions

[0089] Another aspect of the present invention is to provide a pharmaceutical composition comprising a rAAV comprising a recombinant adeno-associated virus according to the present invention and a pharma- ceutically acceptable diluent, carrier or excipient.

[0090] Suitable diluents, carriers or excipients can be readily selected by one skilled in the art. As used herein, carriers or excipients include any solvent, dispersion medium, carrier, coating, diluent, antibacterial and antifungal agents, isotonicity agents, absorption delaying agents, buffers, carrier solutions, suspensions, colloids, preservatives or chemical stabilizers, and the like.

[0091] Exemplary carriers or excipients include sterile saline, lactose, sucrose, calcium phosphate, gelatin, glucan, agar, pectin, peanut oil, sesame oil, and water. For example, suitable excipients or carriers include saline, which can be formulated with various buffers (e.g., phosphate buffer). The action of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Absorption of the injectable composition can be prolonged by using in the composition an absorption-delaying agent, such as aluminum monostearate and gelatin.

[0092] Buffers may be suitably prepared, if necessary, and the liquid diluent may first be made isotonic with sufficient saline or glucose to provide injectable aqueous solutions. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.

[0093] The method for preparing a sterile injectable solution is to mix the active rAAV with various other ingredients mentioned in the present invention in the required amount in an appropriate solvent, followed by filter sterilization. Usually, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile carrier which contains the dispersion medium and other desired ingredients.

[0094] The rAAV compositions of the present invention can also be prepared in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts formed from inorganic acids (e.g., hydrochloric acid or phosphoric acid) or organic acids (e.g., acetic acid, oxalic acid, tartaric acid, mandelic acid). Salts formed from free carboxyl groups can also be produced from inorganic bases such as sodium, potassium, ammonium, calcium, and ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, and purcaine. Once prepared, the solution is administered to suit the dosage form and in a therapeutically effective amount. The formulations can be easily administered in a variety of dosage forms, such as injectable solutions, drug release capsules, and the like.

[0095] The compositions of the present invention can be delivered to suitable host cells via delivery vectors, such as, for example, liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like.

[0096] Injectable, oral or topical gastrointestinal compositions are advantageously prepared in unit dosage form for ease of administration. As used herein, unit dosage form is a physically discrete unit suitable for use as a single dose for a subject to be treated. Each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with a desired drug carrier. The specifications of the unit dosage form of the present invention are determined by the inherent characteristics of the active compound and the particular therapeutic effect to be achieved, and the inherent limitations in the art when mixing such active compounds for use in treating individuals. For example, a unit dosage may be an amount of vector genome, or may contain an amount of vector genome per milliliter, or may contain a unit dosage that measures the pharmaceutical composition using a multiplicity of infection (MOI), which refers to the ratio or multiple of vector or virus genome to cells to which the nucleic acid can be delivered.

[0097] The pharmaceutical compositions may be included in a container, package, or dispenser, such as a syringe, together with instructions for administration. Recombinant adeno-associated virus (rAAV) delivery

[0098] The present invention relates to a method for preventing or treating a disease or condition, comprising delivering a therapeutically effective amount of a rAAV virus or pharmaceutical composition according to the present invention to a subject. The rAAV according to the present invention can be delivered to a subject according to any suitable method known in the art. For example, it is preferred to administer the rAAV suspended in a physiologically compatible carrier (e.g., in a composition) to a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or non-human primate (e.g., rhesus monkey). In some embodiments, the host animal does not include a human. In some embodiments, the subject is a human.

[0099] A "therapeutically effective amount" refers to an amount effective at a dosage and for a period of time necessary to obtain a desired therapeutic effect. A therapeutically effective amount of a rAAV virus or pharmaceutical composition can vary depending on factors such as the disease state, age, sex, and weight of the patient being treated, and the ability of the rAAV virus or pharmaceutical composition to induce a desired response in the patient. The administration scheme can be adjusted to provide an optimal therapeutic response. A therapeutically effective amount is also generally an amount in which the therapeutically beneficial effects outweigh any toxic or harmful effects of the rAAV virus or pharmaceutical composition. A "prophylactically effective amount" refers to an amount effective at a dosage and for a period of time necessary to obtain a desired prophylactic effect, such as prevention or suppression of various diseases. Prophylactic doses can be used in subjects before or at the beginning of the disease, and in some cases, the prophylactically effective amount can be greater or less than the therapeutically effective amount. The dosage depends largely on the condition and size of the subject being treated, the treatment regimen, the frequency of treatment, and the route of administration. Protocols for continued treatment, including dose, formulation, and frequency, may be dictated by initial response and clinical judgment.

[0100] In some embodiments, the rAAV virus or pharmaceutical composition is administered to a subject once a day, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months, once a year or every two years, once every five years, or once a lifetime.

[0101] Exemplary administration and delivery routes include intravenous (IV), intraarticular, intraperitoneal (IP), intraarterial, intramuscular, parenteral, subcutaneous, intrathoracic, dermal, transdermal, parenteral, e.g., transmucosal, intracranial, intraspinal, oral (digestive), mucosal, respiratory, intranasal, intubation, intrapulmonary, intrapulmonary infusion, buccal, sublingual, intravascular, intrathecal, intracavitary, iontophoretic, intraocular, intraglandular, intravisceral, intrafallopian tube, and the like.

[0102] In some embodiments, delivery of rAAV to a mammalian subject is accomplished by affecting the mammalian eye, for example, by intraocular injection, subretinal injection, choroidal injection (e.g., suprachoroidal injection), or topical administration (e.g., eye drops), or by injection into the affected tissue (e.g., intravitreal injection). By "ocular tissue" is meant any tissue derived from or contained in the eye. Non-limiting examples of ocular tissues include neurons, retina (e.g., photoreceptor cells), sclera, choroid, retina, vitreous, macula, fovea, optic nerve head, lens, pupil, iris, aqueous humor, cornea, conjunctiva ciliary body, and optic nerve. The retina is located at the back of the eye and is composed of photoreceptor cells. These photoreceptor cells (e.g., rods, cones) provide vision by distinguishing colors and contrasts within the field of view. In some examples, the rAAV or compositions described herein are administered by intraocular injection. In some examples, the rAAV or compositions described herein are administered by intravitreal injection. In some embodiments, a rAAV or composition described in the invention is administered by subretinal injection. In some embodiments, a rAAV or composition described in the invention is administered by intrachoroidal (e.g., suprachoroidal) injection. In some embodiments, a rAAV or composition described in the invention is administered by intravenous injection.

[0103] In some embodiments, rAAV delivery to a mammalian subject can enter the mammalian subject, for example, by intramuscular injection.

[0104] In some embodiments, rAAV delivery to a mammalian subject can, for example, enter the mammalian subject by intravenous injection.

[0105] In some embodiments, rAAV delivery to a mammalian subject can be, for example, by intra-articular injection into the mammalian subject. "Intra-articular injection" is defined herein as an injection or injection into a joint. Intra-articular injection is often used to administer drugs to joints affected by inflammation.

[0106] The effect of administering the rAAV of the present invention or a pharmaceutical composition comprising the rAAV may be to prevent the progression of the disease, to stop the progression of the disease, to reverse the progression of the disease, etc. The disease or illness is related to the gene of interest loaded by the rAAV and the expressed gene product. For example, if the gene product is a VEGF antagonist, the disease or illness to be prevented or treated may be a VEGF-related disease. For example, "VEGF-related disease" refers to a group of diseases associated with abnormal VEGF activity / signaling. Many studies have confirmed that an abnormal excess of VEGF can stimulate and induce pathological angiogenesis, leading to angiogenesis-related eye diseases. Non-limiting exemplary angiogenesis-associated eye diseases include angiogenesis-dependent cancers, angiogenesis-associated eye diseases, solid tumors (e.g., lung cancer, breast cancer, kidney cancer, liver cancer, pancreatic cancer, head and neck cancer, colon cancer, melanoma), hematogenous tumors (e.g., leukemia, metastatic tumors), benign tumors (e.g., hemangioma, acoustic neuroma, neurofibroma, tracheitis, pyogenic granuloma), rheumatoid arthritis, psoriasis, erythema, Osier-Webber syndrome, myocardial angiogenesis, plaque, telangiectasia, hemophilic joints, or angiofibromas.

[0107] In some embodiments, angiogenesis-related eye diseases include diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma and retrolental fibroplasia, epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overuse, atopic keratitis, bacterial keratitis, ulcers, primary keratosis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis nodosa, trauma, Wegener's sarcoidosis, scleritis, Steven Johnson disease, radial keratotomy for pemphigus, corneal transplant rejection, and corneal graft rejection. These include, but are not limited to, refractory disease, sickle cell anemia, sarcoidosis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid artery occlusive disease, chronic uveitis / vitreous inflammation, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eales' disease, Behcet's disease, retinitis or choroiditis infections, presumed ocular histoplasmosis, Bests' disease, myopia, fovea, Stargardt's disease, pars planitis, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, complications after trauma or laser.

[0108] In some embodiments, the ocular disease is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascular ocular disease, non-infectious uveitis, or glaucoma.

[0109] In some embodiments, the ocular disease is selected from age-related macular degeneration, macular edema, diabetic macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, macular edema due to branch retinal vein occlusion, diabetic retinal edema, diabetic retinopathy, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoidal choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia, or retinopathy of prematurity. Specific forms of macular degeneration may include acute macular degeneration, nonexudative age-related macular degeneration, and nonexudative age-related macular degeneration.

[0110] For example, when the gene product is a TNF-α antagonist, the disease or condition to be prevented or treated may be an arthritis disease or related disease. Currently, it is estimated that there are more than 100 different forms of arthritis. Joint disorders are usually called arthropathy, and when inflammation is involved in one or more joints, the disorder is called arthritis. Arthritis is understood herein to mean "joint pain" or "joint disease". In a preferred embodiment, the arthritic disease is selected from the group consisting of adult Still's disease, ankylosing spondylitis, arthritis, low back pain, Behget's disease, blunt trauma, bursitis, calcium pyrophosphate deposition disease (CPD), carpal tunnel syndrome, chondromalacia of the patella, chronic fatigue syndrome, complex regional pain syndrome, cryopyrin-associated periodic syndromes (CAPS), degenerative disc disease, developmental dysplasia of the hip, L-Danlos, familial Mediterranean fever, fibromyalgia, fifth disease, giant cell arteritis, gout, hemochromatosis, infectious arthritis, inflammatory arthritis, inflammatory bowel disease, joint replacement surgery, juvenile arthritis, juvenile dermatomyositis (JD), juvenile idiopathic arthritis (JIA), juvenile rheumatoid arthritis, juvenile scleroderma, Kawasaki disease, lupus, childhood and adolescent Selected from lupus, Lyme disease, mixed connective tissue disease, myositis (including polymyositis, dermatomyositis), osteoarthritis (OA), osteoporosis, pagets, Hench-Rosenberg syndrome, patellofemoral pain syndrome, childhood rheumatic diseases, childhood SLE, polymyalgia rheumatica, pseudogout, psoriatic arthritis, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, Reiter's syndrome, rheumatic fever, rheumatism, rheumatoid arthritis, scleroderma, septic arthritis, Sjogren's disease, spinal stenosis, spondyloarthritis, Still's disease, systemic juvenile idiopathic arthritis and systemic lupus erythematosus, systemic lupus erythematosus of children and adolescents, systemic sclerosis, temporal arteritis, tendonitis, vasculitis and Wegener's granulomatosis.

[0111] In another preferred embodiment, the arthritic disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, gout, pseudogout, spondylitis, Crohn's disease, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease.

[0112] In a more preferred embodiment, the arthritic disease is a joint disorder related to one or more joint inflammation. Preferably, said arthritic disease is selected from rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, childhood idiopathic arthritis, and Still's disease. The present invention will be further described below with reference to examples, but these examples are not intended to limit the present invention in any way.

[0113] The experimental methods used in the following examples are conventional methods unless otherwise indicated. EXAMPLES

[0114] Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Example 1. Construction of mutants

[0115] Site-directed mutagenesis and recombinant DNA techniques are used to generate AAV8 variants that contain a polypeptide substitution of 5-14 amino acids located at amino acids Q588-A592 (QQNTA) of the parent AAV8, or further contain a mutation at a position corresponding to amino acids N262-N272 of the parent AAV8 capsid protein. The mutated amino acid sequence is SQSGASNDNH (SEQ ID NO: 10), as shown in Table 1, or further contains D80N, V125A, and V125G mutations. An exemplary No. 12 capsid mutant plasmid is shown in FIG. 2.

[0116] [Table 1]

[0117] [Table 2]

[0118] [Table 3]

[0119] Another group of capsid protein mutants have a polypeptide sequence inserted after amino acid Q589 of the parental AAV8, as shown in Table 4.

[0120] [Table 4]

[0121] Viral packaging of AAV8 mutants was generated by co-transfecting three plasmids into cells: the first plasmid (pAAV-CAG-EGFP) with ITRs flanking an expression cassette for the gene of interest (GFP or luciferase), the mutant plasmid (second plasmid) encoding the Rep / Cap genes constructed in Example 1, and the third plasmid containing adenovirus helper function genes. After transfection, cells were harvested and lysed to release the virus, and the viral titer was measured by PCR for subsequent use. Example 2 Transfection experiment by injection into mouse vitreous

[0122] 2.1 EGFP mRNA expression levels in mutants Purified AAV8 mutant viruses were injected into the vitreous of mice at 7E+8vg / eye. 21 days after intravitreal injection, mouse eyes were enucleated and pulverized using a freeze homogenizer, RNA was extracted using a total RNA extraction kit, cDNA was synthesized using a reverse transcription kit, and the specific EGFP primer probe set was determined by qPCR (wherein the sequence of the upstream primer EGFP-F is 5'-CACATGAAGCAGCACGACTT-3' (SEQ ID NO:26), the sequence of the downstream primer EGFP-R is 5'-TCGTCCTTGAAGAAGATGGT-3' (SEQ ID NO:27), and the probe is 5'-AGTCCGCCATGCCCGAAGGCT-3'-TAMRA, and the sequence included in the probe is shown in SEQ ID NO:28). The normalized EGFP mRNA levels of AAV8 mutants were calculated, that is, the effects of various AAV8 mutants on vitreous transfection were compared, and the results are shown in Figure 3.

[0123] 2.2 Preparation of whole mount retinas Purified AAV8 mutant viruses were injected into the vitreous of mice at 7E+8vg / eye. 21 days after intravitreal injection, mouse eyes were enucleated and fixed overnight in 4% paraformaldehyde. Mouse retinas were peeled off using surgical scissors under an upright microscope to prepare whole mount retinas, and EGFP fluorescent signals were captured by confocal imaging. The results are shown in Figure 4. Example 3 Transfection experiment by injection into rat vitreous body

[0124] Purified AAV8 mutant viruses were injected into the vitreous of rats at 1.92E+9vg / eye. 21 days after intravitreal injection, rat eyeballs were enucleated. The rat eyeballs were fixed overnight in 4% paraformaldehyde. The rat retina was peeled off using surgical microscope scissors under an upright microscope. Rat whole mount retinas were prepared, and EGFP fluorescent signals were captured by scanning fluorescent microscopy, and the results are shown in Figure 5. Example 4 Transfection experiment by injection into the suprachoroidal space of rabbits

[0125] The purified AAV8 mutant virus was injected into the suprachoroidal space of New Zealand rabbits at 5E+10vg / eye. 14 days after the suprachoroidal injection, the eyes of the New Zealand rabbits were enucleated. The eyes of the New Zealand rabbits were fixed overnight in 4% paraformaldehyde. Dehydration and tissue embedding were performed on the fixed eyes. Frozen sections were prepared, followed by nuclear staining (DAPI). A confocal microscope was used to scan the entire image and capture the EGFP fluorescent signal and DAPI signal, and the results are shown in Figure 6 (Figure 5 shows EGFP: autofluorescence, DAPI: nucleus, RGC: ganglion cell, INL: inner retinal nuclear layer, ONL: outer retinal nuclear layer, RPE: retinal epithelial cell). Judging from the fluorescent images, the wild type does not have a high transduction efficiency into the photoreceptor layer and RPE layer, but the mutant has a significantly improved transduction efficiency into the photoreceptor layer and RPE layer. Example 5 ARPE19 cell infection experiment

[0126] ARPE19 cells were seeded in 12-well plates and cultured until the cell confluence reached 70-80% (usually the number of cells was 1.0E+5). Viral supernatant (4.5E+9Vg) was added according to the required ratio so that the total volume of viral supernatant + medium was 1000 μL. 16 hours after infection, the medium was changed every day, and the culture was continued for 72 hours, and the EGFP fluorescent signal was captured by confocal to observe the expression of fluorescent protein. The infection status of ARPE19 cells for 48 hours is shown in Figure 7.

[0127] We also used flow cytometry to count the number of EGFP-positive cells. We cultured cells at a number of 2E+5 in a 6-well cell dish, added 1E+10vg packaging mutant virus, and cultured them at 37°C for 48h. We digested the cells with trypsin, collected the cells, and resuspended the cells in 300μL of sterile PBS. Using a Roche flow cytometer, we selected the FITC (488nm) channel for measurement, set the total number of cells to be measured to 100,000, and counted the number of EGFP-positive cells. The results are shown in Figures 8A and 8B. Example 6 Transfection experiment by injection into the suprachoroidal space of rabbits

[0128] According to the three-plasmid co-transfection method described in Example 1, viruses containing different mutants AAV-mCherry (mCherry is a protein isolated from coral, used here as a red fluorescent dye tracer) were prepared, and the genome titer of each virus was measured by ddPCR. The genome titer of each mutant virus was diluted to 1.0E12 vg / mL with buffer, and 100 μL per eye was injected into the suprachoroidal space 4 mm above the temporal part of the rabbit's eye, and two rabbits were injected into four eyes per virus. After 5 days, the New Zealand rabbits were killed, the eyes were enucleated, the retina / choroid tissues were separated, and cryo-tissue homogenates were made. The supernatants were collected and the virus genome DNA was extracted using a virus genome DNA extraction kit, and the relative content of the transduced virus genome DNA in the retina / choroid for each virus was measured by qPCR, and the results are shown in Table 5 and Figure 9.

[0129] [Table 5] Example 7 Protein expression experiments in infected APRE cells

[0130] AAV8-aflibercept virus and AAV No.12-aflibercept mutant virus were produced by three-plasmid co-transfection as described in Example 1, and the genome titer of each virus was measured by ddPCR. The genome titer of each mutant virus was diluted to 3.0E12 vg / mL with buffer, and the cell number was 3.0E5. 10 μL of virus was added to each well, and after 16 h of culture, the liquid was replaced, and after 72 h, the cells and cell supernatant were collected. Cell lysis was performed by freezing in liquid nitrogen and repeating freezing and thawing three times. The expression level of Trap protein was measured by ELISA. The results showed that the protein expression level of AAV8-aflibercept virus was 97 ng / ml, and the protein expression level of AAV No.12-aflibercept virus was 1662 ng / ml. Example 8 Protein expression experiment by suprachoroidal injection in rhesus monkeys

[0131] AAV No.128-aflibercept virus was produced by three-plasmid co-transfection as described in Example 1, and the genome titer of each virus was measured by ddPCR. The genome titer of the virus was diluted to 1.0E13 vg / mL in buffer and injected into the suprachoroidal space 4 mm above the temporal region of the monkey's eye, 100 μL per eye, into two eyes of one monkey. After 56 days, the rhesus monkey was killed, the eyeball was enucleated, and various tissues of the monkey's eye (including aqueous humor, choroid, conjunctiva, iris ciliary body, retina, sclera, and vitreous body) were isolated. After adding protease inhibitors, cold tissue homogenate was performed, the supernatant was collected, and the expression level of aflibercept protein (ng / ml) was measured by ELISA, and the results are shown in Table 6.

[0132] [Table 6] BLQ * :Not detected. Example 9 Transfection experiment by intramuscular injection into mice

[0133] AAV8-luciferase and AAV No.128-luciferase viruses were produced by three-plasmid co-transfection as described in Example 1, and the genome titer of the virus was measured by ddPCR. The genome titer of the virus was diluted to 1.62E11 vg / mL with buffer and injected into the gastrocnemius muscle of the right leg of the mouse in a volume of 50 μL, and five mice (Balb / c) were injected per virus. At 7, 14, 28, and 160 days after virus injection, a certain amount of fluorescein sodium substrate was injected intraperitoneally into the mouse, and in vivo imaging of the mouse was performed, and fluorescence intensity analysis was performed with an in vivo imager, and the results are shown in Table 7 and Figure 10.

[0134] [Table 7] Example 10 Protein expression experiments in transfected 293F cells

[0135] AAV8-No.128-etanercept and AAV No.128-adalimumab plasmids were constructed respectively. 2μg of plasmid was selected and transfected into 293F cells with single plasmid. After 72h, the protein expression level of the plasmid was detected by ELISA. The result was that the expression level of AAV No.128-adalimumab plasmid was 1010ng / mL, and the expression level of AAV No.128-etanercept plasmid was 2020ng / mL. Example 11 Efficacy study of intraarticular / intramuscular injection in mice

[0136] 1) Preparation of sample solution Preparation of test samples: AAV No. 128-etanercept virus was prepared according to the method described in Example 1, the viral genome titer was measured, and then it was diluted to 9.98E12 vg / mL for use. Positive control: Adaximab stock solution 40 mg / 400 μl was taken and diluted to 1.6 μg / μl.

[0137] 2) Modeling First modeling on day 0: 6-week-old DBA / 1 male mice were selected. First, 0.6 ml of bovine type II collagen (CII) solution (2 mg / ml) and 0.6 ml (4 mg / ml) of complete Freund's adjuvant were put into a film extruder to prepare about 1 ml of milky white emulsion. 100 μl of emulsion was subcutaneously injected into the base of the tail of each mouse. After examination, swelling was observed at the base of the tail, no leakage of emulsion was observed, and no mice died, suggesting that the administration was basically successful. Secondary modeling on day 21: 0.6 ml (2 mg / ml) of bovine type II collagen (CII) and 0.6 ml (4 mg / ml) of incomplete Freund's adjuvant were placed in a film extruder to prepare approximately 1 ml of milky white emulsion, and 100 μl of the emulsion was subcutaneously injected at the base of the tail of each mouse.

[0138] 3) Administration Administration was performed on day 22, and on day 0 of administration, 10 μl / ankle of the AAV No. 128-etanercept virus sample solution was administered into the articular cavity (both feet). Administration was performed on day 22, and on day 0 of administration, 100 μl / ankle of the AAV No. 128-etanercept virus sample solution (both legs) was intramuscularly injected near the joint cavity. On day 22, 160 μg of adalimumab (40 mg / 400 μl of stock solution diluted to 1.6 μg / μl, and 100 μl of the solution was injected subcutaneously into the back using an insulin needle) was administered to the adalimumab group once every 2 weeks on the back.

[0139] 4) Observation of results From day 23 to day 2 of administration, the thickness of the sole of the foot was monitored using an electronic caliper, and the clinical score of arthritis was measured every two days. The evaluation criteria are shown in Table 8 below.

[0140] [Table 8]

[0141] 5) Results

[0142] The results of the measurements of plantar thickness and clinical scores of arthritis are shown in FIG. 11 and FIG.

[0143] The above-mentioned embodiments only represent some embodiments of the present invention, and the description is more specific and detailed, but this should not be understood as limiting the patent scope of the present invention. However, those skilled in the art can make some modifications and improvements without departing from the concept of the present invention, which are within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention should be determined based on the attached claims.

Claims

1. A modified adeno-associated virus (AAV) capsid protein, comprising a polypeptide substitution of about 5 to 14 amino acids compared to a parent AAV capsid protein, wherein an AAV virus comprising said modified capsid protein has improved ability to infect retinal cells compared to an AAV virus comprising the corresponding parent AAV capsid protein.

2. The polypeptide may be RGNRQ (SEQ ID NO:1), QQNTARGNRQ (SEQ ID NO:2), RGNRQAAQQNTA (SEQ ID NO:3), RGNRQQNTA (SEQ ID NO:4), RGNRQQQNTA (SEQ ID NO:5), SGNTQ (SEQ ID NO:6), RGNQQNTARQ (SEQ ID NO:7), RGNQQPRPTSRQ (SEQ ID NO:8), RGNRQAAQQPTPTS (SEQ ID NO:9) or RGNRQQQPTPTS (SEQ ID NO:10). 19), wherein the substitution is at amino acid positions 588-592 of the parent AAV8 or the corresponding position of another serotype capsid protein.

3. The capsid protein of claim 2, wherein the polypeptide comprises an amino acid sequence selected from RGNRQ (SEQ ID NO:1), QQNTARGNRQ (SEQ ID NO:2), RGNRQAAQQNTA (SEQ ID NO:3), RGNRQQNTA (SEQ ID NO:4), SGNTQ (SEQ ID NO:6), RGNQQNTARQ (SEQ ID NO:7) or RGNRQQQPTPTS (SEQ ID NO:19).

4. 4. The capsid protein of claim 2 or 3, further comprising a mutation to amino acids 262-272 of the parent AAV8 capsid protein or the corresponding positions of another serotype capsid protein, wherein the mutated amino acid sequence is SQSGASNDNH (SEQ ID NO: 10).

5. 5. The capsid protein of any one of claims 1 to 4, wherein the capsid protein comprises a polypeptide substitution at amino acid positions 588-592 of the parent AAV8 or the corresponding positions of another serotype capsid protein and a mutation to positions 262-272 of the parent AAV8 capsid protein or the corresponding positions of another serotype capsid protein, wherein the polypeptide is RGNRQ (SEQ ID NO: 1) and the mutated amino acid sequence is SQSGASNDNH (SEQ ID NO: 10).

6. The capsid protein of any one of claims 1 to 5, characterized in that the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, and AAVrhlO.

7. The capsid protein of claim 6, wherein the AAV serotype is AAV8.

8. The capsid protein of any one of claims 1 to 7, further comprising a mutation to an amino acid at position D80 and / or V125 of the parent AAV8 capsid protein.

9. The capsid protein of claim 8, wherein the mutation is D80N and / or V125A.

10. The capsid protein of claim 8, wherein the mutation is D80Q and / or V125G.

11. 10. The capsid protein of claim 8 or 9, wherein the capsid protein comprises a polypeptide substitution at amino acid positions 588-592 of the parent AAV8 and mutations to amino acids at positions D80 and V125 of the parent AAV8 capsid protein, the polypeptide being RGNQQNTARQ (SEQ ID NO: 7) and the mutations being D80N and V125A.

12. 11. The capsid protein of claim 8 or 10, wherein the capsid protein comprises a polypeptide substitution at amino acid positions 588-592 of the parent AAV8 and mutations to amino acids at positions D80 and V125 of the parent AAV8 capsid protein, the polypeptide being RGNQQNTARQ (SEQ ID NO: 7) and the mutations being D80Q and V125G.

13. 1. A modified adeno-associated virus (AAV) capsid protein, comprising: A modified adeno-associated virus (AAV) capsid protein, characterized in that the capsid protein comprises an insertion of a polypeptide after the amino acid at position 589 of the parent AAV8 or the corresponding position of another serotype capsid protein, and an AAV virus comprising the modified capsid protein has improved retinal cell infectivity compared to an AAV virus comprising the corresponding parent AAV capsid protein.

14. 14. The capsid protein of claim 13, wherein the polypeptide comprises an amino acid sequence selected from RGDLTTPQQ (SEQ ID NO: 20), RGDLNTPQQ (SEQ ID NO: 21), and RGDVSSPQQ (SEQ ID NO: 22).

15. The capsid protein of claim 13 or 14, characterized in that the AAV serotype is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-DJ, AAV-DJ8, AAV-DJ9, AAVrh8, AAVrh8R, and AAVrhlO.

16. The capsid protein of claim 15, wherein the AAV serotype is AAV8.

17. i. a modified capsid protein according to any one of claims 1 to 16; ii. A recombinant adeno-associated virus (rAAV) comprising a heterologous nucleic acid comprising an encoded gene product.

18. The recombinant adeno-associated virus of claim 17, wherein the gene product is a VEGF antagonist or a TNF-α antagonist.

19. The recombinant adeno-associated virus of claim 18, wherein the VEGF antagonist is selected from aflibercept, conbercept, ranibizumab, and brolucizumab.

20. The recombinant adeno-associated virus of claim 19, wherein the VEGF antagonist is selected from aflibercept.

21. The recombinant adeno-associated virus of claim 18, wherein the TNF-α antagonist is selected from etanercept, infliximab, adalimumab, pecelizumab, and golimumab.

22. The recombinant adeno-associated virus of claim 21, wherein the TNF-α antagonist is selected from etanercept.

23. a) a recombinant adeno-associated virus according to any one of claims 17 to 22; b) a pharma- ceutically acceptable excipient.

24. Use of a recombinant adeno-associated virus according to any one of claims 17 to 22 in the preparation of a medicament for preventing or treating an eye disease.

25. A method for preventing or treating an eye disease, comprising administering an effective amount of the recombinant adeno-associated virus according to any one of claims 17 to 22 or the pharmaceutical composition according to claim 23 to an individual in need thereof.

26. 26. The method of claim 25, wherein the administration is by vitreous, retinal or choroidal injection.

27. 27. The method of claim 26, wherein the administration is by suprachoroidal injection.

28. 28. The use according to claim 24 or the method according to any one of claims 25 to 27, wherein the ocular disease is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascular ocular disease, non-infectious uveitis, or glaucoma.

29. 28. The use according to claim 24 or the method according to any one of claims 25 to 27, wherein the ocular disease is selected from age-related macular degeneration, macular oedema, diabetic macular oedema, macular oedema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, macular oedema due to branch retinal vein occlusion, diabetic retinal oedema, diabetic retinopathy, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoidal choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia, or retinopathy of prematurity.

30. Use of a recombinant adeno-associated virus according to any one of claims 17 to 22 in the preparation of a medicament for preventing or treating an arthritic disease or related condition.

31. A method for treating an arthritic disease or related condition, comprising administering an effective amount of a recombinant adeno-associated virus according to any one of claims 17 to 22 or a pharmaceutical composition according to claim 23 to an individual in need thereof.

32. 32. The method of claim 31, wherein the method is administered by intravenous, subcutaneous, intramuscular, or intraarticular injection.

33. 33. The method of claim 32, wherein the administration is by intramuscular or intraarticular injection.

34. 34. The use according to claim 30 or the method according to any one of claims 31 to 33, wherein the arthritic disease or related condition is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, gout, pseudogout, spondylitis, Crohn's disease, plaque psoriasis, psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement or Still's disease.

Images