Gene delivery vector for delivering human VEGF receptor fusion protein, and use thereof

Abstract

The present invention provides a gene delivery vector for delivering a modified human VEGF receptor fusion protein, in particular a recombinant adeno-associated virus vector, as well the use thereof in treating a VEGF-induced disease. The present invention further provides said modified human VEGF receptor fusion protein, an encoding nucleic acid thereof, a vector, a cell, and an expression cassette.

Description

Gene delivery vector for delivering human VEGF receptor fusion protein and its application Technical Field

[0001] The present invention relates to a gene delivery vector for delivering a human VEGF receptor fusion protein, in particular a recombinant adeno-associated virus. The present invention also relates to the human VEGF receptor fusion protein, a nucleic acid encoding the fusion protein, a vector comprising the nucleic acid, and a host cell comprising 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 gene delivery vector, fusion protein, nucleic acid, vector, host cell, expression cassette, and recombinant plasmid in treating diseases caused by VEGF, in particular ophthalmic diseases, and methods of using the same to treat such diseases. Background Art

[0002] VEGF (vascular endothelial growth factor) plays a major role in the pathogenesis of various eye diseases. During onset, increased intraocular VEGF concentrations lead to abnormal, highly bleedable neovascularization, which can subsequently cause severe complications such as massive hemorrhage, fibrosis, tractional retinal detachment, and neovascular glaucoma. These diseases can also cause significant vascular leakage, leading to persistent and severe tissue edema. These eye diseases include: exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) combined with macular edema (DME), retinal static occlusion (RVO) combined with macular edema (ME), central exudative chorioretinopathy, polypoidal choroidal vasculopathy (PCV), choroidal neovascularization secondary to high myopia, and choroidal neovascularization (CNV) secondary to other diseases. Among these, wAMD and DME are relatively common and have a higher incidence.

[0003] Based on the mechanism of neutralizing VEGF, a number of antibody drugs for inhibiting angiogenesis in ophthalmology are currently on the market, such as Ranibizumab, Bevacizumab, Aflibercept, and Conbercept. 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, binding to VEGF-A, VEGF-B, and placental growth factor (PlGF), preventing them from binding to the receptors and blocking their downstream biological effects. Currently, this drug has been widely used in the clinical treatment of ophthalmic diseases. The FDA has approved the clinical use of Aflibercept in multiple indications, including wet AMD, RVO, DME, etc.

[0004] These drugs have been successful in treating ophthalmic diseases associated with angiogenesis. However, existing ophthalmic anti-VEGF therapies also have limitations. Generally, anti-VEGF drugs are currently the first-line treatment for wAMD and DME, with several anti-VEGF drugs, such as ranibizumab, conbercept, and aflibercept, widely approved for marketing. Currently, these first-line anti-VEGF drugs are all antibody-based macromolecules. These drugs have short metabolic cycles and half-lives, preventing long-term efficacy in the body and necessitating frequent intravitreal injections. For example, the recommended dosing regimen for aflibercept in neovascular AMD is monthly intravitreal injection (2 mg / dose) for the first three months, followed by an intravitreal injection every eight weeks (the "3+every-8-week regimen"). Alternatively, monthly intravitreal injection (2 mg / dose) for the first three months, followed by a treat and extend (T&E) regimen (the "3+T&E regimen"). Existing treatments increase the burden on patients due to their higher dosing frequency and also impose stricter requirements on medication compliance. Summary of the Invention

[0005] By using gene delivery vectors (such as adeno-associated viruses, adenoviruses, lentiviruses, lipid nanoparticles, etc.), genes encoding drug proteins (such as antibody drugs, fusion protein drugs, etc.) are delivered to the site of disease. Drug proteins can be directly and efficiently expressed in the body for a long time, and can exert long-term and stable therapeutic effects in the patient's body, reducing the frequency of dosing. In theory, it can even achieve long-term effective effects with a single dose, thereby effectively reducing the burden on patients.

[0006] The present invention performs site-directed mutagenesis of the human IgG1-Fc fragment of the aflibercept protein molecule using YTE (M252Y / S254T / T256E) to obtain a modified aflibercept protein drug molecule (abbreviated as "Fc-YTE-aflibercept" or "Fc-YTE-Aflibercept"). This gene therapy drug (abbreviated as "rAAV-Fc-YTE" or "rAAV-Fc-YTE-aflibercept") is then produced by encoding the gene for the Fc-YTE-aflibercept protein molecule via a gene delivery vector. The inventors surprisingly discovered that this gene therapy drug can significantly improve the therapeutic efficacy of the target disease without changing the expression level and distribution of the drug molecule in the target tissue.

[0007] Therefore, in one aspect, the present invention provides 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 the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2 and the 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.

[0008] In some embodiments, the human VEGF receptor fusion protein further comprises the immunoglobulin-like domain 4 of human VEGFR2.

[0009] In some embodiments, the hinge-Fc domain of the human immunoglobulin is the hinge-Fc domain of human IgG1.

[0010] In some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: an 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 as shown in SEQ ID NO.1 or 6.

[0011] 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.

[0012] 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% sequence identity with the amino acid sequence shown in SEQ ID NO.7.

[0013] In some embodiments, the hinge-Fc domain of the human immunoglobulin comprises or is: an 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 as shown in SEQ ID NO. 3 or 8.

[0014] 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 as shown in SEQ ID NO. 5 or 9.

[0015] In some embodiments, the expression cassette comprises: a nucleotide sequence as shown in SEQ ID NO. 10 or 11, or a degenerate sequence of either one of them.

[0016] In some embodiments, the expression cassette further comprises a promoter located upstream of the nucleotide sequence encoding the human VEGF receptor fusion protein and operably linked thereto, and the promoter is preferably selected from the group consisting of a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, an MMT promoter, an EF-1α promoter, a U6 promoter, a chicken β-actin promoter, a CAG promoter, a CBA promoter, an RPE65 promoter, a VMD2 promoter, a RPGR promoter, an IRBP promoter, a hGRK1 promoter, a CAR promoter, a RHO promoter, a Grm6 promoter, a GRK1 promoter, and a GFAP promoter.

[0017] 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 globulin polyA, bGH polyA and HSV TK polyA.

[0018] In some embodiments, the gene delivery vector is (a) a viral vector, preferably a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a poxvirus, a papovavirus, a baculovirus or a papillomavirus, more preferably an adeno-associated virus or a lentivirus; or (b) a non-viral vector, preferably a plasmid, a liposome, a nanoparticle, a polymer, a transposon, an exosome or a bacterial vector.

[0019] In some embodiments, the vector is an adeno-associated viral vector comprising: (a) a recombinant adeno-associated viral capsid, and (b) the nucleic acid packaged within the recombinant adeno-associated viral capsid.

[0020] 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-mentioned capsid proteins as the 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.

[0021] In some embodiments, the genome of the adeno-associated viral vector is in single-stranded or double-stranded form, preferably scAAV, ssAAV or cceAAV.

[0022] In some embodiments, a recombinant adeno-associated virus is provided, comprising: (a) an rAAV capsid; and (b) a nucleic acid packaged in the rAAV capsid, wherein the nucleic acid comprises, in 5' to 3' order: (i) a 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) a 3'AAV ITR.

[0023] In some embodiments, the rAAV capsid comprises the capsid protein of AAV1, AAV2, AAV5, AAV7, or AAV8, preferably the capsid protein of AAV8.

[0024] 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.

[0025] In some embodiments, the polyA is SV40 late polyA or rabbit globulin polyA, preferably SV40 late polyA.

[0026] In some embodiments, the 5' AAV ITR and / or 3' AAV ITR is selected from the group consisting of ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6.

[0027] In some embodiments, the present invention provides an expression cassette comprising, in 5' to 3' order: (i) a promoter; (ii) a polynucleotide encoding the human VEGF receptor fusion protein provided by the present invention; and (iii) polyA.

[0028] 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.

[0029] In some embodiments, the polyA is SV40 late polyA or rabbit globulin polyA, preferably SV40 late polyA.

[0030] In some embodiments, the expression cassette comprises a 5' AAV ITR at the 5' end of the promoter and a 3' AAV ITR at the 3' end of the polyA, respectively.

[0031] Another aspect of the present invention provides a human VEGF receptor fusion protein, which comprises the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2 and the 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.

[0032] In some embodiments, the human VEGF receptor fusion protein further comprises the 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.

[0033] 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.

[0034] In some embodiments, the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: an 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 as shown in SEQ ID NO.1 or 6.

[0035] 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.

[0036] 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 as shown in SEQ ID NO. 5 or 9.

[0037] 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 either of them.

[0038] In some embodiments, the present invention provides a vector, preferably a plasmid, comprising the polynucleotide; preferably, the vector is constructed to form ssAAV, scAAV or cceAAV.

[0039] In some embodiments, the present invention provides a host cell comprising the vector, preferably a HEK293 cell or an sf9 cell.

[0040] Other aspects and advantages of the present invention will become apparent from the following detailed description and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG1 shows a plasmid map of the master plasmid used to form the rAAV-Fc-YTE-aflibercept of the present invention according to one embodiment of the present invention.

[0042] FIG2 shows a schematic structural diagram of rAAV-Fc-YTE-aflibercept prepared according to one embodiment of the present invention.

[0043] FIG3 shows the FFA spot scores, *p<0.05, **p<0.01 indicate that there are significant differences among the rAAV-Eylea group, rAAV-Fc-YTE group, positive drug molecule group and negative control reagent group.

[0044] Figure 4 shows the FFA spot area. *p<0.05, **p<0.01 indicate that there are significant differences between the rAAV-Fc-YTE group and the rAAV-Eylea group and the positive drug molecule group; there is a significant difference between the negative control reagent group and the positive drug molecule group; there is no significant difference between the rAAV-Eylea group and the positive drug molecule group.

[0045] FIG5 shows the expression distribution levels of drug protein molecules in each group detected by ELISA method.

[0046] FIG6 shows the expression distribution of the target drug protein gene in cells of various retinal layers detected by BaseScope technology.

[0047] Figure 7 shows the comparison of FFA spot scores after intraocular administration of protein drugs IVT.

[0048] Figure 8 shows the comparison of FFA spot areas after intraocular administration of protein drugs IVT. DETAILED DESCRIPTION

[0049] definition

[0050] 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 comprises a recombinant viral genome having a heterologous polynucleotide encoding a human VEGF receptor fusion protein of the present invention and, optionally, a transcriptional regulatory region.

[0051] The terms "adeno-associated viral vector," "AAV vector," "adeno-associated virus," "AAV virus," "AAV viral particle," "AAV viral particle," and "AAV particle" are used interchangeably herein to 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 the recombinant viral genome, which has a heterologous polynucleotide (encoding the human VEGF receptor fusion protein of the present invention) and a transcriptional regulatory region, which contains at least a promoter. The transcriptional regulatory region may also contain polyA.

[0052] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which an exogenous nucleic acid and / or a recombinant vector has been introduced. It should be understood that "recombinant host cell" and "host cell" refer not only to the specific subject cell, but also to the progeny of such a cell. Due to mutations or environmental influences, certain modifications may occur in the progeny, and thus such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0053] The term "recombinant viral genome" refers to a viral genome or portion thereof into which at least one expression cassette is inserted. As used herein, the term "AAV recombinant viral genome" refers to an AAV genome into which at least one expression cassette polynucleotide is inserted. The minimal "genome" of an AAV genome according to the present invention typically includes cis-acting 5' and 3' inverted terminal repeats (ITRs) and an expression cassette.

[0054] As used herein, the term "expression cassette" refers to a nucleic acid construct recombinantly or synthetically produced with a series of specific nucleic acid elements that allows 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 present invention can include a transcriptional regulatory region operably linked to a region encoding the human VEGF receptor fusion protein of the present invention.

[0055] As used herein, the term "transcriptional regulatory region" refers to a nucleic acid fragment that can regulate the expression of one or more genes. The transcriptional regulatory region according to the present invention includes a promoter and an optional enhancer. The term "promoter" used herein is a nucleic acid fragment located upstream of a polynucleotide sequence, the function of which is to control the transcription of one or more polynucleotides. Any type of promoter can be used in the present invention, including inducible promoters, constitutive promoters, and tissue-specific promoters. The term "inducible promoter" used herein refers to a promoter that is physiologically or developmentally regulated, for example, by applying a chemical inducer. For example, it can be a tetracycline inducible promoter, a mifepristone (RU-486) ​​inducible promoter, etc. The term "constitutive promoter" used herein refers to a promoter whose activity remains at a relatively constant level in all cells of an organism or at most developmental stages, with little or no consideration of cellular environmental conditions. Examples of constitutive promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol 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 virus thymidine kinase promoter.

[0056] 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 include, but are not limited to, RSV enhancers, CMV enhancers, HCR enhancers, and the like.

[0057] As used herein, the term "operably linked" 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 a coding sequence). Typically, an operably linked promoter is adjacent to the sequence of interest. However, an enhancer need not be adjacent to the sequence of interest to control its expression. In another embodiment, the promoter is adjacent to the sequence encoding the human VEGF receptor fusion protein of the present invention.

[0058] The term "therapeutically effective amount" refers to a non-toxic but sufficient amount of a viral vector encoding a human VEGF receptor fusion protein of the present invention to provide a desired biological outcome. This outcome 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 a desired biological outcome.

[0059] The term "Cap protein" as used herein refers to a polypeptide having at least one functional activity of a natural AAV Cap protein (e.g., VP1, VP2, VP3). Examples of the functional activities of Cap proteins include the ability to induce capsid formation, promote single-stranded DNA accumulation, promote AAV DNA packaging into capsids, bind to cell receptors, and promote viral particle entry 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 the viral genome. The capsid is composed of several oligomeric structural subunits composed of proteins. For example, AAV has an icosahedral capsid formed by the interaction of three capsid proteins VP1, VP2, and VP3.

[0060] As used herein, the term "Rep protein" refers to a polypeptide having at least one functional activity of a native AAV Rep protein (e.g., Rep40, 52, 68, 78). The "functional activity" of a Rep protein refers to any activity associated with the physiological function of the protein. Other functions include regulating transcription from an AAV (or other heterologous) promoter and site-specific integration of AAV DNA into a host chromosome. In a specific embodiment, the AAV rep gene is derived from serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and hybrids thereof, as well as capsid protein mutants modified with the above-mentioned capsid proteins as the backbone.

[0061] As used herein, "viral proteins on which AAV replication depends" refer to polypeptides on which AAV replication depends (i.e., "accessory function polypeptides"). Accessory functions include those required for AAV replication, including but not limited to those involved in activating AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Virus-based accessory functions are derived from any known helper virus, such as adenovirus, herpes virus (except herpes simplex virus type 1), and vaccinia virus. Auxiliary functions include but are not limited to adenovirus E1, E2a, VA, and E4 or herpes virus UL5, ULB, UL52, and UL29, as well as herpes virus polymerase. In another embodiment, the viral protein on which AAV replication depends is derived from an adenovirus.

[0062] 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 DNA strand of the adeno-associated virus genome. ITR sequences are required for efficient propagation of the AAV genome. Another property of these sequences is their ability to form hairpins. This property contributes to their self-priming, thereby allowing independent synthesis of the second DNA strand. Procedures for modifying these ITR sequences are known in the art.

[0063] As used herein, the term "polyadenylation signal" or "polyA" refers to a nucleic acid sequence that mediates the attachment of a polyadenylation 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 globulin polyA, bGH polyA, and the HSV thymidine kinase (TK) polyA signal.

[0064] The term "nucleotide or nucleic acid sequence" is used interchangeably herein with "polynucleotide" and refers to any polymeric form of nucleotides of any length.

[0065] The term "signal peptide" as used herein refers to an amino acid residue sequence (ranging from 10 to 30 residues in length) that is bound 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 cleaved by a signal peptidase after endoplasmic reticulum transport.

[0066] As used herein, the term "subject" or "object" refers to an individual mammal, such as a human, non-human primate (e.g., chimpanzee and other ape and monkey species), farm animal (e.g., birds, fish, cattle, sheep, pigs, goats, and horses), domestic mammal (e.g., dogs and cats), or laboratory animal (e.g., rodents, such as mice, rats, and guinea pigs). The term includes subjects of any age or sex. In another embodiment, the subject is a mammal, preferably a human.

[0067] As used herein, the term "Fc" refers to human IgG (immunoglobulin) Fc domain. IgG subtypes such as IgG1, IgG2, IgG3 and IgG4 can all be used as Fc domains. As used herein, "Fc region" and "Fc domain" are portions of the IgG molecule associated with the crystallizable fragments obtained by papain 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 FcRn receptors). The Fc region comprises the entire second constant domain CH2 (according to the EU numbering system, hereinafter the same, residues 231-340 of human IgG1) and the entire third constant domain CH3 (residues 341-447). The term "hinge" comprises all or a fragment (e.g., 221-230) of the hinge region (residues 216-230) extending from the N-terminus of the Fc region. "IgG hinge-Fc region" or "hinge-Fc domain" refers to the region of an IgG molecule consisting of the Fc region (residues 231-447) and the hinge region or fragments thereof extending from the N-terminus of the Fc region.

[0068] As used herein, single-stranded AAV (ssAAV) refers to rAAV that has the coding sequence of the transgene expression cassette on a separate strand and is packaged into a viral capsid, which requires a process of conversion from single-stranded to double-stranded. The synthesis of the second strand of viral DNA has been shown to be the rate-limiting step for viral gene expression.

[0069] As used herein, self-complementary AAV (scAAV) is a virus that has a D sequence (packaging signal) deleted from the right ITR of the ssAAV genome and a terminal melting site mutation (Δtrs). This prevents Rep protein from modifying the melting site and enhancing the packaging of self-complementary double-stranded DNA. After entering cells, double-stranded AAV virus does not need to undergo a single-strand to double-strand conversion process and can be directly expressed at relatively high levels.

[0070] As used herein, covalently closed end AAV (cceAAV) is a recently emerged rAAV system that is formed by blocking one end of the complementary double-stranded DNA of the AAV genome by, for example, 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.

[0071] Gene delivery vectors

[0072] The first aspect of the present invention provides 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 the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2 and the 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.

[0073] In some embodiments, the human VEGF receptor fusion protein further comprises the immunoglobulin-like domain 4 of human VEGFR2. Therefore, in these embodiments, the present invention provides 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 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 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.

[0074] In some embodiments, the hinge-Fc domain of the human immunoglobulin is the hinge-Fc domain of human IgG1. Therefore, in these embodiments, the present invention provides 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 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 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 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, the immunoglobulin-like domain 4 of human VEGFR2, and the hinge-Fc domain of human IgG1; and the hinge-Fc domain of human IgG1 comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0075] 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 sequentially from 5' to 3'. 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 from 5' to 3', 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.

[0076] In any of the above embodiments, preferably, the domains are directly connected. In other embodiments, the domains can be connected via peptide linkers. Suitable peptide linkers are known in the art and are typically composed of multiple glycine and serine residues. The present invention contemplates that any suitable peptide linker can be used to connect the domains of the human VEGF receptor fusion protein.

[0077] In any of the above embodiments, the hinge-Fc domain of the human IgG1 may further comprise one or more of M428L and N434S substitutions, in addition to M252Y, S254T, and T256E substitutions according to EU numbering, which are known to enhance interaction with the FcRn receptor. For example, in some embodiments, the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions and M428L substitutions according to EU numbering. In other embodiments, the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions and N434S substitutions according to EU numbering. In other embodiments, the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions and M428L and N434S substitutions according to EU numbering. The present invention contemplates that the hinge-Fc domain of the human IgG1 may comprise more substitutions.

[0078] 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. Therefore, 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 set forth 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 to the amino acid sequence set forth 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 removed from the N-terminus and / or C-terminus of the amino acid sequence set forth 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 to the N-terminus of the amino acid sequence set forth in SEQ ID NO. 1, and the added amino acid residues may be the amino acid residues at the corresponding positions in 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 to the C-terminus of the amino acid sequence shown in SEQ ID NO. 6, and the added amino acid residues can be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 2 of wild-type human VEGFR1.

[0079] In any of the above embodiments, the immunoglobulin-like domain 3 of human VEGFR2 may comprise or be: the amino acid sequence set forth in SEQ ID NO. 2, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2. Therefore, in some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: the amino acid sequence set forth 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 to the amino acid sequence set forth in SEQ ID NO. 2. In some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 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 set forth in SEQ ID NO. 2. For example, in some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: 1 to 3 amino acid residues are added to the N-terminus of the amino acid sequence shown in SEQ ID NO. 2, and the added amino acid residues can be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 3 of wild-type human VEGFR2.

[0080] In any of the above embodiments, the immunoglobulin-like domain 4 of human VEGFR2 may comprise or be: the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 7. Therefore, in some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: the amino acid sequence set forth 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 to the amino acid sequence set forth in SEQ ID NO. 7. In some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 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 set forth in SEQ ID NO. 7. For example, in some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: 1 to 3 amino acid residues are added to the N-terminus of the amino acid sequence shown in SEQ ID NO.7, and the added amino acid residues can be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 4 of wild-type human VEGFR2.

[0081] Therefore, in some embodiments, the present invention provides 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, from N-terminus to 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 thereto, 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 thereto, 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.

[0082] In other embodiments, the present invention provides 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, from N-terminus to 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 thereto, 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 thereto, 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 thereto, 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.

[0083] 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 to the amino acid sequence as shown in SEQ ID NO. 3 or 8, and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering. Therefore, 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 to the amino acid sequence as shown in SEQ ID NO. 3, and the hinge-Fc domain of the human immunoglobulin comprises 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 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 M252Y, S254T and T256E substitutions according to EU numbering.

[0084] Therefore, in a preferred embodiment, the present invention provides 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, from N-terminus to 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity thereto; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0085] In a preferred embodiment, the present invention provides 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, from N-terminus to 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 thereto, 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity thereto; and the hinge-Fc domain of the human immunoglobulin comprises 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 comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises, from N-terminus to 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or having at least 80% sequence identity thereto; 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 comprising an expression cassette for expressing a human VEGF receptor fusion protein, wherein the human VEGF receptor fusion protein comprises, from N-terminus to 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 thereto, 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.8 or having at least 80% sequence identity thereto; 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 human VEGF receptor fusion protein further comprises a signal peptide at the N-terminus. An example of a signal peptide comprises or is the amino acid sequence shown in SEQ ID NO.4.

[0089] In a preferred embodiment, the present invention provides 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 the hinge-Fc domain of a human immunoglobulin, and the human VEGF receptor fusion protein comprises or is 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.

[0090] In a preferred embodiment, the present invention provides 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 the hinge-Fc domain of a human immunoglobulin, and the human VEGF receptor fusion protein comprises or is 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.

[0091] In a preferred embodiment, the present invention provides 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 the hinge-Fc domain of a human immunoglobulin, 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.

[0092] In a preferred embodiment, the present invention provides 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 the hinge-Fc domain of a human immunoglobulin, 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.

[0093] In a preferred embodiment, the present invention provides a gene delivery vector comprising a 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 either one of them.

[0094] In a preferred embodiment, the present invention provides a gene delivery vector comprising a 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 a codon degenerate sequence thereof.

[0095] In a preferred embodiment, the present invention provides a gene delivery vector comprising a 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 a codon degenerate sequence thereof.

[0096] In the present invention, a codon degenerate sequence refers to a variant of a reference sequence (such as the nucleotide sequence shown in SEQ ID NO. 10 or 11) obtained based on codon degeneracy. As is known in the art, the variant can be obtained by optimizing the reference sequence in one or more aspects including but not limited to codon frequency, mRNA secondary structure, GC content, RNase splice sites, and repetitive sequences, without changing the amino acid sequence obtained by translation based on the reference sequence.

[0097] 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 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 a CMV promoter.

[0098] 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 retinoschisis 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 a 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., a chicken β-actin promoter) comprises an enhancer sequence, such as a 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 (or vice versa).

[0099] 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 can be used to direct selective expression in ocular cells or ocular tissues. For example, a promoter, sequence element, or regulatory sequence found in a specific ocular cell type (such as retinal pigment epithelial cells) can be used in a suitable expression construct (e.g., RPE65 or VMD2 promoter). In some embodiments, the intron is a synthetic or artificial (e.g., heterologous) intron. Examples of synthetic introns include intron sequences derived from SV-40 (referred to as SV-40T intron sequences) and intron sequences derived from the chicken beta-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 can refer to any sequence that can be transcribed but not translated. In some cases, an intron can refer to any sequence that is transcribed in a cell and removed from a mature RNA transcript. In some cases, an intron can comprise approximately at least 100, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000 nucleotides. In some cases, an intron can be approximately 300 nucleotides. In some cases, an intron can be approximately 200 to 400 nucleotides. In some cases, a chimeric intron can be approximately 100 to 500 nucleotides. In some cases, an intron can be a complete naturally occurring intron or a chimeric intron. In some aspects, an intron can include but is not limited to the intron described in CN104994882A, and its disclosure is fully incorporated herein by reference.

[0100] In any of the above embodiments, the expression cassette for 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 globulin polyA, bGH polyA, and HSV TK polyA. In a more preferred embodiment, the polyadenylation signal is SV40 late polyA.

[0101] Therefore, in some embodiments, the present invention provides a gene delivery vector comprising a nucleic acid, wherein the nucleic acid comprises an expression cassette for expressing a human VEGF receptor fusion protein, wherein the expression cassette comprises, from 5' to 3' direction, a promoter, a nucleic acid encoding a human VEGF receptor fusion protein, and polyA, wherein the human VEGF receptor fusion protein comprises, from N-terminus to C-terminus, the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2, and the 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.

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

[0103] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, wherein the nucleic acid comprises an expression cassette for expressing a human VEGF receptor fusion protein, wherein the expression cassette comprises, in order from 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 a hinge-Fc domain of a 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 as 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.

[0104] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, wherein the nucleic acid comprises an expression cassette for expressing a human VEGF receptor fusion protein, wherein the expression cassette comprises, in order from 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 a hinge-Fc domain of a 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 as 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.

[0105] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, wherein the nucleic acid comprises an expression cassette for expressing a human VEGF receptor fusion protein, wherein the expression cassette comprises, from 5' to 3' direction, a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, and polyA, wherein the nucleotide sequence encoding the human VEGF receptor fusion protein is as shown in SEQ ID NO.10 or a codon degenerate sequence thereof.

[0106] In a preferred embodiment, the present invention provides a gene delivery vector comprising a nucleic acid, wherein the nucleic acid comprises an expression cassette for expressing a human VEGF receptor fusion protein, wherein the expression cassette comprises, from 5' to 3' direction, a promoter, a nucleic acid encoding the human VEGF receptor fusion protein, and polyA, wherein the nucleotide sequence encoding the human VEGF receptor fusion protein is as shown in SEQ ID NO.11 or its codon degenerate sequence.

[0107] The gene delivery vector that can be used for 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 a most preferred embodiment, the non-viral vector is a lipid nanoparticle (LNP).

[0108] In some embodiments, the gene delivery vector is a viral vector. In a preferred embodiment, the viral vector is a lentiviral, retroviral, adenoviral, adeno-associated virus, herpes virus, poxvirus, papovavirus, baculovirus or papillomavirus vector.

[0109] Retroviral vectors include Moloney murine leukemia virus and HIV-based viruses. In some cases, HIV-based viral vectors can be used, wherein 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 pox vectors such as orthopox or avipox vectors, herpes virus vectors such as herpes simplex virus 1 (HSV-1) vectors. HSV-1 vectors lacking one or more immediate early genes (IE) are advantageous because they are generally non-cytotoxic, persist in a state similar to latent in target cells, and provide effective target cell transduction. Recombinant HSV vectors can incorporate about 30kb 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 effective in transducing human epithelial cells. Lentiviral vectors for use in the present invention can be derived from human and non-human (including SIV) lentiviruses. Examples of lentiviral vectors include nucleic acid sequences required for vector propagation and tissue-specific promoters operably linked to the human VEGF receptor fusion protein gene. The nucleic acid sequence may include viral LTR, primer binding site, polypurine tract, att site and encapsidation site. Lentiviral vectors can be packaged into any suitable lentiviral shell. Poxvirus vectors can introduce genes into the cytoplasm of cells. Fowlpox virus vectors can only result in short-term expression of genes or nucleic acids. Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus (HSV) vectors can be used with the nucleic acid encoding the human VEGF receptor fusion protein disclosed herein. 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 a most preferred embodiment, the viral vector is an adeno-associated virus vector.

[0110] 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 a 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 in the recombinant adeno-associated virus capsid, wherein the nucleic acid comprises 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. The above description of the nucleic acid and expression cassette contained in the gene delivery vector is applicable to part (b) of rAAV and will not be repeated.

[0111] 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 in 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, from N-terminus to 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity thereto; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0112] 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 in 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, from N-terminus to 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 thereto, 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity thereto; 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 comprising: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged in 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, from N-terminus to 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or having at least 80% sequence identity thereto; 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 comprising: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged in 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, from N-terminus to 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 thereto, 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.8 or having at least 80% sequence identity thereto; 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 comprising: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged in 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 the hinge-Fc domain of a human immunoglobulin, the human VEGF receptor fusion protein comprising or being 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 as 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.

[0116] In a preferred embodiment, the present invention provides an adeno-associated viral vector comprising: (a) a recombinant adeno-associated viral capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated viral 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 a human immunoglobulin, 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.

[0117] In a preferred embodiment, the present invention provides an adeno-associated viral vector comprising: (a) a recombinant adeno-associated viral capsid, and (b) a nucleic acid packaged within the recombinant adeno-associated viral 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 a human immunoglobulin, 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.

[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 in the recombinant adeno-associated virus capsid, wherein the nucleic acid comprises 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 either one of them.

[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 in the recombinant adeno-associated virus capsid, wherein the nucleic acid comprises an expression cassette for expressing a human VEGF receptor fusion protein, and the expression cassette comprises, from 5' to 3' direction, 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, from N-terminus to C-terminus, the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2 and the 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.

[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 in the recombinant adeno-associated virus capsid, wherein the nucleic acid comprises an expression cassette for expressing a human VEGF receptor fusion protein, and the expression cassette comprises, from 5' to 3' direction, 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, from N-terminus to 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 a human immunoglobulin; and the hinge-Fc domain of the 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 comprising: (a) a recombinant adeno-associated virus capsid, and (b) a nucleic acid packaged in the recombinant adeno-associated virus capsid, wherein the nucleic acid comprises an expression cassette for expressing a human VEGF receptor fusion protein, and the expression cassette comprises, from 5' to 3' direction, a 5'AAV ITR, a promoter, a nucleic acid encoding a human VEGF receptor fusion protein, polyA and a 3'AAV ITR, wherein the human VEGF receptor fusion protein comprises a hinge-Fc domain of a 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 as 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.

[0122] 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 in the recombinant adeno-associated virus capsid, wherein the nucleic acid comprises an expression cassette for expressing a human VEGF receptor fusion protein, and the expression cassette comprises, from 5' to 3' direction, a 5'AAV ITR, a promoter, a nucleic acid encoding a human VEGF receptor fusion protein, polyA and a 3'AAV ITR, 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.

[0123] In any of the above embodiments of the adeno-associated virus vector, the recombinant adeno-associated virus capsid may comprise a capsid protein selected from the following serotypes of adeno-associated viruses: 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 the backbone. In a preferred embodiment, the recombinant adeno-associated virus capsid may comprise or be composed of a capsid protein selected from the group consisting of AAV1, AAV2, AAV5, AAV7, AAV8 and hybrids thereof. In a more preferred embodiment, the recombinant adeno-associated virus capsid may comprise or be composed of an AAV8 capsid protein.

[0124] Examples of genomic sequences of different AAV serotypes can be found in the literature or in public databases such as GenBank. For example, 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 viral vector according to the present invention comprises a capsid derived from a serotype selected from the group consisting of AAV2, AAV5, AAV7, AAV8, AAV9, AAV10, and AAVrh10 serotypes. In another embodiment, the serotype of AAV is AAV8. If the viral vector contains a sequence encoding a capsid protein, it can be modified to include exogenous sequences to direct the AAV to a specific cell type or cells, or to increase the efficiency of targeted vector delivery to cells, or to facilitate purification or detection of the AAV, or to reduce host responses.

[0125] In some embodiments, the AAV capsid protein has a tropism for ocular tissue or muscle tissue. In some embodiments, ocular tissue includes ocular neurons, retina, sclera, choroid, retina, vitreous, macula, fovea, optic disc, lens, pupil, iris, aqueous humor, cornea, conjunctiva ciliary body or optic nerve. In some embodiments, the AAV capsid protein targets ocular cell types (e.g., photoreceptor cells, retinal cells, etc.).

[0126] In any of the above embodiments of the adeno-associated viral vector, the genome of the adeno-associated viral vector is single-stranded or double-stranded, preferably any one of scAAV, ssAAV, or cceAAV. Those skilled in the art can select any one of these systems to form the adeno-associated viral vector of the present invention according to actual needs.

[0127] In some embodiments, the present invention provides a recombinant adeno-associated virus comprising: (a) an rAAV capsid; and (b) a nucleic acid packaged in the rAAV capsid, wherein the nucleic acid comprises 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) polyA; and (v) a 3'AAV ITR, wherein for the promoter, the nucleic acid encoding the human VEGF receptor fusion protein, and polyA, the above description of the corresponding components of the gene delivery vector is applicable and will not be repeated.

[0128] In a preferred embodiment, the rAAV capsid comprises or consists of a capsid protein of AAV1, AAV2, AAV5, AAV7 or AAV8, preferably a capsid protein of AAV8.

[0129] In a preferred embodiment, the 5' AAV ITR and / or 3' AAV ITR are selected from the group consisting of ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6.

[0130] In a specific embodiment, the present invention provides a recombinant adeno-associated virus comprising: (a) an rAAV8 capsid; and (b) a nucleic acid packaged in the rAAV8 capsid, wherein the nucleic acid comprises 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 an amino acid sequence as shown in SEQ ID NO. 5 or 9.

[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 in the rAAV8 capsid, wherein the nucleic acid comprises in 5' to 3' order: (i) 5'AAV ITR; (ii) a CMV promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) rabbit globin polyA; and (v) 3'AAV ITR, wherein the human VEGF receptor fusion protein comprises or is an 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 rAAV2 capsid; and (b) a nucleic acid packaged in the rAAV2 capsid, wherein the nucleic acid comprises 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 an 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) an rAAV2 capsid; and (b) a nucleic acid packaged in the rAAV2 capsid, wherein the nucleic acid comprises 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 an amino acid sequence as shown in SEQ ID NO. 5 or 9.

[0134] In a specific embodiment, the present invention provides a recombinant adeno-associated virus comprising: (a) an rAAV1 capsid; and (b) a nucleic acid packaged in the rAAV1 capsid, wherein the nucleic acid comprises in 5' to 3' order: (i) 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) 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) an rAAV1 capsid; and (b) a nucleic acid packaged within the rAAV1 capsid, wherein the nucleic acid comprises, 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) an SV40 late polyA; and (v) a 3'AAV ITR, wherein the human VEGF receptor fusion protein comprises or is an amino acid sequence as 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.

[0136] In some embodiments, the present invention provides an expression cassette comprising, in 5' to 3' order: (i) a promoter; (ii) a nucleic acid encoding a human VEGF receptor fusion protein; and (iii) polyA, wherein the description of the corresponding components of the gene delivery vector above is applicable to the promoter, the nucleic acid encoding a human VEGF receptor fusion protein, and polyA, and no further details are given.

[0137] In some embodiments, the present invention provides an expression cassette comprising, in 5' to 3' order: (i) 5'AAV ITR; (ii) a promoter; (iii) a nucleic acid encoding a human VEGF receptor fusion protein; (iv) polyA; and (v) 3'AAV ITR, wherein for the ITR, promoter, nucleic acid encoding a human VEGF receptor fusion protein, and polyA, the above description of the corresponding components of the gene delivery vector or recombinant adeno-associated virus is applicable and will not be repeated.

[0138] Recombinant AAV can be produced using a triple transfection (three-plasmid transfection) method. Typically, recombinant AAV is produced by transfecting host cells with an AAV vector (comprising a transgene flanked by ITR elements), an AAV helper function vector, and an accessory function vector to be packaged into AAV particles. 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 encapsulation. The accessory function vector encodes nucleotide sequences for non-AAV-derived viral and / or cellular functions, and AAV relies on these functions for replication (e.g., "accessory functions"). Accessory functions include those 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, cap expression product synthesis, and AAV capsid assembly. 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.

[0139] Encoding human VEGF receptor fusion protein and its encoding nucleic acid

[0140] Another aspect of the present invention provides a human VEGF receptor fusion protein, which comprises the immunoglobulin-like domain 2 of human VEGFR1, the immunoglobulin-like domain 3 of human VEGFR2 and the 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.

[0141] In some embodiments, the human VEGF receptor fusion protein further comprises the immunoglobulin-like domain 4 of human VEGFR2. Therefore, 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 a human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering.

[0142] In some embodiments, the hinge-Fc domain of the human immunoglobulin is the hinge-Fc domain of human IgG1. Therefore, 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 human IgG1 comprises substitutions M252Y, S254T, and T256E 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 human IgG1 comprises substitutions M252Y, S254T, and T256E according to EU numbering.

[0143] 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 N-terminus to 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 N-terminus to C-terminus, and the hinge-Fc domain of the human immunoglobulin is located at the most C-terminus.

[0144] In any of the above embodiments, preferably, the domains are directly connected. In other embodiments, the domains can be connected via peptide linkers. Suitable peptide linkers are known in the art and are typically composed of multiple glycine and serine residues. The present invention contemplates that any suitable peptide linker can be used to connect the domains of the human VEGF receptor fusion protein.

[0145] In any of the above embodiments, the hinge-Fc domain of the human IgG1 may further comprise one or more of M428L and N434S substitutions, in addition to M252Y, S254T, and T256E substitutions according to EU numbering, which are known to enhance interaction with the FcRn receptor. For example, in some embodiments, the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions and M428L substitutions according to EU numbering. In other embodiments, the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions and N434S substitutions according to EU numbering. In other embodiments, the hinge-Fc domain of the human IgG1 comprises M252Y, S254T, and T256E substitutions and M428L and N434S substitutions according to EU numbering. The present invention contemplates that the hinge-Fc domain of the human IgG1 may comprise more substitutions.

[0146] 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. Therefore, 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 set forth 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 to the amino acid sequence set forth 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 removed from the N-terminus and / or C-terminus of the amino acid sequence set forth 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 to the N-terminus of the amino acid sequence set forth in SEQ ID NO. 1, and the added amino acid residues may be the amino acid residues at the corresponding positions in 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 to the C-terminus of the amino acid sequence shown in SEQ ID NO. 6, and the added amino acid residues can be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 2 of wild-type human VEGFR1.

[0147] In any of the above embodiments, the immunoglobulin-like domain 3 of human VEGFR2 may comprise or be: the amino acid sequence set forth in SEQ ID NO. 2, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 2. Therefore, in some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: the amino acid sequence set forth 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 to the amino acid sequence set forth in SEQ ID NO. 2. In some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 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 set forth in SEQ ID NO. 2. For example, in some embodiments, the immunoglobulin-like domain 3 of human VEGFR2 comprises or is: 1 to 3 amino acid residues are added to the N-terminus of the amino acid sequence shown in SEQ ID NO. 2, and the added amino acid residues can be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 3 of wild-type human VEGFR2.

[0148] In any of the above embodiments, the immunoglobulin-like domain 4 of human VEGFR2 may comprise or be: the amino acid sequence set forth in SEQ ID NO. 7, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO. 7. Therefore, in some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: the amino acid sequence set forth 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 to the amino acid sequence set forth in SEQ ID NO. 7. In some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 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 set forth in SEQ ID NO. 7. For example, in some embodiments, the immunoglobulin-like domain 4 of human VEGFR2 comprises or is: 1 to 3 amino acid residues are added to the N-terminus of the amino acid sequence shown in SEQ ID NO.7, and the added amino acid residues can be the amino acid residues at the corresponding positions of the immunoglobulin-like domain 4 of wild-type human VEGFR2.

[0149] Therefore, in some embodiments, the present invention provides a human VEGF receptor fusion protein, which comprises, from N-terminus to 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 thereto, 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 thereto, 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.

[0150] In other embodiments, the present invention provides a human VEGF receptor fusion protein, which comprises, from N-terminus to 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 thereto, 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 thereto, 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 thereto, 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.

[0151] 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 to the amino acid sequence as shown in SEQ ID NO. 3 or 8, and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T, and T256E substitutions according to EU numbering. Therefore, 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 to the amino acid sequence as shown in SEQ ID NO. 3, and the hinge-Fc domain of the human immunoglobulin comprises 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 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 M252Y, S254T and T256E substitutions according to EU numbering.

[0152] Therefore, in a preferred embodiment, the present invention provides a human VEGF receptor fusion protein, which comprises, from N-terminus to 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.3 or 8 or having at least 80% sequence identity thereto; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

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

[0155] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein, which comprises, from N-terminus to 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 thereto, 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 thereto, 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 thereto, and a hinge-Fc domain of a human immunoglobulin having an amino acid sequence as shown in SEQ ID NO.8 or having at least 80% sequence identity thereto; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

[0156] In a preferred embodiment, the human VEGF receptor fusion protein further comprises a signal peptide at the N-terminus. An example of a signal peptide comprises or is the amino acid sequence shown in SEQ ID NO.4.

[0157] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein comprising a hinge-Fc domain of a human immunoglobulin, wherein 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 as 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.

[0158] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein comprising a hinge-Fc domain of a human immunoglobulin, wherein 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 as 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.

[0159] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein comprising a hinge-Fc domain of a human immunoglobulin, wherein 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.

[0160] In a preferred embodiment, the present invention provides a human VEGF receptor fusion protein comprising a hinge-Fc domain of a human immunoglobulin, wherein 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.

[0161] In some embodiments, the present invention provides a polynucleotide, such as a DNA or RNA, encoding the human VEGF receptor fusion protein according to any of the above embodiments. 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 either of these. In a preferred embodiment, the polynucleotide is the nucleotide sequence shown in SEQ ID NO. 10 or 11.

[0162] 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 described in any one of the above expression cassette embodiments. In some aspects, an antibiotic resistance gene is introduced into the plasmid. Antibiotic resistance markers can be used to identify positive transgenic cells in the generation of recombinant viruses. In some aspects, antibiotic markers comprise sequences encoding antibiotic resistance genes. For example, markers that confer resistance may include, but are not limited to, kanamycin, gentamicin, 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 to form ssAAV, scAAV or cceAAV. In one embodiment, the plasmid is, for example, the plasmid shown in Figure 1, which is used to form cceAAV.

[0163] In some embodiments, the present invention provides a host cell comprising a vector comprising the polynucleotide, wherein the host cell is non-human, for example, HEK293 cells. Other cells are also feasible, for example, CHO cells. In some embodiments, the cell is transfected with the vector comprising the polynucleotide. In addition, the cell is also transfected with a helper plasmid and an accessory plasmid, for example, as described above, wherein the helper plasmid provides, for example, the rep and cap genes of AAV, and the accessory plasmid provides, for example, genes such as E2, E4a, and / or VA for AAV replication.

[0164] Pharmaceutical compositions, therapies, and indications

[0165] Another aspect of the present invention provides a pharmaceutical composition comprising any one of the gene delivery vectors described in the present invention (such as rAAV) and pharmaceutically acceptable excipients.

[0166] Another aspect of the present invention provides a pharmaceutical composition comprising any one of the human VEGF receptor fusion proteins provided by the present invention; and pharmaceutically acceptable excipients.

[0167] In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly in the presence of high rAAV concentrations (e.g., ˜10 13 GC / mL or higher). Methods for reducing rAAV aggregation are well known in the art and include, for example, the addition of surfactants, pH adjustment, salt concentration adjustment, etc. The preparation of pharmaceutically acceptable excipient and adjuvant solutions, as well as the development of appropriate dosing and treatment regimens for use of the specific compositions described herein in a variety of treatment regimens, are well known to those skilled in the art.

[0168] In some cases, it is desirable to deliver the rAAV-based therapeutic constructs disclosed herein in a suitably formulated pharmaceutical composition by one of intravitreal, intraocular, subretinal, subcutaneous, intrapancreatic, intranasal, parenteral, intravenous, intramuscular, intrathecal, oral, intraperitoneal, or inhalation. In some embodiments, the preferred mode of administration is by intravenous injection.

[0169] Suitable pharmaceutical forms 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 glycol, and mixtures thereof, and in oils. Under general storage and use conditions, these preparations contain preservatives to prevent microbial growth. In many cases, the form is sterile, and the fluidity reaches a level that allows easy injection. It must remain stable under manufacturing and storage conditions, and its preservation must prevent the contamination of microorganisms such as bacteria and fungi. Excipients can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Suitable fluidity can be maintained, for example, by using a coating agent such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. The effects of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0170] For example, for the administration of injectable aqueous solutions, the solution can be suitably buffered, if desired, and the liquid diluent can first be made isotonic with enough saline or glucose. These specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this respect, usable sterile aqueous media are well known to those skilled in the art.

[0171] Sterile injectable solutions are prepared by incorporating the desired amount of active rAAV into a suitable solvent with the various other ingredients listed herein (as needed) and then sterilizing by filtration. Typically, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from those listed 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 produce a powder of the active ingredient plus any other desired ingredients from a previously sterile-filtered solution thereof.

[0172] The rAAV compositions disclosed herein can also be formulated into 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. The salts formed with the 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 preparation is easy to administer in a variety of dosage forms, such as injectable solutions, drug release capsules, etc.

[0173] As used herein, "excipient" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients may also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a host.

[0174] 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 in lipid particles, liposomes, vesicles, nanospheres or nanoparticles, etc. for delivery. Such formulations can be preferably used to introduce pharmaceutically acceptable formulations of 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 circulation half-life have been developed.

[0175] In some cases, targeting ocular (e.g., corneal) tissue by intrastromal 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 intrastromal injection (IS). In some embodiments, the injection is topical administration (e.g., topical administration to the eye). In some cases, multiple doses of rAAV are administered.

[0176] In some embodiments, the administration of rAAV as described herein results in the delivery of transgene (e.g., KH902) to eye tissue. rAAV can be delivered to the eye tissue of a mammalian subject by, for example, intraocular injection, subretinal injection, topical application (e.g., eye drops) or by injection into the eyes of a mammalian subject (e.g., intravitreal injection). As used herein, "eye tissue" refers to any tissue derived from or contained in an eye. Non-limiting examples of eye tissue include neurons, retina (e.g., photoreceptor cells), sclera, choroid, retina, vitreous body, macula, central fovea, optic disc, lens, pupil, iris, aqueous humor, cornea (e.g., keratinocytes, corneal endothelial cells, corneal basal cells, corneal wing cells, and corneal squamous cells), conjunctival ciliary body, and optic nerve. The retina is located at the back of the eye and includes photoreceptor cells. These photoreceptor cells (e.g., rods, cones) impart visual acuity and contrast in the visual field by distinguishing colors.

[0177] Alternatively, rAAV can be delivered to a mammalian subject by intramuscular injection or by administration into the bloodstream of a mammalian subject. Administration into the bloodstream can be by injection into a vein, artery, or any other vascular conduit. Non-limiting exemplary methods of intramuscular administration of rAAV include intramuscular (IM) injection and intravascular infusion. In some embodiments, rAAV or compositions as described in the present disclosure are administered by intravitreal injection. In some embodiments, rAAV or compositions as described in the present disclosure are administered by intraocular injection. In some embodiments, rAAV or compositions as described in the present disclosure are administered by subretinal injection. In some embodiments, rAAV or compositions as described in the present disclosure are administered by intravenous injection. In some embodiments, rAAV or compositions as described in the present disclosure are administered by intramuscular injection.

[0178] 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 extent 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, VEGF (e.g., VEGF activity) activity in a subject that has received an anti-VEGF agent (e.g., injected with a rAAV 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, 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 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 121%, 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).

[0179] In some embodiments, administration of an anti-VEGF agent (e.g., a rAAV 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 months and 3 months, between 2 months and 4 months, between 2 months and 5 months, between 2 months and 6 months, between 2 months and 7 months, between 2 months and 8 months, between 2 months and 9 months, between 2 months and 10 months, between 2 months and 11 months, between 2 months and 12 months), longer than 3 months (e.g., between 3 months and 4 months, between 3 months and 5 months, between 3 months and 6 months, between 3 months and 7 months, between 3 months and 8 months, between 3 months and 9 months, between 3 months and 10 months, between 3 months and 11 months), between 4 months and 5 months, between 4 months and 6 months, between 4 months and 7 months, between 4 months and 8 months, between 4 months and 9 months, between 4 months and 10 months, between 4 months and 11 months, between 4 months and 12 months), longer than 5 months (e.g., between 5 months and 6 months, between 5 months and 7 months, between 5 months and 8 months, between 5 months and 8 months, between 5 months and 9 months, between 5 months and 10 months, between 5 months and 11 months, between 5 months and 12 months) , longer than 6 months (e.g., between 6 months and 7 months, between 6 months and 8 months, between 6 months and 9 months, between 6 months and 10 months, between 6 months and 11 months, between 6 months and 12 months), longer than 7 months (e.g., between 7 months and 8 months, between 7 months and 9 months, between 7 months and 10 months, between 7 months and 11 months, between 7 months and 12 months), longer than 8 months (e.g., between 8 months and 9 months, between 8 months and 10 months, between 8 months and 11 months, between 8 months and 12 months), longer than 9 months (e.g., 9 12 months, longer than 1 year (e.g., 1 to 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 than 20 years.

[0180] The compositions of the present disclosure can comprise rAAV alone, or in combination with one or more other viruses (e.g., encoding a second rAAV with 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 with one or more different transgenes.

[0181] An effective amount of rAAV or composition is an amount sufficient to target an animal for infection, targeting a target tissue (e.g., muscle tissue, eye tissue, etc.). In some embodiments, the effective amount will depend primarily on factors such as species, age, weight, health of the subject, and the tissue to be targeted, and thus may 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 genome copies (e.g., 1 x 10 6 to 1x 10 16 In some embodiments, the effective amount of rAAV is in the range of 1×10 9 to 1x10 14 In some cases, about 10 11 to 10 12 A dosage of between 10 and 100 rAAV genome copies is suitable. 11 to 10 13 A dosage of between 10 and 100 rAAV genome copies is suitable. 11 to 10 14 A dosage of between 10 and 100 rAAV genome copies is suitable. 11 to 10 15 A dosage of between 10 and 100 rAAV genome copies is suitable. 12 to 10 14 A dosage of about 10 rAAV genome copies is suitable. 13 to 10 14 In some embodiments, about 1 x 10 rAAV genome copies are suitable. 12 , about 1.1x 10 12 , about 1.2x10 12 , about 1.3x 10 12 , about 1.4x 10 12 , about 1.5x10 12 , about 1.6x 10 12 , about 1.7x 10 12 , about 1.8x 10 12 , about 1.9x 10 12 , about 1x 10 13 , about 1.1x10 13 , about 1.2x 10 13 , about 1.3x 10 13 , about 1.4x 10 13 , about 1.5x 1013 , about 1.6x 10 13 , about 1.7x 10 13 , about 1.8x 10 13 , about 1.9x 10 13 or approximately 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 Up to 2x 10 13 In some embodiments, a dose of about 1.5 x 10 rAAV genome copies is appropriate. 13 In some embodiments, 10 12 -10 13 In certain embodiments, 10 rAAV genome copies are effective for target tissues (e.g., eye). 13 -10 14 rAAV genome copies are effective against target tissues (e.g., the eye).

[0182] In some embodiments, the rAAV is injected into the subject. In other embodiments, the rAAV is administered to the subject by topical administration (e.g., eye drops). In some embodiments, the effective amount of rAAV is an amount sufficient to express an effective amount of the human VEGF receptor fusion protein in the subject's target tissue (e.g., eye).

[0183] In some embodiments, the effective amount of rAAV delivered by injection (e.g., delivering a rAAV encoding a human VEGF receptor fusion protein) is an amount sufficient to express an effective amount of the human VEGF receptor fusion protein in the target tissue. In some embodiments, the effective amount of rAAV encoding a human VEGF receptor fusion protein delivered is sufficient to deliver 10 μg to 10 mg, or any intermediate amount therebetween, of the human VEGF receptor fusion protein to a subject per eye via a suitable route of administration (e.g., intraocular injection, iv injection, intraperitoneal injection, and intramuscular injection). In some embodiments, the rAAV encoding a human VEGF receptor fusion protein is sufficient to deliver 20 μg to 5 mg, or any intermediate amount therebetween, of the human VEGF receptor fusion protein to a subject per eye. Fusion Protein. In some embodiments, the rAAV encoding the 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 per eye to a subject.

[0184] In some embodiments, rAAV encoding a human VEGF receptor fusion protein is administered to a subject daily, weekly, biweekly, monthly, every 2 months, every 3 months, every 6 months, annually, or throughout the subject's lifetime.

[0185] 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 human VEGF receptor fusion protein in the target tissue. In some embodiments, eye drops containing rAAV encoding human VEGF receptor fusion protein are administered to the subject once a week, once a month, once every three months, once every six months, or once a year.

[0186] In some embodiments, the eye drops contain enough rAAV encoding human VEGF receptor fusion protein to deliver a concentration of 1 mg / ml to 20 mg / ml of human VEGF receptor fusion protein. In some embodiments, the eye drops contain enough rAAV encoding human VEGF receptor fusion protein to deliver a concentration of 2.5 mg / ml to 10 mg / ml of human VEGF receptor fusion protein. In some embodiments, the eye drops contain enough rAAV encoding human VEGF receptor fusion protein to deliver 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 of human VEGF receptor fusion protein. In some embodiments, the eye drops are administered in 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.

[0187] In some embodiments, the methods described herein further include a step of inducing immunosuppression in the subject (e.g., administering one or more immunosuppressants) before the subject is administered rAAV (e.g., rAAV or pharmaceutical composition described herein). In some embodiments, the subject is immunosuppressed (e.g., induced immunosuppression in the subject) between about 30 days and about 0 days prior to administration of rAAV to the subject (e.g., any time within 30 days prior to administration of rAAV, including the endpoint). In some embodiments, the subject is pretreated with an immunosuppressant (e.g., rituximab, sirolimus, and / or prednisone) for at least 7 days.

[0188] In some embodiments, the method described in the present disclosure further includes co-administering or pre-administering a medicament to a subject to whom the rAAV of the present disclosure or a pharmaceutical composition comprising rAAV is administered. In some embodiments, the medicament is selected from miglustat, Keppra, lansoprazole, clonazepam, and any combination thereof. In some embodiments, rAAV and additional medicaments can be delivered to the subject in any order. In some embodiments, rAAV and additional medicaments (e.g., miglustat, Keppra, lansoprazole, clonazepam) are delivered to the subject simultaneously. In some embodiments, rAAV and additional medicaments (e.g., miglustat, Keppra, lansoprazole, clonazepam) are co-administered to the subject (e.g., in a composition or in different compositions). In some embodiments, rAAV is delivered before additional medicaments (e.g., miglustat, Keppra, lansoprazole, clonazepam). In some embodiments, rAAV is delivered after an additional agent (e.g., miglustat, Keppra, lansoprazole, clonazepam). In some embodiments, rAAV and an additional agent (e.g., miglustat, Keppra, lansoprazole, clonazepam) are delivered to a subject at different frequencies, for example, a subject receives rAAV every month, every two months, every six months, every year, every two years, every three years, every five years, or longer, but receives an additional agent (e.g., miglustat, Keppra, lansoprazole, clonazepam) every day, every week, every two weeks, every month, twice a day, three times a day, or twice a week.

[0189] In some embodiments, the subject is immunosuppressed during and / or after administration of the rAAV or pharmaceutical composition. In some embodiments, the subject is immunosuppressed (e.g., administered one or more immunosuppressants) for a period of between 1 day and 1 year after administration of the rAAV or pharmaceutical composition.

[0190] Another aspect of the present invention provides a method for treating a VEGF-induced disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a rAAV or pharmaceutical composition of the present invention. In a preferred embodiment, the VEGF-induced disease is a disease caused by VEGF overexpression.

[0191] In a preferred embodiment, the disease caused by VEGF is selected from exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) combined with macular edema (DME), retinal static occlusion (RVO) combined 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 disease caused by VEGF is selected from exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) combined with macular edema (DME), or retinal static occlusion (RVO) combined with macular edema (ME).

[0192] Accordingly, the present invention provides the use of the gene delivery vector or pharmaceutical composition in the preparation of a medicament for treating a disease caused by VEGF. In a preferred embodiment, the disease caused by VEGF is selected from exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) combined with macular edema (DME), retinal static occlusion (RVO) combined 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 disease caused by VEGF is selected from exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) combined with macular edema (DME), or retinal static occlusion (RVO) combined with macular edema (ME).

[0193] Sequence Listing Example

[0194] Example 1. Preparation of rAAV-Fc-YTE-Aflibercept

[0195] The obtained Fc-YTE-aflibercept gene sequence (SEQ ID NO. 10) was loaded into the AAV master plasmid (GOI plasmid) (see Figure 1 for the plasmid map), 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 (e.g., Helper plasmid, R / C plasmid). In this embodiment, the master plasmid backbone adopts the cceAAV genome configuration, the promoter element adopts the CMV promoter sequence, the PolyA element adopts the SV40 late PolyA sequence, and the viral capsid adopts the AAV8 serotype.

[0196] AAV virus production was achieved by transient transfection of HEK293 cells using a three-plasmid system. The three plasmids and their functions are described below.

[0197] Main plasmid (GOI plasmid): As mentioned above, the vector genomic DNA containing the Fc-YTE-aflibercept gene sequence expression cassette can express the Fc-YTE-aflibercept protein molecule (amino acid sequence as shown in SEQ ID NO. 5) in the target tissue in vivo after administration.

[0198] R / C plasmid (helper plasmid): provides the Rep and Cap proteins in rAAV in trans, and separates the main plasmid genes into two independent plasmid systems, which will allow Rep and Cap to express structures only in rAAV, while deleting the rAAV replication function.

[0199] Helper plasmids (accessory plasmids): Containing gene segments essential for initiating AAV replication, such as E2, E4a, and VA, these plasmids are co-transfected with the GOI main plasmid and R / C plasmid into HEK293 cells. Together, these plasmids package and produce the rAAV-Fc-YTE-aflibercept virus, the structure of which is shown in Figure 2. Subsequently, relevant quality tests are performed, primarily including viral genome titer (Vg), capsid protein purity, viral particle count (Vp), residual host cell DNA (HCD), residual host cell protein (HCP), and bacterial endotoxins, to confirm that all meet the relevant requirements.

[0200] Example 2. rAAV-Fc-YTE-Aflibercept significantly reduces FFA spot score and area

[0201] The laser-induced CNV model of C57BL / 6J mice (male, about 6 weeks) was used to verify, evaluate, and compare the efficacy of the rAAV-Fc-YTE-aflibercept provided in Example 1.

[0202] 1) Grouping and dosing regimen

[0203] Negative control: negative control reagent (solvent reagent Buffer)

[0204] Positive control 1: Positive drug molecule (purchased clinically used aflibercept protein injection)

[0205] Positive control 2: rAAV-Eylea

[0206] Experimental group: rAAV-Fc-YTE

[0207] Compared to the rAAV-Eylea group and the rAAV-Fc-YTE group, the rAAV-Eylea (trade name for aflibercept) IgG1 Fc fragment in the rAAV-Eylea group was wild-type Fc, without YTE site-directed mutagenesis optimization. Both groups used the same master plasmid expression framework and regulatory elements and were prepared according to the method described in Example 1.

[0208] Grouping and dosing regimen

[0209] Note: The model was established 4 weeks after drug administration, and the day of model establishment was recorded as Day 0.

[0210] On Day 28, animals were anesthetized with Zotai (25-50 mg / kg, ip) plus xylazine hydrochloride injection (5 mg / kg, ip) before administration. Animals in groups G1 to G3 received a single subretinal injection of varying doses of the test substance and buffer at 1 μL / eye. Animals in group G4 received a 0.5 μL / eye intravitreal injection of the positive drug on Day 3 after modeling.

[0211] Subretinal Injection: Disinfect the ocular surface with iodine. Under an ophthalmic surgical microscope, puncture the mouse sclera just medial to the limbus with a 30G disposable needle. A microinjector with a 35G flat needle is then inserted through the puncture, bypassing the lens and reaching the vitreous humor, avoiding major blood vessels. The needle is then gradually advanced to the subretinal space and injected slowly. Immediately after withdrawing the syringe, apply pressure to the injection site with a cotton swab for 5 seconds. Immediately after administration, optical coherence tomography (OCT) examination confirms successful injection. Successful administration is determined by a noticeable retinal bulge observed under OCT.

[0212] Post-drug care: After drug administration, both eyes of all 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.

[0213] 2) Modeling:

[0214] On Day 0 (4 weeks after administration), animals were anesthetized with Zota (25-50 mg / kg, ip) and xylazine hydrochloride injection (5 mg / kg, ip). A YAG laser photocoagulator (VITRA, Quantel Medical) was used to burn three laser spots on the RPE / Bruch's membrane of both eyes with a 532 nm laser of the same energy parameters (three laser spots were evenly distributed around the optic disc at a distance of 1-1.5 PD from the optic disc). The laser spot should be located away from the large retinal blood vessels and the injection site to prevent intraocular hemorrhage.

[0215] Laser success sign: OCT detection of the laser spot position immediately after laser treatment to confirm whether the laser burning and visible rupture of Bruch's membrane are successful.

[0216] 3) Main test indicators and test results:

[0217] 3.1) Clinical Observation

[0218] Observe the animals once a day in their cages to see if they are dead, their mental state, and their behavioral activities.

[0219] Results: The animals were observed in their cages once a day, and no obvious abnormal clinical symptoms were observed.

[0220] 3.2)SD-OCT:

[0221] OCT was performed using ultra-high-resolution spectral-domain optical coherence tomography (SD-OCT). Mice were anesthetized, 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 of 5 frames) and full-field volume scans (300 frames) were captured.

[0222] SD-OCT scans were performed on both eyes of the animals 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).

[0223] Conclusion: No abnormal clinical symptoms were observed in the enrolled animals throughout the experiment. OCT scans showed that immediately after drug administration on Day 28 (rAAV-Eylea, rAAV-Fc-YTE, and negative control groups), retinal bulges were observed in all animals, indicating successful subretinal injection. Immediately after modeling on Day 0 (rAAV-Eylea, rAAV-Fc-YTE, negative control, and positive drug groups), laser burns and Bruch's membrane rupture were observed in all animals, indicating successful laser modeling.

[0224] 3.3) FFA spot score and spot area:

[0225] On Day 7 after modeling, all animals underwent FFA testing. Animals were anesthetized with Zota (25-50 mg / kg, ip) and xylazine hydrochloride injection (5 mg / kg, ip). Continuous angiographic images were captured after sodium fluorescein injection. Leakage scores were recorded early and late after sodium fluorescein injection. Leakage grades were I-IV, as shown in the table below.

[0226] FFA Grading Details Table

[0227] result:

[0228] On Day 7 after modeling, all animals underwent FFA testing. The FFA scores and area measurements are shown in Figures 3 and 4, respectively, and are summarized below. FFA spot area was used to assess drug efficacy. The smaller the spot area, the better the drug efficacy.

[0229] FFA spot score (Mean±SEM)

[0230] FFA spot area (mm 2 , Mean ± SEM)

[0231] Summary: The results of fundus fluorescein angiography (FFA) on Day 7 after modeling showed:

[0232] a, Compared with the negative control reagent group, the spot score and spot area of ​​the positive drug molecule group (positive control 1), rAAV-Eylea group (positive control 2), and rAAV-Fc-YTE group (experimental group) were significantly reduced.

[0233] b, The FFA spot area of ​​the rAAV-Fc-YTE group (experimental group) was significantly reduced compared with the positive drug molecule group (positive control 1) and the rAAV-Eylea group (positive control 2).

[0234] 3.4) Elisa Assay: On Day 14 after model establishment (42 days after injection), three eyes were randomly sampled from each of the negative control group, the rAAV-Eylea group (positive control 2), and the rAAV-Fc-YTE group (experimental group). Elisa assays were used to assess the expression and distribution of the drug protein in each group. The results are shown in Figure 5 and summarized below.

[0235] summary:

[0236] Both the rAAV-Eylea group (positive control 2) and the rAAV-Fc-YTE group (experimental group) had high protein expression levels in the target tissue of mouse eyeballs, and the protein expression levels of the two groups were comparable.

[0237] 3.5) Statistical analysis

[0238] Experimental data are expressed as mean ± standard error (SEM). Data were analyzed using Graphpad Prism or SPSS statistical methods. FFA spot scores and spot area data were analyzed using the Kruskal-Wallis / Dunnett's test (for multiple group comparisons) and the Mann-Whitney test (for two-group comparisons). Protein expression data were analyzed using one-way ANOVA. P < 0.05 was considered significant.

[0239] 3.6) Conclusions

[0240] A. In the implementation case, both the experimental group and the two positive control groups showed significant differences and efficacy compared with the negative control group; the efficacy between positive control 1 (vitreal injection of Eylea) and positive control 2 (subretinal injection of rAAV-Eylea) was consistent, further demonstrating the success of this modeling experimental system.

[0241] B. There was no difference in the expression level of drug protein in the target tissue between the experimental group drug rAAV-Fc-YTE group (experimental group) and the positive control 2 (subretinal injection of rAAV-Eylea) of the example of the present invention, but both groups showed significant and better efficacy compared with the two positive control groups.

[0242] Example 3. Expression and distribution experiment of drug molecules in target tissues

[0243] 1) Experimental design: The same test sample 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 injected into C57BL / 6J mice, and samples were collected on Day 33. The mouse eyeball samples were paraffin-embedded, and the BaseScope technology at the mRNA level was used to detect the expression distribution of the target drug protein gene in the cells of each layer of the retina (the target protein is a secreted protein, therefore, the BaseScope technology was used to detect the transcription level instead of the IHC technology to detect the protein level).

[0244] Negative control: negative control reagent (solvent reagent Buffer)

[0245] Positive control: rAAV-Eylea

[0246] Experimental group: rAAV-Fc-YTE (CRG-B191)

[0247] The BaseScope representative result is shown in Figure 6.

[0248] Conclusion: The results showed that both the positive control (rAAV-Eylea) and the experimental group (rAAV-Fc-YTE) were highly expressed in target tissues compared to the negative control (negative control reagent). Furthermore, the expression distribution of the positive control drug gene and the experimental group drug gene was identical across retinal layers: expression in the RPE layer, IS / OS layer, and outer nuclear layer cells, with the highest expression levels in the RPE cells.

[0249] The above results indicate that the modification of drug molecules by the present invention can significantly improve the therapeutic effect on the target disease without changing the expression level and expression distribution of the drug molecules in the target tissue.

[0250] Example 4. Recombinant protein drug CNV modeling and FFA detection experiments

[0251] Modeling, FFA scoring, and testing were performed according to the methods described in Example 2. The protein drug grouping and dosing regimen are shown in the table below. The results showed that the efficacy of Fc-YTE-aflibercept protein administered directly into the eye via intraocular IVT was not inferior to that of aflibercept.

[0252] The FFA spot scoring results (Mean±SEM) on Day 7 after model establishment are shown in the following table and Figure 7.

[0253] Day 7 FFA spot area results after modeling (mm 2 , Mean ± SEM) are shown in the following table and Figure 8.

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.

2. The gene delivery vector according to claim 1, wherein the human VEGF receptor fusion protein further comprises the immunoglobulin-like domain 4 of human VEGFR2. The gene delivery vector according to claim 1 or 2, wherein the hinge-Fc domain of the human immunoglobulin is the hinge-Fc domain of human IgG1.

4. The gene delivery vector according to any one of claims 1 to 3, wherein the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: an 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 as shown in SEQ ID NO.1 or 6.

5. The gene delivery vector according to any one of claims 1 to 4, wherein 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.

6. The gene delivery vector according to any one of claims 1 to 5, wherein 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.

7. The gene delivery vector according to any one of claims 1 to 6, wherein the hinge-Fc domain of the human immunoglobulin comprises or is: an 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 as shown in SEQ ID NO. 3 or 8.

8. The gene delivery vector according to claim 1, wherein 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 as shown in SEQ ID NO. 5 or 9.

9. The gene delivery vector according to claim 8, wherein the expression cassette comprises: a nucleotide sequence as shown in SEQ ID NO. 10 or 11, or a degenerate sequence of either of them.

10. The gene delivery vector according to any one of claims 1 to 9, wherein the expression cassette further comprises a promoter located upstream of the nucleotide sequence encoding the human VEGF receptor fusion protein and operably linked thereto, the promoter preferably being selected from the group consisting of a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, an MMT promoter, an EF-1α promoter, a U6 promoter, a chicken β-actin promoter, a CAG promoter, a CBA promoter, an RPE65 promoter, a VMD2 promoter, a RPGR promoter, an IRBP promoter, a hGRK1 promoter, a CAR promoter, a RHO promoter, a Grm6 promoter, a GRK1 promoter, and a GFAP promoter.

11. The gene delivery vector according to any one of claims 1 to 10, wherein 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, the polyadenylation signal preferably being selected from SV40 early polyA, SV40 late polyA, rabbit globulin polyA, bGH polyA and HSV TK polyA.

12. The gene delivery vector according to any one of claims 1 to 11, wherein the gene delivery vector is (a) a viral vector, preferably a lentivirus, a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a poxvirus, a papovavirus, a baculovirus or a papillomavirus, more preferably an adeno-associated virus or a lentivirus; or (b) Non-viral vectors, preferably plasmids, liposomes, nanoparticles, polymers, transposons, exosomes or bacterial vectors.

13. The gene delivery vector according to any one of claims 1 to 12, wherein the vector is an adeno-associated virus vector comprising: (a) a recombinant adeno-associated virus capsid, and (b) the nucleic acid packaged in the recombinant adeno-associated virus capsid.

14. The gene delivery vector according to claim 13, wherein 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, and capsid protein mutants modified with the above capsid proteins as the backbone; preferably, the recombinant adeno-associated virus capsid comprises a capsid protein of an adeno-associated virus selected from the group consisting of AAV1, AAV2, AAV5, AAV7, AAV8 and hybrids thereof.

15. The gene delivery vector according to claim 13 or 14, wherein the genome of the adeno-associated virus vector is in a single-stranded or double-stranded form, preferably scAAV, ssAAV or cceAAV. 16 . A pharmaceutical composition comprising the gene delivery vector according to any one of claims 1 to 15 ; and pharmaceutically acceptable excipients; preferably, the pharmaceutical composition is an injection.

17. Use of the gene delivery vector according to any one of claims 1 to 15 or the pharmaceutical composition according to claim 16 in the preparation of a medicament for treating a disease caused by VEGF, preferably, the disease caused by VEGF is selected from exudative (wet) age-related macular degeneration (AMD), diabetic retinopathy (DR) combined with macular edema (DME), retinal static occlusion (RVO) combined with macular edema (ME), central exudative chorioretinopathy, polypoidal choroidal vasculopathy (PCV), choroidal neovascularization secondary to high myopia, and secondary macular choroidal neovascularization (CNV).

18. 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 a human immunoglobulin; and the hinge-Fc domain of the human immunoglobulin comprises M252Y, S254T and T256E substitutions according to EU numbering.

19. The human VEGF receptor fusion protein according to claim 18, wherein the human VEGF receptor fusion protein further comprises the 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.

20. The human VEGF receptor fusion protein according to claim 18 or 19, wherein 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.

21. The human VEGF receptor fusion protein according to any one of claims 18 to 20, wherein: (a) the immunoglobulin-like domain 2 of human VEGFR1 comprises or is: an 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 as shown in SEQ ID NO.1 or 6; and / or (b) 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.

22. The human VEGF receptor fusion protein according to claim 18, wherein 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 as shown in SEQ ID NO. 5 or 9.

23. A polynucleotide encoding the human VEGF receptor fusion protein according to any one of claims 18 to 22; preferably, the polynucleotide comprises or is: a nucleotide sequence as shown in SEQ ID NO. 10 or 11, or a degenerate sequence of either of them.

24. A vector, preferably a plasmid, comprising the polynucleotide of claim 23; preferably, the vector is constructed to form ssAAV, scAAV or cceAAV.

25. A host cell comprising the vector of claim 24, preferably a HEK293 cell or a sf9 cell.

26. A recombinant adeno-associated virus, comprising: (a) rAAV capsid; and (b) A nucleic acid packaged in an rAAV capsid, the nucleic acid comprising in 5' to 3' order: (i) 5'AAV ITR; (ii) a promoter; (iii) the polynucleotide of claim 23; (iv) polyA; and (v) 3'AAV ITR.

27. The recombinant adeno-associated virus according to claim 26, wherein: (a) the rAAV capsid comprises the capsid protein of AAV1, AAV2, AAV5, AAV7 or AAV8, preferably the capsid protein of AAV8; (b) 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; (c) the polyA is SV40 late polyA or rabbit globulin polyA, preferably SV40 late polyA; (d) the 5' AAV ITR and / or the 3' AAV ITR are selected from the ITRs of AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6; and / or (e) The recombinant adeno-associated virus is scAAV, ssAAV or cceAAV.

28. An expression cassette comprising, in 5' to 3' order: (i) a promoter; (ii) the polynucleotide of claim 23; (iii) polyA.

29. The expression cassette of claim 28, wherein: (a) 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; and / or (b) The polyA is SV40 late polyA or rabbit globulin polyA, preferably SV40 late polyA.

30. The expression cassette according to claim 28 or 29, wherein the 5' end of the promoter and the 3' end of the polyA comprise a 5' AAV ITR and a 3' AAV ITR, respectively.

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