AAV viral vectors expressing anti-vegf fusion proteins and uses thereof

Abstract

Provided are an AAV viral vector expressing an anti-VEGF fusion protein or antibody and applications thereof. Specifically, provided are a recombinant AAV viral vector comprising a nucleotide sequence encoding an anti-VEGF fusion protein or antibody and applications thereof. The recombinant AAV virus has excellent infection effects in human cells and mouse cells, is stably and abundantly expressed in the cells, can effectively inhibit intraocular VEGF, reduce leakage and angiogenesis caused by laser damage, and can deliver the anti-VEGF fusion protein or anti-VEGF antibody by subretinal space injection, achieve the purpose of long-term effectiveness with one injection, and thus has a wide market prospect as a drug for treating VEGF-related diseases.

Description

AAV viral vector expressing anti-VEGF fusion protein and its use Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a gene expression vector, in particular to a vector for mediating efficient expression of exogenous genes in cells and its application. Background Art

[0002] Age-related macular degeneration (AMD) is a serious eye disease that has become increasingly prevalent in an aging society. It is a common eye disease in people over 50 and a leading cause of vision loss. Clinically, it is categorized into two types: atrophic, or dry, AMD, and exudative, or wet AMD (wAMD), depending on the presence of abnormal neovascularization. Dry AMD causes gradual vision loss, accounting for 90% of cases. Wet AMD progresses rapidly and is one of the leading causes of vision loss, accounting for only 10% of cases, but it causes over 90% of all blindness caused by AMD. Data indicates that by 2020, the number of people with macular degeneration worldwide was approximately 200 million, and this number is projected to increase to 300 million by 2040, making it the leading cause of blindness among the elderly. The prevalence of AMD in people over 45 in China ranges from 2.44% to 18.98%, with a higher incidence among urban intellectual workers. With the accelerating aging of the population, the total number of people with macular degeneration in China is expected to reach approximately 55 million by 2050. Furthermore, high-energy blue light from the electronic world penetrates the cornea and lens of the eye to reach the retina, initially stimulating the brain without causing fatigue. Long-term vision loss can induce eye fatigue, dry eye, and disrupted circadian rhythms. Direct penetration of the lens to the retina accelerates damage to the macula, leading to accelerated damage to the macula and increasing the age and severity of AMD. AMD has become the third leading cause of blindness and is a subject of global research.

[0003] Wet AMD is primarily characterized by abnormal new blood vessel growth beneath the retina in the macula, known as choroidal neovascularization (CNV). This growth leads to localized edema or hemorrhage in the macula, causing swelling and localized pigment epithelial detachment. This ultimately leads to scarring, damage to retinal photoreceptors, and consequently, vision loss. Therefore, inhibiting vascular endothelial growth factor (VEGF) and blocking the growth of these new blood vessels can be an effective treatment for fundus disease.

[0004] The recognized first-line treatment for wet AMD is intravitreal injection of protein-based VEGF inhibitors. Currently, the main protein-based VEGF inhibitors used to treat wet AMD include Roche's bevacizumab, Roche / Novartis' Fab antibody fragment Lucentis, Regeneron's VEGFR-Fc (VEGF-trap) fusion protein aflibercept, and Sichuan Kanghong Pharmaceutical's VEGFR-Fc fusion protein Conbercept. VEGF is the most critical regulator of angiogenesis. The use of VEGF antibodies or neutralizing proteins has the potential to improve vision and anatomical structure. Anti-VEGF therapy has become the first-line treatment for the treatment and stabilization of most cases of neovascular AMD.

[0005] However, VEGF neutralizing proteins do not suppress the continued expression of VEGF. When the concentration of VEGF neutralizing proteins injected into the eye is too low, angiogenesis and leakage will recur. This necessitates continuous injections to maintain the visual improvement benefits. This poses a challenge to patient compliance and significantly increases the financial burden on patients.

[0006] Therefore, new anti-VEGF drugs are still needed.

[0007] Summary of the Invention

[0008] In the first aspect, the present invention has developed an expression vector or expression cassette with excellent properties, which can ensure the long-term high-level expression of exogenous genes, such as anti-VEGF fusion protein genes. In the second aspect, the present invention uses adeno-associated virus vector (AAV) to express VEGF inhibitors. Compared with the positive drug RGX-314, the protein expression level reaches 157 times the positive drug protein concentration, which greatly inhibits angiogenesis and avoids leakage. In the third aspect, in terms of inhibiting angiogenesis and abnormalities, it is comparable to the positive drug Eylea ○R In comparison, the medicine of the present invention has more significant effects.

[0009] Through extensive experimentation, the present invention has developed an excellent expression system that can improve the efficiency of exogenous gene expression and enable continuous and efficient expression of VEGF inhibitors in the eye. This provides a preventive and therapeutic drug for nAMD patients that provides permanent benefits with a single injection.

[0010] The first aspect of the present invention protects an expression cassette comprising:

[0011] (a) a promoter selected from any one of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or any sequence having 85%, 90%, 95%, or 99% or more identity with the sequence of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, or a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter;

[0012] (b) a coding sequence selected from a nucleotide sequence encoding an anti-VEGF fusion protein or an anti-VEGF antibody, preferably, the coding sequence is a nucleotide sequence encoding an anti-VEGF fusion protein, and the coding sequence is operably linked to a promoter; and

[0013] (c) a polyadenylation region (polyA) selected from any one of the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence, and the rabbit globin (rbGlob) polyA sequence, or any sequence that is 85%, 90%, 95%, or 99% or more identical to the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence, or the rabbit globin (rbGlob) polyA sequence; and operably linked to the coding sequence.

[0014] In some embodiments, the expression cassette further comprises an enhancer, wherein the enhancer is selected from any one of CMV enhancer and EF1α enhancer, or any sequence having 85%, 90%, 95% or 99% or more identity with the sequence of CMV enhancer or EF1α enhancer.

[0015] In some embodiments, the expression cassette further comprises an intron, wherein the intron is selected from any one of an SV40 intron, an elongation factor 1 alpha (EF1α) intron, an actin intron, a CMV intron, and a bGlob intron, or any sequence having 85%, 90%, 95%, or 99% or more identity to an SV40 intron, an elongation factor 1 alpha (EF1α) intron, an actin intron, a CMV intron, or a bGlob intron.

[0016] In some embodiments, the expression cassette comprises other regulatory elements, and the other regulatory elements are selected from any one of an expression enhancer sequence EES and a WHP virus post-transcriptional regulatory element (WPRE).

[0017] In some embodiments, the expression cassette comprises

[0018] (a) a promoter selected from any one of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or any sequence having 85%, 90%, 95%, or 99% or more identity with the sequence of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, or a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter;

[0019] (b) an intron selected from any one of the group consisting of an SV40 intron, an elongation factor 1α (EF1α) intron, an actin intron, a CMV intron, and a bGlob intron, or any sequence having 85%, 90%, 95%, or 99% or greater identity to an SV40 intron, an elongation factor 1α (EF1α) intron, an actin intron, a CMV intron, or a bGlob intron; and operably linked to a promoter;

[0020] (c) a coding sequence operably linked to an intron; and

[0021] (d) a polyadenylation region (polyA) selected from any one of the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence, and the rabbit globin (rbGlob) polyA sequence, or any sequence that is 85%, 90%, 95%, or 99% or more identical to the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence, or the rabbit globin (rbGlob) polyA sequence; and operably linked to the coding sequence.

[0022] In some embodiments, the expression cassette further comprises an enhancer selected from any one of a CMV enhancer and an EF1α enhancer, or any sequence having 85%, 90%, 95% or 99% or greater identity to a CMV enhancer or an EF1α enhancer.

[0023] In some embodiments, the expression cassette comprises

[0024] (a) CMV promoter or chicken β-actin promoter;

[0025] (b) bGlob_int intron or chickenβ-actin intron;

[0026] (c) a coding sequence; and

[0027] (d) human growth hormone (HGH or hGH) polyA sequence;

[0028] In some embodiments, the expression cassette further comprises a CMV enhancer or a WPRE regulatory sequence or an EES regulatory sequence.

[0029] In some embodiments, the expression cassette has the structure of formula (A) in 5' to 3' order: A1-A2-A3-A4-A5-A6 (A)

[0030] In the formula, each "-" is independently a bond or a nucleotide linking sequence;

[0031] A1 is an enhancer or is absent, wherein the enhancer is selected from any one of the CMV enhancer and the EF1α enhancer, or any sequence having 85%, 90%, 95% or 99% or greater identity with the sequence of the CMV enhancer or the EF1α enhancer;

[0032] A2 is a promoter, the promoter selected from any one of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or selected from any sequence having 85%, 90%, 95%, or 99% or more identity with the sequence of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, or a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter;

[0033] A3 is an intron, the intron being selected from any one of an SV40 intron, an elongation factor 1α (EF1α) intron, an actin intron, a CMV intron, and a bGlob intron, or any sequence having 85%, 90%, 95%, or 99% or more identity with the sequence of an SV40 intron, an elongation factor 1α (EF1α) intron, an actin intron, a CMV intron, or a bGlob intron;

[0034] A4 is the coding sequence;

[0035] A5 is a regulatory sequence or is absent, wherein the regulatory sequence is selected from any one of an expression enhancer sequence EES and a WHP virus post-transcriptional regulatory element (WPRE); and

[0036] A6 is a polyadenylation region (polyA), the polyadenylation region (polyA) being selected from any one of the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence, and the rabbit globin (rbGlob) polyA sequence, or any sequence having 85%, 90%, 95%, or 99% or greater identity to the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence, or the rabbit globin (rbGlob) polyA sequence; the polyadenylation region (polyA) being operably linked to the coding sequence;

[0037] The positions of A1 and A2 in formula (A) can be independently exchanged.

[0038] In some embodiments, the expression cassette is selected from the group consisting of:

[0039] (a) CMV enhancer-CMV promoter-bGlob intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (b) CMV promoter-bGlob intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (c) CMV enhancer-actin promoter-bGlob intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (d) CMV enhancer-CMV promoter-chickenβ-actin intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (e) CMV enhancer-chickenβ-actin promoter-chickenβ-actin intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (f) CMV promoter-bGlob intron-coding sequence-WPRE-human growth hormone (HGH or hGH) polyA sequence; and

[0040] (g) CMV promoter-bGlob intron-coding sequence-EES-human growth hormone (HGH or hGH) polyA sequence;

[0041] The chicken β-actin intron comprises or is the sequence shown in SEQ ID NO: 5, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto; and the chicken β-actin promoter comprises or is the sequence shown in SEQ ID NO: 3, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto.

[0042] In some embodiments, the coding sequence is a nucleotide sequence encoding a preventive and / or therapeutic protein. Preferably, the preventive and / or therapeutic protein is an antibody, a fusion protein or a polypeptide.

[0043] In some embodiments, the coding sequence is a nucleotide sequence encoding a protein for preventing and / or treating a disease or condition selected from the following: a disease or condition associated with the central nervous system, the peripheral nervous system, a disease or condition associated with neurodegeneration, a gastrointestinal disease or condition associated with smooth muscle contraction, a respiratory disease or condition, an endocrine disease or condition, a disease or condition associated with inflammation, a treatment for tumors, a disease or condition that terminates withdrawal symptoms caused by chemical abuse, an ophthalmic disease, anesthesia, septic shock, and biliary colic.

[0044] In some embodiments, the coding sequence is a nucleotide sequence that encodes a protein that prevents and / or treats a disease or condition selected from the group consisting of Stargardt disease, retinoschisis, age-related macular degeneration (AMD), diabetic retinopathy, Leber congenital amaurosis (LCA), retinal detachment, cysts, cystoid macular edema, retinitis pigmentosa, corneal dystrophy, glaucoma, and cataracts.

[0045] In some embodiments, the coding sequence is a nucleotide sequence encoding an anti-VEGF fusion protein or antibody.

[0046] In some embodiments, the coding sequence is selected from the following nucleotide sequences:

[0047] A nucleotide sequence encoding Conbercept; a nucleotide sequence encoding Aflibercept; a nucleotide sequence V3 as shown in SEQ ID NO: 15; a nucleotide sequence encoding Faricimab; a nucleotide sequence encoding Bevacizumab; and a nucleotide sequence encoding Ranibizumab.

[0048] In some embodiments, the coding sequence is selected from the following nucleotide sequences:

[0049] (a) the nucleotide sequence encoding the C11 protein as shown in SEQ ID NO: 16, the amino acid sequence of the C11 protein as shown in SEQ ID NO: 17;

[0050] (b) a nucleotide sequence encoding Conbercept;

[0051] (c) the nucleotide sequence V3 as shown in SEQ ID NO: 15;

[0052] (d) nucleotide sequence encoding Faricimab;

[0053] (e) a nucleotide sequence encoding Bevacizumab; and

[0054] (f) Nucleotide sequence encoding Ranibizumab.

[0055] In some embodiments, the expression cassette is selected from the group consisting of:

[0056] (a) CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-human growth hormone (HGH or hGH) polyA sequence, wherein the amino acid sequence of the C11 protein is shown in SEQ ID NO: 17;

[0057] (b) CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-human growth hormone (HGH or hGH) polyA sequence;

[0058] (c) CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-WPRE-human growth hormone (HGH or hGH) polyA sequence, wherein the amino acid sequence of the C11 protein is shown in SEQ ID NO: 17;

[0059] (d) CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-WPRE-human growth hormone (HGH or hGH) polyA sequence;

[0060] (e) CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-EES-human growth hormone (HGH or hGH) polyA sequence;

[0061] (f) CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-EES-human growth hormone (HGH or hGH) polyA sequence;

[0062] (g) CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence V3 as shown in SEQ ID NO: 15-human growth hormone (HGH or hGH) polyA sequence;

[0063] (h) CMV promoter-bGlob intron-nucleotide sequence V3-WPRE-human growth hormone (HGH or hGH) polyA sequence as shown in SEQ ID NO: 15; and

[0064] (i) CMV promoter-bGlob intron-nucleotide sequence V3-EES-human growth hormone (HGH or hGH) polyA sequence as shown in SEQ ID NO: 15.

[0065] In some embodiments, the expression cassette is selected from the group consisting of:

[0066] CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-human growth hormone (HGH or hGH) polyA sequence;

[0067] CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-human growth hormone (HGH or hGH) polyA sequence;

[0068] CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding aflibercept-human growth hormone (HGH or hGH) polyA sequence;

[0069] CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-WPRE-human growth hormone (HGH or hGH) polyA sequence;

[0070] CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-WPRE-human growth hormone (HGH or hGH) polyA sequence;

[0071] CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-EES-human growth hormone (HGH or hGH) polyA sequence;

[0072] CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-EES-human growth hormone (HGH or hGH) polyA sequence;

[0073] CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence V3 as shown in SEQ ID NO: 15-human growth hormone (HGH or hGH) polyA sequence;

[0074] CMV promoter-bGlob intron-nucleotide sequence V3-WPRE-human growth hormone (HGH or hGH) polyA sequence as shown in SEQ ID NO: 15; and

[0075] CMV promoter-bGlob intron-nucleotide sequence V3-EES-human growth hormone (HGH or hGH) polyA sequence as shown in SEQ ID NO: 15,

[0076] The amino acid sequence of the C11 protein is shown in SEQ ID NO: 17, the sequence of WPRE is shown in SEQ ID NO: 13, and the nucleotide sequence of EES is shown in SEQ ID NO: 12.

[0077] In another embodiment of the present invention, an expression cassette is provided, which comprises, in order from 5' to 3':

[0078] An enhancer, wherein the enhancer is a CMV enhancer or an EF1α enhancer, or any sequence having 85%, 90%, 95% or 99% or greater identity to the sequence of the CMV enhancer or the EF1α enhancer;

[0079] A promoter, wherein the promoter is selected from the group consisting of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or any sequence having 85%, 90%, 95%, or 99% or more identity with the sequence of the cytomegalovirus (CMV) promoter, the actin promoter, the elongation factor 1α (EF1α) promoter, the CB7 promoter, the ubiquitin promoter, or the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter;

[0080] An intron, wherein the intron is selected from the group consisting of an SV40 intron, an elongation factor 1α (EF1α) intron, an actin intron, a CMV intron, and a bGlob intron, or any sequence having 85%, 90%, 95%, or 99% or more identity with the sequence of the SV40 intron, the elongation factor 1α (EF1α) intron, the actin intron, the CMV intron, or the bGlob intron;

[0081] a coding sequence encoding a protein of interest; and

[0082] A polyadenylation region (polyA), wherein the polyadenylation region (polyA) is selected from the group consisting of the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence, and the rabbit globin (rbGlob) polyA sequence, or any sequence that is 85%, 90%, 95%, or 99% or more identical to the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence, or the rabbit globin (rbGlob) polyA sequence.

[0083] In another embodiment of the present invention, an expression cassette is provided, which comprises, in order from 5' to 3':

[0084] CMV enhancer;

[0085] Cytomegalovirus (CMV) promoter;

[0086] bGlob intron;

[0087] A coding sequence, wherein the coding sequence is a nucleotide sequence encoding an anti-VEGF fusion protein or an anti-VEGF antibody; preferably, the coding sequence is a nucleotide sequence encoding Conbercept, Aflibercept, Faricimab, Bevacizumab or Ranibizumab; and

[0088] Human growth hormone (HGH or hGH) polyA sequence.

[0089] The sixth aspect of the present invention provides an expression cassette, characterized in that the expression cassette comprises, in order from 5' to 3':

[0090] A CMV enhancer comprising the sequence of SEQ ID NO: 4, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto; preferably, the CMV enhancer is the sequence of SEQ ID NO: 4, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto;

[0091] A cytomegalovirus (CMV) promoter, comprising the sequence of SEQ ID NO: 1, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto; preferably, the cytomegalovirus (CMV) promoter is the sequence of SEQ ID NO: 1, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto;

[0092] A bGlob intron, comprising the sequence of SEQ ID NO: 6, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto; preferably, the bGlob intron is the sequence of SEQ ID NO: 6, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto;

[0093] A coding sequence comprising or encoding a nucleotide sequence of Conbercept, Ranibizumab, or Aflibercept, or a sequence having 85%, 90%, 95%, or 99% or greater identity thereto; preferably, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein as set forth in SEQ ID NO: 16 or the nucleotide sequence as set forth in SEQ ID NO: 15, or a sequence having 85%, 90%, 95%, or 99% or greater identity thereto; preferably, the coding sequence comprises or is the nucleotide sequence encoding the C11 protein as set forth in SEQ ID NO: 16; and

[0094] A human growth hormone (HGH or hGH) polyA sequence, wherein the human growth hormone (HGH or hGH) polyA sequence comprises or is the sequence set forth in SEQ ID NO: 9, or a sequence having 85%, 90%, 95%, or 99% or greater identity thereto; preferably, the human growth hormone (HGH or hGH) polyA sequence is the sequence set forth in SEQ ID NO: 9, or a sequence having 85%, 90%, 95%, or 99% or greater identity thereto;

[0095] The amino acid sequence of the C11 protein is shown in SEQ ID NO: 17.

[0096] The second aspect of the present invention provides a vector, characterized in that the vector contains the expression cassette described in the first aspect.

[0097] The third aspect of the present invention provides a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises the expression cassette described in the first aspect; preferably, the rAAV further comprises an AAV 5' inverted terminal repeat sequence (L-ITR) located upstream of the 5' end of the nucleotide sequence of the expression cassette and an AAV 3' inverted terminal repeat sequence (R-ITR) located downstream of the 3' end of the nucleotide sequence of the expression cassette; preferably, the AAV 5' inverted terminal repeat sequence (L-ITR) comprises or is the sequence shown in SEQ ID NO: 19 or a sequence having 85%, 90%, 95% or 99% or more identity thereto, and the AAV 3' inverted terminal repeat sequence (R-ITR) comprises or is the sequence shown in SEQ ID NO: 20 or a sequence having 85%, 90%, 95% or 99% or more identity thereto.

[0098] In another embodiment of the present invention, a recombinant adeno-associated virus (rAAV) is provided, wherein the rAAV comprises a nucleic acid comprising, in 5' to 3' order:

[0099] AAV 5' inverted terminal repeat (L-ITR), preferably the AAV 5' inverted terminal repeat (L-ITR) comprises or is the sequence shown in SEQ ID NO: 19 or a sequence having 85%, 90%, 95% or 99% or greater identity thereto;

[0100] A CMV enhancer comprising the sequence of SEQ ID NO: 4, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto; preferably, the CMV enhancer is the sequence of SEQ ID NO: 4, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto;

[0101] A cytomegalovirus (CMV) promoter, comprising the sequence of SEQ ID NO: 1, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto; preferably, the cytomegalovirus (CMV) promoter is the sequence of SEQ ID NO: 1, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto;

[0102] A bGlob intron, comprising the sequence of SEQ ID NO: 6, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto; preferably, the bGlob intron is the sequence of SEQ ID NO: 6, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto;

[0103] A coding sequence, wherein the coding sequence is a nucleotide sequence encoding an anti-VEGF fusion protein or an anti-VEGF antibody; preferably, the coding sequence comprises or is a nucleotide sequence encoding Conbercept, Ranibizumab or Aflibercept, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the coding sequence comprises or is a nucleotide sequence encoding a C11 protein as set forth in SEQ ID NO: 16 or a nucleotide sequence as set forth in SEQ ID NO: 15, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the coding sequence comprises or is a nucleotide sequence encoding a C11 protein as set forth in SEQ ID NO: 16; wherein the amino acid sequence of the C11 protein is set forth in SEQ ID NO: 17;

[0104] A human growth hormone (HGH or hGH) polyA sequence, wherein the human growth hormone (HGH or hGH) polyA sequence comprises or is the sequence shown in SEQ ID NO: 9, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto; preferably, the human growth hormone (HGH or hGH) polyA sequence is the sequence shown in SEQ ID NO: 9, or a sequence having 85%, 90%, 95% or 99% or greater identity thereto; and

[0105] The AAV 3' inverted terminal repeat sequence (L-ITR), preferably the AAV 3' inverted terminal repeat sequence (R-ITR) comprises or is the sequence shown in SEQ ID NO: 20 or a sequence having 85%, 90%, 95% or 99% or more identity thereto.

[0106] In some embodiments, the recombinant adeno-associated virus (rAAV), wherein the nucleotide sequence of the nucleic acid contained in the rAAV is shown as SEQ ID NO:18.

[0107] In some embodiments, the recombinant adeno-associated virus (rAAV), wherein the rAAV further comprises an AAV capsid.

[0108] In some embodiments, the recombinant adeno-associated virus (rAAV), wherein the adeno-associated virus is selected from AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV;

[0109] Preferably, the adeno-associated virus is AAV type 6 (AAV-6) or AAV type 8 (AAV-8).

[0110] In another embodiment of the present invention, a recombinant adeno-associated virus (rAAV) is provided, wherein the rAAV comprises:

[0111] AAV capsid; and

[0112] A nucleic acid comprising, in 5' to 3' order:

[0113] AAV 5' inverted terminal repeat (L-ITR), wherein the AAV 5' inverted terminal repeat (L-ITR) is the sequence shown in SEQ ID NO: 19;

[0114] CMV enhancer, wherein the CMV enhancer is the sequence shown in SEQ ID NO: 4;

[0115] Cytomegalovirus (CMV) promoter, wherein the cytomegalovirus (CMV) promoter is the sequence shown in SEQ ID NO: 1;

[0116] bGlob intron, wherein the bGlob intron is the sequence shown in SEQ ID NO: 6;

[0117] A coding sequence, wherein the coding sequence is a nucleotide sequence encoding Conbercept, Ranibizumab, or Aflibercept; preferably, the coding sequence is a nucleotide sequence encoding the C11 protein as shown in SEQ ID NO: 16 or a nucleotide sequence as shown in SEQ ID NO: 15, wherein the amino acid sequence of the C11 protein is shown in SEQ ID NO: 17;

[0118] Human growth hormone (HGH or hGH) polyA sequence, wherein the human growth hormone (HGH or hGH) polyA sequence is the sequence shown in SEQ ID NO: 9; and

[0119] AAV 3' inverted terminal repeat (R-ITR), wherein the AAV 3' inverted terminal repeat (R-ITR) is the sequence shown in SEQ ID NO: 20;

[0120] Wherein, the adeno-associated virus is AAV type 8 (AAV-8).

[0121] In another embodiment of the present invention, a recombinant adeno-associated virus (rAAV) is provided having an AAV capsid suitable for intraocular injection, wherein the AAV comprises a vector genome packaged in the capsid, the vector genome comprising:

[0122] (a) AAV 5′ inverted terminal repeat (L-ITR);

[0123] (b) the expression cassette as described in the first aspect of the present invention;

[0124] (c) AAV 3′ inverted terminal repeat (R-ITR).

[0125] In some embodiments, the recombinant adeno-associated virus, wherein the adeno-associated virus is selected from AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV;

[0126] Preferably, the adeno-associated virus is selected from AAV type 6 (AAV-6) or AAV type 8 (AAV-8).

[0127] In some embodiments, the L-ITR sequence comprises the sequence shown in SEQ ID NO: 19, or a sequence at least 85%, at least 90%, at least 95%, or at least 99% identical thereto.

[0128] In some embodiments, the R-ITR sequence comprises the sequence shown in SEQ ID NO: 20, or a sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical thereto.

[0129] The fourth aspect of the present invention provides a pharmaceutical composition comprising the expression cassette according to the first aspect of the present invention, the vector according to the second aspect of the present invention or the adeno-associated virus according to the third aspect of the present invention, and a pharmaceutically acceptable excipient.

[0130] The fifth aspect of the present invention provides an isolated host cell transfected or transduced with the expression cassette of the first aspect of the present invention, the vector of the second aspect of the present invention, or the adeno-associated virus of the third aspect of the present invention.

[0131] The sixth aspect of the present invention provides a method for expressing a transgene in a mammalian cell, the method comprising contacting one or more mammalian cells with a certain amount of an adeno-associated virus as described in the third aspect of the present invention, wherein the target protein is expressed at a certain level in the one or more mammalian cells.

[0132] The seventh aspect of the present invention provides a method for treating or preventing a disease in a mammal in need of treatment or prevention of the disease, the method comprising administering to the mammal an effective amount of the recombinant adeno-associated virus according to the third aspect of the present invention, the pharmaceutical composition according to the fourth aspect of the present invention, and / or the host cell according to the fifth aspect of the present invention;

[0133] Preferably, the disease is an eye disease, further preferably, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization and diabetic retinopathy;

[0134] Preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal, and further preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal by intraocular injection, intravitreal injection or subretinal space.

[0135] In an eighth aspect, the present invention provides a use of the recombinant adeno-associated virus according to the third aspect of the present invention, the pharmaceutical composition according to the fourth aspect of the present invention, and / or the host cell according to the fifth aspect of the present invention in the preparation of a medicament for treating or preventing a disease in a mammal in need of such treatment or prevention.

[0136] Preferably, the disease is an eye disease, further preferably, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization and diabetic retinopathy;

[0137] Preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal, and further preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal by intraocular injection, intravitreal injection or subretinal space.

[0138] The ninth aspect of the present invention provides the recombinant adeno-associated virus according to the third aspect of the present invention, the pharmaceutical composition according to the fourth aspect of the present invention, and / or the host cell according to the fifth aspect of the present invention, for use in treating or preventing a disease in a mammal in need of such treatment or prevention.

[0139] Preferably, the disease is an eye disease, further preferably, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization and diabetic retinopathy;

[0140] Preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal, and further preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal by intraocular injection, intravitreal injection or subretinal space.

[0141] The tenth aspect of the present invention provides a product comprising: (a) a first container, wherein the first container comprises the recombinant adeno-associated virus as described in the third aspect of the present invention or the pharmaceutical composition as described in the fourth aspect of the present invention; and (b) an injection needle.

[0142] definition

[0143] For a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0144] The term "vector" refers to a nucleic acid vector into which a polynucleotide can be inserted. When a vector allows the expression of a protein encoded by the polynucleotide inserted therein, the vector is called an expression vector. The vector can be transformed, transduced or transfected into a host cell to express the genetic material elements carried in the host cell. Vectors are well known to those skilled in the art and include but are not limited to plasmids, phages, artificial chromosomes such as yeast artificial chromosomes, bacterial artificial chromosomes, and viruses; phages such as lambda phage or M13 phage. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). The vector may contain multiple elements for controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain an origin of replication.

[0145] The term "host cell" refers to a cell system that can be engineered to produce a target protein, protein fragment or peptide. Host cells include, but are not limited to, cultured cells, such as cultured mammalian cells derived from rodents (rat, mouse, guinea pig or hamster), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells and insect cells, as well as cells contained in transgenic animals or cultured tissues. The term covers not only specific test cells, but also the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may be different from the parent cell, but are still included within the scope of the term "host cell".

[0146] The term "transfection" refers to the uptake of foreign or exogenous DNA by a cell. A cell is "transfected" when the exogenous DNA is introduced into the cell membrane. Various transfection techniques are well known in the art. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

[0147] The term "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. The term encompasses all subtypes and naturally occurring and recombinant forms, unless otherwise required. The term "AAV" includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infects primates, "non-primate AAV" refers to AAV that infects non-primate mammals, "bovine AAV" refers to AAV that infects bovine mammals, etc.

[0148] "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically composed of all capsid proteins of wild-type AAV) and an encapsidated polynucleotide. If the particle includes a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as a recombinant AAV vector or rAAV. Typically, the heterologous polynucleotide is flanked by AAV inverted terminal repeats (ITRs).

[0149] The term "packaging" refers to the series of intracellular events that lead to the assembly and encapsidation of AAV particles.

[0150] The term "three-plasmid packaging system" refers to a technique commonly used in genetic engineering research that allows exogenous DNA to be converted into plasmids and expressed through the cellular biosynthetic machinery. This system consists of a transfer plasmid, a helper plasmid, and a helper plasmid. The transfer plasmid (rAAV plasmid) contains the target gene sequence to be delivered, typically located between two AAV inverted terminal repeats (ITRs). The ITRs are the only sequences borrowed from wild-type AAV and are essential for rAAV packaging and integration into the host cell genome. The helper plasmid (Rep-Cap plasmid) carries the essential rep and cap genes, which encode the AAV replication (Rep) and capsid (Cap) proteins. The Rep protein is responsible for replication of the rAAV plasmid, while the Cap protein is responsible for forming the viral capsid that encapsulates the rAAV plasmid DNA. The helper plasmid (AdHelper plasmid) provides additional Adenovirus proteins that are essential for the packaging process but are not included in the final rAAV particles. These proteins function to enhance viral replication, assembly, and cellular release.

[0151] The term "coding sequence" refers to a nucleotide sequence in vitro or in vivo that encodes a gene product. In some cases, a gene consists of or essentially consists of a coding sequence, i.e., a sequence that encodes a gene product. In other cases, a gene includes additional non-coding sequences.

[0152] The term "prophylactic and / or therapeutic protein" refers to a protein used for human or veterinary treatment, which can be for acute or chronic administration. In particular, a "prophylactic and / or therapeutic protein" is a protein used to prevent and / or treat a mammal suffering from a disease or pathological condition. Prophylactic and / or therapeutic proteins include, but are not limited to, any polypeptide, protein, antibody, or fusion protein that can be administered to a mammal.

[0153] The term "promoter" is a DNA sequence that guides RNA polymerase to bind and thereby promote RNA synthesis. Promoters and corresponding protein or polypeptide expression can be ubiquitous (meaning that they are strongly active in a wide range of cells, tissues, and species) or cell type-specific, tissue-specific, or species-specific. Promoters can be "constitutive" (meaning that they are continuously active) or "inducible" (meaning that they can be activated or inactivated by the presence or absence of biological factors or abiotic factors). Common promoters include, for example, cytomegalovirus promoter (CMV), actin promoter, elongation factor 1α (EF1α) promoter, CB7 promoter, ubiquitin promoter, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter.

[0154] The term "enhancer" encompasses cis-acting elements that stimulate or inhibit transcription of adjacent genes. Enhancers that inhibit transcription are also referred to as "silencers." Enhancers can act in any orientation at a distance of several thousand base pairs (kb) downstream of the coding sequence and the transcribed region (i.e., can be associated with the coding sequence). The enhancer sequence may or may not be adjacent to a promoter sequence. Enhancer sequences affect promoter-dependent gene expression and can be located in the 5' or 3' region of a native gene.

[0155] The term "intron", also known as intervening sequence, refers to a non-coding segment in a gene or mRNA molecule. It is an intervening sequence in the DNA of eukaryotic cells. These sequences are transcribed in the precursor RNA, removed by splicing, and ultimately do not exist in the mature RNA molecule. The alternating arrangement of introns and exons constitutes a split gene. Introns in precursor RNA are often called "intervening sequences". In post-transcriptional processing, it has more mutations than exons. Introns are a special DNA sequence. Among them, bGlob_int represents bGlob intron, and hGH / polyA signal represents the human growth hormone (HGH or hGH) polyA sequence.

[0156] The term "polyadenylation region (polyA)" or "polyadenylation signal sequence" encompasses the recognition region required for endonuclease cleavage of RNA transcripts, followed by the polyadenylation consensus sequence AATAAA. The polyadenylation signal sequence provides a "polyA site," i.e., a site on the RNA transcript where adenine residues are added by post-transcriptional polyadenylation. A representative example of a "polyadenylation region (polyA)" is the human growth hormone (HGH or hGH) polyA sequence, which may also be represented as hGH / polyA signal.

[0157] The term "operably linked" refers to the juxtaposition of genetic elements (e.g., promoters, enhancers, termination signal sequences, polyadenylation sequences, etc.) in a relationship that permits them to operate in their intended manner. For example, a promoter is operably linked to a coding region if it helps initiate transcription of the coding sequence. Intervening residues may exist between the promoter and the coding region as long as this functional relationship is maintained.

[0158] The terms "identity" or "homology" refer to the sequence similarity between two nucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the total number of positions compared × 100. For example, if 6 out of 10 positions in the two sequences match or are homologous, the two sequences are 60% homologous when the sequences are optimally aligned; if 95 out of 100 positions in the two sequences match or are homologous, the two sequences are 95% homologous.

[0159] The term "fusion protein" refers to a protein product obtained by linking the coding regions of two or more genes by genetic recombination, chemical methods or other appropriate methods, and expressing the gene recombination under the control of the same regulatory sequence. Unless otherwise specified, the first block polypeptide at the N-terminus of the fusion protein is connected to the N-terminus of the next block (or connecting fragment) polypeptide at the C-terminus of the polypeptide, and so on; therefore, the N-terminus of the polypeptide located in the N-terminal block of the fusion protein is the N-terminus of the fusion protein, and the C-terminus of the polypeptide located in the C-terminal block of the fusion protein is the C-terminus of the fusion protein.

[0160] The term "antibody" refers to an immunoglobulin molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions are further divided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminal to carboxyl-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, wherein the three CDRs of VH are HCDR1, HCDR2, and HCDR3, and the three CDRs of VL are LCDR1, LCDR2, and LCDR3. The amino acid assignments for each domain are generally consistent with the following definitions: Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987); Chothia et al., Nature, 342:878-883 (1989).

[0161] The term "ocular disease" refers to a disease, ailment, or condition that affects or involves the eye or one or more parts or regions of the eye. Thus, ocular diseases include retinal diseases or diseases that affect the light-sensitive layer of tissue at the back of the eye. The eye includes the eyeball and the tissues and fluids that make up the eyeball, the muscles around the eye (such as the oblique and rectus muscles), and part of the optic nerve within or near the eyeball.

[0162] The term "treatment" includes therapeutic treatment, prophylactic treatment, and use in reducing the risk of a subject developing a disease or other risk factors. Treatment includes, but is not limited to, complete cure of the disease, as well as alleviation of symptoms or mitigation of potential risks.

[0163] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to: humans and non-human primates, including apes and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figures 1a-1e are schematic diagrams of the vector structures numbered #1, #2, #3, #4, and #5, respectively (wherein: transgene represents the coding sequence, and the L-ITR and R-ITR portions are not shown);

[0165] Figures 2a-2c show the fluorescence results of five GFP coding sequence vectors transfected into 293T cells, HeLa cells, and ARPE-19 cells, respectively. The vertical axis represents the fluorescence intensity.

[0166] Figure 3 shows the protein expression concentration results of four vector structures encoding the V3 sequence transfected into ARPE-19 cells. The vertical axis is the protein concentration (μg / ml) and the horizontal axis is the vector number.

[0167] Figures 4a-4c show the protein concentrations of 293T cells, HeLa cells, and ARPE-19 cells transfected with three vector structures encoding the coding sequence of protein C11, respectively. The vertical axis is protein concentration (μg / ml), and the horizontal axis is vector number.

[0168] Figure 5a shows the expression level of C11 protein after #3AAV, #4AAV, and #5AAV infected 293T cells. Figure 5b shows the expression level of C11 protein after #3AAV, #4AAV, and #5AAV infected HeLa cells. The horizontal axis represents the vector type, and the vertical axis represents the protein concentration of C11 (μg / ml).

[0169] Figure 6 shows the activity of C11 protein expressed in cells infected with #3AAV and commercial Activity results: The horizontal axis is protein concentration (pM), and the vertical axis is relative light units.

[0170] Figure 7a shows the expression levels of C11 protein and V3 gene-expressed proteins in the entire eye of C57BL / 6J wild-type mice after #3AAV and the positive drug RGX-314 were injected into the subretinal space. Figure 7b shows the expression levels of C11 protein and V3 gene-expressed proteins in the vitreous of C57BL / 6J wild-type mice after #3AAV and the positive drug RGX-314 were injected into the subretinal space. The horizontal axis represents the drug type and the vertical axis represents the protein concentration (μg / ml).

[0171] Figure 8 shows that hRHO-hVEGFA mice were injected Figure 3 FFA results after RGX-314 and #3 AAV.

[0172] Figure 9 shows the laser-induced mice injected with different doses of #3FFA results after AAV.

[0173] Figure 10 shows the laser-induced mice injected with different doses of #3Statistical graph of FFA results after AAV.

[0174] FIG11 shows the results of laser angiography in cynomolgus macaques after injection of solvent, RGX-314, and different doses of #3AAV.

[0175] Figure 12 is a graph showing the pharmacokinetic results of laser-induced cynomolgus macaques injected with different doses of #3AAV, with the horizontal axis representing time (0 days, 15 days, 29 days, 59 days, 75 days, and 89 days) and the vertical axis representing concentration (ng / ml).

[0176] FIG13 shows the laser angiography results of macaques injected with solvent, low-dose #3AAV, medium-dose #3AAV, and high-dose #3AAV, respectively.

[0177] FIG14 is a graph showing the pharmacokinetic results of mice in each group after being administered different doses of #3AAV, wherein the horizontal axis represents time (3, 7, 14 days, and 4, 8, 12, and 24 weeks), and the vertical axis represents concentration (ng / eye). DETAILED DESCRIPTION

[0178] The embodiments of the present invention will be described in detail below with reference to the examples, but those skilled in the art will appreciate that the following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0179] Abbreviations

[0180] PBS refers to phosphate buffered saline (PBS, Phosphate Buffer Solution).

[0181] ELISA refers to enzyme linked immunosorbent assay.

[0182] GFP stands for green fluorescent protein (GFP)

[0183] PEI is Polyethylenimine (PEI)

[0184] CDS is the protein coding region or coding sequence (CDS)

[0185] FFA is fundus fluorescein angiography (FFA)

[0186] MOI is the multiplicity of infection (MOI)

[0187] EES is the expression enhancer sequence (EES)

[0188] OCT is Optical Coherence Tomograph (OCT)

[0189] Example 1 Construction of expression vector or expression cassette

[0190] The present invention constructs five exemplary expression vectors, the structures of which are shown in FIG1 . The 5′ to 3′ sequences of the expression vectors are shown in Table 1 .

[0191] Table 1 Combinations of sequence elements used in exemplary AAV expression vectors of the present invention

[0192] Among them, the coding sequence in the exemplary AAV expression vector of the present invention is selected from the gene sequence of GFP, the gene sequence V3 of the anti-VEGF protein, and the gene sequence of the anti-VEGF fusion protein (protein C11). The sequence information of the exemplary AAV expression vector of the present invention and its exemplary elements, as well as the encoded protein is shown in Table 2:

[0193] Table 2 Sequence information

[0194] Example 2 Protein Expression

[0195] 2.1 GFP expression

[0196] The CDS regions of the five vector structures in Example 1 were loaded with GFP gene and transfected into 293T cells, Hela cells, and ARPE-19 cells respectively using PEI. The proportion of GFP-positive cells was detected 48 hours later.

[0197] Plasmid transient transfection and flow cytometry experiments

[0198] (1) Cells were plated in 6-well plates, with a cell seeding density / well of 293T: 8×10 5 cells; HeLa: 3×10 5 cells; ARPE-19: 1.2×10 6 cells;

[0199] (2) Transfection was performed 24 h later (transfection system / well: 100 μL opti-MEM; 4 μg plasmid; PEI:DNA = 1:2);

[0200] (3) Remove half of the culture medium in the well and add the transfection complex;

[0201] (4) After 12 h, add 2 mL of fresh culture medium;

[0202] (5) 48 h after transfection, the culture medium in the wells was aspirated and washed once with PBS. 300 μL of 0.25% Trypsin-EDTA was added to each well to digest the cells. 1 mL of complete culture medium was then added to terminate the digestion. After mixing, 100 μL of the solution was added to a 96-well plate.

[0203] (6) The cells were loaded into a flow cytometer, the FITC channel was selected, and the proportion of GFP-positive cells was analyzed.

[0204] The results are shown in Figures 2a to 2c. Flow cytometry results showed that plasmid #2 had the lowest GFP-positive cell ratio, plasmids #3 and #4 had the highest GFP fluorescence intensity, followed by plasmids #1 and #5, and the results were relatively consistent among the three cell types.

[0205] 2.2 Protein Amount Expressed by Coding Sequence V3

[0206] The CDS regions of the four vectors (#1, #3, #4, and #5) described in Example 1 were loaded with the V3 gene and transfected into ARPE-19 cells using PEI. 48 hours after transfection, the cell culture supernatant and total cell protein were harvested. Protein expressed by the V3 gene in the ARPE-19 cell supernatant was analyzed by ELISA.

[0207] Elisa test

[0208] (1) Coating: ELISA plates were coated with 100 μL of human VEGF-A165 (1 μg / mL) in carbonate / bicarbonate buffer overnight at 4°C. The next day, the solution in the wells was discarded and the plates were washed four times with PBST (0.1% Tween in PBST);

[0209] (2) Blocking: Add 200 μL of blocking solution to each well and incubate at 37°C for 21 h;

[0210] (3) Washing: Remove the sealing film, place the plate in a plate washer, and wash 3-5 times;

[0211] (4) Sample addition: Add 100 μL of the appropriately diluted sample to the coated reaction wells;

[0212] (5) Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 h;

[0213] (6) Washing: Same as step 3;

[0214] (7) Adding antibodies: Add 100 μL of the corresponding diluted (1:15,000) antibody working solution to each well;

[0215] (8) Incubation: Seal the plate with a sealing film and incubate at 37°C for 1 h;

[0216] (9) Washing: Same as step 3.

[0217] (10) Add color development substrate: Add 100 μL of TMB substrate solution to each well and develop color at room temperature for 6-7 min;

[0218] (11) Stop reaction: Add 100 μL of ELISA stop solution to each reaction well;

[0219] (12) Result determination: Read the plate at 450 nm on a microplate reader within 10 min.

[0220] The results of Elisa assay are shown in FIG3 . The protein contents of vectors #3, #4, and #5 in ARPE-19 cells are comparable and relatively high, indicating that the vectors of the present invention all exhibit relatively high expression rates.

[0221] 2.3 Expression of protein C11

[0222] The same experimental method as 2.2 was used to load the nucleotide sequence encoding protein C11 (SEQ ID NO: 16) into 4 vectors respectively, and then transiently transfected into 3 cells. After 48 hours of transfection, the cell culture supernatant and total cell protein were harvested respectively. ELISA was used to analyze the C11 protein in the supernatant of 293T cells, Hela cells, and ARPE-19 cells. The Elisa detection results are shown in Figures 4a to 4c. The efficiency of the expression of C11 protein by the #3 vector structure plasmid in 293T cells, Hela cells, and ARPE-19 cells is the highest, and the efficiency of the expression of C11 protein by the #4 and #5 vector structure plasmids is second, but also shows a high expression efficiency, indicating that the vectors of the present invention all show a high expression rate.

[0223] Example 3 Virus infection of cells

[0224] Three different vector sequences, #3, #4, and #5, carrying the gene sequence of the C11 protein, were packaged in vitro into #3AAV, #4AAV, and #5AAV, respectively, and then infected into 293T cells and Hela cells. The four different vectors were packaged as follows:

[0225] Using a three-plasmid packaging system and purified recombinant AAV8 virus, helper plasmid (phelper), AAV Cap and Rep protein expression plasmids, expression vector target plasmids in a mass ratio of 2:1:1, and PEI promoter (purchased from polysciences) to form a transfection complex, transfect HEK293T cells (purchased from ATCC) and Hela cells (purchased from ATCC) to package AAV viruses containing coding sequences. The cells were collected 60 hours after transfection, and AAV virus particles containing coding sequences were obtained by repeated freeze-thaw disruption. Density gradient centrifugation was performed using different gradients of iodixanol (15%, 25%, 40% and 60%) to obtain purified AAV virus. The purified AAV virus obtained was used to infect cells according to the following method:

[0226] (1) Cells were plated in 24-well plates. Cell seeding density / well: 293T: 2.5×10 5 cells; HeLa: 6×10 4 cells;

[0227] (2) After 24 h, perform virus infection. Dilute the virus sample using complete medium at the desired MOI (multiplicity of infection). Discard the old medium in the well and add 500 μL of the diluted sample.

[0228] (3) After 12 h, add 500 μL of fresh complete culture medium;

[0229] (6) 72 hours after infection, the cell supernatant was collected and used for subsequent ELISA testing;

[0230] Elisa assays were used to measure C11 protein concentrations in the cell supernatant, with the results shown in Figures 5a and 5b. The results showed that after infection of 293T and HeLa cells with the three vectors, the #3 AAV vector expressed the highest concentration of C11 protein, followed by the #4 AAV and #5 AAV vectors, but also exhibited high expression efficiencies.

[0231] Example 4 C11 protein activity detection

[0232] #3AAV vector infected 293T cells to express C11 protein. Serial dilutions of VEGF Trap were prepared in resistant culture medium. Protein activity was detected using HEK293 cells overexpressing VEGFR2. As a control, a Luciferase experiment was performed. The experimental steps are as follows:

[0233] (1) VEGFR2 cells were plated in 96-well plates, with 3 replicate wells for each sample, and 5×10 cells were plated in each well with 100 μL. 4 cells, and Luciferase assay was performed 24 h after plating;

[0234] (2) Prepare C11 and Serial dilutions of , ranging from 13 pM to 5000 pM;

[0235] (3) Remove the old culture medium in the 96-well plate and add 80 μL of culture medium containing different concentrations of C11 and different concentrations of At the same time, add 80 μL of blank culture medium to the control wells. After addition to the VEGFR2 cell line, 20 μL of VEGF165 working solution (8 ng / mL) was immediately added;

[0236] (4) Equilibrate the cell lysate and firefly luciferase detection reagent to room temperature in advance. It is recommended to thaw at room temperature and avoid light.

[0237] d) Incubate in a 37°C, 5% CO2 incubator for 4 hours, equilibrate at room temperature for 30 minutes, centrifuge at 500g for 5 minutes at room temperature, and discard the supernatant;

[0238] (5) After thoroughly mixing the reporter gene cell lysate, add 100 μL of lysate to each well and shake the plate on a shaker at 600 rpm for 5 min to completely lyse the cells. After pipetting evenly, pipette 70 μL of the solution and add it to an opaque white plate.

[0239] (6) Add 100 μL of firefly luciferase detection reagent to each well, shake the plate at 600 rpm for 1 min, mix well, and measure the relative light unit (RLU);

[0240] (7) Use a Tecan multifunctional enzyme-labeled microplate reader, use a detection module for detecting firefly luciferase, choose an opaque white plate for the well plate, set the detection time at 2000 ms, and read the reading.

[0241] The activity results are shown in Figure 6. There was no difference in activity.

[0242] Example 5 Intraocular injection of virus and detection of C11 protein expression

[0243] Experimental subjects: C57BL / 6J wild-type mice (Source: Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.)

[0244] The positive drug RGX-314 and #3AAV were injected into the subretinal space of mice, with an injection volume of 1 μL and an injection concentration of 1E+9 vg / μL. Two weeks after injection, whole-eye protein or vitreous humor was extracted to measure VEGF protein expression. The positive drug RGX-314 was homemade, and #3AAV was prepared according to the method in Example 3.

[0245] The ELISA results are shown in Figures 7a-7b. It can be seen from the results that: when the whole eye protein is detected, the expression level of the target protein C11 in the #3AAV vector is significantly higher than the expression level of ranibizumab in RGX-314. By calculation, the concentration of protein C11 reaches 157 times the concentration of the protein expressed by the V3 gene. When detected in the vitreous body, the expression level of the viral protein C11 in the #3 vector is significantly higher than the expression level of the protein expressed by the V3 gene of RGX-314. By calculation, the concentration of the C11 protein reaches 25 times the concentration of the protein expressed by the V3 gene. Comparing the two, the expression level of the target protein C11 in the #3AAV viral vector of the present invention is significantly better than the expression level of the protein expressed by the V3 gene of the positive drug RGX-314.

[0246] Example 6 Pharmacodynamic Study

[0247] 6.1 #3AAV Inhibitory Effect on Angiogenesis in hRHO-hVEGFA Mice

[0248] Experimental Subjects: hRHO-hVEGFA mice (Source: Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) express human VEGF165 using the human rhodopsin promoter. These mice naturally develop choroidal neovascularization without induction, making them an excellent model for studying ophthalmic diseases caused by vascular proliferation in neonatal mice and useful for screening and evaluating drug efficacy and mechanism studies for related diseases.

[0249] The positive drug RGX-314 is made in the laboratory. This is an anti-VEGF protein drug for treating macular degeneration and diabetic macular edema, purchased from Bayer. #3 AAV was prepared according to the method of Example 3.

[0250] Experimental groups

[0251] hRHO-hVEGFA mice were divided into three groups, with five mice in each group. The left eye was injected with PBS as a negative control, while the right eye was injected with 1 μL / eye of each test article or control. Grouping and dosing information are shown in Table 3. Fluorescein sodium angiography was used 14 days after injection to observe fundus vascular status in the mice: sodium fluorescein (100 mg / ml, 0.02 ml / mouse) was injected intraperitoneally. Fundus images were collected and recorded 3 minutes after injection.

[0252] Table 3 Dosage regimen

[0253] The FFA results are shown in Figure 8. At the time of the photos, there was no significant difference in vascular leakage and neovascularization between the right and left eyes in Group A; there was no significant difference in fundus vascular leakage and neovascularization between the right and left eyes in Group B; the number of neovascular spots in the right and left eyes of Group C was significantly reduced, and fundus vascular leakage was significantly inhibited. The above results show that the test product #3AVV and the positive drug Compared with RGX-314, it has outstanding efficacy and obvious advantages in inhibiting angiogenesis and abnormalities.

[0254] 6.2 #3AAV Inhibitory Effect on Neovascularization in C57BL / 6J Mice

[0255] Experimental subjects: C57BL / 6J mice (Source: Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.)

[0256] Experimental groups

[0257] C57BL / 6J mice were divided into 9 groups, with 6-11 mice in each group. On the first day of the experiment, the corresponding drug dosage in the subretinal cavity of experimental groups 1, 2, 3, 4, and 5 was 1 μL. The grouping and drug administration information is shown in Table 4. On the 14th day of the experiment, PBS was injected into the vitreous cavity of the mice in the negative control group, and 2.5 μg / μL and 25 μg / μL of PBS were injected into the vitreous cavity of the mice in the positive control groups 1 and 2, respectively. All mice were irradiated with a 1μL laser (power: 300mV, duration: 100ms) at the retina, centered on the optic disc, in the superior, inferior, temporal, and nasal quadrants, penetrating Brunch's membrane and inducing choroidal neovascularization. On day 21 of the experiment, fundus angiogenesis was assessed using FFA.

[0258] Table 4 Dosage regimen

[0259] One week after laser injury, the fundus of mice was observed by sodium fluorescein angiography. The results are shown in Figures 9 and 10. In the negative control group, about 90% of the laser-irradiated areas showed clear neovascularization spots, of which about 43% showed progressive neovascularization spots. The number of clear and progressive neovascularization spots in the eyes of mice in the positive control group 1 was no different from that in the PBS group, indicating that Low dose (2.5μg / μL) had no inhibitory effect on angiogenesis caused by laser damage; the positive control group 2 showed that about 20% of the laser irradiated points had clear and progressive angiogenesis spots, and about 80% of the laser irradiated points showed suspected angiogenesis or no angiogenesis spots, indicating that High dose (25μg / μL) has a significant inhibitory effect on angiogenesis caused by laser damage.

[0260] #3AAV experimental groups 2, 3, 4, and 5 all showed positive therapeutic effects on laser damage. In experimental group 2, the number of clear and progressive neovascularization spots in the eyes of mice was about 20%, which was significantly lower than that of mice with high doses (25 μg / μL). The therapeutic effect is comparable. The mice in experimental groups 3 to 5 showed that the eye can completely inhibit the development of clear and progressive angiogenesis after laser damage. The above results show that the #3AVV drug of the present invention is superior to the positive drug in inhibiting angiogenesis and abnormalities.

[0261] 6.3 Inhibitory Effect of #3AAV on Choroidal Neovascularization in Cynomolgus Monkeys

[0262] Experimental animals: Crab-eating macaques (Source: Chengdu Huaxi Haiqi Pharmaceutical Technology Co., Ltd.)

[0263] Before the experiment, cynomolgus macaques were divided into two groups: Group 1 received RGX-314 and Group 2 received #3AAV, with one macaque in each group. On the first day of the experiment, subretinal injections were administered to both eyes, according to the dosing schedule shown in Table 5. Following administration, the animals were observed continuously. Immunization agents (prednisolone acetate injection) were administered once daily from 3 days before to 3 days after administration, at a dose of 1 mg / kg.

[0264] Table 5 Dosage regimen *The RGX-314 group received the same volume of vehicle in the left eye.

[0265] Laser photocoagulation was performed 45 days after the experimental administration; fundus camera laser angiography was performed before the experiment, 2 days before laser photocoagulation, and 2 weeks after laser photocoagulation.

[0266] The macular area was photocoagulated by panretinal microscopy, and the results are shown in Figure 11. Photocoagulation avoided the fovea and irradiated 9 points in each eye.

[0267] There was obvious leakage at the laser point in the solvent-treated eyes; there was no significant difference in the leakage at the laser irradiation point between the RGX-314-injected eyes (1.3E+9 vg / eye) and the control eyes, indicating that this dose of RGX-314 had no inhibitory effect on choroidal neovascularization in monkeys; there was no obvious leakage at the laser point in the low-dose eyes injected with the same dose (1.3E+9 vg / eye) of #3AAV, indicating that #3AAV had a significant inhibitory effect on choroidal neovascularization at a dose of 1.3E+9 vg / eye, which was significantly better than RGX-314; the leakage at the laser point in the high-dose #3AAV eyes (1.3E+11 vg / eye) was further reduced than that in the low-dose eyes.

[0268] The above test results show that #3AAV can significantly and effectively inhibit choroidal angiogenesis in the crab-eating macaque model.

[0269] Aqueous humor samples were collected on days 15, 29, 59, 75, and 89 after administration. The concentration of C11 protein in the aqueous humor was detected by ELISA. The results of C11 protein expression in the #3AAV group are shown in Figure 12. The protein levels in the aqueous humor of the #3AAV low-dose eyes (1.3E+9 vg / eye) at each time point were 5.65 ng / ml, 5.06 ng / ml, 9.1 ng / ml, 7.84 ng / ml, and 9.50 ng / ml, respectively; the C11 protein levels in the aqueous humor of the #3AAV high-dose eyes (1.3E+11 vg / eye) at days 15 and 29 were 605 ng / ml, 492 ng / ml, 698 ng / ml, 499 ng / ml, and 868 ng / ml, respectively.

[0270] The above test results show that #3AAV can normally express the target protein in situ and enter the intraocular circulation after injection into the subretinal space; 15 days after injection, the intraocular concentration of C11 protein has remained stable, and the change in the concentration of C11 protein in the aqueous humor is dose-dependent with #3AAV.

[0271] 6.4 Inhibitory Effect of #3AAV on Choroidal Neovascularization in Rhesus Monkeys

[0272] Experimental animals: Macaques (Source: Chengdu Huaxi Haiqi Pharmaceutical Technology Co., Ltd.)

[0273] Before the experiment, macaques were divided into four groups: Group 1 (solvent group), Group 2 (low-dose #3AAV group), Group 3 (medium-dose #3AAV group), and Group 4 (high-dose #3AAV group), with two macaques in each group. On the first day of the experiment, subretinal injections were administered bilaterally, according to the dosing schedule shown in Table 6. The animals were observed for 8 weeks after administration.

[0274] Table 6 Dosage regimen *Administer the same volume of solvent to both eyes.

[0275] Laser photocoagulation modeling was performed 30 days after administration; fundus photography and sodium fluorescein angiography were performed before the experiment, 29 days after administration, and 6 and 8 weeks after administration.

[0276] The macular area was photocoagulated by panretinal microscopy, and the results are shown in Figure 13. Nine spots were irradiated in each eye.

[0277] The laser spot leakage was obvious in the monkey eyes of the solvent group; there was no obvious leakage in the laser spot of the monkey eyes of the monkey eyes in all dose groups of #3AAV, indicating that #3AAV had a significant inhibitory effect on choroidal neovascularization at a dose of 1E+9vg / eye.

[0278] The above test results show that #3AAV can significantly and effectively inhibit choroidal angiogenesis in the macaque model.

[0279] Example 7 Pharmacokinetic Study

[0280] Experimental subjects: C57BL / 6J mice (Source: Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.).

[0281] Experimental Grouping: C57BL / 6J mice were randomly divided into four groups based on body weight. Group 1 was the vehicle group, consisting of three animals, and Groups 2, 3, and 4 were the drug-treated groups, each consisting of 24 animals. The dosing schedule for each group is shown in Table 7. The drug was administered via subretinal injection into the left eye. After a single dose, the animals were observed for up to one year. The day of drug administration was defined as Day 1 (D1) of the experimental period.

[0282] Table 7 Dosage regimen

[0283] After administration, a single-line OCT scan of the injection site was performed using a small animal retinal imaging system to confirm the success of the injection and locate the injection area. Successful administration was indicated by a visible bulge in the retina observed under a microscope during the injection process and by the presence of retinal neuroepithelial detachment and subretinal fluid accumulation at the injection site on the OCT scan.

[0284] Drug concentration samples were collected 3, 7, 14 days, and 4, 8, 12, and 24 weeks after administration. The method was as follows: mice were euthanized by carbon dioxide, the injected eyes were removed, ground and crushed at low temperature, and the supernatant was collected by high-speed centrifugation and transported to below -60°C for storage.

[0285] After 24 weeks of observation, the results of each group are shown in Figure 14 and are as follows:

[0286] In the 1.5E+6 vg dose group, C11 protein expression was detectable 7 days after intraocular injection of #3AAV. After 2 weeks, the intraocular C11 protein level stabilized, ranging from 0.2 ng / eye to 1.5 ng / eye at each time point. At the 24th week, the intraocular C11 protein level was 1.3 ng / eye.

[0287] In the 1.5E+7 vg dose group, C11 protein expression was detectable 3 days after intraocular injection of #3AAV. After 2 weeks, the protein level stabilized, ranging from 13 ng / eye to 42 ng / eye at each time point. At 24 weeks, the protein level was 35 ng / eye.

[0288] In the 1.5E+9 vg dose group, C11 protein expression was detected 3 days after intraocular injection of #3AAV. The intraocular protein concentration stabilized after 2 weeks, ranging from 1795 ng / eye to 3495 ng / eye at each time point. At week 24, the intraocular C11 protein content was 1873 ng / eye.

[0289] The above test results show that in the mouse model, #3AAV can normally express the target protein in situ after injection into the subretinal space, and there is a dose-dependent relationship with #3AAV; 24 weeks after injection, the intraocular concentration of the target protein can remain stable.

[0290] The present invention has been described through the above-described embodiments. However, it should be understood that the above-described embodiments are for illustrative and illustrative purposes only and are not intended to limit the present invention to the described embodiments. Furthermore, it will be understood by those skilled in the art that the present invention is not limited to the above-described embodiments and that further variations and modifications may be made based on the teachings of the present invention, all of which fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

[0291] sequence:

Claims

1. An expression cassette comprising: (a) a promoter selected from any one of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or any sequence having 85%, 90%, 95% or 99% or more identity with the sequence of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, or a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter; (b) a coding sequence selected from a nucleotide sequence encoding an anti-VEGF fusion protein or an anti-VEGF antibody, preferably, the coding sequence is a nucleotide sequence encoding an anti-VEGF fusion protein, and the coding sequence is operably linked to a promoter; and (c) a polyadenylation region (polyA) selected from any one of the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence and the rabbit globin (rbGlob) polyA sequence, or any sequence having 85%, 90%, 95% or 99% or more identity with the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence or the rabbit globin (rbGlob) polyA sequence; which is operably linked to the coding sequence.

2. The expression cassette of claim 1, further comprising an enhancer, wherein the enhancer is selected from any one of the CMV enhancer and the EF1α enhancer, or is selected from any sequence having 85%, 90%, 95% or 99% or more identity with the sequence of the CMV enhancer or the EF1α enhancer.

3. The expression cassette of claim 1, further comprising an intron, wherein the intron is selected from any one of an SV40 intron, an elongation factor 1 alpha (EF1α) intron, an actin intron, a CMV intron, and a bGlob intron, or is selected from any sequence having 85%, 90%, 95%, or 99% or more identity with an SV40 intron, an elongation factor 1 alpha (EF1α) intron, an actin intron, a CMV intron, or a bGlob intron.

4. The expression cassette according to any one of claims 2 or 3, comprising other regulatory elements, wherein the other regulatory elements are selected from any one of an expression enhancer sequence EES and a WHP virus post-transcriptional regulatory element (WPRE).

5. An expression cassette comprising (a) a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, and a glyceraldehyde 3-phosphate dehydrogenase promoter any of the promoters of cytomegalovirus (CMV) promoter, actin promoter, elongation factor 1α (EF1α) promoter, CB7 promoter, ubiquitin promoter or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or any sequence having 85%, 90%, 95% or 99% or more identity with the sequence of cytomegalovirus (CMV) promoter, actin promoter, elongation factor 1α (EF1α) promoter, CB7 promoter, ubiquitin promoter or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter; (b) an intron selected from any one of the SV40 intron, the elongation factor 1α (EF1α) intron, the actin intron, the CMV intron and the bGlob intron, or any sequence having 85%, 90%, 95% or 99% or more identity with the SV40 intron, the elongation factor 1α (EF1α) intron, the actin intron, the CMV intron or the bGlob intron; It is operably linked to a promoter; (c) a coding sequence operably linked to an intron; and (d) a polyadenylation region (polyA) selected from any one of the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence and the rabbit globin (rbGlob) polyA sequence, or any sequence having 85%, 90%, 95% or 99% or more identity with the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence or the rabbit globin (rbGlob) polyA sequence; It is operably linked to the coding sequence.

6. The expression cassette of claim 5, further comprising an enhancer, wherein the enhancer is selected from any one of the CMV enhancer and the EF1α enhancer, or selected from any sequence having 85%, 90%, 95% or 99% or more identity with the CMV enhancer or the EF1α enhancer.

7. The expression cassette of claim 5, comprising (a) CMV promoter or chicken β-actin promoter; (b) bGlob_int intron or chickenβ-actin intron; (c) a coding sequence; and (d) human growth hormone (HGH or hGH) polyA sequence; Preferably, the expression cassette further comprises a CMV enhancer or a WPRE regulatory sequence or an EES regulatory sequence.

8. An expression cassette, characterized in that The expression cassette has the structure of formula (A) in the order from 5' to 3': A1-A2-A3-A4-A5-A6 (A) In the formula, each "-" is independently a bond or a nucleotide linking sequence; A1 is an enhancer or does not exist, and the enhancer is selected from CMV enhancer and EF1α enhancer. or any sequence having 85%, 90%, 95% or 99% or more identity with the sequence of CMV enhancer or EF1α enhancer; A2 is a promoter, which is selected from any one of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or any sequence having 85%, 90%, 95%, or 99% or more identity with the sequence of a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, or a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter; A3 is an intron, and the intron is selected from any one of SV40 intron, elongation factor 1α (EF1α) intron, actin intron, CMV intron and bGlob intron, or any sequence having 85%, 90%, 95% or 99% or more identity with the sequence of SV40 intron, elongation factor 1α (EF1α) intron, actin intron, CMV intron or bGlob intron; A4 is the coding sequence; A5 is a regulatory sequence or does not exist, and the regulatory sequence is selected from any one of the expression enhancer sequence EES and the WHP virus post-transcriptional regulatory element (WPRE); and A6 is a polyadenylation region (polyA), the polyadenylation region (polyA) is selected from any one of the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence and the rabbit globin (rbGlob) polyA sequence, or is selected from any sequence having 85%, 90%, 95% or 99% or more identity with the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence or the rabbit globin (rbGlob) polyA sequence; the polyadenylation region (polyA) is operably linked to the coding sequence; The positions of A1 and A2 in formula (A) can be independently exchanged.

9. The expression cassette of any one of claims 1 to 8, wherein the expression cassette is selected from the group consisting of: (a) CMV enhancer-CMV promoter-bGlob intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (b) CMV promoter-bGlob intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (c) CMV enhancer-actin promoter-bGlob intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (d) CMV enhancer-CMV promoter-chicken β-actin intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (e) CMV enhancer-chicken β-actin promoter-chicken β-actin intron-coding sequence-human growth hormone (HGH or hGH) polyA sequence; (f) CMV promoter-bGlob intron-coding sequence-WPRE-human growth hormone (HGH or hGH) polyA sequence; and (g) CMV promoter-bGlob intron-coding sequence-EES-human growth hormone (HGH or hGH) polyA sequence; in, The chicken β-actin intron comprises or is a sequence shown in SEQ ID NO:5, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; the chicken β-actin promoter comprises or is a sequence shown in SEQ ID NO:3, or a sequence having 85%, 90%, 95% or 99% or more identity thereto.

10. The expression cassette according to any one of claims 5 to 9, wherein The coding sequence is a nucleotide sequence encoding a preventive and / or therapeutic protein. Preferably, the preventive and / or therapeutic protein is an antibody, a fusion protein or a polypeptide.

11. The expression cassette according to any one of claims 5 to 10, wherein The coding sequence is a nucleotide sequence encoding a protein for preventing and / or treating diseases or conditions selected from the following diseases or conditions: diseases or conditions associated with the central nervous system, peripheral nervous system, diseases or conditions associated with neurodegeneration, gastrointestinal diseases or conditions associated with smooth muscle contraction, respiratory diseases or conditions, endocrine diseases or conditions, diseases or conditions associated with inflammation, treatment of tumors, diseases or conditions causing withdrawal symptoms caused by chemical abuse, ophthalmic diseases, anesthesia, septic shock and biliary colic.

12. The expression cassette of any one of claims 5 to 11, wherein the coding sequence is a nucleotide sequence encoding a protein for preventing and / or treating a disease or condition selected from the group consisting of Stargardt's disease, retinoschisis, age-related macular degeneration (AMD), diabetic retinopathy, Leber congenital amaurosis (LCA), retinal detachment, cysts, cystoid macular edema, retinitis pigmentosa, corneal dystrophy, glaucoma and cataract.

13. The expression cassette according to any one of claims 5 to 12, wherein the coding sequence is a nucleotide sequence encoding an anti-VEGF fusion protein or antibody.

14. The expression cassette according to any one of claims 5 to 13, wherein the coding sequence is selected from the following nucleotide sequences: Nucleotide sequence encoding Conbercept; Nucleotide sequence encoding Aflibercept nucleotide sequence; nucleotide sequence V3 as shown in SEQ ID NO: 15; a nucleotide sequence encoding Faricimab; a nucleotide sequence encoding Bevacizumab; and a nucleotide sequence encoding Ranibizumab.

15. The expression cassette according to any one of claims 1 to 15, wherein the coding sequence is selected from the following nucleotide sequences: (a) a nucleotide sequence encoding a C11 protein as shown in SEQ ID NO: 16, wherein the amino acid sequence of the C11 protein is shown in SEQ ID NO: 17; (b) a nucleotide sequence encoding Conbercept; (c) the nucleotide sequence V3 as shown in SEQ ID NO: 15; (d) a nucleotide sequence encoding Faricimab; (e) a nucleotide sequence encoding Bevacizumab; and (f) Nucleotide sequence encoding Ranibizumab.

16. The expression cassette of any one of claims 1 to 15, wherein the expression cassette is selected from the group consisting of: (a) CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-human growth hormone (HGH or hGH) polyA sequence, wherein the amino acid sequence of the C11 protein is shown in SEQ ID NO: 17; (b) CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-human growth hormone (HGH or hGH) polyA sequence; (c) CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-WPRE-human growth hormone (HGH or hGH) polyA sequence, wherein the amino acid sequence of the C11 protein is shown in SEQ ID NO: 17; (d) CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-WPRE-human growth hormone (HGH or hGH) polyA sequence; (e) CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-EES-human growth hormone (HGH or hGH) polyA sequence; (f) CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-EES-human growth hormone (HGH or hGH) polyA sequence; (g) CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence V3 as shown in SEQ ID NO: 15-human growth hormone (HGH or hGH) polyA sequence; (h) CMV promoter-bGlob intron-nucleotide sequence as shown in SEQ ID NO: 15 V3-WPRE-human growth hormone (HGH or hGH) polyA sequence; and (i) CMV promoter-bGlob intron-nucleotide sequence V3-EES-human growth hormone (HGH or hGH) polyA sequence as shown in SEQ ID NO:

15.

17. The expression cassette of any one of claims 1 to 16, wherein the expression cassette is selected from the group consisting of: CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-human growth hormone (HGH or hGH) polyA sequence; CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-human growth hormone (HGH or hGH) polyA sequence; CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence encoding aflibercept-human growth hormone (HGH or hGH) polyA sequence; CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-WPRE-human growth hormone (HGH or hGH) polyA sequence; CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-WPRE-human growth hormone (HGH or hGH) polyA sequence; CMV promoter-bGlob intron-nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16-EES-human growth hormone (HGH or hGH) polyA sequence; CMV promoter-bGlob intron-nucleotide sequence encoding Conbercept-EES-human growth hormone (HGH or hGH) polyA sequence; CMV enhancer-CMV promoter-bGlob intron-nucleotide sequence V3 as shown in SEQ ID NO: 15-human growth hormone (HGH or hGH) polyA sequence; CMV promoter-bGlob intron-nucleotide sequence V3-WPRE-human growth hormone (HGH or hGH) polyA sequence as shown in SEQ ID NO: 15; and CMV promoter-bGlob intron-nucleotide sequence V3 as shown in SEQ ID NO: 15-EES-human growth hormone (HGH or hGH) polyA sequence, in, The amino acid sequence of the C11 protein is shown in SEQ ID NO: 17, the sequence of WPRE is shown in SEQ ID NO: 13, and the nucleotide sequence of EES is shown in SEQ ID NO:

12.

18. An expression cassette, characterized in that The expression cassette comprises, in order from 5' to 3': An enhancer, wherein the enhancer is a CMV enhancer or an EF1α enhancer, or any sequence having 85%, 90%, 95% or 99% or more identity with the sequence of the CMV enhancer or the EF1α enhancer; A promoter, wherein the promoter is selected from a cytomegalovirus (CMV) promoter, an actin promoter, an elongation factor 1α (EF1α) promoter, a CB7 promoter, a ubiquitin promoter, and a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, or is any sequence having 85%, 90%, 95%, or 99% or more identity with the sequence of the cytomegalovirus (CMV) promoter, the actin promoter, the elongation factor 1α (EF1α) promoter, the CB7 promoter, the ubiquitin promoter, or the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter; An intron, wherein the intron is selected from the group consisting of an SV40 intron, an elongation factor 1α (EF1α) intron, an actin intron, a CMV intron, and a bGlob intron, or any sequence having 85%, 90%, 95%, or 99% or more identity with the sequence of the SV40 intron, the elongation factor 1α (EF1α) intron, the actin intron, the CMV intron, or the bGlob intron; A coding sequence, wherein the coding sequence encodes a target protein; and A polyadenylation region (polyA), wherein the polyadenylation region (polyA) is selected from the group consisting of a human growth hormone (HGH or hGH) polyA sequence, a bovine growth hormone (BGH or bGH) polyA sequence, a β-globin (β-globin) polyA sequence, and a rabbit globin (rbGlob) polyA sequence, or is any sequence having 85%, 90%, 95% or 99% or more identity with the human growth hormone (HGH or hGH) polyA sequence, the bovine growth hormone (BGH or bGH) polyA sequence, the β-globin (β-globin) polyA sequence, or the rabbit globin (rbGlob) polyA sequence.

19. An expression cassette, characterized in that The expression cassette comprises, in order from 5' to 3': CMV enhancer; Cytomegalovirus (CMV) promoter; bGlob intron; A coding sequence, wherein the coding sequence is a nucleotide sequence encoding an anti-VEGF fusion protein or an anti-VEGF antibody; preferably, the coding sequence is a nucleotide sequence encoding Conbercept, Aflibercept, Faricimab, Bevacizumab or Ranibizumab; and Human growth hormone (HGH or hGH) polyA sequence.

20. An expression cassette, characterized in that The expression cassette comprises, in order from 5' to 3': A CMV enhancer, the CMV enhancer comprising the sequence shown in SEQ ID NO:4, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the CMV enhancer is the sequence shown in SEQ ID NO:4, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; A cytomegalovirus (CMV) promoter, the cytomegalovirus (CMV) promoter comprising the sequence shown in SEQ ID NO: 1, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the cytomegalovirus (CMV) promoter is the sequence shown in SEQ ID NO: 1, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; bGlob intron, the bGlob intron comprising the sequence shown in SEQ ID NO:6, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the bGlob intron is the sequence shown in SEQ ID NO:6, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; A coding sequence, wherein the coding sequence comprises or is a nucleotide sequence encoding Conbercept, Ranibizumab or Aflibercept, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the coding sequence comprises or is a nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16 or a nucleotide sequence as shown in SEQ ID NO: 15, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the coding sequence comprises or is a nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16; and A human growth hormone (HGH or hGH) polyA sequence, wherein the human growth hormone (HGH or hGH) polyA sequence comprises or is the sequence shown in SEQ ID NO:9, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the human growth hormone (HGH or hGH) polyA sequence is the sequence shown in SEQ ID NO:9, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; Wherein, the amino acid sequence of the C11 protein is shown in SEQ ID NO:

17.

21. A carrier, characterized in that The vector contains the expression cassette according to any one of claims 1 to 20.

22. A recombinant adeno-associated virus (rAAV), wherein: The rAAV comprises the expression cassette of any one of claims 1 to 20; preferably, the rAAV further comprises an AAV5' inverted terminal repeat sequence (L-ITR) located upstream of the 5' end of the nucleotide sequence of the expression cassette and an AAV3' inverted terminal repeat sequence (R-ITR) located downstream of the 3' end of the nucleotide sequence of the expression cassette; preferably, the AAV5' inverted terminal repeat sequence (L-ITR) comprises or is the sequence shown in SEQ ID NO: 19 or a sequence having 85%, 90%, 95% or 99% or more identity thereto, and the AAV3' inverted terminal repeat sequence (R-ITR) comprises or is the sequence shown in SEQ ID NO: 20 or a sequence having 85%, 90%, 95% or 99% or more identity thereto.

23. A recombinant adeno-associated virus (rAAV), wherein: The rAAV comprises a nucleic acid comprising, in 5' to 3' order: AAV5' inverted terminal repeat sequence (L-ITR), preferably the AAV5' inverted terminal repeat sequence (L-ITR) comprises or is the sequence shown in SEQ ID NO:19 or a sequence having 85%, 90%, 95% or 99% or more identity thereto; A CMV enhancer, the CMV enhancer comprising the sequence shown in SEQ ID NO:4, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the CMV enhancer is the sequence shown in SEQ ID NO:4, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; A cytomegalovirus (CMV) promoter, the cytomegalovirus (CMV) promoter comprising the sequence shown in SEQ ID NO: 1, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the cytomegalovirus (CMV) promoter is the sequence shown in SEQ ID NO: 1, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; bGlob intron, the bGlob intron comprising the sequence shown in SEQ ID NO:6, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the bGlob intron is the sequence shown in SEQ ID NO:6, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; A coding sequence, wherein the coding sequence is a nucleotide sequence encoding an anti-VEGF fusion protein or an anti-VEGF antibody; preferably, the coding sequence comprises or is a nucleotide sequence encoding Conbercept, Ranibizumab or Aflibercept, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the coding sequence comprises or is a nucleotide sequence encoding a C11 protein as shown in SEQ ID NO:16 or a nucleotide sequence as shown in SEQ ID NO:15, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the coding sequence comprises or is a nucleotide sequence encoding a C11 protein as shown in SEQ ID NO:16; wherein the amino acid sequence of the C11 protein is as shown in SEQ ID NO:17; A human growth hormone (HGH or hGH) polyA sequence, wherein the human growth hormone (HGH or hGH) polyA sequence comprises or is the sequence shown in SEQ ID NO: 9, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; preferably, the human growth hormone (HGH or hGH) polyA sequence is the sequence shown in SEQ ID NO: 9, or a sequence having 85%, 90%, 95% or 99% or more identity thereto; and AAV3' inverted terminal repeat sequence (L-ITR), preferably the AAV3' inverted terminal repeat sequence (R-ITR) comprises or is a sequence shown in SEQ ID NO: 20 or has 85%, 90%, Sequences with 95% or more identity.

24. The recombinant adeno-associated virus (rAAV) of claim 17, wherein: The nucleotide sequence of the nucleic acid contained in the rAAV is shown in SEQ ID NO:

18.

25. The recombinant adeno-associated virus (rAAV) according to any one of claims 17 to 18, wherein The rAAV further comprises an AAV capsid.

26. The recombinant adeno-associated virus (rAAV) according to any one of claims 17 to 18, wherein The adeno-associated virus is selected from AAV1 (AAV-1), AAV2 (AAV-2), AAV3 (AAV-3), AAV4 (AAV-4), AAV5 (AAV-5), AAV6 (AAV-6), AAV7 (AAV-7), AAV8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV; Preferably, the adeno-associated virus is AAV6 type (AAV-6) or AAV8 type (AAV-8).

27. A recombinant adeno-associated virus (rAAV), wherein: The rAAV comprises: AAV capsid; and A nucleic acid comprising, in 5' to 3' order: AAV5' inverted terminal repeat sequence (L-ITR), wherein the AAV5' inverted terminal repeat sequence (L-ITR) is the sequence shown in SEQ ID NO: 19; CMV enhancer, wherein the CMV enhancer is the sequence shown in SEQ ID NO: 4; A cytomegalovirus (CMV) promoter, wherein the cytomegalovirus (CMV) promoter is the sequence shown in SEQ ID NO: 1; bGlob intron, wherein the bGlob intron is the sequence shown in SEQ ID NO: 6; A coding sequence, wherein the coding sequence is a nucleotide sequence encoding Conbercept, Ranibizumab or Aflibercept; preferably, the coding sequence is a nucleotide sequence encoding C11 protein as shown in SEQ ID NO: 16 or a nucleotide sequence as shown in SEQ ID NO: 15, wherein the amino acid sequence of the C11 protein is as shown in SEQ ID NO: 17; A human growth hormone (HGH or hGH) polyA sequence, wherein the human growth hormone (HGH or hGH) polyA sequence is the sequence shown in SEQ ID NO: 9; and AAV3' inverted terminal repeat sequence (R-ITR), wherein the AAV3' inverted terminal repeat sequence (R-ITR) is the sequence shown in SEQ ID NO: 20; Wherein, the adeno-associated virus is AAV8 type (AAV-8).

28. A recombinant adeno-associated virus (rAAV) having an AAV capsid suitable for intraocular injection, wherein the AAV comprises a vector genome packaged within the capsid, the vector genome comprising: (a) AAV5' inverted terminal repeat sequence (L-ITR); (b) the expression cassette of any one of claims 1 to 16; (c) AAV 3' inverted terminal repeat (R-ITR).

29. The recombinant adeno-associated virus according to claim 28, wherein The adeno-associated virus is selected from AAV1 (AAV-1), AAV2 (AAV-2), AAV3 (AAV-3), AAV4 (AAV-4), AAV5 (AAV-5), AAV6 (AAV-6), AAV7 (AAV-7), AAV8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV and ovine AAV; Preferably, the adeno-associated virus is selected from AAV6 (AAV-6) or AAV8 (AAV-8).

30. The recombinant adeno-associated virus according to claim 28 or 29, characterized in that The L-ITR sequence comprises the sequence shown in SEQ ID NO: 19 or a sequence having at least 85%, at least 90%, at least 95% or at least 99% identity thereto.

31. The recombinant adeno-associated virus of any one of claims 28-30, wherein the R-ITR sequence comprises the sequence shown in SEQ ID NO: 20 or a sequence having at least 85%, at least 90%, at least 95% or at least 99% identity thereto.

32. A pharmaceutical composition comprising the expression cassette of any one of claims 1 to 20, the vector of claim 21 or the adeno-associated virus of any one of claims 22 to 31, and a pharmaceutically acceptable excipient.

33. An isolated host cell transfected or transduced with the expression cassette of any one of claims 1-20, the vector of claim 21, or the adeno-associated virus of any one of claims 22-31.

34. A method for expressing a transgene in a mammalian cell, the method comprising contacting one or more mammalian cells with a certain amount of the recombinant adeno-associated virus of any one of claims 22-31, wherein the target protein is expressed at a certain level in the one or more mammalian cells.

35. A method for treating or preventing a disease in a mammal in need of treatment or prevention of the disease, the method comprising administering to the mammal an effective amount of the recombinant adeno-associated virus according to any one of claims 22 to 31, the pharmaceutical composition according to claim 32, and / or the host cell according to claim 33; Preferably, the disease is an eye disease, and further preferably, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization and diabetic retinopathy; Preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal, and further preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal by intraocular injection, intravitreal injection or subretinal space.

36. Use of the recombinant adeno-associated virus according to any one of claims 22 to 31, the pharmaceutical composition according to claim 32 and / or the host cell according to claim 33 in the preparation of a medicament for treating or preventing a disease in a mammal in need of such treatment or prevention; Preferably, the disease is an eye disease, further preferably, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization and diabetic retinopathy; Preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal, and further preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal by intraocular injection, intravitreal injection or subretinal space.

37. The recombinant adeno-associated virus according to any one of claims 22 to 31, the pharmaceutical composition according to claim 32 and / or the host cell according to claim 33, for use in treating or preventing a disease in a mammal in need of such treatment or prevention; Preferably, the disease is an eye disease, further preferably, the eye disease is selected from age-related macular degeneration (AMD), wet AMD, dry AMD, retinal neovascularization, choroidal neovascularization and diabetic retinopathy; Preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal, and further preferably, the recombinant adeno-associated virus, pharmaceutical composition or host cell is administered to the eye of the mammal by intraocular injection, intravitreal injection or subretinal space.

38. A product comprising: (a) a first container, wherein the first container comprises the recombinant adeno-associated virus according to any one of claims 22 to 31 or the pharmaceutical composition according to claim 32; and (b) an injection needle.

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