Expression cassette comprising codon-optimized polynucleotide encoding aflibercept and plasmid for producing raav comprising the same

Abstract

Provided are an expression cassette for AAV gene therapy of a neovascular ophthalmic disease, a plasmid for producing rAAV including the expression cassette, an rAAV produced by the plasmid, and use of the rAAV. By the expression cassette and the plasmid according to aspects, rAAV virus particles having reduced immunogenicity can be produced with high productivity and high quality, and an rAAV thus produced can reach a therapeutic concentration of aflibercept in the retina even at a low viral dose, thereby minimizing capsid-induced immune responses.

Description

[0001] EXPRESSION CASSETTE COMPRISING CODON-OPTIMIZED POLYNUCLEOTIDE ENCODING AFLIBERCEPT AND PLASMID FOR PRODUCING rAAV COMPRISING THE SAME

[0002] Cross-Reference to Related Applications

[0003] This application claims the benefit of Korean Patent Application No. KR 10-2024- 0086318, filed on July 1 , 2024, which is hereby incorporated by reference in its entirety.

[0004] Reference to an Electronic Sequence Listing

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 30, 2025, is named 99CR-414334-WO.xml and is 35,107 bytes in size.

[0006] Technical Field

[0007] The disclosure relates to an expression cassette including a codon-optimized polynucleotide encoding aflibercept, a plasmid for producing a recombinant adeno- associated virus (rAAV) including the expression cassette, an rAAV produced by the plasmid, and use of the rAAV for a neovascular ophthalmic disease.

[0008] Background

[0009] Abnormal growth of blood vessels (neovascularization) is a key feature of serious eye diseases that can lead to blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular angiogenesis. Currently, neovascular ocular disease is treated by drug therapy that involves injecting a drug such as ranibizumab, bevacizumab, aflibercept, or brolucizumab directly into the eye (into the vitreous body) during a clinical visit once every 1 to 3 months. It is inconvenient for patients to visit a hospital periodically to get injections into the eye, and thus many pharmaceutical companies are developing techniques to shorten the dosing interval. A rapidly developing field is gene therapy, which may offer significant therapeutic benefits. Delivery of transgenes via viral vectors offers the potential for sustained production of antiangiogenic proteins, which may avoid repeated anti-VEGF injections. In particular, adeno-associated viruses (AAVs) have been chosen as the most promising viral gene delivery tool as they are capable of infecting various types of tissues and are considered safe gene delivery vectors.

[0010] Recombinant AAV-based gene therapy agents have recently been developed for a variety of ocular diseases due to their promising therapeutic efficacy and one-and- done treatment advantages. Given the nature of the eye as an affected area, strategies such as subretinal injection or suprachoroidal injection, and Intravitreal injection are being selected. Meanwhile, subretinal injection involves surgically removing the vitreous body from the eye and then detaching the retina to inject a drug therebetween. Retinal detachment is a disease itself and a phenomenon of aging, and thus rAAV formulations administered by subretinal injection have a fundamental limitation of carrying the risk of retinal detachment. Also, suprachoroidal injection, which is currently being tested in clinical trials, involves administering a drug in a narrow area called the suprachoroidal space between the sclera and choroid, but it has the disadvantage of being a recently developed method and lacking successful treatment cases or established guidelines for clinical application. Accordingly, some rAAV formulations have opted for an intravitreal injection strategy, but these have been limited by low expression of therapeutic genes and the triggering of immune responses. In addition, low AAV productivity and poor quality due to low % full capsid ratio have been pointed out as problems in designing a plasmid for producing rAAVs.

[0011] Therefore, there is a need for the development of rAAV gene therapy agents for a neovascular ophthalmic disease and production systems that can improve the aforementioned problems.

[0012] Summary

[0013] An aspect is to provide a nucleic acid expression cassette for enhanced expression of aflibercept in the retina, comprising a codon-optimized polynucleotide encoding aflibercept which is operably linked to an expression regulatory element, wherein the codon-optimized polynucleotide encoding aflibercept includes a nucleotide sequence consisting of SEQ ID NO: 8 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0014] Another aspect is to provide a plasmid for producing recombinant adeno- associated virus (rAAV), comprising: (a) an AAV 5' inverted terminal repeat (ITR); (b) the nucleic acid expression cassette according to an aspect; and (c) an AAV 3' ITR.

[0015] Another aspect is to provide an isolated host cell transfected or transduced with the plasmid for producing rAAV according to an aspect.

[0016] Another aspect is to provide rAAV comprising an AAV capsid protein and a vector genome, wherein the vector genome comprises: (a) an AAV 5' ITR; (b) the nucleic acid expression cassette according to an aspect; and (c) an AAV 3' ITR.

[0017] Another aspect is to provide a pharmaceutical composition for preventing or treating a neovascular ophthalmic disease, comprising: the rAAV according to an aspect; and a pharmaceutically acceptable carrier.

[0018] Another aspect is to provide a method of preventing or treating a neovascular ophthalmic disease, comprising administering an effective amount of the rAAV according to an aspect or the pharmaceutical composition according to an aspect, to a subject in need thereof.

[0019] Another aspect is to provide use of the rAAV according to an aspect for the manufacture of a medicament for prevention or treatment of a neovascular ophthalmic disease.

[0020] Another aspect is to provide a nucleic acid for encoding an inhibitor of VEGF-A, comprising a codon-optimized polynucleotide encoding the inhibitor of VEGF-A, wherein the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 90 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid further comprises a polynucleotide encoding a signal peptide of dermcidin. In some embodiments, the signal peptide comprises an amino acid sequence consisting of SEQ ID NO: 11. In some embodiments, the polynucleotide encoding the signal peptide is located at the 5'- terminus of the codon-optimized polynucleotide encoding aflibercept. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the codon-optimized polynucleotide encoding the inhibitor of VEGF-A is operably linked to an expression regulatory element. In some embodiments, the expression regulatory element includes at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the nucleic acid further comprises a Kozak sequence.

[0021] Another aspect is to provide a nucleic acid for encoding an inhibitor of VEGF-A, comprising polynucleotide sequences encoding an inhibitor of VEGF-A and a signal peptide, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 11 , or an amino acid sequence having at least 90 % sequence identity with SEQ ID NO: 11. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide is at 5’- end of the polynucleotide sequence encoding the inhibitor of VEGF-A. In some embodiments, the nucleic acid further comprises one or more expression regulatory elements operably linked to the polynucleotide. In some embodiments, the expression regulatory element(s) are selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the nucleic acid further comprises a Kozak sequence.

[0022] Another aspect is to provide a plasmid for producing a recombinant adeno- associated virus (rAAV), comprising (a) an AAV 5’ inverted terminal repeat (ITR), (b) the nucleic acid according to an aspect disclosed herein, and (c) an AAV 3’ ITR. In some embodiments, the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh , AAV11 , AAV12, and AAV13. In some embodiments, the ITR is derived from AAV2. In some embodiments, the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10. In some embodiments, the plasmid further comprises the following genes: (a) a helper virus gene required for producing AAV; (b) a Rep gene of AAV; and (c) a Cap gene of AAV. In some embodiments, a nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus. In some embodiments, a nucleotide sequence of the helper virus gene is derived from an adenovirus. In some embodiments, a nucleotide sequence of the helper virus gene is derived from adenovirus 2 (Ad2). In some embodiments, the helper virus gene comprises one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DNA-binding protein (DBP), and a variant thereof. In some embodiments, the helper virus gene comprises E2a, E4, and VA. In some embodiments, a nucleotide sequence of the Rep gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13. In some embodiments, a nucleotide sequence of the Rep gene is a Rep gene of wild-type AAV2. In some embodiments, a nucleotide sequence of the Cap gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, AAV11 , AAV12, and AAV13. In some embodiments, a nucleotide sequence of the Cap gene is a Cap gene of wild-type AAV2.

[0023] Another aspect is to provide an isolated host cell transfected or transduced with the plasmid according to an aspect disclosed herein. In some embodiments, the host cell is a mammalian cell or an insect cell. In some embodiments, the mammalian cell is selected from a HEK293 cell, a HEK293F cell, a HEK293T cell, and cells derived therefrom. In some embodiments, the insect cell is an Sf9 cell, an Sf21 cell, a TN-5B1- 4 cell, or a High Five cell.

[0024] Another aspect is to provide a recombinant adeno-associated virus (rAAV) comprising the plasmid according to an aspect disclosed herein.

[0025] Another aspect is to provide a recombinant adeno-associated virus (rAAV) comprising the nucleic acid according to an aspect disclosed herein.

[0026] Another aspect is to provide a recombinant adeno-associated virus (rAAV) comprising a transgene plasmid comprising the nucleic acid according to an aspect disclosed herein, and one or more additional plasmids comprising at least one of (a) a helper virus gene required for producing AAV, (b) a Rep gene of AAV, and (c) a Cap gene of AAV.

[0027] Another aspect is to provide a nucleic acid for encoding an inhibitor of VEGF-A, comprising a codon-optimized polynucleotide sequence encoding the inhibitor of VEGF- A, wherein the polynucleotide further comprises (a) a helper virus gene required for producing AAV; (b) a Rep gene of AAV; and (c) a Cap gene of AAV. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 90% to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid further comprises a polynucleotide sequence encoding a signal peptide of dermcidin. In some embodiments, the signal peptide comprises an amino acid sequence consisting of SEQ ID NO: 11. In some embodiments, the polynucleotide sequence encoding the signal peptide is located at the 5'-terminus of the codon- optimized polynucleotide sequence encoding the inhibitor of VEGF-A. In some embodiments, the codon-optimized polynucleotide encoding the inhibitor of VEGF-A is operably linked to an expression regulatory element. In some embodiments, the expression regulatory element includes at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the nucleic acid further comprises a Kozak sequence. In some embodiments, a nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus. In some embodiments, a nucleotide sequence of the helper virus gene is derived from an adenovirus. In some embodiments, a nucleotide sequence of the helper virus gene is derived from adenovirus 2 (Ad2). In some embodiments, the helper virus gene comprises one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DNA-binding protein (DBP), and a variant thereof. In some embodiments, the helper virus gene comprises E2a, E4, and VA. In some embodiments, a nucleotide sequence of the Rep gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13. In some embodiments, a nucleotide sequence of the Rep gene is a Rep gene of wild-type AAV2. In some embodiments, a nucleotide sequence of the Cap gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, AAV11 , AAV12, and AAV13. In some embodiments, a nucleotide sequence of the Cap gene is a Cap gene of wild-type AAV2. In some embodiments, the nucleic acid further comprises an AAV 5’ inverted terminal repeat (ITR) and an AAV 3’ ITR. In some embodiments, the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV1 1 , AAV12, and AAV13. In some embodiments, the ITR is derived from AAV2. In some embodiments, the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10.

[0028] Another aspect is to provide a plasmid comprising the nucleic acid according to an aspect disclosed herein. Another aspect is to provide a recombinant adeno- associated virus (rAAV) comprising the plasmid according to an aspect disclosed herein. Brief Description of Drawings

[0029] FIG. 1 is a schematic diagram of an aflibercept transgene expression cassette flanked by AAV2 inverted terminal repeats (ITRs), according to an embodiment.

[0030] FIG. 2 is a diagram for describing an all-in-one vector designed to include a helper virus gene, a Rep gene, a Cap gene, and a transgene in one vector, according to an embodiment.

[0031] FIG. 3 is a vector map of an all-in-one ssAAV2-aflibercept vector according to an embodiment.

[0032] FIG. 4 is a vector map of a vector including a GOI (including a portion corresponding to from a 5 'ITR to a 3' ITR) of a triple transfection (3TF) ssAAV2- aflibercept vector, according to an embodiment.

[0033] FIG. 5 shows results of measuring the amount of aflibercept production with the Octet® BLI Label-Free after introducing, into ARPE-19 cells by a transfection method using lipofectamine, all-in-one ssAAV2-aflibercept vectors (Example 2, Comparative Examples 1 to 3, and Comparative Examples 6 to 8) prepared by varying the type of signal peptides.

[0034] FIG. 6 shows results of in silico MHC Class I and II binding prediction performed to evaluate immunogenicity of each signal peptide used in all-in-one ssAAV2-aflibercept vectors (Example 2 and Comparative Examples 1 to 8) prepared by varying the type of signal peptides.

[0035] FIG. 7 shows results of evaluating AAV productivity (viral genome / L) of all-in-one ssAAV2-aflibercept vectors (Example 2 and Comparative Examples 2 and 4) prepared by varying the type of signal peptides.

[0036] FIG. 8 shows results of the quality (%full / empty) of AAV particles produced by all-in-one ssAAV2-aflibercept vectors (Example 2, Comparative Example 2, and Comparative Example 4) prepared by varying the type of signal peptides.

[0037] FIG. 9 shows results of analyzing the aflibercept concentration by electrochemiluminescence (ECL) after treating ARPE-19 cells with recombinant AAVs produced by all-in-one ssAAV2-aflibercept vectors (Example 2, Comparative Example 2, and Comparative Example 4) prepared by varying the type of signal peptides.

[0038] FIG. 10 shows results of VEGF neutralization assays performed to evaluate the biological activity of aflibercept expressed by treating ARPE-19 cells with recombinant AAVs produced by all-in-one ssAAV2-aflibercept vectors (Example 2, Comparative Example 2, and Comparative Example 4) prepared by varying the type of signal peptides.

[0039] FIG. 11 shows results of evaluating expression levels of aflibercept according to administered doses at 6 weeks after intravitreal injection (IVT) of an rAAV produced by an all-in-one vector of Example 2 and an rAAV produced by a triple vector of Example 3 into an animal model (rabbit).

[0040] FIG. 12 shows results of observing ocular inflammatory responses according to administered doses at 6 weeks after IVT of an rAAV produced by an all-in-one vector of Example 2 and an rAAV produced by a triple vector of Example 3 into an animal model (rabbit). Detailed Description

[0041] All technical terms as used in the present specification, unless otherwise specified, have the same meaning as commonly understood by those of ordinary skill in the relevant art. In addition, suitable methods or samples are described in the present specification, but similar or equivalent ones are also within the scope of the present specification. Also, although not explicitly stated, numerical values described in the present specification are considered to include the meaning of "about". The contents of all publications cited as references in the present specification are incorporated herein by reference in their entirety.

[0042] In the present specification, the term "about" or "approximately" may be generally interpreted to mean a value or range that is within 10 %, 5 %, 4 %, 3 %, 2 %, or 1 % above or below a given value or range.

[0043] An aspect provides a nucleic acid expression cassette for enhanced expression of aflibercept in the retina, including: a codon-optimized polynucleotide encoding aflibercept which is operably linked to an expression regulatory element, wherein the codon-optimized polynucleotide encoding aflibercept includes a nucleotide sequence consisting of SEQ ID NO: 8 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0044] The "aflibercept" is, as an anti-vascular endothelial growth factor (anti-VEGF) agent, a recombinant fusion protein that acts as a decoy receptor for VEGF subtypes A and B (i.e., VEGF-A and VEGF-B) and a placenta growth factor (PGF). By binding to these ligands, aflibercept may prevent these ligands from binding to VEGF receptors (VEGFRs), such as VEGFR-1 and VEGFR-2, thereby inhibiting neovascularization and reducing vascular permeability. Aflibercept consists of the second domain of VEGFR-1 and the third domain of VEGFR-2, fused to the Fc fragment of lgG1 .

[0045] In an embodiment, the polynucleotide sequence encoding aflibercept may be codon-optimized for expression in a mammal or human subject. Codon optimization may be achieved by any method known in the art. The codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression of a gene in a target or host cell of interest, e.g., a human retinal cell, by replacing at least one codon (e.g., about 1 , 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, or more codons or even more) of the native sequence with a more frequently used or most frequently used codon in the host cell while maintaining the native amino acid sequence.

[0046] The term "polynucleotide" may refer to any form of nucleic acids, including DNA and RNA, and oligonucleotides. The polynucleotide may include a naturally occurring, synthetic, and intentionally modified or altered polynucleotide. A sequence or structure of a particular polynucleotide may be described according to the convention of providing the sequence in the 5' to 3' direction.

[0047] The "polypeptide," "protein," and "peptide" encoded by the polynucleotide sequence may include functional subsequences, modified forms, or sequence variants, as long as they retain the functionality of the native protein. The term "modified or "variant" refers that a polynucleotide sequence or a peptide sequence deviates from a reference sequence. Thus, a modified sequence or a variant sequence may have substantially the same or greater or less activity or function than the reference sequence, but retain at least partial activity or function of the reference sequence.

[0048] Non-limiting examples of modifications may include a substitution, an insertion, and / or a deletion of one or more nucleotides or amino acids. An example of an amino acid substitution may be a conservative amino acid substitution. Examples of the conservative amino acid substitution have been known in the art. The "conservative substitution" is replacement of an amino acid by a biologically, chemically, or structurally similar residue. Biological similarity refers that a substitution does not destroy biological activity. Structural similarity refers that amino acids are similar in length (e.g., alanine, glycine, and serine) or in size. Chemical similarity refers that residues have the same charge, the same hydrophilic or hydrophobic properties, or both. For example, the conservative amino acid substitution may include substitution within the following groups: glycine / alanine, valine / isoleucine / leucine, aspartic acid / glutamic acid, asparagine / glutamine, serine / threonine, lysine / arginine, and phenylalanine / tyrosine.

[0049] In the present specification, the polynucleotide or the polypeptide may include gene or protein variants possessing one or more biological activities (e.g., VEGF- neutralizing ability, etc.).

[0050] At a nucleotide sequence level, naturally occurring and non-naturally occurring variant genes may have at least 50 % or more, 70 % or more, 80 % or more, 85 % or more, 90 % or more, 95 % or more, 96% or more, 97% or more, 98% or more, 99 % or more, 99.5 % or more, or 99.9 % or more sequence identity to a reference gene.

[0051] An amino acid sequence level, naturally occurring and non-naturally occurring variant proteins may have at least 70 % or more, 80 % or more, 85 % or more, 90 % or more, 95 % or more, 96 % or more, 97 % or more, 98 % or more, 99 % or more, 99.5 % or more, or 99.9 % or more sequence identity to a reference protein.

[0052] The term "sequence identity" refers to a degree of identity of amino acid residues or bases between sequences after aligning two sequences to achieve maximum match over a particular comparison region. The sequence identity may be confirmed by methods known in the art. The percentage of sequence identity may be determined by using a sequence comparison program known in the art, such as BLAST of NCBI.

[0053] The codon-optimized polynucleotide encoding aflibercept may include or consist of a nucleotide sequence consisting of SEQ ID NO: 8 or a nucleotide sequence having 90 % or more, 95 % or more, 96% or more, 97 % or more, 98 % or more, 99 % or more, 99.5 % or more, 99.9 % or more, or 100 % sequence identity thereto.

[0054] The codon-optimized polynucleotide encoding aflibercept may include or consist of a nucleotide sequence of SEQ ID NO: 8.

[0055] The codon-optimized polynucleotide may be expressed at a higher level than a non-codon-optimized sequence. The polynucleotide may be expressed at a higher level than existing codon-optimized sequences.

[0056] The term "expression cassette" refers to a nucleic acid molecule including an aflibercept gene and other regulatory elements therefor. The expression cassette may be delivered to a packaging host cell via a genetic element (e.g., a plasmid) and packaged into a capsid of a viral vector (e.g., virus particles). The expression cassette for producing a viral vector may include the aflibercept sequence described herein, flanked by packaging signals of the virus genome, and other expression regulatory elements such as those described herein.

[0057] The term "expression regulatory element" may be used interchangeably with the term "expression regulatory sequence", and may be an element involved in the regulation of gene expression. The expression regulatory element may include at least one selected from a promoter, a transcription factor, an enhancer, a silencer, an insulator, an intron, a splicing donor and acceptor, an engineered splicing donor and acceptor, a riboswitch, an amino acid, a microRNA (miRNA), a short hairpin RNA (shRNA), a 5'- or 3'-untranslated region (UTR), a Kozak sequence, an initiation codon, a signal peptide, a polyadenylation signal sequence, and the like. The expression regulatory element may be "operably linked" to the aflibercept genetic sequence.

[0058] The term "operably linked" refers to both an expression regulatory element that is adjacent to a gene-of-interest and an expression regulatory element that acts in trans or at a distance to control the gene-of-interest.

[0059] The expression regulatory element may include at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

[0060] The term "enhancer" may refer to a regulatory region that promotes transcription of a gene.

[0061] The term "promoter" may refer to a sequence that drives gene expression. The promoter may control an expression level and an expression time of each gene.

[0062] The term "intron" refers to a nucleotide sequence that is removed by RNA splicing from the final gene product. Use of an intron downstream of an enhancer / promoter region and upstream of a cDNA insert may increase a gene expression level.

[0063] The term "polyadenylation signal sequence" is also known as "polyA sequence", and refers to, for example, a polyadenylation signal sequence positioned at the 3'- terminus of a transgene, which allows addition of a poly-A tail to the end of a nascent mRNA during transcription. The poly-A tail consists of up to 300 adenosine ribonucleotides that stabilize the mRNA by protecting it from enzymatic degradation and aid in translation.

[0064] In an embodiment, the enhancer may include or consist of a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having 90 % or more, 95 % or more, 96 % or more, 97 % or more, 98 % or more, 99 % or more, 99.5 % or more, 99.9 % or more, or 100 % sequence identity thereto. The nucleic acid expression cassette may include a CMV immediate early enhancer to enable strong expression given the locally injectable nature of an affected area. The CMV immediate early enhancer may be a human CMV immediate early enhancer.

[0065] In an embodiment, the promoter may include or consist of a CMV immediate early promoter sequence or a nucleotide sequence having 90 % or more, 95 % or more, 96 % or more, 97 % or more, 98 % or more, 99 % or more, 99.5 % or more, 99.9 % or more, or 100% sequence identity thereto. The nucleic acid expression cassette may include a CMV immediate early promoter to enable strong expression given the locally injectable nature of an affected area. The CMV immediate early promoter may be a human CMV immediate early promoter.

[0066] In an embodiment, the expression regulatory element may include or consist of a CMV immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having 90 % or more, 95 % or more, 96 % or more, 97 % or more, 98 % or more, 99 % or more, 99.5 % or more, 99.9 % or more, or 100 % sequence identity thereto.

[0067] In an embodiment, the intron may include or consist of a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having 90 % or more, 95 % or more, 96 % or more, 97 % or more, 98 % or more, 99 % or more, 99.5 % or more, 99.9 % or more, or 100 % sequence identity thereto. The nucleic acid expression cassette may have reduced immunogenicity by using a human-derived intron sequence.

[0068] In an embodiment, the polyadenylation signal sequence may include or consist of a human growth hormone poly A (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having 90 % or more, 95 % or more, 96 % or more, 97 % or more, 98 % or more, 99 % or more, 99.5 % or more, 99.9 % or more, or 100 % sequence identity. The nucleic acid expression cassette may have reduced immunogenicity by using a human-derived polyadenylation signal sequence.

[0069] In an embodiment, the nucleic acid expression cassette may further include a polynucleotide encoding a signal peptide of dermcidin, wherein the polynucleotide encoding the signal peptide may be located at the 5'-terminus of the codon-optimized polynucleotide encoding aflibercept.

[0070] The term "signal peptide" may be used interchangeably with the terms "leader peptide" and "secretion signal peptide". The signal peptide may be an exogenous peptide from aflibercept. The signal peptide may be a human signal peptide of dermcidin, and may include or consist of an amino acid sequence consisting of SEQ ID NO: 11. The nucleic acid expression cassette may have reduced immunogenicity by using a human-derived signal peptide. The nucleic acid expression cassette may produce rAAV virus particles having reduced immunogenicity at high productivity and high quality by using the human-derived signal peptide of dermcidin, and may enhance the expression of aflibercept in the eye. The polynucleotide encoding the signal peptide of dermcidin may include or consist of SEQ ID NO: 7.

[0071] In an embodiment, the nucleic acid expression cassette may further include a Kozak sequence. The kozak sequence may include or consist of 5’-GCCGCCACC-3’.

[0072] In an embodiment, rAAV produced using the nucleic acid expression cassette was shown to be able to express and secrete aflibercept in the retina at the therapeutic concentrations even at low viral administration doses, thereby minimizing capsid- induced immune responses due to low viral doses.

[0073] Another aspect provides a plasmid for producing recombinant AAV (rAAV), including: (a) an AAV 5' inverted terminal repeat (ITR); (b) the nucleic acid expression cassette according to an aspect; and (c) an AAV 3' ITR. In the plasmid for producing rAAV, the nucleic acid expression cassette is as described above.

[0074] In an embodiment, the plasmid for producing rAAV may be for triple transfection, which introduces three plasmids of a vector including a helper gene, a vector including Rep / Cap genes, and a vector including a GOI (including a portion from L-ITR to R-ITR) into a host cell. Specifically, the plasmid for producing rAAV may be a GOI expression vector in a triple transfection system.

[0075] The plasmid for producing rAAV may include (b) the nucleic acid expression cassette according to an aspect including a transgene.

[0076] The transgene refers to a gene that is transferred from one organism to another. The transgene may be a heterologous polynucleotide. The transgene may be a gene- of-interest (GOI) to be packaged into an rAAV capsid. The transgene may be a therapeutic gene. Therefore, the plasmid for producing rAAV may be used for the production of clinical materials of gene therapy agents for the treatment of a patient. The number of transgenes may be 1 or 2 or more.

[0077] The term "packaging" refers to a series of intracellular events that lead to the assembly and encapsulation of rAAV particles.

[0078] The transgene may include the codon-optimized polynucleotide encoding aflibercept according to an aspect. Therefore, the plasmid for producing rAAV may produce rAAV that can be used as a gene therapy agent for treating a neovascular ophthalmic disease.

[0079] The transgene may be arranged between ITRs. The transgene may be flanked by ITRs. The transgene may be flanked on sides by two ITRs. The transgene may be arranged between (a) a 5' ITR (also referred to as L-ITR or first ITR) and (c) 3 ' ITR (also referred to as R-ITR or second ITR). For example, an L-ITR (first ITR), a transgene, and an R-ITR (second ITR) may be arranged sequentially in the 5' to 3' direction.

[0080] The ITR may be involved in replication of the AAV genome and packaging of AAV particles. The ITR may include a Rep-binding element (RBE), RBE', and A, A, B, B', C, C, and D regions. The ITR consists of two arm palindromes (B-B1and C-C) embedded in a larger system palindrome. Consequently, the ITR may have a T-shaped stem-loop structure. The ITR may have two configurations, i.e., flip and flop. The flip configuration and the flop configuration may have a B-B' palindrome and a C-C palindrome that are closest to the 3'-terminus, respectively. In addition, two ITRs on both sides may be referred to: a first ITR and a second ITR; a left (L)-ITR and a right (R)-ITR; or a 5'-ITR and a 3'-ITR. The D region is only present once at each end, and thus remains single-stranded. The ITR structure and sequence are known in the art.

[0081] The ITR may be derived from a virus belonging to the genus Dependovirus in the family Parvoviridae. The ITR may be derived from AAV. The ITR may be derived from any AAV serotype. The ITR may be derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13. The AAV serotype may also include an artificial AAV serotype. The two ITRs on both sides may be derived from the same or different AAV serotypes.

[0082] In an embodiment, the ITR may be derived from AAV2. Both the 5' ITR and the 3' ITR may be derived from AAV2. Specifically, the ITR may be an ITR of wild-type AAV2. The 5' ITR may consist of SEQ ID NO: 3, and the 3' ITR may consist of SEQ ID NO: 10. The rAAV produced by the plasmid may be single-stranded AAV (ssAAV).

[0083] In one or more embodiments, the plasmid for producing rAAV may further include: (d) a helper virus gene required for producing AAV; (e) a Rep gene of AAV; and (f) a Cap gene of AAV, thereby forming an all-in-one vector system capable of producing rAAV by using a single plasmid. More specifically, the plasmid for producing rAAV may be to provide a nucleic acid molecule including, in a single nucleic acid molecule, (a) an AAV 5' ITR; (b) the nucleic acid expression cassette according to an aspect; (c) an AAV 3' ITR; (d) a nucleotide sequence of the helper virus gene required for producing AAV; (e) a nucleotide sequence of the Rep gene of AAV; and (f) a nucleotide sequence of the Cap gene of AAV. In other words, the plasmid for producing rAAV may be an all-in-one vector in which all of (a) to (f) may be integrated into a single plasmid. The term "all-in- one vector" may be used interchangeable with "single vector system" and "all-in-one plasmid". The plasmid for producing rAAV may be one-plasmid.

[0084] The following description applied commonly to the plasmid according to an aspect.

[0085] The "adeno-associated virus (AAV)" is a virus belonging to the genus Dependovirus in the family Parvoviridae. The AAV does not have the ability to replicate on its own, and thus it requires the coexistence of a helper virus for replication. The AAV has a single-stranded DNA (ssDNA) of approximately 4.7 kb.

[0086] The "recombinant AAV (rAAV)" may be used interchangeably with "AAV vector", "AAV particles", "AAV vector particles", "rAAV particles", and "rAAV vector particles", and may refer to an AAV vector capable of expressing a target protein in a target cell. The term "recombinant" refers an AAV or sequence which has been engineered in a way that does normally occur in nature. For example, a recombinant vector such as an AAV vector may refer to a case where a polynucleotide that is not normally present in the wild-type AAV genome has been inserted into the viral genome. Therefore, the rAAV produced by the plasmid for producing rAAV may be applied as a gene therapy agent. For example, the rAAV produced by the plasmid may express the aflibercept protein, and thus may be used as a gene therapy agent for a neovascular ophthalmic disease.

[0087] The term "plasmid for producing rAAV" may refer to a plasmid capable of producing rAAV in a host cell into which the plasmid has been introduced. The plasmid may refer to a plasmid vector.

[0088] The term "vector" may refer to a vehicle capable of artificially transporting a heterologous genetic material into another cell.

[0089] The plasmid may be a DNA molecule. The plasmid may be single-stranded (ss) or double-stranded (ds). The plasmid may be a linear DNA molecule or a circular DNA molecule. The plasmid may include the gene, specifically, a sequence of the gene, more specifically, a nucleotide sequence of the gene, and more specifically, a nucleotide sequence encoding the gene. The term "nucleotide sequence" may be used interchangeably with terms "nucleic acid sequence" and "DNA sequence".

[0090] The plasmid may exist in various forms. For example, the plasmid may be linear or circular. In an embodiment, the plasmid may be a circular plasmid.

[0091] The plasmid for producing rAAV may include (d) a helper virus gene required for producing AAV.

[0092] The helper virus may refer to a virus that assists proliferation of a virus, which cannot reproduce through single infection, through simultaneous infection. The AAV does not have the ability to replicate on its own, and thus it requires a helper virus gene for AAV replication.

[0093] The helper virus gene may be a gene of the helper virus required for producing AAV. In the present specification, the term "helper virus gene" may be used interchangeably with the term "helper gene".

[0094] A nucleotide sequence of the helper virus gene may be derived from one or more selected from adenovirus, herpes simplex virus (HSV), baculovirus, papillomavirus, and bocavirus, but embodiments are not limited thereto.

[0095] 50 or more serotypes of the adenovirus are known in the art. In an embodiment, the nucleotide sequence of the helper virus may be derived from the adenovirus. The adenovirus may be selected from adenovirus 2 (Ad2) and adenovirus 5 (Ad5). The adenovirus may be adenovirus 2 (Ad2). For example, the nucleotide sequence of the helper virus gene may be derived from wild-type adenovirus 2 (Ad2).

[0096] The HSV may be HSV type 1 or HSV type 2.

[0097] The papillomavirus may be papilloma virus or human papilloma virus (HPV). The HPV is known to have 150 or more types. For example, the HPV may be HPV-16, but embodiments are not limited thereto.

[0098] The helper virus gene may include one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA (or referred to as "VA RNA gene"), DNA-binding protein (DBP), and a variant thereof. The helper virus gene is a gene encoding the helper protein.

[0099] The variant may be an engineered helper virus gene.

[0100] In the present specification, term "engineered" may refer that genes have been intentionally modified and manipulated by using genetic engineering techniques. The genetic engineering techniques for engineering genes are known in the art. In the present specification, 'engineered A' may include a variant including one or more mutations in the wild-type sequence of A, a variant in which a part of the sequence of A is truncated, and the like. The mutation may include an insertion, a substitution, a deletion, or a combination thereof.

[0101] The helper virus gene may include a helper virus gene of adenovirus 2 (Ad2). In an embodiment, the helper virus gene may include E2a, E4, and VA.

[0102] The plasmid for producing rAAV may include (e) the Rep gene of AAV.

[0103] The Rep (replication) gene may be a gene required for producing AAV. The Rep gene may be derived from any AAV serotype. The Rep gene may be derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV1 1 , AAV12, and AAV13. When there are two or more of the Rep gene, each gene may be derived from the same or different AAV serotypes. In an embodiment, a nucleotide sequence of the Rep gene may be derived from AAV2.

[0104] The Rep gene may include a gene encoding a known Rep protein or a variant thereof.

[0105] The Rep gene may include one or more selected from Rep78, Rep68, Rep52, Rep40, and a variant thereof. The gene may include Rep68. The Rep gene may include Rep78 or Rep68. The Rep gene may include: (i) Rep78; and (ii) one or more selected from Rep68, Rep52, and Rep40. The Rep gene may include: (i) Rep68; and (ii) one or more selected from Rep78, Rep52, and Rep40. The Rep gene may include: (i) Rep68; and (ii) one or more selected from Rep52 and Rep40. The Rep gene may include all of Rep78, Rep68, Rep52, and Rep40.

[0106] In an embodiment, the Rep gene may include one or more (one, two, or three) selected from Rep68, Rep52, and Rep40. When Rep78 is overexpressed, the cytotoxicity may increase while the AAV productivity may decrease. Therefore, the Rep gene may include a Rep gene except for Rep78.

[0107] In an embodiment, the Rep gene may include a Rep gene of AAV2. In an embodiment, the Rep gene may include Rep78, Rep68, Rep52, and Rep40.

[0108] The variant may be an engineered Rep (ERep) protein. The variant may be a variant having one or more mutations in the wild-type sequence, or a truncated variant.

[0109] In an embodiment, a nucleotide sequence of the Rep gene may be a Rep gene of wild-type AAV2.

[0110] The plasmid for producing rAAV may include (f) the Cap gene of AAV.

[0111] The Cap (Capsid) gene may be a gene encoding a virus capsid protein.

[0112] The Cap gene may be derived from any AAV serotype. The Cap gene may be derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhl 0, AAV11 , AAV12, and AAV13. When there are two or more of the Cap gene, each gene may be derived from the same or different AAV serotypes. In an embodiment, a nucleotide sequence of the Cap gene may be derived from AAV2.

[0113] The Cap gene may include a gene encoding a known Cap protein or a variant thereof.

[0114] The Cap gene may encode one or more selected from a capsid protein, a VP1 protein, a VP2 protein, a VP3 protein, and a variant thereof, but embodiments are not limited thereto. The Cap gene may encode all of the VP1 , VP2, and VP3 proteins.

[0115] The variant may be an engineered Cap (ECap) protein. The variant may be a variant having one or more mutations in the wild-type sequence, or a truncated variant.

[0116] In an embodiment, a nucleotide sequence of the Cap gene may be a Cap gene of wild-type AAV2.

[0117] Nucleotide sequences of the Rep gene and the Cap gene may be derived from the same or different AAV serotypes. In an embodiment, both the Rep gene and the Cap gene may be derived from AAV2.

[0118] The plasmid for producing rAAV may include a plasmid backbone. The "plasmid backbone" refers to a non-genomic portion, which is important for cloning and amplification of the plasmid, processes necessary for proliferation and recombinant of viruses, but is not packaged or encapsulated into virus particles. The plasmid backbone may include one or more (one or two) selected from a replication origin and a selectable marker. The plasmid backbone may not include a replication origin.

[0119] The plasmid may not include a replication origin, or may additionally include a replication origin. The replication origin may be a known replication origin or a variant thereof. The replication origin may be a pUC origin, a pBR322 origin, a pMB1 origin, a pSC101 origin, a p15A origin, or a synthetic origin, embodiments are not limited thereto.

[0120] In an embodiment, the plasmid may include a pBR322 origin. The plasmid may additionally include a selectable marker or a reporter capable of providing selection or identification of a host cell into which the plasmid has been introduced. The selectable marker or the reporter is known in the art. Non-limiting examples of the selectable marker may include genes providing resistance to ampicillin, streptavidin, kanamycin, hygromycin, neomycin, puromycin, blasticidin, zeocin, and the like. Non-limiting examples of the reporter may include luciferase, a green fluorescent protein (GFP), a red fluorescent protein (RFP), a blue fluorescent protein (BFP), and the like. In an embodiment, the plasmid may additionally include a selectable marker.

[0121] In an embodiment, the plasmid may additionally include kanamycin.

[0122] "Gene amplification" may refer to the process by which a particular DNA sequence (i.e., a gene) in the genome is replicated disproportionately with respect to other sequences in the genome, such that the amplified DNA sequence is present in a higher copy number than was initially present. In the present specification, the term "amplified" or "amplification" may refer to a gene or nucleic acid sequence present in two or more copies in a host cell by gene amplification.

[0123] An "amplifiable selectable marker" may refer to a gene that allows amplification of the gene under appropriate growth conditions.

[0124] When the plasmid is an all-in-one vector system further including (d), (e), and (f), (a) to (c), (d), (e), and (f) may be randomly arranged in the 5' to 3' direction or in the 3' to 5' direction. In the plasmid, (a) to (c), (d), (e), and (f) may be arranged in any order in the 5' to 3' direction or in the 3' to 5' direction. In the plasmid, (a) to (c), (d), (e), and (f) may be arranged in any order in the 5' to 3' direction.

[0125] In the plasmid, (a) to (c), (d), (e), and (f) may each independently be arranged in any order in the forward orientation or in the reverse orientation.

[0126] The forward orientation may refer to an arrangement in which a gene is inserted in the 5' to 3' direction in the 5'— >3' strand ((+) strand). The reverse orientation may refer to an arrangement in which a gene is inserted in the 3' to 5' direction in the 5'— >3' strand.

[0127] In the plasmid, (a) to (c), (d), (e), and (f) may be arranged in the following order: (d) - (e) - (f) - (a) - (b) - (c).

[0128] In an embodiment, the plasmid backbone may be arranged between (d) and (e). Therefore, the arrangement order of the components in the nucleic acid molecule may be (d) - plasmid backbone - (e) - (f) - (a) - (b) - (c). The plasmid backbone may include one or more selected from the replication origin and the selectable marker.

[0129] In an embodiment, the components of the plasmid may be arranged in the following order: (d) - plasmid backbone - (e) - (f) - (a) - enhancer - promoter - intron - polynucleotide encoding signal peptide - codon-optimized polynucleotide encoding aflibercept - polyadenylation signal sequence - (c).

[0130] The plasmid and the vector may be prepared by any suitable techniques, and such techniques are well known in the art.

[0131] When the plasmid for producing rAAV is an all-in-one vector system further including (d), (e), and (f), all components necessary for producing rAAV are linked within a single molecule, so that each component may be introduced into a host cell at the same ratio. A host cell into which a single plasmid is introduced may enable balanced gene expression. Balanced gene expression may improve the rAAV productivity and / or may increase the %F / E capsid ratio of rAAV. The improved rAAV productivity may reduce unit production costs. In detail, the improved rAAV productivity may enable improvements in the total yield of a purification process, which accounts for 60 % or more of drug production costs.

[0132] In addition, when such an all-in-one vector system is used, only one single plasmid needs to be produced instead of the independent production of three plasmids in the existing triple transfection system, and thus the cost of raw materials may be reduced. In addition, since the all-in-one vector system only requires the introduction of a single plasmid without the need to introduce three plasmids separately as in the existing triple transfection system, the input amount of individual plasmid may be reduced by 50% or more.

[0133] Another aspect provides an isolated host cell transfected or transduced with the plasmid for producing rAAV according to an aspect. In the host cell, the plasmid for producing rAAV is the same as described herein.

[0134] The host cell may be a mammalian cell. The mammalian cell may include cells derived from any origin or tissue of humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cows, and anthropoids. The mammalian cell may be selected from HEK293 cells, CHO cells, Jurkat cells, KS62 cells, PerC6 cells, HeLa cells, MDCK cells, C127 cells, A549 cells, Vero cells, WI38 cells, MRC5 cells, HT1080 cells, or deviates or functional equivalents thereof, but embodiments are not limited thereto.

[0135] In an embodiment, the mammalian cells may be selected from cells derived from HEK293 cells, HEK293F cells, HEK293T cells, and cells derived therefrom. The HEK293 cells may be human embryonic kidney 293 cell line commonly used in biotechnology. 'Cells derived from HEK293 cells, HEK293F cells, or HEK293T cells' may refer to cells derived from parent cell lines, such as HEK293 cells, HEK293F cells, or HEK293T cells, and may include all of commercially available cells and cells to be developed in the future. Examples of cells derived from the HEK293 parent cell line may include Expi293F (manufacturer: ThermoFisher), HEK293F (manufacturer: ThermoFisher), HEK293.2 (ATCC), and the like. Examples of cells derived from the HEK293T parent cell line may include HEK293FT (manufacturer: ThermoFisher) and the like. Examples of the HEK293F parent cell line may include Viral Production Cells 1 .0 (VPC1 .0) (manufacturer: ThermoFisher), Viral Production Cells 2.0 (VPC2.0) (manufacturer: ThermoFisher), and the like. VPC2.0 is a clonal cell derived from the HEK293F parent cell line, and is a host cell suitable for producing AAV. In an embodiment, the mammalian cell may be a HEK293F cell or a cell derived therefrom.

[0136] The host cell may be an insect cell. The insect cell may include cells derived from Spodoptera frugiperda or Trichoplusia ni. The insect cell may be selected from Sf9 cells, Sf21 cells, TN-5B1-4 cells, High Five cells, or deviates or functional equivalents thereof, but embodiments are not limited thereto.

[0137] Another aspect provides a method of producing rAAV. The method may be a method using the plasmid for producing rAAV, which is an all-in-one vector system further including (d), (e), and (f).

[0138] The method may include: introducing the plasmid for producing rAAV, which is an all-in-one system further including (d), (e), and (f), into a host cell; and isolating the rAAV from the host cell.

[0139] In the method, the amount of plasmid DNA (pDNA) introduced per 0.25X106to 3x106host cells may be, in a range of about 0.1 pg to about 10 pg, about 0.1 pg to about 5 pg, about 0.1 pg to about 4 pg, about 0.1 pg to about 3 pg, about 0.5 pg to about 10 pg, about 0.5 pg to about 5 pg, about 0.5 pg to about 4 pg, about 0.5 pg to about 3 pg, about 1 pg to about 10 pg, about 1 pg to about 5 pg, about 1 pg to about 4 pg, or about 1 pg to about 3 pg, but embodiments are not limited thereto. In an embodiment, the amount of pDNA introduced per 3x6host cells may be, in a range of about 0.1 pg to about 10 pg, about 0.1 pg to about 5 pg, about 0.1 pg to about 4 pg, about 0.1 pg to about 3 pg, about 0.5 pg to about 10 pg, about 0.5 pg to about 5 pg, about 0.5 pg to about 4 pg, about 0.5 pg to about 3 pg, about 1 pg to about 10 pg, about 1 pg to about 5 pg, about 1 pg to about 4 pg, or about 1 pg to about 3 pg, but embodiments are not limited thereto.

[0140] The introducing may be used without limitation by any known method capable of inserting the plasmid for producing rAAV into a host cell. The introducing may be performed by transfection, transformation, or transduction. The terms "transfection", "transformation", and "transduction" may be used to describe insertion of a nonmammalian vector or a viral vector into a target cell. Insertion of a vector is generally called transformation for bacterial cells and transfection for eukaryotic cells, and insertion of a viral vector is also called transduction. By using methods known in the art, those skilled in the art may introduce the plasmid into a host cell. Non-limiting examples of the introducing may include a physical method (e.g., electroporation, cell compression, sonoporation, optical transfection, protoplast fusion, imperfection, magnetofection, gene gun, or particle impact), a method using a chemical reagent (e.g., calcium phosphate, a highly branched organic compound, or a cationic polymer), or a cationic lipid (e.g., lipofection), and the like, but embodiments are not limited thereto. The cationic polymer may include polyethyleneimine (PEI), but embodiments are not limited thereto. A transfection method may additionally require contacting a cell with a pDNA solution, growing the cell, and selecting the cell by expression of a marker gene.

[0141] The method may further include, after the introducing, culturing the cell.

[0142] The culturing may be culturing the host cell under conditions under which the rAAV may be produced. Appropriate culturing methods are well known to those skilled in the art. For example, the cell may be cultured in suspension and / or under animal component-free conditions.

[0143] The method may further include, after the culturing, selecting the cell into which the plasmid has been introduced by using a selectable marker.

[0144] The separating may be performed by a method known in the art. In an embodiment, the separating may be performed by centrifugation or chromatography.

[0145] The centrifugation may be cesium chloride (CsCI)-based ultrahigh-speed centrifugation, but embodiments are not limited thereto.

[0146] The chromatography may include one or more selected from affinity chromatography, ion-exchange chromatography, column chromatography, gel-filtration chromatography, thin-layer chromatography, radial flow chromatography, interference chromatography, and reverse-phase chromatography, but embodiments are not limited thereto.

[0147] The method may also be easily scaled up to industrial production because it requires only single transfection of the host cell by the plasmid for producing rAAV having excellent rAAV productivity.

[0148] According to the method, unlike the existing triple transfection of three plasmids, only a single plasmid needs to be introduced, so that the rAAV production process is simple, and thus costs may be reduced. In addition, unlike the triple transfection method in which the ratios of three plasmids introduced into a cell cannot be precisely controlled, the method according to an aspect allows each component to be introduced at the same ratio because each component is integrated into a single plasmid. Accordingly, the rAAV productivity may be improved. rAAV produced by the method may have an increased full capsid ratio (%full capsid) compared to rAAV produced by the triple transfection. Therefore, high-quality rAAV may be produced by the method.

[0149] Another aspect provides rAAV including an AAV capsid protein and a vector genome, wherein the vector genome includes: (a) an AAV 5' ITR; (b) the nucleic acid expression cassette according to an aspect; and (c) an AAV 3' ITR. In the rAAV, the nucleic acid expression cassette and the ITR are the same as described herein.

[0150] In an embodiment, the AAV capsid may be any one AAV capsid selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV1 1 , AAV12, and AAV13.

[0151] In an embodiment, the AAV capsid may be a wild-type AAV2 capsid.

[0152] The AAV capsid may include a VP1 protein, a VP2 protein, and a VP3 protein.

[0153] The "vector genome" of AAV may refer to a sequence that is ultimately packaged or encapsulated to form a virus particle.

[0154] The vector genome may be linear single-stranded DNA.

[0155] The vector genome may include, in the 5' to 3' direction, a 5' ITR, an enhancer, a promoter, an intron, a polynucleotide encoding a signal peptide, a codon-optimized polynucleotide encoding aflibercept, a polyadenylation signal sequence, and a 3' ITR.

[0156] Another aspect provides a pharmaceutical composition for preventing or treating a neovascular ophthalmic disease, including: the rAAV according to an aspect; and a pharmaceutically acceptable carrier. In the pharmaceutical composition, the rAAV is the same as described herein.

[0157] The pharmaceutical composition may be used for gene therapy fora neovascular ophthalmic disease. The pharmaceutical composition may be a composition for delivering an aflibercept protein for gene therapy for a neovascular ophthalmic disease.

[0158] The term "gene therapy" may refer to a treatment that uses genes to treat or prevent a disease. The rAAV for delivering a therapeutic gene into a cell may be used as a gene therapy agent. Diseases to which gene therapy can be applied may include diseases caused by defects in a single gene, but embodiments are not limited thereto.

[0159] The pharmaceutically acceptable carrier may be used as a term including an excipient, a diluent, or an auxiliary agent. For the pharmaceutically acceptable carrier, one suitable for delivering rAAV into a living body may be used. Specifically, for the pharmaceutically acceptable carrier, one suitable for formulation into a parenteral formulation (e.g., an injection formulation) may be used. For example, for the pharmaceutically acceptable carrier, one suitable for formulation as an intraocular injection or intravitreal injection formulation may be used. The pharmaceutically acceptable carrier may be an aqueous solution, such as water or a buffered saline solution.

[0160] The pharmaceutical composition may be prepared in any dosage form according to a conventional method. The pharmaceutical composition may be formulated in a form suitable for delivering rAAV to a subject. The pharmaceutical composition may be formulated in an aqueous solution, for example, in water or a buffered saline solution. The pharmaceutical composition may be formulated as an injection formulation suitable for administration by any suitable route, such as an intraocular, intravitreal, intraretinal, intrachoroidal, or subretinal route. For example, the pharmaceutical composition may be formulated as an injection formulation suitable for administration by an intravitreal route. The pharmaceutical composition may be prepared as a systemic formulation or a topical formulation. In an embodiment, the pharmaceutical composition may be used for intravitreal injection.

[0161] The pharmaceutical composition may additionally include an additional second therapeutic agent having a preventive or therapeutic effect on a neovascular ophthalmic disease. The pharmaceutical composition may be a single composition or an individual composition.

[0162] The pharmaceutical composition may include the rAAV in a pharmaceutically effective amount. The effective amount may be appropriately selected by those skilled in the art according to an individual.

[0163] In an embodiment, the neovascular ophthalmic disease may be any one selected from the group consisting of age-related macular degeneration (AMD), diabetic retinopathy (DR), retinal vein occlusion (RVO), choroidal neovascularization, diabetic macular edema (DME), corneal neovascularization, and retinopathy of prematurity. The AMD may be wet AMD.

[0164] The AMD is a degenerative eye disease that affects the macula, a small area of light sensitivity within the center of the retina that is responsible for reading and precise vision. Conditions that affect the macula may decrease central vision while leaving peripheral vision intact. In several cases, such a disease may lead to central blindness. Neovascular or exudative or wet AMD (nAMD, wAMD or nwAMD) is an advanced form of AMD. The feature of wAMD is choroidal neovascularization (CNV) characterized by infiltration of abnormal blood vessels in the retina from the basal layer of the choroid, leading to retinal cell damage and central blindness. Such an abnormal neovascularization process may be mediated by a growth factor, particularly a VEGF. The standard management for wAMD is a class of molecules that bind to a VEGF and isolate it, such as ranibizumab (Lucentis) and aflibercept (Eylea).

[0165] DR is a major complication of diabetes, and is a leading cause of vision loss in the working-age population. DR may be non-proliferative DR (NPDR), which does not have new growth of blood vessels, or proliferative DR (PDR), which has abnormal growth of blood vessels within the retina or choroid. Diabetic macular edema (DME) is a complication of DR, and is another example of an eye disease that affects the macula. DME affects up to 10 % of people with diabetes, and is caused by fluid accumulation within the macula. DME is the most common cause of vision loss in people with DR. Available therapies to treat DME may include lasers and anti-VEGF drugs, such as aflibercept.

[0166] The term "prevention" refers to any action that prevents the occurrence of a disease in advance, inhibits a disease, or delays the progression of a disease. For example, this term refers to preventing or interrupting the occurrence of a disease or characteristic features thereof, or defending or protecting against the occurrence of a disease or characteristic features thereof.

[0167] The term "treatment" refers to alleviating, attenuating, or ameliorating a symptom, ameliorating or preventing a basal metabolic cause of a symptom, inhibiting a neovascular ophthalmic disease, for example, stopping the occurrence of a neovascular ophthalmic disease, alleviating a neovascularophthalmic disease, causing degeneration of a neovascular ophthalmic disease, or stopping a symptom of a neovascular ophthalmic disease.

[0168] Another aspect provides a method of preventing or treating a neovascular ophthalmic disease, including administering an effective amount of the rAAV according to an aspect or the pharmaceutical composition according to an aspect, to a subject in need thereof. In the method, the rAAV, the pharmaceutical composition, the neovascular ophthalmic disease, the prevention, and the treatment may be the same as described herein.

[0169] Regarding the aspects, the subject may be a subject requiring expression of aflibercept that is delivered by the rAAV. The subject may be an individual suffering from or at high risk of suffering from a neovascular ophthalmic disease. The subject may be an individual suffering from or at high risk of suffering from a disease that can be treated by expression of aflibercept that is delivered by the rAAV. The subject may be a patient with a neovascular ophthalmic disease. The subject may be a mammal. The mammal may include humans, mice, rats, hamsters, monkeys, rabbits, donkeys, horses, sheep, cows, and the like, but embodiments are not limited thereto.

[0170] In an embodiment, the subject may be a human. The human may include a fetus, a newborn, an infant, an adolescent, or an adult.

[0171] The term "administration" may refer to a method used to deliver a therapeutic agent or a pharmaceutical composition to the site of desired biological action. The administration route may be determined by those skilled in the art, and may include intraocular, intravitreal, intraretinal, intrachoroidal, or subretinal injection. The administration route may be a combination of two or more routes, if necessary. In an embodiment, the administration may be done by intravitreal injection (IVT). The administration may be done in an amount sufficient to infect the subject and in an amount sufficient to provide a sufficient level of introduction and expression of aflibercept.

[0172] The term "effective amount" may refer to a "therapeutically effective amount", and refers to a dosage administered to achieve a therapeutic effect.

[0173] The dosage administered to achieve a therapeutic effect, e.g., dosage of vector genome / eye (vg / eye), may vary based on several factors including the administration route, an expression level of heterologous polynucleotide required to achieve a therapeutic effect, a particular disease being treated, any host immune response by the viral vector, a host immune response to a heterologous polynucleotide or an expressed product (protein), and stability of the expressed protein. Those skilled in the art may determine a dosage range of the rAAV for the treatment of a patient with a particular disease or disorder, based on factors other than the aforementioned factors. A therapeutically effective amount of the rAAV may refer to an amount sufficient to convert a severe neovascular ophthalmic disease to a moderate or mild neovascular ophthalmic disease, or to completely treat a neovascular ophthalmic disease, when administered to a subject with a neovascular ophthalmic disease.

[0174] In an embodiment, the administration may be to administer a unit dose of rAAV particles in a range of about 1 x 109to about 1 .7 x 1011per eye of the subject, for example, about 1 x 109to about 1 x 1011, about 1 x 109to about 9 x 1010, about 1 x 109to about 8 x 1010, about 1 x 109to about 7 x 1010, about 1 x 109to about 6 x 1010, about 1 x 109to about 5 x 1010, about 1 x 109to about 4 x 1010, about 1 x 109to about 3 x 1010, about 1 x 109to about 2 x 1010, about 1 x 109to about 1 x 1010, about 1 x 109to about 9 x 109, about 1 x 109to about 8 x 109, about 1 x 109to about 7 x 109, about 1 x 109to about 6 x 109, about 1 x 109to about 5 x 109, about 1 x 109to about 4 x 109, about 1 x 109to about 3 x 109, about 1 x 109to about 2 x 109, about 3.3 x 109to about 1 .7 x 1011, about 3.3 x 109to about 1 x 1011, about 3.3 x 109to about 9 x 1010, about 3.3 x 109to about 8 x 1010, about 3.3 x 109to about 7 x 1010, about 3.3 x 109to about 6 x 1010, about 3.3 x 109to about 5 x 1010, about 3.3 x 109to about 4 x 1010, about 3.3 x 109to about 3 x 1010, about 3.3 x 109to about 2 x 1010, about 3.3 x 109to about 1 x 1010, about 3.3 x 109to about 9 x 109, about 3.3 x 109to about 8 x 109, about 3.3 x 109to about 7 x 109, about 3.3 x 109to about 6 x 109, about 3.3 x 109to about 5 x 109, or about 3.3 x 109to about 4 x 109vg / eye. When the subject is a rabbit, rAAV particles may be administered per eye of the subject in a unit dose of about 1 x 109to about 5 x 1010vg / eye. When the subject is a human, rAAV particles may be administered at a dosage calculated by multiplying the unit dose of the rabbit by the human conversion factor (vitreous volume ratio), 3.33. For example, per eye of the subject, rAAV particles may be administered at a unit dose of about 3.3 x 109to about 1.7 x 1011vg / eye. The administration may include administering, to one eye of the subject, rAAV particles at a unit dose of 1.7 x 1011vg / eye or less, 1 x 1011vg / eye or less, 9 x 1010vg / eye or less, 8 x 1010vg / eye or less, 7 x 1010vg / eye or less, 6 x 1010vg / eye or less, 5.5 x 1010vg / eye or less, 5 x 1010vg / eye or less, 4.5 x 1010vg / eye or less, 4 x 1010vg / eye or less, 3.5 x 1010vg / eye or less, 3 x 1010vg / eye or less, 2.5 x 1010vg / eye or less, 2 x 1010vg / eye or less, 1.5 x 1010vg / eye or less, 1 x 1010vg / eye or less, 9.5 x 109vg / eye or less, 9 x 109vg / eye or less, 8.5 x 109vg / eye or less, 8 x 109vg / eye or less, 7.5 x 109vg / eye or less, 7 x 109vg / eye or less, 6.5 x 109vg / eye or less, 6 x 109vg / eye or less, 5.5 x 109vg / eye or less, 5 x 109vg / eye or less, 4.5 x 109vg / eye or less, 4 x 109vg / eye or less, 3.5 x 109vg / eye or less, 3.3 x 109vg / eye or less, 3 x 109vg / eye or less, 2.5 x 109vg / eye or less, 2 x 109vg / eye or less, 1 .5 x 109vg / eye or less, or 1 x 109vg / eye or less.

[0175] The rAAV produced by the expression cassette according to an aspect or the plasmid including the same can be administered via IVT, making it safer without the risk of retinal detachment as in subretinal (SR) administration, and an expression level of aflibercept is confirmed to reach a therapeutic level starting from a significantly lower dose interval (3.3E09 vg / eye). Thus, compared to ADVM-022 (Adverum Biotechnologies Inc., IVT administration), in which the expression level of aflibercept reaches a therapeutic level starting from a dose interval of 2E11 vg / eye, or RGX-314 (Regenxbio Inc., SR administration), in which the expression level of aflibercept reaches a therapeutic level starting from a dose interval of 6E10 vg / eye, it is possible to treat with lower viral doses, and at the same time, the immunogenicity may be reduced due to the low doses. By monitoring the expression level of the heterologous polynucleotide, an administration method and an administration frequency may be determined.

[0176] Another aspect provides use of the rAAV according to an aspect for the manufacture of a medicament for prevention or treatment of a neovascular ophthalmic disease. Regarding the use, the rAAV, the neovascular ophthalmic disease, the prevention, and the treatment may be the same as described herein.

[0177] Nucleic Acids, Plasmids, Host Cells, and rAAVs

[0178] Disclosed herein, in some embodiments, are nucleic acids encoding an inhibitor of VEGF-A, comprising a codon-optimized polynucleotide encoding the inhibitor of VEGF-A, wherein the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 90 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having at least 91 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having at least 92 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having at least 93 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having at least 94 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having at least 95 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having at least 96 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having at least 97 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having at least 98 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having at least 99 % sequence identity to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide consists of the nucleotide sequence of SEQ ID NO: 8.

[0179] In some embodiments, the nucleic acid further comprises the polynucleotide encoding a signal peptide of dermcidin. In some embodiments, the signal peptide comprises an amino acid sequence consisting of SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 90 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 91 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 92 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 93 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 94 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 95 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 96 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 97 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 98 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 99 % sequence identity to SEQ ID NO: 11. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the signal peptide consists of the amino acid sequence of SEQ ID NO: 11. In some embodiments, the polynucleotide encoding the signal peptide is located at the 5'-terminus of the codon-optimized polynucleotide encoding aflibercept.

[0180] In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 91 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 92 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 93 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF- A comprises an amino acid sequence having at least 94 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 96 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 97 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 99 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A consists of the amino acid sequence of SEQ ID NO: 20.

[0181] SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDS RKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLV LNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRS DQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 20)

[0182] In some embodiments, the codon-optimized polynucleotide encoding the inhibitor of VEGF-A is operably linked to an expression regulatory element. In some embodiments, the expression regulatory element includes at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the enhancer comprises a cytomegalovirus (CMV) immediate early enhancer sequence. In some embodiments, the enhancer consists of a cytomegalovirus (CMV) immediate early enhancer sequence.

[0183] In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the promoter comprises a cytomegalovirus (CMV) immediate early promoter sequence. In some embodiments, the promoter consists of a cytomegalovirus (CMV) immediate early promoter sequence.

[0184] In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence comprising SEQ ID NO: 4. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4.

[0185] In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the intron includes a human betaglobin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 94% sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence comprising SEQ ID NO: 5. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5.

[0186] In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence comprising SEQ ID NO: 9. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9.

[0187] In some embodiments, the nucleic acid further comprises a Kozak sequence.

[0188] Further disclosed herein, in some embodiments, are nucleic acids encoding an inhibitor of VEGF-A, comprising polynucleotide sequences encoding an inhibitor of VEGF-A and a signal peptide, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 1 , or an amino acid sequence having at least 90 % sequence identity with SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 91 % sequence identity with SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 92 % sequence identity with SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 93 % sequence identity with SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 94 % sequence identity with SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 95 % sequence identity with SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 96 % sequence identity with SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 97 % sequence identity with SEQ ID NO: 1 1. In some embodiments, the signal peptide comprises an amino acid sequence having at least 98 % sequence identity with SEQ ID NO: 11. In some embodiments, the signal peptide comprises an amino acid sequence having at least 99 % sequence identity with SEQ ID NO: 11. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 11. In some embodiments, the signal peptide consists of the amino acid sequence of SEQ ID NO: 11.

[0189] In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 91 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 92 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 93 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF- A comprises an amino acid sequence having at least 94 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 95 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 96 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 97 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 98 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 99 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A includes an amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 20.

[0190] In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 90 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises a nucleotide sequence having at least 91 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises a nucleotide sequence having at least 92 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises a nucleotide sequence having at least 93 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises a nucleotide sequence having at least 94 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises a nucleotide sequence having at least 95 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises a nucleotide sequence having at least 96 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises a nucleotide sequence having at least 97 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises a nucleotide sequence having at least 98 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises a nucleotide sequence having at least 99 % sequence identity with SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encoding the inhibitor of VEGF-A consists of the nucleotide sequence of SEQ ID NO: 8.

[0191] In some embodiments, the polynucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 90 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 91 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 92 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 93 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 94 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 95 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 96 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 97 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 98 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises a nucleotide sequence having at least 99 % sequence identity with SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the polynucleotide sequence encoding the signal peptide consists of the nucleotide sequence of SEQ ID NO: 7.

[0192] In some embodiments, the polynucleotide sequence encoding the signal peptide is at 5’-end of the polynucleotide sequence encoding the inhibitor of VEGF-A.

[0193] In some embodiments, the nucleic acid further comprises one or more expression regulatory elements operably linked to the polynucleotide. In some embodiments, the expression regulatory element(s) are selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the enhancer comprises a cytomegalovirus (CMV) immediate early enhancer sequence. In some embodiments, the enhancer consists of a cytomegalovirus (CMV) immediate early enhancer sequence.

[0194] In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the promoter comprises a cytomegalovirus (CMV) immediate early promoter sequence. In some embodiments, the promoter consists of a cytomegalovirus (CMV) immediate early promoter sequence

[0195] In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 93 % sequence identity thereto, In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the intron includes a human betaglobin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5.

[0196] In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9. In some embodiments, the nucleic acid further comprises a Kozak sequence.

[0197] Further disclosed herein, in some embodiments, are plasmids for producing a recombinant adeno-associated virus (rAAV), comprising (a) an AAV 5’ inverted terminal repeat (ITR), (b) the nucleic acid according to an embodiment disclosed herein; and (c) an AAV 3’ ITR. In some embodiments, the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13. In some embodiments, the ITR is derived from AAV1 . In some embodiments, the ITR is derived from AAV2. In some embodiments, the ITR is derived from AAV3A. In some embodiments, the ITR is derived from AAV3B. In some embodiments, the ITR is derived from AAV4. In some embodiments, the ITR is derived from AAV5. In some embodiments, the ITR is derived from AAV6. In some embodiments, the ITR is derived from AAV7. In some embodiments, the ITR is derived from AAV8. In some embodiments, the ITR is derived from AAV9. In some embodiments, the ITR is derived from AAV10. In some embodiments, the ITR is derived from AAVrhI O. In some embodiments, the ITR is derived from AAV11. In some embodiments, the ITR is derived from AAV12. In some embodiments, the ITR is derived from AAV13. In some embodiments, the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10.

[0198] In some embodiments, the plasmid further comprises the following genes: (a) a helper virus gene required for producing AAV; (b) a Rep gene of AAV; and (c) a Cap gene of AAV. In some embodiments, a nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus. In some embodiments, a nucleotide sequence of the helper virus gene is derived from an adenovirus. In some embodiments, a nucleotide sequence of the helper virus gene is derived from adenovirus 2 (Ad2). In some embodiments, the helper virus gene comprises one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DNA-binding protein (DBP), and a variant thereof. In some embodiments, the helper virus gene comprises E1. In some embodiments, the helper virus gene comprises E2. In some embodiments, the helper virus gene comprises E2a. In some embodiments, the helper virus gene comprises E4. In some embodiments, the helper virus gene comprises E4orf1. In some embodiments, the helper virus gene comprises E4orf2. In some embodiments, the helper virus gene comprises E4orf3. In some embodiments, the helper virus gene comprises E4orf4. In some embodiments, the helper virus gene comprises E4orf5. In some embodiments, the helper virus gene comprises E4orf6. In some embodiments, the helper virus gene comprises E4orf7. In some embodiments, the helper virus gene comprises VA. In some embodiments, the helper virus gene comprises DNA-binding protein (DBP). In some embodiments, the helper virus gene comprises E2a, E4, and VA.

[0199] In some embodiments, a nucleotide sequence of the Rep gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV1 . In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV2. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV3A. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV3B. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV4. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV5. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV6. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV7. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV8. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV9. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV10. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAVrhI O. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV11 . In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV12. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV13. In some embodiments, a nucleotide sequence of the Rep gene is a Rep gene of wild-type AAV2.

[0200] In some embodiments, a nucleotide sequence of the Cap gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7,

[0201] AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV1 . In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV2. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV3A. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV3B. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV4. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV5. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV6. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV7. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV8. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV9. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV10. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAVrhI O. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV11 . In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV12. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV13. In some embodiments, a nucleotide sequence of the Cap gene is a Cap gene of wild-type AAV2.

[0202] Further disclosed herein, in some embodiments, are isolated host cells transfected or transduced with the plasmid of any one of the embodiments disclosed herein. In some embodiments, the host cell is a mammalian cell or an insect cell. In some embodiments, the mammalian cell is selected from a HEK293 cell, a HEK293F cell, a HEK293T cell, and cells derived therefrom. In some embodiments, the insect cell is an Sf9 cell, an Sf21 cell, a TN-5B1-4 cell, or a High Five cell.

[0203] Further disclosed herein, in some embodiments, is a recombinant adeno- associated virus (rAAV) comprising the plasmid of any one of the embodiments disclosed herein.

[0204] Further disclosed herein, in some embodiments, is a recombinant adeno- associated virus (rAAV) comprising the nucleic acid of any one of the embodiments disclosed herein.

[0205] Further disclosed herein, in some embodiments, is a recombinant adeno- associated virus (rAAV) comprising a transgene plasmid comprising the nucleic acid of any one of the embodiments disclosed herein, and one or more additional plasmids comprising at least one of (a) a helper virus gene required for producing AAV, (b) a Rep gene of AAV, and (c) a Cap gene of AAV.

[0206] Disclosed herein, in some embodiments, are nucleic acids encoding an inhibitor of VEGF-A, comprising a codon-optimized polynucleotide sequence encoding the inhibitor of VEGF-A, wherein the polynucleotide further comprises (a) a helper virus gene required for producing AAV; (b) a Rep gene of AAV; and (c) a Cap gene of AAV. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 91 % sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 92% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 93% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 94% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF- A comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 96% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 97% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 98% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises an amino acid sequence having at least 99% sequence identity with SEQ ID NO: 20. In some embodiments, the inhibitor of VEGF-A comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, inhibitor of VEGF-A consists of the amino acid sequence of SEQ ID NO: 20. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 90 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 91 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 92 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 93 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 94 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 95 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 96 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 97 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 98 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 99 % to SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8. In some embodiments, the codon-optimized polynucleotide consists of the nucleotide sequence of SEQ ID NO: 8.

[0207] In some embodiments, the nucleic acid further comprises a polynucleotide sequence encoding a signal peptide of dermcidin. In some embodiments, the signal peptide comprises an amino acid sequence consisting of SEQ ID NO: 11. In some embodiments, the polynucleotide sequence encoding the signal peptide is located at the 5'-terminus of the codon-optimized polynucleotide sequence encoding the inhibitor of VEGF-A.

[0208] In some embodiments, the codon-optimized polynucleotide encoding the inhibitor of VEGF-A is operably linked to an expression regulatory element. In some embodiments, the expression regulatory element includes at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the enhancer comprises a cytomegalovirus (CMV) immediate early enhancer sequence. In some embodiments, the enhancer consists of a cytomegalovirus (CMV) immediate early enhancer sequence.

[0209] In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the promoter comprises a cytomegalovirus (CMV) immediate early promoter sequence. In some embodiments, the promoter consists of a cytomegalovirus (CMV) immediate early promoter sequence

[0210] In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4.

[0211] In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the intron includes a human betaglobin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 ora nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5.

[0212] In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 91 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 92 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 93 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 94 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 95 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 96 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 97 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 98 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 99 % sequence identity thereto. In some embodiments, the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9. In some embodiments, the nucleic acid further comprises a Kozak sequence.

[0213] In some embodiments, a nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus. In some embodiments, a nucleotide sequence of the helper virus gene is derived from an adenovirus. In some embodiments, a nucleotide sequence of the helper virus gene is derived from adenovirus 2 (Ad2). In some embodiments, the helper virus gene comprises one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DNA-binding protein (DBP), and a variant thereof. In some embodiments, the helper virus gene comprises E1. In some embodiments, the helper virus gene comprises E2. In some embodiments, the helper virus gene comprises E2a. In some embodiments, the helper virus gene comprises E4. In some embodiments, the helper virus gene comprises E4orf1. In some embodiments, the helper virus gene comprises E4orf2. In some embodiments, the helper virus gene comprises E4orf3. In some embodiments, the helper virus gene comprises E4orf4. In some embodiments, the helper virus gene comprises E4orf5. In some embodiments, the helper virus gene comprises E4orf6. In some embodiments, the helper virus gene comprises E4orf7. In some embodiments, the helper virus gene comprises VA. In some embodiments, the helper virus gene comprises DNA-binding protein (DBP). In some embodiments, the helper virus gene comprises E2a, E4, and VA.

[0214] In some embodiments, a nucleotide sequence of the Rep gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7,

[0215] AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV1 . In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV2. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV3A. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV3B. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV4. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV5. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV6. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV7. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV8. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV9. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV10. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAVrhI O. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV11 . In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV12. In some embodiments, a nucleotide sequence of the Rep gene is derived from AAV13. In some embodiments, a nucleotide sequence of the Rep gene is a Rep gene of wild-type AAV2.

[0216] In some embodiments, a nucleotide sequence of the Cap gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7,

[0217] AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV1 . In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV2. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV3A. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV3B. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV4. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV5. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV6. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV7. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV8. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV9. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV10. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAVrhI O. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV11 . In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV12. In some embodiments, a nucleotide sequence of the Cap gene is derived from AAV13. In some embodiments, a nucleotide sequence of the Cap gene is a Cap gene of wild-type AAV2.

[0218] In some embodiments, the nucleic acid further comprises an AAV 5’ inverted terminal repeat (ITR) and an AAV 3’ ITR. In some embodiments, the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6,

[0219] AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13. In some embodiments, the ITR is derived from AAV1 . In some embodiments, the ITR is derived from AAV2. In some embodiments, the ITR is derived from AAV3A. In some embodiments, the ITR is derived from AAV3B. In some embodiments, the ITR is derived from AAV4. In some embodiments, the ITR is derived from AAV5. In some embodiments, the ITR is derived from AAV6. In some embodiments, the ITR is derived from AAV7. In some embodiments, the ITR is derived from AAV8. In some embodiments, the ITR is derived from AAV9. In some embodiments, the ITR is derived from AAV10. In some embodiments, the ITR is derived from AAVrhI O. In some embodiments, the ITR is derived from AAV11. In some embodiments, the ITR is derived from AAV12. In some embodiments, the ITR is derived from AAV13. In some embodiments, the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10.

[0220] Further disclosed herein, in some embodiments, is a plasmid comprising the nucleic acid sequence of any one of the embodiments disclosed herein. Further disclosed herein, in some embodiments, is a recombinant adeno-associated virus (rAAV) comprising the plasmid of any one of the embodiments disclosed herein.

[0221] Redundancies are omitted in consideration of the complexity of the present specification, and terms not otherwise defined herein may have the meanings commonly used in the technical field to which the present invention pertains.

[0222] Advantageous Effects of Invention

[0223] The expression cassette and the plasmid for AAV gene therapy of a neovascular ophthalmic diseases according to an aspect enable high productivity and high-quality production of rAAV virus particles having reduced immunogenicity, by applying a codon- optimized aflibercept sequence in combination with various human-derived expression regulatory elements including specific signal peptides, and an rAAV thus produced can reach therapeutic concentrations even at low viral administration doses due to enhanced expression of the aflibercept in the retina so that capsid-induced immune responses can be minimized.

[0224] The plasmid for producing rAAV according to an aspect can be applied as an all- in-one vector system as well as a triple vector system for triple transfection. In the case of an all-in-one vector system, transfection is performed by using one plasmid so that there are advantages of simplifying the rAAV production process and reducing production costs by producing a small number of plasmids.

[0225] Other Embodiments

[0226] Embodiment 1 comprises a nucleic acid expression cassette for enhanced expression of aflibercept in the retina, comprising a codon-optimized polynucleotide encoding aflibercept which is operably linked to an expression regulatory element, wherein the codon-optimized polynucleotide encoding aflibercept includes a nucleotide sequence consisting of SEQ ID NO: 8 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0227] Embodiment 2 comprises the nucleic acid expression cassette of embodiment 1 , further comprising a polynucleotide encoding a signal peptide of dermcidin, wherein the polynucleotide encoding the signal peptide is located at the 5'-terminus of the codon- optimized polynucleotide encoding aflibercept.

[0228] Embodiment 3 comprises the nucleic acid expression cassette of embodiment 2, wherein the signal peptide comprises an amino acid sequence consisting of SEQ ID NO: 11.

[0229] Embodiment 4 comprises the nucleic acid expression cassette of embodiment 1 , wherein the expression regulatory element includes at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

[0230] Embodiment 5 comprises the nucleic acid expression cassette of embodiment 4, wherein the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

[0231] Embodiment 6 comprises the nucleic acid expression cassette of embodiment 4, wherein the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

[0232] Embodiment 7 comprises the nucleic acid expression cassette of embodiment 1 , wherein the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0233] Embodiment 8 comprises the nucleic acid expression cassette of embodiment 4, wherein the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0234] Embodiment 9 comprises the nucleic acid expression cassette of embodiment 4, wherein the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0235] Embodiment 10 comprises the nucleic acid expression cassette of embodiment 1 , further comprising a Kozak sequence.

[0236] Embodiment 11 comprises a plasmid for producing a recombinant adeno- associated virus (rAAV), comprising: (a) an AAV 5' inverted terminal repeat (ITR); (b) the nucleic acid expression cassette of any one of embodiments 1 to 10; and c) an AAV 3' ITR. Embodiment 12 comprises the plasmid of embodiment 11 , wherein the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhW, AAV11 , AAV12, and AAV13.

[0237] Embodiment 13 comprises the plasmid of embodiment 11 , wherein the ITR is derived from AAV2.

[0238] Embodiment 14 comprises the plasmid of embodiment 11 , wherein the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10.

[0239] Embodiment 15 comprises the plasmid of embodiment 11 , further comprising the following genes: (d) a helper virus gene required for producing AAV; e) a Rep gene of AAV; and (f) a Cap gene of AAV.

[0240] Embodiment 16 comprises the plasmid of embodiment 15, wherein a nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus.

[0241] Embodiment 17 comprises the plasmid of embodiment 15, wherein a nucleotide sequence of the helper virus gene is derived from an adenovirus.

[0242] Embodiment 18 comprises the plasmid of embodiment 15, wherein a nucleotide sequence of the helper virus gene is derived from adenovirus 2 (Ad2).

[0243] Embodiment 19 comprises the plasmid of embodiment 15, wherein the helper virus gene comprises one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DNA-binding protein (DBF), and a variant thereof.

[0244] Embodiment 20 comprises the plasmid of embodiment 15, wherein the helper virus gene comprises E2a, E4, and VA.

[0245] Embodiment 21 comprises the plasmid of embodiment 15, wherein a nucleotide sequence of the Rep gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhW, AAV11 , AAV12, and AAV13.

[0246] Embodiment 22 comprises the plasmid of embodiment 15, wherein a nucleotide sequence of the Rep gene is a Rep gene of wild-type AAV2.

[0247] Embodiment 23 comprises the plasmid of embodiment 15, wherein a nucleotide sequence of the Cap gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhW, AAV11 , AAV12, and AAV13. Embodiment 24 comprises the plasmid of embodiment 15, wherein a nucleotide sequence of the Cap gene is a Cap gene of wild-type AAV2.

[0248] Embodiment 25 comprises an isolated host cell transfected or transduced with the plasmid of any one of embodiments 11 to 24.

[0249] Embodiment 26 comprises the host cell of embodiment 25, wherein the host cell is a mammalian cell or an insect cell.

[0250] Embodiment 27 comprises the host cell of embodiment 26, wherein the mammalian cell is selected from a HEK293 cell, a HEK293F cell, a HEK293T cell, and cells derived therefrom.

[0251] Embodiment 28 comprises the host cell of embodiment 26, wherein the insect cell is an Sf9 cell, an Sf21 cell, a TN-5B1-4 cell, or a High Five cell.

[0252] Embodiment 29 comprises a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome, wherein the vector genome comprises: (a) an AAV 5' inverted terminal repeat (ITR); (b) the nucleic acid expression cassette of any one of embodiments 1 to 10; and (c) an AAV 3' ITR.

[0253] Embodiment 30 comprises the rAAV of embodiment 29, wherein the AAV capsid is any one AAV capsid selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13.

[0254] Embodiment 31 comprises the rAAV of embodiment 29, wherein the AAV capsid is a wild-type AAV2 capsid.

[0255] Embodiment 32 comprises the rAAV of embodiment 29, wherein the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13.

[0256] Embodiment 33 comprises the rAAV of embodiment 29, wherein the ITR is derived from AAV2.

[0257] Embodiment 34 comprises the rAAV of embodiment 29, wherein the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10.

[0258] Embodiment 35 comprises a pharmaceutical composition for preventing or treating a neovascular ophthalmic disease, comprising: the rAAV of any one of embodiments 29 to 34; and a pharmaceutically acceptable carrier.

[0259] Embodiment 36 comprises the pharmaceutical composition of embodiment 35, wherein the neovascular ophthalmic disease is any one selected from the group consisting of age-related macular degeneration (AMD), diabetic retinopathy (DR), retinal vein occlusion (RVO), choroidal neovascularization, diabetic macular edema (DME), corneal neovascularization, and retinopathy of prematurity.

[0260] Embodiment 37 comprises the pharmaceutical composition of embodiment 36, wherein the AMD is wet AMD.

[0261] Embodiment 38 comprises a method of preventing or treating a neovascular ophthalmic disease, comprising administering an effective amount of the rAAV of any one of embodiments 29 to 34 or the pharmaceutical composition of any one of embodiments 35 to 37, to a subject in need thereof.

[0262] Embodiment 39 comprises the method of embodiment 38, wherein the administering is performed by intravitreal injection (IVT).

[0263] Embodiment 40 comprises the method of embodiment 38, wherein the administering is administration of a unit dose of rAAV particles in a range of about 1 x 109to about 1 .7 x 1011vector genome per eye (vg / eye) of the subject.

[0264] Embodiment 41 comprises a use of the rAAV of any one of embodiments 29 to 34 for the manufacture of a medicament for preventing or treating a neovascular ophthalmic disease.

[0265] Embodiment 42 comprises a nucleic acid for encoding an inhibitor of VEGF-A, comprising a codon-optimized polynucleotide encoding the inhibitor of VEGF-A, wherein the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 90 % sequence identity to SEQ ID NO: 8.

[0266] Embodiment 43 comprises the nucleic acid of embodiment 42, wherein the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.

[0267] Embodiment 44 comprises the nucleic acid of embodiment 42 or 43, further comprising a polynucleotide encoding a signal peptide of dermcidin.

[0268] Embodiment 45 comprises the nucleic acid of embodiment 44, wherein the signal peptide comprises an amino acid sequence consisting of SEQ ID NO: 11 .

[0269] Embodiment 46 comprises the nucleic acid of embodiment 44 or 45, wherein the polynucleotide encoding the signal peptide is located at the 5'-terminus of the codon- optimized polynucleotide encoding aflibercept.

[0270] Embodiment 47 comprises the nucleic acid of any one of embodiments 42-46, wherein the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20.

[0271] Embodiment 48 comprises the nucleic acid of any one of embodiments 42-47, wherein the inhibitor of VEGF-A comprises the amino acid sequence of SEQ ID NO: 20.

[0272] Embodiment 49 comprises the nucleic acid of any one of embodiments 42-48, wherein the codon-optimized polynucleotide encoding the inhibitor of VEGF-A is operably linked to an expression regulatory element.

[0273] Embodiment 50 comprises the nucleic acid of embodiment 49, wherein the expression regulatory element includes at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

[0274] Embodiment 51 comprises the nucleic acid of embodiment 50, wherein the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

[0275] Embodiment 52 comprises the nucleic acid of embodiment 50, wherein the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

[0276] Embodiment 53 comprises the nucleic acid of embodiment 50, wherein the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0277] Embodiment 54 comprises the nucleic acid of embodiment 50, wherein the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0278] Embodiment 55 comprises the nucleic acid of embodiment 50, wherein the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0279] Embodiment 56 comprises the nucleic acid of any one of embodiments 42-55, further comprising a Kozak sequence.

[0280] Embodiment 57 comprises a nucleic acid for encoding an inhibitor of VEGF-A, comprising: polynucleotide sequences encoding an inhibitor of VEGF-A and a signal peptide, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 11 , or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11.

[0281] Embodiment 58 comprises the nucleic acid of embodiment 57, wherein the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20.

[0282] Embodiment 59 comprises the nucleic acid of embodiment 57 or 58, wherein the polynucleotide sequence encoding the inhibitor of VEGF-A comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 8.

[0283] Embodiment 60 comprises the nucleic acid of embodiment 59, wherein the polynucleotide sequence encoding the inhibitor of VEGF-A comprises the nucleotide sequence of SEQ ID NO: 8.

[0284] Embodiment 61 comprises the nucleic acid of any one of embodiments 57-60, wherein the polynucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 7.

[0285] Embodiment 62 comprises the nucleic acid of embodiment 61 , wherein the polynucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 7.

[0286] Embodiment 63 comprises the nucleic acid of any one of embodiments 57-62, wherein the polynucleotide sequence encoding the signal peptide is at 5’-end of the polynucleotide sequence encoding the inhibitor of VEGF-A.

[0287] Embodiment 64 comprises the nucleic acid of any one of embodiments 57-63, further comprising one or more expression regulatory elements operably linked to the polynucleotide.

[0288] Embodiment 65 comprises the nucleic acid of embodiment 64, wherein the expression regulatory element(s) are selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

[0289] Embodiment 66 comprises the nucleic acid of embodiment 65, wherein the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

[0290] Embodiment 67 comprises the nucleic acid of embodiment 65, wherein the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

[0291] Embodiment 68 comprises the nucleic acid of embodiment 65, wherein the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0292] Embodiment 69 comprises the nucleic acid of embodiment 65, wherein the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0293] Embodiment 70 comprises the nucleic acid of embodiment 65, wherein the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0294] Embodiment 71 comprises the nucleic acid of any one of embodiments 57-70, further comprising a Kozak sequence.

[0295] Embodiment 72 comprises a plasmid for producing a recombinant adeno- associated virus (rAAV), comprising (a) an AAV 5’ inverted terminal repeat (ITR), (b) the nucleic acid of any one of embodiments 42-71 ; and (c) an AAV 3’ ITR.

[0296] Embodiment 73 comprises the plasmid of embodiment 72, wherein the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh , AAV11 , AAV12, and AAV13.

[0297] Embodiment 74 comprises the plasmid of embodiment 72 or 73, wherein the ITR is derived from AAV2.

[0298] Embodiment 75 comprises the plasmid of any one of embodiments 72-74, wherein the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10.

[0299] Embodiment 76 comprises the plasmid of any one of embodiments 72-75, further comprising the following genes: (a) a helper virus gene required for producing AAV; (b) a Rep gene of AAV; and (c) a Cap gene of AAV.

[0300] Embodiment 77 comprises the plasmid of embodiment 76, wherein a nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus.

[0301] Embodiment 78 comprises the plasmid of embodiment 76 or 77, wherein a nucleotide sequence of the helper virus gene is derived from an adenovirus. Embodiment 79 comprises the plasmid of any one of embodiments 76-78, wherein a nucleotide sequence of the helper virus gene is derived from adenovirus 2 (Ad2).

[0302] Embodiment 80 comprises the plasmid of any one of embodiments 76-79, wherein the helper virus gene comprises one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DNA-binding protein (DBP), and a variant thereof.

[0303] Embodiment 81 comprises the plasmid of any one of embodiments 76-80, wherein the helper virus gene comprises E2a, E4, and VA.

[0304] Embodiment 82 comprises the plasmid of any one of embodiments 76-81 , wherein a nucleotide sequence of the Rep gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV1 1 , AAV12, and AAV13.

[0305] Embodiment 83 comprises the plasmid of any one of embodiments 76-82, wherein a nucleotide sequence of the Rep gene is a Rep gene of wild-type AAV2.

[0306] Embodiment 84 comprises the plasmid of any one of embodiment 76-83, wherein a nucleotide sequence of the Cap gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13.

[0307] Embodiment 85 comprises the plasmid of any one of embodiments 76-84, wherein a nucleotide sequence of the Cap gene is a Cap gene of wild-type AAV2.

[0308] Embodiment 86 comprises an isolated host cell transfected or transduced with the plasmid of any one of embodiments 76-85.

[0309] Embodiment 87 comprises the host cell of embodiment 86, wherein the host cell is a mammalian cell or an insect cell.

[0310] Embodiment 88 comprises the host cell of embodiment 87, wherein the mammalian cell is selected from a HEK293 cell, a HEK293F cell, a HEK293T cell, and cells derived therefrom.

[0311] Embodiment 89 comprises the host cell of embodiment 87, wherein the insect cell is an Sf9 cell, an Sf21 cell, a TN-5B1-4 cell, or a High Five cell.

[0312] Embodiment 90 comprises a recombinant adeno-associated virus (rAAV) comprising the plasmid of any one of embodiments 72-85. Embodiment 91 comprises a recombinant adeno-associated virus (rAAV) comprising the nucleic acid of any one of embodiments 42-71 .

[0313] Embodiment 92 comprises a recombinant adeno-associated virus (rAAV) comprising a transgene plasmid comprising the nucleic acid of any one of embodiments 42-71 , and one or more additional plasmids comprising at least one of (a) a helper virus gene required for producing AAV; (b) a Rep gene of AAV; and (c) a Cap gene of AAV.

[0314] Embodiment 93 comprises a nucleic acid for encoding an inhibitor of VEGF-A, comprising a codon-optimized polynucleotide sequence encoding the inhibitor of VEGF- A, wherein the polynucleotide further comprises (a) a helper virus gene required for producing AAV; (b) an Rep gene of AAV; and (c) a Cap gene of AAV.

[0315] Embodiment 94 comprises the nucleic acid of embodiment 93, wherein the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20.

[0316] Embodiment 95 comprises the nucleic acid of embodiment 93 or 94, wherein the inhibitor of VEGF-A comprises the amino acid sequence of SEQ ID NO: 20.

[0317] Embodiment 96 comprises the nucleic acid of any one of embodiments 93-95, wherein the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 90% to SEQ ID NO: 8.

[0318] Embodiment 97 comprises the nucleic acid of any one of embodiments 93-96, wherein the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.

[0319] Embodiment 98 comprises the nucleic acid of any one of embodiments 93-97, further comprising a polynucleotide sequence encoding a signal peptide of dermcidin.

[0320] Embodiment 99 comprises the nucleic acid of embodiment 95, wherein the signal peptide comprises an amino acid sequence consisting of SEQ ID NO: 11 .

[0321] Embodiment 100 comprises the nucleic acid of any one of embodiments 93-99, wherein the polynucleotide sequence encoding the signal peptide is located at the 5'- terminus of the codon-optimized polynucleotide sequence encoding the inhibitor of VEGF-A.

[0322] Embodiment 101 comprises the nucleic acid of any one of embodiments 93-100, wherein the codon-optimized polynucleotide encoding the inhibitor of VEGF-A is operably linked to an expression regulatory element. Embodiment 102 comprises the nucleic acid of embodiment 101 , wherein the expression regulatory element includes at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

[0323] Embodiment 103 comprises the nucleic acid of embodiment 102, wherein the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

[0324] Embodiment 104 comprises the nucleic acid of embodiment 102, wherein the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

[0325] Embodiment 105 comprises the nucleic acid of embodiment 102, wherein the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0326] Embodiment 106 comprises the nucleic acid of embodiment 102, wherein the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0327] Embodiment 107 comprises the nucleic acid of embodiment 102, wherein the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto.

[0328] Embodiment 108 comprises the nucleic acid of any one of embodiments 93-107, further comprising a Kozak sequence.

[0329] Embodiment 109 comprises the nucleic acid of any one of embodiments 93-108, wherein a nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus.

[0330] Embodiment 110 comprises the nucleic acid of any one of embodiments 93-109, wherein a nucleotide sequence of the helper virus gene is derived from an adenovirus.

[0331] Embodiment 111 comprises the nucleic acid of any one of embodiments 93-110, wherein a nucleotide sequence of the helper virus gene is derived from adenovirus 2 (Ad2).

[0332] Embodiment 112 comprises the nucleic acid of any one of embodiments 93-110, wherein the helper virus gene comprises one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DNA-binding protein (DBF), and a variant thereof.

[0333] Embodiment 113 comprises the nucleic acid of any one of embodiments 93-112, wherein the helper virus gene comprises E2a, E4, and VA.

[0334] Embodiment 114 comprises the nucleic acid of any one of embodiments 93-113, wherein a nucleotide sequence of the Rep gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV1 1 , AAV12, and AAV13.

[0335] Embodiment 115 comprises the nucleic acid of any one of embodiments 93-114, wherein a nucleotide sequence of the Rep gene is a Rep gene of wild-type AAV2.

[0336] Embodiment 116 comprises the nucleic acid of any one of embodiments 93-115, wherein a nucleotide sequence of the Cap gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, AAV1 1 , AAV12, and AAV13.

[0337] Embodiment 117 comprises the nucleic acid of any one of embodiments 93-116, wherein a nucleotide sequence of the Cap gene is a Cap gene of wild-type AAV2.

[0338] Embodiment 118 comprises the nucleic acid of any one of embodiments 93-117, further comprising an AAV 5’ inverted terminal repeat (ITR) and an AAV 3’ ITR.

[0339] Embodiment 119 comprises the nucleic acid of any one of embodiments 93-118, wherein the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13.

[0340] Embodiment 120 comprises the nucleic acid of any one of embodiments 93-119, wherein the ITR is derived from AAV2.

[0341] Embodiment 121 comprises the nucleic acid of any one of embodiments 93-120, wherein the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10.

[0342] Embodiment 122 comprises a plasmid comprising the nucleic acid of any one of embodiments 93-121 .

[0343] Embodiment 123 comprises a recombinant adeno-associated virus (rAAV) comprising the plasmid of embodiment 122. Hereinafter, the invention will be explained in more detail by Examples below. However, Examples below are for illustrating the invention, and accordingly, the scope of the present invention is not limited thereto.

[0344] Examples

[0345] Example 1. Construction of aflibercept transqene expression cassette

[0346] (1.1) Construction of transgene expression cassette

[0347] An expression cassette including a polynucleotide encoding a therapeutic gene, aflibercept, was constructed. The structure of the expression cassette for aflibercept transgene includes the following in the 5'-terminus to 3'-terminus, as shown in FIG. 1 : a CMV immediate early enhancer / promoter (SEQ ID NO: 4); a human beta-globin (hBG) intron (SEQ ID NO: 5); a Kozak sequence (5’-GCCGCCACC-3’); a polynucleotide encoding a human Dermcidin signal peptide (SEQ ID NO: 7); a codon-optimized polynucleotide encoding aflibercept as a transgene (SEQ I D NO: 8); and a human growth hormone polyA (hGH pA) (SEQ ID NO: 9).

[0348] For use as the enhancer / promoter, an enhancer / promoter (CMV) capable of inducing strong expression was used due to the local injectable nature of a lesion. For the gene-of-interest (GOI) used as the transgene, a codon-optimized nucleotide sequence of aflibercept prepared by using a GenSmart Codon Optimization Tool (Genscript) was used.

[0349] FIG. 1 is a schematic diagram of the aflibercept transgene expression cassette flanked by AAV2 ITRs, according to an embodiment.

[0350] The sequence information used to construct the aflibercept expression cassette is shown in Table 1.

[0351] Example 2. Preparation of all-in-one vector for producing rAAV including aflibercept expression cassette

[0352] A vector for producing rAAV was prepared, in which the aflibercept expression cassette of Example 1 flanked by AAV2 ITRs was packaged within an AAV2 capsid. For the vector, an all-in-one vector system designed to include a helper virus gene, an Rep gene, a Cap gene, and a transgene within a single vector was used.

[0353] The gene sequence information of each component of the all-in-one vector was obtained based on the existing documents.

[0354] Helper E2a, E4, and VA genes were obtained from wild-type (WT) adenovirus 2 (Ad2) (Gene Bank Accession No. AC_000007.1 ).

[0355] Rep, Cap, L-ITR (5' ITR), and R-ITR (3' ITR) genes were obtained from WT AAV serotype 2 (Gene Bank Accession No. NC_001401 .2).

[0356] For the aflibercept transgene expression cassette, the one constructed in Example 1 was used.

[0357] Primers were prepared based on the sequence information of the corresponding genes of each component and used for PCR amplification to secure the components, which were then inserted in 1 copy each into the pMG-Kan vector (Macrogen). According to Ausubel et al. (Ausubel et al. (ed.), J. Wiley & Sons, 1997, Curr. Protocols of Molecular Biology.). The expression vector was prepared using molecular genetic technology. Genes and primers used for PCR amplification were synthesized and prepared by Macrogen Co., Ltd. (Seoul, Korea). The genetic sequence of the cloned expression vector was confirmed by request to Macrogen Co., Ltd. (Seoul, Korea). The prepared vector was named "all-in-one (AIO) ssAAV2-alibercept vector".

[0358] FIG. 2 is a diagram for describing the AIO vector designed to include a helper virus gene, an Rep gene, a Cap gene, and a transgene in one vector, according to an embodiment.

[0359] FIG. 3 is a vector map of the AIO ssAAV2-aflibercept vector according to an embodiment.

[0360] The AIO ssAAV2-alibercept vector of FIG. 3 includes each component in the following order: a helper gene, a plasmid backbone, a Rep gene, a Cap gene, a 5' ITR, an enhancer, a promoter, an intron, a Kozak sequence, a signal peptide, a GOI, a polyadenylation signal sequence, and 3' ITR.

[0361] In the AIO ssAAV2-alibercept vector of FIG. 3, the following were used: E2a, E4, and VA of WT adenovirus 2 as helper genes (SEQ ID NO: 1); WT AAV2 Rep as an Rep gene and WT AAV2 Cap as a Cap gene (SEQ ID NO: 2); a WT AAV2 5' ITR (SEQ ID NO: 3); a CMV immediate early enhancer / promoter (SEQ ID NO: 4); a human betaglobin (hBG) intron (SEQ ID NO: 5); a Kozak sequence (5’-GCCGCCACC-3’); a polynucleotide encoding a human Dermcidin signal peptide (SEQ ID NO: 7); a codon- optimized polynucleotideencoding aflibercept as a transgene (SEQ ID NO: 8); a human growth hormone polyA (hGH pA) (SEQ ID NO: 9); and a WT AAV2 3' ITR (SEQ ID NO: 10). In addition, pBR322 was used as the replication origin and kanamycin (kan) was used as the selectable marker.

[0362] The sequence information used to construct the AIO ssAAV2-alibercept vector is as shown in Table 1.

[0363] [Table 1]

[0364] Example 3. Preparation of triple (3TF) vector for producing rAAV including aflibercept expression cassette

[0365] A vector for producing rAAV was prepared, in which the aflibercept expression cassette of Example 1 flanked by AAV2 ITRs was packaged within an AAV2 capsid. For use as the vector, a triple vector system for the existing triple transfection was used. In detail, for the triple vector, a vector including a helper gene, a vector including a Rep / Cap gene, and a vector including a GOI (including a portion corresponding to from a 5' ITR to a 3' ITR) were each prepared. Each component was secured by PCR amplification and inserted into the pMG-Kan vector (Macrogen). The genes included in each vector are the same type as in Example 2. The genetic sequence of the cloned expression vector was confirmed by request to Macrogen Co., Ltd. (Seoul, Korea). The vector thus prepared was named "triple transfection (3TF) ssAAV2-alibercept vector".

[0366] FIG. 4 is a vector map of the vector including the GOI (including a portion corresponding to from a 5 'ITR to a 3' ITR) of the 3TF ssAAV2-aflibercept vector, according to an embodiment.

[0367] [Comparative Examples]

[0368] Comparative Example 1. Preparation of AIO vector for producing rAAV using Clusterin signal peptide

[0369] An AIO vector for producing rAAV was prepared in the same manner as in Example 2, except that a human Clusterin signal peptide was used as a signal peptide instead of the human Dermcidin signal peptide used in the AIO ssAAV2-alibercept vector of Example 2. Comparative Example 2. Preparation of AIO vector for producing rAAV using Serotransferrin signal peptide

[0370] An AIO vector for producing rAAV was prepared in the same manner as in Example 2, except that a human Serotransferrin signal peptide was used as a signal peptide instead of the human Dermcidin signal peptide used in the AIO ssAAV2- alibercept vector of Example 2.

[0371] Comparative Example 3. Preparation of AIO vector for producing rAAV using Albumin signal peptide

[0372] An AIO vector for producing rAAV was prepared in the same manner as in Example 2, except that a human Albumin signal peptide was used as a signal peptide instead of the human Dermcidin signal peptide used in the AIO ssAAV2-alibercept vector of Example 2.

[0373] Comparative Example 4. Preparation of AIO vector for producing rAAV using IL-2 signal peptide

[0374] An AIO vector for producing rAAV was prepared in the same manner as in Example 2, except that a human IL-2 signal peptide, which was known to be used in RGX-314 (Regenxbio Inc.), was used as a signal peptide instead of the human Dermcidin signal peptide used in the AIO ssAAV2-alibercept vector of Example 2.

[0375] Comparative Example 5. Preparation of AIO vector for producing rAAV using VEGFR-1 signal peptide

[0376] An AIO vector for producing rAAV was prepared in the same manner as in Example 2, except that a human VEGFR-1 signal peptide, which was known to be used in ADVM-022 (Adverum Biotechnologies Inc.), was used as a signal peptide instead of the human Dermcidin signal peptide used in the AIO ssAAV2-alibercept vector of Example 2.

[0377] Comparative Example 6. Preparation of AIO vector for producing rAAV using mlLC signal peptide

[0378] An AIO vector for producing rAAV was prepared in the same manner as in Example 2, except that a murine IgG light chain (mlLC) signal peptide, which was known to have high productivity in Chinese hamster ovary (CHO) cells, was used as a signal peptide instead of the human Dermcidin signal peptide used in the AIO ssAAV2- alibercept vector of Example 2. Comparative Example 7. Preparation of AIO vector for producing rAAV using synthetic SP signal peptide

[0379] An AIO vector for producing rAAV was prepared in the same manner as in Example 2, except that synthetic SP was used as a signal peptide instead of the human Dermcidin signal peptide used in the AIO ssAAV2-alibercept vector of Example 2. The synthetic SP is a synthetic peptide in which the hydrophilic amino acids of the mlLC signal peptide was replaced with hydrophobic amino acids to enhance the function of the signal peptide.

[0380] Comparative Example 8. Preparation of AIO vector for producing rAAV using synthetic-0.93 signal peptide

[0381] An AIO vector for producing rAAV was prepared in the same manner as in Example 2, except that synthetic-0.93 was used as a signal peptide instead of the human Dermcidin signal peptide used in the AIO ssAAV2-alibercept vector of Example 2. The synthetic-0.93 signal peptide has the same amino acid sequence as the synthetic SP (CAI=0.78), but the codon adaptation index (CAI) value was adjusted to 0.93 to enhance the rAAV productivity.

[0382] The signal peptides used in the vectors of Example 2 and Comparative Examples 1 to 8 are shown in Table 2.

[0383] [Table 2]

[0384] [Experimental Examples]

[0385] Experimental Example 1. Signal peptide screening

[0386] (1.1) Experiments to evaluate in-vitro aflibercept productivity in human- derived retinal cells

[0387] The signal peptides were cleaved from the secretory system of cells, and thus do not co-exist with aflibercept, but there is still the potential for the signal peptides to remain in the cells and cause immunogenicity. To screen a signal peptide having low immunogenicity and high productivity of aflibercept due to its intrinsic function of secretion among four candidate proteins (Dermcidin, Clusterin, Serotransferrin, and Albumin) that are predicted to be highly expressed in the retina, ARPE-19 that is a human retinal pigment epithelial cell line was transfected with the vector of Examples or Comparative Examples by using lipofectamine, and the amount of aflibercept protein produced was evaluated.

[0388] The vectors of Example 2 and Comparative Examples 1 to 3 and Comparative Examples 7 and 8 were used as the experimental group, and the vector of Comparative Example 6 using the mlLC signal peptide, which is known to exhibit high productivity in CHO cells, was used as the vector of the control group.

[0389] In detail, a target cell, the ARPE-19 that is a human retinal pigment epithelial cell line, was cultured in a DMEM / F12 medium supplemented with 10 % fetal bovine serum (FBS) under conditions of 37 °C and 5 % CO2. One day before treatment with the AIO vectors of Example 2 and Comparative Examples 1 to 3 and Comparative Examples 6 to 8, the ARPE-19 cell was applied onto a 6-well plate at a concentration of 2.5E05 cells / well. pDNA of each AIO vector was mixed with a cationic lipid transfection reagent (lipofectamine LTX and plus, Invitrogen) at a ratio of 1 :1 , incubated at room temperature for 25 minutes, and then slowly added dropwise to the target cell. Here, the concentration of the pDNA treated in the cell was 0.75 pg / well. After the transfection, the cell was incubated for 4 hours under conditions of 37 °C and 5 % CO2 Afterwards, all culture solution was removed and replaced with a culture medium. After 48 hours of culture, the ARPE-19 culture solution was collected to measure the expression level of aflibercept by using the Octet® BLI Label-Free detection system.

[0390] FIG. 5 shows the results of measuring the amount of aflibercept production with the Octet® BLI Label-Free after introducing, into the ARPE-19 cell by a transfection method using lipofectamine, the AIO ssAAV2-aflibercept vectors (Example 2 and Comparative Examples 1 to 3 and 6 to 8) prepared by varying the type of signal peptides.

[0391] As a result, as shown in FIG. 5, the vectors of Example 2 and Comparative Examples 1 to 3 using the signal peptides of the four candidate proteins (Dermcidin, Clusterin, Serotransferrin, and Albumin) showed high expression levels of aflibercept compared to the vectors of Comparative Examples 7 and 8 using the synthetic signal peptides, but did not show a statistically significant difference compared to the control group.

[0392] (1.2) in silico immunogenicity evaluation

[0393] For the signal peptides of the four proteins of Section (1.1 ), the signal peptides were additionally evaluated by using a bioinformatics method that can predict immunogenicity.

[0394] In detail, each signal peptide of the vectors of Example 2 and Comparative Examples 1 to 5 and Comparative Examples 7 and 8 was used as the experimental group, and the mlLC signal peptide of the vector of Comparative Example 6 was used as the control group. As a result of analyzing the MHC-1 binding score based on the Immune Epitope Database (IEDB) DB, the mlLC was predicted to exhibit high immunogenicity due to high possibility of HLA-A02 type binding (percentile rank and binding score). By using the mlLC as the control group, signal peptides having low MHC- 1 and MHC-II binding scores were determined to be suitable. The MHC-I and MHC-II binding prediction was performed by using the Immune Epitope Database and Analysis Resource and EpiScan predictor (Peter et al. Nat. Biotech, 2023).

[0395] FIG. 6 shows the results of in silico MHC Class I and II binding prediction performed to evaluate immunogenicity of each signal peptide used in the AIO ssAAV2- aflibercept vectors (Example 2 and Comparative Examples 1 to 8) prepared by varying the type of signal peptides.

[0396] As a result, as shown in FIG. 6, the signal peptides of two candidate proteins (Dermcidin and Serotransferrin) used in the vectors of Example 2 and Comparative Example 2 passed the rank as candidates compared to the control group, mlLC, and were used for subsequent experiments.

[0397] (1.3) Evaluation of rAAV productivity and quality (VG, VG / VP)

[0398] One of the hurdles of the AAV gene therapeutic agent is the high production cost, and since there is a possibility that the signal peptide may affect the productivity of rAAV, the productivity of rAAV was evaluated for the evaluation of the signal peptides.

[0399] The vectors of Example 2 and Comparative Example 2 that have passed the immunogenicity evaluation were used as the experimental group, and the vector of Comparative Example 4 using the human IL-2 signal peptide, which is known to be used in RGX-314 (Regenxbio Inc.), was used as the control group.

[0400] In detail, the HEK293 cell line was transfected with the AIO vectors for producing rAAV of Example 2, Comparative Example 2, and Comparative Example 4 by using VPM and FectoVIR. After 72 hours of the transfection, cells were lysed to confirm the AAV productivity, and the viral genome (VG) of the produced AAV was measured through qPCR using ITR-specific primers by using the AAVpro® Titration Kit (TAKARA). In addition, the viral protein (VP) of the produced AAV was measured through AAV Titration ELISA (PROGEN). Through the measured VGA / P ratio value, the quantity (%full / empty) of the produced AAV particles was confirmed. FIG. 7 shows the results of evaluating the AAV productivity (viral genome / L) of the AIO ssAAV2-aflibercept vectors (Example 2 and Comparative Examples 2 and 4) prepared by varying the type of signal peptides.

[0401] FIG. 8 shows the results of the quality (%full / empty) of the AAV particles produced by the AIO ssAAV2-aflibercept vectors (Example 2 and Comparative Examples 2 and 4) prepared by varying the type of signal peptides.

[0402] As a result, as shown in FIGS. 7 and 8, the signal peptide of Dermcidin showed the highest productivity of 2.03E13 vg / L, and was also shown to have the best quality as calculated by the VGA / P.

[0403] (1.4) Evaluation of aflibercept protein expression and biological activity

[0404] The aflibercept expression and in vitro potency (VEGF neutralization) are the experiments that can most directly estimate the efficacy of rAAV. To determine how signal peptides affect the aflibercept protein expression, the human retinal ARPE-19 cell was transduced with each candidate AAV, and then the aflibercept concentration was measured. In addition, to determine whether aflibercept thus produced actually had potency, VEGF neutralization assay was performed.

[0405] The rAAV produced by using the vectors of Example 2 and Comparative Example 2 that have passed the immunogenicity evaluation was used as the experimental group, and the rAAV produced by using the vector of Comparative Example 4 using the human IL-2 signal peptide, which is known to be used in RGX-314 (Regenxbio Inc.), was used as the control group. In addition, when evaluating VEGF neutralization ability, the purified SB15 (aflibercept) protein was used as the control group.

[0406] In detail, each of the AIO vectors for producing rAAV of Example 2, Comparative Example 2, and Comparative Example 4 was used for transient transfection using polyethylenimine (PEI) on the mammalian cell (HEK294) as a host cell. After 48 hours of the transfection, the cell was lysed, and highly pure AAV was obtained by CsCI ultracentrifugation. A target cell, the ARPE-19 that is a human retinal pigment epithelial cell line, was cultured in a DMEM / F12 medium supplemented with 10 % fetal bovine serum (FBS) under conditions of 37 °C and 5 % CO2. The day before AAV injection, the ARPE- 19 cell was applied onto a 96-well plate at a concentration of 1.0E4 cell / cm2. The obtained AAV was diluted in a FBS-free DMEM / F12 medium to be treated at 1.37E03, 4.12E03, 1.23E04, 3.70E04, 1.11 E05, and 3.33E05 vg / cell, and then the virus was treated in the 96-well plate coated with the ARPE-19 cell from which the culture solution has been completely removed. After 2 hours of the virus treatment, a DMEM / F12 medium supplemented with 10 % FBS was added to the virus-treated 96-well plate. After 72 hours of the virus treatment, the ARPE-19 culture solution was collected, and the concentration of the protein expressed by the AAV was analyzed by electrochemiluminescence (ECL).

[0407] Aflibercept binds VEGF and consequently prevents neovascularization. Therefore, to confirm the biological activity of aflibercept in inhibiting cell proliferation, the VEGF-dependent proliferation inhibitory effect on human umbilical vein endothelial cells (HUVECs) was measured by using the Cell Titer-Blue® system. Upon the treatment of HUVECs with aflibercept, fluorescence signals appear to be proportional or inversely proportional to the antibody concentration as an indicator of proliferation inhibition, and thus the relative anti-proliferative capacity of aflibercept relative to the reference was determined by using the Cell Titer-Blue® system, thereby confirming the biological activity in cell proliferation inhibition.

[0408] FIG. 9 shows the results of analyzing the aflibercept concentration by ECL after treating the ARPE-19 cells with the rAAVs produced by the AIO ssAAV2-aflibercept vectors (Example 2, Comparative Example 2, and Comparative Example 4) prepared by varying the type of signal peptides.

[0409] FIG. 10 shows the results of VEGF neutralization assays performed to evaluate the biological activity of aflibercept expressed by treating the ARPE-19 cells with the rAAVs produced by the AIO ssAAV2-aflibercept vectors (Example 2, Comparative Example 2, and Comparative Example 4) prepared by varying the type of signal peptides.

[0410] As a result, as shown in FIG. 9 and FIG. 10, all candidate groups and the control group (IL-2) showed statistically insignificant differences in the expression level of aflibercept, and similarly, it was confirmed that the signal peptide did not affect the biological activity of aflibercept.

[0411] Based on the results of Sections (1.1) to (1.4), the signal peptide of Dermcidin is expected to have the lowest immunogenicity, and due to excellent rAAV productivity and quality, it is selected as the final signal peptide. Experimental Example 2. In-vivo evaluation of expression level of aflibercept protein according to administered doses

[0412] As being able to lower the viral dose for the same efficacy could reduce immunogenicity which is one of the problems found with the AAV gene therapeutic agent, the ability of rAAV produced by the vectors of the Examples to achieve a therapeutic level of the aflibercept expression concentration even at low doses was evaluated by using a rabbit animal model from New Zealand. Furthermore, to investigate ocular inflammatory responses in the animal model, ophthalmic examinations for conjunctival injection, keratitis, aqueous humor cells, aqueous flare, anterior and posterior lens, vitreous opacity, and iris invasion were performed by using an ophthalmotonometer (PRACTITIONE or PanOptic) and a slit lamp (Kowa SL-15).

[0413] The rAAVs produced by using the AIO vector of Example 2 and the 3TF vector of Example 3 were used as the experimental group, and the purified SB15 (aflibercept) protein was used as the control group.

[0414] In detail, to produce a non-clinical animal experiment sample, the AIO vector of Example 2 and the 3TF vector of Example 3 were used for transient transfection using PEI on the mammalian cell (HEK293) as a host cell in a 50 L bioreactor. Clarification (depth filtration), 1st UF / DF, affinity chromatography, AEX chromatography, and 2nd UF / DF were sequentially performed on the crude lysate obtained from the 50 L bioreactor. Then, the 2nd UF / DF purified product and the formulated DS were processed with a target VG titer of 6.0E12 VG / mL, and diluted to titers of 5.5E12, 2.2E11 , 2.2E10, and 1.1 E09 VG / mL. The final DS sample was aliquoted in 150 uL aliquots into autoclaved ep-tubes and stored in a -70 °C deep freezer.

[0415] Afterwards, the rAAV samples prepared by producing the AIO vector of Example 2 and the 3TF vector of Example 3 were administered into the vitreous cavity of the New Zealand White (NZW) rabbit to conduct an ocular pharmacokinetic study. The NZW rabbit is widely recognized as an appropriate species for ophthalmic research due to its similarity to the human eye and its suitability for ocular pharmacokinetic studies involving intravitreal administration of drugs. Moreover, considerable background data exists on the NZW rabbit.

[0416] On the last day of the isolation adaptation period (group assignment date), 101 males with a body weight similar to the average body weight were selected. The selected animals were randomly assigned to 11 groups in a stratified manner based on the recent body weight (9 groups: 10 animals / group, 1 group: 8 animals / group, 1 group: 3 animals / group). A drug was administered into 101 19-week-old male rabbits.

[0417] A thawed test agent (about 0.1 mL / vial) was tapped 10 times before administration, transferred to a syringe, and kept on ice until immediately before administration. A dosing formulation was administered once to the vitreous of each eye (about 0.05 and 0.03 mL / eye, G2-G9 and G10-G11 , respectively) in all animals. Before injecting the dosing formulation, both an anesthetics (Alfaxalone) and a sedative (Medetomidine) were mixed according to the ratios suggested in the SOP. The animals were the anesthetized by intravenous injection of the mixed solution. Additional anesthesia was performed depending on the condition of the animals.

[0418] Ophthalmic examinations of all surviving animals were performed before administration and at 6 weeks before sacrifice. Before examination, a miotic agent was instilled into the eyes of all animals (Tropherine® eye erops, Hanmi Pharmaceutical Co., Ltd.), and the pupillary light reflex and blink reflex tests were macroscopically performed. Then, macroscopic examination of the anterior chamber, transparent media, and ocular fundus was performed by using an ophthalmoscope (PRACTITIONER, Keeler, UK or PanOptic Ophthalmoscope, Welch Allyn, USA). Examination of aqueous humor cells and aqueous flare was performed by using a slit lamp (Kowa SL-15, KOWA, Japan). Symptoms were observed without distinguishing between the left and right eyes, and each eye was observed independently without giving any significance to lateral position. Criteria for scoring the severity of each symptom were established based on a substantial reference document (Eaton JS et al. J Ocul Pharmacol Ther. 2017 Dec;33(10):718-734). Evaluations were performed by using a scoring system based on the severity of symptoms such as conjunctival injection, corneal precipitates, aqueous humor cells, aqueous flare, anterior and posterior lenticule, vitreous clouding, and iris invasion. Blood samples (about 3 mL) were collected from the jugular vein from all surviving animals during the pre-dose period and at the time of final sacrifice. On the day of organ harvesting, the animals were bled under sodium thiopental anesthesia for the scheduled autopsy. The optic nerves, eyeballs, and brain of the scarified animal were harvested, and the optic nerve and eyeballs were subdivided into substructures, and the retina was harvested and measured for volume and weight. Analysis of number of viral gene copies (vg) and gene product (aflibercept) in the ocular tissue / fluid sample and serum was performed by using qPCR and ECL analysis methods.

[0419] FIG. 1 1 shows the results of evaluating the expression level of aflibercept according to the administered doses at 6 weeks after intravitreal injection (IVT) of the rAAV produced by the AIO vector of Example 2 and the rAAV produced by the 3TF vector of Example 3 into the animal model (rabbit).

[0420] FIG. 12 shows the results of observing ocular inflammatory responses according to the administered doses at 6 weeks after IVT of the rAAV produced by the AIO vector of Example 2 and the rAAV produced by the 3TF vector of Example 3 into the animal model (rabbit).

[0421] As a result, as shown in FIG. 11 , the rAAV produced by the AIO vector of Example 2 was found to secure a therapeutic level of the aflibercept expression concentration at an administration dose of 1 E09 to 5E10 vg / eye. Here, when the human conversion factor (vitreous volume ratio) of 3.33 is applied, the administration dose becomes 3.3E09-1.7E11 vg / eye, indicating that the therapeutic concentration can be reached starting from 3.3E09 vg / eye. Therefore, it was confirmed that the therapeutic concentrations could be reached even at significantly low doses compared to the previously reported clinical dose of 2E11 to 6E11 for ADVM-022 (Adverum Biotechnologies Inc.) which is an rAAV product administered via IVT, or the previously reported clinical dose of 6E10 to 2.5E11 for RGX-314 (Regenxbio Inc.) which is an rAAV product administered via subretinal injection (SR). This means that the viral dose can be lowered for the same efficacy, thereby reducing the possibility of inducing an immune response by a capsid. In addition, the rAAV produced by the AIO vector of Example 2 and the rAAV produced by the 3TF vector of Example 3 showed no significant difference (p>0.05) in the expression concentration of aflibercept, confirming that there was no difference in the expression concentration of aflibercept according to the production system in a target area (retina).

[0422] In addition, as shown in FIG. 12, compared to the group administered with the purified SB15 (aflibercept) protein, inflammatory responses were observed to occur in a dose-dependent manner in the group administered with the rAAV produced by the AIO vector of Example 2, and inflammatory responses were also observed in the group administered with the rAAV produced by the 3FT vector of Example 3. However, despite of the administration of the virus, the level remained at a mild level of 1 and a mild inflammatory responses was observed in the concentration range that showed efficacy, thereby confirming that the safety of the rAAV therapeutic agent was high.

[0423] The foregoing descriptions are only for illustrating the disclosure, and it will be apparent to a person having ordinary skill in the art to which the present invention pertains that the embodiments disclosed herein can be easily modified into other specific forms without changing the technical spirit or essential features. Therefore, it should be understood that Examples described herein are illustrative in all respects and are not limited.

Claims

1. Claims1. A nucleic acid for encoding an inhibitor of VEGF-A, comprising a codon-optimized polynucleotide encoding the inhibitor of VEGF-A, wherein the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8 or a nucleotide sequence having at least 90 % sequence identity to SEQ ID NO: 8.

2. The nucleic acid of claim 1 , wherein the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.

3. The nucleic acid of claim 1 or 2, further comprising a polynucleotide encoding a signal peptide of dermcidin.

4. The nucleic acid of claim 3, wherein the signal peptide comprises an amino acid sequence consisting of SEQ ID NO: 11 .

5. The nucleic acid of claim 3 or 4, wherein the polynucleotide encoding the signal peptide is located at the 5'-terminus of the codon-optimized polynucleotide encoding aflibercept.

6. The nucleic acid of any one of claims 1-5, wherein the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20.

7. The nucleic acid of any one of claims 1-6, wherein the inhibitor of VEGF-A comprises the amino acid sequence of SEQ ID NO: 20.

8. The nucleic acid of any one of claims 1-7, wherein the codon-optimized polynucleotide encoding the inhibitor of VEGF-A is operably linked to an expression regulatory element.

9. The nucleic acid of claim 8, wherein the expression regulatory element includes at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

10. The nucleic acid of claim 9, wherein the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

11. The nucleic acid of claim 9, wherein the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

12. The nucleic acid of claim 9, wherein the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto.

13. The nucleic acid of claim 9, wherein the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto.

14. The nucleic acid of claim 9, wherein the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto.

15. The nucleic acid of any one of claims 1-14, further comprising a Kozak sequence.

16. A nucleic acid for encoding an inhibitor of VEGF-A, comprising: polynucleotide sequences encoding an inhibitor of VEGF-A and a signal peptide, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 11 , or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 11.

17. The nucleic acid of claim 16, wherein the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20.

18. The nucleic acid of claim 16 or 17, wherein the polynucleotide sequence encoding the inhibitor of VEGF-A comprises the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 8.

19. The nucleic acid of claim 18, wherein the polynucleotide sequence encoding the inhibitor of VEGF-A comprises the nucleotide sequence of SEQ ID NO: 8.

20. The nucleic acid of any one of claims 16-19, wherein the polynucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 7, or a nucleotide sequence having at least 90% sequence identity with SEQ ID NO: 7.21 . The nucleic acid of claim 20, wherein the polynucleotide sequence encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 7.

22. The nucleic acid of any one of claims 16-21 , wherein the polynucleotide sequence encoding the signal peptide is at 5’ -end of the polynucleotide sequence encoding the inhibitor of VEGF-A.

23. The nucleic acid of any one of claims 16-22, further comprising one or more expression regulatory elements operably linked to the polynucleotide.

24. The nucleic acid of claim 23, wherein the expression regulatory element(s) are selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

25. The nucleic acid of claim 24, wherein the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

26. The nucleic acid of claim 24, wherein the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

27. The nucleic acid of claim 24, wherein the expression regulatory element comprises a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto.

28. The nucleic acid of claim 24, wherein the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto.

29. The nucleic acid of claim 24, wherein the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto.

30. The nucleic acid of any one of claims 16-29, further comprising a Kozak sequence.

31. A plasmid for producing a recombinant adeno-associated virus (rAAV), comprising (a) an AAV 5’ inverted terminal repeat (ITR), (b) the nucleic acid of any one of claims 1-30; and (c) an AAV 3’ ITR.

32. The plasmid of claim 31 , wherein the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhIO, AAV11 , AAV12, and AAV13.

33. The plasmid of claim 31 or 32, wherein the ITR is derived from AAV2.

34. The plasmid of any one of claims 31-33, wherein the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10.

35. The plasmid of any one of claims 31-34, further comprising the following genes:(a) a helper virus gene required for producing AAV;(b) a Rep gene of AAV; and(c) a Cap gene of AAV.

36. The plasmid of claim 35, wherein a nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus.

37. The plasmid of claim 35 or 36, wherein a nucleotide sequence of the helper virus gene is derived from an adenovirus.

38. The plasmid of any one of claims 35-37, wherein a nucleotide sequence of the helper virus gene is derived from adenovirus 2 (Ad2).

39. The plasmid of any one of claims 35-38, wherein the helper virus gene comprises one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DNA-binding protein (DBP), and a variant thereof.

40. The plasmid of any one of claims 35-39, wherein the helper virus gene comprises E2a, E4, and VA.

41. The plasmid of any one of claims 35-40, wherein a nucleotide sequence of the Rep gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhI O, AAV11 , AAV12, and AAV13.

42. The plasmid of any one of claims 35-41 , wherein a nucleotide sequence of the Rep gene is a Rep gene of wild-type AAV2.

43. The plasmid of any one of claims 35-42, wherein a nucleotide sequence of the Cap gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh , AAV11 , AAV12, and AAV13.

44. The plasmid of any one of claims 35-43, wherein a nucleotide sequence of the Cap gene is a Cap gene of wild-type AAV2.

45. An isolated host cell transfected or transduced with the plasmid of any one of claims 35 to 44.

46. The host cell of claim 45, wherein the host cell is a mammalian cell or an insect cell.

47. The host cell of claim 46, wherein the mammalian cell is selected from a HEK293 cell, a HEK293F cell, a HEK293T cell, and cells derived therefrom.

48. The host cell of claim 46, wherein the insect cell is an Sf9 cell, an Sf21 cell, a TN- 5B1-4 cell, or a High Five cell.

49. A recombinant adeno-associated virus (rAAV) comprising the plasmid of any one of claims 31-44.

50. A recombinant adeno-associated virus (rAAV) comprising the nucleic acid of any one of claims 1-30.

51. A recombinant adeno-associated virus (rAAV) comprising a transgene plasmid comprising the nucleic acid of any one of claims 1-30, and one or more additional plasmids comprising at least one of (a) a helper virus gene required for producing AAV;(b) a Rep gene of AAV; and(c) a Cap gene of AAV.

52. A nucleic acid for encoding an inhibitor of VEGF-A, comprising a codon-optimized polynucleotide sequence encoding the inhibitor of VEGF-A, wherein the polynucleotide further comprises (a) a helper virus gene required for producing AAV; (b) a Rep gene of AAV; and (c) a Cap gene of AAV.

53. The nucleic acid of claim 52, wherein the inhibitor of VEGF-A comprises an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 20.

54. The nucleic acid of claim 52 or 53, wherein the inhibitor of VEGF-A comprises the amino acid sequence of SEQ ID NO: 20.

55. The nucleic acid of any one of claims 52-54, wherein the codon-optimized polynucleotide comprises a nucleotide sequence having sequence identity of at least 90% to SEQ ID NO: 8.

56. The nucleic acid of any one of claims 52-55, wherein the codon-optimized polynucleotide comprises the nucleotide sequence of SEQ ID NO: 8.

57. The nucleic acid of any one of claims 52-56, further comprising a polynucleotide sequence encoding a signal peptide of dermcidin.

58. The nucleic acid of claim 54, wherein the signal peptide comprises an amino acid sequence consisting of SEQ ID NO: 11 .

59. The nucleic acid of any one of claims 52-58, wherein the polynucleotide sequence encoding the signal peptide is located at the 5'-terminus of the codon-optimized polynucleotide sequence encoding the inhibitor of VEGF-A.

60. The nucleic acid of any one of claims 52-59, wherein the codon-optimized polynucleotide encoding the inhibitor of VEGF-A is operably linked to an expression regulatory element.61 . The nucleic acid of claim 60, wherein the expression regulatory element includes at least one selected from the group consisting of an enhancer, a promoter, an intron, and a polyadenylation signal sequence.

62. The nucleic acid of claim 61 , wherein the enhancer includes a cytomegalovirus (CMV) immediate early enhancer sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

63. The nucleic acid of claim 61 , wherein the promoter includes a cytomegalovirus (CMV) immediate early promoter sequence or a nucleotide sequence having at least 90 % sequence identity thereto.

64. The nucleic acid of claim 61 , wherein the expression regulatory element includes a cytomegalovirus (CMV) immediate early enhancer / promoter sequence consisting of SEQ ID NO: 4 or a nucleotide sequence having at least 90 % sequence identity thereto.

65. The nucleic acid of claim 61 , wherein the intron includes a human beta-globin (hBG) intron sequence consisting of SEQ ID NO: 5 or a nucleotide sequence having at least 90 % sequence identity thereto.

66. The nucleic acid of claim 61 , wherein the polyadenylation signal sequence includes a human growth hormone polyadenylation (hGH pA) signal sequence consisting of SEQ ID NO: 9 or a nucleotide sequence having at least 90 % sequence identity thereto.

67. The nucleic acid of any one of claims 52-66, further comprising a Kozak sequence.

68. The nucleic acid of any one of claims 52-67, wherein a nucleotide sequence of the helper virus gene is derived from one or more selected from adenovirus, herpes simplex virus, baculovirus, papillomavirus, and bocavirus.

69. The nucleic acid of any one of claims 52-68, wherein a nucleotide sequence of the helper virus gene is derived from an adenovirus.

70. The nucleic acid of any one of claims 52-69, wherein a nucleotide sequence of the helper virus gene is derived from adenovirus 2 (Ad2).

71. The nucleic acid of any one of claims 52-70, wherein the helper virus gene comprises one or more selected from E1 , E2, E2a, E4, E4orf1 , E4orf2, E4orf3, E4orf4, E4orf5, E4orf6, E4orf7, VA, DNA-binding protein (DBP), and a variant thereof.

72. The nucleic acid of any one of claims 52-71 , wherein the helper virus gene comprises E2a, E4, and VA.

73. The nucleic acid of any one of claims 52-72, wherein a nucleotide sequence of the Rep gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhW, AAV11 , AAV12, and AAV13.

74. The nucleic acid of any one of claims 52-73, wherein a nucleotide sequence of the Rep gene is a Rep gene of wild-type AAV2.

75. The nucleic acid of any one of claims 52-74, wherein a nucleotide sequence of the Cap gene is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhW, AAV11 , AAV12, and AAV13.

76. The nucleic acid of any one of claims 52-75, wherein a nucleotide sequence of the Cap gene is a Cap gene of wild-type AAV2.

77. The nucleic acid of any one of claims 52-76, further comprising an AAV 5’ inverted terminal repeat (ITR) and an AAV 3’ ITR.

78. The nucleic acid of any one of claims 52-77, wherein the ITR is derived from one or more selected from AAV1 , AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrhW, AAV11 , AAV12, and AAV13.

79. The nucleic acid of any one of claims 52-78, wherein the ITR is derived from AAV2.

80. The nucleic acid of any one of claims 52-79, wherein the 5' ITR consists of SEQ ID NO: 3, and the 3' ITR consists of SEQ ID NO: 10.81 . A plasmid comprising the nucleic acid of any one of claims 52-80.

82. A recombinant adeno-associated virus (rAAV) comprising the plasmid of claim 81 .

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