CN117143920A

CN117143920A - Gene medicine for treating neovascular eye diseases

Info

Publication number: CN117143920A
Application number: CN202311102696.4A
Authority: CN
Inventors: 李桂忱; 吴理达; 靳瑾; 李航; 张巍; 顾雨春
Original assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Current assignee: Chengnuo Regenerative Medical Technology Beijing Co ltd
Priority date: 2023-08-30
Filing date: 2023-08-30
Publication date: 2023-12-01

Abstract

The invention discloses a gene medicine for treating neovascular eye diseases, which not only can effectively inhibit the formation of neovascular, but also has the protection and repair effects on fundus cells, has the effect of inhibiting inflammation, can fundamentally treat all types of neovascular eye diseases, and has good clinical application prospect.

Description

Gene medicine for treating neovascular eye diseases

Technical Field

The invention belongs to the field of gene therapy, and in particular relates to a gene medicine for treating neovascular eye diseases.

Background

Neovascular ocular diseases are a disease with higher incidence rate and causing vision impairment, and ocular fundus can regenerate unnecessary blood vessels with the increase of age, the aggravation of myopia or the development of related metabolic diseases. These new blood vessels invade the otherwise normal ocular fundus tissues such as choroid and retina photoreceptor cell layers, and cause ocular fundus diseases such as age-related macular degeneration (age-related macular degeneration, AMD), pathological myopia (pathological myopia, PM) and diabetic retinopathy (diabetic retinopathy, DR), and the like, so that the visual function is impaired, and serious blindness is caused. Among them, age-related macular degeneration and diabetic retinopathy are two more common types of neovascular ocular diseases. At present, the number of patients suffering from the neovascular eye diseases is over 4000 ten thousand, and the number of patients is continuously increased along with the aging degree of population.

Studies have shown that the primary signaling pathway responsible for ocular physiology and pathologic angiogenesis is the vascular endothelial growth factor (vascular endothelial growth factor, VEGF) pathway, and that expression of VEGF and its receptors is significantly upregulated in human neovascular ocular tissue. Thus, intravitreal injection of anti-VEGF drugs is currently the first line treatment modality for neovascular ocular diseases. Common anti-VEGF drugs are ranibizumab, combretastatin and aflibercept. The ranibizumab is Sub>A recombinant humanized monoclonal anti-VEGF-A drug imported by Switzerland, and is the anti-VEGF drug with the most extensive coverage age group and the most indication. The combretastatin and the aflibercept are fusion protein anti-VEGF drugs which are independently developed in China and imported in Germany respectively. The vitreous cavity injection of the VEGF resisting medicine can improve the baseline vision of wet AMD patients, relieve macular edema of diabetic macular edema patients, inhibit retinal vein occlusion of new blood vessels of patients, and inhibit choroidal new blood vessels and leakage of pathological myopia CNV patients, so as to achieve the effect of improving vision. However, these drugs are difficult to be concentrated on the fundus focus for a long time, and it is usually required to inject once a month in clinical treatment, and even so, the effective rate is only 40% -60%. The gene therapy method can provide long-term and stable anti-VEGF activity, and becomes a current research hotspot.

However, current gene therapy approaches for neovascular ocular diseases have the following drawbacks: fewer indications, mostly only for macular degeneration; longer sequence, limited AAV virus capacity, and lower packaging efficiency; the treatment of the disease is only aimed at inhibiting the formation of new blood vessels, and has no protective and repair effects on cells at the bottom of eyes, and no inflammation inhibiting effect. In view of the above, the present invention aims to provide a gene drug capable of simultaneously inhibiting angiogenesis, protecting retinal ganglion cells, and reducing inflammatory reactions.

Disclosure of Invention

In order to overcome the technical defects existing in the prior art, the invention aims to provide a gene medicine for treating neovascular eye diseases.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

in a first aspect the invention provides a construct for use in the treatment of a neovascular ocular disease.

Further, the construct comprises a nucleotide encoding sFLT1 (1-4) or sFLT1 (2-3); the nucleotide sequence of the coding sFLT1 (1-4) is shown as SEQ ID NO. 2; the nucleotide sequence of the coding sFLT1 (2-3) is shown as SEQ ID NO. 5.

Further, the construct further comprises a nucleotide encoding CNTF; the nucleotide sequence of the coded CNTF is shown as SEQ ID NO. 9; the nucleotide sequence encoding sFLT1 (1-4) or sFLT1 (2-3) and the nucleotide sequence encoding CNTF are connected through T2A; preferably, the construct comprises AAV-sFLT1 (1-4), AAV-sFLT1 (2-3), AAV-sFLT1 (1-4) -T2A-CNTF, AAV-sFLT1 (2-3) -T2A-CNTF; more preferably, the construct is AAV-sFLT1 (1-4) -T2A-CNTF.

In some embodiments, the neovascular ocular disease includes wet macular degeneration, diabetic macular edema, diabetic retinopathy, outer exudative retinopathy, retinal vein occlusion, corneal neovascularization, pathological myopia CNV, neovascular glaucoma, uveitis, polypoidal choroidal vasculopathy, radial optic neuropathy. In the present invention, the neovascular ocular diseases are not limited to the specific types of diseases listed above, and diseases occurring in any part of the eye (e.g., cornea, iris, retina, vitreous body and choroid) to generate new blood vessels in a pathogenic manner are within the scope of the present invention.

In a second aspect, the invention provides a recombinant viral vector for use in the treatment of a neovascular ocular disease.

Further, the recombinant viral vector comprises the construct of the first aspect of the invention and a viral vector backbone.

Further, the viral vectors include adeno-associated viral vectors, adenovirus vectors, lentiviral vectors, retrovirus vectors, herpes simplex viral vectors, baculovirus vectors, sendai viral vectors, poxvirus vectors, geminivirus vectors;

Preferably, the viral vector is an adeno-associated viral vector; more preferably, the adeno-associated viral vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof; most preferably, the adeno-associated viral vector is AAV2.

In some embodiments, recombinant vectors comprising the construct of the first aspect of the invention and a DNA vector backbone are also included within the scope of the invention, including, but not limited to: DNA plasmid vectors, liposomes that bind DNA plasmids, molecular conjugates that bind DNA plasmids, multimers that bind DNA plasmids, and the like.

In some embodiments, the recombinant viral vector may be administered alone or in a pharmaceutical composition comprising the recombinant viral vector according to the second aspect of the invention, one or more pharmaceutically acceptable carriers and/or excipients, and/or one or more other therapeutic agents that can be used to treat neovascular ocular diseases, wherein the one or more pharmaceutically acceptable carriers and/or excipients must be compatible with the other components of the pharmaceutical composition and not deleterious to the subject.

In some embodiments, the pharmaceutically acceptable carrier and/or adjuvant refers to a substance suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers and/or excipients useful in the present invention are conventional, remington's Pharmaceutical Sciences, mack Publishing co., easton, PA, 15 th edition (1975) written by e.w. martin, describe compositions and formulations suitable for drug delivery of one or more therapeutic compounds, molecules or agents. Generally, the nature of the carrier will depend on the particular mode of administration employed.

In some embodiments, the adeno-associated viral vector is not particularly limited as long as it can be used to deliver sFLT1 (1-4) or sFLT1 (2-3)) and CNTF genes, thereby functioning to effectively inhibit ocular fundus vascular proliferation and vascular leakage, and to restore bipolar cell function, and the adeno-associated viral vector is within the scope of the present invention.

In a third aspect, the present invention provides a method for preparing a recombinant viral vector according to the second aspect of the present invention.

Further, the method comprises the following steps:

(1) Construction of AAV-sFLT1 (1-4), AAV-sFLT1 (2-3), AAV-sFLT1 (1-4) -T2A-CNTF or AAV-sFLT1 (2-3) -T2A-CNTF plasmid vectors;

(2) Co-transfecting the plasmid vector constructed in the step (1) with AAV2 and pHelper into cells, and culturing the cells;

(3) And collecting cell supernatant to obtain infectious viral particles, namely the recombinant viral vector in the second aspect of the invention.

Further, the nucleotide sequence of the AAV-sFLT1 (1-4), AAV-sFLT1 (2-3), AAV-sFLT1 (1-4) -T2A-CNTF or AAV-sFLT1 (2-3) -T2A-CNTF plasmid vector for coding sFLT1 (1-4) is shown as SEQ ID NO. 2, the nucleotide sequence of the coding sFLT1 (2-3) is shown as SEQ ID NO. 5, and the nucleotide sequence of the coding CNTF is shown as SEQ ID NO. 9; preferably, the construction of the AAV-sFLT1 (1-4) or AAV-sFLT1 (2-3) plasmid vector of step (1) comprises the steps of: synthesizing sFLT1 (1-4) or sFLT1 (2-3) coding cDNA sequence by taking pAAV-SFFV plasmid as a skeleton vector for gene expression, inserting the cDNA sequence into pAAV-SFFV plasmid through homologous recombination, transforming into escherichia coli DH5 alpha, and picking up monoclonal to obtain AAV-sFLT1 (1-4) or AAV-sFLT1 (2-3) plasmid vector; preferably, the construction of the AAV-sFLT1 (1-4) -T2A-CNTF or AAV-sFLT1 (2-3) -T2A-CNTF plasmid vector described in step (1) comprises the following steps: synthesizing sFLT1 (1-4) or sFLT1 (2-3) coding cDNA sequence, T2A coding sequence and CNTF coding cDNA sequence by taking pAAV-SFFV plasmid as a skeleton vector for gene expression, inserting the pAAV-SFFV plasmid into the pAAV-SFFV plasmid through homologous recombination, transforming into escherichia coli DH5 alpha, and picking up monoclonal to obtain AAV-sFLT1 (1-4) -T2A-CNTF or AAV-sFLT1 (2-3) -T2A-CNTF plasmid vector;

Preferably, the cells in step (2) are HEK293FT cells; preferably, the conditions of the culture in step (2) are 37℃and 5% CO ₂ ；

Preferably, the collecting of the cell supernatant in step (3) is from the beginning of the transfection of 48H, 72H, 96H.

In a fourth aspect, the present invention provides a gene therapy agent for the treatment of neovascular ocular diseases.

Further, the gene therapy agent comprises the recombinant viral vector according to the second aspect of the present invention; preferably, the gene therapy drug further comprises a pharmaceutically acceptable carrier and/or adjuvant; preferably, the neovascular ocular disease comprises wet macular degeneration, diabetic macular edema, diabetic retinopathy, outer exudative retinopathy, retinal vein occlusion, corneal neovascularization, pathologic myopia CNV, neovascular glaucoma, uveitis, polypoidal choroidal vasculopathy, radiation optic neuropathy; preferably, the administration mode of the gene therapy drug comprises intravitreal injection and subretinal injection; more preferably, the gene therapy drug is administered by intravitreal injection.

In some embodiments, pharmaceutical formulations suitable for topical administration to the eye include eye drops wherein the active ingredient (recombinant viral vector according to the second aspect of the invention) is dissolved or suspended in a suitable carrier, in particular an aqueous solvent. Formulations to be applied to the eye will have an ophthalmically compatible pH and osmolality. One or more ophthalmically acceptable pH adjusting agents and/or buffers may be included in the compositions of the present invention, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, acetic acid/sodium lactate; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. These acids, bases, and buffers may be included in amounts that maintain the pH of the composition within an ophthalmically acceptable range. One or more ophthalmically acceptable salts may be included in the composition in an amount sufficient to bring the osmolality of the composition within an ophthalmically acceptable range. These salts include those containing sodium, potassium or ammonium cations and hydrochloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfide anions.

In some embodiments, the pharmaceutical formulation is adapted for intraocular administration by intraocular injection or other means for intraocular delivery. Examples of ocular devices that may be used in the methods of the present invention include periocular or intra-crystalline devices, contact lenses, liposomes, and the like. See, for example, U.S. Pat. nos. 3,416,530;3,828,777;4,014,335;4,300,557;4,327,725;4,853,224;4,946,450;4,997,652;5,147,647;5,164,188;5,178,635;5,300,114;5,322,691;5,403,901;5,443,505;5,466,466;5,476,511;5,516,522;5,632,984;5,679,666;5,710,165;5,725,493;5,743,274;5,766,242;5,766,619;5,770,592;5,773,019;5,824,072;5,824,073;5,830,173;5,836,935;5,869,079,5,902,598;5,904,144;5,916,584;6,001,386;6,074,661;6,110,485;6,126,687;6,146,366;6,251,090;6,299,895;6,331,313;6,416,777;6,649,184;6,719,750;6,660,960; and U.S. patent publication nos. 2003/0064088, 2004/024645 and 2005/0110206; each of which is incorporated herein by reference for the purpose of their teachings regarding ophthalmic devices.

In some embodiments, the ocular delivery device may be designed to control the release of one or more therapeutic agents, have different prescribed release rates and maintain dose kinetics and permeability. The choice of biodegradable/bioerodible polymers (e.g., polyethylene-vinyl acetate (EVA), super hydrolyzed PVA), hydroxyalkyl cellulose (HPC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), polycaprolactone, polyglycolic acid, polylactic acid, polyanhydrides, and the choice of polymer molecular weight, polymer crystallinity, copolymer ratio, processing conditions, surface finish, geometry, excipient addition, and polymer coating, etc., will enhance drug diffusion, erosion, dissolution, and permeation by designing the polymer matrix to achieve controlled release, including different choices and properties.

In some embodiments, a formulation for delivering a drug using an ophthalmic device may incorporate one or more active agents and adjuvants appropriate for the intended route of administration. For example, the active agent may be mixed with any pharmaceutically acceptable excipient, lactose, sucrose, starch powder, cellulose alkanoates, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, acacia, gelatin, sodium alginate, polyvinylpyrrolidone and/or polyvinyl alcohol and formulated into tablets or capsules for conventional administration. Alternatively, the compound may be dissolved in polyethylene glycol, propylene glycol, carboxymethyl cellulose gum solution, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth, and/or various buffers. The compounds may also be mixed with both biodegradable and non-biodegradable polymers, and carrier or diluent components which may have delay properties. Illustrative examples of biodegradable components include albumin, gelatin, starch, cellulose, dextran, polysaccharides, poly (D, L-lactide), D, L-lactide-co-glycolide, polyglycolides, polyhydroxybutyrate, polyalkylcarbonate, and polyorthoesters, and mixtures thereof. Illustrative examples of the biodegradable polymer may include EVA copolymer, silicone rubber, and polymethyl acrylate, and mixtures thereof.

In some embodiments, the pharmaceutical composition for ocular delivery further comprises an aqueous composition that is gellable in situ. Such compositions include a gelling agent at a concentration effective to promote gelling when contacted with the eye or tear fluid. Suitable gelling agents include, but are not limited to, thermosetting polymers. Wherein the "in situ gellable" includes not only low viscosity liquids that form gels upon contact with the eye or tear fluid, but also more viscous liquids, such as semi-fluids and thixotropic gels that have substantially increased viscosity or gel stiffness when applied to the eye. See, for example, ludwig (2005) adv. Drug deliv. Rev.3;57:1595-639, which is incorporated herein by reference for the purpose of its teachings regarding examples of polymers used in ocular drug delivery.

In a fifth aspect the invention provides a pharmaceutical composition for use in the treatment of neovascular ocular diseases.

Further, the pharmaceutical composition comprises the gene therapy drug of the fourth aspect of the present invention; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant; preferably, the pharmaceutical composition further comprises a second therapeutic agent for treating a neovascular ocular disease.

Further, the second therapeutic agent comprises a hypoglycemic agent, an anticoagulant agent, a vascular protective agent; preferably, the hypoglycemic agent comprises sulfonylurea agent, glinide agent, DPP-4 inhibitor, biguanide agent, thiazolidinedione agent, alpha-glycosidase inhibitor; preferably, the anticoagulants include aspirin, brivudine, heparin anticoagulants and warfarin; preferably, the vascular protection drug comprises a hypolipidemic drug and an antiplatelet drug; preferably, the neovascular ocular disease includes wet macular degeneration, diabetic macular edema, diabetic retinopathy, outer exudative retinopathy, retinal vein occlusion, corneal neovascularization, pathological myopia CNV, neovascular glaucoma, uveitis, polypoidal choroidal vasculopathy, radial optic neuropathy.

In some embodiments, the gene therapy agents or pharmaceutical compositions of the present invention may be presented in unit dosage form containing a predetermined amount of active ingredient per unit dose (recombinant viral vector of the second aspect of the present invention). Depending on the type and/or severity of the disease being treated, the route of administration, the age, weight and condition of the patient, or the gene therapy drug or pharmaceutical composition of the invention may be presented in unit dosage form containing a predetermined amount of active ingredient per unit dose. In certain embodiments, unit dosage formulations are those containing a daily dose or sub-dose, or an appropriate fraction thereof, of the active ingredient. In addition, these pharmaceutical formulations may be prepared by any of the methods well known in the pharmaceutical arts.

In some embodiments, the gene therapy agents or pharmaceutical compositions of the present invention may be administered by any suitable route. In certain embodiments, the suitable route of administration includes intraocular administration. In certain embodiments, the means of intraocular administration includes, but is not limited to: intravitreal administration, subretinal administration, subscleral administration, intracoronary administration, subconjunctival administration, etc., it being understood that the preferred route of administration for a particular subject may depend on the condition of the subject.

In some embodiments, the gene therapy agents or pharmaceutical compositions of the present invention may also be combined with other therapeutic agents that can be used to treat ocular neovascular disorders, where combination therapy is employed, the therapeutic agents may be administered together or separately. The same mode of administration may be used for more than one therapeutic agent in combination therapy; alternatively, different therapeutic agents in combination therapy may be administered in different ways. When the therapeutic agents are administered separately, they may be administered simultaneously or sequentially in any order (closer or farther apart in time). The relative time of administration of the gene therapy drug or pharmaceutical composition and/or other therapeutic agent or agents of the present invention may be selected in accordance with the desired combination therapeutic effect.

In addition, the present invention provides a method of treating a neovascular ocular disease, the method comprising the steps of: administering to a subject in need thereof an effective amount of a recombinant viral vector according to the second aspect of the invention, a gene therapy drug according to the fourth aspect of the invention, and/or a pharmaceutical composition according to the fifth aspect of the invention.

In some embodiments, the effective amount refers to an amount that has a therapeutic effect or an amount required to produce a therapeutic effect in a subject. For example, a pharmaceutically or pharmaceutically effective amount refers to the amount of drug required to produce a desired therapeutic effect, which can be reflected by the results of a clinical trial, a model animal study, and/or an in vitro study. The pharmaceutically effective amount depends on several factors, including, but not limited to, the subject's characteristic factors (e.g., height, weight, sex, age, and history of administration), the severity of the disease.

In some embodiments, the subject refers to any animal, and also refers to human and non-human animals. The term non-human animal includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any domestic animals or pets; and non-mammals, such as chickens, amphibians, reptiles, etc., in certain embodiments, the subject is preferably a human.

A sixth aspect of the invention provides any one of the following applications:

(1) Use of a construct according to the first aspect of the invention for the preparation of a recombinant viral vector for the treatment of a neovascular ocular disease;

(2) The recombinant viral vector of the second aspect of the present invention is used in preparing gene therapeutic medicine for treating ocular neovascular diseases;

(3) The use of a gene therapy agent according to the fourth aspect of the invention in the manufacture of a pharmaceutical composition for the treatment of neovascular ocular diseases;

(4) Use of sFLT1 (1-4) or sFLT1 (2-3) for the preparation of a recombinant viral vector for the treatment of neovascular ocular diseases;

(5) Use of sFLT1 (1-4) or sFLT1 (2-3) in the manufacture of a gene therapy medicament for the treatment of neovascular ocular disorders;

(6) Use of sFLT1 (1-4) or sFLT1 (2-3) for the manufacture of a pharmaceutical composition for the treatment of a neovascular ocular disease;

(7) Use of sFLT1 (1-4) or a combination of sFLT1 (2-3) and CNTF for the preparation of a recombinant viral vector for the treatment of a neovascular ocular disease;

(8) Use of sFLT1 (1-4) or sFLT1 (2-3) in combination with CNTF for the preparation of a gene therapy medicament for the treatment of neovascular ocular diseases;

(9) Use of sFLT1 (1-4) or a combination of sFLT1 (2-3) and CNTF for the preparation of a pharmaceutical composition for the treatment of a neovascular ocular disease;

the nucleotide sequence of the coding sFLT1 (1-4) is shown as SEQ ID NO. 2;

the nucleotide sequence of the coding sFLT1 (2-3) is shown as SEQ ID NO. 5;

the nucleotide sequence for coding the CNTF is shown as SEQ ID NO. 9.

Compared with the prior art, the invention has the advantages and beneficial effects that:

(1) The invention provides a novel gene therapy drug for neovascular eye diseases, which comprises recombinant virus vectors AAV2-sFLT1-T2A-CNTF, wherein sFLT1 is sFLT1 (1-4) or sFLT1 (2-3) obtained after sFLT1 is truncated, and the obtained sequence is shorter and can normally perform biological functions, thereby being more beneficial to virus packaging. The gene therapy medicine provided by the invention not only can effectively inhibit the formation of new blood vessels, but also has the effects of protecting and repairing fundus cells, inhibiting inflammation and having obvious therapeutic effects on new vascular eye diseases;

(2) The gene therapy medicine provided by the invention can effectively inhibit fundus blood vessel hyperplasia and blood vessel leakage, and restore bipolar cell function, can fundamentally treat all types of neovascular eye diseases, and can also treat fundus diseases such as diabetic macular edema, diabetic retinopathy, neovascular glaucoma, pathologic myopia CNV, cornea neovascular, outer exudative retinopathy, retinal vein occlusion and the like besides macular degeneration. Overcomes the technical defects of few indications, low packaging efficiency, no protection and repair effects on ocular fundus cells and the like existing in the current gene therapy method for the neovascular ocular diseases, provides a new thought for the treatment of various neovascular ocular diseases, and has very good clinical application prospect.

Drawings

FIG. 1 is a vector map of AAV-sFLT1 (1-3), AAV-sFLT1 (1-4), AAV-sFLT1 (1-5), AAV-sFLT1 (1-6), AAV-sFLT1 (2-3), AAV-sFLT1 (2-4), AAV-sFLT1 (2-5), AAV-sFLT1 (2-6);

FIG. 2 is a graph showing the analysis of GFP expression after infection of HUVEC cells with AAV-GFP virus at 48H, with a scale of 100. Mu.m; FIG. 3 is a graph of results of testing the capacity of HUVEC cells in each treatment group for tube formation, wherein, panel A: the HUVEC cells of each treatment group had a tube formation at 4H, 6H and 8H, and the scale bar was 200 μm; b, drawing: after culturing 4H, 6H, 8H, total length of each group of HUVEC cell junctions; c, drawing: after incubation for 4H, 6H, 8H, the number of HUVEC cell connections for each group, data expressed as mean ± SD of three independent experiments, P < 0.05, P < 0.01, P < 0.001 compared to AAV-GFP group;

FIG. 4 is a graph of results of CCK8 testing the proliferation potency of HUVEC cells in each treatment group, as indicated by mean+ -SD of three independent experiments comparing P < 0.05, P < 0.01, P < 0.001 with AAV-GFP;

FIG. 5 is a graph of results of testing HUVEC cell migration capacity for each treatment group, wherein, panel A: scratch growth at 0H, 2H, 4H, 6H, 8H, 10H for HUVEC cells of each treatment group was rated at 200 μm; b, drawing: after culturing 0H, 2H, 4H, 6H, 8H, 10H, each group of HUVEC cells was analyzed for scratch healing rate, and data were expressed as mean ± SD of three independent experiments;

FIG. 6 is a vector map of AAV-sFLT1 (1-4) -CNTF;

FIG. 7 is a graph of results of testing the capacity of HUVEC cells in each treatment group for tube formation, wherein, panel A: the HUVEC cells of each treatment group had a tube formation at 4H, 6H and 8H, and the scale bar was 200 μm; b, drawing: after culturing 4H, 6H, 8H, total length of each group of HUVEC cell junctions; c, drawing: after incubation for 4H, 6H, 8H, the number of HUVEC cell connections for each group, data expressed as mean ± SD of three independent experiments, P < 0.05, P < 0.01, P < 0.001 compared to AAV-GFP group;

FIG. 8 is a graph of the results of CCK8 testing the proliferation capacity of HUVEC cells in each treatment group, the proliferation rate of HUVEC cells in each treatment group at 24H and 48H, data expressed as mean+ -SD of three independent experiments, P < 0.05, P < 0.01 compared to AAV-GFP;

FIG. 9 is a graph of results of testing HUVEC cell migration capacity for each treatment group, wherein, panel A: scratch growth at 0H, 2H, 4H, 6H, 8H, 10H for HUVEC cells of each treatment group was rated at 200 μm; b, drawing: after culturing 0H, 2H, 4H, 6H, 8H, 10H, each group of HUVEC cells was analyzed for scratch healing rate, and data were expressed as mean ± SD of three independent experiments;

FIG. 10 is a graph showing the results of sFLT1 (1-4) in cell supernatants inhibiting the ability of HUVECs to form tubes, proliferate and migrate, wherein, panel A: ELISA (enzyme Linked immunosorbent assay) for detecting the sFLT1 content in culture supernatant after the transient HEK293FT cells of the plasmid AAV-GFP and AAV-sFLT1 (1-4) are 48H; b, drawing: statistical analysis of tube length of each group of cells at 4H, 6H and 8H after plating after treatment of HUVEC cells with the culture supernatants described above, respectively; c, drawing: after HUVEC cells are treated by the culture supernatant respectively, the number of the tube forming nodes of each group of cells is statistically analyzed at 4H, 6H and 8H after seed placement; d, drawing: proliferation rate analysis of each group of cells at 24H and 48H after the respective treatment of HUVEC cells with the culture supernatants; e, drawing: statistical analysis of cell mobility at scratches 0H, 2H, 4H, 6H, 8H and 10H for each group of cells after treatment of HUVEC cells with the culture supernatants described above, respectively. Data are expressed as mean value of three independent experiments, SD. * P < 0.05, P < 0.01, P < 0.001 compared to AAV-GFP group;

Fig. 11 is a statistical graph showing the recovery rate of vascular network and vascular leakage of the fundus of a mouse by fluorescein sodium radiography, wherein, the graph a: observing the conditions of the eyeground blood vessel network and vascular leakage of AAV-SFFV, afliberceptp, AAV-CNTF, AAV-sFLT1 (1-4) -CNTF and Afliibercept groups by sodium fluorescein radiography before and 7 days, 14 days, 21 days and 29 days after the administration; representative images of each group are shown with a scale of 200 μm; b, drawing: 4 mice and 8 eyes are used for each experimental group, the vascular leakage recovery rate at 7 days, 14 days and 21 days is calculated by taking the vascular leakage rate of 0 day as a standard, the vascular leakage recovery rate calculation formula is The recovery rate = (leakage area on D0-leakage area on D7/D14/D21)/leakage area on D0 multiplied by 100%, and the data are expressed as average value of 8 eyes recovery rate +/-SD;

FIG. 12 is a graph of analysis of retinal paving and IB4 staining showing the distribution of blood vessels and leakage of blood vessels from the fundus of each group when the retinal paving and IB4 antibody staining was performed for 29 days after administration; representative images of each group are shown with a scale of 50 μm;

fig. 13 is an electroretinogram analysis chart, wherein, a chart: after dark adaptation, the flash intensity was 0.01 Cd.s/m ² Statistical analysis of a-wave and b-wave amplitudes in each set of waveforms; b, drawing: after dark adaptation, the flash intensity was 3 Cd.s/m ² Statistical analysis of a-wave and b-wave amplitudes in each set of waveforms; c, drawing: after explicit adaptation, the flash intensity was 3 Cd.s/m ² Is a statistical analysis of the amplitudes of the a-wave and b-wave in each set of waveforms. Data are expressed as mean ± SD of 8 eyes, P < 0.05 compared to pAAV-SFFV group;

FIG. 14 is a frozen section analysis chart showing the expression distribution of GFP in each cell layer of the fundus of mice after intravitreal injection of AAV2/2-GFP virus suspension for 21 days; the scale bar is 100 μm.

Detailed Description

The invention provides a novel treatment method for neovascular eye diseases, which comprises a recombinant AAV virus vector, wherein the vector has good targeting property on fundus cells, comprises the coding sequences of truncated sFLT1 and CNTF, can deliver the coding sequences of truncated sFLT1 and CNTF carried by AAV virus to fundus of neovascular eye diseases patients, and enables the functional sFLT1 and CNTF to be expressed in fundus cells, so that photoreceptor cells are protected and repaired on the basis of inhibiting neovascular formation, and vision of the neovascular eye diseases patients is improved. The experimental materials and instruments used in the examples of the present invention are shown in the following table.

The invention is further illustrated below in conjunction with specific examples, which are intended to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

EXAMPLE 1 construction of sFLT1 truncated structural adeno-associated Virus expression vector

Naturally occurring truncated splice variant sFLT1 has 6N-terminal Ig-like extracellular motifs. Wherein the first 3 Ig-like domains are responsible for binding ligands and the last 3 Ig-like domains are responsible for forming receptor dimers. AAV viruses have limited packaging capacity, so we have truncated sFLT1 to varying degrees, resulting in 8 truncated variants: AAV-sFLT1 (1-3), AAV-sFLT1 (1-4), AAV-sFLT1 (1-5), AAV-sFLT1 (1-6), AAV-sFLT1 (2-3), AAV-sFLT1 (2-4), AAV-sFLT1 (2-5), AAV-sFLT1 (2-6), and the corresponding vector structures are shown in FIG. 1, and the vector sequences are as follows: the coding sequence of sFLT1 (1-3) is shown as SEQ ID NO. 1, the coding sequence of sFLT1 (1-4) is shown as SEQ ID NO. 2, the coding sequence of sFLT1 (1-5) is shown as SEQ ID NO. 3, the coding sequence of sFLT1 (1-6) is shown as SEQ ID NO. 4, the coding sequence of sFLT1 (2-3) is shown as SEQ ID NO. 5, the coding sequence of sFLT1 (2-4) is shown as SEQ ID NO. 6, the coding sequence of sFLT1 (2-5) is shown as SEQ ID NO. 7, and the coding sequence of sFLT1 (2-6) is shown as SEQ ID NO. 8.

The specific construction method of the AAV-sFLT1 recombinant vector with different truncated structures is as follows:

(1) The pAAV-SFFV plasmid (purchased from general organisms) is taken as a skeleton vector for gene expression;

(2) AAV-sFLT1 different truncated structural vector construction: the cDNA sequences of different truncated structure codes of sFLT1 are respectively synthesized, inserted into pAAV-SFFV vectors (namely pAAV-SFFV plasmids) through homologous recombination, transformed into escherichia coli DH5 alpha, and monoclonal is selected for sequencing to ensure that the coding sequences of different truncated structure codes of sFLT1 have no mutation after insertion.

EXAMPLE 2 Effect of the recombinant vector constructed in example 1 on in vitro tube inhibition of HUVEC cells

1. Detection of HUVEC infectivity by adeno-associated virus AAV2/2

The invention selects a human umbilical vein endothelial cell (human umbilical vein endothelial cell, HUVEC) model to investigate the influence of AAV-sFLT1 (1-3), AAV-sFLT1 (1-4), AAV-sFLT1 (1-5), AAV-sFLT1 (1-6), AAV-sFLT1 (2-3), AAV-sFLT1 (2-4), AAV-sFLT1 (2-5) and AAV-sFLT1 (2-6) constructed in example 1 on the vascular biological behavior of HUVEC in vitro. First, the present invention uses AAV2/2 capsids for viral packaging of plasmid AAV-GFP, and then uses AAV-GFP virus at an MOI of 1:10 ⁵ HUVEC cells were infected and fluorescent observations were made at 48H post infection.

The results showed that AAV2/2 virus had higher infection efficiency on HUVEC cells (FIG. 2).

2. Comparison of in vitro tube inhibition of HUVEC cells by AAV-sFLT1 (1-3), AAV-sFLT1 (1-4), AAV-sFLT1 (1-5), AAV-sFLT1 (1-6), AAV-sFLT1 (2-3), AAV-sFLT1 (2-4), AAV-sFLT1 (2-5), AAV-sFLT1 (2-6)

The present invention separately constructs the vector AAV-sFLT constructed in example 11 (1-3), AAV-sFLT1 (1-4), AAV-sFLT1 (1-5), AAV-sFLT1 (1-6), AAV-sFLT1 (2-3), AAV-sFLT1 (2-4), AAV-sFLT1 (2-5), AAV-sFLT1 (2-6) were packaged with AAV2/2 housing, followed by viral packaging at an MOI of 1:10 ⁵ HUVEC cells are infected respectively, and after 48H infection, the cells are collected for tube forming experiments, the tube forming experiments are needed to be carried out by using a 48-pore plate, the 48-pore plate is put into a refrigerator for precooling in advance, and a Matrigel stock solution is paved, wherein 200 mu L of Matrigel stock solution is paved in each pore. Note that the plate is laid in the hole in the middle of the plate, which is convenient for later photographing. Placing the culture box for 30min. Note that there are no bubbles, and a proper amount of Matrigel is sucked up to prevent bubbles from being generated, which affects the tube forming effect. After cell counting, 8 ten thousand cells per well were plated in a plated 48-well plate without washing out Matrigel. At the time of plating 4H, 6H and 8H, drawing was performed under an inverted microscope, and the tube length and the number of nodes were counted by ImageJ.

The results showed that the tube length of AAV-sFLT1 (1-4), AAV-sFLT1 (2-3) and AAV-sFLT1 (2-5) groups were significantly lower than that of AAV-GFP control groups, and that the tube node numbers of AAV-sFLT1 (1-4) and AAV-sFLT1 (2-3) groups were significantly lower than that of AAV-GFP control groups, indicating that the inhibition effect of AAV-sFLT1 (1-4) and AAV-sFLT1 (2-3) on the tube-forming ability of HUVEC cells was more significant (FIGS. 3A-C).

EXAMPLE 3 Effect of the recombinant vector constructed in example 1 on in vitro proliferation inhibition of HUVEC cells

The HUVEC cells infected in example 2 were plated on average into 6 wells of a 96-well cell culture plate of 30000 cells per group, 5000 cells per well, and 100. Mu.L of ECM medium was added, respectively. Placing the culture plate into an incubator, adding 10 mu L of CCK-8 solution into each hole of 24H and 48H, and adding the solution in a liquid-changing mode, wherein the error can be caused by the fact that the added CCK-8 is less and the reagent is adhered to the hole wall, and the culture plate is suggested to be gently shaken to help uniform mixing after the reagent is added, or the culture medium containing 10% of CCK-8 is directly prepared. It is also noted that no bubbles were generated during the sample addition. After incubation of the plates in an incubator for 4h, the absorbance (OD) at 450nm was measured by a microplate reader and the proliferation activity of each group of HUVEC cells was analyzed.

The results showed that AAV-sFLT1 (1-3), AAV-sFLT1 (1-4), AAV-sFLT1 (1-5), AAV-sFLT1 (1-6), AAV-sFLT1 (2-3), AAV-sFLT1 (2-4) groups had significantly lower proliferation rates at 24H and 48H than the AAV-GFP virus infected group, indicating that AAV-sFLT1 (1-3), AAV-sFLT1 (1-4), AAV-sFLT1 (1-5), AAV-sFLT1 (1-6), AAV-sFLT1 (2-3), AAV-sFLT1 (2-4) could significantly inhibit the proliferation capacity of HUVEC cells (FIG. 4).

EXAMPLE 4 comparison of the in vitro migration inhibition of HUVEC cells by the recombinant vector constructed in example 1

The HUVEC cells infected in example 2, 30W cells per group, were plated into 1 well of a 6-well cell culture plate, 3 horizontal lines were drawn in advance at the bottom of the 6-well plate, and 2mL of ECM complete medium was added to each well. The next day, the growth of HUVEC cells in the 6-well plate was observed under a microscope, when the HUVEC cells were expanded to a cell polymerization degree of about 90%, 2-3 vertical lines were drawn by 200. Mu.L of gun head and yesterday's horizontal line for each group, the medium was discarded, 2mL of DPBS was added to wash the scraped cells, fresh ECM complete medium was added, and the cells were placed in an incubator for static culture. Drawing is carried out at 0, 2, 4, 6, 8 and 10 hours, 10 sheets are taken for each group, drawing is carried out at the same locating point at different time points, scratch areas are analyzed through imageJ, and scratch healing rates of the groups at different time points are counted.

The results showed that the scratch healing rate was significantly lower for the sFLT1 truncated group than for the AAV-GFP group, indicating that different truncation structures of sFLT1 could significantly inhibit the migration capacity of HUVEC cells, but there was no significant difference between the groups (fig. 5A-B).

EXAMPLE 5 construction of sFLT1 (1-4) -CNTF adeno-associated Virus expression vector

The results show that the AAV-sFLT1 (1-4) and AAV-sFLT1 (2-3) have remarkable inhibition effects on various capacities of HUVEC cells, and under comprehensive consideration, we select sFLT1 (1-4) as an optimal structure for subsequent experiments. We linked sFLT1 (1-4) to CNTF via T2A to construct pSFFV-sFLT1 (1-4) -CNTF vector, which was functionally analyzed with AAV-GFP, AAV-sFLT1 (1-4), and AAV-CNTF. The vector structure is shown in FIG. 6, wherein the coding sequence of sFLT1 (1-4) is shown in SEQ ID NO. 2, and the coding sequence of CNTF is shown in SEQ ID NO. 9.

The specific construction method of the AAV-sFLT1 (1-4) -CNTF recombinant vector is as follows:

(2) AAV-sFLT1 (1-4) -CNTF vector construction: and respectively synthesizing a cDNA sequence for sFLT1 (1-4), a T2A coding sequence and a CNTF coding cDNA sequence, inserting the cDNA sequences into a pAAV-SFFV vector (namely the pAAV-SFFV plasmid) through homologous recombination, transforming the cDNA sequences into escherichia coli DH5 alpha, picking up a monoclonal, and sequencing to ensure that the sFLT1 (1-4) -T2A-CNTF sequence has no mutation after insertion.

EXAMPLE 6 comparison of the in vitro tube inhibition of HUVEC cells by the recombinant vector constructed in example 5

We virus-packaged AAV-GFP, AAV-sFLT1 (1-4), AAV-CNTF and AAV-sFLT1 (1-4) -CNTF, respectively, using AAV2/2 capsids, followed by MOI at 1:10 ⁵ HUVEC cells were infected separately. In addition, 2 mu M Bevacizumab is added to the positive control group to treat HUVEC cells, and after infection or treatment for 48H, the cells are collected to perform a tube forming experiment, the tube forming experiment is required to be performed by a 48-hole plate, the 48-hole plate is put into a refrigerator for pre-cooling in advance, and a Matrigel stock solution is paved for 200 mu L per hole. Note that the plate is laid in the hole in the middle of the plate, which is convenient for later photographing. Placing the culture box for 30min. Note that there are no bubbles, and a proper amount of Matrigel is sucked up to prevent bubbles from being generated, which affects the tube forming effect. After cell counting, 8 ten thousand cells per well were plated in a plated 48-well plate without washing out matrigel. At the time of plating 4H, 6H and 8H, drawing was performed under an inverted microscope, and the tube length and the number of nodes were counted by ImageJ.

The results show that the number of the tube forming nodes and the tube forming length of the AAV-sFLT1 (1-4) and AAV-sFLT1 (1-4) -CNTF virus infection groups and the Bevacizumab treatment groups at 4H, 6H and 8H are all obviously lower than those of the AAV-GFP and AAV-CNTF virus infection groups, and the AAV-sFLT1 (1-4) and AAV-sFLT1 (1-4) -CNTF and Bevacizumab can obviously inhibit the tube forming capacity of HUVEC cells without obvious difference. Notably, at 8H, the number of tube forming nodes and tube forming length were lower for AAV-sFLT1 (1-4) -CNTF groups than for AAV-sFLT1 (1-4), indicating that AAV-sFLT1 (1-4) -CNTF inhibited HUVEC cells more strongly than AAV-sFLT1 (1-4) (FIGS. 7A-C).

EXAMPLE 7 comparison of the in vitro proliferation inhibition of HUVEC cells by the recombinant vector constructed in example 5

HUVEC cells infected or treated in example 6, in addition 30000 cells per group, were plated on average into 6 wells of a 96 well cell culture plate, 5000 cells per well, with 100. Mu.L of ECM medium added, respectively. Placing the culture plate into an incubator, adding 10 mu L of CCK-8 solution into each hole of 24H and 48H, and adding the solution in a liquid-changing mode, wherein the error can be caused by the fact that the added CCK-8 is less and the reagent is adhered to the hole wall, and the culture plate is suggested to be gently shaken to help uniform mixing after the reagent is added, or the culture medium containing 10% of CCK-8 is directly prepared. It is also noted that no bubbles were generated during the sample addition. After incubation of the plates in an incubator for 4H, the absorbance (OD) at 450nm was measured by a microplate reader and the proliferation activity of each group of HUVEC cells was analyzed.

The results showed that the proliferation rates at 24H and 48H were significantly lower for AAV-sFLT1 (1-4), AAV-sFLT1 (1-4) -CNTF virus-infected groups and Bevacizumab-treated groups than for AAV-GFP virus-infected groups, indicating that AAV-sFLT1 (1-4), AAV-sFLT1 (1-4) -CNTF and Bevacizumab significantly inhibited the proliferation capacity of HUVEC cells, with no significant differences between the three (FIG. 8).

EXAMPLE 8 comparison of the in vitro migration inhibition of HUVEC cells by the recombinant vector constructed in example 5

HUVEC cells infected or treated in example 6, 30W cells per group were plated into 1 well of a 6-well cell culture plate, 3 horizontal lines were drawn in advance at the bottom of the 6-well plate, and 2mL of ECM complete medium was added to each well. The next day, the growth of HUVEC cells in the 6-well plate is observed under a lens, when the HUVEC cells are amplified to about 90% of the cell polymerization degree, 2-3 vertical lines are drawn by 200 mu L of gun heads and yesterday transverse lines of each group, the culture medium is discarded, 2mL of DPBS is added to wash the scraped cells, fresh ECM complete culture medium is added, and the cells are placed in an incubator for static culture. Drawing is carried out at 0, 2, 4, 6, 8 and 10 hours, 10 sheets are taken for each group, drawing is carried out at the same locating point at different time points, scratch areas are analyzed through imageJ subsequently, and scratch healing rates of the groups at different time points are counted.

The results showed that the scratch healing rate of AAV-sFLT1 (1-4), AAV-sFLT1 (1-4) -CNTF virus-infected groups was significantly lower than that of AAV-GFP, AAV-CNTF and Bevacizumab groups, indicating that AAV-sFLT1 (1-4), AAV-sFLT1 (1-4) -CNTF could significantly inhibit the migration ability of HUVEC cells (FIG. 9).

EXAMPLE 9 sFLT1 (1-4) in cell supernatant inhibited the ability of HUVECs to form tubes, proliferate and migrate

In vivo, sFLT1 functions primarily through a paracrine mechanism, and to verify whether sFLT1 (1-4) is so, we transiently transfected 293T cells with plasmid AAV-sFLT1 (1-4) (constructed in example 1), and collected cell supernatants after 48 hours of culture. ELISA results showed that the concentration of sFLT1 (1-4) in the culture supernatant was about 1000pg/mL. To verify the inhibitory effect of the culture supernatant of AAV-sFLT1 (1-4) transiently transfected 293T cells on the ability of HUVECs to tube, proliferate and migrate, we treated HUVEC cells with the culture supernatant of plasmid AAV-sFLT1 (1-4) or AAV-GFP transiently transfected 293T cells for 48 hours. Cell plating and analysis were performed as described in example 2, example 3 and example 4.

The results show that 293T cell culture supernatant is effective in inhibiting HUVEC tube formation, proliferation and migration capacity. These findings indicate that the expression of sFLT1 (1-4) results in its secretion into the extracellular environment, thereby exerting an inhibitory effect on the tube formation, proliferation and migration of HUVECs (fig. 10A-E).

EXAMPLE 10 study of the inhibition Effect of the recombinant vector constructed in example 5 on vascular proliferation of VEGF over-expression mouse model

The animal experiment is carried out by taking a hVEGF mouse (B6-Rho-hVEGFA-Tg mouse, NO. C001395, sai-Xie) as an animal model, wherein the eyeground of the hVEGF mouse has a phenotype of abnormal hyperplasia of blood vessels and leakage of blood vessels due to the overexpression of VEGF. AAV-SFFV, aflibercept, AAV-CNTF, AAV-sFLT1 (1-4) and pSFFV-sFLT1 (1-4) -CNTF were grouped, wherein AAV-SFFV group was used as negative control, and known antibody drug Aflibecept was used as positive control, and AAV-2 capsids were used for AAV virus packaging by four groups of plasmids (AAV-SFFV, AAV-sFLT1 (1-4), AAV-CNTF and AAV-sFLT1 (1-4) -CNTF). Since sFLT1 (1-4) works by virtue of paracrine mechanisms, we use intravitreal injection to more effectively infect a variety of cell types. Taking AAV-sFLT1 (1-4) -CNTF recombinant viral vectors as an example, the AAV-sFLT1 (1-4) -CNTF uses AAV2/2 capsids for AAV viral packaging specifically comprises the following steps:

(1) AAV-sFLT1 (1-4) -CNTF plasmid vectors were constructed using the methods described in example 5;

(2) The plasmid vector AAV-sFLT1 (1-4) -CNTF (7. Mu.g) and AAV2/2 (7. Mu.g) obtained in the step (1) and pHelper (10. Mu.g) were CO-transfected into HEK293FT cells cultured in T175 cell culture flask, and then the cells were cultured under the conditions of 37℃and 5% CO ₂ ；

(3) After transfection for 6-8H, the whole supernatant was aspirated, and 25mL DMEM-reduced serum medium containing 3% fetal bovine serum and 1% diabody was added; after 48H, 72H and 96H transfection, collecting cell supernatant to obtain infectious virus particles, and transferring the infectious virus particles into a new DMEM serum-reduced medium;

(4) The collected virus supernatant is filtered by a 0.45 mu m filter membrane, 7.5mL 5 XPEG-8000+NaCl is added to each 30mL of the filtered supernatant, and the mixture is placed in a refrigerator at 4 ℃ after being uniformly mixed, and the mixture is mixed for 3 to 5 times every 20 to 30 minutes. After standing overnight at 4deg.C in a refrigerator, centrifuging at 4deg.C at 8000g for 45min, discarding supernatant, adding 1mL DPBS, and re-suspending to obtain recombinant virus vector AAV2/2-sFLT1 (1-4) -CNTF.

AAV was administered in a single injection at a frequency of 1.5. Mu.L virus by monocular injection, a total of 10 ¹⁰ vg; aflibecept was administered weekly at 1.5. Mu.L by monocular injection and 3. Mu.g total. Sodium fluorescein contrast (FFA) measurements were performed before and 7, 14, 21 and 29 days after dosing, and vascular leak areas were counted and the leak area recovery rates of each group at 7, 14, 21 and 29 days were analyzed.

FFA results showed that the ocular fundus of each group of mice had severe fluorescent leakage prior to dosing, indicating severe vascular leakage, and that the vascular leakage areas were significantly reduced for the afiibrept and AAV-sFLT1 (1-4), AAV-CNTF, AAV-sFLT1 (1-4) -CNTF groups after dosing for 7 days, 14 days, 21 days, and 29 days (fig. 11A). The following statistics of the recovery rate of the vascular leakage area show that the recovery rate of each group is gradually increased along with the extension of the administration time, wherein the recovery rate of the AAV-sFLT1 (1-4) -CNTF group is obviously higher than that of the positive control Aflibecept group, and the recovery rates of the AAV-sFLT1 (1-4) -CNTF and the Aflibecept group are obviously higher than that of the AAV-sFLT1 (1-4) and the AAV-CNTF independent expression group and the negative control group (figure 11B). The AAV-sFLT1 (1-4) -CNTF has the best recovery effect on fundus vascular proliferation and vascular leakage. The restoration of these vascular functions was also evident in retinal vascular staining analysis, as a leak reversal was clearly observed in the AAV-sFLT1 (1-4) -CNTF and afflibrept groups, and progress was also more pronounced in the AAV-CNTF and AAV-sFLT1 (1-4) groups (fig. 12). Taken together, these results underscore that our AAV-based gene therapy, in particular AAV-sFLT1 (1-4) -CNTF, can rapidly and substantially alleviate retinal neovascularization.

In addition, the nutrition effect of CNTF on optic nerve cells such as cone rods is analyzed in this example, and the function of optic nerve cells of mice in each group is reflected by recording retinal membrane potential of mice in each group by Electroretinogram (ERG) and counting amplitudes of a wave and b wave. After dark adaptation, the flash intensity was 0.01 Cd.s/m ² B wave reflects the function of the bipolar cell connected to the rod. The results showed that the amplitudes of the b waves of AAV-sFLT1 (1-4) -CNTF and Aflibecept groups were significantly increased relative to the negative control groups and AAV-CNTF and AAV-sFLT1 (1-4) groups, indicating that the functions of the bipolar cells of AAV-sFLT1 (1-4) -CNTF and Aflibecept groups were partially restored. After dark adaptation, the flash intensity was 3 Cd.s/m ² The a-wave reflects the function of the rod and cone cells and the b-wave reflects the function of the bipolar cells connected to the rod cone. The results showed no significant difference between the a-wave amplitudes and no significant increase in the b-wave amplitudes of the Aflibercept group relative to the negative control group and the AAV-sFLT1 (1-4) -CNTF and AAV-sFLT1 (1-4) groups, and the AAV-sFLT1 (1-4) -CNTF group also had higher amplitudes than the negative control group and the AAV-CNTF and AAV-sFLT1 (1-4) groups, indicating that the function of the bipolar cells of the AAV-sFLT1 (1-4) -CNTF and Aflibercept groups was partially restored (fig. 13).

Since the present example uses AAV virus of the serotype AAV2/2, in order to evaluate the infectivity of AAV2/2 capsid packaging virus on ocular cells, intravitreal injection of AAV-GFP was used. After 21 days of injection, eye tissue was frozen and sectioned. The results show that AAV-GFP can infect retinal multilayers, including ganglion cell layers, inner core layers, and outer core layers (fig. 14).

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. A construct for use in the treatment of a neovascular ocular disease, wherein the construct comprises a nucleotide encoding sFLT1 (1-4) or sFLT1 (2-3);

the nucleotide sequence of the coding sFLT1 (1-4) is shown as SEQ ID NO. 2;

the nucleotide sequence of the coding sFLT1 (2-3) is shown as SEQ ID NO. 5.

2. The construct of claim 1, further comprising a nucleotide encoding CNTF;

the nucleotide sequence of the coded CNTF is shown as SEQ ID NO. 9;

The nucleotide sequence encoding sFLT1 (1-4) or sFLT1 (2-3) and the nucleotide sequence encoding CNTF are connected through T2A;

preferably, the construct comprises AAV-sFLT1 (1-4), AAV-sFLT1 (2-3), AAV-sFLT1 (1-4) -T2A-CNTF, AAV-sFLT1 (2-3) -T2A-CNTF;

more preferably, the construct is AAV-sFLT1 (1-4) -T2A-CNTF.

3. A recombinant viral vector for use in the treatment of a neovascular ocular disease, comprising the construct of claim 1 or 2 and a viral vector backbone.

4. The recombinant viral vector according to claim 3, wherein the viral vector comprises an adeno-associated viral vector, an adenovirus vector, a lentiviral vector, a retrovirus vector, a herpes simplex viral vector, a baculovirus vector, a sendai viral vector, a poxvirus vector, a geminivirus vector;

preferably, the viral vector is an adeno-associated viral vector;

more preferably, the adeno-associated viral vector is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any combination thereof;

most preferably, the adeno-associated viral vector is AAV2.

5. A method of producing the recombinant viral vector according to claim 3 or 4, comprising the steps of:

(3) Collecting the cell supernatant to obtain infectious viral particles, which are the recombinant viral vectors of claim 3 or 4.

6. The method according to claim 5, wherein the AAV-sFLT1 (1-4), AAV-sFLT1 (2-3), AAV-sFLT1 (1-4) -T2A-CNTF or AAV-sFLT1 (2-3) -T2A-CNTF plasmid vector has the nucleotide sequence encoding sFLT1 (1-4) shown in SEQ ID NO:2, the nucleotide sequence encoding sFLT1 (2-3) shown in SEQ ID NO:5, and the nucleotide sequence encoding CNTF shown in SEQ ID NO: 9;

preferably, the construction of the AAV-sFLT1 (1-4) or AAV-sFLT1 (2-3) plasmid vector of step (1) comprises the steps of: synthesizing sFLT1 (1-4) or sFLT1 (2-3) coding cDNA sequence by taking pAAV-SFFV plasmid as a skeleton vector for gene expression, inserting the cDNA sequence into pAAV-SFFV plasmid through homologous recombination, transforming into escherichia coli DH5 alpha, and picking up monoclonal to obtain AAV-sFLT1 (1-4) or AAV-sFLT1 (2-3) plasmid vector;

Preferably, the construction of the AAV-sFLT1 (1-4) -T2A-CNTF or AAV-sFLT1 (2-3) -T2A-CNTF plasmid vector described in step (1) comprises the following steps: synthesizing sFLT1 (1-4) or sFLT1 (2-3) coding cDNA sequence, T2A coding sequence and CNTF coding cDNA sequence by taking pAAV-SFFV plasmid as a skeleton vector for gene expression, inserting the pAAV-SFFV plasmid into the pAAV-SFFV plasmid through homologous recombination, transforming into escherichia coli DH5 alpha, and picking up monoclonal to obtain AAV-sFLT1 (1-4) -T2A-CNTF or AAV-sFLT1 (2-3) -T2A-CNTF plasmid vector;

preferably, the cells in step (2) are HEK293FT cells;

preferably, the conditions of the culture in step (2) are 37℃and 5% CO ₂ ；

7. A gene therapy drug for treating a neovascular ocular disease, characterized in that the gene therapy drug comprises the recombinant viral vector of claim 3 or 4;

preferably, the gene therapy drug further comprises a pharmaceutically acceptable carrier and/or adjuvant;

preferably, the neovascular ocular disease comprises wet macular degeneration, diabetic macular edema, diabetic retinopathy, outer exudative retinopathy, retinal vein occlusion, corneal neovascularization, pathologic myopia CNV, neovascular glaucoma, uveitis, polypoidal choroidal vasculopathy, radiation optic neuropathy;

Preferably, the administration mode of the gene therapy drug comprises intravitreal injection and subretinal injection;

more preferably, the gene therapy drug is administered by intravitreal injection.

8. A pharmaceutical composition for treating a neovascular ocular disease, comprising the gene therapy agent of claim 7;

preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant;

preferably, the pharmaceutical composition further comprises a second therapeutic agent for treating a neovascular ocular disease.

9. The pharmaceutical composition of claim 8, wherein the second therapeutic agent comprises a hypoglycemic agent, an anticoagulant agent, a vascular protective agent;

preferably, the hypoglycemic agent comprises sulfonylurea agent, glinide agent, DPP-4 inhibitor, biguanide agent, thiazolidinedione agent, alpha-glycosidase inhibitor;

preferably, the anticoagulants include aspirin, brivudine, heparin anticoagulants and warfarin;

preferably, the vascular protection drug comprises a hypolipidemic drug and an antiplatelet drug;

preferably, the neovascular ocular disease includes wet macular degeneration, diabetic macular edema, diabetic retinopathy, outer exudative retinopathy, retinal vein occlusion, corneal neovascularization, pathological myopia CNV, neovascular glaucoma, uveitis, polypoidal choroidal vasculopathy, radial optic neuropathy.

10. An application according to any one of the following, characterized in that the application comprises:

(1) Use of a construct according to claim 1 or 2 for the preparation of a recombinant viral vector for the treatment of a neovascular ocular disease;

(2) Use of the recombinant viral vector of claim 3 or 4 in the manufacture of a gene therapy drug for the treatment of neovascular ocular diseases;

(3) Use of the gene therapy drug of claim 7 for the preparation of a pharmaceutical composition for the treatment of neovascular ocular diseases;

the nucleotide sequence of the coding sFLT1 (1-4) is shown as SEQ ID NO. 2;

the nucleotide sequence of the coding sFLT1 (2-3) is shown as SEQ ID NO. 5;

the nucleotide sequence for coding the CNTF is shown as SEQ ID NO. 9.