AAV drug for treating angiogenesis-related fundus diseases

Abstract

The present invention belongs to the technical field of recombinant adeno-associated virus (rAAV) gene therapy, and relates to the delivery by rAAV, the preparation of an Fc gene-modified VEGF receptor fusion protein or an anti-VEGF antibody, and the use in the treatment of angiogenesis-related fundus diseases such as age-related macular degeneration, wet macular degeneration, diabetic retinopathy, etc. Based on the aflibercept-expressing gene, the present invention achieves long-term stable expression of the target gene in the RPE layer by recombinant AAV delivery through gene modification and vector optimization, and delivers the optimized target gene sequence to the fundus cells of patients, realizing "long-term effects with a single administration".

Description

Technical Field

[0001] The present invention belongs to the technical field of recombinant adeno-associated virus (rAAV) gene therapy, and relates to the delivery by rAAV, the preparation of an Fc gene-modified VEGF receptor fusion protein or an anti-VEGF antibody, and the use in the treatment of angiogenesis-related fundus diseases such as age-related macular degeneration, wet macular degeneration, diabetic retinopathy, and the like.

Background Art

[0002] Angiogenesis occurs in intraocular tissues such as the retina, choroid, macula, optic disc, cornea, iris, and ciliary body, and may cause pathological changes such as bleeding, exudation, and proliferation of the tissues in these parts. It includes age-related macular degeneration (AMD), diabetic retinopathy (DR), retinopathy of prematurity (ROP), etc., which have a significant impact on vision and are the main cause of blindness in the elderly.

[0003] AMD is mainly divided into two types: non-neovascular (dry AMD) and neovascular (wet AMD). Vascular endothelial growth factor (VEGF) is one of the important proteins that promote angiogenesis. Currently, anti-VEGF drug therapy (including Ranibizumab, Aflibercept, Bevacizumab, Conbercept, Brolucizumab, etc.) has become the standard treatment protocol in the treatment of wet AMD.

[0004] Diabetic retinopathy (DR) is a common microvascular complication of diabetes, with a high blindness rate and a complex pathogenesis. The pathological features of DR are the formation of retinal neovascularization and the disruption of the blood-retinal barrier. The use of anti-VEGF drug therapy is also relatively common.

[0005] In recent years, with the progress of gene delivery systems and editing technologies, the field of gene therapy has been developing rapidly. Currently, recombinant adeno-associated virus (rAAV) has become a major platform for in vivo gene therapy. In the genome packaged by rAAV, the AAV protein coding sequence is deleted while a therapeutic gene expression cassette is added. The only virus-derived sequence is the ITR, which is necessary to direct genome replication and packaging during vector production. AAV has the advantages of a broad host range, high safety, low immunogenicity, tissue affinity, and stable long-term expression, so it is widely used in basic research and clinical trials.

[0006] Currently, anti-VEGF therapy has become the first choice for the treatment of neovascular eye diseases. Clinically approved drugs include antibodies and antibody fragments (Bevacizumab, Ranibizumab, and Brolucizumab), fusion proteins (Aflibercept and Conbercept), and the nucleic acid aptamer Pegaptanib, and the bispecific antibody Vabysmo (Faricimab-svoa).

[0007] Eylea (Aflibercept) is a recombinant fusion protein composed by fusing domain 2 of VEGFR-1, domain 3 of VEGFR-2, and the Fc fragment of IgG1. By binding to vascular endothelial growth factor isoforms A and B (VEGF-A and VEGF-B) and placental growth factor (PlGF), it inhibits angiogenesis and reduces vascular permeability. The US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) respectively approved Aflibercept as a therapeutic drug for intravitreal injection for three indications, namely macular edema secondary to retinal vein occlusion, wet age-related macular degeneration, and diabetic macular edema, before August 2014.

[0008] Conbercept (Lumitin, Conbercept) is a Chinese biological product that has obtained the international nonproprietary name from the World Health Organization. This drug is a recombinant antibody drug, similar in structure to aflibercept, but different in that Conbercept contains the Ig-like region of the VEGF receptor. Such a structure can improve the affinity for VEGF, block all isoforms of VEGF-A, VEGF-B, and placental growth factor, increase the binding rate, extend the half-life of the drug in vivo, and has a molecular weight of 142 KD. Since the end of 2013, it has been approved by the China National Food and Drug Administration as a therapeutic drug for wAMD.

[0009] Protein drug therapies such as aflibercept are currently the standard treatment for neovascular eye diseases. However, long-term anti-VEGF therapy may increase the incidence of RPE atrophy, choroidal atrophy, and geographic atrophy. In a multi-center clinical study, after 7 years of anti-VEGF treatment, it was found that the vision of some patients decreased to or below the baseline level, and furthermore, the occurrence of macular atrophy and fibrosis was accompanied.

[0010] In addition, the standard treatment every 4 to 8 weeks is actually difficult to maintain. Repeated injections may increase the risk of inflammation, infections, and other side effects in some patients, the treatment cost is high, and the patient compliance is poor.

[0011] Therefore, in order to improve the treatment effect of neovascular eye disease patients, it is necessary to develop alternative or complementary treatment methods that can reduce the dosing frequency, or bring lifelong effects with a single dose, and improve patient compliance. Summary of the Invention

[0012] Based on the aflibercept-expressing gene, through gene modification and vector optimization, long-term stable expression of the target gene in the RPE layer is achieved by recombinant AAV delivery, and the optimized target gene sequence is delivered to the fundus cells of patients. It realizes the "long-term effect with a single administration", provides patients with a better treatment method that is safer, more economical, and more convenient, reduces the burden of existing clinical treatments for neovascular eye diseases, and meets unmet clinical needs.

[0013] Considering its excellent clinical performance, aflibercept is a candidate molecule for delivery in gene therapy, which can express stably in vivo for a long time, maintain efficacy, and have low risk.

[0014] Choroidal neovascularization (CNV) is a characteristic lesion of exudative nAMD and an important cause of vision loss in the elderly. Currently, laser-induced CNV animal models are commonly used to evaluate the efficacy of AMD therapeutic drugs, including protein pharmaceuticals such as bevacizumab, aflibercept, and ranibizumab. In the CNV models of laser-induced nAMD in rodents and NHPs, the transient vascular leakage and neovascular reaction in the choroidal vascular system persist for 2-3 weeks (rodents) or 6-8 weeks (NHPs) after laser disruption of Bruch's membrane and then resolve spontaneously.

[0015] By establishing models of persistent and recurrent vascular leakage and angiogenesis, the evaluation of long-acting interventions for addressing multiple clinical manifestations of pathological vascular instability and angiogenesis is significantly facilitated and accelerated. The DL-α-aminoadipic acid (DL-AAA) model is a chronic leakage model reported in rats and rabbits and is commonly used for candidate drug screening in these species. DL-AAA is a selective gliotoxin that inhibits the action of glutamine synthetase, impairs the retinal homeostasis function of extensive Müller cells, causes glial dysfunction and death, and thereby has been reported to cause disruption of the blood-retinal barrier. Two months after injection of D-LAAA, the subretinal blood-retinal barrier in rats is disrupted, and vascular leakage and tortuosity increase. According to several recent studies, 2 - 36 months after intravitreal injection (IVT) of DL-AAA in rabbits, vascular leakage and RNV increased, but the anti-VEGF drugs bevacizumab, ranibizumab, aflibercept, and DARPins targeting VEGF-A165 suppressed this type of disease. Although the anatomical structures of retinal blood vessels and neurons are different between rats and rabbits and humans, they are basically the same between monkeys and humans. The retinal vascular structure, the boundary of retinal segmentation and the basal layer, the abundance ratio of subtypes of retinal neuronal cells and glial cells, and the presence of the macula are homologous between monkeys and humans. Also, preclinical models of chronic retinal vascular leakage and neovascularization can screen the effectiveness of short-acting and long-acting anti-angiogenic compounds at multiple stages of disease onset. In the present invention, the effect is verified using a chronic vascular leakage NHP model by DL-AAA.

[0016] The first aspect provided by the present invention is a Fc fragment variant of human IgG1. The variant is obtained by the occurrence of at least one mutation among T250A, L251A, M252L, I253A / D / P, S254A, T256A, L309A, H310L / V / A / D / E / Q, Q311A, L314A, M428L / I, H433L / V / A, N434L / V / A, H435L / V / A, Y436A. Those numbers are numbered according to the EU index.

[0017] Preferably, the variant has a single mutation of one of H310A, H310L, H435A, H435L, I253A, I253D, I253P, H310D, H310E or H310Q.

[0018] More preferably, the variant has a single point mutation of one of H310A, H310E or H435A.

[0019] Preferably, the variant has a double mutation of one of M252L / M482L, M252L / M482I, M252L / M482L, T250A / H310L, L251A / H310L, I253A / H310L, S254A / H310L, T256A / H310L, L309A / H310L, H310L / Q311A, H310L / L314A, H310L / H433A, H310L / N434A, H310L / H435A, H310L / Y436A, I253A / H310A, I253A / H435A or H310A / H435A.

[0020] More preferably, the variant has a double mutation of H310A / H435A.

[0021] Preferably, the variant has a triple mutation of one of M252L / H310L / M482L, M252L / H310V / M482L, M252L / M482L / H433L, M252L / M482L / H433V, M252L / M482L / N434L or M252L / M482L / H435V.

[0022] Preferably, the variant has a quadruple mutation of one of M252L / M482L / N434V / H435L, M252L / H310L / M482L / H433L, M252L / H310L / M482L / N434L or M252L / H310L / M482L / H435L.

[0023] More preferably, the Fc fragment variant has the sequence shown in SEQ ID NO: 1, 2, 3 or 4.

[0024] The second aspect provided by the present invention is a VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody.

[0025] The VEGF receptor recombinant fusion protein is a fusion of domain 2 of VEGFR-1, domain 3 of VEGFR-2, and the Fc fragment variant of IgG1 described in the first aspect.

[0026] Furthermore, domain 2 of the VEGFR-1 is shown in SEQ ID NO: 5.

[0027] Furthermore, domain 3 of the VEGFR-2 is shown in SEQ ID NO: 6.

[0028] Preferably, the Fc fragment variant of IgG1 is H310A (SEQ ID NO: 1), H310E (SEQ ID NO: 2), H435A (SEQ ID NO: 3), H310A / H435A (SEQ ID NO: 4).

[0029] More preferably, the amino acid sequence of the VEGF receptor recombinant fusion protein is shown in SEQ ID NO: 7, 8, 9 or 10.

[0030] The anti-VEGF recombinant antibody is obtained by replacing the Fc fragment of a conventional anti-VEGF antibody with the Fc fragment variant of IgG1 described in the first aspect.

[0031] Furthermore, conventional anti-VEGF antibodies include, but are not limited to, Bevacizumab, Ranibizumab, and Brolucizumab.

[0032] Furthermore, the anti-VEGF recombinant antibody is obtained by replacing the Fc of Bevacizumab with the Fc fragment variant of IgG1 described in the first aspect, and more preferably has the amino acid sequence shown in SEQ ID NO: 11.

[0033] A third aspect provided by the present invention is an AAV viral vector expression cassette that expresses the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody described in the second aspect. The expression cassette includes a structure represented by the following formula I from the 5'-end to the 3'-end. ITR-E1-E2-E3-E4-ITR Formula (I) In the formula, ITR is an inverted terminal repeat sequence, E1 is a promoter, E2 is a signal peptide, E3 is a nucleotide sequence encoding the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody described in the second aspect, E4 is a Poly A sequence.

[0034] Furthermore, the ITR (inverted terminal repeat sequence) is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9, and preferably, the ITR sequence is selected from AAV2.

[0035] Furthermore, the promoter is a DNA sequence capable of initiating transcription of a target gene. This sequence can be recognized by RNA polymerase and start transcription and synthesis of RNA. The promoter includes, but is not limited to, natural, optimized or combined promoters.

[0036] Furthermore, the promoter is preferably a CMV, CBA, EF1a, SV40, PGK1, Ubc, CAG, TEF1, U6 or H1 promoter.

[0037] Furthermore, the signal peptide is derived from, but not limited to, Human OSM, Gaussia luc or Albumin (HSA).

[0038] Furthermore, the Poly A sequence is selected from bGH PolyA, SV40 PolyA or hGH PolyA.

[0039] Furthermore, the above expression cassette further includes regulatory elements. The expression regulatory elements include, but are not limited to, (1) regulatory elements for regulating the expression of the target protein (e.g., IRES for initiating the translation of downstream genes), (2) regulatory elements for expressing miRNA and siRNA sequences, (3) introns, (4) localization sequences for localizing and expressing the target protein in the cell nucleus, cytoplasm or various cell organelles and secreting it extracellularly, (5) some kozak sequences (the kozak sequence is G / N-C / N-C / N-ANNAUGG, such as GCCACCAUGG, etc.), (6) enhancers (the enhancers may be derived from SV40 virus, CMV virus or adenovirus, etc.), and (7) functional regulatory elements selected from WPRE.

[0040] Furthermore, the above expression cassette further includes a label element. The label element includes, but is not limited to, FLAG, HA, MYC, fluorescent proteins, luciferase, SUMO proteins, ubiquitin proteins, GST, etc.

[0041] Preferably, the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody is one that expresses the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody described in the second aspect on the AAV viral vector.

[0042] Furthermore, the AAV viral vector includes, but is not limited to, pAAV-CMV, pX601, pX551, pAAV-MCS plasmid, etc.

[0043] Preferably, the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody includes ITR-CMV promoter-CMV enhancer-intron-signal peptide-Kozak sequence-VEGF receptor recombinant fusion protein / anti-VEGF recombinant antibody coding sequence-PolyA-ITR.

[0044] More preferably, the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein has the sequence shown in SEQ ID NO: 12.

[0045] More preferably, the AAV viral vector expression cassette expressing the anti-VEGF recombinant antibody has the sequence shown in SEQ ID NO: 13.

[0046] The third aspect provided by the present invention is an adeno-associated virus packaging vector system. The packaging vector system includes an AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein or the anti-VEGF recombinant antibody described in the third aspect, a vector carrying the AAV rep and cap genes, and a helper virus vector. The above vectors become AAV viruses by packaging.

[0047] Furthermore, the vector carrying the AAV rep and cap genes includes, but is not limited to, AAV1, AAV2, AAV5, AAV8, AAV9, AAV-R100, AAV-NN, AAV-GL, AAV8-Y447F, AAV8-Y733F, AAV8-Y447F / Y733F, AAV-DJ or AAV7m8 vectors, etc.

[0048] Furthermore, the helper virus vector is an adenovirus or herpesvirus helper virus vector, and preferably, it is a pHelper plasmid.

[0049] The fifth aspect provided by the present invention is a method for packaging adeno-associated virus. In this method, the adeno-associated virus packaging vector system described in the fourth aspect is introduced into a host cell to perform virus packaging.

[0050] Furthermore, an AAV viral vector expression cassette expressing a VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody in the packaging vector system according to the fourth aspect, a vector carrying the AAV rep and cap genes, and a helper viral vector are introduced into a host cell for virus packaging.

[0051] Furthermore, the host cell is a cell line capable of virus replication and stable inheritance, including but not limited to cells such as Hela-S3, HEK-293, HEK-293T, HEK-293FT, Expi293F, A549, and Sf9.

[0052] Preferably, the host cell is HEK-293, HEK-293T cell, or Expi293F cell.

[0053] The sixth aspect provided by the present invention is an adeno-associated virus obtained by the packaging method according to the fifth aspect.

[0054] Preferably, it is obtained by introducing an AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein shown in SEQ ID NO: 12, an AAV8 Rep-Cap plasmid, and a pHelper plasmid into a host cell Expi293F cell for virus packaging.

[0055] Preferably, it is obtained by introducing an AAV viral vector expression cassette expressing the anti-VEGF recombinant antibody shown in SEQ ID NO: 13, an AAV8 Rep-Cap plasmid, and a pHelper plasmid into a host cell Expi293F cell for virus packaging.

[0056] The seventh aspect provided by the present invention is a formulation, prescription, or drug containing an AAV viral vector expression cassette expressing a VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody according to the third aspect, or an adeno-associated virus according to the sixth aspect.

[0057] Furthermore, the formulation, prescription or drug may be in any dosage form, including but not limited to injection dosage forms and ointment dosage forms.

[0058] Furthermore, in the formulation, prescription or drug, the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody, or the adeno-associated virus, is the only active ingredient.

[0059] The eighth aspect provided by the present invention is the use of the AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody described in the third aspect, or the adeno-associated virus described in the sixth aspect, in the preparation of a formulation, prescription or drug for use in angiogenesis-related fundus diseases, particularly diseases such as age-related macular degeneration, wet macular degeneration, and diabetic retinopathy.

[0060] Furthermore, the administration method of the formulation, prescription or drug is unilateral administration or bilateral administration.

[0061] Furthermore, the administration forms include intravitreal injection, subretinal injection, suprachoroidal injection, etc.

[0062] Furthermore, it may be a single administration or multiple administrations throughout life, and the total dose is 1×10 8 -1×10 13 virus genomes / eye.

[0063] Beneficial effects 1. The present invention reduces the affinity with FcRn due to Fc mutation, improves the PK behavior in the eye, reduces the entry into the blood, does not affect the stability, significantly increases the accumulation in the eye, and extends the half-life. Even if it enters the blood, due to the reduced FcRn affinity, the half-life in the circulatory system is shortened, and other systemic safety risks are avoided. 2. Through the combined design of Fc mutation and vector elements, continuous and stable expression in vivo is achieved, which has obvious advantages compared with the in vivo protein expression data of existing similar AAV products. 3. The combination of delivery by the rAAV vector element designed in the present invention and the Fc-mutated and modified VEGF antagonist has high durability compared to the control drug of existing commercially available protein drugs. 4. The AAV virus prepared in the present invention can achieve lifelong remission or cure of angiogenesis-related fundus diseases with a single administration, or can reduce the number of administrations of protein drugs. 5. The same combination design can use various administration forms such as intravitreal injection, subretinal administration, suprachoroidal administration, etc.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0065] To make the object, technical means and advantages of the present invention clearer, the present invention will be further described in more detail by specific examples below. The following specific examples are for explaining the present invention and do not limit the present invention.

[0066] In the present invention, a VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody optimized by AAV is delivered, and an optimized target gene sequence is delivered to the eye of a patient by subretinal injection, intravitreal injection or suprachoroidal injection, etc., to achieve long-term stable expression of the target gene in the retina, enabling lifelong treatment with a single low-dose administration, providing a safer, more convenient and excellent treatment method for patients, reducing the clinical treatment burden of existing angiogenesis-related fundus diseases, solving the problems of low patient compliance and persistence, and improving the treatment effect of patients.

[0067] An Fc fusion protein refers to a novel recombinant protein produced by fusing a protein molecule with specific biological activity and the Fc fragment of an immunoglobulin (such as IgG, IgA, etc.) through techniques such as genetic engineering. It not only retains the biological activity of the functional protein molecule but also has the properties of an antibody, such as binding to related Fc receptors to extend the half-life and inducing antibody-dependent cell-mediated cytotoxicity effects, and is very important in the diagnosis and treatment of diseases. FcRn is an IgG antibody receptor located on the cell membrane surface. Its protein structure is similar to that of MHC-I molecules and is mainly expressed in endothelial cells (it can also be detected in other tissues or cells), and its structure includes a heterodimer composed of an α chain and β2 microglobulin. FcRn binds to the Fc portion of IgG to prevent the degradation of IgG molecules by lysosomes, can extend the half-life of IgG in vivo, and is involved in the transport, maintenance, distribution, and metabolism of IgG in vivo. The Fc portion of IgG interacts with the neonatal Fc receptor (FcRn, Brambell receptor) to prevent degradation within lysosomes and enable the long-term retention of antibodies in the serum. Other biological roles of FcRn include perinatal IgG transport, antibody-mediated antigen presentation, and IgG transport across epithelial and endothelial barriers. Modification of the IgG:FcRn interaction is being increasingly studied to improve the therapeutic or diagnostic applications of antibodies. The in vivo action pathway of intravitreal injection of IgG or Fc fusion proteins mainly passes through the blood-retinal barrier via FcRn on retinal RPE and endothelial cells, enters the systemic circulation, and is gradually eliminated. Therefore, in ocular metabolism, FcRn mediates the blood entry and clearance of IgG or Fc fusion proteins.

[0068] The optimized VEGF receptor recombinant fusion protein described in the present invention comprises a recombinant protein of Domain 2 of human vascular endothelial growth factor (VEGF) receptor 1 and Domain 3 of receptor 2 and a variant of the Fc region of human immunoglobulin. In one embodiment of the present invention, multiple groups of single-point or multiple-point mutants of Fc are designed. The selection of the mutation sites of the present invention preferably takes into account the stability and persistence of the in vivo and in vitro target protein expression of the rAAV composition in addition to the FcRn affinity. Specific mutation sites of the Fc variant include T250A, L251A, M252L, I253A / D / P, S254A, T256A, L309A, H310L / V / A / D / E / Q, Q311A, L314A, M428L / I, H433L / V / A, N434L / V / A, H435L / V / A, Y436A, etc. In one embodiment of the present invention, the preferred mutation sites are single-point mutations, including H310A, H310L, H435A, H435L, I253A, I253D, I253P, H310D, H310E, H310Q. In another embodiment of the present invention, the preferred mutation sites are double-point mutations, including M252L and M482L, M252L and M482I, M252L and M482L, T250A and H310L, L251A and H310L, I253A and H310L, S254A and H310L, T256A and H310L, L309A and H310L, H310L and Q311A, H310L and L314A, H310L and H433A, H310L and N434A, H310L and H435A, H310L and Y436A, I253A and H310A, I253A and H435A, H310A and H435A, etc. In another embodiment of the present invention, the preferred mutation sites are triple-point mutations, including M252L, H310L and M482L, M252L, H310V and M482L, M252L, M482L and H433L, M252L, M482L and H433V, M252L, M482L and N434L, M252L, M482L and H435V.In another embodiment of the present invention, the preferred mutation site is a quadruple mutation, including M252L, M482L, N434V and H435L, M252L, H310L, M482L and H433L, M252L, H310L, M482L and N434L, M252L, H310L, M482L and H435L. In another embodiment of the present invention, the more preferred single-point mutation sites are H310A, H310E, H435A, and the obtained Fc mutants have the amino acid sequences shown in SEQ ID NO: 1, 2 or 3. In another embodiment of the present invention, the more preferred double-point mutation site is H310A / H435A, and the Fc mutant has the amino acid sequence shown in SEQ ID NO: 4.

[0069] The above Fc mutants are mutations (SEQ ID NO: 15) generated based on the following wild-type Fc fragment of human IgG1. DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.

[0070] In the present invention, the numbers of amino acid residues in the Fc fragment are the numbers of immunoglobulin heavy chains, and are based on the EU index in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991) (incorporated herein by reference expressly). The "EU index / number" in this specification refers to the EU code number of the constant region residues of the human IgG1 antibody. This number is known to those skilled in the art and is commonly used in the art. You may refer to the URL https: / / www.imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html

[0071] More preferably, in another embodiment of the present invention, the amino acid sequence of the VEGF receptor recombinant fusion protein is shown in SEQ ID NOs: 7-10.

[0072] The anti-VEGF recombinant antibody described in the present invention is one in which the Fc fragment of a conventional anti-VEGF antibody is replaced with the Fc fragment variant of IgG1 described above. In another embodiment of the present invention, the conventional anti-VEGF antibodies include, but are not limited to, Bevacizumab, Ranibizumab, and Brolucizumab. In another embodiment of the present invention, the anti-VEGF recombinant antibody is one in which the Fc fragment of Bevacizumab is replaced with the Fc fragment variant H310E of IgG1 described in the first aspect, and preferably includes the amino acid sequence shown in SEQ ID NO: 11.

[0073] The present invention further provides a vector expression cassette. The expression cassette has the structure described in Formula I from the 5' end to the 3' end. ITR-E1-E2-E3-E4-ITR (I) Wherein, ITR is an inverted terminal repeat sequence, E1 is a promoter (including natural, optimized or combinatorial promoters), E2 is a signal peptide, E3 is a nucleotide sequence encoding the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody of the present invention, and E4 is a Poly A sequence.

[0074] In some embodiments, the ITR sequence (inverted terminal repeat sequence) is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9, preferably derived from AAV2.

[0075] In some embodiments, the nucleic acid sequence has a promoter. The promoter is used to initiate the expression of the nucleic acid sequence to which it is bound. The promoter may be constitutive or inducible, and the promoter may be a promoter specifically expressed in retinal pigment epithelial cells or a ubiquitous promoter. Preferred promoters include various eukaryotic promoters such as CMV, EF1a, CBA, SV40, PGK1, Ubc, CAG, TEF1, U6, H1, etc. Most recombinant AAV vector promoters are broad-spectrum CMV (CMV enhancer and CMV promoter), CBA (CMV enhancer and chicken β9-actin promoter), PGK promoter and EF1a promoter. More preferably, the promoter is the CMV promoter.

[0076] A signal peptide is a short peptide chain (5 - 30 amino acids in length) that induces the movement of a newly synthesized protein into the secretory pathway. In many cases, it refers to the N-terminal amino acid sequence of a newly synthesized polypeptide chain (not necessarily at the N-terminus) that is used to guide the transmembrane movement (positioning) of the protein. After the start codon, there is an RNA region encoding a hydrophobic amino acid sequence, which is called the signal peptide sequence and plays a role in guiding the protein to organelles within the cell, including different membrane structures. It contains three regions. The positively charged N-terminus is called the basic amino terminus. The middle hydrophobic sequence consists mainly of neutral amino acids and can form an α-helix structure, which is the main functional region of the signal peptide. The negatively charged long C-terminus contains low molecular weight amino acids and is the cleavage site of the signal sequence, also called the processing region. After the signal peptide sequence is synthesized, it is recognized by the signal recognition particle (SRP), protein synthesis is temporarily stopped or slowed down, the signal recognition particle transports the ribosome to the endoplasmic reticulum, and protein synthesis resumes. Guided by the signal peptide, the newly synthesized protein enters the lumen of the endoplasmic reticulum. The signal peptide sequence is cleaved by the action of signal peptidase. If the transport termination sequence is located at the C-terminus of the nascent peptide chain, it may not be cleaved by signal peptidase.

[0077] In some embodiments of the present invention, a commonly used eukaryotic expression signal peptide (see Table 1) is selected, and SP1 to SP13 signal peptides are inserted into the N-terminus of the target gene by PCR and recombinated homologously into the PTT5 vector digested with EcoRI and HindIII. After transforming Escherichia coli and sequencing to confirm the correct sequence, endotoxin-free plasmids are extracted in large quantities and transiently transfected into HEK293E cells by the PEI method. After culturing for 5 days using CD05 medium containing glutamine, cell supernatants are collected to detect protein expression, and target proteins in the cell culture supernatants are detected by protein A affinity chromatography. Protein quantification is performed by the bicinchoninic acid (BCA) method. Based on the expression levels of the target proteins, preferred signal peptide sequences are obtained.

[0078] In another embodiment, the preferred signal peptide is derived from Human OSM, Gaussia luc or Albumin (HSA). In another embodiment, the more preferred signal peptide is derived from Gaussia luc.

[0079] Polyadenylation refers to the covalent attachment of polyadenylic acid to messenger RNA (mRNA) molecules. In the process of protein biosynthesis, this is part of the way in which mature mRNA ready for translation is generated. In eukaryotes, polyadenylation is a mechanism that interrupts at the 3'-end of the mRNA molecule. The polyadenylate tail (or poly-A tail) protects the mRNA from exonuclease attack, terminates transcription, and is very important for the transport and translation of mRNA from the cell nucleus.

[0080] In some embodiments, the main options for the PolyA sequence are the bGH PolyA (derived from pCMV3) and the SV40 PolyA (derived from pCGS3) sequences. The interaction between different PolyA sequences and other expression elements can improve the transcription level. In one embodiment of the present invention, three different PolyA sequences, bGH PolyA, SV40 PolyA, and hGH PolyA, are tested to obtain optimal gene expression and virus production. In another embodiment of the present invention, the preferred PolyA sequences are bGH PolyA and hGH PolyA. In another embodiment of the present invention, the preferred PolyA sequence is bGH PolyA.

[0081] In some embodiments, the above expression cassette further includes regulatory elements. The expression regulatory elements include (1) regulatory elements for regulating the expression of the target protein (e.g., IRES for initiating the translation of downstream genes), (2) regulatory elements for expressing miRNA and siRNA sequences, (3) introns (Introns are also called spacer sequences and refer to non-coding fragments of genes or mRNA molecules), (4) localization sequences for localizing and expressing the target protein in the cell nucleus, cytoplasm, or various cell organelles and secreting it extracellularly, (5) some Kozak sequences (The Kozak sequence is a nucleic acid sequence located behind the cap structure at the 5' end of eukaryotic mRNA, usually GCCACCAUGG, which can bind to translation initiation factors and mediate the initiation of translation of mRNA containing the 5' end cap structure, corresponding to the SD sequence of prokaryotes, and is a sequence present in eukaryotic mRNA, playing an important role in the initiation of translation. The Kozak sequence is G / N-C / N-C / N-ANNAUGG, for example, GCCACCAUGG), (6) enhancers (The enhancers may be derived from, for example, SV40 virus, CMV virus, or adenovirus), (7) functional regulatory elements selected from WPRE, but are not limited thereto.

[0082] In some embodiments, the expression cassette further includes a label element. The label element includes, for example, but is not limited to, FLAG, HA, MYC, fluorescent protein, luciferase, SUMO protein, ubiquitin protein, GST, etc.

[0083] In one embodiment, the present invention discloses a recombinant viral vector. It includes the following elements: (a) the first AAV2 inverted terminal repeat (ITR) sequence, (b) the CMV enhancer and promoter, (c) the chimeric intron, (d) the Kozak sequence, (e) the signal peptide sequence, (f) the VEGF receptor recombinant fusion protein / anti-VEGF recombinant antibody coding sequence, (g) the WPRE sequence, (h) the bGH polyA sequence, and (i) the second AAV2 ITR.

[0084] In one embodiment, the above-optimized VEGF receptor recombinant fusion protein / anti-VEGF recombinant antibody is expressed on an AAV viral vector. The AAV viral vector includes, but is not limited to, pAAV-CMV, pAAV-MCS plasmid. Preferably, the AAV viral vector is pAAV-CMV.

[0085] In the present invention, an AAV virus packaged by a vector expressing the above-optimized VEGF receptor fusion protein / anti-VEGF recombinant antibody is further provided. The AAV virus is prepared by standard methods disclosed in the art (see 《AAV Production Everywhere: A Simple, Fast, and Reliable Protocol for In-house AAV Vector Production Based on Chloroform Extraction》).

[0086] Replication-deficient recombinant AAV can be prepared by co-transfecting three plasmids into a cell line infected with a human helper virus (such as adenovirus). The flanking of the target nucleic acid sequence to be included is a plasmid with two AAV inverted terminal repeat (ITR) regions, which are a helper packaging plasmid and a plasmid containing the AAV capsid formation genes (rep and cap genes), respectively. Subsequently, the AAV recombinant virus produced by standard techniques is purified. Preferably, the recombinant adeno-associated virus is single-stranded AAV.

[0087] In some embodiments, the optimized coding gene fragment of the VEGF receptor recombinant fusion protein / anti-VEGF recombinant antibody is packaged into virus particles (including, but not limited to, AAV serotype virus particles such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV7m8, AAV PHP.eB). Accordingly, the present invention includes recombinant virus particles containing any of the vectors described herein. Preferably, the serotype of the recombinant adeno-associated virus is AAV2 and AAV8. More preferably, the serotype of the recombinant adeno-associated virus is AAV8.

[0088] A preferred viral vector for delivering the nucleic acid sequence encoding the VEGF receptor recombinant fusion protein / anti-VEGF recombinant antibody is an AAV vector, such as self-complementary AAV (scAAV), single-stranded AAV (ssAAV), pAAV-CMV, pAAV-MCS, etc.

[0089] Selective targeting can also be achieved by using a specific AAV serotype (AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV7m8, AAV PHP.eB) or any other modified serotype.

[0090] In the present invention, the host cells used for the assembly of the above viruses are used for AAV vector-transduced cells to express the Fc-modified VEGF receptor recombinant fusion protein and the anti-VEGF antibody. Preferably, the host cells are mammalian cells (preferably human-derived cells, more preferably human optic nerve cells or photoreceptor cells). The expression levels of the VEGF receptor fusion protein and the anti-VEGF antibody are increased, indicating the specific expression of the AAV vector molecule of the present invention. The host cells include, but are not limited to, cells such as Hela-S3, HEK-293, HEK-293T, HEK-293FT, Expi293F, A549, and Sf9. In some embodiments of the present invention, preferred host cells are HEK-293, HEK-293T cells, or Expi293F cells.

[0091] In one embodiment, the present invention discloses the following recombinant adeno-associated virus: AAV8-Afli Fc Variant10. This recombinant virus is an AAV8 serotype virus, the vector carrying the transgene has the sequence shown in SEQ ID NO: 12, the transgene carried is the gene encoding the VEGF receptor recombinant fusion protein, and it has been subjected to H310E mutation modification at the 310th position of the Fc region of the Aflibercept fusion protein gene.

[0092] In one embodiment, the present invention discloses the following recombinant virus: AAV8-Bevacizumab Fc Variant10. This recombinant virus is an AAV8 serotype virus, the vector carrying the transgene has the sequence shown in SEQ ID NO: 13, the transgene carried is the mutant gene of the Bevacizumab monoclonal antibody, and it has been subjected to H310E mutation modification at the 310th position of the Fc region of the Bevacizumab gene.

[0093] The recombinant virus constructed in the present invention is applied in vivo or ex vivo, and preferably, it is preferably used by single administration into the vitreous body, subretinal space or suprachoroidal space of patients with angiogenesis-related fundus diseases (age-related macular degeneration, wet macular degeneration, diabetic macular edema). Preferably, the safe and effective dose range for mouse administration is 1×10 6 -1×10 11 viral genomes / eye, and the safe and effective dose range for NHP administration is 1×10 8 -1×10 13 viral genomes / eye, and the safe and effective dose range for human administration is 1×10 8 -1×10 13 viral genomes / eye (viral genome, genomic copies, abbreviation: GC).

[0094] In one embodiment, the present invention provides a composition comprising the polynucleotide, viral vector or adeno-associated virus of the present invention.

[0095] In one embodiment, the present invention provides a method for preparing a composition comprising the polynucleotide, viral vector or adeno-associated virus of the present invention.

[0096] In one embodiment, the present invention provides an administration form of the composition comprising the polynucleotide, viral vector or adeno-associated virus of the present invention into the subretinal space.

[0097] In one embodiment, the present invention provides an administration form of intravitreal injection of the composition comprising the polynucleotide, viral vector or adeno-associated virus of the present invention.

[0098] In one embodiment, the present invention provides an administration form of the composition of the polynucleotide, viral vector or adeno-associated virus of the present invention into the suprachoroidal space.

[0099] In one embodiment, the present invention provides a method of observation after administration to mice / NHP / humans, including optical coherence tomography (OCT) and fluorescein fundus angiography (FFA).

[0100] In one embodiment, the present invention provides a method of examining the expression of aflibercept protein in ocular tissues after administration to mice.

[0101] Formulations and Compositions The present invention provides a formulation or composition. The formulation or composition includes the adeno-associated virus vector or virus described in the present invention, and a pharmaceutically acceptable vector or excipient.

[0102] In another preferred example, the pharmaceutical formulation is used for the treatment of eye diseases. Preferably, the pharmaceutical formulation is used for the treatment of VEGF-related eye diseases, preferably for the treatment of macular degeneration and / or diabetic retinopathy, more preferably for the treatment of wet age-related macular degeneration or wet macular degeneration.

[0103] For the convenience of clinical application, the pharmaceutical composition of the present invention can be filled into a sealed vial or a prefilled syringe. From the perspective of convenience in storage and use, the vial or prefilled syringe can be packaged in a pharmaceutical packaging box. The pharmaceutical formulations and compositions of the present invention are stored in an ultra-low temperature refrigerator at <-60°C and use dry ice cold chain transportation during transportation.

[0104] The dosage form of the pharmaceutical formulation or formulation composition may be liquid or solid, for example, powder, gel or paste. Preferably, the composition is liquid, more preferably an injection.

[0105] The titer of the AAV vector formulation provided by the present invention is 1×10 10 -1×10 14 GC / ml, and the preferred titer is 1×10 12 -1×10 13 GC / ml.

[0106] The preparation contains pharmaceutically acceptable excipients. In some cases, the excipients include surfactants or stabilizers. In the present invention, the surfactants are selected from polysorbate, sodium dodecyl sulfate, sodium lauryl sulfate, lauryl dimethyl oxide gum, polyethoxylated alcohol, polyoxyethylene sorbitol, octylphenol polyvinyl, Brij, pluronic (registered trademark), and polyoxyl caster oil. In some cases, the pharmaceutically acceptable excipients include phenol, mannitol, sorbitol, sucrose, or sodium chloride.

[0107] The "active ingredient" in the pharmaceutical composition described in the present invention refers to the vector or AAV virus described in the present invention. The "active ingredient", preparation and / or composition described in the present invention can be used for the treatment of eye diseases. The "safe and effective amount" refers to the amount of the active ingredient sufficient to significantly improve the condition or symptoms without causing serious side effects. The "pharmaceutically acceptable vector or excipient" refers to one or more compatible solid or liquid fillers or gel substances suitable for human use and having sufficient purity and low enough toxicity. The "compatibility" refers to the property that each component of the composition can be mixed with the active ingredient of the present invention and with each other without significantly reducing the medicinal effect of the active ingredient.

[0108] Treatment method The present invention provides a method for treating VEGF-mediated angiogenic eye diseases. The method includes introducing into the eye a virus or viral vector containing a nucleic acid sequence encoding an Fc-modified VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody. The method may include injecting the nucleic acid vector into the subretinal space, vitreous body, or suprachoroidal space. The nucleic acid vector targets RPE cells or photoreceptor cells.

[0109] The present invention provides a nucleic acid vector for use in a method of treating an angiogenic eye disease by delivering a nucleic acid sequence encoding a therapeutic protein to retinal cells. The nucleic acid vector contains a nucleic acid sequence encoding an Fc-modified VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody. The compositions of the present invention may be administered alone or in combination with other therapeutic agents (e.g., formulated in the same pharmaceutical composition).

[0110] As described in the present invention, treating a disease means administering the nucleic acid, vector or virus described herein to improve or reduce one or more symptoms of the disease (including reduction of vascular leakage, reduction of angiogenesis, etc.).

[0111] Preferably, the method of the present invention includes introducing a nucleic acid sequence encoding an Fc-modified VEGF receptor recombinant fusion protein or a recombinant antibody against VEGF into the subretinal space of the eye.

[0112] Preferably, the method of the present invention includes contacting cells with a vector (preferably a virus, more preferably an adeno-associated virus) containing a nucleic acid sequence encoding an Fc-modified VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody. Preferably, the cells are retinal cells, preferably including pigment epithelial cells, cone cells, rod cells, bipolar cells, horizontal cells, ganglion cells, and / or amacrine cells.

[0113] The recombinant adeno-associated virus vector of the present invention can effectively suppress VEGF expression in the animal eye, reduce leakage and angiogenesis caused by laser injury, and reduce retinal vascular lesions caused by DL-AAA. Therefore, it can be used in the preparation of drugs for treating VEGF-related diseases to treat human VEGF-related eye diseases and has a broad market prospect.

[0114] When using a viral vector containing an effective amount of the nucleic acid sequence provided by the present invention for the treatment of human VEGF-related eye diseases, the clinical dosage is 1×10 8 -1×10 13It is a GC virus, the ocular injection volume is 50 to 300 μl, and the administration route is subretinal, intravitreal or suprachoroidal injection.

[0115] Hereinafter, the present invention will be further described with specific examples. The following examples are some examples of the present invention, not all examples. As can be understood, the following examples are for providing a complete disclosure and explanation of the methods and compositions of the present invention to those skilled in the art, and do not limit the scope of the present invention. Based on the examples in the present invention, all other examples obtained by those skilled in the art without creative efforts are included in the protection scope of the present invention.

[0116] The names and information of the variants in the present invention and the examples are shown in the following table. Variant comparison table JPEG2025523593000002.jpg98157Note: In the table, variants Variant1-12 are Aflibercept variants obtained by mutating the Fc fragment based on Eylea (Aflibercept) shown in SEQ ID NO: 14.

[0117] Example 1: Promoter screening of gene expression cassette Most recombinant AAV vector promoters use the broad-spectrum CMV (CMV enhancer and CMV promoter), CBA (CMV enhancer and chicken-β actin promoter). According to literature reports, optimized retinal cell-specific promoters may express target proteins more efficiently in specific tissues. In the experiment, based on the pAAV-MCS plasmid, the promoter element was changed. The CBA, CMV+TPL-MLP (TPL-MLP sequence derived from the pTT5 vector), RPE65 (GenBank: AF304008.1), and IRBPe / GNAT2 (GenBank:X53044.1,GenBank:U66698.1) promoters were selected for research. The amino acid sequence of aflibercept was derived from the DrugBank database (registration number DB08885), codon optimization was performed according to the codon usage preference of Homo sapiens, and the sequence was cloned into the pTT5 expression vector.

[0118] The specific experimental procedures are as follows. 1) Optimization synthesis of the aflibercept sequence, and synthesis of the RPE65 and IRBPe / GNAT2 promoter sequences. 2) Amplify by PCR to obtain the RPE65 and IRBPe / GNAT2 promoter sequences, and perform homologous recombination with the pAAV-MCS vector digested by NcoI and SacI, MluI and SacI in sequence. After Escherichia coli transformation, verify by sequencing and enzymatic digestion that the pAAV-RPE65-MCS and pAAV-IRBPe / GNAT2-MCS vectors are obtained. 3) Digest pAAV-RPE65-MCS, pAAV-IRBPe / GNAT2-MCS, and pAAV-MCS with EcoRI and HindIII, amplify by PCR to obtain the target gene sequence of aflibercept, and then insert the aflibercept sequence into the digested above vectors by homologous recombination method. After Escherichia coli transformation, verify by sequencing and enzymatic digestion that the pRPE65-AAV-Afli YB, pGNAT2-AAV-Afli, and pAAV-Afli YB vectors are obtained. 4) Amplify the CBA and aflibercept sequences by PCR, perform homologous recombination of the inserted sequence with the pAAV-MCS vector digested by NcoI and HindIII after overlap PCR. After Escherichia coli transformation, verify by sequencing and enzymatic digestion that the pCBA-AAV-Afli YB vector is obtained. 5) Amplify the CMV+TPL / MLP and aflibercept sequences by PCR, perform homologous recombination of the inserted sequence with the pAAV-MCS vector after overlap PCR. After Escherichia coli transformation, verify by sequencing and enzymatic digestion that the pMPL-AAV-Afli YB vector is obtained. 6) Remove endotoxin from the plasmids obtained in steps 3, 4, and 5, perform large-scale extraction, transiently transfect ARPE-19 cells, and measure the expression level of Aflibercept in the cell supernatant by ELISA 72 hours later. The experimental results are shown in the promoter optimization experimental results of Figure 1. As can be seen from the results, the expression level of the protein containing the CMV promoter sequence is significantly higher than that of other promoter sequences.

[0119] Example 2: Screening of signal peptide sequences Using the commonly used eukaryotic expression signal peptides (Table 1), the signal peptides SP1 - SP13 were inserted into the N-terminus of the Aflibercept target gene shown in SEQ ID NO: 14 by PCR, and homologous recombination was performed in the PTT5 vector digested with EcoRI and HindIII. After verifying the correct sequence by sequencing after E. coli transformation, endotoxin was removed from the plasmid and extracted in large quantities, transiently transfected into HEK293E cells by the PEI method, cultured in CD05 medium containing glutamine for 5 days, then the cell supernatant was taken, the expression level of Aflibercept was measured by ELISA, and the target protein in the cell culture supernatant was purified by Protein A affinity chromatography. Protein quantification was measured by the BCA method (Pierce TM Rapid Gold BCA Protein Assay Kit). Based on the expression levels of the target proteins, preferred signal peptide sequences were obtained. In this study, the effects of 13 different signal peptides SP1 - SP13 on the expression levels of target proteins were compared, with Gaussia luc as a control. The results are shown in Figure 2. As can be seen from the results, the protein expression level of the SP1 signal peptide is the highest.

[0120] Table 1: Eukaryotic expression signal peptides JPEG2025523593000003.jpg86170

[0121] Example 3: Screening for Fc region mutants of the target protein In the experiment, multiple groups of Fc single-point and multi-point mutants were designed based on Aflibercept (SEQ ID NO: 14). After designing site-directed mutagenesis primers, the coding genes of each mutant fragment Variant1 - 12 were obtained by PCR. After purifying the PCR products, they were digested with endonuclease DpnI for 30 minutes. After transforming the digested products into E. coli, clones were selected and sequenced to verify the sequences.

[0122] After expanding and culturing correctly arrayed clones, endotoxin was removed and plasmids were extracted in large quantities. Then, transient transfection was performed on Expi293F cells by the PEI method, and after culturing in CD05 medium containing glutamine for 5 days, the cell supernatant was collected, the expression level of aflibercept was measured by ELISA, and the target protein in the cell culture supernatant was purified by Protein A affinity chromatography. Protein quantification was performed by the BCA method. The supernatant expression level and FcRn affinity of the mutant molecules were investigated, and the results are shown in Figure 3 and Table 2. After mutating the Fc region of the aflibercept molecule, Variants 1, 2, 6, and 10 did not bind to FcRn, and there was no significant difference in protein expression level compared to before the mutation. Among them, Variant 12 is a positive affinity mutant in the Fc region and increased the affinity with FcRn.

[0123] Table 2: Measurement results of the affinity of Fc mutant proteins for FcRn JPEG2025523593000004.jpg84169Note: ND indicates that it was not detected.

[0124] Example 4: Screening of polyadenylation sequence (PolyA) To achieve efficient mRNA pretreatment, it is necessary to construct an efficient polyadenylation sequence behind the transgene to form an appropriate PolyA tail at the 3' end of the RNA. The main options for the PolyA sequence in the rAAV vector are bGH PolyA and SV40 PolyA sequences. The interaction between different PolyA sequences and other expression elements may improve the transcription level. Therefore, in the experiment, three different PolyA sequences were tested to obtain optimal gene expression and virus production.

[0125] Selected the preferred Fc mutant aflibercept mutant molecules and the non-mutant aflibercept molecule (abbreviation: Afli), inserted these target gene fragments into the pAAV-MCS vector (containing hGH PolyA), and changed the PolyA sequences to bGH PolyA and SV40 PolyA sequences respectively. Transiently transfected each constructed AAV vector into ARPE-19 cells and HEK293 cells with lipofectamine2000 transfection reagent, and measured the expression level of the target protein 72 h later.

[0126] Inserted three different PolyA sequences into four molecules: Afli, Variant2, 6, and 10, and tested the effect on the expression level of the target protein. The results are shown in Figure 4 (results of transient transfection in ARPE-19 cells) and Figure 5 (results of transient transfection in HEK293 cells). As can be seen from the results, there are differences in the expression efficiency of PolyA for different mutant molecules, and it is preferred that different molecules use different PolyA elements.

[0127] Example 5: Thermal stability analysis of fusion proteins In this study, the thermal stability of the Aflibercept mutant protein molecule under different heating conditions was measured. The SEC analysis column used was MAbPac SEC-1 from Thermo fisher. The results are shown in Figure 6 (results of thermal stability analysis of the candidate Fc gene-modified VEGF receptor recombinant fusion protein at 37 °C) and Figure 7 (results of thermal stability analysis of the candidate Fc gene-modified VEGF receptor recombinant fusion protein at 40 °C). As can be seen from the results, after storage at 37 °C and 40 °C for a certain period of time, the SEC purity of each protein molecule tended to decrease, and there was no significant difference in the thermal stability of each protein molecule except for Variant12.

[0128] Example 6: Pharmacokinetics of single intravitreal injection of candidate proteins To provide data support for the design and optimization of the following AAV virus packaging vector genes, the PK behaviors after single intravitreal injection of different genetically modified Variants 2 and 10 and the control group aflibercept (hereinafter referred to as Afli; SEQ ID NO: 14) into both eyes of New Zealand rabbits were investigated. The dose of each protein was 1.25 mg / eye, and the administration volume was 50 μL / eye. After anesthetizing New Zealand rabbits with sodium pentobarbital, the injection eyes were disinfected with povidone iodine solution. For both eyes, the candidate protein was injected into the vitreous of each eye at a predetermined dose at a volume of 50 μL / eye. Before injection, 1-2 drops of oxybuprocaine hydrochloride eye drops were instilled into the injection eyes for surface anesthesia. After intravitreal injection, to moisten the cornea and resist infection, about 1-2 drops of ofloxacin ophthalmic ointment were instilled into both eyes of the rabbits in each group. At 1 h to 672 h after administration, eye tissues were taken at different time points, the retina / choroid plexus, aqueous humor and vitreous were separated, the tissues were homogenized to obtain supernatants, and venous blood samples were collected. The concentrations of the protein drug in the eye tissues and serum were measured by ELISA method.

[0129] For the three tissue samples of vitreous, aqueous humor and retinal choroid plexus, drug time curves were created for the average concentration values of each group of animals versus time points, PK was analyzed using a one-compartment model, and the results are shown in Table 3. The elimination half-life t 1 / 2 and the area under the concentration-time curve AUC 0-t of each molecule were compared. As a result, the half-life t 1 / 2 of the Variant 10 molecule was 1.03 - 1.76 times that of Afli, and the exposure AUC was 1.19 - 1.84 times that of Afli. The half-life t 1 / 2 of the Variant 2 molecule was 1.15 - 1.40 times that of Afli, and the exposure AUC was 1.02 - 1.78 times that of Afli. Drug time curves were created for the average concentration values of serum samples versus time points, and the drug exposure AUC after each protein molecule entered the blood from the eye was calculated using a non-compartment model. The results (Table 4) showed that the average AUC of Variant 10 was 74.2% lower than that of Afli, and the average AUC of Variant 2 was 54.9% lower than that of Afli.

[0130] Table 3: PK analysis parameters of ocular tissues of each mutant molecule (one-compartment model) JPEG2025523593000005.jpg77170

[0131] Table 4: Summary of serum exposure AUC of each mutant molecule (non-compartment model) JPEG2025523593000006.jpg17170

[0132] Example 7: Preparation of AAV8-Afli Fc Variant10 AAV virus sample 1. Construction of Variant 10 vector (1) The amino acid sequence of aflibercept was obtained from the DrugBank database (registration number DB08885, SEQ ID NO: 14). Using synthetic aflibercept optimized by codon as a template, the aflibercept fragment was amplified by PCR method, and the signal peptide derived from Gaussia luc (see Table 1 for sequence information) was added. This fragment was inserted into the PTT5 vector digested with NotI and BamHI. After verifying the correctness by sequencing, the Aflibercept-PTT5 vector was obtained.

[0133] (2) Using the Aflibercept-PTT5 vector as a template, site-directed mutagenesis primers for H310E of the Fc fragment were designed. Then, the Variant10 mutant coding fragment was obtained by PCR method. After purifying the PCR product, it was digested with endonuclease DpnI for 30 minutes. After transforming the digested product into Escherichia coli, clones were selected and the sequence was verified by sequencing. The Variant10-PTT5 vector was verified to be correct by sequencing.

[0134] (3) Using Variant10-PTT5 as a template, after amplifying the Variant10 coding fragment by the PCR method, it was inserted into the pAAV-MCS plasmid digested by EcoRI and HindIII by homologous recombination. After Escherichia coli transformation, clones were selected and the sequence was verified by sequencing. The vector verified to be correct by sequencing was digested with SmaI to verify whether there was a deletion in the ITR, and the vector verified to be correct was named Afli Fc Variant10.

[0135] (4) The AAV vector verified to be correct in step (3) was digested with HindIII and RsrII, and the bGHpolyA (the template was derived from pCMV3-3-Flag) fragment was obtained by the PCR method. After purifying the double-digested above vector and the bGHpolyA fragment obtained by PCR, homologous recombination ligation was performed. After Escherichia coli transformation, clones were selected and the sequence was verified by sequencing. Also, the vector determined to be correct by sequencing was digested with SmaI to verify whether there was a deletion in the ITR. After verification, the target plasmid Variant 10-pAAV (SEQ ID NO: 12) was obtained.

[0136] 2. AAV virus packaging AAV virus was packaged by co-transfecting three plasmids into Expi293F cells. The three plasmids were a helper packaging plasmid, an AAV8 rep-cap plasmid, and a transgene plasmid, respectively.

[0137] (1) Plasmid amplification and extraction The Variant 10-pAAV vector, AAV8 rep-cap plasmid, and pHelper helper plasmid constructed in step 1 were extracted in large quantities. For virus packaging, the concentration needs to be higher than 1 μg / μL and the A260 / 280 needs to be 1.8 - 2.0.

[0138] (2) Virus packaging Expi293F cells were cultured in suspension in serum-free medium so that the cell density reached 1E+6 cells / ml. The two extracted plasmids were mixed at a molar ratio of 1:1:1, and further, the plasmid DNA was uniformly mixed with the PEIpro transfection reagent at a mass ratio of 1:2 in terms of the total mass. After incubating at room temperature for 20 minutes, it was slowly added to the cell suspension (the ratio of cells to the three plasmids was 1 mL:1 μg) and uniformly mixed. The cells were cultured for 3 days in a shaker at 37 °C and 8% CO2, and the cell suspension was collected.

[0139] (3) Concentration of AAV virus 1) The cell suspension was centrifuged at 10,000 g for 10 min. The obtained centrifuged supernatant was transferred to a centrifuge tube. The obtained cell pellet was resuspended in a small amount of PBS solution, and then the cells were lysed by repeating freezing and thawing. After freezing and thawing, the cells were centrifuged at 10,000 g for 10 min, and the obtained supernatant was collected. 2) The supernatants collected after two centrifugations were mixed and filtered through a 0.45 μm filter to remove impurities. 3) 1 / 2 volume of 1 M NaCl and 10% PEG8000 solution were added, uniformly mixed, and left standing at 4 °C overnight. 4) Centrifuged at 12,000 rpm for 2 h, the supernatant was discarded, the virus pellet was dissolved in an appropriate amount of PBS solution, and after complete dissolution, it was filtered through a 0.22 μm filter for sterilization. 5) Benzonase nuclease was added for digestion to remove the remaining plasmid DNA (final concentration 50 U / ml). The lid was put on and inverted several times to mix uniformly. Incubated at 37 °C for 30 minutes. 6) Filtered through a 0.45 μm syringe filter, and the filtrate was taken out to obtain concentrated AAV virus.

[0140] (4) Purification of AAV 1) Solid CsCl was added to the virus concentrate until the density reached 1.41 g / ml (refractive index 1.372). 2) The sample was put into an ultracentrifuge tube and the remaining space in the centrifuge tube was filled with the pre-prepared 1.41 g / ml CsCl solution. 3) A density gradient was formed by centrifuging at 175,000 g for 24 hours. Samples with different densities were collected step by step in order, sampled, and the titer was measured. The fraction rich in AAV particles was collected. 4) The above process was repeated once. 5) The virus was placed in a 100 kDa dialysis bag and dialyzed overnight at 4 °C for desalting. The components of the dialysis buffer were PBS (pH 7.2) containing 0.001% Pluronic F68. The obtained post-dialysis sample was the purified AAV virus AAV8-Afli Fc Variant10, which could be used for in vitro and in vivo analysis tests. 6) The purified virus was stored in a -80 °C ultra-low temperature freezer.

[0141] All AAV viruses used in the present invention were prepared by the above method. If necessary, specific AAV vectors, expression elements, and VEGF receptor recombinant fusion protein variants or VEGF recombinant antibodies can be selected. Here, after changing Aflibercept to Bevacizumab and the H310E mutation in the Fc fragment occurred, the plasmid constructed by the same method as above is shown in SEQ ID NO: 13. Using this plasmid, the AAV virus constructed by the same method as above was named AAV8-Bevacizumab Fc Variant10.

[0142] Example 8: Biological Activity of the Protein Expressed by the AAV Vector Molecule Verification of the biological activity of the candidate vector molecule expression product is an important consideration in the pharmaceutical evaluation of gene therapy. In this study, HEK293T cells were infected with AAV8-Afli Fc Variant 10 and AAV8 empty vector prepared in Example 7 at an MOI of 2E+5, respectively, and the cell supernatant was collected 72 h later. 6E+3 HUVEC cells were seeded in a 96-well plate (50 μL / well) and cultured in a basal medium containing 100 ng / ml VEGF165. After 4 - 6 h, 50 μL of the virus-infected cell supernatant was added, and the cells were continuously cultured for 72 h. 10 μL of CCK-8 solution was added to each well, incubated at 37 °C, and read at OD450 nm after 3 h. The results are shown in Figure 8. As can be seen from the results, the expression product of the candidate vector molecule has an inhibitory effect on the proliferation of HUVEC cells. Specifically, the expression product can bind to VEGF and inhibit the proliferation effect of VEGF on HUVEC cells.

[0143] Example 9: Transduction and expression of different administration forms of AAV vector molecules SPF-grade C57BL / 6J male mice aged 8 - 10 weeks were bred in the laboratory for 3 - 5 days. Before grouping, general ophthalmic examinations, fundus photography (FP), and fundus fluorescein angiography (FFA) were performed on the animals to screen animals eligible for the experiment. The qualified mice were randomly divided into 4 groups according to their body weight before administration. The AAV virus AAV8-Afli Fc Variant10 prepared in Example 7 was used as the test article. The groups of animals and administration treatments are shown in Table 7 below. Table 7: Groups of mice and dosages of different administration routes JPEG2025523593000007.jpg42170 Note: All solvents used in the examples of the present invention are PBS (pH 7.2) containing 0.001% (W / V) Pluronic F68.

[0144] The test article may be diluted with a solvent according to the dosage requirements when in use.

[0145] Each mouse was administered once in both eyes. The administration methods are as follows. It is necessary to anesthetize the animals before administration. Calculate the dosage of anesthesia according to the weight of the animals measured most recently. During the period from anesthesia to recovery from anesthesia, it is necessary to take measures to keep the animals warm (such as covering them with a blanket).

[0146] Connect the syringe needle (using different syringe needles for animals in each group), aspirate the required amount of test article / control article solution and administer it. The administration is carried out under a surgical microscope.

[0147] Subretinal injection: Cover the cornea with a coverslip, use carbomer eye drops to remove air, use a disposable insulin needle to puncture the sclera and choroid at a small angle to form a channel, withdraw the insulin needle, replace it with a microinjector, and insert it from the channel opening to the subretinal space for injection.

[0148] Vitreous cavity injection: Cover the cornea with a coverslip, use carbomer eye drops to remove air, use a disposable insulin needle to puncture the sclera and choroid at a small angle to form a channel, withdraw the insulin needle, replace it with a microinjector, and insert it from the channel opening to the vitreous cavity for injection.

[0149] Supraciliary injection: Use a disposable insulin needle to puncture the sclera to form a channel, withdraw the insulin needle, replace it with a microinjector, and insert it from the channel opening to the supraciliary space for injection.

[0150] After the administration, levofloxacin eye drops and / or ofloxacin eye ointment were administered 2 - 3 times a day for 3 consecutive days.

[0151] On the 8th day after administration, the eyeballs of both eyes of the animals were collected, 200 μL of PBS containing protease inhibitor was added to each eye, homogenized using a tissue homogenizer, centrifuged (5000 g, 5 min, 4 °C), and then the supernatant was taken and stored at -60 °C or lower.

[0152] The deep well plates were coated with VEGF165 protein, washed, and then HRP-conjugated goat anti-human IgG (Fc specific) antibody was added to detect the aflibercept captured on the plates. After the final wash, TMB substrate solution was added, and HRP catalyzed the color reaction of the substrate. Finally, a stop solution was added to stop the reaction, and the solution changed from blue to yellow. The depth of color was positively correlated with the concentration of aflibercept protein in the sample. A microplate reader was used to detect the solution after the color reaction, and the amount of aflibercept protein in the supernatant of the eye tissue homogenate was calculated.

[0153] As can be seen from the detection results, high aflibercept protein expression was shown in mice with all three different administration forms. JPEG2025523593000008.jpg41170

[0154] Example 10: Long-term expression test after injecting AAV vector molecules into mice SPF-grade C57BL / 6J male mice aged 8 - 10 weeks were bred in the laboratory for 3 - 5 days. Before grouping, general ophthalmic examinations, fundus photography (FP), and fundus fluorescein angiography (FFA) were performed on the animals to screen for eligible animals for the experiment. Twenty-eight qualified mice were randomly divided into 7 groups according to their body weight before administration. The AAV virus AAV8-Afli Fc Variant10 prepared in Example 7 was used as the test article. The groups of animals and administration treatments are shown in Table 8 below.

[0155] Table 8: Groups and dosage table of mice JPEG2025523593000009.jpg45170

[0156] Each mouse was administered once in both eyes, and the same subretinal injection administration method and post-treatment method as in Example 9 were adopted. The expression level of aflibercept protein in the eyes was measured at different time points after administration. The measurement results of the amount of aflibercept protein are shown in Table 9. The time-expression curve is shown in Figure 9.

[0157] Table 9: Measurement results of protein expression levels at different time points after injecting AAV into mice JPEG2025523593000010.jpg23170

[0158] As can be seen from the test results, the candidate vector molecule maintained high expression for 12 weeks at a dose of 3E+8 vg / eye. In contrast, according to the literature report, the protein drug Eylea could only be maintained for 4 to 8 weeks after administration.

[0159] Example 11: AAV vector molecule dose escalation test in mice SPF-grade C57BL / 6J male mice aged 8 - 10 weeks were purchased and bred in the laboratory for 3 - 5 days. Before grouping, general ophthalmic examinations, fundus photography (FP), OCT, and fundus fluorescein angiography (FFA) were performed on the animals to screen for animals eligible for the experiment. 30 qualified mice were randomly divided into 6 groups according to their body weight before administration. The AAV virus AAV8-Afli Fc Variant10 prepared in Example 7 was used as the test article. The animal groups and administration treatments are shown in Table 10 below (the administration volume for each group is 1 μL / eye). Table 10: Measurement results of protein expression levels after injecting different doses of AAV into mice JPEG2025523593000011.jpg46170

[0160] A single administration was given to both eyes of each mouse, and the solvent and different doses of the AAV8-Afli Fc Variant10 test article were administered by subretinal injection. The administration method was the same as in Example 9.

[0161] In general ophthalmic examinations, fundus photography (FP), OCT, and FFA, there were no significant ocular adverse reactions. As can be seen from the protein expression results at different doses, the protein expression showed an obvious dose-dependent correlation.

[0162] Example 12: Laser-induced mouse CNV model of candidate vector molecule - efficacy test 8 - 10 - week - old SPF - grade C57BL / 6J male mice were purchased and bred in the laboratory for 3 - 5 days. Before grouping, general ophthalmic examinations, fundus photography (FP), and fundus fluorescein angiography (FFA) were performed on the animals to screen for animals eligible for the experiment. Thirty qualified mice were randomly divided into 6 groups according to their body weight before administration. The AAV virus AAV8 - Afli Fc Variant10 and Eylea® protein drug prepared in Example 7 were used as test articles respectively. The groups of animals and administration treatments are shown in Table 11 below (the administration volume for each group is 1 μL / eye). Table 11: Groups and Dosages of Mice JPEG2025523593000012.jpg57170 Note: 1) The start of administration is denoted as D1. 2) Since the effect of the Eylea® protein drug appears immediately after administration, it is administered after model establishment. AAV8 - Afli Fc Variant10 reaches a stable expression level about 1 - 2 weeks after administration and maintains high expression for more than 12 weeks, so it is administered after model establishment. 3) The Eylea® protein drug is the standard treatment for treating AMD, and its administration route is intravitreal injection.

[0163] Both eyes of each mouse were administered once, and the solvent and different dosages of the AAV8 - Afli Fc Variant10 test article were administered by subretinal injection respectively. The administration method is the same as that in Example 9. The second group is the protein control group, and 80 μg / eye was injected intravitreally.

[0164] On D17, the choroidal neovascularization model was constructed by irradiating the fundus of both sides with a laser, and the planned number of laser burns for each eye was 4. The specific procedure is as follows. 1) Mydriasis: 1 - 2 drops of 0.5% compound tropicamide eye drops were instilled into both eyes of the animal to dilate the pupils. 2) Anesthesia: The animal was anesthetized by intramuscular injection of Zoletil® 50 at 50 mg / kg and 50 mg / mL. 3) Laser photocoagulation: Before photocoagulating the eyes of the animals, carbomer eye drops were instilled, the fundus laser lens was placed, and after observing the fundus clearly, at a position about 1.5 - 2 PD away from the optic disc, avoiding blood vessels, photocoagulation was performed around the optic disc using a laser with a wavelength of 532 nm (spot diameter approximately 50 μm). 4) Animal care: After laser photocoagulation, ofloxacin eye ointment was applied to both eyes of the animals, and they were placed on a warming blanket to maintain body temperature before waking up, and then returned to the cage after waking up.

[0165] On D31, FFA (fundus fluorescein angiography) examination was performed, and the typical atlas is shown in Figure 10. Before the FFA examination, the animals were intravenously injected with sodium fluorescein injection (10%, 0.02 mL / animal).

[0166] Analysis indicators: Images in the early stage (within 1.5 min) and the late stage (more than 3 min) of fundus fluorescein angiography were compared. According to whether there was fluorescence leakage in the fundus of the animals, the formation and leakage of choroidal neovascularization were judged, the degree of fluorescence leakage was evaluated, and the ratio of leakage spots in each grade and the average score of spot leakage were calculated.

[0167] The evaluation criteria for spot fluorescence leakage are: Grade 0 (no fluorescence leakage), Grade 1 (mild fluorescence leakage; leakage area is 1% - 50% of the size of the laser spot), Grade 2 (moderate fluorescence leakage; leakage area is 50% - 100% of the size of the laser spot), Grade 3 (severe fluorescence leakage; leakage area is larger than the size of the laser spot).

[0168] The ratio (%) of spots in each grade = the total number of spots in the corresponding grade ÷ the total number of four types of spots (i.e., the number of effective spots) × 100%.

[0169] The average score of spot leakage = [(the number of Grade 0 spots × 0) + (the number of Grade 1 spots × 1) + (the number of Grade 2 spots × 2) + (the number of Grade 3 spots × 3)] ÷ the total number of four types of spots (i.e., the number of effective spots) Here, the effective spot refers to a spot that has no severe subretinal or preretinal hemorrhage nearby and appears completely in fluorescein angiography.

[0170] As can be seen from the statistical results of the percentage of grade 3 leakage spots in the solvent control, Eylea® protein control, and 1E+7, 3E+7, 1E+8, 3E+8 GC / eye AAV8-Afli Fc Variant 10 groups in D31 (Table 12), each dosage group of Eylea® and AAV8-Afli Fc Variant was significantly lower than the solvent control group (p≤0.05), and each dosage group of AAV8-Fc variant was significantly lower than the Eylea® protein control group (p≤0.05).

[0171] Table 12: Percentage of leakage spots generated in the laser-induced CNV model after injecting different dosages of AAV into mice JPEG2025523593000013.jpg50170

[0172] The average score of spot leakage is shown in Figure 11. In D31, the average score of the AAV8-Afli Fc Variant 10 group was lower than that of the solvent control and Eylea® protein control groups. Also, compared with D24, the average spot leakage scores of each dosage group of AAV8-Afli Fc Variant 10 decreased, while the average spot leakage score of the Eylea® protein control group increased, indicating that each dosage group of AAV8-Afli Fc Variant 10 can maintain stable expression and has a longer drug efficacy compared with the Eylea® protein drug.

[0173] Example 13: Evaluation of the drug efficacy of AAV vector molecules in non-human primate (cynomolgus monkey) laser model animals The eyes of non-human primates are similar to the ocular structures of humans. Cynomolgus monkeys are non-rodent animal models commonly used in non-clinical research of biopharmaceuticals. A cynomolgus monkey choroidal neovascularization (CNV) model was constructed by laser photocoagulation, and the inhibitory effect on cynomolgus monkey CNV after a single administration of the AAV virus AAV8-Afli Fc Variant10 prepared in Example 7 by subretinal injection was investigated.

[0174] Nine cynomolgus monkeys that had received a normal ophthalmic examination and were confirmed to have no eye diseases were enrolled in the experiment. The AAV virus AAV8-Afli Fc Variant10 prepared in Example 7 was used as the test article. The groups and administration status are shown in Table 13. Table 13: Groups and Dosages of Cynomolgus Monkeys JPEG2025523593000014.jpg39170

[0175] At D0, a single administration was performed through the subretinal cavity. The eye surface was disinfected using povidone-iodine. Under an ophthalmic surgical microscope, a 30G disposable injection needle was used to puncture the sclera of the cynomolgus monkey inside the limbus. A microinjector equipped with a 35G flat needle was used to enter along the puncture site, bypass the lens to reach the vitreous body, avoid the major blood vessels, and gradually advance the needle into the subretinal space and slowly inject at a point above the fovea. Immediately after withdrawing the syringe, the needle entry point was pressed with a cotton swab with povidone-iodine for 5 seconds. 50 μL was administered to each eye, and both eyes received a single administration.

[0176] For D56, animals were anesthetized with Zoletil® (5 - 10 mg / kg, i.m.), and 8 - 9 laser spots were created around the macula with a 532 nm laser (spot diameter was approximately 50 μm). Immediately after laser modeling, IR photos were taken and OCT scans were performed to confirm the appropriateness of the modeling. At the 4th week after laser modeling, the animals were anesthetized with Zoletil® (5 - 10 mg / kg, i.m.), then IR photos were taken, and the degree of healing of the laser spots was examined by SD - OCT scan. Images of early and late fluorescein fundus angiography (FFA) were taken and used for leakage scoring. The details of the scoring are shown in Table 14.

[0177] Table 14: Evaluation criteria for leakage spots JPEG2025523593000015.jpg47170 Note: Spots of grade III - IV are leakage lesions.

[0178] After administration, the content of the target protein in the aqueous humor was continuously measured, and the expression continued to be stable for 12 weeks and is still under monitoring (the Eylea® protein drug could only be maintained for 4 - 8 weeks).

[0179] A typical atlas of fundus angiography (FFA) 4 weeks after laser modeling is shown in Figure 12. As can be seen from Figure 12, in the low - dose group (1E+9 GC / eye) and high - dose group (1E+10 GC / eye) of AAV virus AAV8 - Afli Fc Variant10, the leakage spots after laser modeling were significantly reduced compared with the solvent group, and there were significant differences in both.

[0180] After calculating the spot area and evaluating the spots, the results of Two - way Anova / Dunnett statistical evaluation showed that there were significant differences in both the low - dose group and the high - dose group compared with the control group. The statistical results are shown in Table 15.

[0181] Table 15: Leakage spot statistics after constructing a laser - induced CNV model in cynomolgus monkeys JPEG2025523593000016.jpg 124170 Note: The significance analysis is a significance analysis that compares the realization group with the solvent control group. P < 0.05 indicates a statistical difference, P < 0.01 indicates a significant statistical difference, and P < 0.001 indicates a very significant statistical difference.

[0182] Example 14: Evaluation of the Efficacy of AAV Vector Molecules in Non-Human Primate (Cynomolgus Monkey) DL-AAA Model Animals One challenge of retinal vascular disease models in species whose eyes are very similar to human eyes (e.g., non-human primate animals (NHP)) is the lack of chronic vascular leakage and / or the absence of the angiogenic response characteristics of human eAMD, DME, and PDR. In the NHP laser-induced CNV model of eAMD, transient extravascular leakage and angiogenic responses in the choroidal vasculature persist for 6 - 8 weeks after laser disruption of Bruch's membrane and then resolve spontaneously. Anti-VEGF treatment promotes the resolution of this induced disease state, but in humans, the CNV disease state recurs and persists after removal of the anti-VEGF drug. Therefore, constructing models of persistent and recurrent extravascular leakage and angiogenesis can greatly facilitate and accelerate the evaluation of long-acting interventions and can address many clinical manifestations of pathological vascular instability and angiogenesis. The DL-α-aminoadipic acid (DL-AAA) model is a chronic leakage model reported in rats and rabbits, and screening of common candidate drugs has been carried out in these species. DL-AAA is a selective glial cell toxin and, according to reports, may inhibit the action of glutamine synthetase, impair the retinal homeostasis function of extensive Müller cells, cause glial dysfunction and death, and cause disruption of the blood-retinal barrier. The vascular structure of the retina, the boundary between the retinal segmentation and the basal layer, the proportional abundance of the subtypes of retinal neuron cells and glial cells, and the presence of the macula are homologous between monkeys and humans. The newly induced chronic vascular leakage NHP model by DL-AAA, which is a preclinical model of chronic retinal vascular leakage and angiogenesis, enables the screening of the efficacy of short-acting and long-acting anti-angiogenic compounds at multiple stages of disease onset.

[0183] One cynomolgus monkey (animal number P1) was selected, and 30 μL of 50 mmol / L DL-α-AAA (sigma-aldrich) was injected subretinally into both eyes. For each eye, fundus color photography, fluorescein fundus angiography, macular autofluorescence, optical coherence tomography (OCT), and multifocal electroretinogram were performed at 1 week before injection and 6 weeks and 12 weeks after injection. It was confirmed that the construction of the cynomolgus monkey persistent retinal neovascularization (PRNV) model was successful, and the modeling was started 6 - 8 weeks later.

[0184] One cynomolgus monkey with a persistent retinal neovascularization (PRNV) model (this model has been constructed for more than half a year (one in the bilateral PRNV model group (animal number P1))) was selected. The virus AAV8-Afli Fc Variant10 (1E+10 GC / eye / 50 μL) prepared in Example 7 was injected into the right eye, and AAV diluent (solvent control group, 50 μL / eye) was injected into the left eye. The changes in long-term ophthalmic FFA examinations at Baseline, 2 weeks, 6 weeks, 8 weeks, 10 weeks, and 12 weeks after injection were observed. The results observed so far are as follows.

[0185] As shown in Figure 13 (in the figure, *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistically significant differences), the fluorescence leakage in the PRNV model administration group (right eye) decreased significantly compared to the baseline at 2 weeks and continued to decrease every 2 weeks until the 12th week, and the continuous decreasing effect was obvious. There was no significant change in fluorescence leakage in the PRNV model monkey solvent group (left eye) compared to the baseline from 2 weeks to 12 weeks.

[0186] The sustained relaxation time of the AAV8-Afli Fc Variant10 test article exceeds that of the CNV laser modeling, indicating the superiority of this model when observing the long-term efficacy of AAV. As can be seen from the statistical analysis, regardless of the leakage area and leakage intensity, the test article is significantly different from the solvent group in all cases, indicating that the AAV8-Afli Fc Variant10 adeno-associated virus has a significant therapeutic effect on the non-human primate PRNV model.

[0187] The above embodiments are only some embodiments of the present invention and are described in detail, but do not limit the scope of the present invention. Those skilled in the art can make various modifications, combinations, and improvements to the above embodiments without departing from the idea of the present invention, and all of these are included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the scope of the claims.

Claims

1. An Fc fragment variant of human IgG1, wherein the variant has at least one mutation among T250A, L251A, M252L, I253A / D / P, S254A, T256A, L309A, H310L / V / A / D / E / Q, Q311A, L314A, M428L / I, H433L / V / A, N434L / V / A, H435L / V / A, Y436A, and the numbering is according to the EU index, an Fc fragment variant of human IgG1.

2. having one single point mutation among H310A, H310E or H435A, or having a double mutation of H310A / H435A, and the amino acid sequence is shown in SEQ ID NO: 1, 2, 3 or 4, an Fc fragment variant of human IgG1 according to Claim 1.

3. A VEGF receptor recombinant fusion protein comprising the Fc fragment variant of human IgG1 according to Claim 1, wherein the VEGF receptor recombinant fusion protein is a fusion of domain 2 of VEGFR-1, domain 3 of VEGFR-2, and the Fc fragment variant according to Claim 1, the domain 2 of VEGFR-1 is shown in SEQ ID NO: 5, and the domain 3 of VEGFR-2 is shown in SEQ ID NO: 6, a VEGF receptor recombinant fusion protein.

4. and the amino acid sequence is shown in SEQ ID NO: 7, 8, 9 or 10, a VEGF receptor recombinant fusion protein according to Claim 3.

5. An anti-VEGF recombinant antibody comprising the Fc fragment variant according to Claim 1, wherein the anti-VEGF recombinant antibody is obtained by replacing the Fc fragment of the anti-VEGF antibody with the Fc fragment variant according to Claim 1, an anti-VEGF recombinant antibody.

6. the anti-VEGF recombinant antibody is obtained by mutating the Fc fragment of bevacizumab to H310E and has the amino acid sequence shown in SEQ ID NO: 11, an anti-VEGF recombinant antibody according to Claim 5.

7. An AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein according to Claim 3 or the anti-VEGF recombinant antibody according to Claim 5, wherein the expression cassette contains the structure of formula I from the 5'-end to the 3'-end, ITR-E1-E2-E3-E4-ITR formula (I) wherein ITR is an inverted terminal repeat sequence, E1 is a promoter, E2 is a signal peptide, E3 is the nucleotide sequence of the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody, E4 is a Poly A sequence, and the vector expression cassette is characterized by this. **Claim 8** The ITR is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9, The promoter is preferably a CMV, CBA, EF1a, SV40, PGK1, Ubc, CAG, TEF1, U6 or H1 promoter, The signal peptide is derived from Human OSM, Gaussia luc or Albumin (HSA), but is not limited thereto, The vector expression cassette according to claim 7, wherein the Poly A sequence is selected from bGH PolyA, SV40 PolyA or hGH PolyA. **Claim 9** The expression cassette further includes a regulatory element, and the expression regulatory element is (1) a regulatory element for regulating the expression of the target protein (for example, an IRES that initiates the translation of a downstream gene), (2) a regulatory element for expressing miRNA and siRNA sequences, (3) an intron, (4) a localization sequence for localizing and expressing the target protein in the cell nucleus, cytoplasm or various organelles and secreting it outside the cell, (5) a Kozak sequence, (6) an enhancer, (7) a functional regulatory element selected from WPRE, but is not limited thereto. The vector expression cassette according to claim 7 is characterized by this. **Claim 10** The expression cassette further includes a label element, and the label element includes, but is not limited to, FLAG, HA, MYC, fluorescent protein, luciferase, SUMO protein, ubiquitin protein, GST, etc. The vector expression cassette according to claim 7 is characterized by this. **Claim 11** Express the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody on an AAV viral vector, The AAV viral vector includes, but is not limited to, pAAV-CMV, pX601, pX551, pAAV-MCS plasmid. The vector expression cassette according to claim 7 is characterized by this. **Claim 12** The AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein has the sequence shown in SEQ ID NO: 12, The AAV viral vector expression cassette expressing the anti-VEGF recombinant antibody is characterized by having the sequence shown in SEQ ID NO: 13, and the vector expression cassette according to claim 7.

13. An adeno-associated virus packaging vector system, The packaging vector system includes an AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein or anti-VEGF recombinant antibody according to claim 7, a vector carrying the AAV rep, cap genes, and a helper virus vector. The vector is characterized by becoming an AAV virus upon packaging, and it is an adeno-associated virus packaging vector system.

14. The vector carrying the AAV rep, cap genes includes, but is not limited to, AAV1, AAV2, AAV5, AAV8, AAV9, AAV-R100, AAV-NN, AAV-GL, AAV8-Y447F, AAV8-Y733F, AAV8-Y447F / Y733F, AAV-DJ or AAV7m8 vector. The helper virus vector is a pHelper plasmid, and it is the adeno-associated virus packaging vector system according to claim 13.

15. The packaging vector system according to claim 13 is introduced into a host cell for virus packaging. The host cell is a cell line capable of virus replication and stable inheritance, including but not limited to cells such as Hela-S3, HEK-293, HEK-293T, HEK-293FT, Expi293F, A549 or Sf9, and it is a method for packaging adeno-associated virus.

16. An adeno-associated virus prepared by packaging according to the method for packaging adeno-associated virus according to claim 15.

17. It is obtained by introducing an AAV viral vector expression cassette expressing the VEGF receptor recombinant fusion protein shown in SEQ ID NO: 12, an AAV8 Rep-Cap plasmid, and a pHelper plasmid into Expi293F cells as host cells for virus packaging, and it is the adeno-associated virus according to claim 16.

18. An adeno-associated virus according to claim 16, which is obtained by introducing an AAV viral vector expression cassette expressing an anti-VEGF recombinant antibody shown in SEQ ID NO: 13, an AAV8 Rep-Cap plasmid, and a pHelper plasmid into Exp293F as a host cell for virus packaging.

19. A preparation, formulation or drug comprising an AAV viral vector expression cassette expressing a VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody according to claim 7, or an adeno-associated virus according to claim 16.

20. The preparation, formulation or drug according to claim 19, which may be in any dosage form, including but not limited to an injection dosage form and an ointment dosage form.

21. The preparation, formulation or drug according to claim 19, wherein the AAV viral vector expression cassette expressing a VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody, or the adeno-associated virus is the only active ingredient.

22. Use of an AAV viral vector expression cassette expressing a VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody according to claim 7, or an adeno-associated virus according to claim 16, in the preparation of a preparation, formulation or drug for treating angiogenesis-related fundus diseases.

23. The use according to claim 22, wherein the angiogenesis-related fundus diseases include age-related macular degeneration, wet macular degeneration, and diabetic retinopathy.

24. The administration method of the preparation, formulation or drug is unilateral administration or bilateral administration, and the administration form is an injection method such as intravitreal injection, subretinal injection, suprachoroidal injection, etc. The total dosage for single administration or multiple administrations during the lifetime is 1×10 ８ -1×10 １３ The use according to claim 22, characterized in that it is 1×10 ８ -1×10 １３ viral genomes / eye.

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