Recombinant adeno-associated virus with capsid mutation and its application

Abstract

The present invention provides a recombinant adeno-associated virus having a capsid mutation and its application, specifically, a mutant adeno-associated virus (AAV) capsid protein, a recombinant adeno-associated virus (rAAV) virus particle containing the mutant AAV capsid protein, and its application for delivering a gene product to a target cell (e.g., a retinal cell).

Description

[Technical field]

[0001] This application claims priority to a Chinese patent application filed on May 28, 2021, bearing application number 202110594986.X.

[0002] The present disclosure relates to the field of recombinant adeno-associated virus (rAAV) technology, specifically to rAAV viral particles having capsid mutations and their application in delivering gene products to target cells (e.g., retinal cells). [Background technology]

[0003] Adeno-associated viruses (AAVs) are small (25 nm), non-enveloped, single-stranded DNA viruses in which nucleic acid is encapsulated in an icosahedral capsid (cap). AAVs contain two open reading frames: rep, which encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for genome replication, and cap, which encodes three structural proteins (VP1 to VP3) required for viral capsid assembly.

[0004] As a vector, AAV has shown great potential for the treatment of genetic and congenital diseases and has been used in gene replacement therapy (also called gene enhancement therapy). By complementing the missing gene function, it restores the loss or dysregulation of gene function due to mutation, restoring the biological function of the target cell to a normal physiological state. Expression of AAV-based vectors in preclinical disease models and human clinical trials shows the promise of several disease treatment applications, for example, studies have shown that efficient transfer and long-term, sustained expression of genes into retinal cells can be achieved (Boye et al. Mol Ther. 2013 Mar, 21(3):509-19. Trapani et al. Prog Retin Eye Res. 2014 Nov, 43:108-28.). AAV vectors currently in clinical use have been genetically engineered to exhibit a latent state without a helper virus, and their safety and long-term expression of transgenes have been extensively tested in rodent models, non-human primates, and multiple human studies (MacLaren et al. Lancet. 2014 Mar 29;383(9923):1129-37; Maguire et al. N Engl J Med. 2008 May 22;358(21):2240-8; Simonelli et al. Mol Ther. 2010 Mar;18(3):643-50; Nathwani et al. N Engl J Med. 2014 Nov 20;371(21):1994-2004).

[0005] AAV capsid proteins occur naturally with different compositions and structures, and different capsids have different tissue tropism. Several homologous primate and non-human primate AAV serotypes have been identified, and different engineered AAV variants (also called AAV serotypes) have been developed. AAV vectors have been shown to have a certain degree of diffuse infection in various organisms and tissues, but many clinical trials, especially in ocular gene therapy applications, use localized injection into the lesion area. The main ocular clinical embodiments at present are subretinal space injection (i.e., the cavity formed after injecting fluid between the RPE and photoreceptor cells) and vitreous space injection. Subretinal space injection allows AAV to fully contact the RPE and photoreceptor cells, and has a high local infection effect, but it is prone to high risks for injection manipulation and retinal dropout. In the case of vitreous space injection, the AAV preparation is first distributed uniformly in the vitreous humor, and then diffusely infects the retina layer by layer. Due to the dense structure and complex cell population composition of the retinal layer, native serotypes of AAV8 and AAV2, which have strong ocular penetration properties, are less effective when injected into the vitreous cavity; however, partially modified capsids (e.g., AAVDJ, AAV2.7M8, etc.) have been shown to have strong infectivity when injected into the vitreous cavity.

[0006] There is still a need for new AAV variants in the field. The present disclosure provides recombinant adeno-associated virus (rAAV) particles with a new capsid protein structure, high retinal tissue infection tropism, and high heterologous gene expression efficiency, providing a more potential method for clinical treatment. Summary of the Invention

[0007] The present disclosure provides mutant AAV capsid proteins and the gene products they may carry, rAAV viral particles comprising said capsid proteins, pharmaceutical compositions, cells (e.g., retinal cells) infected with said rAAV viral particles, methods for treating and preventing diseases (e.g., ocular diseases), and pharmaceutical uses.

[0008] Mutant adeno-associated virus (AAV) capsid proteins The present disclosure provides an adeno-associated virus (AAV) capsid protein comprising an inserted polypeptide relative to a parent AAV capsid protein, the inserted polypeptide being one of the following 1) to 6): 1) LAETTRP (SEQ ID NO: 11) or a polypeptide consisting of SEQ ID NO: 11; 2) LGDTTRP (SEQ ID NO: 12) or a polypeptide consisting of SEQ ID NO: 12; 3) LGETTRN (SEQ ID NO: 13) or a polypeptide consisting of SEQ ID NO: 13; 4) KADTTKN (SEQ ID NO: 14) or a polypeptide consisting of SEQ ID NO: 14; 5) KDDTTRN (SEQ ID NO: 15) or a polypeptide consisting of SEQ ID NO: 15; 6) LADTTKN (SEQ ID NO: 16) or a polypeptide consisting of SEQ ID NO: 16 The present invention includes a polypeptide as set forth in any one of the groups consisting of:

[0009] The present disclosure provides an adeno-associated virus (AAV) capsid protein comprising an inserted polypeptide compared to a parent AAV capsid protein, wherein the polypeptide comprises or consists of X1X2X3TTX4X5 (SEQ ID NO:35), wherein X1 is selected from L or K, X2 is selected from G, D or A, X3 is selected from D or E, X4 is selected from R or K, and X5 is selected from P or N.

[0010] In some embodiments, the polypeptide shown in 1) to 6) above or SEQ ID NO: 35 has 1 to 4 spacer amino acids (Y1 to Y4) at its amino terminus and / or carboxy terminus. In some specific embodiments, the spacer amino acids include, but are not limited to, A, L, G, S, and T.

[0011] The present disclosure provides an adeno-associated virus (AAV) capsid protein comprising an inserted polypeptide compared to a parent AAV capsid protein, the polypeptide comprising or consisting of Y1Y2X1X2X3TTX4X5Y3Y4 (SEQ ID NO:36), wherein X1 is selected from L or K, X2 is selected from G, D or A, X3 is selected from D or E, X4 is selected from R or K, and X5 is selected from P or N, and Y1, Y2, Y3, Y4 may independently be present or absent, and Y1, Y2, Y3, Y4 may independently be selected from A, L, G, S, and T. For example, Y1 is L, Y2 is A, Y3 is A, and Y4 is absent.

[0012] The present disclosure provides an adeno-associated virus (AAV) capsid protein comprising an inserted polypeptide compared to a parent AAV capsid protein, the inserted polypeptide being one of the following: 1-1) A polypeptide comprising LALAETTRPA (SEQ ID NO: 17) or consisting of SEQ ID NO: 17; 2-1) A polypeptide comprising LALGDTTRPA (SEQ ID NO: 18) or consisting of SEQ ID NO: 18; 3-1) A polypeptide comprising LALGET TRNA (SEQ ID NO: 19) or consisting of SEQ ID NO: 19; 4-1) A polypeptide comprising LAKADTTKNA (SEQ ID NO: 20) or consisting of SEQ ID NO: 20; 5-1) A polypeptide comprising LAKDDTTRNA (SEQ ID NO: 21) or consisting of SEQ ID NO: 21; 6-1) A polypeptide comprising LALADTTKNA (SEQ ID NO: 22) or consisting of SEQ ID NO: 22 The present invention includes a polypeptide as set forth in any one of the groups consisting of:

[0013] In some embodiments, the AAV capsid protein is an AAV2 capsid protein or an AAV9 capsid protein.

[0014] In some embodiments, the polypeptide of 1) to 6), 1-1) to 6-1), or SEQ ID NO: 35 or 36 is located in the GH ring or IV ring (cyclic domain IV) of the parent AAV capsid protein, for example in a solvent-accessible portion of the GH ring or IV ring of the AAV capsid protein (see van Vliet et al. (2006) Mol. Ther. 14:809, Padron et al. (2005) J. Virol. 79:5047, and Shen et al. (2007) Mol. Ther. 15:1955). "Parent AAV capsid protein" refers to a capsid protein of the same AAV serotype without the inserted polypeptide (including a wild-type AAV serotype or a mutant capsid protein thereof, such as the AAV2 capsid protein set forth in SEQ ID NO:1 or the AAV9 capsid protein set forth in SEQ ID NO:37 of the present disclosure, which may or may not have the V708I mutation).

[0015] In some embodiments, the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10 (including AAVrh10).

[0016] In some embodiments, the inserted polypeptide is located between amino acid residues 411 and 650, or between 432 and 640, or between 570 and 671, or between 570 and 614, or between 570 and 610, or between 580 and 600, or between 570 and 575, or between 575 and 580, or between 580 and 585, or between 585 and 590, or between 590 and 600, or between 600 and 614 of the parent AAV capsid protein. For example, it is located between amino acid residues 570 and 611 of a parent AAV2 capsid protein, between amino acid residues 571 and 612 of a parent AAV1 capsid protein, between amino acid residues 560 and 601 of a parent AAV5 capsid protein, between amino acid residues 571 and 612 of a parent AAV6 capsid protein, between amino acid residues 572 and 613 of a parent AAV7 capsid protein, between amino acid residues 573 and 614 of a parent AAV8 capsid protein, between amino acid residues 571 and 612 of a parent AAV9 capsid protein, or between amino acid residues 573 and 614 of a parent AAV10 (including AAVrh10) capsid protein.

[0017] In some embodiments, the inserted polypeptide is located between amino acid residues 587 and 588 of the parent AAV2 capsid protein, or between amino acid residues 588 and 589 of the parent AAV9 capsid protein, or at a corresponding position in the capsid protein of another parent AAV serotype. In some embodiments, the inserted polypeptide is located between amino acid residues 587 and 588 of the parent AAV2 capsid protein, or between amino acid residues 588 and 589 of the parent AAV9 capsid protein, or at a corresponding position in the capsid protein of another parent AAV serotype. The other serotype is selected from, for example, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV10 (including AAVrhlO). Sequences in different AAV serotypes corresponding to amino acids 570 to 611 of the capsid protein VP1 of AAV2 are known in the art (see, e.g., Figure 6 of WO2012145601A, and GenBank accession numbers AAV1 at NP_049542, AAV5 at AAD13756, AAV6 at AAB95459, AAV7 at YP_077178, AAV8 at YP_077180, AAV9 at AAS99264, and AAV10 at AAT46337). In some embodiments, the inserted polypeptide is located between amino acid residues 590 and 591 of a parent AAV1 capsid protein, between amino acid residues 575 and 576 of a parent AAV5 capsid protein, between amino acid residues 590 and 591 of a parent AAV6 capsid protein, between amino acid residues 589 and 590 of a parent AAV7 capsid protein, between amino acid residues 590 and 591 of a parent AAV8 capsid protein, or between amino acid residues 588 and 589 of a parent AAV10 (including AAVrh10) capsid protein.

[0018] The number of amino acid residues in the present disclosure is counted in natural numbers from the N-terminus of the amino acid sequence encoding the VP1 of the AAV capsid protein. For example, "the inserted polypeptide is located between the amino acid residues 587 and 588 of the AAV2 capsid protein" means that the polypeptide is located between the amino acid residues 587 and 588 of the amino acid sequence encoding the VP1 of the AAV2 capsid protein, corresponds to the amino acid residues 450 and 451 of the amino acid sequence encoding the VP2, and corresponds to the amino acid residues 385 and 386 of the amino acid sequence encoding the VP3.

[0019] In some embodiments, the inserted polypeptide is located between amino acid residues 450 and 460 of the parent AAV capsid protein, for example before or after amino acid residue 453 of parent AAV2, amino acid residue 454 of parent AAV1, amino acid residue 454 of parent AAV6, amino acid residue 456 of parent AAV7, amino acid residue 456 of parent AAV8, amino acid residue 454 of parent AAV9, or amino acid residue 456 of parent AAV10 (including AAVrhlO). The amino acid residues of the capsid proteins of different AAV serotypes and their correspondence as shown in Figure 17 of WO2012145601A are incorporated herein in their entirety.

[0020] In some embodiments, the present disclosure provides a mutant AAV capsid protein comprising the polypeptide of 1) to 6), 1-1) to 6-1), or SEQ ID NO: 35 or 36 above.

[0021] In some embodiments, the mutant AAV capsid proteins of the present disclosure further comprise point mutations (including substitutions, deletions and / or additions) of one or more amino acid residues.

[0022] In some embodiments, the point mutations in the amino acid residues are located at one or any combination of positions 1, 15, 34, 57, 66, 81, 101, 109, 144, 164, 176, 188, 196, 226, 236, 240, 250, 312, 363, 368, 449, 456, 463, 472, 484, 524, 535, 551, 593, 698, 708, 719, 721, and 735.

[0023] In some embodiments, the point mutations (substitutions) of the amino acid residues are 1L, 15P, 34A, 57D, 66K, 81Q, 101R, 109T, 144K or M, 164K, 176P, 188I, 196Y, 226E, 236V, 240T, 250S, 312K, 363L, 368H, 449D, 456K, 463Y, 472N, 484C, One selected from the group consisting of 524T, 535S, 551S, 593E, 698V, 708I, 719M, 721L and 735Q or any combination thereof, for example one or more of 312K, 449D, 472N, 551S, 698V, 735Q, 273F, 444F, 500F, 730F, 708I, for example 708I.

[0024] In some embodiments, the point mutations (substitutions) of the amino acid residues are selected from the group consisting of M1L, L15P, P34A, N57D, N66K, R81Q, Q101R, S109T, R144K, R144M, Q164K, T176P, L188I, S196Y, G226E, G236V, I240T, P250S, N312K, P363L, D368H, N449D, T456K, S463Y, D472N, R484C, A52 4T, P535S, N551S, A593E, I698V, V708I, V719M, S721L, L735Q, Y273F, Y444F, Y500F, Y730F or any combination thereof, for example, one or more of N312K, N449D, D472N, N551S, I698V, L735Q, Y273F, Y444F, Y500F, Y730F, V708I.

[0025] In some specific embodiments, the point mutations (substitutions) of the amino acid residues are 708I and / or 449D, or V708I and / or N449D. In some specific embodiments, the mutations (substitutions) of the amino acid residues are 273F, 444F, 500F and / or 730F, or Y273F, Y444F, Y500F and / or Y730F.

[0026] The point mutation is relative to the corresponding position in the corresponding parent AAV capsid protein, for example, relative to the corresponding position in the parent AAV2 capsid protein.

[0027] The capsid protein mutations in WO2012145601A, WO2017197355A, and WO2018022905A are incorporated in their entirety into this disclosure.

[0028] In some embodiments, the AAV capsid of the present disclosure is a chimeric capsid. For example, the capsid comprises a portion of a first AAV serotype AAV capsid and a portion of a second AAV serotype AAV capsid, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 (including AAVrhlO). For example, the AAV capsid may be AAV2G9, which comprises sequences from AAV2 and AAV9, and the entire AAV2G9 sequence of US20160017005 is incorporated herein.

[0029] In some embodiments, the mutant AAV capsid proteins of the present disclosure are isolated and / or purified.

[0030] In some embodiments, the disclosure provides a mutant AAV2 capsid protein that comprises a polypeptide in the capsid protein GH ring or IV ring (cyclic domain IV) compared to a corresponding parent AAV2 capsid protein (e.g., as set forth in SEQ ID NO:1), wherein the polypeptide is selected from 1)-6), 1-1)-6-1), SEQ ID NO:35 or 36. In some specific embodiments, the polypeptide is or comprises SEQ ID NO:12 or 18. In some specific embodiments, the polypeptide is located between amino acid residues 587 and 588 of VP1 of the parent AAV2 capsid protein.

[0031] In some embodiments, the disclosure provides a mutant AAV9 capsid protein that comprises a polypeptide in the capsid protein GH ring or IV ring (cyclic domain IV) compared to a corresponding parent AAV9 capsid protein (e.g., as set forth in SEQ ID NO: 37), wherein the polypeptide is selected from 1) to 6), 1-1) to 6-1), SEQ ID NO: 35 or 36. In some specific embodiments, the polypeptide is or comprises SEQ ID NO: 12 or 18. In some specific embodiments, the polypeptide is located between amino acid residues 588 and 589 of VP1 of the parent AAV9 capsid protein.

[0032] In some embodiments, the disclosure provides a mutant AAV capsid protein, the amino acid sequence of which is set forth in any one of SEQ ID NOs: 3-9, 23-27, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0033] In some embodiments, the mutant AAV capsid proteins provided by the present disclosure comprise: (a) increased infectivity of ocular tissues (e.g., retinal cells), particularly increased infectivity of retinal neuronal layers, compared to the infectivity of ocular tissues (e.g., retinal cells) of AAV viral particles containing the corresponding parental AAV capsid protein; (b) altered cellular tropism compared to the tropism of AAV viral particles containing the corresponding parental AAV capsid protein; (c) an increased ability to bind to and / or cross the inner limiting membrane (ILM) compared to AAV viral particles containing the corresponding parent AAV capsid protein; and / or (d) increased expression of the encapsulated or harbored gene product in ocular tissues (e.g., retinal tissue, aqueous humor, vitreous body) compared to AAV viral particles containing the corresponding parent AAV capsid protein; It has the following characteristics.

[0034] Recombinant adeno-associated virus (rAAV) virions The present disclosure provides a recombinant adeno-associated virus (rAAV) viral particle comprising: (a) comprising any of the mutant AAV capsid proteins of the present disclosure; (b) optionally, comprising a heterologous polynucleotide.

[0035] In some embodiments, the heterologous polynucleotide comprises a polynucleotide that expresses or encodes a gene product. In some embodiments, the gene product is heterologous to the AAV. In some embodiments, the gene product is heterologous or endogenous to the target cell. In some embodiments, the gene product is one or more (e.g., two, three, four).

[0036] In some embodiments, the heterologous polynucleotide comprises a regulatory sequence that regulates expression or coding of a gene product.

[0037] Regarding gene products: In some embodiments, the gene product is therapeutic or prophylactic for a disease, condition.

[0038] In some embodiments, the gene product is selected from an interfering RNA (RNAi), an aptamer, and a polypeptide.

[0039] In some specific embodiments, the gene product is an RNAi, for example, the RNAi that reduces or decreases the level of apoptotic or angiogenic factors in cells.For example, the RNAi can be an shRNA or siRNA that reduces the level of gene products that induce or promote apoptosis in cells, and the gene products that promote apoptosis include gene products such as Bax, Bid, Bak and Bad (see US 7,846,730, which is incorporated herein in its entirety). As another example, RNAi can be directed against an angiogenic agent such as VEGF (e.g., Cand5, see US2011 / 0143400, US2008 / 0188437, all of which are incorporated herein), VEGFR1 (e.g., Sirna-027, see Kaiser et al. (2010) Am. J. Ophthalmol. 150:33, and Shen et al. (2006) Gene Ther. 13:225, all of which are incorporated herein), or VEGFR2 (see, e.g., Kou et al. (2005) Biochem. 44:15064, all of which are incorporated herein).

[0040] In some specific embodiments, the gene product is an aptamer, such as a specific aptamer for VEGF (e.g., 5'-cgcaaucagugaaugcuuauacauccg-3', see Ng et al. (2006) Nat. Rev. Drug Discovery 5:123, and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:18902, all of which are incorporated herein), a specific aptamer for PDGF (e.g., E10030, see Ni and Hui (2009) Ophthalmologica 223:401, and Akiyama et al. (2006) J. Cell Physiol. 207:407, all of which are incorporated herein).

[0041] In some specific embodiments, the gene product is a polypeptide.

[0042] In some specific embodiments, the gene product is a neuroprotective polypeptide, an anti-angiogenic polypeptide, or a polypeptide that enhances retinal cell function.

[0043] In some specific embodiments, the polypeptide can enhance the function of a retinal cell, for example, a rod or cone photoreceptor cell, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell.

[0044] In some specific embodiments, the polypeptide comprises or is selected from neuroprotective polypeptides (e.g., GDNF, CNTF, NT4, NGF, and NTN), anti-angiogenic polypeptides (e.g., soluble vascular endothelial growth factor (VEGF) receptors, anti-VEGF antibodies or antigen-binding fragments thereof, endostatin, tumstatin, angiostatin, soluble Flt polypeptides and fusion proteins with Fc regions (see Lai et al. (2005) Mol. Ther. 12:659; Pechan et al. (2009) Gene Ther. 16:10), pigment epithelium morphogenetic factor (PEDF), soluble Tie-2 receptor, and the like), tissue inhibitor of metalloproteinase-3 (TIMP-3), light-responsive opsins (e.g., rhodopsin), anti-apoptotic polypeptides (e.g., Bcl-2, Bcl-Xl), and the like.

[0045] In some specific embodiments, the polypeptides include, but are not limited to, epidermal growth factor, rhodopsin, X-linked inhibitor of apoptosis protein.

[0046] In some specific embodiments, the polypeptides include, but are not limited to, retinoschisin, retinitis pigmentosa GTPase modulator (RGPR) interacting protein-1 (GenBank Accession Nos. Q96KN7, Q9EPQ2, Q9GLM3), peripheral protein-2 (Prph2) (GenBank Accession No. NP_000313), and retinal pigment epithelium specific protein (RPE65) (GenBank Accession No. AAC39660).

[0047] In some specific embodiments, the polypeptide is a polypeptide that, when deficient or deleted, causes achoroidia, such as CHM (choroidermia (Rab escort protein 1)) (Donnelly et al. (1994) Hum. Mol. Genet. 3:1017); a polypeptide that, when deficient or deleted, causes Leber congenital amaurosis, such as debris homolog 1 (CRB1) (GenBank Accession NO. CAM23328) and a polypeptide that causes retinitis pigmentosa; a polypeptide that, when deficient or deleted, causes rod photoreceptor cGMP-gated channel subunit alpha (CNGA3) (GenBank Accession NO. Polypeptides that cause color blindness include, but are not limited to, polypeptides that cause color blindness, such as rod photoreceptor cGMP-gated channel beta subunit (CNGB3), guanine nucleotide binding protein (G protein), alpha transduction active polypeptide 2 (GNAT2) (ACHM4), ACHM5, L-opsin, M-opsin, and S-opsin.

[0048] In some specific embodiments, the gene product provides a site-specific endonuclease for knocking down the site specificity of gene function, for example, the endonuclease knocks out an allele associated with a retinal disease. For example, if a dominant allele encodes a defective copy of a gene that provides a retinal structural protein and / or normal retinal function when it belongs to the wild type, the site-specific endonuclease can target the defective allele and knock out the defective allele. The site-specific endonuclease can be, for example, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), where such site-specific endonucleases are not naturally occurring and are modified to target a specific gene.

[0049] In addition, the sequences and origins of the above gene products in WO2012145601A, WO2017197355A, and WO2018022905A are incorporated in their entirety by this disclosure.

[0050] In some specific embodiments, the gene products of the disclosure are anti-angiogenic agents, including anti-angiogenic polypeptides such as anti-VEGF antibodies or antigen-binding fragments thereof, and, as another example, VEGF antagonists (e.g., VEGF-A, B, C antagonists) or PDGF antagonists.

[0051] In some specific embodiments, the VEGF antagonist is selected from the group consisting of ranibizumab, bevacizumab, aflibercept, KH902 VEGF receptor-Fc fusion protein, 2C3 antibody, ORA102, pegaptanib sodium, bevasiranib, SIRNA-027, decursin, decursinol, picropodophyllin, guggulsterone, PLG101, eicosanoid LXA4, PTK787, pazopanib, axitinib, CDDO-Me, CDDO-Imm, The VEGF antagonists include, but are not limited to, shikonin, beta-hydroxyisovalerylshikonin, or ganglioside GM3, DC101 antibody, Mab25 antibody, Mab73 antibody, 4A5 antibody, 4E10 antibody, 5F12 antibody, VA01 antibody, BL2 antibody, VEGF-related protein, sFLT01, sFLT02, peptide B3, TG100801, sorafenib, sunitnab, G6-31 antibody, or a pharma- ceutically acceptable salt thereof. The VEGF antagonist information in WO2018160686A is incorporated herein in its entirety.

[0052] For sequence information of ranibizumab (Lucentis®), see US 7,060,269 (Figure 1); for sequence information of bevacizumab (Avastin®), see US 6,054,297 (Figure 1); for sequence information of aflibercept (Eyelea®), see Do et al. (Br J Ophthalmol. 2009, 93:144-9), herein incorporated by reference in their entireties.

[0053] In some embodiments, the VEGF antagonist is or includes the naturally occurring protein sFlt-1 or a functional fragment thereof (e.g., sFlt-1 domain 2, see sFlt-1 sequence information in US 5,861,484, sFlt-1 domain 2 sequence information in US 2013 / 0323302, all of which are incorporated herein).

[0054] In some embodiments, the VEGF antagonist is a VEGF-binding fusion protein, the entire sequence information for the VEGF-binding fusion protein of US7,635,474 is incorporated herein.

[0055] In some specific embodiments, the amino acid sequence of aflibercept is set forth in SEQ ID NO: 38, and a polynucleotide sequence encoding SEQ ID NO: 38, e.g., a codon-optimized polynucleotide sequence, is provided and set forth in any one of SEQ ID NOs: 39-41.

[0056] Regarding regulatory sequences: In some embodiments, the polynucleotide comprises one of the following: (a) a 5' inverted repeat sequence (5'ITR) and / or a 3' inverted repeat sequence (3'ITR); (b) a 5' untranslated region (5'UTR) and / or a 3' untranslated region (3'UTR); (c) a promoter; (d) enhancers, (e) intron, (f) post-transcriptional regulatory elements; (g) polyadenylation signal (polyA), (h) Kozak sequence The present invention includes any one of the polynucleotides (regulatory sequences) described above or any combination thereof.

[0057] In some embodiments, the AAV ITRs need not have a wild-type nucleotide sequence, but may be altered by the insertion, deletion or substitution of nucleotides, or the AAV ITRs may be derived from any one of several AAV serotypes, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. In some specific embodiments, the 5' and 3' ITRs are the 5' and 3' ITRs of AAV2.

[0058] In some embodiments, the nucleotide sequence encoding the gene product is operably linked to a tissue-specific or cell type-specific regulatory element, for example, a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), or, as another example, a regulatory element that confers selective expression of the operably linked gene in photoreceptor cells.

[0059] In some specific embodiments, any combination of (a)-(h) can satisfy a function of gene product (e.g., an anti-angiogenic agent (or anti-angiogenic polypeptide), in another example, an anti-VEGF antibody or antigen-binding fragment thereof, aflibercept) expression, e.g., in an ocular tissue (e.g., aqueous humor, retinal tissue) of a subject.

[0060] In some specific embodiments, any one or any combination of the polynucleotides of (a)-(h) above is operably linked to a polynucleotide encoding the gene product.

[0061] In some specific embodiments, the 5'ITR and / or 3'ITR are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ or AAV-DJ8, e.g., AAV2, AAV9.

[0062] In some specific embodiments, the 5'UTR and / or 3'UTR are derived from hemopexin (HPX), hemoglobin subunit beta (HBB), HSPB1, CCL13, Xenopus globin, etc. For example, the sequence of the 5'UTR is shown in SEQ ID NO:30, and the sequence of the 3'UTR is shown in SEQ ID NO:32.

[0063] In some specific embodiments, the promoter may be a constitutive promoter or an inducible promoter.

[0064] In some specific embodiments, the promoter is selected from a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, a UB6 promoter, a chicken β-actin promoter, a CAG promoter, an RPE65 promoter, a CBh promoter, an EFS promoter, an EF1 (e.g., EF-1α) promoter, a PGK promoter, an SV40 promoter, a Ubi promoter, an opsin promoter, or any combination thereof. The opsin promoters include, but are not limited to, rhodopsin promoter, rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076), beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015), retinitis pigmentosa gene promoter (Nicoud et al. (2007) as above), photoreceptor retinoic acid binding protein (IRBP) gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225). For example, the promoter is a CMV promoter, the sequence of which is shown, for example, in SEQ ID NO:29.

[0065] In some specific embodiments, the enhancer is selected from Ubi, CMV, RSV, IRBP gene enhancers (Nicoud et al. (2007) J. Gene Med. 9:1015), or any combination thereof. For example, the enhancer is a CMV enhancer, the sequence of which is shown, for example, in SEQ ID NO: 28.

[0066] In some specific embodiments, the intron is selected from MVM, SV40, β Globin, EF1 (eg, EF-1α), a hybrid intron, or any combination thereof.

[0067] In some specific embodiments, the polyA is selected from PA75 polyA, SV40 polyA, hGH polyA, BGH polyA, rbGlob polyA, or any combination thereof. For example, the polyA is SV40 polyA, the sequence of which is shown, for example, in SEQ ID NO:34.

[0068] In some specific embodiments, the post-transcriptional regulatory element is selected from WPRE, HPRE, or a combination thereof. For example, the post-transcriptional regulatory element is WPRE, the sequence of which is shown, for example, in SEQ ID NO:33.

[0069] In some embodiments, the polynucleotide comprises one of the following: (a) the 5'ITR and / or the 3'ITR derived from AAV2; (b) 5'UTR and / or 3'UTR derived from Xenopus globin; (c) CMV promoter, (d) CMV enhancer, (e)WPRE, (f) Kozak sequence, (g) SV40 polyA The present invention includes any one of the polynucleotides (regulatory sequences) described above or any combination thereof.

[0070] Illustratively, the polynucleotide has, in order from the 5' to 3' end, the following (with optional regulatory sequences in parentheses):

[0071] 5'ITR-enhancer-promoter-5'UTR-(Kozak sequence)-gene product-3'UTR-(WPRE)-polyA-3'ITR, or Enhancer-promoter-5'UTR-(Kozak sequence)-gene product-3'UTR-(WPRE)-polyA, Promoter-(5'UTR)-(Kozak sequence)-gene product-(3'UTR)~(WPRE)-polyA.

[0072] Efficacy of rAAV viral particles: In some embodiments, the rAAV viral particles are infectious, while in other embodiments, the rAAV viral particles are non-infectious.

[0073] In some embodiments, the rAAV viral particles of the present disclosure exhibit enhanced infectivity of cells by at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold or more compared to the infectivity of a cell of an AAV viral particle comprising a corresponding parent AAV capsid protein, and the cell is 1) retinal cells, 2) photoreceptor cells, 3) RPE cells, 4) bipolar cells, 5) amacrine cells, and / or 6) horizontal cell Selected from.

[0074] In some embodiments, the rAAV viral particles of the present disclosure exhibit at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or more than 50-fold enhanced ability to cross the inner limiting membrane (ILM) compared to the ability of an AAV viral particle comprising the corresponding parent AAV capsid protein to cross the ILM.

[0075] In some embodiments, the rAAV viral particles of the present disclosure selectively infect retinal cells, for example, the rAAV viral particles of the present disclosure specifically infect retinal cells 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, or more than 50-fold more strongly than non-retinal cells (e.g., non-ocular tissue cells).

[0076] In some embodiments, compared to rAAV viral particles comprising AAV2.7m8 capsid protein, the rAAV viral particles of the present disclosure (e.g., rAAV viral particles in which the capsid protein comprises seq2) enhance expression of a gene product (e.g., aflibercept) in whole eye tissue by 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold or more than 50-fold.

[0077] In some embodiments, compared to rAAV viral particles comprising AAV2.7m8 capsid protein, the rAAV viral particles of the present disclosure (e.g., rAAV viral particles in which the capsid protein comprises seq2) enhance the expression of a gene product (e.g., aflibercept) in aqueous humor by 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold or more than 50-fold.

[0078] In some embodiments, compared to rAAV viral particles comprising AAV2.7m8 capsid protein, the rAAV viral particles of the present disclosure (e.g., rAAV viral particles in which the capsid protein comprises seq2) enhance the expression of a gene product (e.g., aflibercept) in the vitreous by 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold or more than 50-fold.

[0079] In some embodiments, compared to rAAV viral particles comprising AAV2.7m8 capsid protein, the rAAV viral particles of the present disclosure (e.g., rAAV viral particles in which the capsid protein comprises seq2) enhance expression of a gene product (e.g., aflibercept) in retinal tissue by 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 50-fold or more than 50-fold.

[0080] In some embodiments, the method of administration of the rAAV viral particles is intravitreal injection.

[0081] Polynucleotides and (Expression) Vectors The present disclosure provides a polynucleotide encoding aflibercept, the encoded amino acid sequence of which is set forth in SEQ ID NO:38.

[0082] In some embodiments, the present disclosure provides a codon-optimized polynucleotide encoding aflibercept, set forth in any one of SEQ ID NOs: 39-41, or having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.

[0083] The present disclosure provides polynucleotides encoding any of the above gene products of the present disclosure.

[0084] The present disclosure provides a polynucleotide encoding any of the above AAV capsid proteins of the present disclosure.

[0085] In some embodiments, the polynucleotide may be RNA, DNA, or cDNA.

[0086] In some embodiments, the polynucleotide is an isolated polynucleotide.

[0087] The polynucleotides of the present disclosure may be in the form of a vector, present in and / or part of a vector, which may be, for example, a plasmid, Costello plasmid, YAC, or viral vector. The vector may be, for example, an expression vector, i.e., a vector capable of providing expression of the polynucleotide in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Such expression vectors typically contain at least one polynucleotide of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.).

[0088] Polynucleotides of the present disclosure may be produced or obtained by known methods (eg, automated DNA synthesis and / or recombinant DNA techniques) and / or may be isolated from a suitable natural source.

[0089] The disclosure provides a vector comprising:

[0090] (a) an isolated polynucleotide encoding any of the above mutant AAV capsid proteins of the present disclosure (set forth in any one of SEQ ID NOs: 3-9 and 23-27, or having at least 90% or 95% sequence identity thereto); (b) an isolated polynucleotide encoding any of the mutant AAV capsid proteins of the present disclosure (set forth in any one of SEQ ID NOs: 3-9 and 23-27, or having at least 90% or 95% sequence identity thereto), and / or a heterologous polynucleotide encoding the gene product (a polynucleotide encoding aflibercept set forth in SEQ ID NO: 38, a polynucleotide set forth in any one of SEQ ID NOs: 39-41, or having at least 90% or 95% sequence identity thereto).

[0091] In some embodiments, the polynucleotide encoding the mutant AAV capsid protein and the heterologous polynucleotide encoding a gene product in (b) are present on different vectors.

[0092] host cell The present disclosure provides a host cell comprising any of the above polynucleotides or (expression) vectors of the present disclosure.

[0093] In some embodiments, the host cell may be an isolated cell, such as a cell derived from an in vitro cell culture, that is used to produce any of the rAAV capsid proteins, gene products, or rAAV viral particles of the present disclosure, also referred to as a producer cell.

[0094] In some specific embodiments, the production cells are bacterial cells, fungal cells or mammalian cells. Exemplary production cells include, but are not limited to, HeLa, CHO, 293 (including 293T), Vero, NIH 3T3, Huh-7, BHK, PC12, COS (including COS-7), RAT1, HepG2 cells, and the like. Exemplary mammalian cells include, but are not limited to, 293 (293T), COS, HeLa, Vero, 3T3, C3H10T1 / 2, CHO cells. Production cells may be amphibian cells, insect cells, plant cells, and any other cells used in the art for expressing proteins, viral particles. Exemplary insect cells include, but are not limited to, mosquito cell lines such as Fallopia moth, Drosophila cell lines, or Aedesalbopictus derived cell lines, including, but not limited to, Se301, SeIZD2109, SeUCR1, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAm1, Ha2302, Hz2E5, HighFive (Invitrogen, CA, USA), AO38, and BM-N cells.

[0095] Method for producing rAAV virus particles Methods for producing rAAV viral particles are common in the art. The entirety of the methods for producing and manufacturing rAAV viral particles in WO200028004, WO200123001, WO2004112727, WO2005005610, WO2005072364, WO2013123503, WO2015191508 and US20130195801 are incorporated herein by reference. The rAAV viral particles have properties that improve delivery efficiency, can be efficiently packaged, and can successfully infect target cells (e.g., mammalian or human cells) with high frequency and minimal toxicity.

[0096] The present disclosure provides methods for producing and manufacturing rAAV viral particles comprising packaging any of the polynucleotides of the present disclosure into an AAV capsid.

[0097] In some embodiments, a method for producing and manufacturing rAAV viral particles is provided, comprising introducing a polynucleotide encoding the above-described gene product of the present disclosure or an (expression) vector thereof, a polynucleotide encoding any of the above-described AAV capsid proteins of the present disclosure or an (expression) vector thereof, and a helper function plasmid (e.g., pHelper) into a producer cell (e.g., 293 cell), packaging, and purifying the resulting rAAV viral particles.

[0098] In some embodiments, a method for producing and manufacturing rAAV viral particles is provided, comprising: 1) a polynucleotide or (expression) vector encoding any of the gene products of the present disclosure, a vector expressing Rep and Cap genes comprising a polynucleotide encoding any of the AAV capsid proteins described above of the present disclosure (e.g., pR2C9), and a helper vector (one that provides helper function, e.g., pHelper) are simultaneously co-transfected into mammalian cells (e.g., 293 cells); 2) A method for obtaining and purifying rAAV viral particles containing a polynucleotide encoding a gene product.

[0099] In some embodiments, a system for producing rAAV viral particles for producing any of the rAAV viral particles of the present disclosure is provided, 1) a polynucleotide encoding an AAV capsid protein; and 2) a heterologous polynucleotide encoding any gene product of the present disclosure or its (expression) vector (e.g., a pGOI plasmid); 3) helper elements having sufficient AAV rep and helper functions to package a heterologous polynucleotide encoding a gene product in 2) into an AAV capsid.

[0100] In some specific embodiments, sufficient AAV rep and helper functions are provided by a packaging cell or three plasmids, pHelper, pR2C9, and pGOI, which may comprise the three plasmids pHelper, pR2C9, and pGOI.

[0101] In some embodiments, the Rep gene encodes a nonstructural protein that regulates functions such as replication of the AAV genome and may be selected from Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are typically transcribed from the p5 promoter, while Rep52 and Rep40 are typically transcribed from the p19 promoter. The Cap gene encodes the structural proteins VP1, VP2, and / or VP3 that assemble to form the viral capsid. The Cap gene is typically transcribed from the p40 promoter. In some embodiments, rAAV viral particles produced by the above-described AAV production method or production system are provided.

[0102] Pharmaceutical Compositions The present disclosure relates to (a) any of the AAV capsid proteins or rAAV viral particles of the present disclosure; (b) one or more pharma- ceutically acceptable vectors, diluents, excipients, or buffers.

[0103] The present disclosure relates to (a) a prophylactically or therapeutically effective amount of an active ingredient; (b) one or more pharma- ceutically acceptable vectors, diluents, excipients, or buffers.

[0104] The active ingredient is selected, for example, from any of the rAAV viral particles of the present disclosure, SEQ ID NOs: 39-41, or a polynucleotide encoding a gene product having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0105] In some embodiments, the unit dose of the pharmaceutical composition may contain 0.01% to 99% by weight of a polynucleotide (e.g., a polynucleotide encoding a VEGF inhibitor, as another example, aflibercept) or rAAV virus particles. In some specific embodiments, the copy amount of a gene product (e.g., a VEGF inhibitor, as another example, aflibercept) contained in the unit dose of the pharmaceutical composition is 0.1 to 10 × 10 13 In some specific embodiments, the concentration of rAAV viral particles in the pharmaceutical composition is 1×10 8 / mL or more, typically 1×10 15 cells / mL or less.

[0106] In some embodiments, any of the rAAV viral particles of the present disclosure can be transfected into cells, which can then be transferred or transplanted into a subject.

[0107] In some embodiments, a pharmaceutical composition comprises any of the rAAV viral particles of the present disclosure, wherein a polynucleotide is encapsulated in the rAAV viral particle, and the polynucleotide has a sequence set forth in any one of SEQ ID NOs: 39-41 or has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity thereto.

[0108] Methods for treating and preventing diseases and pharmaceutical uses The present disclosure provides methods or uses of any of the above AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure for the treatment, amelioration, or amelioration of a disease or condition.

[0109] In some embodiments, a method of delivering a gene product to a subject in need thereof is provided, comprising administering to the subject an effective amount of any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, or polynucleotides encoding aflibercept of the present disclosure.

[0110] In some embodiments, a method for delivering a gene product to a target cell is provided, comprising contacting the target cell with any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure. In some specific embodiments, the target cell is selected from hepatocytes, pancreatic cells, skeletal muscle cells, cardiac muscle cells, fibroblasts, retinal cells, synovial joint cells, lung cells, T cells, neurons, glial cells, stem cells, endothelial cells, and cancer cells. In some specific embodiments, the target cell is outside the body, and in some other specific embodiments, the target cell is inside the body. At the same time, a pharmaceutical use is provided for delivering heterologous nucleic acid to a target cell using any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure.

[0111] In some embodiments, a method for specifically infecting retinal cells is provided, comprising administering a prophylactically or therapeutically effective amount of any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure into the eye, for example by intravitreal injection or subretinal injection. At the same time, a pharmaceutical use is provided in the manufacture of a medicament for specifically infecting retinal cells using any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure.

[0112] In some specific embodiments, there is provided any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure for treating a disease or condition of a retinal cell selected from the group consisting of a photoreceptor cell, a retinal ganglion cell, a Muller cell, a bipolar cell, an amacrine cell, a horizontal cell, or a retinal pigment epithelial cell. In some cases, the retinal cell is a photoreceptor cell, such as a rod cell or a cone cell.

[0113] In some specific embodiments, the present invention is directed to a retinal condition such as acute macular neuroretinal pathology, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, acute macular degeneration, macular degeneration such as nonexudative age-related macular degeneration and exudative age-related macular degeneration, edema such as macular edema, macular cystoid edema and diabetic macular edema, multifocal choroiditis, ocular trauma affecting a site or location at the back of the eye, ocular tumors, retinal pathologies such as central retinal vein occlusion, diabetic retinal pathology (including proliferative diabetic retinal pathology), proliferative vitreous retinal disease (PVR), retinal artery occlusive disease, retinal detachment and uveitic retinal disease, sympathetic ophthalmia, Vogt-Koyanagi-Harada disease (Vogt Koyanagi-Harada (VKH) syndrome, uveal diffusion, posterior ocular pathologies caused or affected by ophthalmic laser therapy, posterior ocular pathologies caused or affected by photodynamic therapy, photocoagulation, radioactive retinal pathology, preretinal membrane pathology, branch retinal vein occlusion, anterior ischemic optic nerve pathology, non-retinal pathology, diabetic retinal dysfunction, retinal detachment, retinitis pigmentosa, glaucoma, Usher syndrome, cone-rod malnutrition, Stargardt disease (fundus maculopathy), hereditary macular edema The present invention provides any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure for treating a disease or condition of retinal cells selected from the group consisting of retinal degeneration, choroidal retinal degeneration, Leber's congenital amaurosis, congenital stationary night blindness, choroideremia, Bardet-Biedl syndrome, macular telangiectasia, Leber's hereditary optic nerve pathology, retinal pathology of prematurity, and color vision conditions including total acanthosis nigricans, protanopia, deuteranopia, and tritanopia, and hereditary retinitis pigmentosa.

[0114] In some embodiments, the present disclosure provides a method or use of any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept for the treatment of ophthalmic diseases, including, but not limited to, age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization, diabetic retinal lesions, proliferative diabetic retinal lesions, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinal lesions, or diabetic retinal edema, optionally by intravitreal or subretinal injection.

[0115] In some embodiments, the present disclosure provides a method or use of treating a retina-related disease using any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept.

[0116] Also provided are pharmaceutical uses of any of the above AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure in the manufacture of a medicament for treating or preventing the above diseases.

[0117] Method of administration The present disclosure provides methods of administration of any of the AAV capsid proteins, rAAV viral particles, pharmaceutical compositions, and polynucleotides encoding aflibercept of the present disclosure as a treatment for the above-mentioned ocular diseases (e.g., retina-related diseases), wherein the method is administered by intraocular injection.

[0118] In some embodiments, intraocular injection includes intravitreal injection, subretinal injection, suprachoroidal injection, or any other suitable form or route of administration that results in delivery of rAAV viral particles to the eye, including, but not limited to, intravenous, intraarterial, periocular, intracameral, subconjunctival, and subcapsular injection, as well as topical and intranasal administration. [Brief description of the drawings]

[0119] [Figure 1] Schematic diagram of the AAV2 capsid protein sequence and the initiation sites of VP1, VP2, and VP3, and circular domains I to V. [Diagram 2] FIG. 1 is a schematic diagram of a mutant AAV capsid protein of the present disclosure having a 708 site mutation and a peptide fragment inserted between positions 587 and 588. [Diagram 3] This figure shows the results of a fluorescent signal inspection of the rAAV vector viruses AAV2 seq1, AAV2 seq2, AAV2 seq3, AAV2 seq4, AAV2 seq5, AAV2 seq6, and AAV2 and AAV2.7m8 of the present disclosure infected into mouse retina. [Figure 4A] and [Figure 4B] FIG. 4 shows the results of fluorescent signal examination of the rAAV vector viruses AAV2 seq1, AAV2 seq2, AAV2 seq3, AAV2 seq4, AAV9 seq2, and AAV2 and AAV2.7m8 of the present disclosure infected into mouse retina, where FIG. 4A shows the fundus examination results after 1 week, and FIG. 4B shows the fundus examination results after 4 weeks. [Figure 5A] FIG. 1 shows a vector configuration diagram containing a nucleic acid molecule expressing VEGF Trap (aflibercept). [Figure 5B] FIG. 2 is a schematic diagram of an AAV packaging plasmid. [Figure 6] These are the results of testing the rAAV vector viruses AAV2 seq2 and AAV2.7m8 that express the target gene (aflibercept) in mouse eyes. [Figure 7]Test results of rAAV vector viruses AAV2 seq2 and AAV2.7m8 expressing a target gene (aflibercept) protein in rabbit eyes, which is detection of target gene protein content in aqueous humor 14 days after virus injection. [Figure 8] Test results of rAAV vector viruses AAV2 seq2 and AAV2.7m8 expressing a target gene protein in rabbit eyes, which is the detection of molar concentrations of the target gene protein in the aqueous humor 28 days after virus injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0120] In order that the present disclosure may be more readily understood, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0121] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. Unless otherwise specified, the term encompasses all AAV isoforms and naturally occurring and recombinant forms. "AAV" includes, but is not limited to, AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV10), AAV rh10 (AAVrh10), and various types of avian, bovine, canine, equine, primate, non-primate, and ovine AAV.

[0122] AAV is a nonpathogenic parvovirus composed of a 4.7 kb single-stranded DNA genome contained in a nonenveloped icosahedral capsid. The genome contains three open reading frames (ORFs) flanked by inverted terminal repeats (ITRs) that function as origins of viral replication and packaging signals. The Rep ORFs encode four nonstructural proteins that function in viral replication, transcriptional regulation, site-specific integration, and viral particle assembly. The Cap ORF encodes three structural proteins (VP1-3) that assemble to form the 60-mer viral capsid. Finally, an ORF present as an alternative reading frame within the cap gene produces the assembly activating protein (AAP), a viral protein that localizes AAV capsid proteins in the cell nucleus and functions during capsid assembly.

[0123] The genomic sequences and natural inverted terminal repeats (ITRs), Rep protein sequences and capsid subunit sequences of various AAV serotypes are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. For example, GenBank accession numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3), NC_001729.1 (AAV3), NC_001829.1 (AAV4), U89790. 1 (AAV4), NC_006152.1 (AAV5), AF085716.1 (AAV5), AF028704.1 (AAV6), NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC_006261.1 (AAV8), AY530579.1 (AAV9), and Srivistava et al. (1983) J. Virology 45:555, Chiorini et al. (1998) J. Virology 71: 6823, Chiorini et al. (1999) J. Virology 73: 1309, Bantel-Schaal et al. (1999) J. Virology 73: 939, Xiao et al. al. (1999) J. Virology 73:3994, Muramatsu et al. (1996) Virology 221: 208, Shade et al. (1986) J. Virol. 58: 921, Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99: 11854, Morris et al. (2004) Virology 33:375-383, and WO00 / 28061, WO99 / 61601, WO98 / 11244, US6156303, WO2012145601A, WO2017197355A, WO2018022905A, the contents of which are incorporated by reference in their entirety.

[0124] For the GH ring or ring IV of the AAV capsid, see, e.g., van Vliet et al. (2006) Mol. Ther. 14:809, Padron et al. (2005) J. Virol. 79:5047, and Shen et al. (2007) Mol. Ther. 15:1955.

[0125] An "AAV virion" or "AAV virus particle" refers to a virion composed of at least one AAV capsid protein and an encapsidated AAV polynucleotide.

[0126] "rAAV" refers to recombinant adeno-associated virus, and "recombinant" as applied to a polynucleotide refers to the fact that the polynucleotide is the product of various combinations of cloning, restriction, or ligation, and other processes that result in the construction of a polynucleotide that differs from that found in nature. A recombinant virus is a virion that contains a recombinant polynucleotide.

[0127] When an "AAV viral particle" contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a gene product (e.g., a transgene, RNAi, etc.) to be delivered to a target cell), it is generally referred to as a "recombinant AAV (rAAV) viral particle" or "rAAV virion" or "rAAV vector virus." Generally, the heterologous polynucleotide is flanked by at least one, and generally two, AAV inverted terminal repeats (ITRs).

[0128] "rAAV vector" includes an rAAV viral particle that contains an rAAV polynucleotide, and also includes a polynucleotide encoding an rAAV (e.g., a single-stranded polynucleotide encoding an rAAV (ss-rAAV), a double-stranded polynucleotide encoding an rAAV (ds-rAAV), a plasmid encoding an rAAV, etc.).

[0129] "AAV variant," "AAV mutant," or "capsid-mutated rAAV" means a virion comprising: (a) a variant AAV capsid protein that contains at least one amino acid difference (e.g., an amino acid substitution, insertion, or deletion) compared to a corresponding parent AAV capsid protein and that differs from or does not correspond to the amino acid sequence of a naturally occurring AAV capsid protein, and, optionally, (b) a heterologous polynucleotide encoding a heterologous gene product, wherein the variant AAV capsid protein confers increased binding to heparin-like or heparin sulfate proteoglycans compared to binding to an AAV viral particle comprising the corresponding parent AAV capsid protein.

[0130] "Packaging" refers to a series of intracellular events that result in the assembly and encapsidation of AAV particles.

[0131] "Rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0132] "Helper virus" refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. A variety of helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. Adenoviruses encompass many different subgroups, with adenovirus type 5 of the C subgroup being the most commonly used, and viruses of the herpes family include, for example, herpes simplex virus (HSV) and Escherichia virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV). Many adenoviruses, herpesviruses, etc. from humans, non-human mammals, and birds are known and available from mechanisms such as ATCC.

[0133] "Helper virus functions" or "helper functions" refer to functions encoded in the helper virus genome that allow replication and packaging of AAV (in combination with other requirements for replication and packaging as described herein). In the present disclosure, "helper virus functions" can be provided in a number of ways, including by providing a helper virus or, for example, by providing a polynucleotide sequence encoding the essential functions to the producer cell during transport.

[0134] An "infectious" virus or virion is one that contains a properly assembled viral capsid and is capable of delivering polynucleotide components to a cell of choice for the viral species, and does not necessarily imply that the virus is replication-competent. Measurements that count infectious virions are known in the art. Viral infectivity can be expressed as a ratio of infectious virions to total virions. Methods for determining the ratio of infectious virions to total virions are known in the art. See, for example, Grainger et al. (2005) Mol. Ther. 11:S337 (TCID 50 Infectious titers are measured), see Zolotukhin et al. (1999) Gene Ther. 6:973.

[0135] "Tropism" or "specificity" refers to a virus (e.g., AAV) preferentially targeting cells of a particular host species or a particular cell type within a host species. For example, a virus that can infect cells of the heart, lungs, liver, and muscles has a broader (i.e., increased) tropism than a virus that can only infect cells of the lungs and muscles. Tropism may also include the virus's dependency on a particular type of cell surface molecule of the host. For example, some viruses can only infect cells that have surface glycosaminoglycans, while others can only infect cells that have sialic acid (this dependency may be tested using various cell lines that lack a particular type of molecule as potential host cells for viral infection). In some cases, viral tropism describes the relative preference of the virus. For example, a first virus may be able to infect all cell types, but is more successful in infecting with surface glycosaminoglycans. If the second virus also prefers the same characteristics (e.g., the second virus is also more successful in infecting cells with surface glycosaminoglycans), the second virus is considered to have a similar (or consistent) tropism to the first virus, even if the absolute transduction efficiency is not similar. For example, if the second virus is more effective than the first virus in infecting each given cell type tested, but the relative preferences are similar (or consistent), the second virus may still be considered to have a similar (or consistent) tropism to the first virus. In some embodiments, the tropism of the viral particles comprising the mutant AAV capsid proteins of the present disclosure is unchanged compared to naturally occurring viral particles. In some embodiments, the tropism of the viral particles comprising the mutant AAV capsid proteins of the present disclosure is extended (i.e., broadened) compared to naturally occurring viral particles. In some embodiments, the tropism of the viral particles comprising the mutant AAV capsid proteins of the present disclosure is reduced compared to naturally occurring viral particles.

[0136] "Polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. Modifications to the nucleotide structure may be made before or after assembly of the polymer. Polynucleotides refer interchangeably to double-stranded and single-stranded molecules, and unless otherwise indicated, the polynucleotides of this disclosure encompass the double-stranded form and the two complementary single-stranded forms that make up the double-stranded form.

[0137] "Homology" or "identity" refers to the similarity of sequences between two polynucleotide sequences or between two polypeptides. If the positions in two compared sequences are occupied by the same nucleotide or amino acid monomer subunit, for example, if the positions of each of two DNA molecules are occupied by the same nucleotide, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, if 6 out of 10 positions in two sequences are matched or homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Sequence similarity can be measured in many different ways. To measure sequence identity, sequences can be aligned using methods and computer programs such as BLAST, which can be obtained from the internet address ncbi.nlm.nih.gov / BLAST / . Another alignment algorithm is FASTA, available from the Genetics Computing Group (GCG) package at Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other alignment techniques are described in Enzymology, Vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), Doolittle ed., Academic Press, Inc., division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are comparison procedures that allow gaps in sequences. Smith-Waterman is one type of algorithm that allows gaps in sequence alignment. See Meth. Mol. Biol. 70: 173-187 (1997). The GAP program, which uses the Needleman and Wunsch comparison method, can also be used for sequence alignment. See J. Mol. Biol. 48: 443-453 (1970).

[0138] "Gene" refers to a polynucleotide that contains at least one open reading frame that can encode a particular gene product after transcription and possibly translation. "Gene" or "coding sequence" refers to an exogenous or endogenous nucleotide sequence that encodes a gene product. In some cases, a gene consists of, or consists essentially of, a coding sequence, i.e., a sequence that encodes a gene product. In other cases, a gene includes additional non-coding sequences. For example, a gene may or may not include regions before and after the coding region, such as 5'UTR, 3'UTR, and insertion sequences (introns) between each coding region (exon).

[0139] A "gene product" is a molecule produced by expression of a particular gene, such as a polypeptide, an aptamer, an interfering RNA, an mRNA, etc. In some embodiments, a "gene product" is a polypeptide, a peptide, a protein, or an interfering RNA, including a short interfering RNA (siRNA), miRNA, or a short hairpin RNA (shRNA). In some specific embodiments, the gene product is a therapeutic gene product, such as a therapeutic polypeptide. The therapeutic gene product confers a beneficial effect on the cell, tissue, or mammal in which it is present, including ameliorating a sign or symptom of a condition or disease, preventing or inhibiting a condition or disease, or imparting a desired characteristic. When a gene encodes a polypeptide, the terms "gene product" and "gene expression product of interest" are used interchangeably.

[0140] "RNA interference agent" or "RNAi reagent" includes any reagent (or polynucleotide encoding such a reagent) useful for altering the expression of a gene (as defined above). Examples of RNAi reagents known to those skilled in the art include, but are not limited to, (i) siRNA reagents ("small interfering" or "short interfering RNA" (or siRNA)), (ii) antisense RNA, (iii) CRISPR reagents, (iv) zinc finger nuclease reagents, and (v) transcription activator-like effector nuclease (TALEN) reagents. The entire definitions of (i)-(v) in WO2017197355A are incorporated herein by reference.

[0141] Among them, siRNA reagents are RNA duplexes that target nucleotides of a gene of interest ("target gene"). "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule, thereby forming a region of double-stranded RNA (dsRNA). The nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the target gene, so that the siRNA "targets" the gene. In some embodiments, the length of the siRNA duplex is less than 30 nucleotides. In some embodiments, the duplex may be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides long. In some embodiments, the duplex is 19-25 nucleotides long. The RNA duplex portion of the siRNA may be part of a hairpin structure. An siRNA reagent that includes a hairpin may be called an "shRNA (short hairpin RNA) reagent." The hairpin structure may include a ring portion located between the two sequences forming the duplex in addition to the double-stranded portion. The length of the ring may vary. In some embodiments, the length of the ring is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides. The hairpin structure may further include a 3' or 5' overhang portion. In some embodiments, the overhang is a 3' or 5' overhang of 0, 1, 2, 3, 4, or 5 nucleotides in length. In general, the level of the expression product of a target gene (e.g., mRNA, polypeptide, etc.) is reduced by an siRNA reagent (e.g., siRNA, shRNA, etc.) that includes a specific double-stranded nucleotide sequence that is at least complementary to a 19-25 nucleotide long segment (e.g., a 20-21 nucleotide sequence) of the transcript of the target gene, including the 5' untranslated (UTR) region, the ORF, or the 3'UTR region. In some embodiments, the length of the short interfering RNA is about 19-25 nt. See, e.g., PCT applications WO0 / 44895, WO99 / 32619, WO01 / 75164, WO01 / 92513, WO01 / 29058, WO01 / 89304, WO02 / 16620, and WO02 / 29858, and U.S. Patent Publication No. 20040023390 (for a description of siRNA technology).The siRNA and / or shRNA may be encoded by a nucleic acid sequence, and the nucleic acid sequence may further comprise a promoter. The nucleic acid sequence may further comprise a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal.

[0142] Among them, antisense RNA is RNA that is complementary to gene expression products.For example, antisense RNA that targets specific mRNA is an RNA-based reagent that is complementary to mRNA (or can be modified RNA), and the hybridization of antisense RNA to mRNA changes the expression of mRNA (for example, by changing the stability of RNA, changing the translation of RNA, etc.).Nucleic acid that codes for antisense RNA is also included in "antisense RNA".

[0143] "VEGF" refers to vascular endothelial growth factors that induce angiogenesis or neovascularization processes, including, for example, the various isoforms of VEGF (also called vascular permeability factor (VPF) and VEGF-A), which are produced by alternative splicing of the VEGF-A / VPF gene (see Figures 2(A) and (B) of US Patent Application Publication No. 20120100136), including VEGF121, VEGF165, and VEGF189. Additionally, "VEGF" includes VEGF-related angiogenic factors, such as PIGF (placental growth factor), VEGF-B, VEGF-C, VEGF-D, and VEGF-E, which act through their cognate VEGF receptors (i.e., VEGFR) to induce angiogenesis or neovascularization processes.

[0144] As used herein, "sFlt-1" or "sFlt-1 protein" refers to a polypeptide sequence, or functional fragment thereof, that is at least 90% or more homologous to the naturally occurring human sFLT-1 sequence, such that the sFlt-1 protein or polypeptide binds to VEGF and / or a VEGF receptor.

[0145] "Polypeptide," "peptide," and "protein" refer to polymers of amino acids of any length, and further include polymers of amino acids that have been modified, such as by disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0146] A "regulatory element" or "regulatory sequence" is a nucleotide sequence involved in a molecular interaction that facilitates the functional regulation of a polynucleotide, including replication, repetition, transcription, splicing, translation, or degradation. Regulation affects the frequency, rate, or specificity of a process, and essentially has enhancing or inhibitory properties. Known control elements include transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that is capable of binding RNA polymerase under certain conditions and initiating transcription of a coding region that is usually located downstream (3' direction) of the promoter.

[0147] An "expression vector" is a vector that contains a region encoding a gene product of interest and is used to achieve expression of the gene product in a target cell of interest, the vector comprising a polynucleotide encoding the gene product of interest. The expression vector further comprises a control element operably linked to the coding region to facilitate expression of the gene product in the target. The combination of a control element, such as a promoter, enhancer, UTR, miRNA target sequence, and one or more genes operably linked thereto for expression, may be referred to as an "expression cassette." Many expression cassettes are known and available in the art or can be readily constructed from components available in the art.

[0148] "Operatively linked" or "operably linked" refers to the juxtaposition of genetic elements in a relationship that permits them to operate in their intended manner. For example, a promoter is operably linked to a coding polynucleotide sequence if it helps initiate transcription of the coding polynucleotide sequence; there can be intervening nucleic acid residues between the promoter and the coding polynucleotide sequence so long as this functional relationship is maintained.

[0149] "Administration" or "introduction" refers to the delivery of a vector for expression of a recombinant gene or protein to a cell, a cell and / or organ of a subject, or to a subject. Such administration or introduction can occur in vivo, in vivo, or ex vivo. A vector for expressing a gene product can be introduced into a cell by transfection, which usually means inserting heterologous DNA into a cell by physical means (e.g., calcium phosphate transfection, electroporation, microinjection, or lipotransfection), infection, which usually means introducing by an infectious agent, i.e., virus, stably infecting a cell, or transduction, which usually means transferring genetic material from one microorganism to another via a viral agent (e.g., bacterial phage). "Transformation" is generally used to refer to bacteria containing heterologous DNA, or cells expressing an oncogene and converted into a continuous growth pattern, e.g., tumor cells. The vectors used to "transform" a cell may be plasmids, viruses, or other vehicles. Depending on the method for administering, introducing or inserting the heterologous DNA (i.e., vector) into the cells, the cells are generally referred to as "transduced," "infected," "transfected," or "transformed." "Transduced," "transfected," and "transformed" are used interchangeably herein, without regard to the method of introduction of the heterologous DNA.

[0150] "Host cell" refers to a cell that has been transduced, infected, transfected or transformed with a vector, and includes the original transduced, infected, transfected or transformed cell and its progeny. The vector may be a plasmid, virion, phage, etc. Culture conditions such as temperature, pH, etc. will be apparent to one of skill in the art.

[0151] "Treatment" is generally used to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic in the complete or partial prevention of a disease or its symptoms, or therapeutic in, for example, reducing the likelihood of the onset of the disease or its symptoms in a subject, and / or curing the disease and / or adverse reactions caused by the disease, partially or completely. "Treatment" encompasses any treatment of a disease in a mammal, including (a) preventing the disease from occurring in a subject who may be prone to the disease, but has not been diagnosed as having the disease, (b) inhibiting or halting the progression of the disease, or (c) alleviating the disease (or symptoms resulting therefrom) or regressing the disease. Therapeutic agents can be administered before, during, or after the onset of the disease or injury. Particularly in the case of treatment of an ongoing disease, the treatment stabilizes or reduces the undesirable clinical symptoms of the patient. In some preferred forms, such treatment is administered before the complete loss of function of the affected tissue. In some preferred forms, the treatment of the present disclosure is administered during, and optionally after, the symptomatic phase of the disease.

[0152] An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. Effective amount further refers to an amount sufficient to permit or facilitate diagnosis. The effective amount used for a particular subject can vary depending on factors such as the condition being treated, the overall health of the subject, the method, route and dose of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0153] "Retinal cells" in this disclosure may refer to any one of the cell types that comprise the retina, such as retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells including rods and cones, Muller glial cells, astrocytes (e.g., retinal astrocytes), and retinal pigment epithelial cells.

[0154] An "individual," "subject," and "patient" are used interchangeably herein and include, but are not limited to, humans and non-human primates, such as monkeys, humans and other mammals (e.g., horses, sheep, goats, dogs, cats, and rodents (e.g., mice, rats, etc.)), preferably humans.

[0155] In this disclosure, "polypeptide" and "protein" are used interchangeably. Working Example

[0156] The present disclosure will be further described below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. Experimental methods for which specific conditions are not specified in the examples of the present disclosure generally follow normal conditions or conditions recommended by the manufacturers of raw materials or products. For reagents for which a specific source is not specified, the reagents can be obtained from any molecular biology reagent supplier with the quality / purity for molecular biology applications. Example 1. AAV capsid modification

[0157] Molecular dynamics simulation and protein fingerprinting techniques are widely applied in protein engineering and antibody affinity modification, and through dynamic simulation of the physicochemical properties of antibodies and receptors, the local affinity effects of amino acid modifications can be predicted to a certain extent.

[0158] Heparan sulfate receptors have been proven to be the most important receptors mediating AAV cell infection, which play an important role in the infection of retinal and optic nerve cells. Research has demonstrated that the amino acid sequence of the AAV2 native serotype capsid protein contains five cyclic domains (shown in Figure 1) that are involved in the viral infection ability according to their spatial structure characteristics. Among them, inserting a short peptide of 7 to 12 amino acids between positions 587 and 588 of cyclic domain IV does not affect the construction of the viral capsid protein, but at the same time can significantly change the form of interaction between the AAV capsid and the cell receptor (heparan sulfate), thereby affecting the AAV infection ability of the host cell.

[0159] The present disclosure utilizes an autonomously developed bioinformatics algorithm to insert several random amino acid sequences (7-12 amino acids long) between positions 587 and 588 in the amino acid sequence of AAV2 native serotype capsid protein, and predict the interaction between the capsid protein and the heparan sulfate receptor. The sequences in the algorithm that can greatly improve and predict the infection ability are selected for subsequent validation. According to the algorithm prediction, six amino acid sequences seq1-seq6 shown in Table 1, each of which is 10 amino acids long, were selected. At the same time, the amino acid at position 708 in the amino acid sequence of AAV2 native serotype capsid protein, valine (V), was replaced with isoleucine (I). The method of insertion between positions 587 and 588 and the method of replacing the amino acid at position 708 are shown in FIG. 2, and an engineered AAV capsid protein sequence was obtained. At the same time, a capsid with seq2 inserted into the corresponding position of AAV9 was produced.

[0160] [Table 1] After expression, the AAV capsid protein is constructed of three monomer proteins, VP1, VP2, and VP3, in a certain ratio and has a higher-order structure compared to normal recombinant proteins. Therefore, the functional effects of the polypeptide insertion into the AAV capsid protein are not limited to the changes in the physicochemical properties, but are likely to have a more complex effect on the construction of the three monomer proteins, VP1, VP2, and VP3. Example 2. Detection of retinal function in a mouse model infected with rAAV vector virus

[0161] To verify the function, the present inventors packaged the plasmid pGOI, which contains and can express the EGFP target gene driven by the CMV promoter, in capsids having eight sequences, SEQ ID NO: 3 to SEQ ID NO: 8, AAV2 (SEQ ID NO: 1), and AAV2.7m8 (SEQ ID NO: 2). The AAV vectors packaged in the above different AAV capsids capable of expressing EGFP were injected into the mouse vitreous in equal amounts, and the expression of EGFP was detected by fluorescent fundus photography. The difference in the intensity of the EGFP fluorescent signal was used to evaluate the difference in the infection efficiency of different AAV capsids into retinal tissue.

[0162] The packaging and purification steps of rAAV vector virus included transfecting three plasmid systems, pHelper, pR2C9, and pGOI, into 293T adherent cells, harvesting the cells and the supernatant, purifying them by iogisol centrifugation, concentrating them by ultrafiltration, and replacing the buffer with DPBS. After the packaging and purification of the virus was completed, the titer (unit: vg) was detected by qPCR. Eight viruses expressing EGFP target genes (capsids are AAV2, AAV2.7m8, AAV2 seq1, AAV2 seq2, AAV2 seq3, AAV2 seq4, AAV2 seq5, and AAV2 seq6, respectively) were diluted to the same titer of 1.03E+12 in DPBS buffer. Ten-week-old C57 mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were injected with unilateral intravitreal injections in a volume of 1 μL, three mice (n = 3) per type of AAV vector, and in vivo fluorescent fundus photography was performed weekly from 1 to 4 weeks after injection.

[0163] Some of the results are shown in Figure 3. The AAV2, AAV2 seq5, and AAV2 seq6 groups had significantly weaker fluorescent signal intensities than the other groups. The AAV2 seq1, AAV2 seq2, AAV2 seq3, and AAV2 seq4 groups had fluorescent signal intensities relatively similar to those of AAV2.7m8, indicating that the infection efficiency in retinal tissues was relatively high.

[0164] The "LALGDTTRPA" sequence in Table 1 was inserted into the same position (between positions 588 and 589) in the AAV9 capsid to obtain AAV9 seq2. A total of seven capsids, AAV2, AAV2.7m8, AAV2 seq1, AAV2 seq2, AAV2 seq3, AAV2 seq4, and AAV9 seq2, were packaged with a plasmid vector that contains and can express the EGFP target gene driven by the CMV promoter. The AAV vectors packaged with the above different AAV capsids capable of expressing EGFP were injected into the mouse vitreous in equal amounts, and the expression of EGFP was detected by fluorescent fundus photography. The difference in the intensity of the EGFP signal was used to evaluate the difference in the infection efficiency of different AAV capsids into retinal tissue.

[0165] AAV viral vectors were packaged and purified using the method described above. Seven viruses expressing EGFP target genes (capsids are AAV2, AAV2.7m8, AAV2 seq1, AAV2 seq2, AAV2 seq3, AAV2 seq4, AAV2 seq5, and AAV9 seq2, respectively) were diluted to the same titer of 1.03E+12 in DPBS buffer. Ten-week-old C57 mice were injected with unilateral intravitreal injections in a volume of 1 μL, three mice (n=3) per type of AAV vector, and in vivo fluorescent fundus photography was performed weekly from 1 to 4 weeks after injection.

[0166] The results are shown in Figures 4A and 4B, among which the AAV2 group, the AAV2 seq2 group, and the AAV2 seq3 group had fluorescent signal intensities that were clearly stronger than the other groups and similar to that of AAV2.7m8. Example 3. Detection of biological functions of rAAV vector viruses on retinal infection in a mouse model

[0167] To further verify the infectious ability of seq2 capsid to retinal tissue and their associated biological functions, the target plasmid vectors (AAV2 seq2-aflibercept, AAV2.7m8-aflibercept) containing aflibercept driven by the CMV promoter were packaged in AAV2 seq2 and AAV2.7m8, respectively, and the two viruses were injected into the vitreous of New Zealand rabbits and C57 mice, respectively. Two weeks after injection, the content of aflibercept protein injected into the ocular tissue was detected to evaluate the infection of the corresponding AAV virus to the retina and their associated biological functions.

[0168] The structure of the rAAV-delivered gene expression cassette includes, from its 5' end to its 3' end, a CMV enhancer, a promoter, a 5'UTR, a Kozak sequence (gccacc), a VEGF inhibitor aflibercept, a 3'UTR, a WPRE, and a SV40polyA (shown in FIG. 5A). The base sequence of the CMV enhancer is shown in SEQ ID NO: 28, the base sequence of the CMV promoter is shown in SEQ ID NO: 29, the 5'UTR is shown in SEQ ID NO: 30, the Kozak sequence is shown in SEQ ID NO: 31, the 3'UTR is shown in SEQ ID NO: 32, the WPRE is shown in SEQ ID NO: 33, and the SV40polyA is shown in SEQ ID NO: 34.

[0169] The construction of the expression vector involves constructing an expression cassette containing, in order, a CMV enhancer, a promoter, 5'UTR, a Kozak sequence (gccacc), a VEGF trap (aflibercept), 3'UTR, WPRE and SV40polyA, and constructing an AAV packaging plasmid expressing aflibercept through conventional molecular biology operations such as enzyme cleavage, ligation, transformation and cloning screening identification. Both sides of the expression cassette are inverted terminal repeats (ITRs), the configuration diagram of which is shown in Figure 5B, with EcoRV and BSMI being the enzyme cleavage sites.

[0170] Vector packaging and purification of rAAV vector virus were performed according to the method of Example 2.

[0171] The two purified viruses, AAV seq2-aflibercept and AAV2.7m8-aflibercept, were each diluted to the same titer of 9E+12vg / mL in DPBS buffer, and 4 mice (n=4) were injected per type of rAAV vector virus into C57 mice aged 8 to 10 weeks in a volume of 1 μL via bilateral vitreous injection. Two weeks after injection, the mice were sacrificed, and the ocular tissues were collected and polished, respectively. The tissue polishing process was performed in two different ways for the left and right eyes of each injection group. For the left eye, method 1 was used, which involved polishing with PBS + protease inhibitors, followed by centrifugation and then taking the supernatant. For the right eye, method 2 was used, which involved polishing with RIP Buffer + protease inhibitors, followed by centrifugation and then taking the supernatant. The content of aflibercept protein in the supernatant was detected using enzyme-linked immunoassay (four samples per injection group, three replicates for each sample). The results are shown in Figure 6. Among them, the blank control was the PBS-injected group, and the ordinate, HRAMD / total protein, was the number of ng of aflibercept protein per mg of total tissue protein.

[0172] [Table 2-1] [Table 2-2] The results in Figure 6 and Table 2 showed that the expression level in mice of the AAV seq2-aflibercept group was higher than that of the AAV2.7m8-aflibercept group. Example 4. Detection of biological functions of rAAV vector virus on retinal infection in rabbit model

[0173] Vector packaging and purification of rAAV vector virus were performed according to the method of Example 2.

[0174] The two purified viruses, AAV seq2-aflibercept and AAV2.7m8-aflibercept, were each diluted to 4E+9vg / mL in DPBS buffer, and 8-10 week-old New Zealand rabbits (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were injected with 50 μL of bilateral vitreous injections, with two rabbits (n=2) injected per type of rAAV vector virus. Two weeks after injection, the rabbits were sacrificed, and aqueous humor, vitreous, and whole eye tissues were collected and polished, respectively. The left and right eyes of each injection group were subjected to two different methods of tissue polishing. For the left eye, method 1 was used, which involved polishing with PBS + protease inhibitors, followed by centrifugation and then the supernatant was taken. For the right eye, method 2 was used, which involved polishing with RIP Buffer + protease inhibitors, followed by centrifugation and then the supernatant was taken. Enzyme-linked immunoassay was used to detect the content of aflibercept protein in the supernatant (2 samples per injection group, each sample was detected in triplicate). The results are shown in Figure 7, where the blank control was the PBS injection group, and the ordinate HRAMD / total protein was the ng of aflibercept protein per mg of total tissue protein.

[0175] The results in Figure 7 showed that the expression levels of the AAV seq2-aflibercept group in New Zealand rabbits were higher than those of the AAV2.7m8-aflibercept group. Because rabbits have a large eye volume, aflibercept is diluted into tissue fluid after whole eye polishing, so the concentration is much lower than that of the vitreous and aqueous humor. Since vectors packaged in AAV can only successfully express the target gene protein contained therein after the AAV virus has successfully infected cells, the results in Figure 7 showed that the infection efficiency of AAV2 seq2 capsids to cells was higher than that of AAV2.7m8 capsids.

[0176] In addition, the expression levels of the target gene protein (VEGF inhibitor) delivered by AAV2 seq2 and AAV2.7m8 capsids were further compared. The experimental method was to inject 8 New Zealand rabbits into both eyes according to the doses in Table 3, and 2 rabbits in each dose group were injected into 4 eyes, for a total of 4 groups. On the 28th day after injection, the experimental animals were sacrificed to extract the aqueous humor, and the molar concentration of the target gene protein in the aqueous humor sample was detected using the ELISA method.

[0177] The results, as shown in Figure 8, showed that the molar concentration of the target gene protein expressed by AAV2 seq2 was clearly higher than that of AAV2.7m8 when injected at the same dose.

[0178] [Table 3-1] [Table 3-2] Below are some sequences from this disclosure.

[0179] > AAV2 capsid amino acid sequence MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPA DVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTN VDIEKVMITDEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:1 > AAV2.7m8 capsid amino acid sequence MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSINVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:2 > AAV2 seq1 capsid amino acid sequence MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLALAETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSINVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:3 > AAV2 seq2 capsid amino acid sequence * SEQ ID NO:4 > AAV2 seq3 capsid amino acid sequence MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRNARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSINVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:5 > AAV2 seq4 capsid amino acid sequence MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLAKADTTKNARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSINVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:6 > AAV2 seq5 capsid amino acid sequence MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLAKDDTTRNARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSINVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:7 > AAV2 seq6 capsid amino acid sequence MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLALADTTKNARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSINVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:8 > AAV9 seq2 capsid amino acid sequence MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGNGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQ PIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFM IPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVM ITNEEIKTTNPVATESYGQVATNHQSAQLALGDTTRPAAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL SEQ ID NO:9 > EGFP amino acid sequence MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO:10 > seq1 (without linker) LAETTRP SEQ ID NO:11 > seq2 (without linker) LGDTRP SEQ ID NO:12 > seq3 (without linker) LGETTRN SEQ ID NO:13 > seq4 (without linker) KADTTKN SEQ ID NO:14 > seq5 (without linker) KDDTTRN SEQ ID NO:15 > seq6 (without linker) LADTTKN SEQ ID NO:16 > seq1 LALAETTRPA SEQ ID NO:17 > seq2 LALGDTTRPA SEQ ID NO:18 > seq3 LALGET TRNA SEQ ID NO:19 > seq4 LAKADTTKNA SEQ ID NO:20 > seq5 LAKDDTTRNA SEQ ID NO:21 > seq6 LALADTTKNA SEQ ID NO:22 > AAV2 seq2 capsid amino acid sequence (not including V708I) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGDTTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:23 > AAV2 seq3 capsid amino acid sequence (not including V708I) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLALGETTRNARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:24 > AAV2 seq4 capsid amino acid sequence (not including V708I) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLAKADTTKNARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:25 > AAV2 seq5 capsid amino acid sequence (not including V708I) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLAKDDTTRNARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:26 > AAV2 seq6 capsid amino acid sequence (not including V708I) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQ PLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMV PQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVM ITDEEEIRTTNPVATEQYGSVSTNLQRGNLALADTTKNARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO:27 > CMV enhancer sequence CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG SEQ ID NO:28 >CMV promoter sequence TGCTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTT GACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGAGGCCATCCACGCTGTTTTGACCTCCATAGTGGACACCGGGACCGATCCAGCCTCCGCGTCTCAGGGGAAGCTT SEQ ID NO:29 >5'UTR sequence CTTGTTCTTTTTGCAGAAGCTCAGAATAAACGCTCAACTTTGG SEQ ID NO:30 >Kozak sequence GCCACC SEQ ID NO:31 >3'UTR sequence GTCTAGAAACCAGCCTCAAGAACACCCGAATGGAGTCTCTAAGCTACATAATACCAACTTACACTTTCAAAAATGTTGTCCCCCAAAATGTAGCCATTCGTATCTGCTCCTAATAAAAAGAAAGTTTCTTCAC sequence number 32 >WPRE sequence AATCAACCTCTGGATTACAAAATTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCTTGTATAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCTGTGTGTCACTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATGGCCACCACTGTCAGCTCTTCCGGACTTTCGCTTTCCCC CTCCCTATTGCCCAGGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGTGTTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTCGCGTCTTCGCCTTCCCTCCAGACGAGTCGGATCTCCCTTGGGCCGCCTCCCCGC. sequence number 33 >SV40polyA sequence: CCTAGGTAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTAAAGCTGCAATAAACAAGTTATCGATAGATCT sequence number 34 > AAV9 capsid amino acid sequence MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPD PQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFP ADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDN VDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL SEQ ID NO:37 >Aflibercept protein sequence MPLLLLLPLLWAGALASDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHR QTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:38 >Aflibercept nucleic acid sequence 1 SEQ ID NO:39 >Aflibercept nucleic acid sequence 2 SEQ ID NO:40 >Aflibercept nucleic acid sequence 3 SEQ ID NO:41

Claims

**Claim 1** A mutant adeno-associated virus (AAV) capsid protein comprising a polypeptide inserted as compared to the parental AAV capsid protein, wherein the inserted polypeptide is any one of the following 1) to 6): 1) LGDTTRP (SEQ ID NO: 12) or LALGDTTRPA (SEQ ID NO: 18), 2) LAETTRP (SEQ ID NO: 11) or LALAEETTRPA (SEQ ID NO: 17), 3) LGETTRN (SEQ ID NO: 13) or LALGETTRNA (SEQ ID NO: 19), 4) KADTTKN (SEQ ID NO: 14) or LAKADTTKNA (SEQ ID NO: 20), 5) KDTTTRN (SEQ ID NO: 15) or LAKDTTTRNA (SEQ ID NO: 21), 6) LADTTKN (SEQ ID NO: 16) or LALADTTKNA (SEQ ID NO: 22) and includes a polypeptide shown in any one selected from the group consisting of or any combination thereof, a mutant AAV capsid protein. **Claim 2** The polypeptide of any one of the above 1) to 6) or any combination thereof is located within the GH loop of the AAV capsid protein, preferably, it is located after any amino acid residue between the 570th and 611th positions of the amino acid sequence encoding VP1 of the parental AAV2 capsid protein, more preferably, it is located between the 587th and 588th positions of the amino acid sequence encoding VP1 of the parental AAV2 capsid protein, or between the 588th and 589th positions of the amino acid sequence encoding VP1 of the parental AAV9 capsid protein, or at the corresponding position of other parental AAV serotype capsid proteins, The mutant AAV capsid protein according to claim 1. **Claim 3** It further includes a point mutation of one or more amino acid residues selected from the group consisting of 1L, 15P, 34A, 57D, 66K, 81Q, 101R, 109T, 144K, 144M, 164K, 176P, 188I, 196Y, 226E, 236V, 240T, 250S, 312K, 363L, 368H, 449D, 456K, 463Y, 472N, 484C, 524T, 535S, 551S, 593E, 698V, 708I, 719M, 721L and 735Q, and the position of the mutation is based on the corresponding position of the AAV2 capsid protein shown in SEQ ID NO: 1 or other AAV serotype capsid proteins, preferably, the point mutation is 708I, The mutant AAV capsid protein according to claim 1 or 2. **Claim 4** The amino acid sequence is shown by any one of SEQ ID NOs: 3 to 9 and 23 to 27, or has at least 90% or 95% sequence identity therewith. The mutant AAV capsid protein according to claim 1.

5. A recombinant adeno-associated virus (rAAV) viral particle comprising: (a) the mutant AAV capsid protein according to claim 1; and (b) a heterologous polynucleotide. (rAAV) viral particle.

6. The heterologous polynucleotide comprises a polynucleotide encoding a gene product selected from a polypeptide, an interfering RNA or an aptamer, The polypeptide is preferably an antibody or an antigen-binding fragment thereof, a fusion protein. The rAAV viral particle according to claim 5.

7. The heterologous polynucleotide further comprises one or more selected from the group consisting of the following (a) to (g): (a) a 5' terminal inverted repeat sequence (5' ITR) and / or a 3' terminal inverted repeat sequence (3' ITR); (b) a 5' untranslated region (5' UTR) and / or a 3' untranslated region (3' UTR); (c) a promoter; (d) an enhancer; (e) a post-transcriptional regulatory element; (f) a polyadenylation signal (polyA); and (g) a Kozak sequence Preferably, the heterologous polynucleotide further comprises one or more selected from the group consisting of the following (a) to (g): (a) a 5' ITR and / or a 3' ITR derived from AAV2; (b) a 5' UTR and / or a 3' UTR derived from Xenopus globin; (c) a CMV promoter; (d) a CMV enhancer; (e) a WPRE; (f) an SV40 polyA; and (g) a Kozak sequence Preferably, the heterologous polynucleotide further comprises one or more selected from the group consisting of the following (a) to (g): (a) a 5' ITR and / or a 3' ITR derived from AAV2; The rAAV viral particle according to claim 6.

8. The heterologous polynucleotide is operably linked from the 5' end to the 3' end and comprises a polynucleotide ordered as follows: (a) an enhancer; (b) a promoter; (c) a 5' UTR; (d) a Kozak sequence; (e) a polynucleotide encoding a gene product; (f) a 3' UTR; (g) a WPRE; (h) a polyA. (a) an enhancer; (b) a promoter; (c) a 5' UTR; (d) a Kozak sequence; (e) a polynucleotide encoding a gene product; (f) a 3' UTR; (g) a WPRE; (h) a polyA The rAAV viral particle according to claim 7.

9. The polypeptide is a neuroprotective polypeptide, an anti-angiogenic polypeptide, a polypeptide that enhances retinal cell function, Preferably, the anti-angiogenic polypeptide is a VEGF antagonist. ​ More preferably, the VEGF antagonist is aflibercept. The rAAV viral particles according to claim 6.

10. The heterologous polynucleotide comprises a polynucleotide encoding aflibercept shown in SEQ ID NO:

38. Preferably, the heterologous polynucleotide comprises a polynucleotide shown in any one of SEQ ID NOs: 39 to 41, or a polynucleotide having at least 90% or 95% sequence identity thereto. The rAAV viral particles according to claim 5.

11. An isolated polynucleotide, encoding the mutant AAV capsid protein according to claim 1. Polynucleotide.

12. A vector, (a) the isolated polynucleotide according to claim 11, or (b) the isolated polynucleotide according to claim 11 and the heterologous polynucleotide according to claim 5, Vector.

13. A host cell comprising the vector according to claim 12. Host cell.

14. A pharmaceutical composition, (a) the rAAV viral particles according to claim 5, and (b) one or more pharmaceutically acceptable carriers, diluents, excipients or buffers. Pharmaceutical composition.

15. A method for specifically infecting retinal cells, comprising the step of intraocularly injecting an effective amount of the rAAV viral particles according to claim 5, the heterologous polynucleotide according to claim 5 or the pharmaceutical composition according to claim 14, preferably by intravitreal injection or subretinal injection. Method.

16. A method for treating an eye-related disease, comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of the rAAV viral particles according to claim 5, the heterologous polynucleotide according to claim 5 or the pharmaceutical composition according to claim 14, preferably by intravitreal injection or subretinal injection. Method.

17. The eye-related disease is a retinal cell disease, preferably, the retinal cell disease is a disease selected from photoreceptor cells, retinal ganglion cells, Muller cells, bipolar cells, amacrine cells, horizontal cells or retinal pigment epithelial cells. The method according to claim 16.

18. The eye-related diseases are selected from retinitis pigmentosa, macular degeneration, wet AMD, dry AMD, retinal angiogenesis, choroidal angiogenesis, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy, diabetic retinal edema, retinal detachment, glaucoma, Leber congenital cataract and / or color blindness. The method according to claim 16.

19. A method for delivering a heterologous polynucleotide to target cells in vitro and / or in vivo, comprising contacting the target cells with the rAAV viral particles according to claim 5, the heterologous polynucleotide according to claim 5 or the pharmaceutical composition according to claim 14. Method.

20. An rAAV viral particle production system, comprising (a) the isolated polynucleotide according to claim 11, (b) a heterologous polynucleotide encoding a gene product, (c) a helper element having an AAV rep function and a helper function sufficient to package the heterologous polynucleotide of (b) encoding the gene product into the mutant AAV capsid of (a). System.

21. A method for producing or manufacturing the rAAV viral particles according to claim 5, comprising introducing a vector according to claim 12, a vector containing a helper element into a production cell, and packaging and purifying the rAAV viral particles. Method.