Gene delivery vector for delivering human VEGF (vascular endothelial growth factor) receptor fusion protein and application of gene delivery vector

Abstract

The invention provides a gene delivery vector for delivering a modified human VEGF (vascular endothelial growth factor) receptor fusion protein, particularly a recombinant adeno-associated virus vector, and application of the gene delivery vector and the recombinant adeno-associated virus vector in treatment of diseases caused by VEGF. The invention also provides the modified human VEGF receptor fusion protein as well as a coding nucleic acid, a vector, a cell and an expression cassette thereof.

Description

[0001] This application is a divisional application of the invention patent application with the application number 202410111905.X, the application date of January 26, 2024, and the invention title of "Gene Delivery Vector for Delivering Human VEGF Receptor Fusion Protein and Its Application". Technical Field

[0002] The present invention relates to a gene delivery vector for delivering human VEGF receptor fusion protein, especially recombinant adeno-associated virus. The present invention also relates to the human VEGF receptor fusion protein, the nucleic acid encoding the fusion protein, the vector containing the nucleic acid, and the host cell containing the vector. The present invention also relates to an expression cassette for expressing the human VEGF receptor fusion protein, and a recombinant plasmid for forming the recombinant adeno-associated virus. The present invention also relates to the use of the above gene delivery vector, fusion protein, nucleic acid, vector, host cell, expression cassette and recombinant plasmid in the treatment of diseases caused by VEGF, especially ophthalmic diseases, and the method of treating the diseases using them. Background Art

[0003] VEGF (vascular endothelial growth factor) plays a major role in the pathogenesis of various ophthalmic diseases. During the onset of the disease, the concentration of VEGF in the eye increases, generating abnormal and highly hemorrhagic neovascularization, followed by severe complications such as massive hemorrhage, fibroproliferation, tractional retinal detachment, and neovascular glaucoma. It may also cause obvious vascular leakage, leading to persistent severe tissue edema. Such ophthalmic diseases include: exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) complicated with macular edema (DME), retinal vein occlusion (RVO) complicated with macular edema (ME), central exudative chorioretinopathy, polypoidal choroidal vasculopathy (PCV), choroidal neovascularization secondary to high myopia, choroidal neovascularization (CNV) secondary to other diseases, etc. Among them, the incidence of wAMD and DME is relatively high and more common.

[0004] Based on the mechanism of neutralizing VEGF, several anti-angiogenic antibody drugs for ophthalmology have been marketed, such as Ranibizumab, Bevacizumab, Aflibercept, Conbercept, etc. Among them, Aflibercept is a soluble, fully humanized fusion protein composed of the Ig domain 2 of human VEGFR1, the Ig domain 3 of human VEGFR2, and the hinge-Fc region of human IgG1. It can act as a decoy receptor, bind to VEGF-A, VEGF-B, and placental growth factor (PlGF), prevent their binding to receptors, and block their downstream biological effects. Currently, this drug has been widely used in the clinical treatment of ophthalmic diseases, and the FDA has successively approved the clinical application of Aflibercept in multiple indications, including wet AMD, RVO, DME, etc.

[0005] These drugs have achieved success in the treatment of ophthalmic diseases related to angiogenesis. However, the existing ophthalmic anti-VEGF therapies also have deficiencies: Under normal circumstances, anti-VEGF drug treatment is currently the first-line treatment option suitable for wAMD and DME, and multiple anti-VEGF drugs, such as Ranibizumab, Conbercept, and Aflibercept, have been widely approved and marketed. Currently, these first-line anti-VEGF drugs are all antibody-based macromolecular drugs. Such drugs have a short metabolic cycle and half-life and cannot exert their efficacy in the body for a long time. Therefore, it is necessary to frequently inject drugs into the vitreous cavity of patients. Taking Aflibercept as an example, the recommended dosing regimen for the treatment of neovascular AMD is to inject 1 time into the vitreous cavity every month for the initial 3 months (2 mg / time), and then inject 1 time into the vitreous cavity every 8 weeks, that is, the 3 + every 8 weeks regimen; or inject 1 time into the vitreous cavity every month for the initial 3 months (2 mg / time), and then adopt treat and extend (T&E), that is, the 3 + T&E regimen. The existing treatment methods, due to their high dosing frequency, increase the burden on patients and also put forward more stringent requirements for medication compliance. Summary of the Invention

[0006] By means of a gene delivery vector (such as adeno-associated virus, adenovirus, lentivirus, lipid nanoparticle, etc.), the gene encoding a drug protein (such as an antibody drug, a fusion protein drug, etc.) can be targeted and delivered to the diseased site, which can directly express the drug protein efficiently in the body for a long time, can stably exert its efficacy in the patient's body for a long time, reduce the dosing frequency, and theoretically even achieve the effect of long-term efficacy with a single dose, thus effectively reducing the burden on patients.

[0007] The present invention performs YTE on the human IgG1-Fc fragment in the Aflibercept protein molecule (M252Y / S254T / T256E) Site-directed mutagenesis was performed to obtain a modified aflibercept protein drug molecule (referred to as "Fc-YTE-aflibercept" or "Fc-YTE-Aflibercept"), and a gene therapy drug (referred to as "rAAV-Fc-YTE" or "rAAV-Fc-YTE-aflibercept") was obtained by encoding the gene of the Fc-YTE-aflibercept protein molecule with a gene delivery vector. The inventors surprisingly found that this gene therapy drug can significantly improve the therapeutic effect on the target disease without changing the expression level and expression distribution of the drug molecule in the target tissue.

[0008] Thus, in one aspect, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, and the hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0009] In some embodiments, the human VEGF receptor fusion protein further comprises the immunoglobulin-like domain 4 of human VEGFR2.

[0010] In some embodiments, the hinge-Fc domain of the human immunoglobulin is the hinge-Fc domain of human IgG1.

[0011] In some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: the amino acid sequence as shown in SEQ ID NO.1 or 6, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or 6.

[0012] In some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: the amino acid sequence as shown in SEQ ID NO.2, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.2.

[0013] In some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: the amino acid sequence as shown in SEQ ID NO.7, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.7.

[0014] In some embodiments, the hinge-Fc domain of the human immunoglobulin comprises or is: the amino acid sequence shown in SEQ ID NO.3 or 8, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.3 or 8.

[0015] In some embodiments, the human VEGF receptor fusion protein comprises or is: the amino acid sequence shown in SEQ ID NO.5 or 9, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.5 or 9.

[0016] In some embodiments, the expression cassette comprises: the nucleotide sequence shown in SEQ ID NO.10 or 11, or a degenerate sequence of any one of them.

[0017] In some embodiments, the expression cassette further comprises a promoter located upstream of and operably linked to the nucleotide sequence encoding the human VEGF receptor fusion protein, and the promoter is preferably selected from the cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1α promoter, U6 promoter, chicken β-actin promoter, CAG promoter, CBA promoter, RPE65 promoter, VMD2 promoter, RPGR promoter, IRBP promoter, hGRK1 promoter, CAR promoter, RHO promoter, Grm6 promoter, GRK1 promoter, and GFAP promoter.

[0018] In some embodiments, the expression cassette further comprises a polyadenylation signal located downstream of and operably linked to the nucleotide sequence encoding the human VEGF receptor fusion protein, and the polyadenylation signal is preferably selected from SV40 early polyA, SV40 late polyA, rabbit globin polyA, bGH polyA, and HSV TK polyA.

[0019] In some embodiments, the gene delivery vector is (a) a viral vector, preferably a lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, papovavirus, baculovirus, or papillomavirus, more preferably an adeno-associated virus or a lentivirus; or (b) a non-viral vector, preferably a plasmid, liposome, nanoparticle, polymer, transposon, exosome, or bacterial vector.

[0020] In some embodiments, the vector is an adeno-associated virus vector, which comprises: (a) a recombinant adeno-associated virus capsid, and (b) the nucleic acid packaged within the recombinant adeno-associated virus capsid.

[0021] In some embodiments, the recombinant adeno-associated virus capsid comprises a capsid protein of an adeno-associated virus selected from the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof, as well as capsid protein mutants modified with the above capsid proteins as a backbone; preferably, the recombinant adeno-associated virus capsid comprises a capsid protein of an adeno-associated virus selected from AAV1, AAV2, AAV5, AAV7, AAV8, and hybrids thereof.

[0022] In some embodiments, the genome of the adeno-associated virus vector is in single-stranded or double-stranded form, preferably scAAV, ssAAV, or cceAAV.

[0023] In some embodiments, there is provided a recombinant adeno-associated virus comprising: (a) an rAAV capsid; and (b) a nucleic acid packaged within the rAAV capsid, the nucleic acid comprising, in 5' to 3' order: (i) 5' AAV ITR; (ii) a promoter; (iii) a polynucleotide encoding the human VEGF receptor fusion protein provided by the present invention; (iv) polyA; and (v) 3' AAV ITR.

[0024] In some embodiments, the rAAV capsid comprises a capsid protein of AAV1, AAV2, AAV5, AAV7, or AAV8, preferably a capsid protein of AAV8.

[0025] In some embodiments, the promoter is a cytomegalovirus (CMV) promoter, an EF-1α promoter, a chicken β-actin promoter, a GRK1 promoter, or a CAG promoter, preferably a CMV promoter.

[0026] In some embodiments, the polyA is an SV40 late polyA or a rabbit globin polyA, preferably an SV40 late polyA.

[0027] In some embodiments, the 5' AAV ITR and / or 3' AAV ITR is selected from the ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6.

[0028] In some embodiments, the present invention provides an expression cassette which comprises, in 5' to 3' order: (i) a promoter; (ii) a polynucleotide encoding the human VEGF receptor fusion protein provided by the present invention; (iii) polyA.

[0029] In some embodiments, the promoter is a cytomegalovirus (CMV) promoter, an EF-1α promoter, a chicken β-actin promoter, a GRK1 promoter, or a CAG promoter, preferably a CMV promoter.

[0030] In some embodiments, the polyA is an SV40 late polyA or a rabbit globin polyA, preferably an SV40 late polyA.

[0031] In some embodiments, in the expression cassette, a 5' AAV ITR and a 3' AAV ITR are respectively included at the 5' end of the promoter and the 3' end of the polyA.

[0032] Another aspect of the present invention provides a human VEGF receptor fusion protein, which comprises an immunoglobulin-like domain 2 of human VEGFR1, an immunoglobulin-like domain 3 of human VEGFR2, and a hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin contains M252Y, S254T, and T256E substitutions according to EU numbering.

[0033] In some embodiments, the human VEGF receptor fusion protein further comprises an immunoglobulin-like domain 4 of human VEGFR2; preferably, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.7.

[0034] In some embodiments, the hinge-Fc domain of the human immunoglobulin is the hinge-Fc domain of human IgG1; preferably, the hinge-Fc domain of human IgG1 comprises or is: the amino acid sequence shown in SEQ ID NO.3 or 8, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.3 or 8.

[0035] In some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: the amino acid sequence shown in SEQ ID NO.1 or 6, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or 6.

[0036] In some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.2.

[0037] In some embodiments, the human VEGF receptor fusion protein comprises or is an amino acid sequence as shown in SEQ ID NO.5 or 9, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.5 or 9.

[0038] In some embodiments, the present invention provides a polynucleotide encoding any of the human VEGF receptor fusion proteins. Preferably, the polynucleotide comprises or is: a nucleotide sequence as shown in SEQ ID NO.10 or 11, or a degenerate sequence of any of them.

[0039] In some embodiments, the present invention provides a vector comprising the polynucleotide, preferably a plasmid; preferably, the vector is constructed for forming ssAAV, scAAV or cceAAV.

[0040] In some embodiments, the present invention provides a host cell comprising the vector, preferably HEK293 cells or sf9 cells.

[0041] Other aspects and advantages of the present invention can be seen from the following specific description and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Shows a plasmid map of the master plasmid for forming rAAV-Fc-YTE-Aflibercept of the present invention according to an embodiment of the present invention.

[0043] Figure 2 Shows a schematic structural diagram of rAAV-Fc-YTE-Aflibercept prepared according to an embodiment of the present invention.

[0044] Figure 3 Shows the FFA spot score, *p<0.05, **p<0.01 indicating significant differences between the rAAV-Eylea group, rAAV-Fc-YTE group, positive drug molecule group and negative control reagent group.

[0045] Figure 4 Shows the FFA spot area, *p<0.05, **p<0.01 indicating significant differences between the rAAV-Fc-YTE group and the rAAV-Eylea group and positive drug molecule group; significant differences between the negative control reagent group and the positive drug molecule group; no significant differences between the rAAV-Eylea and positive drug molecule groups.

[0046] Figure 5 Shows the detection of the expression distribution level of the drug protein molecule in each group by the Elisa method.

[0047] Figure 6Show the expression distribution of the target drug protein gene detected by the BaseScope technology in each layer of retinal cells.

[0048] Figure 7 Show the comparison of FFA spot scores after intravitreal administration of protein drugs by IVT.

[0049] Figure 8 Show the comparison of FFA spot areas after intravitreal administration of protein drugs by IVT. Detailed implementation manners

[0050] Definition

[0051] The terms "viral vector" or "viral particle" used interchangeably herein refer to a viral particle composed of at least one enveloped or non-enveloped viral capsid protein and a packaged recombinant viral genome. The viral particle contains a recombinant viral genome having a heterologous polynucleotide, which heterologous polynucleotide encodes the human VEGF receptor fusion protein of the present invention and optionally a transcriptional regulatory region.

[0052] The terms "adeno-associated virus vector", "AAV vector", "adeno-associated virus", "AAV virus", "AAV virus particle", "AAV viral particle" and "AAV particle", used interchangeably herein, refer to a viral particle composed of at least one AAV capsid protein (preferably composed of all capsid proteins of a specific AAV serotype) and a packaged recombinant viral genome. The particle contains a recombinant viral genome having a heterologous polynucleotide (encoding the human VEGF receptor fusion protein of the present invention) and a transcriptional regulatory region, which transcriptional regulatory region at least includes a promoter. The transcriptional regulatory region may also include polyA.

[0053] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which exogenous nucleic acid and / or a recombinant vector have been introduced. It should be understood that "recombinant host cell" and "host cell" refer not only to a particular test cell but also to the progeny of such a cell. Due to mutations or environmental influences, certain modifications may occur in the progeny, so that such progeny may not actually be identical to the parental cell, but are still included within the scope of the term "host cell" as used herein.

[0054] The term "recombinant viral genome" refers to a viral genome or a portion thereof into which at least one expression cassette has been inserted. The term "AAV recombinant viral genome" as used herein refers to an AAV genome into which at least one expression cassette polynucleotide has been inserted. The minimum "genome" of the AAV genome according to the present invention generally includes cis-acting 5' and 3' inverted terminal repeats (ITRs) and an expression cassette.

[0055] As used herein, the term "expression cassette" refers to a nucleic acid construct that is recombinantly or synthetically generated with a series of specific nucleic acid elements and that permits transcription of a specific nucleic acid in a target cell. The expression cassette of the AAV recombinant viral genome of the AAV vector according to the invention can comprise a transcriptional regulatory region operably linked to a coding sequence of the human VEGF receptor fusion protein of the invention.

[0056] As used herein, the term "transcriptional regulatory region" refers to a nucleic acid fragment capable of regulating the expression of one or more genes. The transcriptional regulatory region according to the invention comprises a promoter and optionally an enhancer. The term "promoter" as used herein is a nucleic acid fragment located upstream of a polynucleotide sequence and whose function is to control the transcription of one or more polynucleotides. Any kind of promoter can be used in the present invention, including inducible promoters, constitutive promoters and tissue-specific promoters. The term "inducible promoter" as used herein refers to a promoter that is regulated physiologically or developmentally, for example by the application of a chemical inducer. For example, it can be a tetracycline-inducible promoter, a mifepristone (RU-486)-inducible promoter, etc. The term "constitutive promoter" as used herein refers to a promoter whose activity remains at a relatively constant level in all cells of an organism or in most developmental stages, with little or no regard for the cellular environmental conditions. Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerate kinase (PGK) promoter and the EF1a promoter. Exemplary viral promoters that function constitutively in cells include, for example, the SV40 early promoter region, the promoter contained in the 3′ long terminal repeat sequence of Rous sarcoma virus, or the herpes simplex virus thymidine kinase promoter.

[0057] The term "enhancer" as used herein refers to a DNA sequence element that binds to a transcription factor to increase gene transcription. Examples of enhancers can be, but are not limited to, the RSV enhancer, the CMV enhancer, the HCR enhancer, etc.

[0058] The term "operably linked" as used herein refers to the functional relationship and position of a promoter sequence relative to a polynucleotide of interest (e.g., if a promoter or enhancer affects the transcription of a sequence, it is operably linked to the coding sequence). Generally, an operably linked promoter is adjacent to the sequence of interest. However, an enhancer does not have to be adjacent to the sequence of interest to control its expression. In another embodiment, the promoter and the coding sequence of the human VEGF receptor fusion protein of the invention are adjacent.

[0059] The term "therapeutically effective amount" refers to a non-toxic but sufficient amount of a viral vector encoding the human VEGF receptor fusion protein of the present invention to provide the desired biological result. Such result can be a reduction and / or alleviation of the signs, symptoms or causes of a disease, or any other desired alteration of a biological system. For example, a therapeutically effective amount of an AAV vector according to the present invention is an amount sufficient to produce the desired biological result.

[0060] As used herein, the term "Cap protein" refers to a polypeptide having at least one functional activity of a native AAV Cap protein (such as VP1, VP2, VP3). Examples of the functional activities of the Cap protein include the ability to induce capsid formation, promote single-stranded DNA accumulation, promote AAV DNA packaging into the capsid, bind to cell receptors, and promote entry of viral particles into host cells. In principle, any Cap protein can be used in the context of the present invention. The term "capsid" as used herein refers to the packaging structure of a viral genome. The capsid is composed of several oligomeric structural subunits made of proteins. For example, AAV has an icosahedral capsid formed by the interaction of three capsid proteins, VP1, VP2, and VP3.

[0061] As used herein, the term "Rep protein" refers to a polypeptide having at least one functional activity of a native AAV Rep protein (such as Rep40, 52, 68, 78). The "functional activity" of the Rep protein refers to any activity related to the physiological function of the protein. Other functions include regulating the transcription of AAV (or other heterologous) promoters, and site-specific integration of AAV DNA into the host chromosome. In certain embodiments, the AAV rep gene is derived from serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and their hybrids, as well as capsid protein mutants modified with the above-mentioned capsid proteins as the backbone.

[0062] As used herein, "viral proteins required for AAV replication" refers to polypeptides that perform functions required for AAV replication (i.e., "accessory function polypeptides"). Accessory functions include those functions required for AAV replication, including but not limited to those parts involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of capsid expression products, and AAV capsid assembly. The viral-based accessory functions are derived from any known helper virus, such as adenovirus, herpesvirus (except herpes simplex virus type 1), and vaccinia virus. Helper functions include but are not limited to adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, ULB, UL52, and UL29, as well as herpesvirus polymerase. In another embodiment, the viral proteins required for AAV replication are derived from adenovirus.

[0063] As used herein, the term "adeno-associated virus ITRs" or "AAV ITRs" refers to the inverted terminal repeat sequences present at both ends of the adeno-associated virus genomic DNA strands. The ITR sequences are essential for the efficient propagation of the AAV genome. Another characteristic of these sequences is their ability to form hairpins. This characteristic aids in its self-priming, thus allowing the independent synthesis of the second DNA strand. Procedures for modifying these ITR sequences are known in the art.

[0064] As used herein, the term "polyadenylation signal" or "polyA" refers to a nucleic acid sequence that mediates the ligation of a polyadenosine chain to the 3'-end of an mRNA. Suitable polyA signals include, but are not limited to, the SV40 early polyA signal, the SV40 late polyA signal, rabbit globin polyA, bGH polyA, and the HSV thymidine kinase (TK) polyA signal.

[0065] The term "nucleotide or nucleic acid sequence" is used interchangeably herein with "polynucleotide" and refers to any polymeric form of nucleotides of any length.

[0066] As used herein, the term "signal peptide" refers to a sequence of amino acid residues (ranging in length from 10 to 30 residues) that binds to the amino terminus of a nascent protein of interest during protein translation. The signal peptide is recognized by the signal recognition particle (SRP) and is cleaved by signal peptidase after translocation across the endoplasmic reticulum.

[0067] As used herein, the term "subject" or "individual" refers to an individual mammal, such as a human, a non-human primate (such as a chimpanzee and other ape and monkey species), a farm animal (such as a bird, a fish, a cow, a sheep, a pig, a goat, and a horse), a domestic mammal (such as a dog and a cat), or a laboratory animal (such as a rodent, such as a mouse, a rat, and a guinea pig). The term includes subjects of any age or sex. In another embodiment, the subject is a mammal, preferably a human.

[0068] As used herein, the term "Fc" refers to the Fc domain of human IgG (immunoglobulin). Subtypes of IgG such as IgG1, IgG2, IgG3, and IgG4 can all be used as the Fc domain. As used herein, the "Fc region" or "Fc domain" is the part of the IgG molecule related to the crystallizable fragment obtained by pepsin digestion of the IgG molecule. It has no antigen-binding activity but contains a carbohydrate moiety and binding sites for complement and Fc receptors (including the FcRn receptor). The Fc region contains the entire second constant domain CH2 (residues 231 - 340 of human IgG1 according to the EU numbering system, the same hereinafter) and the entire third constant domain CH3 (residues 341 - 447). The term "hinge" encompasses all or a fragment (e.g., 221 - 230) of the hinge region (residues 216 - 230) extending from the N-terminus of the Fc region. The "IgG hinge-Fc region" or "hinge-Fc domain" refers to the region of the IgG molecule composed of the Fc region (residues 231 - 447) and the hinge region or its fragment extending from the N-terminus of the Fc region.

[0069] As used herein, single-stranded AAV (ssAAV) refers to rAAV having the coding sequence of a transgene expression cassette on a single strand and packaged into a viral capsid, which requires a process of conversion from single-stranded to double-stranded, and the synthesis of the second strand of viral DNA has been shown to be the rate-limiting step for viral gene expression.

[0070] As used herein, self-complementary AAV (scAAV) has a D sequence (packaging signal) deleted from the right ITR of the ssAAV genome and a terminal resolution site mutation (Δtrs), which can prevent the Rep protein from modifying and regulating the resolution nicks and enhance the packaging of self-complementary double-stranded DNA. After the double-stranded AAV virus enters the cell, it does not require a process of conversion from single-stranded to double-stranded and can be directly expressed, and the expression level is relatively high.

[0071] As used herein, covalently closed end AAV (cceAAV) is a newly emerging rAAV system, which is formed by closing one end of the complementary double-stranded DNA of the AAV genome with, such as, shRNA or an oligonucleotide chain. An example of cceAAV is the cceAAV system described in WO2020 / 092904, which is incorporated herein by reference in its entirety.

[0072] Gene delivery vector

[0073] A first aspect of the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises immunoglobulin-like domain 2 of human VEGFR1, immunoglobulin-like domain 3 of human VEGFR2, and a hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0074] In some embodiments, the human VEGF receptor fusion protein further comprises immunoglobulin-like domain 4 of human VEGFR2. Thus, in these embodiments, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises immunoglobulin-like domain 2 of human VEGFR1, immunoglobulin-like domain 3 of human VEGFR2, immunoglobulin-like domain 4 of human VEGFR2, and a hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0075] In some embodiments, the hinge-Fc domain of the human immunoglobulin is the hinge-Fc domain of human IgG1. Thus, in these embodiments, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises immunoglobulin-like domain 2 of human VEGFR1, immunoglobulin-like domain 3 of human VEGFR2, and the hinge-Fc domain of human IgG1; and the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions according to EU numbering. In some embodiments, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises immunoglobulin-like domain 2 of human VEGFR1, immunoglobulin-like domain 3 of human VEGFR2, immunoglobulin-like domain 4 of human VEGFR2, and the hinge-Fc domain of human IgG1; and the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0076] In any of the above embodiments, preferably, in the expression cassette for expressing the human VEGF receptor fusion protein, the nucleic acids encoding the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, the immunoglobulin-like domain 4 of human VEGFR2, and the hinge-Fc domain of the human immunoglobulin are arranged in sequence in the 5'-to-3' direction. In other embodiments, in the expression cassette for expressing the human VEGF receptor fusion protein, the nucleic acids encoding the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, and the immunoglobulin-like domain 4 of human VEGFR2 can be arranged in any order in the 5'-to-3' direction, and the nucleic acid encoding the hinge-Fc domain of the human immunoglobulin is located at their 3' end. In other embodiments, in the expression cassette for expressing the human VEGF receptor fusion protein, the nucleic acids encoding the immunoglobulin-like domain 2 of human VEGFR1 and the immunoglobulin-like domain 3 of human VEGFR2 can be arranged in any order in the 5'-to-3' direction, and the nucleic acid encoding the hinge-Fc domain of the human immunoglobulin is located at their 3' end.

[0077] In any of the above embodiments, preferably, the domains are directly connected. In other embodiments, the domains can be connected by a peptide linker. Suitable peptide linkers are known in the art and generally consist of multiple glycines and serines. The present invention contemplates that any suitable peptide linker can be used to connect the domains of the human VEGF receptor fusion protein.

[0078] In any of the above embodiments, the hinge-Fc domain of human IgG1 may, in addition to containing the M252Y, S254T, and T256E substitutions according to EU numbering, also contain one or more of the M428L and N434S substitutions, which are known to have the effect of enhancing the interaction with the FcRn receptor. For example, in some embodiments, the hinge-Fc domain of human IgG1 contains the M252Y, S254T, and T256E substitutions and the M428L substitution according to EU numbering. In other embodiments, the hinge-Fc domain of human IgG1 contains the M252Y, S254T, and T256E substitutions and the N434S substitution according to EU numbering. In other embodiments, the hinge-Fc domain of human IgG1 contains the M252Y, S254T, and T256E substitutions and the M428L and N434S substitutions according to EU numbering. The present invention contemplates that the hinge-Fc domain of human IgG1 may contain more substitutions.

[0079] In any of the above embodiments, the immunoglobulin-like domain 2 of human VEGFR1 may comprise or be: the amino acid sequence shown in SEQ ID NO.1 or 6, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or 6. Thus, in some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.1. In some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.6. In some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: 1 to 10, 1 to 5 or 1 to 3 amino acid residues are added or deleted at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1 or 6. For example, in some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: 1 to 3 amino acid residues are added at the N-terminus of the amino acid sequence shown in SEQ ID NO.1, and the added amino acid residues may be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 2 of wild-type human VEGFR1. For example, in some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: 1 to 3 amino acid residues are added at the C-terminus of the amino acid sequence shown in SEQ ID NO.6, and the added amino acid residues may be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 2 of wild-type human VEGFR1.

[0080] In any of the above embodiments, the immunoglobulin-like domain 3 of human VEGFR2 may comprise or be: the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.2. Thus, in some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.2. In some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: with 1 to 10, 1 to 5 or 1 to 3 amino acid residues added or deleted at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.2. For example, in some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: with 1 to 3 amino acid residues added at the N-terminus of the amino acid sequence shown in SEQ ID NO.2, and the added amino acid residues may be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 3 of wild-type human VEGFR2.

[0081] In any of the above embodiments, the immunoglobulin-like domain 4 of human VEGFR2 may comprise or be: the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.7. Thus, in some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.7. In some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: with 1 to 10, 1 to 5 or 1 to 3 amino acid residues added or deleted at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.7. For example, in some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: with 1 to 3 amino acid residues added at the N-terminus of the amino acid sequence shown in SEQ ID NO.7, and the added amino acid residues may be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 4 of wild-type human VEGFR2.

[0082] Thus, in some embodiments, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or 6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0083] In other embodiments, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or 6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 4 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.7 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0084] In any of the above embodiments, the hinge-Fc domain of the human immunoglobulin may comprise or be: the amino acid sequence as shown in SEQ ID NO.3 or 8, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.3 or 8, and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T and T256E substitutions according to EU numbering. Thus, in some embodiments, the hinge-Fc domain of the human immunoglobulin comprises or is: the amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.3, and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T and T256E substitutions according to EU numbering. In some embodiments, the hinge-Fc domain of the human immunoglobulin comprises or is: the amino acid sequence as shown in SEQ ID NO.8 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.8, and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T and T256E substitutions according to EU numbering.

[0085] Thus, in a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having the amino acid sequence as shown in SEQ ID NO.1 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having the amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having the amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T and T256E substitutions according to EU numbering.

[0086] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 4 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.7 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0087] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0088] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 4 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.7 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.8 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0089] In a preferred embodiment, the human VEGF receptor fusion protein further comprises a signal peptide at the most N-terminus. An example of the signal peptide comprises or is an amino acid sequence as shown in SEQ ID NO.4.

[0090] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises a hinge-Fc domain of human immunoglobulin, and the human VEGF receptor fusion protein comprises or is an amino acid sequence as shown in SEQ ID NO.5, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.5; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0091] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises a hinge-Fc domain of human immunoglobulin, and the human VEGF receptor fusion protein comprises or is an amino acid sequence as shown in SEQ ID NO.9, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.9; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0092] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises a hinge-Fc domain of human immunoglobulin, the human VEGF receptor fusion protein is aflibercept, and the hinge-Fc domain of human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0093] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises a hinge-Fc domain of human immunoglobulin, the human VEGF receptor fusion protein is conbercept, and the hinge-Fc domain of human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0094] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the nucleotide sequence encoding the human VEGF receptor fusion protein is as shown in SEQ ID NO.10 or 11 or a degenerate sequence of any one of them.

[0095] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the nucleotide sequence encoding the human VEGF receptor fusion protein is as shown in SEQ ID NO.10 or its degenerate codon sequence.

[0096] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the nucleotide sequence encoding the human VEGF receptor fusion protein is as shown in SEQ ID NO.11 or its degenerate codon sequence.

[0097] In the present invention, a degenerate codon sequence refers to a variant of a reference sequence (the nucleotide sequence shown in SEQ ID NO.10 or 11) obtained according to codon degeneracy. As is known in the art, the variant can be obtained by optimizing the reference sequence from one or more aspects including but not limited to codon frequency, mRNA secondary structure, GC content, RNase splicing site, repetitive sequence, etc., without changing the amino acid sequence translated from the reference sequence.

[0098] In any of the above embodiments, the expression cassette of the human VEGF receptor fusion protein may comprise a promoter located upstream of and operably linked to the nucleotide sequence encoding the human VEGF receptor fusion protein. In a preferred embodiment, the promoter is selected from the cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) promoter, MMT promoter, EF-1α promoter, U6 promoter, chicken β-actin promoter, CAG promoter, CBA promoter, RPE65 promoter, VMD2 promoter, RPGR promoter, IRBP promoter, hGRK1 promoter, CAR promoter, RHO promoter, Grm6 promoter, and GFAP promoter. In a more preferred embodiment, the promoter is the CMV promoter.

[0099] In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the tissue-specific promoter is an eye-specific promoter. Examples of eye-specific promoters include the retinoschisin proximal promoter, the interphotoreceptor retinoid-binding protein enhancer (RS / IRBPa), rhodopsin kinase (RK), RPE65, and the human cone opsin promoter. In some embodiments, the promoter is the chicken β-actin (CB) promoter. The chicken β-actin promoter can be a short chicken β-actin promoter or a long chicken β-actin promoter. In some embodiments, the promoter (e.g., the chicken β-actin promoter) comprises an enhancer sequence, such as the cytomegalovirus (CMV) enhancer sequence. The CMV enhancer sequence can be a short CMV enhancer sequence or a long CMV enhancer sequence. In some embodiments, the promoter comprises a long CMV enhancer sequence and a long chicken β-actin promoter. In some embodiments, the promoter comprises a short CMV enhancer sequence and a short chicken β-actin promoter. However, those skilled in the art will appreciate that a short CMV enhancer can be used with a long CB promoter, and a long CMV enhancer can be used with a short CB promoter (and vice versa).

[0100] In some embodiments, the expression cassette may further comprise one or more introns. In some embodiments, at least one intron is located between the promoter / enhancer sequence and the transgene. In some cases, a promoter or regulatory sequence element may be used to direct selective expression in eye cells or eye tissues. For example, a promoter, sequence element, or regulatory sequence found in a particular eye cell type, such as retinal pigment epithelial cells, may be used in a suitable expression construct (e.g., the RPE65 or VMD2 promoter). In some embodiments, the intron is a synthetic or artificial (e.g., heterologous) intron. Examples of synthetic introns include an intron sequence derived from SV-40 (referred to as the SV-40T intron sequence) and an intron sequence derived from the chicken β-actin gene. In some embodiments, the transgene described in the present disclosure comprises one or more (1, 2, 3, 4, 5 or more) artificial introns. In some embodiments, one or more artificial introns are located between the promoter and the nucleic acid sequence encoding the human VEGF receptor fusion protein (or transgene). In some cases, an intron may refer to any sequence that is transcribed but not translated. In some cases, an intron may refer to any sequence that is transcribed in a cell and removed from the mature RNA transcript. In some cases, an intron may comprise about at least 100, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000 nucleotides. In some cases, an intron may be about 300 nucleotides. In some cases, an intron may be about 200 to 400 nucleotides. In some cases, a chimeric intron may be about 100 to 500 nucleotides. In some cases, an intron may be a complete naturally occurring intron or a chimeric intron. In some aspects, the intron may include, but is not limited to, the introns described in CN104994882A, the entire disclosure of which is incorporated herein by reference.

[0101] In any of the above embodiments, the expression cassette of the human VEGF receptor fusion protein may comprise a polyadenylation signal (polyA) located downstream of and operably linked to the nucleotide sequence encoding the human VEGF receptor fusion protein. In a preferred embodiment, the polyadenylation signal is selected from SV40 early polyA, SV40 late polyA, rabbit globin polyA, bGH polyA, and HSV TK polyA. In a more preferred embodiment, the polyadenylation signal is SV40 late polyA.

[0102] Accordingly, in some embodiments, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette comprising, in the 5' to 3' direction, a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, and polyA, wherein the human VEGF receptor fusion protein comprises, from the N-terminus to the C-terminus, the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, and the hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T, and T256E substitutions according to EU numbering.

[0103] In some embodiments, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette comprising, in the 5' to 3' direction, a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, and polyA, wherein the human VEGF receptor fusion protein comprises, from the N-terminus to the C-terminus, the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, the immunoglobulin-like domain 4 of VEGFR2, and the hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T, and T256E substitutions according to EU numbering.

[0104] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette comprising, in the 5' to 3' direction, a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, and polyA, wherein the human VEGF receptor fusion protein comprises the hinge-Fc domain of human immunoglobulin, and the human VEGF receptor fusion protein comprises or is an amino acid sequence as shown in SEQ ID NO.5, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.5; and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T, and T256E substitutions according to EU numbering.

[0105] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette sequentially comprising a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, and polyA from the 5'- to 3'-direction, wherein the human VEGF receptor fusion protein comprises a hinge-Fc domain of human immunoglobulin, the human VEGF receptor fusion protein comprises or is an amino acid sequence as shown in SEQ ID NO.9, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.9; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0106] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette sequentially comprising a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, and polyA from the 5'- to 3'-direction, wherein the nucleotide sequence encoding the human VEGF receptor fusion protein is as shown in SEQ ID NO.10 or its codon-degenerate sequence.

[0107] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette sequentially comprising a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, and polyA from the 5'- to 3'-direction, wherein the nucleotide sequence encoding the human VEGF receptor fusion protein is as shown in SEQ ID NO.11 or its codon-degenerate sequence.

[0108] The gene delivery vector that can be used in the present invention can be a viral vector or a non-viral vector. In some embodiments, the gene delivery vector is a non-viral vector. In a preferred embodiment, the non-viral vector is a plasmid, liposome, nanoparticle, polymer, transposon, exosome, or bacterial vector. In a more preferred embodiment, the non-viral vector is a plasmid, liposome, or nanoparticle. In the most preferred embodiment, the non-viral vector is a lipid nanoparticle (LNP).

[0109] In some embodiments, the gene delivery vector is a viral vector. In a preferred embodiment, the viral vector is a lentivirus, retrovirus, adenovirus, adeno-associated virus, herpes virus, poxvirus, papovavirus, baculovirus, or papillomavirus vector.

[0110] Retroviral vectors include Moloney murine leukemia virus and HIV-based viruses. In some cases, HIV-based viral vectors can be used, where the HIV-based viral vector comprises at least two vectors, wherein the gag and pol genes are from the HIV genome and the env gene is from another virus. DNA viral vectors can be used. These vectors include poxvirus vectors such as orthopox or avipox vectors, and herpesvirus vectors such as herpes simplex virus type I (HSV-1) vectors. HSV-1 vectors lacking one or more immediate early genes (IE) are advantageous because they are generally non-cytotoxic, persist in target cells in a latent-like state, and provide efficient target cell transduction. Recombinant HSV vectors can incorporate approximately 30 kb of heterologous nucleic acid. Lentiviral vectors can be advantageous because they can infect both actively dividing and non-dividing cells. They can also be highly efficient in transducing human epithelial cells. The lentiviral vectors for use in the present disclosure can be derived from human and non-human (including SIV) lentiviruses. Examples of lentiviral vectors include nucleic acid sequences required for vector propagation and a tissue-specific promoter operably linked to the human VEGF receptor fusion protein gene. The nucleic acid sequence can include viral LTRs, primer binding sites, polypurine tracts, att sites, and encapsidation sites. Lentiviral vectors can be packaged into any suitable lentiviral capsid. Poxvirus vectors can introduce genes into the cytoplasm of cells. Avipoxvirus vectors can result in only short-term expression of genes or nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used together with the nucleic acid encoding the human VEGF receptor fusion protein of the present disclosure. Adenovirus vectors can result in shorter-term expression than adeno-associated viruses (e.g., shorter than about 1 month). In a more preferred embodiment, the viral vector is a lentiviral vector or an adeno-associated virus vector. In the most preferred embodiment, the viral vector is an adeno-associated virus vector.

[0111] When the gene delivery vector is an adeno-associated virus vector, the gene delivery vector is also referred to as a recombinant adeno-associated virus vector or recombinant adeno-associated virus (rAAV). In such an embodiment, the present invention provides an adeno-associated virus vector comprising: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises immunoglobulin-like domain 2 of human VEGFR1, immunoglobulin-like domain 3 of human VEGFR2, and the hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering. The above description of the nucleic acid and the expression cassette comprised by the gene delivery vector applies to part (b) of rAAV and will not be repeated here.

[0112] In a preferred embodiment, the present invention provides an adeno-associated virus vector, which comprises: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or 6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0113] In a preferred embodiment, the present invention provides an adeno-associated virus vector, which comprises: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or 6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 4 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.7 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0114] In a preferred embodiment, the present invention provides an adeno-associated virus vector, which comprises: (a) A recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0115] In a preferred embodiment, the present invention provides an adeno-associated virus vector, which comprises: (a) A recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 4 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.7 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.8 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0116] In a preferred embodiment, the present invention provides an adeno-associated virus vector, which comprises: (a) A recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises a hinge-Fc domain of human immunoglobulin, the human VEGF receptor fusion protein comprises or is an amino acid sequence as shown in SEQ ID NO.5 or 9, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.5 or 9; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0117] In a preferred embodiment, the present invention provides an adeno-associated virus vector, comprising: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises a hinge-Fc domain of human immunoglobulin, the human VEGF receptor fusion protein is aflibercept, and the hinge-Fc domain of human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0118] In a preferred embodiment, the present invention provides an adeno-associated virus vector, comprising: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises a hinge-Fc domain of human immunoglobulin, the human VEGF receptor fusion protein is conbercept, and the hinge-Fc domain of human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0119] In a preferred embodiment, the present invention provides an adeno-associated virus vector, comprising: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the nucleotide sequence encoding the human VEGF receptor fusion protein is as shown in SEQ ID NO.10 or 11 or a degenerate sequence of any one of them.

[0120] In a preferred embodiment, the present invention provides an adeno-associated virus vector, comprising: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette comprising in the 5' to 3' direction in sequence 5' AAV ITR, a promoter, a nucleic acid encoding a human VEGF receptor fusion protein, polyA, and 3' AAV ITR, wherein the human VEGF receptor fusion protein comprises in sequence from the N-terminus to the C-terminus an immunoglobulin-like domain 2 of human VEGFR1, an immunoglobulin-like domain 3 of human VEGFR2, and a hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0121] In a preferred embodiment, the present invention provides an adeno-associated virus vector, which comprises: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette sequentially comprising 5' AAV ITR, a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, polyA, and 3' AAV ITR from the 5' to 3' direction, wherein the human VEGF receptor fusion protein sequentially comprises the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, the immunoglobulin-like domain 4 of VEGFR2, and the hinge-Fc domain of human immunoglobulin from the N-terminus to the C-terminus; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0122] In a preferred embodiment, the present invention provides an adeno-associated virus vector, which comprises: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette sequentially comprising 5' AAV ITR, a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, polyA, and 3' AAV ITR from the 5' to 3' direction, wherein the human VEGF receptor fusion protein comprises the hinge-Fc domain of human immunoglobulin, and the human VEGF receptor fusion protein comprises or is: an amino acid sequence as shown in SEQ ID NO.5 or 9, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.5 or 9; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0123] In a preferred embodiment, the present invention provides an adeno-associated virus vector, which comprises: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated virus capsid, the nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, the expression cassette sequentially comprising 5' AAV ITR, a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, polyA, and 3' AAV ITR from the 5' to 3' direction, wherein the nucleotide sequence encoding the human VEGF receptor fusion protein is as shown in SEQ ID NO.10 or 11 or its codon degenerate sequence.

[0124] In any of the above-described embodiments of the adeno-associated virus vector, the recombinant adeno-associated virus capsid may comprise capsid proteins of adeno-associated virus selected from the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof, as well as capsid protein mutants modified based on the above capsid proteins as a backbone. In a preferred embodiment, the recombinant adeno-associated virus capsid may comprise or consist of capsid proteins of adeno-associated virus selected from AAV1, AAV2, AAV5, AAV7, AAV8, and hybrids thereof. In a more preferred embodiment, the recombinant adeno-associated virus capsid may comprise or consist of AAV8 capsid protein.

[0125] Examples of the genomic sequences of different AAV serotypes can be found in the literature or public databases (such as GenBank). For example, the GenBank accession numbers are NC_001401.2 (AAV2), NC_001829.1 (AAV4), NC / 006152.1 (AAV5), AF028704.1 (AAV6), NC-006260.1 (AAV7), NC.006261.1 (AAV8), AX753250.1 (AAV9), and AX753362.1 (AAV10). In some embodiments, the adeno-associated virus vector according to the present invention comprises a capsid derived from a serotype selected from AAV2, AAV5, AAV7, AAV8, AAV9, AAV10, and AAVrh10 serotypes. In another embodiment, the AAV serotype is AAV8. If the viral vector comprises a sequence encoding a capsid protein, it can be modified to comprise a foreign sequence, thereby directing AAV to a specific cell type or types, or increasing the efficiency of targeted vector delivery to cells, or facilitating the purification or detection of AAV, or reducing the host response.

[0126] In some embodiments, the AAV capsid protein has tropism for eye tissue or muscle tissue. In some embodiments, the eye tissue includes eye neurons, retina, sclera, choroid, retina, vitreous, macula, fovea centralis, optic disc, lens, pupil, iris, aqueous humor, cornea, conjunctiva ciliary body, or optic nerve. In some embodiments, the AAV capsid protein targets eye cell types (e.g., photoreceptor cells, retinal cells, etc.).

[0127] In any of the above-described embodiments of the adeno-associated virus vector, the genome of the adeno-associated virus vector is in single-stranded or double-stranded form, preferably any one of scAAV, ssAAV, or cceAAV. Those skilled in the art can select any one of these systems according to actual needs to form the adeno-associated virus vector of the present invention.

[0128] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: (a) an rAAV capsid; and (b) a nucleic acid packaged within the rAAV capsid, the nucleic acid comprising, in 5' to 3' order: (i) a 5' AAV ITR; (ii) a promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) a polyA; and (v) a 3' AAV ITR, wherein for the promoter, the nucleic acid encoding the human VEGF receptor fusion protein, and the polyA, the descriptions of the corresponding components of the gene delivery vector as above apply and will not be repeated.

[0129] In a preferred embodiment, the rAAV capsid comprises or consists of the capsid protein of AAV1, AAV2, AAV5, AAV7 or AAV8, preferably the capsid protein of AAV8 or consists of the same.

[0130] In a preferred embodiment, the 5' AAV ITR and / or the 3' AAV ITR are selected from the ITRs of AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6.

[0131] In a specific embodiment, the present invention provides a recombinant adeno-associated virus comprising: (a) an rAAV8 capsid; and (b) a nucleic acid packaged within the rAAV8 capsid, the nucleic acid comprising, in 5' to 3' order: (i) a 5' AAV ITR; (ii) a CMV promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) an SV40 late polyA; and (v) a 3' AAV ITR, wherein the human VEGF receptor fusion protein comprises or is the amino acid sequence as shown in SEQ ID NO.5 or 9.

[0132] In a specific embodiment, the present invention provides a recombinant adeno-associated virus comprising: (a) an rAAV8 capsid; and (b) a nucleic acid packaged within the rAAV8 capsid, the nucleic acid comprising, in 5' to 3' order: (i) a 5' AAV ITR; (ii) a CMV promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) a rabbit globin polyA; and (v) a 3' AAV ITR, wherein the human VEGF receptor fusion protein comprises or is the amino acid sequence as shown in SEQ ID NO.5 or 9.

[0133] In a specific embodiment, the present invention provides a recombinant adeno-associated virus comprising: (a) rAAV2 capsid; and (b) a nucleic acid packaged within the rAAV2 capsid, the nucleic acid comprising, in 5' to 3' order: (i) a 5' AAV ITR; (ii) a CMV promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) SV40 late polyA; and (v) a 3' AAV ITR, wherein the human VEGF receptor fusion protein comprises or is the amino acid sequence shown in SEQ ID NO. 5 or 9.

[0134] In a specific embodiment, the present invention provides a recombinant adeno-associated virus comprising: (a) rAAV2 capsid; and (b) a nucleic acid packaged within the rAAV2 capsid, the nucleic acid comprising, in 5' to 3' order: (i) a 5' AAV ITR; (ii) a CMV promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) rabbit globin polyA; and (v) a 3' AAV ITR, wherein the human VEGF receptor fusion protein comprises or is the amino acid sequence shown in SEQ ID NO. 5 or 9.

[0135] In a specific embodiment, the present invention provides a recombinant adeno-associated virus comprising: (a) rAAV1 capsid; and (b) a nucleic acid packaged within the rAAV1 capsid, the nucleic acid comprising, in 5' to 3' order: (i) a 5' AAV ITR; (ii) a chicken β-actin promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) rabbit globin polyA; and (v) a 3' AAV ITR, wherein the human VEGF receptor fusion protein comprises or is the amino acid sequence shown in SEQ ID NO. 5 or 9.

[0136] In a specific embodiment, the present invention provides a recombinant adeno-associated virus comprising: (a) rAAV1 capsid; and (b) a nucleic acid packaged within the rAAV1 capsid, the nucleic acid comprising, in 5' to 3' order: (i) a 5' AAV ITR; (ii) a chicken β-actin promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) SV40 late polyA; and (v) a 3' AAV ITR, wherein the human VEGF receptor fusion protein comprises or is the amino acid sequence shown in SEQ ID NO. 5 or 9. In some embodiments of the present invention, the recombinant adeno-associated virus is any one of scAAV, ssAAV or cceAAV.

[0137] In some embodiments, the present invention provides an expression cassette that, in a 5' to 3' order, comprises: (i) a promoter; (ii) a nucleic acid encoding a human VEGF receptor fusion protein; (iii) a polyA, wherein for the promoter, the nucleic acid encoding a human VEGF receptor fusion protein, and the polyA, the descriptions of the corresponding components of the gene delivery vector as described above are applicable and will not be repeated here.

[0138] In some embodiments, the present invention provides an expression cassette that, in a 5' to 3' order, comprises: (i) a 5' AAV ITR; (ii) a promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) a polyA; and (v) a 3' AAV ITR, wherein for the ITR, the promoter, the nucleic acid encoding a human VEGF receptor fusion protein, and the polyA, the descriptions of the corresponding components of the gene delivery vector or recombinant adeno-associated virus as described above are applicable and will not be repeated here.

[0139] The triple transfection (triple plasmid transfection) method can be used to generate recombinant AAV. Generally, recombinant AAV is produced by transfecting a host cell with an AAV vector (containing a transgene flanked by ITR elements) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (e.g., rep and cap), which trans - provide the functional proteins required for AAV replication and encapsidation. The accessory function vector encodes nucleotide sequences for non - AAV - derived viral and / or cellular functions on which AAV depends for replication (e.g., "accessory functions"). Accessory functions include those functions required for AAV replication, including but not limited to those parts involved in AAV gene transcriptional activation, stage - specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. The virus - based accessory functions can be derived from any known helper virus, such as adenovirus, herpes virus (except herpes simplex virus type 1), and vaccinia virus.

[0140] The human VEGF receptor fusion protein and its encoding nucleic acid

[0141] Another aspect of the present invention provides a human VEGF receptor fusion protein that comprises the immunoglobulin - like domain 2 of human VEGFR1, the immunoglobulin - like domain 3 of human VEGFR2, and the hinge - Fc domain of human immunoglobulin; and the hinge - Fc domain of the human immunoglobulin contains M252Y, S254T, and T256E substitutions according to EU numbering.

[0142] In some embodiments, the human VEGF receptor fusion protein further comprises the immunoglobulin-like domain 4 of human VEGFR2. Thus, in these embodiments, the present invention provides a human VEGF receptor fusion protein comprising the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, the immunoglobulin-like domain 4 of human VEGFR2, and the hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0143] In some embodiments, the hinge-Fc domain of the human immunoglobulin is the hinge-Fc domain of human IgG1. Thus, in these embodiments, the present invention provides a human VEGF receptor fusion protein comprising the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, and the hinge-Fc domain of human IgG1; and the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions according to EU numbering. In some embodiments, the present invention provides a human VEGF receptor fusion protein comprising the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, the immunoglobulin-like domain 4 of human VEGFR2, and the hinge-Fc domain of human IgG1; and the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0144] In any of the above embodiments, preferably, the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, the immunoglobulin-like domain 4 of human VEGFR2, and the hinge-Fc domain of the human immunoglobulin are arranged in sequence from the N-terminus to the C-terminus. In other embodiments, the nucleic acids of the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, and the immunoglobulin-like domain 4 of human VEGFR2 can be arranged in any order, and the hinge-Fc domain of the human immunoglobulin is located at the most C-terminus. In other embodiments, the immunoglobulin-like domain 2 of human VEGFR1 and the immunoglobulin-like domain 3 of human VEGFR2 are arranged from the N-terminus to the C-terminus, and the hinge-Fc domain of the human immunoglobulin is located at the most C-terminus.

[0145] In any of the above embodiments, preferably, the domains are directly connected. In other embodiments, the domains may be connected by a peptide linker. Suitable peptide linkers are known in the art and typically consist of multiple glycines and serines. The present invention contemplates that any suitable peptide linker can be used to connect the domains of the human VEGF receptor fusion protein.

[0146] In any of the above embodiments, the hinge-Fc domain of the human IgG1 may further comprise one or more of the M428L and N434S substitutions in addition to the M252Y, S254T, and T256E substitutions according to the EU numbering, which are known to have the effect of enhancing the interaction with the FcRn receptor. For example, in some embodiments, the hinge-Fc domain of the human IgG1 comprises the M252Y, S254T, and T256E substitutions and the M428L substitution according to the EU numbering. In other embodiments, the hinge-Fc domain of the human IgG1 comprises the M252Y, S254T, and T256E substitutions and the N434S substitution according to the EU numbering. In other embodiments, the hinge-Fc domain of the human IgG1 comprises the M252Y, S254T, and T256E substitutions and the M428L and N434S substitutions according to the EU numbering. The present invention contemplates that the hinge-Fc domain of the human IgG1 may comprise more substitutions.

[0147] In any of the above embodiments, the immunoglobulin-like domain 2 of human VEGFR1 may comprise or be: the amino acid sequence shown in SEQ ID NO.1 or 6, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or 6. Thus, in some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.1. In some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.6. In some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: adding or deleting 1 to 10, 1 to 5 or 1 to 3 amino acid residues at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.1 or 6. For example, in some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: adding 1 to 3 amino acid residues at the N-terminus of the amino acid sequence shown in SEQ ID NO.1, and the added amino acid residues may be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 2 of wild-type human VEGFR1. For example, in some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: adding 1 to 3 amino acid residues at the C-terminus of the amino acid sequence shown in SEQ ID NO.6, and the added amino acid residues may be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 2 of wild-type human VEGFR1.

[0148] In any of the above embodiments, the immunoglobulin-like domain 3 of human VEGFR2 may comprise or be: the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.2. Thus, in some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: the amino acid sequence shown in SEQ ID NO.2, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.2. In some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: adding or deleting 1 to 10, 1 to 5 or 1 to 3 amino acid residues at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.2. For example, in some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: adding 1 to 3 amino acid residues at the N-terminus of the amino acid sequence shown in SEQ ID NO.2, and the added amino acid residues may be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 3 of wild-type human VEGFR2.

[0149] In any of the above embodiments, the immunoglobulin-like domain 4 of human VEGFR2 may comprise or be: the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.7. Thus, in some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: the amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.7. In some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: adding or deleting 1 to 10, 1 to 5 or 1 to 3 amino acid residues at the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO.7. For example, in some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: adding 1 to 3 amino acid residues at the N-terminus of the amino acid sequence shown in SEQ ID NO.7, and the added amino acid residues may be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 4 of wild-type human VEGFR2.

[0150] Thus, in some embodiments, the present invention provides a human VEGF receptor fusion protein which sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or 6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0151] In other embodiments, the present invention provides a human VEGF receptor fusion protein which sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or 6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 4 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.7 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0152] In any of the above embodiments, the hinge-Fc domain of the human immunoglobulin may comprise or be: the amino acid sequence as shown in SEQ ID NO.3 or 8, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.3 or 8, and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T, and T256E substitutions according to EU numbering. Thus, in some embodiments, the hinge-Fc domain of the human immunoglobulin comprises or is: the amino acid sequence as shown in SEQ ID NO.3 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.3, and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T, and T256E substitutions according to EU numbering. In some embodiments, the hinge-Fc domain of the human immunoglobulin comprises or is: the amino acid sequence as shown in SEQ ID NO.8 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.8, and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T, and T256E substitutions according to EU numbering.

[0153] Thus, in a preferred embodiment, the present invention provides a human VEGF receptor fusion protein which sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having the amino acid sequence as shown in SEQ ID NO.1 or 6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having the amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having the amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises the M252Y, S254T, and T256E substitutions according to EU numbering.

[0154] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein, which sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or 6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 4 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.7 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to the EU numbering.

[0155] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein, which sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.1 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to the EU numbering.

[0156] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein, which sequentially comprises, from the N-terminus to the C-terminus, an immunoglobulin-like domain 2 of human VEGFR1 having an amino acid sequence as shown in SEQ ID NO.6 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 3 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.2 or having at least 80% sequence identity therewith, an immunoglobulin-like domain 4 of human VEGFR2 having an amino acid sequence as shown in SEQ ID NO.7 or having at least 80% sequence identity therewith, and a hinge-Fc domain of human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.8 or having at least 80% sequence identity therewith; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to the EU numbering.

[0157] In a preferred embodiment, the human VEGF receptor fusion protein further comprises a signal peptide at the most N-terminus. An example of the signal peptide comprises or is an amino acid sequence as shown in SEQ ID NO.4.

[0158] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein, which comprises a hinge-Fc domain of human immunoglobulin, and the human VEGF receptor fusion protein comprises or is, for example: the amino acid sequence shown in SEQ ID NO.5, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.5; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0159] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein, which comprises a hinge-Fc domain of human immunoglobulin, and the human VEGF receptor fusion protein comprises or is, for example: the amino acid sequence shown in SEQ ID NO.9, or an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO.9; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0160] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein, which comprises a hinge-Fc domain of human immunoglobulin, and the human VEGF receptor fusion protein is aflibercept, and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0161] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein, which comprises a hinge-Fc domain of human immunoglobulin, and the human VEGF receptor fusion protein is conbercept, and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0162] In some embodiments, the present invention provides a polynucleotide encoding the human VEGF receptor fusion protein of any one of the above embodiments, such as DNA or RNA. In a preferred embodiment, the polynucleotide comprises or is: the nucleotide sequence shown in SEQ ID NO.10 or 11, or a degenerate sequence of any of them. In a preferred embodiment, the polynucleotide is the nucleotide sequence shown in SEQ ID NO.10 or 11.

[0163] In some embodiments, the present invention provides a vector comprising the polynucleotide, such as a plasmid. In some embodiments, the plasmid comprises the expression cassette as described in any one of the above expression cassette embodiments. In some aspects, an antibiotic resistance gene is introduced into the plasmid. The antibiotic resistance marker can be used to identify positive transgenic cells in recombinant virus production. In some aspects, the antibiotic marker comprises a sequence encoding an antibiotic resistance gene. For example, the markers conferring resistance may include, but are not limited to, kanamycin, gentamycin, ampicillin, chloramphenicol, tetracycline, doxycycline, or hygromycin. In some aspects, the antibiotic resistance gene is a non-beta-lactam antibiotic resistance gene, such as kanamycin. In some embodiments, the vector or plasmid is constructed for forming ssAAV, scAAV, or cceAAV. In one example, the plasmid is, for example, Figure 1 the plasmid shown, which is used for forming cceAAV.

[0164] In some embodiments, the present invention provides a host cell comprising a vector having the polynucleotide, the host cell being non-human, such as HEK293 cells. Other cells are also feasible, such as CHO cells. In some embodiments, the cell is transfected with the vector comprising the polynucleotide. In addition, the cell is also co-transfected with, for example, the helper plasmid and the accessory plasmid as described above, wherein the helper plasmid provides, for example, the rep and cap genes of AAV, and the accessory plasmid provides genes such as E2, E4a, and / or VA for AAV replication.

[0165] Drug Compositions, Therapies, and Indications

[0166] Another aspect of the present invention provides a drug composition comprising any one of the gene delivery vectors (such as rAAV) of the present invention; and a pharmaceutically acceptable excipient.

[0167] Another aspect of the present invention provides a drug composition comprising any one of the human VEGF receptor fusion proteins provided by the present invention; and a pharmaceutically acceptable excipient.

[0168] In some embodiments, the rAAV composition is formulated to reduce aggregation of AAV particles in the composition, especially in the presence of a high rAAV concentration (e.g., ~1013 GC / mL or higher). Methods for reducing rAAV aggregation are well known in the art and include, for example, adding surfactants, adjusting the pH, adjusting the salt concentration, etc. The formulation of pharmaceutically acceptable excipients and auxiliary solutions, and the development of suitable dosing and treatment regimens for using the specific compositions described herein in various treatment regimens are well known to those skilled in the art.

[0169] In certain instances, it is desirable to deliver the rAAV-based therapeutic construct in the pharmaceutically suitable formulated compositions disclosed herein by one of intravitreal, intraocular, subretinal, subcutaneous, intra-pancreatic, intranasal, parenteral, intravenous, intramuscular, intrathecal, oral, intraperitoneal or inhalation. In some embodiments, the preferred mode of administration is by intravenous injection.

[0170] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that easy injection is possible. It must be stable under the conditions of manufacture and storage and its preservation must prevent the contaminating action of microorganisms such as bacteria and fungi. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and / or vegetable oils. Suitable fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable composition can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0171] For example, for the administration of injectable aqueous solutions, if desired, the solution can be suitably buffered and first diluted with sufficient saline or glucose to render the liquid diluent isotonic. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, the sterile aqueous media that can be used are known to those of skill in the art.

[0172] Sterile injectable solutions are prepared by incorporating the required amount of the active rAAV with the various other ingredients enumerated herein (as required) into a suitable solvent and then filtering sterilizing. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any other desired ingredients from its previously sterile-filtered solution.

[0173] The rAAV compositions disclosed herein can also be formulated in neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein), which are formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc. By formulation, the solution will be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, such as injectable solutions, drug-release capsules, etc.

[0174] As used herein, "excipients" include any and all solvents, dispersion media, vehicles, coating agents, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to a host.

[0175] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, etc. can be used to introduce the compositions of the present disclosure into suitable host cells. In particular, the transgenes delivered by rAAV vectors can be formulated for encapsulation and delivery in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc. Such formulations can preferably be pharmaceutically acceptable formulations for introducing the nucleic acids or rAAV constructs disclosed herein. The formation and use of liposomes are generally known to those skilled in the art. Currently, liposomes with improved serum stability and circulating half-life have been developed.

[0176] In some cases, targeting ocular (e.g., corneal) tissues by intra-stromal administration or subcutaneous injection may require a different (e.g., higher or lower) dose than by another method (e.g., systemic administration, topical administration). Thus, in some embodiments, the injection is an intra-stromal injection (IS). In some embodiments, the injection is a topical administration (e.g., topical administration to the eye). In some cases, multiple doses of rAAV are administered.

[0177] In some embodiments, the administration of rAAV as described herein results in a transgene (e.g., Delivery to ocular tissue. rAAV can be delivered to the ocular tissue of a mammalian subject by, for example, intravitreal injection, subretinal injection, topical administration (e.g., eye drops), or by injection into the eye of a mammalian subject (e.g., intravitreal injection). As used herein, "ocular tissue" refers to any tissue derived from or contained within the eye. Non-limiting examples of ocular tissue include neurons, retina (e.g., photoreceptor cells), sclera, choroid, retina, vitreous, macula, fovea centralis, optic disc, lens, pupil, iris, aqueous humor, cornea (e.g., keratinocytes, corneal endothelial cells, corneal basal cells, corneal limbal cells, and corneal squamous cells), conjunctiva ciliary body, and optic nerve. The retina is located at the back of the eye and contains photoreceptor cells. These photoreceptor cells (e.g., rod cells, cone cells) confer visual acuity by discriminating colors, as well as contrast in the visual field.

[0178] Alternatively, rAAV can be delivered to a mammalian subject by intramuscular injection or by administration to the bloodstream of a mammalian subject. Administration to the bloodstream can be by injection into a vein, artery, or any other vascular catheter. Non-limiting exemplary methods of intramuscular administration of rAAV include intramuscular (IM) injection and intravascular infusion. In some embodiments, the rAAV or composition as described in the present disclosure is administered by intravitreal injection. In some embodiments, the rAAV or composition as described in the present disclosure is administered by intraocular injection. In some embodiments, the rAAV or composition as described in the present disclosure is administered by subretinal injection. In some embodiments, the rAAV or composition as described in the present disclosure is administered by intravenous injection. In some embodiments, the rAAV or composition as described in the present disclosure is administered by intramuscular injection.

[0179] In some embodiments, administration of rAAV as described herein results in inhibition of VEGF (e.g., VEGF activity). In some embodiments, administration of rAAV as described herein results in inhibition of VEGF (e.g., VEGF activity) in ocular tissue. The degree of VEGF inhibition can be measured by any suitable known method (e.g., HUVEC angiogenesis assay, retinal vascular development assay, retinal edema assay, laser injury-induced choroidal neovascularization (CNV), etc.). In some embodiments, the VEGF (e.g., VEGF activity) in a subject who has received an anti-VEGF agent (e.g., has been injected with rAAV as described herein) is inhibited by at least 2%, at least 5%, at least 10%, at least 15%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100% compared to a non-injected subject or the same subject prior to receiving the anti-VEGF agent. In some embodiments, the VEGF (e.g., VEGF activity) in a non-injected subject or a subject prior to receiving an anti-VEGF agent is at least 2%, at least 5%, at least 10%, at least 15%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 100%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 10 to 50-fold (e.g., 10-fold, 20-fold, 30-fold, 40-fold, or 50-fold), at least 50 to 100-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold) higher than that in a subject who has received administration of an anti-VEGF agent (e.g., has been injected with rAAV as described herein).

[0180] In some embodiments, administration of an anti-VEGF agent (e.g., rAAV as described herein) results in inhibition of VEGF (e.g., VEGF activity) for longer than 1 day, longer than 2 days, longer than 3 days, longer than 4 days, longer than 5 days, longer than 6 days, longer than 7 days, longer than 1 week (e.g., 8 days, 9 days, 10 days, 11 days, 12 days, 13 days or 14 days), longer than 2 weeks (e.g., 15 days, 16 days, 17 days, 18 days, 19 days, 20 days or 21 days), longer than 3 weeks (e.g., 22 days, 23 days, 24 days, 25 days, 26 days, 27 days or 28 days), longer than 4 weeks (e.g., 29 days, 30 days, 40 days, 50 days, 60 days, 100 days or more), longer than 1 month (e.g., 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more weeks), longer than 2 months (e.g., 2 months to 2.Between 5 months, between 2 and 3 months, between 2 and 4 months, between 2 and 5 months, between 2 and 6 months, between 2 and 7 months, between 2 and 8 months, between 2 and 9 months, between 2 and 10 months, between 2 and 11 months, between 2 and 12 months), longer than 3 months (e.g., between 3 and 4 months, between 3 and 5 months, between 3 and 6 months, between 3 and 7 months, between 3 and 8 months, between 3 and 9 months, between 3 and 10 months, between 3 and 11 months, between 3 and 12 months), longer than 4 months (e.g., between 4 and 5 months, between 4 and 6 months, between 4 and 7 months, between 4 and 8 months, between 4 and 9 months, between 4 and 10 months, between 4 and 11 months, between 4 and 12 months), longer than 5 months (e.g., between 5 and 6 months, between 5 and 7 months, between 5 and 8 months, between 5 and 8 months, between 5 and 9 months, between 5 and 10 months, between 5 and 11 months, between 5 and 12 months), longer than 6 months (e.g., between 6 and 7 months, between 6 and 8 months, between 6 and 9 months, between 6 and 10 months, between 6 and 11 months, between 6 and 12 months), longer than 7 months (e.g., between 7 and 8 months, between 7 and 9 months, between 7 and 10 months, between 7 and 11 months, between 7 and 12 months), longer than 8 months (e.g., between 8 and 9 months, between 8 and 10 months, between 8 and 11 months, between 8 and 12 months), longer than 9 months (e.g., between 9 and 10 months, between 9 and 11 months, between 9 and 12 months), longer than 10 months (e.g., between 10 and 11 months, between 11 and 12 months), longer than 11 months (e.g., between 11 and 12 months), longer than 12 months (e.g., between 12 and 15 months, between 12 and 18 months, between 12 and 21 months, between 12 and 2 months), longer than 1 year (e.g., between 1 and 1.5 years), longer than 2 years, longer than 3 years, longer than 4 years, longer than 5 years, longer than 10 years, longer than 15 years, longer than 20 years or longer.

[0181] The compositions of the present disclosure can comprise rAAV alone, or rAAV in combination with one or more other viruses (e.g., a second rAAV encoding one or more different transgenes). In some embodiments, the composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs, each having one or more different transgenes.

[0182] An effective amount of rAAV or the composition is an amount sufficient to target infection of an animal, a target tissue (e.g., muscle tissue, eye tissue, etc.). In some embodiments, the effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and thus can vary between animals and tissues. For example, an effective amount of rAAV is typically in the range of about 1 ml to about 100 ml of solution containing about 10 6 to 10 16 genomic copies (e.g., 1x10 6 to 1x10 16 , including the endpoints). In some embodiments, the effective amount of rAAV ranges between 1x10 9 and 1x10 14 rAAV genomic copies. In some cases, a dose between about 10 11 and 10 12 rAAV genomic copies is suitable. In some embodiments, a dose between about 10 11 and 10 13 rAAV genomic copies is suitable. In some embodiments, a dose between about 10 11 and 10 14 rAAV genomic copies is suitable. In some embodiments, a dose between about 10 11 and 10 15 rAAV genomic copies is suitable. In some embodiments, a dose between about 10 12 and 10 14 rAAV genomic copies is suitable. In some embodiments, a dose between about 10 13 and 10 14 rAAV genomic copies is suitable. In some embodiments, a dose between about 1x10 12 , about 1.1x10 12 , about 1.2x10 12 , about 1.3x10 12 , about 1.4x10 12 , about 1.5x10 12 , about 1.6x10 12 , about 1.7x10 12 , about 1.8x10 12 , about 1.9x10 12 , about 1x10 13 , about 1.1x10 13 , about 1.2x10 13 , about 1.3x10 13 , about 1.4x10 13 , about 1.5x1013 , about 1.6 x 10 13 , about 1.7 x 10 13 , about 1.8 x 10 13 , about 1.9 x 10 13 or about 2.0 x 10 14 vector genome (vg) copies per kilogram (kg) of body weight is suitable. In some embodiments, about 4 x 10 12 to 2 x 10 13 rAAV genome copies is a suitable dose. In some embodiments, a dose of about 1.5 x 10 13 vg / kg administered intravenously is suitable. In certain embodiments, 10 12 -10 13 rAAV genome copies are effective for a target tissue (e.g., the eye). In certain embodiments, 10 13 -10 14 rAAV genome copies are effective for a target tissue (e.g., the eye).

[0183] In some embodiments, rAAV is injected into a subject. In other embodiments, rAAV is administered to a subject by topical administration (e.g., eye drops). In some embodiments, an effective amount of rAAV is an amount sufficient to express an effective amount of the human VEGF receptor fusion protein in a target tissue (e.g., the eye) of the subject.

[0184] In some embodiments, the effective amount of rAAV delivered by injection (e.g., the effective amount of rAAV delivering a human VEGF receptor fusion protein encoding sequence) is an amount sufficient to express an effective amount of the human VEGF receptor fusion protein in the target tissue. In some embodiments, delivering an effective amount of rAAV encoding a human VEGF receptor fusion protein is sufficient to deliver from 10 μg to 10 mg or any intermediate value therebetween of the human VEGF receptor fusion protein to each eye of a subject by a suitable route of administration (e.g., intravitreal injection, i.v. injection, intraperitoneal injection, and intramuscular injection). In some embodiments, rAAV encoding a human VEGF receptor fusion protein is sufficient to deliver from 20 μg to 5 mg or any intermediate value therebetween of the human VEGF receptor fusion protein to each eye of a subject. In some embodiments, rAAV encoding a human VEGF receptor fusion protein is sufficient to deliver 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg or more of the human VEGF receptor fusion protein to each eye of a subject.

[0185] In some embodiments, rAAV encoding a human VEGF receptor fusion protein is administered to a subject once daily, once weekly, once every two weeks, once monthly, once every two months, once every three months, once every six months, once a year, or once in the lifetime of the subject.

[0186] In some embodiments, the effective amount of rAAV delivered by topical administration such as eye drops is an amount sufficient to express an effective amount of the human VEGF receptor fusion protein in the target tissue. In some embodiments, eye drops containing rAAV encoding a human VEGF receptor fusion protein are administered to a subject once weekly, once monthly, once every three months, once every six months, or once a year.

[0187] In some embodiments, the eye drop contains rAAV encoding a human VEGF receptor fusion protein sufficient to deliver the human VEGF receptor fusion protein at a concentration of 1 mg / ml to 20 mg / ml. In some embodiments, the eye drop contains rAAV encoding a human VEGF receptor fusion protein sufficient to deliver the human VEGF receptor fusion protein at a concentration of 2.5 mg / ml to 10 mg / ml. In some embodiments, the eye drop contains rAAV encoding a human VEGF receptor fusion protein sufficient to deliver the human VEGF receptor fusion protein at a concentration of 1 mg / ml, 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, or 20 mg / ml. In some embodiments, the eye drop is administered at 0.01 ml, 0.02 ml, 0.03 ml, 0.04 ml, 0.05 ml, 0.06 ml, 0.07 ml, 0.08 ml, 0.09 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, or 0.5 ml.

[0188] In some embodiments, the methods described in the present disclosure further include the step of inducing immunosuppression (e.g., administering one or more immunosuppressive agents) in a subject prior to the subject being administered rAAV (e.g., the rAAV or pharmaceutical composition described in the present disclosure). In some embodiments, immunosuppression (e.g., inducing immunosuppression in the subject) is performed on the subject between about 30 days and about 0 days prior to administering rAAV to the subject (e.g., at any time within 30 days prior to administering rAAV, including the endpoints). In some embodiments, the subject is pretreated with an immunosuppressive agent (e.g., rituximab, sirolimus, and / or prednisone) for at least 7 days.

[0189] In some embodiments, the methods described in the present disclosure further comprise co-administering or pre-administering an agent to a subject to whom the rAAV of the present disclosure or a pharmaceutical composition comprising rAAV is administered. In some embodiments, the agent is selected from Miglustat, Keppra, Prevacid, Clonazepam, and any combination thereof. In some embodiments, rAAV and the additional agent can be delivered to the subject in any order. In some embodiments, rAAV and the additional agent (e.g., Miglustat, Keppra, Prevacid, Clonazepam) are delivered to the subject simultaneously. In some embodiments, rAAV and the additional agent (e.g., Miglustat, Keppra, Prevacid, Clonazepam) are co-administered to the subject (e.g., in one composition or in different compositions). In some embodiments, rAAV is delivered before the additional agent (e.g., Miglustat, Keppra, Prevacid, Clonazepam). In some embodiments, rAAV is delivered after the additional agent (e.g., Miglustat, Keppra, Prevacid, Clonazepam). In some embodiments, rAAV and the additional agent (e.g., Miglustat, Keppra, Prevacid, Clonazepam) are delivered to the subject at different frequencies, e.g., the subject receives rAAV monthly, bi-monthly, semi-annually, annually, biennially, triennially, every 5 years or longer, but receives the additional agent (e.g., Miglustat, Keppra, Prevacid, Clonazepam) daily, weekly, bi-weekly, monthly, twice daily, three times daily, or twice weekly.

[0190] In some embodiments, immunosuppression of the subject is maintained during and / or after administration of the rAAV or pharmaceutical composition. In some embodiments, the subject is immunosuppressed (e.g., administered one or more immunosuppressive agents) for a time between 1 day and 1 year after administration of the rAAV or pharmaceutical composition.

[0191] Another aspect of the invention provides a method for treating a VEGF-mediated disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the rAAV or pharmaceutical composition of the invention. In a preferred embodiment, the VEGF-mediated disease is a disease caused by VEGF overexpression.

[0192] In a preferred embodiment, the diseases caused by VEGF are selected from exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) complicated with macular edema (DME), retinal vein occlusion (RVO) complicated with macular edema (ME), central exudative chorioretinopathy, polypoidal choroidal vasculopathy (PCV), choroidal neovascularization secondary to high myopia, and secondary macular choroidal neovascularization (CNV). In a more preferred embodiment, the diseases caused by VEGF are selected from exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) complicated with macular edema (DME), or retinal vein occlusion (RVO) complicated with macular edema (ME).

[0193] Accordingly, the present invention provides the use of the gene delivery vector or the pharmaceutical composition in the preparation of a medicament for treating diseases caused by VEGF. In a preferred embodiment, the diseases caused by VEGF are selected from exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) complicated with macular edema (DME), retinal vein occlusion (RVO) complicated with macular edema (ME), central exudative chorioretinopathy, polypoidal choroidal vasculopathy (PCV), choroidal neovascularization secondary to high myopia, and secondary macular choroidal neovascularization (CNV). In a more preferred embodiment, the diseases caused by VEGF are selected from exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) complicated with macular edema (DME), or retinal vein occlusion (RVO) complicated with macular edema (ME).

[0194] Sequence Listing Examples

[0195] Preparation of rAAV-Fc-YTE-Aflibercept in Example 1

[0196] The obtained Fc-YTE-Aflibercept gene sequence (SEQ ID NO.10) was loaded into the AAV main plasmid (GOI plasmid) (the plasmid map is shown in Figure 1 ), and the corresponding recombinant adeno-associated virus (rAAV) drug rAAV-Fc-YTE-Aflibercept (also referred to as "rAAV-Fc-YTE") was prepared with the help of helper plasmids (such as Helper plasmid, R / C plasmid). In this example, the main plasmid backbone adopted the cceAAV genome configuration, the promoter element adopted the CMV promoter sequence, the PolyA element adopted the SV40 late PolyA sequence, and the viral capsid adopted the AAV8 serotype.

[0197] The AAV virus is packaged and produced using a three-plasmid system, which is completed by transient transfection of the following three-plasmid system in HEK293 cells. The three plasmids and their functions are described as follows.

[0198] Main plasmid (GOI plasmid): As described above, it contains the vector genomic DNA of the Fc-YTE-Aflibercept gene sequence expression cassette, and after administration, it can express the Fc-YTE-Aflibercept protein molecule (the amino acid sequence is shown in SEQ ID NO.5) in the target tissues in vivo.

[0199] R / C plasmid (helper plasmid): Trans-provides the Rep and Cap proteins in rAAV. The main plasmid gene and the R / C plasmid are separated into two independent plasmid systems, which will enable the Rep and Cap to only express the structure in rAAV, while deleting the rAAV replication function.

[0200] Helper plasmid (accessory plasmid): Carries the gene fragments necessary to initiate AAV replication in adenovirus, such as E2, E4a, VA, etc. It is co-transfected with the GOI main plasmid and the R / C plasmid in HEK293 cells, and under their combined action, the rAAV-Fc-YTE-Aflibercept virus is packaged and prepared. Its structural schematic diagram is as Figure 2 shown. Subsequently, relevant quality inspections are carried out, mainly including virus genome titer (Vg), capsid protein purity, virus particle number (Vp), host cell DNA residue (HCD), host cell protein residue (HCP), and bacterial endotoxin, etc., to confirm that they all meet the corresponding requirements.

[0201] Example 2. rAAV-Fc-YTE-Aflibercept significantly reduces the FFA spot score and area

[0202] The rAAV-Fc-YTE-Aflibercept provided in Example 1 was used to verify the efficacy, evaluate the efficacy, and make comparisons using a laser-induced CNV model of C57BL / 6J mice (male, about 6 weeks).

[0203] 1) Grouping and dosing regimen

[0204] Negative control: Negative control reagent (solvent reagent Buffer)

[0205] Positive control 1: Positive drug molecule (purchased Aflibercept protein injection for clinical use)

[0206] Positive control 2: rAAV-Eylea

[0207] Experimental group: rAAV-Fc-YTE

[0208] In the rAAV-Eylea group, the Fc fragment of IgG1 of Eylea (trade name of aflibercept) is wild-type Fc without YTE site-directed mutation optimization compared with the rAAV-Fc-YTE group. Both groups use the same main plasmid expression framework and regulatory elements and are prepared according to the method described in Example 1.

[0209] Grouping and dosing regimen

[0210] Note: The model was established 4 weeks after dosing, and the day of model establishment was recorded as Day0.

[0211] On Day -28, before dosing, the animals were anesthetized with Zoletil (25 - 50 mg / kg, i.p.) + Xylazine Hydrochloride Injection (5 mg / kg, i.p.). Animals in groups G1 - G3 received a single subretinal injection of different doses of the test article and Buffer in both eyes, 1 μL / eye; animals in group G4 received an intravitreal injection of the positive drug on Day 3 after model establishment, 0.5 μL / eye.

[0212] Subretinal injection: The ocular surface was disinfected with povidone-iodine. Under a special ophthalmic surgical microscope, a 30G disposable injection needle was used to puncture the sclera of the mouse medial to the limbus corneae. A microsyringe with a 35G flat needle was inserted along the puncture site and reached the vitreous body after bypassing the lens, avoiding the main blood vessels, and then gradually inserted into the subretinal space, and the injection was slowly pushed. Immediately after the syringe was withdrawn, the injection site was compressed with a cotton swab for 5 s. Immediately after dosing, OCT examination was performed to confirm successful injection. The criterion for successful dosing was that obvious elevation of the retina was observed under OCT.

[0213] Post-dosing care: After dosing, both eyes of all enrolled animals were cared for with levofloxacin eye drops and ofloxacin eye ointment, once in the morning and once in the afternoon for three consecutive days.

[0214] 2) Model establishment:

[0215] On Day 0 (4 weeks after dosing), the animals were anesthetized with Zoletil (25 - 50 mg / kg, i.p.) and Xylazine Hydrochloride Injection (5 mg / kg, i.p.). Three laser spots were burned on the RPE / Bruch membrane of both eyes using a 532 nm laser of the same energy parameters with a YAG laser photocoagulator (VITRA, Quantel Medical) (three laser points were evenly distributed around the optic disc at a distance of 1 - 1.5 PD from the optic disc). The positions of the laser spots should avoid the major retinal blood vessels and the injection sites to prevent intraocular hemorrhage.

[0216] Criterion for successful laser: Immediately after laser, OCT was used to detect the position of the laser spots to confirm whether the laser burn and visible rupture of the Bruch membrane were successful.

[0217] 3) Main detection indicators and detection results:

[0218] 3.1) Clinical observation:

[0219] Observe the animals beside the cage once a day to check for any deaths, mental state, behavioral activities, etc.

[0220] Result: The animals included in the study were observed beside the cage once a day, and no obvious abnormal clinical symptoms were found.

[0221] 3.2) SD-OCT:

[0222] OCT was performed using spectral domain optical coherence tomography with ultra-high resolution (SD-OCT). The mice were anesthetized, the pupils were dilated, and the mice were positioned to allow the optic nerve head (ONH) to appear in the center of the image. B-scans (average 5 frames) and full-field volume scans (300 frames) were captured.

[0223] Immediately after administration on Day - 28 (rAAV-Eylea group, rAAV-Fc-YTE group, negative control reagent group), and immediately after modeling on Day 0 (rAAV-Eylea group, rAAV-Fc-YTE group, negative control reagent group, positive drug molecule group), SD-OCT scans were performed on both eyes of the animals.

[0224] Conclusion: No abnormal clinical symptoms were found in the animals included in the study during the entire experimental process. The OCT scan results showed that elevations were visible in the retinas of the animals immediately after administration on Day - 28 (rAAV-Eylea group, rAAV-Fc-YTE group, negative control reagent group), indicating successful subretinal injection; laser burns and Bruch's membrane rupture were visible in all animals immediately after modeling on Day 0 (rAAV-Eylea group, rAAV-Fc-YTE group, negative control reagent group, positive drug molecule group), indicating successful laser modeling.

[0225] 3.3) FFA spot score and spot area:

[0226] On Day 7 after modeling, FFA was performed on all animals. Before the detection, the animals were anesthetized with Zoletil (25 - 50 mg / kg, i.p.) and xylazine hydrochloride injection (5 mg / kg, i.p.). After the injection of sodium fluorescein, sequential angiographic images were taken, and leakage scores were recorded at the early and late stages after sodium fluorescence injection. The leakage grades were I - IV, as shown in the following table.

[0227] Details table of FFA score Grade I No strong fluorescence. Grade II The lesion shows high fluorescence in the early or middle stage without leakage. Grade III The lesion shows high fluorescence in the early stage and leakage in the late stage. Grade IV The lesion shows bright high fluorescence in the early stage and leakage beyond the boundary of the burn area in the late stage.

[0228] Result:

[0229] On Day 7 after model establishment, all animals were subjected to FFA detection. The FFA scores and area measurement results are shown in Figure 3 and 4 respectively, and are summarized as follows. The FFA spot area was used to evaluate the drug effect. The smaller the spot area, the better the drug effect.

[0230] FFA Spot Score (Mean±SEM) Group Test Substance Number of Eyes Spot Score on Day 7 after Modeling rAAV-Eylea n＝12 2.31±0.10 rAAV-Fc-YTE n＝12 1.94±0.08 Negative Control Reagent n＝12 3.56±0.11 Positive Drug Molecule n＝12 2.39±0.14

[0231] FFA Spot Area (mm 2 , Mean±SEM) Test Substance Group Number of Eyes Spot Area on Day 7 after Modeling rAAV-Eylea n＝12 1.90±0.63 rAAV-Fc-YTE n＝12 0.08±0.08 Negative Control Reagent n＝12 5.22±0.50 Positive Drug Molecule n＝12 2.15±0.57

[0232] Summary: The results of fundus fluorescein angiography (FFA) on Day 7 after model establishment showed that:

[0233] a. Compared with the negative control reagent group, the positive drug molecule group (positive control 1), rAAV-Eylea group (positive control 2), and rAAV-Fc-YTE group (experimental group) had significantly lower spot scores and spot areas.

[0234] b. Compared with the positive drug molecule group (positive control 1) and rAAV-Eylea group (positive control 2), the FFA spot area of the rAAV-Fc-YTE group (experimental group) was significantly lower.

[0235] 3.4) ELISA detection: On Day 14 after model establishment (42 days after injection), 3 eyeballs were randomly selected from each of the negative control reagent group, rAAV-Eylea group (positive control 2), and rAAV-Fc-YTE group (experimental group), and the expression distribution levels of drug protein molecules in each group were detected by ELISA method. The results are shown in Figure 5 and are summarized as follows.

[0236] Summary:

[0237] Both the rAAV-Eylea group (positive control 2) and the rAAV-Fc-YTE group (experimental group) had relatively high protein expression levels in the target tissue of mouse eyeballs, and the protein expression levels of the two were comparable.

[0238] 3.5) Statistical analysis:

[0239] Experimental data were expressed as mean ± standard error of the mean (mean ± S.E.M.). The data were analyzed using appropriate statistical methods with Graphpad Prism or SPSS. For FFA spot scores and spot area data, the Kruskal-Wallis test / Dunnett’s (for multiple groups comparison) and Mann-Whitney test (for two groups comparison) were used; for protein expression data, One-Way ANOVA was used for comparative analysis, and a P < 0.05 was considered statistically significant.

[0240] 3.6) Summary of conclusions:

[0241] A. In the implementation case, compared with the negative control group, both the experimental group and the two positive control groups showed significant differences and drug effects; the drug effects were consistent between positive control 1 (intravitreal injection of Eylea) and positive control 2 (subretinal injection of rAAV-Eylea), further indicating that the modeling experimental system in this study was successful.

[0242] B. In the experimental group of the embodiment of the present invention, the rAAV-Fc-YTE group (experimental group) showed no difference in the expression level of the drug protein in the target tissue compared with positive control 2 (subretinal injection of rAAV-Eylea), but showed significant and better drug effects compared with the two positive control groups.

[0243] Example 3. Experiment on the expression and distribution of drug molecules in target tissues

[0244] 1) Experimental design: The same test article as in Example 2 was used, and the same subretinal injection method and injection dose (1×10 9 vg / μL / eye, SRI, both eyes) were used to inject C57BL / 6J mice. The samples were taken on Day 33, and the mouse eye samples were paraffin-embedded. The BaseScope technology at the mRNA level was used to detect the expression and distribution of the target drug protein gene in each layer of retinal cells (since the target protein is a secreted protein, the BaseScope technology was used to detect the transcriptional level instead of using the IHC technology to detect the protein level).

[0245] Negative control: Negative control reagent (solvent reagent Buffer)

[0246] Positive control: rAAV-Eylea

[0247] Experimental group: rAAV-Fc-YTE (CRG-B191)

[0248] The representative results of BaseScope are as Figure 6 shown.

[0249] Conclusion: The results showed that both the positive control (rAAV-Eylea) and the experimental group (rAAV-Fc-YTE) had high expression in the target tissue compared with the negative control (negative control reagent). Moreover, the expression distributions of the positive control drug gene and the experimental group drug gene in the various retinal layers were the same: that is, they were expressed in the retinal RPE layer, IS / OS layer, and outer nuclear layer cells, with the highest expression level in the RPE cells.

[0250] The above results indicate that the modification of the drug molecule in the present invention can significantly improve the therapeutic effect on the target disease without changing the expression level and expression distribution of the drug molecule in the target tissue.

[0251] Example 4. Recombinant protein drug CNV modeling and FFA detection experiment

[0252] Modeling, FFA scoring, and detection were performed according to the method described in Example 2. The grouping and dosing regimens of the protein drugs are shown in the following table. The results showed that the efficacy of intravitreal IVT administration of the Fc-YTE-ranibizumab protein was not inferior to that of ranibizumab. Grouping and dosing regimen

[0253] The FFA spot score results (Mean±SEM) on Day 7 after modeling are as shown in the following table and Figure 7 shown. Group Test Substance Number of Eyes Spot Score on Day 7 after Modeling G1: Negative Control (PBS) n＝12 3.03±0.16 G2: Aflibercept n＝12 2.50±0.18 G3: Test Substance (Fc-YTE-Aflibercept) n＝12 2.69±0.18

[0254] The FFA spot area results (mm 2 , Mean±SEM) on Day 7 after modeling are as shown in the following table and Figure 8 shown. Test Substance Group Number of Eyes Spot Area on Day 7 after Modeling G1: Negative Control (PBS) n＝12 5.25±0.82 G2: Aflibercept n＝12 2.99±0.60 G3: Test Substance (Fc-YTE-Aflibercept) n＝12 2.94±0.55

Claims

1. A gene delivery vector comprising a nucleic acid comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises an immunoglobulin-like domain 2 of human VEGFR1, an immunoglobulin-like domain 3 of human VEGFR2, and a hinge-Fc domain of a human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

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