Fusion polypeptide, expression cassette containing coding gene of fusion polypeptide, gene delivery vector, pharmaceutical composition and use

Abstract

Provided are a fusion polypeptide, an expression cassette containing a coding gene of the fusion polypeptide, a gene delivery vector, a pharmaceutical composition and a use. Specifically, provided is a fusion polypeptide, containing a secretion signal peptide and an aflibercept polypeptide, wherein the secretion signal peptide is selected from any one among a human albumin secretion signal peptide, a human Opticin protein secretion signal peptide, a human interleukin-2 secretion signal peptide and a mouse receptor tyrosine kinase-like orphan receptor 1 secretion signal peptide. The provided fusion polypeptide, a polynucleotide encoding same, a gene expression cassette containing the polynucleotide, and a gene delivery vector containing the expression cassette can improve the expression of aflibercept and improve the effect of treating ocular diseases by using aflibercept.

Description

Fusion polypeptide, expression cassette containing its encoding gene, gene delivery vector, pharmaceutical composition and use Priority and related applications The present disclosure claims the priority of PCT international application PCT / CN2023 / 110563, filed on August 1, 2023, entitled “Fusion polypeptide, expression cassette comprising its encoding gene, gene delivery vector, pharmaceutical composition and use”, the entire contents of which including the appendix are incorporated into the present disclosure as a reference. Technical Field The present disclosure belongs to the technical field of gene therapy, and specifically relates to a fusion polypeptide, an expression cassette comprising a gene encoding the fusion polypeptide, a gene delivery vector, a pharmaceutical composition and uses. Background Art Eye diseases related to pathological angiogenesis, such as wet age-related macular degeneration (Wet AMD), also known as wet AMD, develop rapidly, causing patients' vision to decline or even be lost in a short period of time. Although Wet AMD only accounts for 20% of all AMD cases, the blindness rate is as high as 80%, which has a huge impact on the quality of life of patients and their families. It is the third leading cause of blindness in the world and the main cause of vision loss in the elderly. The incidence rate in my country has also increased year by year. There are about 9 million people with Wet AMD in my country. The proportion of adults over 50 years old suffering from AMD in my country is 15.5%, and wet AMD accounts for 11.9% of the total number of AMD. Currently, there is no effective cure for Wet AMD. The mainstream treatment at this stage is anti-angiogenesis. This treatment mainly uses regular vitreous injections of anti-VEGF recombinant proteins or antibodies. This traditional treatment method has obvious pain points. Repeated administration is required, which causes great damage to patients, poor compliance, and large side effects. Therefore, it is very urgent to develop a gene therapy drug that can treat the disease once. Aflibercept is a recombinant fusion protein that acts as a decoy receptor for vascular endothelial growth factor subtypes A and B (VEGF-A and VEGF-B) and placental growth factor PIGF. By binding to these ligands, aflibercept is able to prevent these ligands from binding to vascular endothelial growth factor receptors (VEGFR) VEGFR-1 and VEGFR-2 to inhibit neovascularization and reduce vascular permeability. Aflibercept is composed of domain 2 of VEGFR-1 and domain 3 of VEGFR-2 fused to the Fc fragment of IgG1. Aflibercept is marketed under the trade name (Aflibercept) is commercially marketed as an intravitreal aflibercept fusion protein injection for ophthalmic use. In recent years, gene therapy has gradually matured and become safe, the efficiency of gene transfer and delivery has been further improved, and clinical research has also made substantial progress. In 2008, the case of Leber's congenital amaurosis (LCA) was successfully cured using adeno-associated virus vectors, bringing gene therapy back into the public eye. In 2012, the adeno-associated virus-derived gene therapy drug Glybera was first approved for marketing by the European Medicines Agency (EMA), opening a new era of gene therapy. In 2017, the FDA approved the gene therapy drug Luxturna, which uses adeno-associated virus vector type 2 adenovirus-associated virus (Adeno-Associated Virus 2, AAV2) to carry the RPE65 gene to treat vision loss caused by inherited retinal degeneration (IRDs). Subsequently, the field of gene therapy has flourished, with more and more clinical cases and related drugs being reported, and the safety and effectiveness of gene therapy being gradually recognized. In gene therapy, AAV viral vectors are the main application mode, and an important direction of gene therapy delivered by recombinant AAV is the central nervous system (CNS), including the eyes and brain. The eye is a relatively closed environment, and direct intraocular injection of AAV gene therapy can achieve the effect of treating a variety of genetic eye diseases. Clinical trials have shown that the use of AAV to deliver gene therapy in the eye does not cause systemic side effects and does not cause significant immune responses. Summary of the invention Problem that the invention aims to solve although (Aflibercept) is the current standard of care for wet AMD in patients, and gene therapy approaches that deliver aflibercept to the eye could offer patients an improved treatment option because gene therapy could provide extended or sustained release of aflibercept in the body without the need for repeated injections, which can increase the risk of inflammation, infection and other side effects in some patients. Solutions for solving problems [1]. A fusion polypeptide comprising a secretion signal peptide and an aflibercept polypeptide, wherein the secretion signal peptide is selected from any one of a human albumin secretion signal peptide, a human Opticin protein secretion signal peptide, a human interleukin-2 secretion signal peptide and a mouse receptor tyrosine kinase-like orphan receptor 1 secretion signal peptide. [2] The fusion polypeptide according to [1], wherein the amino acid sequence of the aflibercept polypeptide comprises the amino acid sequence shown in SEQ ID NO: 48; and / or, The amino acid sequence of the secretory signal peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 44-47. [3] The fusion polypeptide according to [1] or [2], wherein the amino acid sequence of the fusion polypeptide comprises any one selected from the following sequences: (i) an amino acid sequence as shown in any one of SEQ ID NO: 1, 30, 32 or 34; (ii) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34, and which retains the function of the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34, and which retains the function of the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34; or (iv) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34 under stringent conditions, and the amino acid sequence retains the function of the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34, and the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions. [4] An isolated polynucleotide encoding the fusion polypeptide described in any one of [1] to [3]; Preferably, the polynucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NO: 2, 29, 31 or 33, or a nucleotide sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence as shown in any one of SEQ ID NO: 2, 29, 31 or 33. [5]. A gene expression cassette comprising the polynucleotide described in [4]. [6] The gene expression cassette according to [5], wherein the gene expression cassette further comprises one or more of a promoter, an enhancer, an intron and a Kozak sequence upstream of the polynucleotide encoding the fusion polypeptide. [7] The gene expression cassette according to [6], wherein the promoter comprises any one of a CBh promoter, a CB7 promoter and a CMV promoter; Preferably, the CBh promoter comprises the sequence shown in SEQ ID NO: 3 or a sequence having at least 85% identity thereto; and / or, The CB7 promoter comprises the sequence shown in SEQ ID NO:4 or a sequence having at least 85% identity thereto; and / or, The CMV promoter includes the sequence shown in SEQ ID NO: 5 or a sequence having at least 85% identity thereto. [8] The gene expression cassette according to [6] or [7], wherein the intron comprises a human β-globin intron; Preferably, the human β-globin intron comprises the sequence shown in SEQ ID NO: 6 or a sequence having at least 85% identity thereto. [9] The gene expression cassette according to any one of [5] to [8], wherein the gene expression cassette comprises a polyadenylation region downstream of the polynucleotide encoding the fusion polypeptide; Optionally, the polyadenylation region is selected from any one of bovine growth hormone, human growth hormone and β-globin polyadenylation regions; Preferably, the polyadenylation region comprises a human growth hormone polyadenylation region; More preferably, the human growth hormone polyadenylation region comprises the sequence shown in SEQ ID NO: 7, or a sequence having at least 85% identity thereto.

[0010] . The gene expression cassette according to any one of [5] to [9], wherein the gene expression cassette comprises a sequence shown in any one of SEQ ID NOs: 8 to 10, 35 to 43, or a sequence having at least 85% identity with any one of SEQ ID NOs: 8 to 10, 35 to 43.

[0011] . A gene delivery vector comprising the gene expression cassette described in any one of [5] to

[0010] .

[0012] . The gene delivery vector according to

[0011] , wherein the gene delivery vector is a viral vector derived from a virus; Preferably, the gene delivery vector is a recombinant adeno-associated virus.

[0013] . The gene delivery vector according to

[0012] , wherein the recombinant adeno-associated virus comprises a capsid protein, and the gene expression cassette is encapsidated within the capsid protein; Optionally, the capsid protein is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 adeno-associated virus serotypes or variants thereof.

[0014] . The gene delivery vector according to

[0013] , wherein the capsid protein is AAV2 capsid protein or a variant thereof; Preferably, the capsid protein is an AAV2 capsid protein variant; More preferably, the AAV2 capsid protein variant comprises a sequence shown in any one of SEQ ID NOs: 16, 70 to 83, or a sequence having at least 85% identity with any one of SEQ ID NOs: 16, 70 to 83; Further preferably, the AAV2 capsid protein variant comprises the sequence shown in SEQ ID NO: 16 or 82 or a sequence having at least 85% identity with SEQ ID NO: 16 or 82.

[0015] . A pharmaceutical composition comprising the fusion polypeptide described in any one of [1] to [3], the polynucleotide described in [4], the gene expression cassette described in any one of [5] to

[0010] , or the gene delivery vector described in any one of

[0011] to

[0014] , and, optionally, a pharmaceutically acceptable carrier.

[0016] Use of the fusion polypeptide described in any one of [1] to [3], the polynucleotide described in [4], the gene expression cassette described in any one of [5] to

[0010] , or the gene delivery vector described in any one of

[0011] to

[0014] in the preparation of a medicament for preventing and / or treating a disease; Optionally, the disease is an eye disease; Preferably, the eye disease is an eye disease associated with ocular neovascularization, or an eye disease associated with choroidal neovascularization; More preferably, the eye disease is selected from one or more of wet age-related macular degeneration, macular edema secondary to retinal vein occlusion, diabetic macular edema and diabetic retinopathy.

[0017] . A method for preventing and / or treating a disease, comprising administering to a subject a preventive and / or therapeutically effective amount of the fusion polypeptide described in any one of [1] to [3], the polynucleotide described in [4], the gene expression cassette described in any one of [5] to

[0010] , the gene delivery vector described in any one of

[0011] to

[0014] , or the pharmaceutical composition described in

[0015] ; Optionally, the disease is an eye disease; Preferably, the eye disease is an eye disease associated with ocular neovascularization, or an eye disease associated with choroidal neovascularization; More preferably, the eye disease is selected from one or more of wet age-related macular degeneration, macular edema secondary to retinal vein occlusion, diabetic macular edema and diabetic retinopathy. Effects of the Invention The fusion polypeptide provided by the present disclosure, the polynucleotide encoding the fusion polypeptide, the gene expression cassette comprising the polynucleotide, and the gene delivery vector comprising the expression cassette can increase the expression of aflibercept and improve the effect of using aflibercept to treat eye diseases. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A : Schematic diagram of the pssAAV-CMV-Fluc-hGH pA vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CMV promoter, human cytomegalovirus early promoter. Human beta Globin intron, human beta Globin intron. Fluc, firefly luciferase gene coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 1B: Schematic diagram of the pssAAV-CMV-Aflibercept vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CMV promoter, human cytomegalovirus early promoter. Human beta Globin intron, human beta Globin intron. Aflibercept, codon-unoptimized aflibercept coding sequence (including its own secretory signal peptide sequence). hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 1C: Schematic diagram of the pssAAV-CMV-Aflibercept opt2 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CMV promoter, human cytomegalovirus early promoter. Human beta Globin intron, human beta Globin intron. Aflibercept opt2, codon-unoptimized aflibercept coding sequence (including its own secretory signal peptide sequence). hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 1D: Schematic diagram of the pssAAV-CMV-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CMV promoter, human cytomegalovirus early promoter. Human beta Globin intron, human beta Globin intron. Aflibercept opt3, codon-unoptimized aflibercept coding sequence (including its own secretory signal peptide sequence). hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 2: Effect of codon optimization on aflibercept expression. HEK 293T cells were seeded in 6-well plates and grown to 70% to 80% confluence. Lipo3000 was used to transfect the cells with pssAAV-CMV-Aflibercept, pssAAV-CMV-Aflibercept opt2, and pssAAV-CMV-Aflibercept opt3 at 2 μg / well. After 48 hours, the cells and supernatant were collected. Elisa was used to detect the concentration of Aflibercept. FIG. 3A : Schematic diagram of the pssAAV-CBh-Fluc-hGH pA vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CBh promoter, artificially designed promoter, composed of human cytomegalovirus enhancer, chicken β-actin promoter and part of chicken β-actin intron and part of minute virus of mice (mvm) intron, sequence information see SEQ ID NO:3. Fluc, firefly luciferase gene coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. FIG. 3B : Schematic diagram of the pssAAV-CB7-Fluc-hGH pA vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CB7 promoter, artificially designed promoter, spliced ​​by human cytomegalovirus enhancer, chicken β-actin promoter and intron, etc., sequence information is shown in SEQ ID NO:4. Fluc, firefly luciferase gene coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3C: Schematic diagram of the pssAAV-CMV-hAlbumin sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CMV promoter, human cytomegalovirus early promoter. Human beta Globin intron, human beta globin intron. hAlbumin sp, human albumin secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3D: Schematic diagram of the pssAAV-CBh-hAlbumin sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CBh promoter, artificially designed promoter, composed of human cytomegalovirus enhancer, chicken β-actin promoter and part of chicken β-actin intron and part of minute virus of mice (mvm) intron, sequence information see SEQ ID NO:3. hAlbumin sp, human albumin secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3E: Schematic diagram of the pssAAV-CB7-hAlbumin sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CB7 promoter, artificially designed promoter, spliced ​​by human cytomegalovirus enhancer, chicken β-actin promoter and intron, etc., sequence information is shown in SEQ ID NO:4. hAlbumin sp, human albumin secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3F: Schematic diagram of the pssAAV-CMV-hOpticin sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CMV promoter, human cytomegalovirus early promoter. Human beta Globin intron, human beta Globin intron. hOpticin sp, human Opticin protein secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3G: Schematic diagram of the pssAAV-CBh-hOpticin sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CBh promoter, artificially designed promoter, composed of human cytomegalovirus enhancer, chicken β-actin promoter and part of chicken β-actin intron and part of minute virus of mice (mvm) intron. See SEQ ID NO:3 for sequence information. hOpticin sp, human Opticin protein secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3H: Schematic diagram of the pssAAV-CB7-hOpticin sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CB7 promoter, artificially designed promoter, spliced ​​by human cytomegalovirus enhancer, chicken β-actin promoter and intron, etc., sequence information is shown in SEQ ID NO:4. hOpticin sp, human Opticin protein secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3I: Schematic diagram of the pssAAV-CMV-hIL2sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CMV promoter, human cytomegalovirus early promoter. Human beta Globin intron, human beta Globin intron. hIL2sp, human interleukin 2 protein secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3J: Schematic diagram of the pssAAV-CBh-hIL2sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CBh promoter, artificially designed promoter, composed of human cytomegalovirus enhancer, chicken β-actin promoter and part of chicken β-actin intron and part of minute virus of mice (mvm) intron, sequence information see SEQ ID NO:3. hIL2sp, human interleukin 2 protein secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3K: Schematic diagram of the pssAAV-CB7-hIL2sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CB7 promoter, artificially designed promoter, spliced ​​by human cytomegalovirus enhancer, chicken β-actin promoter and intron, etc., sequence information is shown in SEQ ID NO:4. hIL2sp, human interleukin 2 protein secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3L: Schematic diagram of the pssAAV-CMV-mROR1 sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CMV promoter, human cytomegalovirus early promoter. Human beta Globin intron, human beta Globin intron. mROR1 sp, mouse ROR1 protein secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3M: Schematic diagram of the pssAAV-CBh-mROR1 sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CBh promoter, artificially designed promoter, composed of human cytomegalovirus enhancer, chicken β-actin promoter and part of chicken β-actin intron and part of minute virus of mice (mvm) intron, sequence information see SEQ ID NO:3. mROR1 sp, mouse ROR1 protein secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3N: Schematic diagram of the pssAAV-CB7-mROR1 sp-Aflibercept opt3 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CB7 promoter, artificially designed promoter, composed of human cytomegalovirus enhancer, chicken β-actin promoter and intron, etc., sequence information is shown in SEQ ID NO:4. hOpticin sp, mROR1 sp, mouse ROR1 protein secretion signal peptide sequence. Aflibercept opt3, codon-optimized aflibercept (without secretion signal peptide sequence) coding sequence. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 3O: Schematic diagram of the pssAAV-ADVM-022 vector structure. ITR, inverted terminal repeat, reverse terminal repeat sequence (upstream ITR length is about 145bp, downstream ITR length is about 141bp). CMV promoter, human cytomegalovirus early promoter. TPL intron, tripartite leader sequence. Aflibercept, aflibercept coding sequence. human scaffold-attached region, scaffold attachment region. hGH polyA, human growth hormone polynucleotide tailing signal. Kan, kanamycin resistance gene reading frame. Figure 4: Effects of different secretion signal peptide sequences on the secretion of aflibercept. HEK 293T cells were seeded in 6-well plates and grown to 70% to 80% confluence. Lipo3000 was used to transfect the cells with 2 μg / well of pssAAV-CMV-Aflibercept opt3, pssAAV-CMV-hAlbumin sp-Aflibercept opt3, pssAAV-CMV-hOpticin sp-Aflibercept opt3, pssAAV-CMV-hIL2sp-Aflibercept opt3, and pssAAV-CMV-mROR1 sp-Aflibercept opt3. After 48 hours, the cells and supernatant were collected. The cell supernatant was diluted 5 times with sterile PBS. The cells were lysed with Ripa lysis buffer (Biyuntian, Beijing) and the total protein was extracted. After the protein concentration was determined, the total protein concentration of the cells was adjusted to be consistent. Western blot was used to detect aflibercept in the cell supernatant and total cell protein. Figure 5: Expression of different rAAV IVT15-hIL2sp-Aflibercept opt3 in mouse aqueous humor. rAAV IVT15-CMV-hIL2sp-Aflibercept opt3, rAAV IVT15-CBh-hIL2sp-Aflibercept opt3 and rAAV IVT15-CB7-hIL2sp-Aflibercept opt3 were respectively used at 6×10 9 vg / eye (viral genome, vg) was injected into the vitreous cavity of wild-type male c57 mice. Each virus was injected into a group of 9 mice, and the mice were injected in both eyes at 6-8 weeks of age. In addition, a group of mice injected with PBS was added as a control. The four groups of mice were named A1: injected with rAAV IVT15-CMV-hIL2sp-Aflibercept opt3; A2: injected with rAAV IVT15-CBh-hIL2sp-Aflibercept opt3; A3: injected with rAAV IVT15-CB7-hIL2sp-Aflibercept opt3; A4: injected with rAAV2 7m8-ADVM-022 and A5: injected with PBS. After 2 weeks, 4 weeks, and 6 weeks after injection, 3 mice were randomly selected from each group, and after euthanasia, the eyeballs of both eyes were removed, and the expression of aflibercept was detected by Elisa after the aqueous humor was obtained. Figure 6: Establishment of mouse choroidal neovascularization (CNV) model. rAAV IVT15-CMV-hIL2sp-Aflibercept opt3, rAAV IVT15-CB7-hIL2sp-Aflibercept opt3 and rAAV2 7m8-ADVM-022 were respectively used at 6×10 9 Wild-type male c57 mice were injected intravitreally with a dose of 10 vg / eye for each virus, and the mice were injected monocularly (right eye) at 6-8 weeks of age. Two groups of mice were added at the same time, one of which was injected with 1 μL PBS, and the other group was not treated for the time being. Laser CNV modeling was performed on the right eyes of all mice. Three days after modeling, a group of untreated mice were injected with 2.5 μg Eylea (Aflibercept, purchased from Bayer (China) Co., Ltd., Beijing, China) into the vitreous cavity of the modeling eye. The four groups of mice were named G1 group: injected with rAAV IVT15-CMV-hIL2sp-Aflibercept opt3; G2 group: injected with rAAV IVT15-CB7-hIL2sp-Aflibercept opt3; G3 group: injected with rAAV2 7m8-ADVM-022; G4 group: injected with PBS; G5 group: injected with Eylea. After modeling, all mice were examined by spectral domain optical coherence tomography (SD-OCT). FIG. 7A : Vascular leakage in CNV mice treated with different viruses and other drugs 7 days after modeling. In the mice of groups G1 to G5 in FIG6 , 7 days after CNV modeling, the modeling eyes were tested by fluorescein fundus angiography (FFA). FIG. 7B : Vascular leakage in CNV mice treated with different viruses and other drugs 14 days after modeling. In Figure 6 , mice in groups G1 to G5 were subjected to FFA testing of the modeling eyes 14 days after CNV modeling. FIG8 : Vascular leakage in CNV mice treated with different viruses and other drugs 14 days after modeling compared with 7 days after modeling. For mice in groups G1 to G5 in Figure 6, 7 and 14 days after CNV modeling, the photos taken by FFA detection were processed and the fluorescence area was calculated using Image-Pro Plus 6.0. The mean fluorescence area was calculated for each group of mice. Figure 9: Neovascularization leakage scores in CNV mice treated with different viruses and other drugs. The G1-G5 group mice in Figure 6 were scored for spot area and brightness based on the photos taken by FFA detection 14 days after CNV modeling. Three technicians with relevant knowledge of ophthalmology were asked to perform randomized double-blind scoring of neovascularization leakage (fluorescent spot area, brightness) on the photos taken, and the average score was calculated for each group of mice. Figure 10: Vascular leakage in virus- and control-drug-treated cynomolgus monkeys 28 days after CNV modeling. rAAV IVT15-CB7-hIL2sp-Aflibercept opt3 was used at 2.5×10 11 vg / eye was injected into the vitreous cavity of cynomolgus monkeys, 2 monkeys were injected with each virus, and the injection was performed in both eyes. The monkeys were 3-5 years old at the time of injection. At the same time, a group of 2 cynomolgus monkeys were added and injected with 50μL PBS. All cynomolgus monkeys underwent laser CNV modeling in both eyes 28 days after injection. The two groups of mice were named N1 group: injected with rAAV IVT15-CB7-hIL2sp-Aflibercept opt3; N2 group: injected with PBS. 14 and 28 days after modeling, fluorescein fundus angiography (FFA) was performed in both eyes. The results of the 28-day test are shown in Figure 10. Figure 11: The proportion of level 4 spots in cynomolgus monkeys treated with virus and control drugs 14 days (2 weeks) and 28 days (4 weeks) after CNV modeling. In the N1 and N2 groups of cynomolgus monkeys in Figure 10, the spot area and brightness were scored based on the photos taken by FFA detection 14 and 28 days after CNV modeling. Three technicians with relevant knowledge of ophthalmology were asked to perform randomized double-blind neovascularization leakage (fluorescent spot area, brightness) scoring on the photos taken, and the proportion of level 4 spots is shown in Figure 11. DETAILED DESCRIPTION In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present disclosure belongs. According to the present disclosure, a numerical range expressed using "a numerical value A to a numerical value B" means a range including the endpoint numerical values ​​A and B. According to the present disclosure, the use of “substantially” or “essentially” means that the standard deviation from a theoretical model or theoretical data is within 5%, preferably 3%, and more preferably 1%. According to the present disclosure, the meaning of "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process. According to the present disclosure, “optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not. According to the present disclosure, the terms "include" and "have" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment. According to the present disclosure, the term "plurality" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects before and after are in an "or" relationship. According to the present disclosure, references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., refer to specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments, which are included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner. According to the present disclosure, "Vg" refers to the viral genome (Viral Genome); equivalent to the genome copy (Genome Copy). According to the present disclosure, the terms "polypeptide", "protein" and "peptide" are used interchangeably herein to refer to a polymeric form of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with similar peptide backbones. According to the present disclosure, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", and "nucleic acid" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or their analogs. According to the present disclosure, "isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated", but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-natural environment such as, for example, a host cell. According to the present disclosure, the terms "upstream" and "downstream" are relative terms that define the linear position of at least two elements located in a nucleic acid molecule (whether single-stranded or double-stranded) oriented in the 5' to 3' direction. According to the present disclosure, "fusion protein", "fusion polypeptide" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. Any protein provided herein can be produced by any method known in the art. For example, the protein provided herein can be produced via recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known and include those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated herein by reference. According to the present disclosure, the term "amino acid" may include natural amino acids, unnatural amino acids, amino acid analogs and all their D and L stereoisomers. According to the present disclosure, the amino acid three-letter code and one-letter code used are as described in J.biol.chem, 243, p3558 (1968). The amino acids and their abbreviations and English abbreviations in the present disclosure are as follows: histidine (His, H); serine (Ser, S); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); threonine (Thr, T); phenylalanine (Phe, F); aspartic acid (Asp, D); tyrosine (Tyr, Y); leucine (Leu, L); isoleucine (Ile, I); arginine (Arg, R); alanine (Ala, A); valine (Val, V); tryptophan (Trp, W); methionine (Met, M); asparagine (Asn, N); cysteine ​​(Cys, C); lysine (Lys, K); proline (Pro, P). According to the present disclosure, amino acid "addition" refers to adding amino acids at the C-terminus or N-terminus of an amino acid sequence. According to the present disclosure, amino acid "deletion" refers to deleting 1, 2 or 3 or more amino acids from an amino acid sequence. According to the present disclosure, amino acid "insertion" refers to inserting an amino acid residue at an appropriate position in an amino acid sequence, and the inserted amino acid residues may also be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other. According to the present disclosure, amino acid "substitution" refers to replacing an amino acid residue at a certain position in an amino acid sequence with other amino acid residues; wherein, the "substitution" may be a conservative amino acid substitution. According to the present disclosure, "conservative modification", "conservative substitution" or "conservative replacement" refers to the replacement of an amino acid in a protein with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacements of amino acids with similar structure or function are unlikely to destroy biological activity. Exemplary conservative replacements are set forth in the following "Exemplary amino acid conservative replacements". Exemplary conservative amino acid substitutions According to the present disclosure, "moderate to very high stringency conditions" include "moderate stringency conditions", "moderate-high stringency conditions", "high stringency conditions" or "very high stringency conditions", which describe conditions for nucleic acid hybridization and washing. Guidance for conducting hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Aqueous and non-aqueous methods are described in this document, and either can be used. For example, specific hybridization conditions are as follows: (1) low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by two washes in 0.2×SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the washing temperature can be increased to 55°C); (2) moderate stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 60°C; (3) high stringency hybridization conditions are in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C and preferably; (4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2×SSC, 1% SDS at 65°C. According to the present disclosure, "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, the comparison is made when the two sequences are aligned to obtain the maximum percent identity. According to the present disclosure, "administering," "giving," and "treating" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also means in vitro and ex vivo treatment of, for example, a cell by an agent, a diagnostic agent, a binding composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic, prophylactic or preventative measures, research, and diagnostic applications. According to the present disclosure, "treatment" means administering an internal or external therapeutic agent, such as any of the antibodies disclosed herein, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in the treated patient or population, either by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical test method commonly used by physicians or other health care professionals to evaluate the severity or progression of the symptom. According to the present disclosure, the term "prevention" refers to the preventive treatment of subjects who do not have a disease now and in the past but are at risk of developing a disease or who have had a disease in the past and do not have a disease now but are at risk of recurrence of the disease. In certain embodiments, the subject has a higher risk of developing a disease or a higher risk of recurrence of the disease compared to the average healthy member of the subject population. According to the present disclosure, an "effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. According to the present disclosure, a "therapeutically effective amount" is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or sufficient to delay or minimize one or more symptoms associated with a condition. A therapeutically effective amount refers to an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment of a condition. The term "therapeutically effective amount" can include an amount that improves overall therapy; reduces or avoids symptoms, signs, or causes of a condition; and / or enhances the therapeutic efficacy of another therapeutic agent. According to the present disclosure, a "prophylactically effective amount" is an amount sufficient to prevent a condition or one or more symptoms associated with a condition or to prevent its recurrence. A prophylactically effective amount refers to an amount of a therapeutic agent, alone or in combination with other agents, that provides a prophylactic benefit in preventing a condition. The term "prophylactically effective amount" may include an amount that improves overall prevention or enhances the prophylactic efficacy of another prophylactic agent. According to the present disclosure, the term "subject" refers to a human (i.e., a male or female of any age, e.g., a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young, middle-aged, or elderly person)) or a non-human animal. In certain embodiments, the non-human animal is a mammal (e.g., a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, pig, horse, sheep, goat, cat, or dog), or a bird. The non-human animal can be male or female at any stage of development. The non-human animal can be a transgenic animal or a genetically engineered animal. According to the present disclosure, "vector" refers to a macromolecule or an association of macromolecules that includes or is associated with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell. Illustrative vectors include, for example, plasmids, viral vectors (i.e., viruses such as adeno-associated viruses), liposomes, and other gene delivery vehicles. According to the present disclosure, "expression vector" encompasses vectors, such as plasmids, minicircles, viral vectors, liposomes, etc., that include a gene expression cassette encoding a gene product of interest and are used to deliver the gene expression cassette to intended target cells. According to the present disclosure, the term "AAV" is an abbreviation for adeno-associated virus, and can be used to refer to the virus itself or its derivatives. The term covers all subtypes and naturally occurring and recombinant forms unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, etc. According to the present disclosure, "AAV virus" or "AAV virus particle" or "rAAV vector particle" refers to a virus particle composed of at least one AAV capsid protein (usually composed of all capsid proteins of wild-type AAV) and an encapsidated polynucleotide. If the particle includes a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is generally referred to as a recombinant AAV vector or rAAV. Typically, the heterologous polynucleotide is flanked by AAV inverted terminal repeats (ITRs). According to the present disclosure, the term "replication defective" with respect to the AAV viral vector of the present disclosure means that the AAV vector cannot replicate and package its genome independently. For example, when the cells of a subject are infected with rAAV viral particles, the heterologous gene is expressed in the infected cells, however, due to the fact that the infected cells lack the AAV rep and cap genes and auxiliary function genes, the rAAV cannot replicate further. According to the present disclosure, "AAV variant" or "AAV mutant" refers to a viral particle composed of a variant AAV capsid protein, wherein the variant AAV capsid protein includes at least one amino acid difference (e.g., amino acid substitution, amino acid insertion, amino acid deletion) relative to the corresponding parent AAV capsid protein, and wherein the variant capsid protein confers increased infectivity to retinal cells compared to the infectivity of retinal cells of AAV viral particles including the corresponding parent AAV capsid protein, wherein the AAV capsid protein does not include an amino acid sequence present in a naturally occurring AAV capsid protein. The polynucleotide expression cassette of the present disclosure can be packaged in a variant AAV particle to facilitate delivery of the cassette to a specific cell type (e.g., retinal cells) in a target tissue. According to the present disclosure, the term "gene", "coding sequence" or "coding gene" refers to a nucleotide sequence that encodes a gene product in vitro or in vivo. The term "transgene" refers to a coding sequence or gene delivered into a cell by a vector. The coding sequence or gene can encode a peptide or polypeptide molecule. According to the present disclosure, the term "operably linked" refers to the juxtaposition of genetic elements (e.g., promoters, enhancers, termination signal sequences, polyadenylation sequences, etc.), wherein the elements are in a relationship that permits them to operate in the intended manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of a coding sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained. According to the present disclosure, the term "heterologous" refers to an entity that is derived from a different genotype than the rest of the entity to which it is being compared. For example, a polynucleotide introduced into a plasmid or vector derived from a different species by genetic engineering techniques is a heterologous polynucleotide. As another example, a promoter removed from its native coding sequence and operably linked to a coding sequence to which it is not naturally found is a heterologous promoter. Thus, for example, a rAAV comprising a heterologous nucleic acid encoding a heterologous gene product is a rAAV comprising a nucleic acid that is not normally contained in a naturally occurring wild-type AAV, and the encoded heterologous gene product is a gene product that is not normally encoded by a naturally occurring wild-type AAV. According to the present disclosure, the term "endogenous" with respect to nucleotide molecules or gene products refers to nucleic acid sequences (eg, genes or genetic elements) or gene products (eg, RNA, proteins) that are naturally present in or associated with a host virus or cell. DETAILED DESCRIPTION OF THE INVENTION <Fusion polypeptide> In some aspects of the present disclosure, a fusion polypeptide is provided, which comprises a secretion signal peptide and an aflibercept polypeptide, wherein the secretion signal peptide is selected from any one of a human albumin secretion signal peptide, a human Opticin protein secretion signal peptide, a human interleukin-2 secretion signal peptide and a mouse receptor tyrosine kinase-like orphan receptor 1 secretion signal peptide. According to the present disclosure, the term "signal peptide", "signal sequence", "signal peptide sequence" or "secretion signal peptide" refers to a short peptide that, when fused to a target protein (e.g., aflibercept), can promote the secretion of the target protein expressed by the cell onto the cell membrane or outside the cell. The signal peptide is usually located at the N-terminus of the target protein. Exemplarily, the secretion signal peptide is selected from the hemagglutinin signal sequence, the human insulin signal sequence, the human albumin secretion signal peptide sequence, the human Opticin protein secretion signal peptide sequence, the human interleukin-2 secretion signal peptide sequence, the mouse receptor tyrosine kinase-like orphan receptor 1 secretion signal peptide sequence, and the like. However, different secretion signal peptides are used for different target proteins, and the effects in different gene expression frames and gene delivery vectors are unknown. In some optional embodiments, the amino acid sequence of the human albumin secretion signal peptide comprises: The amino acid sequence of the secretion signal peptide of human Opticin protein contains: The amino acid sequence of the human interleukin-2 secretion signal peptide comprises: The amino acid sequence of the mouse receptor tyrosine kinase-like orphan receptor 1 secretion signal peptide is: According to the present disclosure, aflibercept is a 115 kDa fusion protein that may be glycosylated. Aflibercept comprises an IgG backbone fused to the extracellular VEGF receptor sequences of human VEGFR-1 and VEGFR-2, and functions similarly to a soluble decoy receptor by binding to VEGF-A with a higher affinity than its native or endogenous receptor. See, e.g., Stewart MW. Aflibercept (VEGF Trap-eye): the newest anti-VEGF drug. Br. J. Ophthalmol. 2012 Sep; 96(9): 1157-8. Aflibercept's high affinity for VEGF interferes with or disrupts subsequent binding and activation of the native or endogenous VEGF receptor. Reduced VEGF activity may result in reduced angiogenesis and reduced vascular permeability. Aflibercept's inhibition of placental growth factor PIGF and VEGF-B may also help treat angiogenic conditions. PIGF has been associated with angiogenesis and may be elevated in a variety of conditions, such as wet AMD. VEGF-B overexpression may be associated with disruption of the blood-retinal barrier and retinal angiogenesis. Therefore, inhibition of VEGF-A, VEGF-B, and PIGF may all contribute to the efficacy of aflibercept. In some specific embodiments, the amino acid sequence of the aflibercept polypeptide comprises: In some specific embodiments, the amino acid sequence of the fusion polypeptide comprises any one of the following sequences: (i) an amino acid sequence as shown in any one of SEQ ID NO: 1, 30, 32 or 34; (ii) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34, and which retains the function of the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34, and which retains the function of the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34; or (iv) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34 under stringent conditions, and the amino acid sequence retains the function of the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34, and the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions. An exemplary amino acid sequence of a fusion polypeptide containing a human interleukin-2 secretion signal peptide and an aflibercept polypeptide (abbreviated as hIL2sp-Aflibercept opt3) (SEQ ID NO: 1): The single underlined portion is human interleukin-2 secretion signal peptide (also referred to as hIL2sp in this specification); the double underlined portion is aflibercept. <Polynucleotide> In some aspects of the present disclosure, an isolated polynucleotide is provided, which encodes a fusion polypeptide provided by the present disclosure. In some embodiments of the present disclosure, the polynucleotide encoding the fusion polypeptide provided by the present disclosure is modified or "codon optimized" to enhance expression by replacing infrequently expressed codons with more frequently expressed codons. The coding sequence is a part of the mRNA sequence encoding the amino acid for translation. During the translation process, each of the 61 trinucleotide codons is translated into one of the 20 amino acids, resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species utilize tRNA (each carrying an anticodon) that encodes the same amino acid at different frequencies. When the gene sequence contains codons that are infrequently expressed by the corresponding tRNA, the ribosome translation mechanism may slow down, thereby hindering effective translation. Expression can be improved by "codon optimization" of a specific species, in which the coding sequence is changed to encode the same protein sequence, and codons (Cid-Arregui et al., 2003; Journal of Virology 77: 4928) that are highly expressed and / or utilized by highly expressed human proteins are utilized. In some preferred embodiments of the present disclosure, the polynucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NO: 2, 29, 31 or 33, or a nucleotide sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence as shown in any one of SEQ ID NO: 2, 29, 31 or 33. Exemplary, a polynucleotide encoding a fusion polypeptide comprising a human interleukin-2 secretion signal peptide and an aflibercept polypeptide provided by the present disclosure (SEQ ID NO: 2) <Gene expression cassette> In some aspects of the present disclosure, a gene expression cassette is provided, which comprises a polynucleotide provided by the present disclosure, that is, comprises a polynucleotide encoding a fusion polypeptide provided by the present disclosure. In some optional embodiments of the present disclosure, the gene expression cassette further comprises one or more of a promoter, an enhancer, an intron and a Kozak sequence upstream of the polynucleotide encoding the fusion polypeptide provided by the present disclosure. According to the present disclosure, a "promoter" encompasses a DNA sequence that directs RNA polymerase binding and thereby promotes RNA synthesis. Promoters and corresponding protein or polypeptide expression can be ubiquitous (meaning that they are strongly active in a wide range of cells, tissues, and species) or cell type-specific, tissue-specific, or species-specific. Promoters can be "constitutive" (meaning that they are continuously active) or "inducible" (meaning that the promoter can be activated or inactivated by the presence or absence of biological or abiotic factors). According to the present disclosure, "enhancer" encompasses cis-acting elements that stimulate or inhibit transcription of adjacent genes. Enhancers that inhibit transcription are also referred to as "silencers". Enhancers can act in any direction at a distance of several thousand base pairs (kb) from the coding sequence and the position downstream of the transcription region (i.e., can be associated with the coding sequence). Exemplary, an example of a suitable enhancer is the CMV enhancer. Other suitable enhancers include enhancers suitable for the desired target tissue indication. According to the present disclosure, "intron" includes a splice donor / acceptor region. The intron is located downstream of the promoter region and upstream of the translation start sequence of the gene. Introns are DNA polynucleotides that are transcribed into RNA by intron splicing and removed during mRNA processing. The expression of gene expression cassettes containing introns is generally higher than that of those gene expression cassettes that do not have introns. In some optional embodiments of the present disclosure, introns can be, for example, chicken β-actin (β-actin) introns, human β-globin introns (Human beta Globin intron). In other optional embodiments of the present disclosure, introns are chimeric introns (CI)-hybrid introns composed of human β-globin splice donors and immunoglobulin G (IgG) splice acceptor elements. Other suitable introns include introns known in the art, for example, introns as described in WO 2011 / 126808. According to the present disclosure, a "Kozak sequence" is a nucleic acid sequence located behind the 5' cap structure of eukaryotic mRNA, which can bind to a translation initiation factor to mediate the translation initiation of mRNA containing a 5' cap structure. In some specific embodiments, the specific sequence of the Kozak sequence is 5'-GCCACC-3'. In some optional embodiments of the present disclosure, in the gene expression cassette, a promoter is included upstream of the polynucleotide encoding the fusion polypeptide provided by the present disclosure, and the promoter includes any one of a CBh promoter, a CB7 promoter and a CMV promoter. The CBh promoter is an artificially designed promoter, which is formed by splicing a human cytomegalovirus enhancer, a chicken β-actin promoter, a part of a chicken β-actin intron, and a part of a minute virus of mice (mvm) intron. In some specific embodiments, the CBh promoter comprises the sequence shown in SEQ ID NO: 3 or a sequence having at least 85% identity thereto. Nucleotide sequence of CBh promoter (SEQ ID NO: 3): The CB7 is an artificially designed promoter, which is spliced ​​from human cytomegalovirus enhancer, chicken β-actin promoter and intron. In some specific embodiments, the CB7 promoter comprises the sequence shown in SEQ ID NO:4 or a sequence having at least 85% identity thereto. Nucleotide sequence of CB7 promoter (SEQ ID NO:4) In some optional embodiments of the present disclosure, in the gene expression cassette, a promoter and an intron are included upstream of the polynucleotide encoding the fusion polypeptide provided by the present disclosure, the promoter is a CMV promoter, and the intron is a human β-globin intron. In some specific embodiments, the CMV promoter comprises the sequence shown in SEQ ID NO: 5 or a sequence having at least 85% identity thereto. Nucleotide sequence of CMV promoter (SEQ ID NO:5): In some specific embodiments, the human β-globin intron comprises the sequence shown in SEQ ID NO: 6 or a sequence having at least 85% identity thereto. Nucleotide sequence of human β-globin intron (SEQ ID NO: 6): In some optional embodiments of the present disclosure, in the gene expression cassette, a polyadenylation region (or polynucleotide tailing signal) is included downstream of the polynucleotide encoding the fusion polypeptide provided by the present disclosure. As understood in the art, RNA polymerase II transcripts are terminated by cleavage and addition of a polyadenylation region, which may also be referred to as a polynucleotide tailing signal, a polyA signal, a polyA region, or a polyA tail. The polyA region contains multiple consecutive adenosine monophosphates, which typically have repeats of the motif AAUAAA. Several effective polyadenylation sites have been identified, including those from SV40, bovine growth hormone, human growth hormone, and rabbit beta-globin. The most effective polyA signal for expressing a transgene in a mammalian cell may depend on the cell type and species of interest, as well as the specific vector used. In some embodiments of the present disclosure, the gene expression cassette includes a polyA region selected from the group consisting of bovine growth hormone (bGH), human growth hormone (hGH), and beta-globin (beta-globin). In some preferred embodiments, the polyA region is a human growth hormone polyA region. In some specific embodiments, the human growth hormone polyA region comprises a sequence as shown in SEQ ID NO: 7 or a sequence having at least 85% identity thereto. Nucleotide sequence of human growth hormone polyA region (SEQ ID NO:7) As will be appreciated by those of ordinary skill in the art, the gene expression cassette may optionally contain other elements, including but not limited to restriction sites to facilitate cloning and regulatory elements for a particular gene expression vector. Examples of regulatory sequences include ITRs of AAV vectors, bacterial sequences of plasmid vectors, attP sites or attB sites of phage integrase vectors, and transposable elements of transposons. In some more preferred embodiments, the gene expression cassette comprises a sequence shown in any one of SEQ ID NOs: 8-10, 35-43, or a sequence having at least 85% identity with any one of SEQ ID NOs: 8-10, 35-43. Exemplary: SEQ ID NO:8(CMV-hIL2sp-Aflibercept opt3-hGH pA) SEQ ID NO:9(CBh-hIL2sp-Aflibercept opt3-hGH pA) SEQ ID NO:10(CB7-hIL2sp-Aflibercept opt3-hGH pA) <Gene delivery vector> In some aspects of the present disclosure, the gene expression cassette of the present disclosure is used to deliver a gene (encoding a fusion polypeptide) to an animal cell, for example, to determine the effect of the gene on cell viability and / or function, to treat a cell disorder, etc. Therefore, in some aspects of the present disclosure, a gene delivery vector is provided, comprising the gene expression cassette of the present disclosure. In some preferred embodiments, the gene delivery vector is used to express a transgene (encoding a fusion polypeptide) in a mammalian cell. The gene delivery vector of the present disclosure encompasses any convenient gene delivery vector for delivering a polynucleotide sequence to a mammalian cell. For example, the vector can include a single-stranded nucleic acid or a double-stranded nucleic acid, such as a single-stranded DNA or a double-stranded DNA. For example, the gene delivery vector can be a DNA, such as a naked DNA, such as a plasmid or a mini-circle, etc. The vector can include a single-stranded RNA or a double-stranded RNA, including a modified form of RNA. In another example, the gene delivery vector can be an RNA, such as an mRNA or a modified mRNA. As another example, the gene delivery vector can be a viral vector derived from a virus, such as an adenovirus, an adeno-associated virus (AAV), a lentivirus, a herpes virus, an alpha virus, or a retrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV) or a lentivirus. Although embodiments covering the use of adeno-associated viruses are described in more detail below, it is expected that the skilled artisan will recognize that similar knowledge and skills in the art can also be applied to non-AAV gene delivery vectors. In some embodiments, the gene delivery vector is a recombinant adeno-associated virus (rAAV). In this embodiment, the gene expression cassette is flanked by functional AAV reverse terminal repeat (ITR) sequences at the 5' and 3' ends. "Functional AAV ITR sequence" refers to the ITR sequence for rescuing, replicating and packaging AAV virus particles as expected. Therefore, the AAV ITR for the gene delivery vector of the present disclosure does not need to have a wild-type nucleotide sequence and can be changed by the insertion, deletion or substitution of nucleotides, or the AAV ITR can be derived from any AAV serotype in several AAV serotypes, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10. Preferred AAV vectors have wild-type Rep genes and Cap genes that are all or partially deleted, but retain functional flanking ITR sequences. In specific embodiments, the AAV viral vector is an AAV variant. In some embodiments, the AAV variant is an AAV viral vector comprising a variant AAV capsid (or referred to as an AAV capsid protein variant). In some embodiments, the gene expression cassette is encapsidated in an AAV capsid, which can be derived from any adeno-associated virus serotype, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc., any of which can serve as a gene delivery vector. For example, the AAV capsid can be a wild-type capsid or a natural capsid. However, like ITR, the capsid does not need to have a wild-type nucleotide sequence, but as long as the capsid is capable of transducing mammalian cells, it can be changed relative to the wild-type sequence by insertion, deletion or substitution of nucleotides in the VP1, VP2 or VP3 sequence. In other words, the AAV capsid can be a variant AAV capsid, which includes one or more amino acid substitutions, deletions or insertions relative to the parent capsid protein or AAV capsid protein derived from. The AAV capsid is an icosahedron, composed of 60 VP capsid protein monomers, including 5 VP1 monomers, 5 VP2 monomers and 50 VP3 monomers. VP1, VP2, and VP3 monomers are all transcribed and translated from the Cap gene of AAV. Among them, VP1 is the longest, containing about 735 amino acids. VP2 and VP3 are "truncated versions" of VP1, which do not contain some amino acids at the N-terminus of the VP1 protein. By convention, the capsid protein modification site is named according to the amino acid sequence of the VP1 protein. The AAV capsid protein variant can be an AAV capsid protein variant containing a targeting peptide that is targeted to a specific tissue. In some specific embodiments, the targeting peptide is a targeting peptide that has good targeting to the eye, and the targeting peptides that can be selected are shown in Table 1 below. Table 1: In some more specific embodiments, the AAV capsid protein variant is an AAV2 capsid protein variant. In some preferred embodiments, the AAV2 capsid protein variant is any one of AAV2 capsid protein variants IVT1 to IVT15. The above-mentioned targeting peptides 1 to 15 and IVT1 to IVT15 are described in PCT International Application No. PCT / CN2023 / 095837, which is incorporated herein by reference. The sequences and specific construction methods of targeting peptides 1 to 15 and IVT1 to IVT15 are also described in Example 5 below. In some preferred embodiments, the AAV2 capsid protein variant is the AAV2 capsid protein variant IVT15, which is obtained by inserting the targeting peptide 15 (AAAGNGRAHAAA (SEQ ID NO: 11)) having good targeting to the eye at the position of the 587th amino acid of AAV2 VP1. That is, the targeting peptide is located between the 587th amino acid and the 588th amino acid of AAV2 VP1, and the AAV2 capsid protein / AAV2 containing the capsid protein is obtained. In other preferred embodiments, the AAV2 capsid protein variant is the AAV2 capsid protein variant IVT13, which is obtained by inserting the targeting peptide 13 (AAARGSLAA (SEQ ID NO: 69)) having good targeting to the eye at the position of the 587th amino acid of AAV2 VP1. That is, the targeting peptide is located between the 587th amino acid and the 588th amino acid of AAV2 VP1, and the AAV2 capsid protein / AAV2 containing the capsid protein is obtained. Preferably, rAAV is replication-defective, as the AAV vector is unable to independently replicate and package its genome. For example, when cone cells are transduced with rAAV viral particles, genes are expressed in the transduced cone cells, however, due to the fact that the transduced cone cells lack the AAV rep and cap genes and the auxiliary function genes, the rAAV cannot replicate. Standard methods can be used to produce gene delivery vectors (e.g., rAAV virus particles) that encapsidate the gene expression cassette of the present disclosure. For example, in the case of rAAV virus particles, the AAV expression vector according to the present disclosure can be introduced into the production cell, and then the AAV auxiliary construct is introduced, wherein the auxiliary construct comprises an AAV coding region that can be expressed in the production cell and the AAV coding region can supplement the AAV auxiliary function that does not exist in the AAV vector. Then the helper virus and / or another vector are introduced into the production cell, wherein the helper virus and / or another vector provide auxiliary functions that can support effective rAAV virus production. Then, the production cell is cultured to produce rAAV. These steps are performed using standard methods. The replication-defective AAV virus particles that encapsidate the recombinant AAV vector of the present disclosure are prepared using AAV packaging cells and packaging technology by standard techniques known in the art. Any concentration of viral particles suitable for effective transduction of mammalian cells can be prepared to contact mammalian cells in vitro or in vivo. Similarly, any total number of viral particles suitable for providing appropriate cell transduction to impart the desired effect or treat disease can be administered to a mammal. Any suitable number of vectors can be administered to the eye of a mammal or primate. The viral vector can be formulated into a pharmaceutical composition comprising any suitable unit dosage of the vector, which can be administered to a subject to produce a change in the subject or to treat a disease in the subject. In some cases, the multiplicity of infection (MOI) can be used to measure the unit dose of a pharmaceutical composition. MOI refers to the ratio or number of cells to which a vector or viral genome can be delivered to a nucleic acid. When preparing rAAV compositions, any host cell for producing rAAV viral particles can be used, including but not limited to mammalian cells (e.g., 293 cells), insect cells (e.g., SF9 cells), microorganisms and yeast. The host cell can also be a packaging cell or a production cell, in which the AAV rep gene and the cap gene are stably maintained in the host cell, and the AAV vector genome is stably maintained and packaged in the production cell. Exemplary packaging and production cells are derived from SF-9, 293, A549 or HeLa cells. AAV vectors are purified and formulated using standard techniques known in the art. <Pharmaceutical Composition> As disclosed herein, in some aspects of the present disclosure, a pharmaceutical composition is provided, comprising a fusion polypeptide, a polynucleotide, a gene expression cassette or a gene delivery vector provided by the present disclosure, and, optionally, a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition described in the present disclosure contains the above-mentioned fusion polypeptide, polynucleotide, gene expression cassette or the above-mentioned gene delivery vector as an active ingredient. In some specific embodiments, the pharmaceutical composition of the present disclosure contains a gene delivery vector as an active ingredient, and the gene delivery vector is a recombinant adeno-associated virus. In these embodiments, the pharmaceutical composition includes about 1×10 8 To about 1×10 15 Viral genome (vg), about 1×10 9 To about 1×10 14 vg, about 1×10 9 To about 1×10 13 vg, for example 6.0×10 9 vg. Individual doses are usually not less than the amount required to produce a measurable effect on the subject and can be determined based on the pharmacokinetics and pharmacology of absorption, distribution, metabolism and excretion ("ADME") of the pharmaceutical composition or its byproducts and thus based on the disposition of the composition in the subject. This includes consideration of route of administration and dosage. Effective doses and / or dosage regimens can be readily determined empirically based on preclinical assays, based on safety and escalation and dose range trials, individual clinician-patient relationships, and in vitro and in vivo assays. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The pharmaceutical composition is usually formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intraarterial, subcutaneous, intraperitoneal, intrathecal, intramuscular, or injection or infusion. Therefore, delivery can be systemic or local. For example, in order to deliver to the retina, subretinal or intravitreal injection can be used (see, for example, Ochakovski et al., Front Neurosci. 2017; 11: 174; Xue et al., Eye (Lond). 2017 Sep; 31 (9): 1308-1316). In some embodiments, the pharmaceutical compositions of the present disclosure are designed, engineered, or adapted for administration to primates (e.g., non-human primates and human subjects) by intravitreal or subretinal injection. Methods of formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). <Use and method> In some aspects of the present disclosure, the present disclosure provides a method for treating or preventing a disease (e.g., an ocular disease) in a subject in need thereof, the method comprising administering an effective amount of the fusion polypeptide, polynucleotide, gene expression cassette, gene delivery vector or pharmaceutical composition of the present disclosure to a subject in need thereof. In other aspects of the present disclosure, the present disclosure also provides use of the fusion polypeptide, polynucleotide, gene expression cassette or gene delivery vector of the present disclosure in the preparation of a medicament for treating a disease (eg, an ocular disease). In some specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition of the present disclosure can at least partially improve eye diseases associated with ocular neovascularization, or associated with choroidal neovascularization (CNV). In some embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition (coding sequence of fusion polypeptide) of the present disclosure can be delivered to the eye of a human subject. Approved indications for aflibercept fusion protein include neovascular (wet) age-related macular degeneration (AMD), macular edema secondary to retinal vein occlusion (RVO), diabetic macular edema (DME) and diabetic retinopathy (DR). In some cases, the methods and gene expression cassettes, gene delivery vectors or pharmaceutical compositions disclosed herein can be used to prevent or treat eye diseases for which aflibercept has been approved or indicated. In some specific embodiments, eye disease can be choroidal neovascularization, also referred to as wet AMD. Choroidal neovascularization can be related to the rupture of new blood vessels from choroid through Bruch's membrane to subretinal pigment epithelium (sub-RPE) or subretinal space, which can be the main cause of vision loss. CNV can cause sudden deterioration of central vision, which is obvious in a few weeks. Other symptoms can include color interference and visual distortion (wherein straight line appears wavy distortion). The bleeding of new blood vessels can accelerate the onset of the symptoms of CNV. CNV can also include a sense of pressure behind the eye. In some embodiments, the method as disclosed herein and gene expression cassette, gene delivery vector or pharmaceutical composition are used to treat CNV or the eye conditions related to neovascularization. The late stage " wet " form (neovascular or exudative) of AMD is less common, but can frequently cause the rapid and often large-scale loss of patient's central vision.In the wet form of AMD, choroidal neovascularization forms and develops into a vascular network that can be below the retinal pigment epithelium and through its growth.Because this is accompanied by plasma leakage and / or hemorrhage in the subretinal space, if this occurs in the macula, serious sudden loss of central vision can occur.Term " AMD ", if there is no additional explanation, can be dry AMD or wet AMD.The present disclosure considers treating or preventing AMD, wet AMD and / or dry AMD.In some embodiments, by as disclosed method and gene expression cassette, gene delivery vector or pharmaceutical composition for treating AMD. In some embodiments, the methods and gene expression cassettes, gene delivery vectors or pharmaceutical compositions as disclosed herein are used to prevent or treat an ocular disease or condition that is responsive to aflibercept in vivo. In some specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition can be administered parenterally by intravenous injection or oral infusion. In some more specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition is applied to the eye by injection, for example, to the retina, lower retina or vitreous. In other more specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition is applied by retinal injection, lower retinal injection or intravitreal injection. In other more specific embodiments, the gene expression cassette, gene delivery vector or pharmaceutical composition is applied locally or directly to the tissue or organ of interest, for example, by injection into the liver. In some embodiments, the method includes a single administration; in other embodiments, when the attending clinician considers it appropriate, multiple administrations can be performed over time. The subject can be a mammal, including, for example, a human subject in need of treatment for a particular disease (eg, an ocular disease associated with ocular neovascularization, or associated with choroidal neovascularization). In some embodiments, a gene delivery vector (recombinant adeno-associated virus) is administered to a subject, and the therapeutically effective amount for effecting a change or producing a therapeutic effect in the subject may be about 1×10 8 Viral genomes (vg) or more, in some cases, about 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 or 1×10 13 Viral genome or more. Example The present disclosure is further described in detail below in conjunction with specific embodiments, and the examples given are only for illustrating the present disclosure, rather than for limiting the scope of the present disclosure. The examples provided below can be used as a guide for further improvements by ordinary technicians in the technical field, and do not constitute a limitation of the present disclosure in any way. The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels. Materials and methods HEK 293T cell line (ATCC, CRL-3216); HEK 293 cell line (purchased from the Chinese Academy of Sciences); Polyethylenimine Linear (PEI) MW40000 (YEASEN, 40816ES03); Pipettes (Serological Pipets, Jet Bio-Filtration); Benzonase nuclease (Benzonase, Sigma-Aldrich, E8263-25KU); C57 mice (purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.). Example 1 Construction of codon-optimized aflibercept plasmid vector In order to construct an AAV vector plasmid (i.e., a gene delivery vector) carrying an aflibercept expression cassette (gene expression cassette), this example first constructs an aflibercept expression vector and optimizes the codons. The Aflibercept non-optimized sequence (Aflibercept opt) and two artificially synthesized codon-optimized Aflibercept coding sequences (Aflibercept opt2 and Aflibercept opt3) were used to replace the Fluc sequence in the AAV vector plasmid pssAAV-CMV-Fluc-hGH pA (Figure 1A) carrying the firefly luciferase (Fluc) reporter gene expression frame preserved by the applicant to obtain pssAAV-CMV-Aflibercept (Figure 1B), pssAAV-CMV-Aflibercept opt2 (Figure 1C), and pssAAV-CMV-Aflibercept opt3 (Figure 1D). The specific construction process is as follows: (1) Codon-optimized Aflibercept sequence According to the patent CN114375195A disclosed by Adverum Biotechnologies, Inc., the aflibercept expression frame and the coding sequence of aflibercept used by the company to construct the AAV vector were obtained as the unoptimized sequence, namely the Aflibercept sequence SEQ ID NO: 20. The protein coding region sequence was further codon optimized to obtain two optimized Aflibercept (Coding optimized Recombined Aflibercept, Aflibercept opt) sequences, namely the Aflibercept opt ​​2 sequence SEQ ID NO: 22 and the Aflibercept opt3 sequence SEQ ID NO: 23. SEQ ID NO:20 (Aflibercept nucleotide sequence) SEQ ID NO:21 (Aflibercept amino acid sequence) SEQ ID NO:22 (Aflibercept opt2 nucleotide sequence) SEQ ID NO:23 (Aflibercept opt3 nucleotide sequence) (2) Construction of AAV vector carrying Aflibercept / Aflibercept opt2 / Aflibercept opt3 expression frame After adding the "5'-GCCACC-3'" sequence (Kozak sequence) to the 5' end of Aflibercept (SEQ ID NO:20), Aflibercept opt2 (SEQ ID NO:22) and Aflibercept opt3 (SEQ ID NO:23), the sequences were sent to General Biotechnology (Anhui) Co., Ltd. for synthesis, and seamlessly cloned into the vector plasmid pssAAV-CMV-Fluc-hGH pA preserved by the applicant to replace Fluc therein, thereby obtaining pssAAV-CMV-Aflibercept, pssAAV-CMV-Aflibercept opt2 and pssAAV-CMV-Aflibercept opt3. The expression cassettes (i.e., gene expression cassettes) carried by the above three vector plasmids are CMV-Aflibercept-hGH pA (SEQ ID NO: 24), CMV-Aflibercept opt2-hGH pA (SEQ ID NO: 12), and CMV-Aflibercept opt3-hGH pA (SEQ ID NO: 56). SEQ ID NO:24 (CMV-Aflibercept-hGH pA nucleotide sequence) SEQ ID NO:12 (CMV-Aflibercept opt2-hGH pA nucleotide sequence) SEQ ID NO:56 (CMV-Aflibercept opt3-hGH pA nucleotide sequence) Example 2 Comparison of expression levels of two codon optimized To determine whether codon optimization can improve the expression level of Aflibercept, this example used lipo3000 (ThermoFisher) to transfect pssAAV-CMV-Aflibercept, pssAAV-CMV-Aflibercept opt2 and pssAAV-CMV-Aflibercept opt3 constructed in Example 1 into HEK293 cells in vitro. After 48 hours of transfection, the cell supernatant was collected and the Aflibercept in the cell supernatant was detected by Elisa. The kit was purchased from ImmunoGuide (Turkey), catalog number: IG-AA115. The test results showed that the Aflibercept opt3 sequence increased the expression level of Aflibercept (Figure 2). Example 3 Construction of Aflibercept Plasmid Vector with Modified Secretion Signal The results of Example 2 showed that Aflibercept opt3 had the highest expression level, and Aflibercept opt3 was used for further research in subsequent examples. In order to improve the secretion of Aflibercept, we replaced its own secretion signal peptide. The secretion signal peptide sequence of Aflibercept itself in the above plasmids was replaced with artificially synthesized sequences of human albumin secretion signal peptide (hAlbuminSP or hAlbumin sp), human Opticin protein secretion signal peptide (hOpticinSP or hOpticin sp), human interleukin-2 secretion signal peptide (hIL-2SP or hIL2sp), and mouse receptor tyrosine kinase-like orphan receptor 1 secretion signal peptide (mouse ROR1 protein secretion signal peptide, mROR1SP or mROR1 sp), respectively, to obtain the following sequences: hAlbumin sp-Aflibercept opt3, hOpticin sp-Aflibercept opt3, hIL2sp-Aflibercept opt3, and mROR1 sp-Aflibercept opt3. The above sequences were used to replace the Fluc sequences in the pssAAV-CMV-Fluc-hGH pA (Figure 1A), pssAAV-CBh-Fluc-hGH pA (Figure 3A) and pssAAV-CB7-Fluc-hGH pA (Figure 3B) preserved by the applicant, respectively, to obtain the following vector plasmids: pssAAV-CMV-hAlbumin sp-Aflibercept opt3 (Figure 3C), pssAAV-CBh-hAlbumin sp-Aflibercept opt3 (Figure 3D), pssAAV-CB7-hAlbumin sp-Aflibercept opt3 (Figure 3E); pssAAV-CMV-hOpticin sp-Aflibercept opt3 (Figure 3F), pssAAV-CBh-hOpticin sp-Aflibercept opt3 (Figure 3G), pssAAV-CB7-hOpticin sp-Aflibercept opt3 (Figure 3H); pssAAV-CMV-hIL2sp-Aflibercept opt3 (Figure 3I), pssAAV-CBh-hIL2sp-Aflibercept opt3 (Figure 3J), pssAAV-CB7-hIL2sp-Aflibercept opt3 (Figure 3K); pssAAV-CMV-mROR1 sp-Aflibercept opt3 (Figure 3L), pssAAV-CBh-mROR1sp-Aflibercept opt3 (Figure 3M), and pssAAV-CB7-mROR1 sp-Aflibercept opt3 (Figure 3N). Finally, according to patent CN114375195A disclosed by Adverum Biotechnologies, Inc., the aflibercept expression cassette used by the company to construct an AAV vector was obtained. The expression cassette was used to replace the CMV-Fluc-hGH pA in pssAAV-CMV-Fluc-hGH pA to obtain pssAAV-ADVM-022 ( FIG. 3O ). The specific construction process is as follows: (1) Changing the aflibercept sequence of the secretory signal peptide Aflibercept is a secretory protein, and the first 26 amino acids at the N-terminus are the secretion signal peptide of Aflibercept. This example uses the human albumin secretion signal peptide (hAlbuminSP) sequence (5' end-ATGAAGTGGGTGACCTTCATCAGCCTGCTGTTCCTGTTTTCTTCAGCCTACAGC-3' end (SEQ ID NO: 25), the human Opticin protein secretion signal peptide (hOpticinSP) sequence (5' end-ATGAGACTGCTGGCCTTCCTGAGCCTGCTGGCCCTGGTGCTGCAGGAGACAGGCACC-3' end (SEQ ID NO: 26), and the human interleukin-2 secretion signal peptide (hIL-2SP) sequence (5' end-ATGTACAGAATGCAGCTGCTGAGCTGCATAGCCCTGTCTCTGGCCCTGGTGACCAA CAGC-3' end (SEQ ID NO:27), mouse receptor tyrosine kinase-like orphan receptor 1 secretory signal peptide (mROR1SP) sequence (5' end-ATGCACAGACCCAGAAGGAGGGGCACCAGACCTCCACCTCTGGCTCTGCTGGCAGCCCTGCTGCTGGCAGCTAGAGGAGCAGATGCC-3' end SEQ ID NO:28) respectively replaced the secretory signal peptide gene sequence of Aflibercept opt3 to obtain four recombinant Aflibercept sequences, namely hAlbumin sp-Aflibercept opt3 (SEQ ID NO:29), the encoded amino acid sequence is SEQ ID NO:30; hOpticin sp-Aflibercept opt3 (SEQ ID NO:31), the encoded amino acid sequence is SEQ ID NO:32; hIL2sp-Aflibercept opt3 (SEQ ID NO:2), the encoded amino acid sequence is SEQ ID NO:1 and mROR1 sp-Aflibercept opt3 (SEQ ID NO:33). NO:33), and the encoded amino acid sequence is SEQ ID NO:34. SEQ ID NO:29 (hAlbumin sp-Aflibercept opt3 nucleotide sequence) SEQ ID NO:30 (hAlbumin sp-Aflibercept opt3 amino acid sequence) SEQ ID NO:31 (hOpticin sp-Aflibercept opt3 nucleotide sequence) SEQ ID NO:32 (hOpticin sp-Aflibercept opt3 amino acid sequence) SEQ ID NO:33 (mROR1 sp-Aflibercept opt3 nucleotide sequence) SEQ ID NO:34 (mROR1 sp-Aflibercept opt3 amino acid sequence) (2) Construction of AAV vectors carrying expression frames of hAlbumin sp-Aflibercept opt3, hOpticin sp-Aflibercept opt3, hIL2sp-Aflibercept opt3, and mROR1 sp-Aflibercept opt3 After adding the "5'-GCCACC-3'" sequence to the 5' end of hAlbumin sp-Aflibercept opt3 (SEQ ID NO: 29), hOpticin sp-Aflibercept opt3 (SEQ ID NO: 31), hIL2sp-Aflibercept opt3 (SEQ ID NO: 2) and mROR1 sp-Aflibercept opt3 (SEQ ID NO: 33), the sequences were sent to General Biotechnology (Anhui) Co., Ltd. for synthesis, and seamlessly cloned into three vector plasmids preserved by our company, pssAAV-CMV-Fluc-hGH pA, pssAAV-CBh-Fluc-hGH pA and pssAAV-CB7-Fluc-hGH pA, respectively, replacing Fluc therein to obtain pssAAV-CMV-hAlbumin sp-Aflibercept opt3, pssAAV-CBh-hAlbumin sp-Aflibercept opt3, pssAAV-CB7-hAlbumin sp-Aflibercept opt3 and pssAAV-CMV-hOpticin sp-Aflibercept opt3, pssAAV-CBh-hOpticin sp-Aflibercept opt3, pssAAV-CB7-hOpticin sp-Aflibercept opt3 and pssAAV-CMV-hIL2sp-Aflibercept opt3, pssAAV-CBh-hIL2sp-Aflibercept opt3, pssAAV-CB7-hIL2sp-Aflibercept opt3 and pssAAV-CMV-mROR1 sp-Aflibercept opt3, pssAAV-CBh-mROR1 sp-Aflibercept opt3 and pssAAV-CB7-mROR1 sp-Aflibercept opt3. The expression cassettes (i.e., gene expression cassettes) carried by the above 12 vector plasmids are CMV-hAlbumin sp-Aflibercept opt3-hGH pA (SEQ ID NO: 35), CBh-hAlbumin sp-Aflibercept opt3-hGH pA (SEQ ID NO: 36), CB7-hAlbumin sp-Aflibercept opt3-hGH pA (SEQ ID NO: 37), CMV-hOpticin sp-Aflibercept opt3-hGH pA (SEQ ID NO: 38), CBh-hOpticin sp-Aflibercept opt3-hGH pA (SEQ ID NO: 39), CB7-hOpticin sp-Aflibercept opt3-hGH pA (SEQ ID NO: 40), CMV-hIL2sp-Aflibercept opt3-hGH pA (SEQ ID NO: 8), CBh-hIL2sp-Aflibercept opt3-hGH pA (SEQ ID NO: 9). NO:9), CB7-hIL2sp-Aflibercept opt3-hGH pA (SEQ ID NO:10) and CMV-mROR1 sp-Aflibercept opt3-hGH pA (SEQ ID NO:41), CBh-mROR1 sp-Aflibercept opt3-hGH pA (SEQ ID NO:42), CB7-mROR1 sp-Aflibercept opt3-hGH pA (SEQ ID NO:43). (3) Construction of AAV vector carrying the aflibercept expression cassette for comparative study In order to package the AAV for comparative study in the subsequent embodiments, this embodiment obtained the aflibercept expression frame (SEQ ID NO: 52) for constructing the AAV vector according to the patent CN114375195A disclosed by Adverum Biotechnologies, Inc., and named ADVM-022 herein. The sequence was sent to General Bio (Anhui) Co., Ltd. for synthesis and seamlessly cloned into the pssAAV-CMV-Fluc-hGH pA plasmid preserved by the applicant. The expression frame replaced the CMV-Fluc-hGH pA in the pssAAV-CMV-Fluc-hGH pA to obtain pssAAV-ADVM-022 (Figure 3O). SEQ ID NO:52 (ADVM-022 expression cassette nucleotide sequence) Example 4 Comparison of four secretion signal peptides In order to determine whether the recombinant aflibercept with a changed secretion signal peptide can increase the secretion level, this example uses lipo3000 (ThermoFisher) to transfect pssAAV-CMV-Aflibercept opt3, pssAAV-CMV-hAlbumin sp-Aflibercept opt3, pssAAV-CMV-hOpticin sp-Aflibercept opt3, pssAAV-CMV-hIL2sp-Aflibercept opt3 and pssAAV-CMV-mROR1 sp-Aflibercept opt3 constructed in Example 1 or Example 3 into HEK293 cells in vitro. After 48 hours of transfection, the cell supernatant (diluted 5 times with sterile PBS) and cells were collected. After the cells were lysed with Ripa lysis buffer (Biyuntian, Beijing), the total protein was extracted. After the protein concentration was determined, the total cell protein concentration was adjusted to be consistent. Western blot was used to detect aflibercept in the cell supernatant and total cell protein. Since Aflibercept has the Fc structure of an antibody, WB only uses an anti-Fc antibody with an HRP label (Jackson ImmunoResearch, USA) for detection, without the need to add a secondary antibody. The test results showed that the replacement of the four secretion signals can significantly increase the secretion of Aflibercept; among them, the secretion signal peptides of human interleukin-2 and mouse ROR1 protein are more favorable (Figure 4). Example 5 Design and construction of AAV capsid protein expression plasmid pAAV-RC2_IVT 1. Construction of intermediate plasmid RC2_IVB-NotI plasmid In this example, a reverse P5 promoter sequence was added upstream of the Rep sequence on pAAV-RC2 (purchased from CellBiolabs, catalog number: VPK-410-SER2), and a NotI restriction endonuclease site was inserted at 1752 bp of the Cap2 sequence. The intermediate plasmid RC2_IVB-NotI plasmid was commissioned to Anhui General Biotechnology to complete the construction, and the specific sequence is as follows: RC2_IVB-NotI plasmid vector sequence (SEQ ID NO: 13) 2. Construction of AAV capsid protein expression plasmid encoding the capsid protein of interest The AAV capsid protein expression plasmid encoding the capsid protein of interest was constructed using the Gibson assembly method (for specific steps, see Gibson Chemical Transformation Protocol (E2611), Gibson assembly of the PCR fragment and the intermediate plasmid RC2_IVB-NotI plasmid constructed in step 1 of NotI linearization, and then obtain different AAV capsid plasmids encoding capsid proteins of interest. In the process of obtaining the PCR fragment, PCR has no template, and the primers can be paired by themselves for PCR amplification. The primer sequences are shown in Table 2: Table 2: Experimental results: In this example, the plasmid DNA to be constructed was subjected to restriction enzyme digestion and Sanger sequencing identification, proving that the expression plasmid of the corresponding capsid protein was successfully constructed. The amino acid sequence of the AAV capsid protein expressed in the obtained AAV capsid protein expression plasmid is shown in Table 3 below. Table 3: Example 6 Virus packaging and identification In this example, the sequence of hIL2sp-Aflibercept opt3 with the best secretion effect verified in Example 4 was selected for further study and virus packaging. Reference was made to the literature (Xiao X, et al. J Virol. 1998; 72(3): 2224-2232.), and some adjustments were made on this basis. The three-plasmid packaging system was used to package recombinant AAV, and all AAVs used IVT15 constructed in Example 5 as the shell. HEK 293T cells were inoculated in a 100 mm culture dish and grown to a confluence of 70% to 80% for plasmid transfection. 5 μg of pssAAV-CMV-hIL2sp-Aflibercept opt3 plasmid (or pssAAV-CBh-hIL2sp-Aflibercept opt3, pssAAV-CB7-hIL2sp-Aflibercept opt3 plasmid) constructed in Example 3, 5 μg of RepCap plasmid pAAV-RC2_IVT15 with serotype IVT15 constructed in Example 5, and 10 μg of phepler plasmid (purchased and stored by the applicant from Cell Biolabs. The plasmid contains the adenovirus-derived auxiliary function genes E2A, E4 and VA RNA required for co-transfection of three plasmids into HEK293 (or HEK293T) cells to prepare recombinant AAV viruses.) were co-transfected into HEK293T cells using polyethyleneimine (PEI), and the cells and supernatant were harvested 72 hours after transfection. The cells were pelleted by low-speed centrifugation, and lysed by adding 0.5% (w / v) sodium deoxycholate, 50U / mL Benzonase nuclease and 2mM MgCl2 were added, and the free DNA molecules were digested by incubation at 37°C for 2 hours. At the same time, the supernatant was precipitated on ice with a 1:5 volume of 40% PEG8000, 2.5M NaCl solution, the cell lysate and supernatant were mixed and centrifuged, and the supernatant was taken and purified by iodixanol ultracentrifugation purification method to finally obtain rAAV IVT15-CMV-hIL2sp-Aflibercept opt3, rAAV IVT15-CBh-hIL2sp-Aflibercept opt3, and rAAV IVT15-CB7-hIL2sp-Aflibercept opt3. The purified AAV titer was then determined by qPCR and stored in a -80°C refrigerator. The specific determination method is as follows: The physical titer of the prepared AAV viral genome was determined by qPCR. The specific process is as follows: Design primers and probes for qPCR detection based on the exact same sequence of hIL2sp-Aflibercept opt3: Aflibercept opt3-QF: 5'-GCTTCTACCCCTCTGACATTGC-3' (SEQ ID NO: 17) Aflibercept opt3-QR: 5'-CAGGAAGAATGAGCCATCAGAGT-3' (SEQ ID NO: 18) Aflibercept opt3-QP: 5'-CCAGCCTGAGAACAACTACAAGACAACCC-3' (SEQ ID NO: 19) Aflibercept opt3-QF and Aflibercept opt3-QR were used as primers, and Aflibercept opt3-QP was used as a probe. The 5' end of the probe was labeled with FAM fluorescent protein, and the 3' end was connected to BlackBerry quencher. The primers and probe were synthesized by ThermoFisher Scientific. Aflibercept opt3-F and Aflibercept opt3-R were used as primers to specifically amplify a 107 bp fragment in the hIL2sp-Aflibercept opt3 sequence. The TaqMan probe binding method was used to amplify the fragment at 1.0×10 8 The pssAAV-CB7-hIL2sp-Aflibercept opt3 plasmid with a copy / mL and its 10-fold gradient dilution samples were used as standards, and the physical titer of the viral genome was detected using a fluorescence quantitative PCR instrument (model: Q5, ThermoFisher) using Premix Ex Taq (Probe qPCR) reagent (TaKaRa, Beijing, China). The operation process is described in the instructions of the reagents used. The virus processing method is described in the literature (Aurnhammer C, et al. Hum Gene Ther Methods. 2012; 23 (1): 18-28.). The applicant used the same method as in this example to package the virus rAAV2 7m8 ADVM-022 used for comparative studies in other examples. 5 μg of pssAAV-ADVM-022 plasmid, 5 μg of RepCap plasmid pAAV-2 7m8 with serotype AAV2 7m8 preserved by the applicant, and 10 μg of phepler plasmid were co-transfected into HEK293T cells to package rAAV2 7m8 ADVM-022. The purified AAV titer was then measured by qPCR and stored in a -80°C refrigerator. The specific determination method is as follows: The physical titer of the prepared AAV viral genome was determined by qPCR. The specific process is as follows: Design primers and probes for qPCR detection based on the exact same sequence of ADVM-022: ADVM-022-QF:5'-ACCAAGCCGAGAGAAGAACA-3'(SEQ ID NO:53) ADVM-022-QR:5'-TGTTGGACACCTTGCACTTG-3'(SEQ ID NO:54) ADVM-022-QP:5'-CAGCACCGACACCACCCGGT-3'(SEQ ID NO:55) ADVM-022-QF and ADVM-022-QR were used as primers, and ADVM-022-QP was used as a probe. The 5' end of the probe was labeled with FAM fluorescent protein, and the 3' end was connected to BlackBerry quencher. The primers and probe were synthesized by ThermoFisher Scientific. ADVM-022-F and ADVM-022-R were used as primers to specifically amplify a 112 bp fragment in the ADVM-022 sequence. The TaqMan probe binding method was used to amplify the fragment at 1.0×10 8 The pssAAV-ADVM-022 plasmid with a copy / mL and its 10-fold serial dilution samples were used as standards, and the physical titer of the viral genome was detected using a fluorescence quantitative PCR instrument (model: Q5, ThermoFisher) using Premix Ex Taq (Probe qPCR) reagent (TaKaRa, Beijing, China). Example 7 Virus Expression in Mice To detect the expression of several viruses carrying the aflibercept expression cassette, 45 wild-type C57 mice aged 6-8 weeks were randomly divided into 5 groups, with 9 mice in each group. 9vg dose, rAAV IVT15-CMV-hIL2sp-Aflibercept opt3, rAAV IVT15-CBh-hIL2sp-Aflibercept opt3, rAAV IVT15-CB7-hIL2sp-Aflibercept opt3, rAAV2 7m8-ADVM-022 (as control virus) or 1 μL PBS (as negative control) were injected into the vitreous of both eyes and named as group A1, group A2, group A3, group A4 and group A5. At three time points after 2 weeks, 4 weeks and 6 weeks of injection, the aqueous humor of the left and right eyes of 3 mice were separated for Elisa detection of the concentration (i.e., expression level) of secreted aflibercept. The kit for aflibercept detection was purchased from ImmunoGuide (Turkey), catalog number: IG-AA115. The results showed that mice injected with three viruses carrying the hIL2sp-Aflibercept opt3 expression frame could continuously express Aflibercept for 2-6 weeks (Figure 5). Among them, group A3, i.e. mice injected with rAAV IVT15-CB7-hIL2sp-Aflibercept opt3, had the highest expression level at several time points. In addition, the control virus rAAV2 7m8-ADVM-022 (group A4) could also continuously express Aflibercept for 2-6 weeks, but its average expression level was lower than that of groups A1 to A3. Example 8 Establishment of Choroidal Neovascularization (CNV) Model in Mice Injected with Virus rAAV IVT15-CMV-hIL2sp-Aflibercept opt3 and rAAV IVT15-CB7-hIL2sp-Aflibercept opt3 were selected for further functional studies. Forty wild-type c57 mice aged 6-8 weeks were randomly divided into four groups, with 10 mice in each group. Each group was treated with 6.0×10 9vg dose, and rAAV IVT15-CMV-hIL2sp-Aflibercept opt3 or rAAV IVT15-CB7-hIL2sp-Aflibercept opt3, rAAV2 7m8-ADVM-022 (as control virus) or 1μL PBS (as negative control) was injected into the vitreous of the right eye (monocular). And named as G1 group, G2 group, G3 group and G4 group. The above mice were laser modeled 28 days after injection to prepare choroidal neovascularization (CNV) mouse model. The preparation process is as follows: the mouse is placed in a suitable position of the slit lamp, the surface of the eyeball is perpendicular to the slit lamp beam, a hand-held cover glass is used as a lens, and the optic disc is moved to the center of the visual field so that the red blood vessels are clearly visible. Ar laser with a wavelength of 532nm was used to induce photocoagulation to destroy the bilateral Bruch's membrane of the mouse. Four laser photocoagulation spots were formed at the posterior pole of the retina, with a power of 100mW, a duration of 100ms, and a spot size of 50μm. For all mice, only the drug-administered eye (i.e., the right eye) was used for modeling. Ten wild-type c57 mice of the same age (10-12 weeks old) were used for CNV modeling in the right eye. 2.5 μg of Eylea (aflibercept, purchased from Bayer (China) Co., Ltd., Beijing, China) was injected into the vitreous cavity of the right eye 3 days after modeling, and the group was named G5 (because the half-life of aflibercept protein in the vitreous is only 4.7 days, in order to exert its best anti-angiogenic effect, according to the usual practice of modeling of CNV mouse model, the modeling is done 2-3 days after drug administration. Therefore, in order to exert the best effect of G5 group treatment, G5 group was given drug 3 days after modeling). After modeling, all mice were tested by OCT, and each burn site was tested to determine whether the modeling was successful. The results showed that the modeling of mice in groups G1 to G5 was successful (Figure 6). Example 9 Viruses carrying the aflibercept expression cassette reduce neovascularization leakage in CNV model mice The mice in the G1-G5 groups described in Example 8 were subjected to fluorescein fundus angiography (FFA) 7 and 14 days after CNV modeling. The specific process is as follows: After the mice are anesthetized, they are transferred to the observation platform, the ophthalmoscope is adjusted to observe the fundus field, and sodium fluorescein is injected into the tail vein. Fundus photography begins immediately after the injection of sodium fluorescein, and lasts for 0-5 minutes. FA images at specific time points are intercepted for fluorescence leakage scoring and grading (grades I-IV), that is, the severity of neovascular leakage is evaluated. The captured images are processed using Image-Pro Plus 6.0 software to calculate the area of ​​the imaging spot, that is, the neovascular leakage area. The results of FFA showed that the spot area and brightness of mice in G1 and G2 groups, i.e., the severity of neovascularization leakage, were significantly lower than those in G3-G5 groups 7 and 14 days after modeling (Figure 7A, 7B). The results of fluorescence area measurement by Image-Pro Plus 6.0 showed that the spot area of ​​mice in G1 and G2 groups decreased significantly from 7 to 14 days after modeling, and the decrease in G2 group was more significant (Figure 8), i.e., the improvement of neovascularization leakage was better. In this embodiment, technicians with relevant knowledge of ophthalmology were asked to perform randomized double-blind neovascularization leakage scoring and grading (based on the area and brightness of the fluorescent spot) on the images taken by FFA detection. Scoring was performed according to the standards described in the reference (Song, JH, et al. J. Mol. Sci. 2020; 21 (8), 2842.). Three technicians were asked to score and grade all fluorescent spots separately, and the average score was calculated for each spot. The results showed that the neovascularization leakage scores of mice in the G1 and G2 groups were lower 14 days after modeling, that is, less leakage (Figure 9). Example 10 Viruses carrying the aflibercept expression cassette reduce neovascularization leakage in the cynomolgus monkey CNV model rAAV IVT15-CB7-hIL2sp-Aflibercept opt3 was selected for further functional studies. Four male cynomolgus monkeys aged 3-5 years were randomly divided into two groups, with two monkeys in each group. One group received an intravitreal injection of 2.5E+11 (i.e., 2.5×10 11)vg of rAAV IVT15-CB7-hIL2sp-Aflibercept opt3, the volume was 50μL (N1); another group was injected with 50μL PBS into the vitreous cavity of both eyes (N2). The two groups of animals were recorded as N1 and N2 groups respectively. The above animals were laser modeled 28 days after injection to prepare the choroidal neovascularization (CNV) model of cynomolgus monkeys. The specific process is as follows: 1) Mydriasis: Compound tropicamide eye drops were dropped into both eyes of the animal for mydriasis; 2) Anesthesia: Anesthesia was first performed by intramuscular injection (Sutane 50, 5mg / kg, concentration of 50mg / mL; combined anesthesia was performed with xylazine hydrochloride injection, 0.4mg / kg, concentration of 20mg / mL). Anesthetics were supplemented according to the anesthesia state of the animals; an equal amount of benzothiazine hydrochloride injection and xylazine hydrochloride were injected to help the animals wake up faster. 3) Laser photocoagulation: Carbomer gel was applied to the corneal contact lens and placed in front of the animal's cornea. After the fundus was clearly seen, photocoagulation was performed at a distance of about 1.5 to 2 PD from the fovea, avoiding large blood vessels. The laser parameters were: wavelength 532 nm, power 400 to 600 mW, spot diameter 50 μm, exposure time 100 ms. 4) OCT and FP examinations were performed immediately after modeling to confirm that Bruch's membrane was penetrated and no severe bleeding was observed, and the modeling was considered successful. Fluorescein Fundus Angiography (FFA) was performed 14 and 28 days after CNV modeling. The specific process is as follows: After the monkey is anesthetized, it is transferred to the observation platform, the fundus scope is adjusted to observe the fundus field, and sodium fluorescein is injected into the tail vein. Fundus photography was started immediately after the injection of sodium fluorescein, which lasted for 0 to 5 minutes. FA images at specific time points were intercepted for fluorescence leakage scoring (I to IV grades), that is, the severity of neovascular leakage was evaluated. The results of FFA test showed that the spot area and brightness of the N1 group of animals, that is, the severity of neovascularization leakage, were significantly lower than those of the N2 group of mice 14 and 28 days after modeling (Figure 10), indicating that rAAV IVT15-CB7-hIL2sp-Aflibercept opt3 inhibited the formation of CNV. In this example, technicians with relevant knowledge of ophthalmology were asked to perform randomized double-blind neovascularization leakage scoring and grading (based on the area and brightness of the fluorescent spot) on the images taken by FFA detection. Scoring was performed according to the standards described in the reference (Song, JH, et al. J. Mol. Sci. 2020; 21(8), 2842.). All fluorescent spots were scored and graded by 3 technicians. The results showed that the proportion of grade 4 spots in animals in the N1 group was lower 14 and 28 days after modeling (Figure 11). Nucleotide sequences of some gene expression cassettes and vectors in Examples 1 and 3 SEQ ID NO:35(CMV-hAlbumin sp-Aflibercept opt3-hGH pA) SEQ ID NO:36(CBh-hAlbumin sp-Aflibercept opt3-hGH pA) SEQ ID NO:37(CB7-hAlbumin sp-Aflibercept opt3-hGH pA) SEQ ID NO:38(CMV-hOpticin sp-Aflibercept opt3-hGH pA) SEQ ID NO:39(CBh-hOpticin sp-Aflibercept opt3-hGH pA) SEQ ID NO:40(CB7-hOpticin sp-Aflibercept opt3-hGH pA) SEQ ID NO:41(CMV-mROR1 sp-Aflibercept opt3-hGH pA) SEQ ID NO:42(CBh-mROR1 sp-Aflibercept opt3-hGH pA) SEQ ID NO:43(CB7-mROR1 sp-Aflibercept opt3-hGH pA) pssAAV-CMV-Fluc-hGH pA expression vector (SEQ ID NO: 49; wherein Fluc is marked with bold, italics, and underline; the upstream ITR is marked with a wavy line; and the downstream ITR is marked with a double underline.) pssAAV-CBh-Fluc-hGH pA expression vector (SEQ ID NO: 50; Fluc is marked with bold, italic, and underline; the upstream ITR is marked with a wavy line; the downstream ITR is marked with a double underline.) pssAAV-CB7-Fluc-hGH pA expression vector (SEQ ID NO: 51; Fluc is marked with bold, italic, and underline; the upstream ITR is marked with a wavy line; the downstream ITR is marked with a double underline.)

Claims

1. A fusion polypeptide comprising a secretion signal peptide and an aflibercept polypeptide, wherein: The secretion signal peptide is selected from any one of human albumin secretion signal peptide, human Opticin protein secretion signal peptide, human interleukin-2 secretion signal peptide and mouse receptor tyrosine kinase-like orphan receptor 1 secretion signal peptide.

2. The fusion polypeptide according to claim 1, wherein The amino acid sequence of the aflibercept polypeptide comprises the amino acid sequence shown in SEQ ID NO: 48; and / or, The amino acid sequence of the secretory signal peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 44-47.

3. The fusion polypeptide according to claim 1 or 2, wherein The amino acid sequence of the fusion polypeptide comprises any one selected from the following sequences: (i) an amino acid sequence as shown in any one of SEQ ID NO: 1, 30, 32 or 34; (ii) an amino acid sequence that is at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34, and which retains the function of the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34; (iii) an amino acid sequence in which one or more amino acid residues are added, substituted, deleted or inserted into the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34, and which retains the function of the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34; or (iv) an amino acid sequence encoded by a nucleotide sequence, which hybridizes with a polynucleotide sequence encoding the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34 under stringent conditions, and the amino acid sequence retains the function of the amino acid sequence shown in any one of SEQ ID NOs: 1, 30, 32 or 34, and the stringent conditions are medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions.

4. An isolated polynucleotide encoding the fusion polypeptide according to any one of claims 1 to 3; Preferably, the polynucleotide comprises a nucleotide sequence as shown in any one of SEQ ID NO: 2, 29, 31 or 33, or a nucleotide sequence having at least 80%, 82%, 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence as shown in any one of SEQ ID NO: 2, 29, 31 or 33. A gene expression cassette comprising the polynucleotide according to claim 4 .

6. The gene expression cassette according to claim 5, wherein In the gene expression cassette, upstream of the polynucleotide encoding the fusion polypeptide, one or more of a promoter, an enhancer, an intron and a Kozak sequence are further included.

7. The gene expression cassette according to claim 6, wherein The promoter includes any one of a CBh promoter, a CB7 promoter and a CMV promoter; Preferably, the CBh promoter comprises the sequence shown in SEQ ID NO: 3 or a sequence having at least 85% identity thereto; and / or, The CB7 promoter comprises the sequence shown in SEQ ID NO:4 or a sequence having at least 85% identity thereto; and / or, The CMV promoter includes the sequence shown in SEQ ID NO: 5 or a sequence having at least 85% identity thereto.

8. The gene expression cassette according to claim 6 or 7, wherein The intron includes a human β-globin intron; Preferably, the human β-globin intron comprises the sequence shown in SEQ ID NO: 6 or a sequence having at least 85% identity thereto.

9. The gene expression cassette according to any one of claims 5 to 8, wherein In the gene expression cassette, a polyadenylation region is included downstream of the polynucleotide encoding the fusion polypeptide; Optionally, the polyadenylation region is selected from any one of bovine growth hormone, human growth hormone and β-globin polyadenylation region; Preferably, the polyadenylation region comprises a human growth hormone polyadenylation region; More preferably, the human growth hormone polyadenylation region comprises the sequence shown in SEQ ID NO: 7, or a sequence having at least 85% identity thereto.

10. The gene expression cassette according to any one of claims 5 to 9, wherein The gene expression cassette comprises a sequence shown in any one of SEQ ID NOs: 8 to 10, 35 to 43, or a sequence having at least 85% identity with any one of SEQ ID NOs: 8 to 10, 35 to 43. 11 . A gene delivery vector comprising the gene expression cassette according to any one of claims 5 to 10.

12. The gene delivery vector according to claim 11, wherein The gene delivery vector is a viral vector derived from a virus; Preferably, the gene delivery vector is a recombinant adeno-associated virus.

13. The gene delivery vector according to claim 12, wherein The recombinant adeno-associated virus comprises a capsid protein, and the gene expression cassette is encapsidated within the capsid protein; Optionally, the capsid protein is selected from any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 adeno-associated virus serotypes or variants thereof.

14. The gene delivery vector according to claim 13, wherein The capsid protein is AAV2 capsid protein or a variant thereof; Preferably, the capsid protein is an AAV2 capsid protein variant; More preferably, the AAV2 capsid protein variant comprises a sequence shown in any one of SEQ ID NOs: 16, 70 to 83, or a sequence having at least 85% identity with any one of SEQ ID NOs: 16, 70 to 83; Further preferably, the AAV2 capsid protein variant comprises the sequence shown in SEQ ID NO: 16 or 82 or a sequence having at least 85% identity with SEQ ID NO: 16 or 82.

15. A pharmaceutical composition comprising the fusion polypeptide according to any one of claims 1 to 3, the polynucleotide according to claim 4, the gene expression cassette according to any one of claims 5 to 10, or the gene delivery vector according to any one of claims 11 to 14, and, optionally, a pharmaceutically acceptable carrier.

16. Use of the fusion polypeptide according to any one of claims 1 to 3, the polynucleotide according to claim 4, the gene expression cassette according to any one of claims 5 to 10, or the gene delivery vector according to any one of claims 11 to 14 in the preparation of a medicament for preventing and / or treating a disease; Optionally, the disease is an eye disease; Preferably, the eye disease is an eye disease associated with ocular neovascularization, or an eye disease associated with choroidal neovascularization; More preferably, the eye disease is selected from one or more of wet age-related macular degeneration, macular edema secondary to retinal vein occlusion, diabetic macular edema and diabetic retinopathy.

17. A method for preventing and / or treating a disease, comprising administering to a subject a preventive and / or therapeutically effective amount of the fusion polypeptide according to any one of claims 1 to 3, the polynucleotide according to claim 4, the gene expression cassette according to any one of claims 5 to 10, the gene delivery vector according to any one of claims 11 to 14, or the pharmaceutical composition according to claim 15; Optionally, the disease is an eye disease; Preferably, the eye disease is an eye disease associated with ocular neovascularization, or an eye disease associated with choroidal neovascularization; More preferably, the eye disease is selected from one or more of wet age-related macular degeneration, macular edema secondary to retinal vein occlusion, diabetic macular edema and diabetic retinopathy.

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