Modified AAV capsid proteins and their use

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, and specifically, to a modified AAV capsid protein and its use.

Background Art

[0002] Vectors used in gene therapy are classified into viral vectors and non-viral vectors. Among viral vectors, the most commonly used ones are adenovirus vectors, lentivirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, etc.

[0003] Adeno-associated virus (AAV) belongs to the Parvoviridae family and the Dependovirus genus. The virion is composed of a 25 nm icosahedral capsid containing a 4.7 kb single-stranded DNA genome with two open reading frames, Rep and Cap. The non-structural Rep gene encodes four regulatory proteins required for virus replication, and Cap encodes three structural proteins (VP1 - 3) assembled into a 60-subunit capsid. This viral capsid mediates the ability of the AAV vector to overcome many biological barriers to virus transduction, such as cell surface receptor binding, endocytosis, intracellular transport, and uncoating in the nucleus.

[0004] AAV is widely used in many fields such as gene transduction, gene therapy, vaccination, and oncolytic therapy. It has many advantages, such as low pathogenicity, a wide range of infected tissues, a wide range of host cells (infectable and expressible in both proliferating and non-proliferating cells), low immunogenicity, long-term expression of foreign genes in vivo, and no integration into the host cell genome, and is widely used in experimental and clinical studies. The US FDA approved the first AAV-mediated gene therapy in 2017 for the treatment of rare genetic eye diseases. In patients who received a single subretinal injection of an rAAV vector delivery therapeutic agent, the expression of the therapeutic agent was consistently observed for more than 4 years.

[0005] However, there are still several challenges to the current application of AAV, such as improving the targeting and transduction efficiency of vectors to tissues and cells, and improving the packaging efficiency of specific vectors to adapt them to industrial production. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The present invention involves delivering a viral vector containing a target gene (e.g., an AAV viral vector) to a desired cell or tissue. Specifically, the present invention comprises a modified capsid protein, the modified capsid protein comprising one or more modifications (e.g., substitution, insertion, or mutation) in the amino acid sequence compared to the parent AAV capsid protein, and when present in the AAV viral vector, it enhances infectivity to target tissues or target cells (e.g., retina, muscle, or joint cavity) compared to an AAV viral vector containing an 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, comprising approximately 5 to 14 amino acid polypeptide substitutions compared to the parent AAV capsid protein, wherein the AAV virus containing the modified capsid protein exhibits improved retinal cell infectivity compared to an AAV virus containing 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), wherein the substitution is located at amino acids between positions 588 and 592 of the parent AAV8 or at the corresponding position of 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 at the amino acids between positions 262 and 272 of the parent AAV8 capsid protein or at the corresponding position of another serotype capsid protein, wherein the mutated amino acid sequence is SQSGASNDNH (SEQ ID NO: 10). In one preferred embodiment, the capsid protein comprises a polypeptide substitution at the amino acids between positions 588 and 592 of the parent AAV8 or at the corresponding position of another serotype capsid protein, and a mutation at positions 262 and 272 of the parent AAV8 capsid protein or at the corresponding position 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).

[0010] In some specific embodiments, the AAV of the present invention may be derived from any serotype AAV, for example, the AAV serotype may be 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 mutations to the amino acids at positions D80 and / or V125 of the parent AAV8 capsid protein. In one preferred embodiment, the mutations are D80N and / or V125A. In another preferred embodiment, the mutations are D80Q and / or V125G. In one preferred specific embodiment, the capsid protein comprises a polypeptide substitution at amino acids between positions 588 and 592 of the parent AAV8 and mutations 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 mutations are D80N and V125A. In another preferred specific embodiment, the capsid protein comprises a polypeptide substitution at amino acids 588-592 of parent AAV8 and mutations to amino acids at positions D80 and V125 of 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 present invention, another modified adeno-associated virus (AAV) capsid protein is provided, comprising a polypeptide insertion located after the 589th amino acid of parent AAV8 or the corresponding position of another serotype capsid protein, wherein the AAV virus comprising the modified capsid protein exhibits improved retinal cell infectivity compared to the 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 derived from any serotype AAV, 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, i. The modified capsid protein according to the present invention, ii. Provided are recombinant adeno-associated viruses (rAAV) comprising heterologous nucleic acids containing encoded gene products.

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

[0016] In some specific embodiments, the VEGF antagonist is selected from aflibercept, convercept, ranibizumab, and brolucizumab, but is preferably aflibercept.

[0017] In some specific embodiments, the TNF-α antagonist is selected from etanercept, infliximab, adalimumab, pecerizumab, or golimumab, but is preferably etanercept.

[0018] Another aspect of the present invention is, a) The recombinant adeno-associated virus according to the present invention, b) To provide a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

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

[0020] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the present invention is administered by intravitreal, retinal, choroidal injection, intravenous, subcutaneous, intramuscular or intra-articular 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 intra-articular injection. Another aspect of the present invention provides the recombinant adeno-associated virus or pharmaceutical composition according to the present invention for therapeutic use.

[0021] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition according to the present invention is administered by intravitreal, 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.

[0022] Another aspect of the present invention provides the use of the recombinant adeno-associated virus in the preparation of a drug for preventing or treating an eye disease.

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

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

[0025] In some specific embodiments, the eye diseases described in the present invention are selected from retinal angiogenesis, choroidal angiogenesis, iris angiogenesis, corneal angiogenic eye diseases, non-infectious uveitis, or glaucoma. In some specific embodiments, the eye diseases described in the present invention are 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 angiogenesis secondary to degenerative myopia, or retinopathy of prematurity.

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

[0027] In some specific embodiments, the recombinant adeno-associated virus or pharmaceutical composition described in the present invention is administered by intravitreal, retinal or choroidal injection. In one preferred embodiment, the recombinant adeno-associated virus or pharmaceutical composition described in the present invention is administered by suprachoroidal injection.

[0028] In some specific embodiments, the eye diseases described in the present invention are selected from retinal angiogenesis, choroidal angiogenesis, iris angiogenesis, corneal angiogenic eye diseases, non-infectious uveitis, or glaucoma. In some specific embodiments, the eye diseases described in the present invention are 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 angiogenesis secondary to degenerative myopia, or retinopathy of prematurity.

[0029] In another aspect of the present invention, there is provided the use of the recombinant adeno-associated virus in the preparation of a drug for preventing or treating an arthritis disease or related disorder.

[0030] Another aspect of the present invention provides a method for preventing or treating arthritis or related diseases, comprising administering an effective amount of the recombinant adeno-associated virus or pharmaceutical composition described in the present invention to an individual in need.

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

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

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

[0034] In another aspect of the present invention, recombinant adeno-associated virus or pharmaceutical composition according to the present invention is provided for a method of treating arthritis or related diseases.

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

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

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

[0038] [Figure 1A] This shows the amino acid sequence alignment of the GH loop region of the AAV capsid protein. [Figure 1B] This shows the amino acid sequence alignment of the GH loop region of the AAV capsid protein. [Figure 2] This is a schematic diagram of the construction of the AAV8 capsid protein mutant plasmid. [Figure 3] This shows the EGFP mRNA expression levels in mice injected intravitreously with the AAV8 mutant virus. [Figure 4] This image shows the EGFP fluorescence signal from the whole-mount retina of mice injected intravitreously with the AAV8 mutant virus. [Figure 5] This image shows the EGFP fluorescence signal in the whole-mount retina of rats injected intravitreously with the AAV8 mutant virus. [Figure 6] This shows the EGFP fluorescence signal and DAPI signal from rabbits injected into the epichoroidal space with AAV8 mutant virus. [Figure 7] This shows the EGFP fluorescence signal of AAV8 mutant virus cultured in ARPE19 cells. [Figure 8A] This shows the number of EGFP-positive cells after infection of ARPE19 cells with AAV8 mutant virus, as determined by flow cytometry. [Figure 8B] This shows the number of EGFP-positive cells after infection of ARPE19 cells with AAV8 mutant virus, as determined by flow cytometry. [Figure 9] This shows the relative content of mCherry genomic DNA in rabbits injected with AAV8 mutant virus into the epichoroidal space. [Figure 10] This shows the luciferase fluorescence intensity of mice injected intramuscularly with the AAV8 mutant virus. [Figure 11] The clinical scores of mice injected with AAV8 mutant virus intraarticularly / intramuscularly are shown. [Figure 12] This shows the thickness of the sole of the foot of a mouse injected intra-articular / intramuscularly with the AAV8 mutant virus. [Modes for carrying out the invention]

[0039] The present invention relates to a capsid protein that exhibits enhanced infectivity to target tissues or target cells (e.g., retina, muscle, or joint cavity) compared to an AAV virus vector containing an unmodified parental AAV capsid protein, to rAAV containing this capsid protein and expressing a target gene (e.g., a VEGF antagonist, a TNF-α antagonist), to a pharmaceutical composition thereof, and to its use. Capsid protein

[0040] The present invention provides a modified AAV capsid protein comprising one or more amino acid modifications compared to the corresponding wild-type AAV or parental AAV. The modifications include one or more amino acid substitutions, deletions, or insertions compared to the parental capsid protein or AAV capsid protein. The modifications result in improved infectivity to target tissues or cells (e.g., retina, muscle, or joint cavity) compared to AAV viral vectors containing unmodified parental AAV capsid protein.

[0041] In certain embodiments, the modified AAV capsid protein includes capsid protein serotypes belonging to AAV 1, AAV 2, AAV 3, AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, AAVrh10, 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, such as heterologous polypeptide substitutions within VP1. In some embodiments, the capsid mutant may be a chimeric capsid mutant. The sequence of the chimeric capsid mutant may include parts of two or more AAV capsid serotypes or their variants. 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 contains approximately 5 to 14 heterogeneous polypeptide substitutions. In some specific embodiments, the modified capsid protein of the present invention contains approximately 5 to 14 heterogeneous polypeptide substitutions at the amino acids between positions 588 and 592 of parent AAV8, or at the corresponding positions of another serotype capsid protein. Amino acid sequences such as AAV1 to AAV9 are described in patents US8962330B, US10041090B, etc. In certain embodiments, the modified capsid protein of the present invention contains approximately 5 to 14 heterogeneous polypeptide substitutions at the amino acids between positions 588 and 592 of parent AAV8. The amino acids between positions 588 and 592 of the parent / wild-type AAV8 capsid protein are "QQNTA". Therefore, a particular embodiment of the present invention is a modified capsid protein in which the amino acid "QQNTA" at positions 588-592 of the parental / wild-type AAV8 capsid protein is replaced with a heterologous polypeptide of 5-14 amino acids. For example, as shown in Figure 1A, the substitution site corresponding to the amino acids at positions 588-592 of AAV8 is located at the corresponding position in other capsid proteins. For example, this is at amino acids 586-590 of AAV1, 585-589 of AAV2, 586-590 of AAV3, 584-588 of AAV4, 575-579 of AAV5, 586-590 of AAV6, 587-591 of AAV7, 586-590 of AAV9, and 588-592 of AAV10.

[0043] The lengths of the above-mentioned substituted heteronucleotides are 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, or 11 amino acids. In some specific embodiments, the polypeptide described in the present 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 polypeptide described in the present invention comprises 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 includes mutations compared to the amino acids at positions 262–272 of the parental AAV8 capsid protein or the corresponding positions of another serotype capsid protein. The amino acids at positions 262–272 of the parental / wild-type AAV8 capsid protein are "SNGTSGGATND". Thus, in certain embodiments of the present invention, the modified capsid protein is mutated at the amino acid "SNGTSGGATND" at positions 262–272 of the parental / wild-type AAV8 capsid protein. The mutation site corresponding to amino acids 262-272 of AAV8 is located at the corresponding position on other capsid proteins, for example, amino acids 261-271 of AAV1, 261-271 of AAV2, 261-271 of AAV3, 255-265 of AAV4, 251-261 of AAV5, 261-271 of AAV6, 262-272 of AAV7, 261-271 of AAV9, and 262-272 of AAV10. The mutation site corresponding to amino acids 262-272 of AAV8 is located at the corresponding position on other capsid proteins, for example, as shown in Figure 1B. In some specific embodiments, the mutant amino acid sequence described in the present invention is SQSGASNDNH (SEQ ID NO: 10).

[0045] In some specific embodiments, the modified capsid protein described in the present invention comprises a polypeptide substitution at amino acids at positions 588-592 of parent AAV8 or at the corresponding position of another serotype capsid protein, and a mutation at positions 262-272 of parent AAV8 capsid protein or at the corresponding position 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 in the present invention is AAV8.

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

[0047] In some other embodiments, the modified AAV capsid protein of the present invention includes a polypeptide insertion that lies after the 589th amino acid of parent AAV8 or the corresponding position of another serotype capsid protein. The insertion site is after the 589th amino acid of AAV8 or the corresponding position of other capsid proteins, for example, after the 589th amino acid of AAV1, the 586th amino acid of AAV2, the 587th amino acid of AAV3, the 585th amino acid of AAV4, the 576th amino acid of AAV5, the 587th amino acid of AAV6, the 588th amino acid of AAV7, the 587th amino acid of AAV9, and the 589th amino acid of AAV10. In some specific embodiments, the inserted polypeptide includes 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 located after the 589th amino acid 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 described in the present invention comprises a sequence having at least 85%, 90%, 95%, 98%, 99%, or 100% homology to the amino acid sequence of the above-mentioned capsid protein subjected to amino acid substitution, mutation, and / or insertion.

[0049] In some embodiments, AAVs containing modified capsid proteins exhibit at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or even greater infectivity of retinal cells compared to AAV viruses containing parental / wild-type capsid proteins. The retinal cells include RPE-19 cells (human retinal pigment epithelial cell line), retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, cone cells, rod cells, Muller glial cells, and retinal pigment epithelial cells.

[0050] In some specific embodiments, AAVs containing modified capsid proteins exhibit at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or even greater infectivity of RPE cells compared to AAV viruses containing the corresponding parental / wild-type capsid protein.

[0051] In some specific embodiments, AAVs containing modified capsid proteins exhibit at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or even greater infectivity to retinal cells after epichoroidal injection compared to AAV viruses containing the corresponding parental / wild-type capsid proteins.

[0052] In some specific embodiments, AAVs containing modified capsid proteins exhibit at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or even greater infectivity to retinal cells after intravitreous injection compared to AAV viruses containing the corresponding parental / 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 an rAAV having the coding sequence and complementary sequence of the target gene on separate strands, and packaged in separate viral capsids.

[0054] In some embodiments, the recombinant adeno-associated virus (rAAV) described in the present invention comprises i) a modified capsid protein described in the present invention, and ii) a heterogeneous nucleic acid containing an encoding gene product.

[0055] In some embodiments, the 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 ability to specifically target cells or tissues (e.g., eye tissue or cells, muscle tissue or joint cavities) or infectivity compared to an rAAV containing the parent or wild-type capsid protein.

[0056] In some embodiments, the rAAV described in the present invention comprises a heterogeneous nucleic acid encoding a gene product and a polynucleotide expression cassette of regulatory elements. The term “regulatory elements” means segments of nucleic acid sequences that regulate the expression of operably linked gene products. Thus, expression regulatory elements may include promoters, enhancers, internal ribosome entry sites (IRESs), transcriptional terminators, start codons before protein-coding genes, intron splicing signals, and terminator codons. The term may also include nucleic acid sequence designs from which potentially undesirable start codons inside and outside the frame have been removed from the sequence. It may also include nucleic acid sequence designs from which potentially undesirable splicing sites have been removed. It may also include sequences that direct polyadenylation or polyA addition, and so on.

[0057] In the polynucleotide expression cassette of the present invention, the heterologous nucleic acid comprises a nucleotide sequence encoding the gene product of a target gene, for example, a therapeutic gene product. In some embodiments, the gene product is 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, etc.

[0058] In some embodiments, the gene products described in the present invention include VEGF antagonists, TNF-α antagonists, PD-1 / PD-L1 inhibitors, Ang-2 inhibitors, plasma kallikrein inhibitors, endostatins, tumor statins, angiostatins, pigment epithelial-inducing factor (PEDF), soluble Tie-2 receptor, Ang-2 antagonists, CD tissue metalloproteinase inhibitor-3 (TIMP-3), photoresponsive opsins (e.g., rhodopsin), anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl), glial-derived neurotrophic factor (GDNF), fibroblast growth factor 2, and neurturin (NTN). Selected from: 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 with any of these proteins or polypeptides, and functional variants or fragments containing 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 their variants.

[0059] In one embodiment, the gene product described in 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), for example, VEGFR-1 extracellular domain 2 (Flt d2) and / or VEGFR-2 extracellular domain 3 or 4.

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

[0061] Here, SEQ ID NO: 12 is the sequence shown below. GRPFVEMYSEIPEIIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGI DFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEV SERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPGDKTHTCPLCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN 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. MEIVMTQSPSTLSASVGDRVIITCQASEIIHSWLAWYQQKPGKAPKLLIYLASTLASGVPSRFSGSGSGAEFTLISSLQPDDFATYYCQNVYLASTNGANFGQGTKLTVLG

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

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

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

[0068] In a particular embodiment, the TNF-α antagonist comprises a heavy chain variable region sequence described in SEQ ID NO: 17 and a light chain variable region sequence described 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 described in SEQ ID NO: 24. LPAQVAFTPYAPEPGSTCRLREYYDQTAQMCCSKCSPGQHAKVFCTKTSDVCDSCEDSTYTQLWNWVPECLSCGSRCSSDQVETQACTREQNRICTCRPGWYCALSKQEGCRLCAP LRKCRPGFGVARPGTETSDVVCKPCAPGTFSDTTSSTDICRPHQICNVVAIPGDASMDAVCTSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGD EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 24)

[0072] In a particular embodiment, the nucleotide encoding the TNF-α antagonist has the sequence described 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 above amino acid sequence.

[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 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 a Faricimab biantibody.

[0076] In some embodiments of the present invention, coding sequences may be designed using codon optimization to better express the target gene. The present invention can optimize nucleotide codons encoding polypeptides, fusion proteins, antibodies, or functional fragments thereof to improve the protein expression level of the target gene in target cells, tissues, or species. A coding sequence is a portion of an mRNA sequence that encodes amino acids used for transcription and translation. In the transcription and translation process, every three nucleotide codons out of 61 nucleotide codons are transcribed and translated into one of 20 amino acids. However, the frequency of tRNAs encoding the same amino acids varies depending on the cell type and animal species. If a gene sequence contains codons that are not frequently expressed, the ribosome transcription mechanism may be impaired, potentially inhibiting effective transcription. Therefore, those skilled in the art can improve the expression level of the target gene by "codon optimization." Simply put, codon optimization involves modifying the nucleotide sequence encoding a product with synonymous codon sequences, for example, by substituting a low-frequency expressed codon with a codon that is more frequently expressed in the target cell, thereby increasing the expression level of a target substance in a specific cell, tissue, or substance to, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or above 100%. Codon sequences for codon optimization can be designed in various ways. This optimization can be performed using methods available online, publicly disclosed 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 full length of the open read frame (ORF) of the product is modified as appropriate. However, in some embodiments, only one fragment of the ORF can be modified. One of these methods is used to produce codon-optimized coding nucleotides encoding 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 includes a constitutive promoter region, a nucleotide sequence encoding a gene product is operably linked to the constitutive promoter, and the promoter is widely present and active in cells, tissues, or species and promotes the expression of a target gene in specific cells or tissues in vivo or in vitro. In some embodiments, examples of promoters include the chicken β-actin promoter (CBA), cytomegalovirus promoter (CMV), CMV early enhancer / chicken β-actin (CBA) promoter / rabbit β-globin intron (CAG), elongation factor 1α promoter (EF1α), human phosphoglycerate kinase promoter (PGK), MNT promoter, UB6 promoter, CAG promoter, RPE65 promoter, opsin promoter, mitochondrial heavy chain promoter, ubiquitin promoterson, and the like.

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

[0079] The expression regulatory sequences in the polynucleotide expression cassette described in the present invention may also include polyadenylation signals. These polyadenylation signals are also called polyadenylation sites, polyadenylate tails, Poly(A) sites, Poly(A) signals, or Poly(A) tails. A polyadenylation region refers to the covalent bond between polyadenylate and a messenger RNA (mRNA) molecule. It is part of the process of protein biosynthesis, generating mature mRNA ready for translation. In eukaryotes, polyadenylation is a mechanism that blocks the 3' end of the mRNA molecule. Polyadenylation signals protect mRNA from exonuclease attack and are important for transcription termination, mRNA output from the cell nucleus, and translation. Polyadenosination signals contain multiple consecutive adenosine monophosphates, typically including an AAUAAA repeat sequence. Some exemplary polyadenylation signals include monkey vacuolar virus 40 (SV40), human growth hormone (HGH), bovine growth hormone (BGH), or betaglobin.

[0080] The polynucleotide expression cassette described in the present invention may also include a reverse terminal repeat (ITR). A functional adenovirus reverse terminal repeat (ITR) refers to an ITR sequence for, for example, incorporating, replicating, and packaging AAV virus particles. Typically, the length of an ITR sequence is about 145 bp. Preferably, substantially the entire sequence encoding the ITR, and modifications made to these ITR sequences by conventional means of those skilled in the art, are used intramolecularly. An example of the use of an ITR molecule in this application is a “cis” plasmid containing the target gene sequence and associated regulatory elements flanking the 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 nucleotide encoding the target gene is located on the side of the AAV ITR (e.g., oriented 5'-ITR-target-gene-ITR-3'). In some embodiments, the AAV ITR is 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 described in the present invention further includes 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 regulator that can enhance the output of RNA from the cell nucleus. Exemplary RNA output sequences include, but are not limited to, sequences from hepatitis B virus post-transcriptional regulators (HPREs) and guinea pig hepatitis virus post-transcriptional regulators (WPREs).

[0082] Those skilled in the art will understand that the polynucleotide expression cassette described in the present invention includes, but is not limited to, restriction sites that facilitate cloning, regulatory elements operably linked to individual elements, or regulatory elements that regulate gene expression, and may optionally include other elements. Examples include bacterial sequences in plasmid vectors, attp sites, attB sites, promoter linkers, and polyadenylated sequence linkers in phage integrase vectors.

[0083] In some embodiments, the polynucleotide expression cassette described in the present invention includes those prepared by those skilled in the art from combinations of two or more of the elements described above and the target gene, wherein the target gene coding sequence and associated regulatory element aspects in the polynucleotide expression cassette are 5' and 3' AAV ITR sequences.

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

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

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

[0087] A recombinant viral vector containing a modified capsid protein as described in the present invention can be produced using a standard method. For example, the method comprises culturing a host cell containing an artificial genome and recovering a recombinant AAV encapsulating the artificial genome from the cell culture, wherein the artificial genome comprises a cis-polynucleotide expression cassette adjacent to an AAV ITR containing a coding sequence for a gene product operably linked to an expression regulatory element that controls the expression of the gene product in human cells; a trans-expression cassette lacking an AAV ITR, the trans-expression cassette encoding an AAV rep and a capsid protein, the AAV rep and capsid protein driving the expression of the AAV rep and capsid protein in the host cell in the culture and the trans-expression cassette operably linked to an expression regulatory element that provides the AAV rep and capsid protein in a trans manner; and an adenovirus helper having sufficient function to enable replication and packaging of the artificial genome via the AAV capsid protein.

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

[0089] Another aspect of the present invention is to provide a pharmaceutical composition comprising rAAV, which comprises recombinant adeno-associated virus as described in the present invention and a pharmaceutically acceptable diluent, carrier, or excipient.

[0090] Suitable diluents, carriers, or excipients can be easily selected by those skilled in the art. As used herein, carriers or excipients include any solvent, dispersion medium, carrier, coating, diluent, antimicrobial and antifungal agent, isotonic agent, absorption retarder, buffer, carrier solution, suspension, colloid, preservative, or chemical stabilizer.

[0091] Exemplary carriers or excipients include sterile saline, lactose, sucrose, calcium phosphate, gelatin, glucan, agar, pectin, peanut oil, sesame oil, and water. For example, a suitable excipient or carrier includes saline solution that can be prepared with various buffers (e.g., phosphate buffer). Microbial activity can be prevented by various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include sugars and isotonic agents such as sodium chloride. The absorption of injectable compositions can be extended by using absorption retarders such as aluminum monostearate and gelatin in the composition.

[0092] To provide an injectable aqueous solution, buffers are prepared as needed, and the liquid diluent may be first isopermated with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.

[0093] The method for preparing a sterile injection solution involves mixing various other components listed in the present invention with the required amount of active rAAV in a suitable solvent, followed by sterilization by filtration. Typically, the dispersion is prepared by incorporating various sterilizing active components into a sterile carrier containing a dispersion medium and other desired components.

[0094] The rAAV composition of the present invention can also be prepared in neutral or salt form. The 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 iron hydroxide, as well as organic bases such as isopropylamine, trimethylamine, histidine, and prukaine. After preparation is complete, the solution is administered in a dosage form suitable for the drug and in a therapeutically effective dose. The formulation can be easily administered in various dosage forms, such as injectable solutions and drug-release capsules.

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

[0096] Compositions for injection, oral use, or topical gastrointestinal use are advantageous to be prepared in unit dosage forms for ease of administration. As used herein, a unit dosage form is a physically discrete unit suitable for use as a single dose to a subject under treatment. Each unit contains a predetermined amount of an active compound calculated to produce a desired therapeutic effect in relation to a desired drug carrier. The specifications of the unit dosage forms of the present invention are determined by the inherent properties of the active compound and the specific therapeutic effect to be achieved, and the inherent limitations in the art when mixing such active compounds for use in the treatment of an individual. For example, a unit dose may be a certain amount of vector genome, or may contain a certain amount of vector genome per milliliter, or may contain a unit dose that measures the pharmaceutical composition using the multiplicity of infection (MOI), which means the ratio or multiplier of the vector or viral genome to the cells to which the nucleic acid can be delivered.

[0097] The pharmaceutical composition may be contained in a container, package, or dispenser such as a syringe, along with instructions for administration. Delivery of recombinant adeno-associated virus (rAAV)

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

[0099] "Therapeutic dose" refers to the amount effective in the dose and duration required to obtain the desired therapeutic effect. The therapeutic dose of rAAV virus or pharmaceutical composition may vary depending on factors such as the patient's disease state, age, sex, and weight, and the ability of the rAAV virus or pharmaceutical composition to induce the desired response in the patient. The administration scheme may be adjusted to provide the optimal therapeutic response. The therapeutic dose is also generally the amount in which the therapeutically beneficial effect outweighs any toxic or adverse effects of the rAAV virus or pharmaceutical composition. "Preventive dose" refers to the amount effective in the dose and duration required to obtain the desired preventive effect, such as the prevention or suppression of various diseases. Preventive doses can be used in subjects before or in the early stages of disease, and in some cases, the preventive dose may be greater or less than the therapeutic dose. The dosage depends heavily on the patient's condition and size, the treatment prescription, the frequency of treatment, and the route of administration. Protocols for sustained treatment, including dose, formulation, and frequency, may be indicated by the initial response and clinical judgment.

[0100] In some embodiments, the rAAV virus or pharmaceutical composition is administered to a subject once daily, once weekly, once every two weeks, once monthly, once every two months, once every three months, once every six months, once annually, or once every two years, once every five years, or once in a lifetime.

[0101] Similar routes of administration and delivery include intravenous (IV), intra-articular, intraperitoneal (IP), intra-arterial, intramuscular, extra-gastrointestinal, subcutaneous, intrathoracic, cutaneous, percutaneous, extra-gastrointestinal, such as transmucosal, intracranial, intraspinal, oral (digestive system), mucosa, respiratory system, nasal cavity, intubation, intrapulmonary, intrapulmonary infusion, oral cavity, sublingual, intravascular, subarachnoid space, intracavitary, iontophoresis, intraocular, intraglandular, intraorgan, and intrafallopian tube.

[0102] In some embodiments, delivery of rAAV to a mammalian subject is achieved by affecting the eye of the mammal, for example, by intraocular injection, subretinal injection, choroidal injection (e.g., suprachorionic injection), or topical administration (e.g., eye drops), or by injection into the affected tissue (e.g., intravitreal injection). "Ocular tissue" means any tissue originating from or contained within the eye. Non-limiting examples of ocular tissue include neurons, retina (e.g., photoreceptor cells), sclera, choroid, retina, vitreous humor, macula, fovea, optic disc, lens, pupil, iris, aqueous humor, cornea, conjunctival 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 visual field. In some embodiments, the rAAV or compositions described in the present invention are administered by intraocular injection. In some embodiments, the rAAV or compositions described in the present invention are administered by intravitreal injection. In some embodiments, the rAAV or composition described in the present invention is administered by subretinal injection. In some embodiments, the rAAV or composition described in the present invention is administered by intrachoroidal (e.g., suprachoroidal) injection. In some embodiments, the rAAV or composition described in the present invention is administered by intravenous injection.

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

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

[0105] In some embodiments, rAAV delivery to a mammalian subject can be achieved by, for example, intra-articular injection. “Intra-articular injection” is defined here as an injection or infusion into a joint. Intra-articular injection is commonly used to administer a drug to a joint affected by inflammation.

[0106] The effects of administering rAAV or a pharmaceutical composition containing rAAV according to the present invention may include preventing disease progression, halting disease progression, or reversing disease progression. The disease or illness is related to the target gene loaded by rAAV and the gene product expressed. For example, if the gene product is a VEGF antagonist, the disease or illness being 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. Numerous studies have confirmed that abnormal excess VEGF can stimulate and induce pathological angiogenesis, potentially leading to angiogenesis-related eye diseases. Non-exclusive exemplary angiogenesis-related 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., hemangiomas, acoustic neuromas, neurofibromas, tracheitis, pyogenic granulomas), rheumatoid arthritis, psoriasis, erythema, Osier-Webber syndrome, myocardial angiogenesis, plaques, telangiectasia, hemophilic arthritis, or angiofibroma.

[0107] In some embodiments, neovascularization-related eye diseases include diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma and posterior lens fibrosis, epidemic keratoconjunctivitis, vitamin A deficiency, excessive contact lens wear, atopic keratitis, bacterial keratitis, ulcers, primary keratosis, rheumatoid arthritis, systemic lupus erythematosus, polyarteritis, trauma, Wegener's sarcoidosis, scleritis, Stevens-Johnson disease, radial keratotomy for pemphigus, and corneal graft rejection. This includes, but is not limited to, dysreactions, sickle cell anemia, sarcoidosis, pseudoxanthoma elasticum, Paget's disease, venous occlusion, arterial occlusion, carotid artery occlusion, chronic uveitis / vitritis, mycobacterial infections, Lyme disease, systemic lupus erythematosus, retinopathy of prematurity, Eales disease, Behçet's disease, retinitis or choroiditis infection, presumed ocular histoplasmosis, Bests disease, myopia, fovea, Stargardt's disease, squamous cell carcinoma, chronic retinal detachment, hyperviscosity syndrome, toxoplasmosis, and complications following trauma or laser treatment.

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

[0109] In some embodiments, the eye 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 retinal damage, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoid choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia, or retinopathy of prematurity. Specific forms of macular degeneration may include acute macular degeneration, non-exudative age-related macular degeneration, and non-exudative age-related macular degeneration.

[0110] For example, if the gene product is a TNF-α antagonist, the disease or illness being prevented or treated may be arthritis or related diseases. Currently, it is estimated that there are more than 100 different forms of arthritis. Generally, joint disorders are 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 preferred embodiments, arthritis diseases include: 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 patellarum, chronic fatigue syndrome, complex regional pain syndrome, cryopyrin-associated periodic syndromes (CAPS), intervertebral disc degeneration, developmental hip dysplasia, El Danlos disease, familial Mediterranean fever, fibromyalgia, disease V, giant cell arteritis, gout, hemochromatosis, infectious arthritis, inflammatory arthritis, inflammatory bowel disease, joint replacement, juvenile arthritis, juvenile dermatomyositis (JD), juvenile idiopathic arthritis (JIA), juvenile rheumatoid arthritis, juvenile scleroderma, Kawasaki disease, lupus, childhood and adolescence. The following conditions are selected: lupus, Lyme disease, mixed connective tissue disease, myositis (including polymyositis and dermatomyositis), osteoarthritis (OA), osteoporosis, pageats, 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, Sjögren's disease, spinal stenosis, spondyloarthritis, Still's disease, systemic juvenile idiopathic arthritis and systemic lupus erythematosus, systemic lupus erythematosus in children and adolescents, systemic sclerosis, temporal arteritis, tendinitis, vasculitis, and Wegener's granulomatosis.

[0111] In another preferred embodiment, the arthritis disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, gout, pseudogout, spondylitis, Crohn's disease, psoriasis plaque, 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 arthritis disease is a joint disorder relating to one or more joint inflammations. Preferably, the arthritis disease is selected from rheumatoid arthritis (RA), juvenile rheumatoid arthritis, osteoarthritis (OA), gout, pseudogout, spondyloarthritis (SpA), psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, and Still's disease. The present invention will be further described below with reference to examples, but these examples do not limit the present invention in any way.

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

[0114] Unless otherwise specified, the materials, reagents, etc. used in the following examples can be obtained commercially. Example 1: Construction of a mutant

[0115] Site-directed mutagenesis and recombinant DNA techniques were used to generate AAV8 variants, which consist of 5-14 amino acid polypeptide substitutions located at amino acids Q588-A592 (QQNTA) of the parent AAV8, or further include mutations at positions 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 include D80N, V125A, and V125G mutations. An exemplary No. 12 capsid mutant plasmid is shown in Figure 2.

[0116] [Table 1]

[0117] [Table 2]

[0118] [Table 3]

[0119] Another group of capsid protein mutants, as shown in Table 4, have polypeptide sequences inserted after the amino acid at position Q589 of the parent AAV8.

[0120] [Table 4]

[0121] The viral packaging of the AAV8 mutant was generated by simultaneously transfecting cells with three plasmids. The ITR consists of a first plasmid (pAAV-CAG-EGFP) adjacent to the expression cassette of the target gene (GFP or luciferase), a mutant plasmid (second plasmid) encoding the Rep / Cap gene constructed in Example 1, and a third plasmid containing the adenovirus helper function gene. After transfection, cells were collected, lysed to release the virus, and viral titers were measured by PCR for subsequent use. Example 2: Transfection experiment by injection into the mouse vitreous humor.

[0122] 2.1 EGFP mRNA expression levels of mutants Purified AAV8 mutant virus was injected into the vitreous humor of mice at a dose of 7E+8vg / eye. Twenty-one days after intravitreous injection, the mouse eyeballs were excised, pulverized using a freeze homogenizer, RNA was extracted using a total RNA extraction kit, cDNA was synthesized using a reverse transcription kit, and a specific EGFP primer probe set was determined by qPCR (where 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, with the sequence contained in the probe indicated by SEQ ID NO:28). The EGFP mRNA levels of normalized AAV8 mutants were calculated, i.e., the effects of various AAV8 mutants on vitreous transfection were compared, and the results are shown in Figure 3.

[0123] 2.2 Creation of a whole-mount retina Purified AAV8 mutant virus was injected into the vitreous humor of mice at a dose of 7E+8vg / eye. Twenty-one days after intravitreous injection, the mouse eyeballs were extracted and fixed overnight with 4% paraformaldehyde. The mouse retina was then detached using surgical microscope scissors under an upright microscope to create a whole-mount retina, and the EGFP fluorescence signal was confocally captured. The results are shown in Figure 4. Example 3 Transfection experiment by injection into rat vitreous humor

[0124] Purified AAV8 mutant virus was injected into the vitreous humor of rats at a dose of 1.92E+9vg / eye. Twenty-one days after intravitreous injection, the rat eyeballs were extracted. The rat eyeballs were fixed overnight with 4% paraformaldehyde. The rat retina was detached using surgical microscope scissors under an upright microscope. Whole-mount rat retinas were prepared, and the EGFP fluorescence signal was captured using a scanning fluorescence microscope; the results are shown in Figure 5. Example 4 Transfection experiment by injection into the suprachoroidal space of rabbits

[0125] Purified AAV8 mutant virus was injected into the epichoroidal space of New Zealand rabbits at a dose of 5E+10vg / eye. Fourteen days after epichoroidal injection, the eyeballs of the New Zealand rabbits were excised. The eyeballs of the New Zealand rabbits were fixed overnight with 4% paraformaldehyde. Dehydration and tissue embedding were performed on the fixed eyeballs. Frozen sections were prepared, followed by nuclear staining (DAPI). The entire image was scanned using a confocal microscope to capture EGFP fluorescence signals and DAPI signals, 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 fluorescence images, the wild type did not have high transduction efficiency into the photoreceptor layer and RPE layer, but the mutant showed 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 cell confluence reached 70-80% (typically 1.0E+5 cells). Viral supernatant (4.5E+9Vg) was added according to the required proportion until the total volume of viral supernatant + medium reached 1000 μL. After 16 hours of infection, the medium was changed daily, and culture was continued for 72 hours. EGFP fluorescence signals were captured confocally to observe the expression of the fluorescent protein. Figure 7 shows the infection status of ARPE19 cells after 48 hours.

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

[0128] Following the three-plasmid simultaneous transfection method described in Example 1, AAV-mCherry (mCherry is a protein isolated from coral and used here as a red fluorescent dye tracer) containing different mutants was prepared, and the genomic titer of each virus was measured by ddPCR. The genomic titer of each mutant virus was diluted to 1.0E12 vg / mL in buffer, and 100 μL was injected into the epichoroidal space 4 mm above the temporal region of the rabbit eye, with each virus being injected into two rabbits, or four eyes. After 5 days, the New Zealand rabbits were killed, the eyeballs were extracted, and the retinal / choroidal tissue was separated and subjected to cryogenic tissue homogenation. The supernatant was collected, and viral genomic DNA was extracted using a viral genomic DNA extraction kit. The relative content of transduction viral genomic DNA into 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 experiment of infected APRE cells

[0130] AAV8-afliberceptvirus and AAV No.12-aflibercept mutant viruses were produced by the simultaneous transfection of three plasmids described in Example 1, and the genomic titer of each virus was measured by ddPCR. The genomic titer of each mutant virus was diluted with buffer to 3.0E12 vg / mL, and the cell number was set to 3.0E5. 10 μL of virus was added to each well, and after incubation for 16 hours, the solution was changed, and after 72 hours, the cells and cell supernatant were collected. Cell lysis was performed by freezing in liquid nitrogen and repeating freeze-thaw cycles three times. The expression level of the Trap protein was measured by ELISA. The results showed that the protein expression level of AAV8-afliberceptvirus was 97 ng / ml, and the protein expression level of AAV No.12-afliberceptvirus was 1662 ng / ml. Example 8: Protein expression experiment in rhesus monkeys by epichoroidal injection

[0131] AAV No. 128-afliberceptvirus was produced by simultaneous transfection of three plasmids as described in Example 1, and the genomic titer of each virus was measured by ddPCR. The genomic titer of the virus was diluted with buffer to 1.0E13 vg / mL, and 100 μL was injected into each of the two eyes of one monkey, into the epichoroidal space 4 mm above the temporal region of the eye. After 56 days, the rhesus monkeys were killed, the eyeballs were extracted, and various tissues of the monkey eyes (including aqueous humor, choroid, conjunctiva, iris-ciliary body, retina, sclera, and vitreous humor) were isolated. After adding a protease inhibitor, cryogenic tissue homogenation was performed, the supernatant was collected, and the expression level of aflibercept protein (ng / ml) was measured by ELISA. 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 the simultaneous transfection of three plasmids described in Example 1, and the viral genome titers were measured by ddPCR. The viral genome titers were diluted with buffer to 1.62E11 vg / mL and injected into the gastrocnemius muscle of the right leg of mice in a volume of 50 μL, with each virus being injected into 5 mice (Balb / c). On days 7, 14, 28, and 160 after viral injection, a fixed amount of fluorescein sodium basic was injected into the peritoneal cavity of the mice, and in vivo imaging of the mice was performed. Fluorescence intensity analysis was performed using an in vivo imager, and the results are shown in Table 7 and Figure 10.

[0134] [Table 7] Example 10: Protein expression experiment of transmitted 293F cells

[0135] AAV8-No.128-etanercept and AAV No.128-adalimumab plasmids were constructed. A 2 μg plasmid was selected and transmitted to 293F cells as a single plasmid. After 72 hours, the protein expression level of the plasmid was detected by ELISA. The results showed that the expression level of the AAV No.128-adalimumab plasmid was 1010 ng / mL, and the expression level of the AAV No.128-etanercept plasmid was 2020 ng / mL. Example 11: Efficacy experiment of intra-articular / intramuscular injection in mice

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

[0137] 2) Modeling Initial modeling on day 0: A 6-week-old DBA / 1 male mouse was 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 placed in a film extruder to prepare approximately 1 ml of milky emulsion. 100 μl of the emulsion was subcutaneously injected into the tail base of each mouse. Examination revealed swelling at the tail base, no leakage of the emulsion, and no death of the mice, 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 into the tail base of each mouse.

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

[0139] 4) Observation of results From day 23 to day 2 of administration, the thickness of the soles of the feet was monitored using electronic calipers, and the clinical score for 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 measurement of foot sole thickness and the clinical score for arthritis are shown in Figures 11 and 12.

[0143] The embodiments described above represent only a few embodiments of the present invention, and while the description is more specific and detailed, this should not be understood as limiting the scope of the patent for the present invention. Furthermore, those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these fall within the scope of protection. Therefore, the scope of patent protection for the present invention should be based on the appended claims.

Claims

1. Compared to the wild-type parental AAV capsid protein, the modified capsid protein has 9 to 14 amino acid polypeptide substitutions, and compared to AAV viruses containing the corresponding wild-type parental AAV capsid protein, the AAV virus containing the modified capsid protein has improved retinal cell infectivity. The polypeptides are QQNTARGNRQ (SEQ ID NO: 2), RGNRQAAQQNTA (SEQ ID NO: 3), RGNRQQNTA (SEQ ID NO: 4), RGNRQQQNTA (SEQ ID NO: 5), RGNQQNTARQ (SEQ ID NO: 7), RGNQQPRPTSRQ (SEQ ID NO: 8), RGNRQAAQQPTPTS (SEQ ID NO: 9), or RGNRQQQPTPTS (SEQ ID NO: A modified adeno-associated virus (AAV) capsid protein comprising an amino acid sequence selected from 19), wherein the substitution is located at the amino acids between positions 588 and 592 of parent AAV8 or at the corresponding position of another serotype capsid protein.

2. The capsid protein according to claim 1, characterized in that the polypeptide comprises an amino acid sequence selected from QQNTARGNRQ (SEQ ID NO: 2), RGNRQAAQQNTA (SEQ ID NO: 3), RGNRQQNTA (SEQ ID NO: 4), RGNQQNTARQ (SEQ ID NO: 7), or RGNRQQQPTPTS (SEQ ID NO: 19).

3. The capsid protein according to claim 1, further comprising a mutation at the amino acids between positions 262 and 272 of the parent AAV8 capsid protein or at the corresponding position of another serotype capsid protein, wherein the mutated amino acid sequence is SQSGASNDNH (SEQ ID NO: 10).

4. The capsid protein according to claim 1, characterized in that the capsid protein comprises a polypeptide substitution at the amino acids between positions 588 and 592 of parent AAV8 or at the corresponding position of another serotype capsid protein, and a mutation at positions 262 to 272 of parent AAV8 capsid protein or at the corresponding position 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).

5. The capsid protein according to claim 1, 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 AAVrh10.

6. The capsid protein according to claim 5, characterized in that the AAV serotype is AAV8.

7. The capsid protein according to claim 1, further comprising mutations in the amino acids at the D80 and / or V125 positions of the parent AAV8 capsid protein.

8. The capsid protein according to claim 7, characterized in that the mutation is D80N and / or V125A.

9. The capsid protein according to claim 7, characterized in that the mutation is D80Q and / or V125G.

10. The capsid protein according to claim 7, wherein the capsid protein comprises a polypeptide substitution at the amino acids between positions 588 and 592 of parent AAV8 and mutations to the amino acids at positions D80 and V125 of parent AAV8 capsid protein, wherein the polypeptide is RGNQQNTARQ (SEQ ID NO: 7) and the mutations are D80N and V125A.

11. The capsid protein according to claim 7, wherein the capsid protein comprises a polypeptide substitution at the amino acids between positions 588 and 592 of parent AAV8 and mutations to the amino acids at positions D80 and V125 of parent AAV8 capsid protein, wherein the polypeptide is RGNQQNTARQ (SEQ ID NO: 7) and the mutations are D80Q and V125G.

12. A modified adeno-associated virus (AAV) capsid protein, The capsid protein comprises an insertion of a polypeptide at position 589 of the wild-type parent AAV8 or at a position later than the corresponding position of another serotype capsid protein, and the AAV virus containing the modified capsid protein has improved retinal cell infectivity compared to the AAV virus containing the corresponding wild-type parent AAV capsid protein. The polypeptide is characterized by comprising an amino acid sequence selected from RGDLTTPQQ (SEQ ID NO: 20), RGDLNTPQQ (SEQ ID NO: 21), and RGDVSSPQQ (SEQ ID NO: 22), thereby being a modified adeno-associated virus (AAV) capsid protein.

13. The capsid protein according to claim 12, 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 AAVrh10.

14. The capsid protein according to claim 13, characterized in that the AAV serotype is AAV8.

15. i. The modified capsid protein according to claim 1 or 12 above, ii. Recombinant adeno-associated virus (rAAV) containing heterologous nucleic acids containing encoded gene products.

16. The recombinant adeno-associated virus according to claim 15, characterized in that the gene product is a VEGF antagonist or a TNF-α antagonist.

17. The recombinant adeno-associated virus according to claim 16, characterized in that the VEGF antagonist is selected from aflibercept, convercept, ranibizumab, and brolucizumab.

18. The recombinant adeno-associated virus according to claim 17, characterized in that the VEGF antagonist is selected from aflibercept.

19. The recombinant adeno-associated virus according to claim 16, characterized in that the TNF-α antagonist is selected from etanercept, infliximab, adalimumab, pecerizumab, and golimumab.

20. The recombinant adeno-associated virus according to claim 19, characterized in that the TNF-α antagonist is selected from etanercept.

21. a) Recombinant adeno-associated virus according to any one of claims 15 to 20, b) A pharmaceutical composition comprising a pharmaceutically acceptable excipient.

22. Use of recombinant adeno-associated virus according to any one of claims 15 to 20 in the preparation of a drug for preventing or treating eye diseases.

23. The use according to claim 22, characterized in that the aforementioned eye disease is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, corneal neovascular eye disease, non-infectious uveitis, or glaucoma.

24. The use according to claim 22, characterized in that the aforementioned eye 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 retinal damage, proliferative diabetic retinopathy, diabetic retinal ischemia, polypoid choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia, or retinopathy of prematurity.

25. Use of recombinant adeno-associated virus according to any one of claims 15 to 20 in the preparation of a drug for preventing or treating arthritis or related diseases.

26. The use according to claim 25, wherein the arthritis disease or related disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, gout, pseudogout, spondylitis, Crohn's disease, macular psoriasis, psoriatic arthritis, ankylosing spondylitis, septic arthritis, arthritis, juvenile idiopathic arthritis, blunt trauma, joint replacement, or Still's disease.

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