Method for treating angiogenesis-associated eye disease in subject

Abstract

Provided is a method for treating an angiogenesis-associated eye disease in a subject, specifically comprising administering to the eye of a subject a pharmaceutical composition containing a recombinant adeno-associated virus, wherein the recombinant adeno-associated virus comprises a nucleotide encoding a fusion protein of an anti-vascular endothelial growth factor, and the dosage of the recombinant adeno-associated virus administered is 108 to 1013 vg per eye.

Description

A method for treating angiogenesis-related eye disease in a subject Technical Field

[0001] The present invention relates to the field of gene therapy, and in particular to a method for treating an eye disease associated with angiogenesis in a subject. Background Art

[0002] Neovascularization is key to the development and spread of many diseases. Numerous ocular diseases involve angiogenesis, including age-related macular degeneration (AMD), retinal vein occlusion (RVO), diabetic retinopathy (DR), and pathological myopia. VEGF is a highly specific vascular endothelial growth factor that promotes increased vascular permeability, extracellular matrix degeneration, endothelial cell migration and proliferation, and angiogenesis. Numerous studies have demonstrated that excessive VEGF expression can induce pathological neovascular eye diseases. Given the importance of VEGF signaling in angiogenesis, blocking VEGF or VEGF receptors to inhibit angiogenesis holds important therapeutic potential for angiogenesis-related diseases, including cancer and retinal vascular lesions. Over the past decade, several anti-VEGF drugs have been developed for the treatment of ocular neovascular diseases, including pegaptanib (Macugen), bevacizumab (Avastin), ranibizumab (Lucentis), and aflibercept (Eylea).

[0003] However, the short half-life of currently marketed drugs necessitates repeated dosing, placing a significant burden on both physicians and patients. Therefore, gene therapy approaches, such as adeno-associated virus (AAV) gene therapy tools, are gaining popularity due to their ability to sustain drug expression and maintain long-term efficacy.

[0004] Summary of the Invention

[0005] The present invention provides an effective method for treating angiogenesis-related ocular diseases in a subject.

[0006] In some embodiments, the method comprises administering to the eye of the subject a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV) comprising nucleotides encoding an anti-vascular endothelial growth factor (VEGF) fusion protein, wherein the administered rAAV dose is 10 8 to 10 13 vg / eye.

[0007] In some specific embodiments, the anti-vascular endothelial growth factor (VEGF) fusion protein comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% homology thereto.

[0008] In some preferred embodiments, the anti-vascular endothelial growth factor (VEGF) fusion protein comprises the amino acid sequence of SEQ ID NO: 1.

[0009] In some preferred embodiments, the recombinant adeno-associated virus (rAAV) comprises:

[0010] (i) rAAV capsid protein, wherein the capsid protein is AAV8 capsid protein or a mutant thereof; and

[0011] (ii) a polynucleotide expression cassette comprising, from 5' to 3' sequence:

[0012] (a) AAV 5′ITR;

[0013] (b) CBA promoter;

[0014] (c) a nucleotide encoding an anti-vascular endothelial growth factor (VEGF) fusion protein, wherein the anti-vascular endothelial growth factor (VEGF) protein has the amino acid sequence as set forth in SEQ ID NO: 1;

[0015] (d) rabbit-globin polyadenylation signal;

[0016] (e) AAV 3'ITR.

[0017] In some specific embodiments, the nucleic acid encoding the anti-vascular endothelial growth factor (VEGF) protein has a nucleotide sequence as shown in SEQ ID NO:2.

[0018] In some more specific embodiments, the polynucleotide expression cassette has a nucleotide sequence as set forth in SEQ ID NO: 3 from 5' to 3'.

[0019] In some specific embodiments, the pharmaceutical composition is provided in the form of a rAAV vector active ingredient dispersed in a buffer comprising a physiologically compatible aqueous buffer, a surfactant, and optionally an excipient.

[0020] In some specific embodiments, the rAAV dose administered is 3*10 8 to 1*10 13 vg / eye.

[0021] In some specific embodiments, the rAAV dose administered is 3*10 8 to 1*10 12 vg / eye.

[0022] In some specific embodiments, the rAAV dose administered is 3*10 9 Up to 5*10 11 vg / eye.

[0023] In some specific embodiments, the rAAV dose administered is 3*10 9 to 1*10 11 vg / eye.

[0024] In some specific embodiments, the administered rAAV dose is 1*10 10 Up to 3*10 10 vg / eye.

[0025] In some specific embodiments, the rAAV dose administered is 3*10 8 / eye, 4*10 8 / eye, 5*10 8 / eye, 6*10 8 / eye, 7*10 8 / eye, 8*10 8 / eye, 9*10 8 / eye, 1*10 9 / eye, 2*10 9 / eye, 3*10 9 / eye, 4*10 9 / eye, 5*10 9 / eye, 6*10 8 / eye, 7*10 9 / eye, 8*10 9 / eye, 9*10 9 / eye, 1*10 10 / eye, 2*10 10 / eye, 3*10 10 / eye, 4*10 10 / eye, 5*10 10 / eye, 6*10 10 / eye, 7*10 10 / eye, 8*10 10 / eye, 9*10 10 / eye, 1*10 11 / eye, 2*10 11 / eye, 3*10 11 / eye, 4*10 11 / eye, 5*10 11 / eye, 6*10 11 / eye, 7*10 11 / eye, 8*10 11 / eye, 9*10 11 / eye, 1*10 12 / eye, 2*1012 / eye, 3*10 12 / eye, 41*10 12 / eye, 5*10 12 / eye, 6*10 12 / eye, 7*10 12 / eye, 8*10 12 / eye, 9*10 12 / eye, 1*10 13 / Eye.

[0026] In some specific embodiments, the method of the present invention is administered intraretina, choroidally or intravitreally, preferably by subretinal administration. Recombinant adeno-associated virus (rAAV) should be administered in a certain volume under the retina in a therapeutically effective dose. In some embodiments, the recombinant adeno-associated virus (rAAV) of a therapeutically effective dose should be administered in a volume of 500ul or less, for example, in a volume of 10-20ul, 20-50ul, 50-100ul, 100-200ul, 200-300ul, 300-400ul or 400-500ul. Subretinal injection is a surgical procedure performed by a trained retinal surgeon, and the surgical procedure involves injecting the subject into the retina under local anesthesia. In some specific embodiments, the rAAV is injected into the subretinal space by using a subretinal drug delivery device.

[0027] In some specific embodiments, the eye diseases or disorders associated with angiogenesis (VEGF) described herein are age-related macular degeneration, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy and diabetic retinal edema.

[0028] In some specific embodiments, the subject of the present invention is a non-human mammal, such as a mouse, cat, dog, cow, sheep, pig, monkey, etc. In some preferred embodiments, the subject of the present invention is a non-human primate, such as a macaque, rhesus monkey, cynomolgus monkey, etc.

[0029] Another aspect of the present invention provides a method for treating age-related macular degeneration (AMD) in a human subject, the method comprising administering to the eye of the human subject a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV), the rAAV comprising a nucleotide encoding an anti-vascular endothelial growth factor (VEGF) fusion protein, the rAAV administered at a dose of 10 8 to 10 13 vg / eye.

[0030] In some specific embodiments, the anti-vascular endothelial growth factor (VEGF) fusion protein comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% homology thereto. In some preferred embodiments, the anti-vascular endothelial growth factor (VEGF) fusion protein comprises the amino acid sequence of SEQ ID NO: 1.

[0031] In some preferred embodiments, the recombinant adeno-associated virus (rAAV) comprises:

[0032] (i) rAAV capsid protein, wherein the capsid protein is AAV8 capsid protein or a mutant thereof; and

[0033] (ii) a polynucleotide expression cassette comprising, from 5' to 3' sequence:

[0034] (a) AAV 5′ITR;

[0035] (b) CBA promoter;

[0036] (c) a nucleotide encoding an anti-vascular endothelial growth factor (VEGF) fusion protein, wherein the anti-vascular endothelial growth factor (VEGF) protein has the amino acid sequence as set forth in SEQ ID NO: 1;

[0037] (d) rabbit-globin polyadenylation signal;

[0038] (e) AAV 3'ITR.

[0039] In some specific embodiments, the nucleic acid encoding the anti-vascular endothelial growth factor (VEGF) protein has the nucleotide sequence of SEQ ID NO: 2. In some more specific embodiments, the polynucleotide expression cassette has the nucleotide sequence of SEQ ID NO: 3 from 5' to 3'.

[0040] In some specific embodiments, the pharmaceutical composition is provided in the form of a rAAV vector active ingredient dispersed in a buffer comprising a physiologically compatible aqueous buffer, a surfactant, and optionally an excipient.

[0041] In some specific embodiments, the rAAV dose administered is 3*10 8 to 1*10 13 In some specific embodiments, the rAAV dose administered is 3*10 8 to 1*10 12In some specific embodiments, the rAAV dose administered is 3*10 9 Up to 5*10 11 In some specific embodiments, the rAAV dose administered is 3*10 9 to 1*10 11 In some specific embodiments, the rAAV dose administered is 1*10 10 Up to 3*10 10 vg / eye.

[0042] In some specific embodiments, the rAAV dose administered is 3*10 8 / eye, 4*10 8 / eye, 5*10 8 / eye, 6*10 8 / eye, 7*10 8 / eye, 8*10 8 / eye, 9*10 8 / eye, 1*10 9 / eye, 2*10 9 / eye, 3*10 9 / eye, 4*10 9 / eye, 5*10 9 / eye, 6*10 8 / eye, 7*10 9 / eye, 8*10 9 / eye, 9*10 9 / eye, 1*10 10 / eye, 2*10 10 / eye, 3*10 10 / eye, 4*10 10 / eye, 5*10 10 / eye, 6*10 10 / eye, 7*10 10 / eye, 8*10 10 / eye, 9*10 10 / eye, 1*10 11 / eye, 2*10 11 / eye, 3*10 11 / eye, 4*10 11 / eye, 5*10 11 / eye, 6*10 11 / eye, 7*10 11 / eye, 8*10 11 / eye, 9*10 11 / eye, 1*10 12 / eye, 2*10 12 / eye, 3*10 12 / eye, 41*1012 / eye, 5*10 12 / eye, 6*10 12 / eye, 7*10 12 / eye, 8*10 12 / eye, 9*10 12 / eye, 1*10 13 / Eye.

[0043] In some specific embodiments, the method of the present invention is administered intraretina, choroidally or intravitreally, preferably by subretinal administration. Recombinant adeno-associated virus (rAAV) should be administered in a certain volume under the retina in a therapeutically effective dose. In some embodiments, the recombinant adeno-associated virus (rAAV) of a therapeutically effective dose should be administered in a volume of 500ul or less, for example, in a volume of 10-20ul, 20-50ul, 50-100ul, 100-200ul, 200-300ul, 300-400ul or 400-500ul. Subretinal injection is a surgical procedure performed by a trained retinal surgeon, and the surgical procedure involves injecting the subject into the retina under local anesthesia. In some specific embodiments, the rAAV is injected into the subretinal space by using a subretinal drug delivery device.

[0044] In some specific embodiments, the anti-vascular endothelial growth factor (VEGF) protein is produced by human photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells and / or retinal pigment epithelial cells in the external limiting membrane of the human subject. Choroidal, subretinal or intravitreal injection should result in the delivery of the transgene product (anti-vascular endothelial growth factor (VEGF) protein) to the retina, vitreous humor and / or aqueous humor. Specifically, expression of the transgene product by retinal cells, such as rods, cones, retinal pigment epithelium, horizontal, bipolar, amacrine, ganglion and / or Müller cells, results in the delivery and maintenance of the transgene product in the retina, vitreous humor and / or aqueous humor.

[0045] In some specific embodiments, the inclusion criteria of the human subjects shall meet at least one of the following conditions: 1) currently receiving anti-VEGF ophthalmic injection treatment and at least one anti-VEGF ophthalmic injection treatment within 6 months before the screening period; 2) previous OCT examination proves that the anti-VEGF ophthalmic injection treatment is effective (CRT value decreases by ≥50μm from the highest value, or subretinal or intraretinal fluid decreases by 30% from the highest value); 3) the best corrected visual acuity (BCVA) of the sentinel subject's study eye is 63-9 letters (approximately equivalent to 20 / 50 to 20 / 640 on the Snellen visual acuity chart), and the best corrected visual acuity (BCVA) of the remaining subjects' study eyes is 78-19 letters (approximately equivalent to 20 / 32 to 20 / 400 on the Snellen visual acuity chart); the best corrected visual acuity (BCVA) of the fellow eye is ≥19 letters (approximately equivalent to 20 / 400 on the Snellen visual acuity chart); 4) at least 1 month after intraocular lens implantation; 5) female subjects must be postmenopausal for at least 1 year.

[0046] In some specific embodiments, the exclusion criteria for the human subjects include: 1) those whose choroidal neovascularization (CNV) in the study eye is secondary to diseases other than AMD (including pathological myopia, etc.); or those whose study eye has non-nAMD diseases that affect macular examination or central vision (including diabetic retinopathy, central vein occlusion, etc.) as determined by the researcher; 2) those whose study eye has implants (except intraocular lenses), opacities of refractive media that affect fundus examination, or miosis; 3) Patients with scars, fibrosis or atrophy under the fovea of ​​the study eye that affect the evaluation of the test results; 4) Patients with a history of or current retinal detachment (including rhegmatogenous retinal detachment, tractional retinal detachment, exudative retinal detachment, etc.) in the study eye; 5) Patients with severe dry eye currently receiving medication for the study eye; 6) Patients with tear drainage system diseases (including tear duct obstruction or tear duct stenosis, etc.) that require medication or surgery for the study eye; 7) Patients with glaucoma or ocular hypertension (intraocular pressure ≥25 mmHg during the screening period) that is not controlled by medication for the study eye; 8) Patients with intraocular tumors (including choroidal melanoma, etc.) in the study eye ) 9) Patients with active eye infection (including viral, bacterial and fungal) in the study eye or fellow eye, such as blepharitis, infectious conjunctivitis, keratitis, scleritis, endophthalmitis, dacryocystitis, etc.; 9) Patients with active eye infection (including viral, bacterial and fungal) in the study eye or fellow eye, such as blepharitis, infectious conjunctivitis, keratitis, scleritis, endophthalmitis, dacryocystitis, etc.; 10) Patients with a history of treatment for nAMD (including intraocular hormone use, etc.) other than anti-VEGF ophthalmic injections within 6 months before the study eye screening; 11) Patients with a history of retinal irritation within 6 months before the study eye screening 1) Patients who have taken toxic drugs (including chloroquine, hydroxychloroquine, etc.); 12) Patients who have received local or systemic gene or cell therapy products or participated in local or systemic gene or cell therapy clinical trials; 13) Patients with a history of intraocular surgery (including surgery on the vitreous, retina, etc.) within 3 months before the screening of the study eye; 14) Patients with allergic reactions or allergic history to sodium fluorescein; 15) Patients who are allergic to two or more drugs and / or non-drug factors; 16) Patients with active disseminated intravascular coagulation within 3 months before the screening; or patients who are currently using anticoagulants or antiplatelet drugs that the researchers believe need to be excluded; 17) Patients with blood pressure examination during the screening period, who have been tested by the lowering 1) Patients with uncontrolled hypertension after antihypertensive treatment: SBP / DBP ≥ 160 / 100 mmHg; 2) Patients with poor blood sugar control during the screening period (glycosylated hemoglobin value ≥ 10%); 3) Patients with liver and kidney function tests during the screening period: ALT, AST > 2 × ULN, or Cr > ULN and clinically significant as determined by the investigator; 4) Patients with a history of thrombotic events (including acute myocardial infarction, ischemic stroke, etc.) within 6 months before screening; 5) Patients with a history of chemotherapy or radiotherapy for malignant tumors within 5 years before screening; 6) Patients with a history of systemic immune diseases or those currently receiving immunosuppressive or immunopotentiator therapy;23) Patients with any other uncontrollable clinical problems (such as severe mental, neurological, cardiovascular, respiratory, digestive, urinary, blood, endocrine, immune and other systemic diseases and malignant tumors); 24) Patients with active hepatitis B virus (surface antigen positive and HBV DNA ≥ 2000 IU / mL), hepatitis C virus, human immunodeficiency virus, and Treponema pallidum pathogen testing during the screening period, or those who are unwilling to undergo human immunodeficiency virus and Treponema pallidum pathogen testing; 25) Patients with active herpes virus infection during the screening period; 26) Patients who cannot take effective contraceptive measures during the trial or have fertility intentions; 27) Pregnant or lactating women (pregnancy in this trial is defined as a positive blood or urine pregnancy test); 28) Patients who have participated in other clinical trials within 3 months before screening (if the half-life of the investigational drug is long, the 7 half-lives are greater than 3 months, then the 7 half-lives are 7 half-lives); 29) Patients who are considered unsuitable for participation in this trial by the investigator. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Percentage of level 4 spots in the negative control group and each dose group in Example 2

[0048] Figure 2 Representative schematic diagrams of fluorescein angiography in the negative control group and each dose group in Example 2

[0049] Figure 3: Changes in retinal thickness in the negative control group and each dose group in Example 2 DETAILED DESCRIPTION

[0050] The present invention will be further described below with reference to specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention.

[0051] Example 1 Preparation of recombinant adeno-associated virus vector (rAAV)

[0052] Conventional DNA recombination technology and cloning technology are used to construct and clone a recombinant plasmid containing the target protein gene of the present invention in Escherichia coli for the subsequent preparation of recombinant AAV virus. The recombinant plasmid contains 5'ITR, CBA promoter, Conbercept fusion protein coding sequence, RGB terminator and 3'ITR in sequence from 5' to 3' between the inverted terminal repeats (ITRs) of adeno-associated virus serotype 2 (AAV2), wherein the Conbercept fusion protein as the target protein has the amino acid sequence as described in SEQ ID NO: 1. The coding sequence of the Conbercept fusion protein has the nucleotide sequence as described in SEQ ID NO: 2. The recombinant plasmid contains the nucleotide sequence as described in SEQ ID NO: 3 from 5' to 3' between the inverted terminal repeats (ITRs).

[0053] Recombinant adeno-associated viral vectors (rAAV) were prepared using a three-plasmid co-transfection process known in the art. Specifically, the three plasmids (pAAV-Conbercept, pAAV 8-RC-Kan, and pHelper-Kan) were first added to HBS buffer and vortexed to mix thoroughly. The transfection reagent was then added, vortexed to mix thoroughly, and incubated at room temperature for 20-30 minutes. The cells were then added to the HEK293 cells to be transfected. After transfection, the cells were cultured at 36.5°C, 5.0% CO2, and 120 rpm. After 72 hours, the cells were harvested and the rAAV genome titer was determined.

[0054] Example 2: Drug efficacy experiment on rhesus monkey retina

[0055] Animal models

[0056] Rhesus monkeys, 24 in total.

[0057] Age: 2.5-5 years old; Weight: 3.70-5.90 kg at purchase, average 4.70 kg.

[0058] Sample solution:

[0059] The rAAV prepared in Example 1 was prepared using a buffer solution at a concentration of 5×10 12 vg / mL, 1×10 12 vg / mL, 3×10 11 vg / mL, 1×10 11 vg / mL, 3×10 10 vg / mL, 1×10 10 vg / mL, 3×10 9 vg / mL of rAAV injection.

[0060] Experimental groups

[0061] One negative control group (Vehicle / buffer group) consisted of 3 animals.

[0062] 7 rAAV dose groups: 5×10 11 vg / eye group, 1×10 11 vg / eye group, 3×10 10 vg / eye group, 1×10 10 vg / eye group, 3×10 9 vg / eye group, 1×10 9 vg / eye group, 3×10 8 vg / eye group, of which 3×10 10 vg / eye group, 1×10 10 vg / eye group. 3×10 10 vg / eye group, 1×1010 In the vg / eye group, one animal was excluded from each group due to drug administration reasons, so only two animals were actually included in the experimental statistics. The other groups had three animals in each group.

[0063] Dosage

[0064] 5×10 were injected subretinally in each eye at a dose of 100 μL / eye. 12 vg / mL, 1×10 12 vg / mL, 3×10 11 vg / mL, 1×10 11 vg / mL, 3×10 10 vg / mL rAAV injection and buffer solution were administered, and 28 days after administration, the Bruch's membrane of the outer retina of rhesus monkeys was damaged by laser in both eyes to establish an animal model similar to human choroidal neovascularization (CNV).

[0065] Modeling Method: Rhesus monkeys were anesthetized with 2.5% pentobarbital (25 mg / kg) and / or 5% ketamine (10 mg / kg) intravenously (the anesthetic dose was adjusted based on the monkey's anesthesia status). Small drops of Diclofenac were administered periodically during anesthesia to maintain corneal moisturization. After mydriasis with Midori (compound tropicamide eye drops) in both eyes, the monkeys' heads were fixed in front of an ophthalmic laser photocoagulation device, and the macula was photocoagulated using a panretinal lens. Six spots were illuminated per eye, avoiding the fovea.

[0066] Laser parameters: spot diameter 50 μm, energy 0.5-0.7 W, exposure time 0.05 s.

[0067] Photocoagulation criteria: Bubbles can be seen, indicating that Bruch's membrane has been ruptured.

[0068] Experimental observation

[0069] 1) Fundus photography and fluorescein angiography

[0070] Testing time: once during the adaptation period, and once every 4, 6, and 8 weeks after administration;

[0071] Examination method: The monkeys to be examined were anesthetized with 5% ketamine (10 mg / kg intramuscularly) and mydriatic with Midori (compound tropicamide eye drops, 1-2 drops / eye). After fundus photography was performed with a fundus camera, sodium fluorescein injection (20 mg / kg) was rapidly injected into the saphenous vein of the monkey's lower limb (or other suitable site). The fundus images were taken with a fundus camera in the early (within about 1 minute) and late (about 5 minutes after) post-contrast. During the test, ketamine (about 5-25 mg / animal) or sodium pentobarbital (about 10-80 mg / animal) was injected intramuscularly or intravenously to maintain anesthesia. After the operation, 1-2 drops of ofloxacin eye ointment were applied to prevent infection.

[0072] Evaluation indicators:

[0073] (1) Fluorescent spot rating

[0074] Grading standards for the light spots photographed by fluorescence angiography after modeling:

[0075] Level 1: The light spot does not show high fluorescence;

[0076] Grade 2: The spot has high fluorescence but no fluorescein leakage;

[0077] Grade 3: The spot has high fluorescence and slight fluorescein leakage, which does not exceed the edge of the spot;

[0078] Level 4: The spot has high fluorescence and significant fluorescein leakage, which exceeds the edge of the spot.

[0079] The number of level 4 laser spots was counted, and the level of fundus laser spots was recorded at each examination after modeling.

[0080] 2) Optical coherence tomography (OCT) examination

[0081] Examination Method: The monkeys were anesthetized with 2.5% pentobarbital sodium (25 mg / kg) and / or 5% ketamine (10 mg / kg) intravenously (the anesthetic dosage can be adjusted appropriately depending on the monkey's anesthesia status). Mydriasis was then dilated with Midori (compound tropicamide eye drops, 1-2 drops / eye), and optical coherence tomography (OCT) was performed on the monkey's fundus. After the procedure, 1-2 drops of ofloxacin eye ointment were applied to prevent infection.

[0082] CNV efficacy evaluation test: A rapid linear scan centered on the neovascular lesion is performed to obtain an image, showing the structure of each retinal layer and measuring the retinal thickness at the highest point of each lesion.

[0083] Evaluation indicators:

[0084] (1) Retinal thickness: the thickness from the nerve fiber layer to Bruch's membrane;

[0085] (2) Change in retinal thickness = retinal thickness after modeling - retinal thickness before modeling.

[0086] Experimental results

[0087] 1) Fundus photography and fluorescein angiography results

[0088] The number and percentage of level 4 spots in each group 14 days (2 weeks after modeling) and 28 days (4 weeks after modeling) after laser modeling are shown in Table 1 and Figure 1 . The representative schematic diagram of fluorescein angiography in each group is shown in Figure 2 .

[0089] The above results show that in the case of buffer control, significant neovascularization fluorescein leakage was observed 14 days after modeling, and fluorescein leakage was slightly reduced from 14 to 28 days after modeling. After administration of different doses of rAAV, the proportion of fourth-order spots decreased significantly, indicating that administration of 3×10 8 vg / eye~5×10 11 vg / eye rAAV has a significant inhibitory effect on laser-induced choroidal neovascularization leakage in crab-eating macaques.

[0090] Table 1

[0091] 2) Optical coherence tomography (OCT) examination

[0092] The changes in retinal thickness levels of each group 14 days (2 weeks after modeling) and 28 days (4 weeks after modeling) after laser modeling are shown in Table 2 and Figure 3.

[0093] The results showed that there was no significant difference in retinal thickness between the groups of animals before modeling. OCT examination of the animals in each group before administration showed 6 laser spots around the macula, continuous destruction of the retinal pigment epithelium at the spot, and a significant increase in retinal thickness compared with before. There was retinal edema, and there was no significant difference in thickness between the groups. In the case of subretinal injection of buffer solution, obvious neuroepithelial edema was seen 14 days after modeling. The edema was slightly reduced from 14 to 28 days after modeling, but there was still a significant change compared with before modeling. The retinal thickness of the groups given different doses of rAAV was significantly reduced compared with the buffer control group, indicating that the administration of 3×10 8 vg / eye~5×10 11 vg / eye rAAV has an inhibitory effect on the growth of new blood vessels.

[0094] Example 3 Phase I / II clinical trial

[0095] Phase 1 of this example mainly evaluates the tolerability of different doses of AAV-VEGF ophthalmic injection injected subretinaly in patients with neovascular (wet) age-related macular degeneration (nAMD), and preliminarily evaluates the efficacy of KH631 ophthalmic injection in the treatment of nAMD, mainly evaluating the overall safety at 24 weeks. Phase 2 mainly selects the Phase 2 recommended dose group to expand the sample size to evaluate the efficacy and safety of KH631 ophthalmic injection in patients with nAMD based on the results of Phase 1 tolerability, safety and preliminary efficacy evaluation, and mainly evaluates the effectiveness at 52 weeks. The purpose of the long-term follow-up period is to evaluate the long-term safety and efficacy of KH631 ophthalmic injection in patients who have received subretinal injection of KH631 ophthalmic injection.

[0096] Subject information: 50 years old (minimum age) to 85 years old (maximum age)

[0097] Gender: Male / Female

[0098] Inclusion criteria: 1) voluntarily signed the informed consent form and was willing to attend the study visit according to the specified time; 2) aged 50 to 85 years (including the cutoff value) and diagnosed with neovascular (wet) age-related macular degeneration (nAMD); 3) currently receiving anti-VEGF ophthalmic injection treatment and at least one anti-VEGF ophthalmic injection treatment within 6 months before the screening period; 4) previous OCT examination proved that the anti-VEGF ophthalmic injection treatment was effective (CRT value decreased by ≥50μm compared with the highest value, or subretinal or intraretinal fluid decreased compared with the highest value) 30%); 5) The best-corrected visual acuity (BCVA) of the study eye of the sentinel subject was 63 to 9 letters (approximately equivalent to 20 / 50 to 20 / 640 on the Snellen chart); the best-corrected visual acuity (BCVA) of the study eye of the remaining subjects was 78 to 19 letters (approximately equivalent to 20 / 32 to 20 / 400 on the Snellen chart); the best-corrected visual acuity (BCVA) of the fellow eye was ≥19 letters (approximately equivalent to 20 / 400 on the Snellen chart); 6) At least 1 month after intraocular lens implantation; 7) Female subjects must have been postmenopausal for at least 1 year.

[0099] Exclusion criteria: 1) Patients with choroidal neovascularization (CNV) secondary to diseases other than AMD (including pathological myopia, etc.) in the study eye; or patients with non-nAMD diseases in the study eye that affect macular examination or central vision (including diabetic retinopathy, central vein occlusion, etc.) as determined by the investigator; 2) Patients with implants (except intraocular lenses) in the study eye, opacities of refractive media that affect fundus examination, or pupil constriction; 3) Patients with scarring, fibrosis, or atrophy under the fovea of ​​the study eye that affects the evaluation of the test results; 4) Patients with a history or current retinal detachment (including rhegmatogenous retinal detachment, tractional retinal detachment, exudative retinal detachment, etc.) in the study eye; 5) Patients with severe dry eye currently receiving medication. ; 6) The study eye currently suffers from tear drainage system diseases that require medication or surgery (including tear duct obstruction or tear duct stenosis, etc.); 7) The study eye currently suffers from glaucoma or ocular hypertension (intraocular pressure ≥ 25 mmHg during the screening period) that is not controlled by medication; 8) The study eye currently suffers from intraocular tumors (including choroidal melanoma, etc.); 9) The study eye or the fellow eye currently suffers from active eye infections (including viruses, bacteria and fungi), such as blepharitis, infectious conjunctivitis, keratitis, scleritis, endophthalmitis, dacryocystitis, etc.; 9) The study eye or the fellow eye currently suffers from active eye infections (including viruses, bacteria and fungi), such as blepharitis, infectious conjunctivitis, keratitis, scleritis, endophthalmitis, dacryocystitis, etc.; 10) The study eye Patients with a history of other nAMD treatments (including intraocular hormones, etc.) other than anti-VEGF ophthalmic injections within 6 months before screening; 11) Patients who used retinal toxic drugs (including chloroquine, hydroxychloroquine, etc.) within 6 months before the study eye screening; 12) Patients who had previously received local or systemic gene or cell therapy products or participated in local or systemic gene or cell therapy clinical trials; 13) Patients with a history of intraocular surgery (including surgery on the vitreous, retina, etc.) within 3 months before the study eye screening; 14) Patients with an allergic reaction or history of allergy to sodium fluorescein; 15) Patients who are allergic to two or more drugs and / or non-drug factors; 16) Patients with active disseminated intravascular coagulation within 3 months before screening; or patients who the researcher believes Patients currently taking anticoagulants or antiplatelet drugs need to be excluded; 17) Patients with hypertension whose blood pressure is still not controlled after antihypertensive treatment during the screening period: SBP / DBP ≥ 160 / 100 mmHg; 18) Patients with poor blood sugar control during the screening period (glycosylated hemoglobin value ≥ 10%); 19) Patients with liver and kidney function tests during the screening period: ALT, AST> 2×ULN, or Cr> ULN and clinically significant as determined by the investigator; 20) Patients with a thrombotic event (including acute myocardial infarction, ischemic stroke, etc.) within 6 months before screening; 21) Patients with a history of chemotherapy or radiotherapy for malignant tumors within 5 years before screening; 22) Patients with a previous or current systemic immune disease or those receiving immunosuppressive or immunopotentiator treatment;23) Patients with any other uncontrollable clinical problems (such as severe mental, neurological, cardiovascular, respiratory, digestive, urinary, blood, endocrine, immune and other systemic diseases and malignant tumors); 24) Patients with active hepatitis B virus (surface antigen positive and HBV DNA ≥ 2000 IU / mL), hepatitis C virus, human immunodeficiency virus, and Treponema pallidum pathogen testing during the screening period, or those who are unwilling to undergo human immunodeficiency virus and Treponema pallidum pathogen testing; 25) Patients with active herpes virus infection during the screening period; 26) Patients who cannot take effective contraceptive measures during the trial or have fertility intentions; 27) Pregnant or lactating women (pregnancy in this trial is defined as a positive blood or urine pregnancy test); 28) Patients who have participated in other clinical trials within 3 months before screening (if the half-life of the investigational drug is long, the 7 half-lives are greater than 3 months, then the 7 half-lives are 7 half-lives); 29) Patients who are considered unsuitable for participation in this trial by the investigator.

[0100] Dosage and administration: Subretinal injection, single dose.

[0101] Primary endpoints and evaluation period: Observe and monitor the frequency and incidence of adverse events (AEs), serious adverse events (SAEs), and dose-limiting toxicities (DLTs) following subretinal injection of different doses of KH631 ophthalmic injection within 24 weeks. Monitor the change in BCVA compared to baseline at week 52.

[0102] Secondary endpoints and evaluation schedules: Changes from baseline in BCVA, CRT, retinal leakage area, number of rescue treatments, visual function scale, biodistribution, detachment assessment, aqueous humor, and blood VEGF-A concentrations, the proportion of subjects with geographic atrophy, and the proportion of subjects without subretinal and intraretinal fluid, will be observed or monitored at Weeks 52 and 104. Cellular and humoral immunity will be monitored at Days 1-7 and Weeks 2, 3, 4, 12, 24, 52, 76, and 104.

Claims

1. A method for treating an eye disease associated with angiogenesis in a subject, comprising administering to the subject's eye a pharmaceutical composition comprising a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises a nucleotide encoding a fusion protein of an anti-vascular endothelial growth factor (VEGF), wherein the administered rAAV dose is 10 8 Up to 10 13 vg / eye.

2. The method according to claim 1, characterized in that The anti-vascular endothelial growth factor (VEGF) fusion protein comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 99% or 100% homology thereto.

3. The method according to claim 1, characterized in that The recombinant adeno-associated virus (rAAV) comprises: (i) rAAV capsid protein, wherein the capsid protein is AAV8 capsid protein or a mutant thereof; and (ii) a polynucleotide expression cassette comprising, from 5' to 3': (a) AAV 5'ITR; (b) CBA promoter; (c) a nucleotide encoding an anti-vascular endothelial growth factor (VEGF) fusion protein, wherein the anti-vascular endothelial growth factor (VEGF) protein has an amino acid sequence as described in SEQ ID NO: 1; (d) rabbit-globin polyadenylation signal; (e) AAV 3'ITR.

4. The method according to claim 1, characterized in that: The nucleic acid encoding the anti-vascular endothelial growth factor (VEGF) protein has a nucleotide sequence as shown in SEQ ID NO:

2.

5. The method according to claim 1, characterized in that The polynucleotide expression cassette has a nucleotide sequence as shown in SEQ ID NO: 3 from 5' to 3'.

6. The method according to claim 1, characterized in that The rAAV dose administered was 3*10 8 Up to 10 13 vg / eye. The method according to claim 1, characterized in that the rAAV dose administered is 3*10 8 Up to 10 12 vg / eye.

7. The method according to claim 1, characterized in that The rAAV dose administered was 3*10 9 Up to 5*10 11 vg / eye or 3*10 9 Up to 1*10 11 vg / eye or 1*10 10 Up to 3*10 10 vg / eye.

8. The method according to claim 1, characterized in that Administration is via the retina, choroid or intravitreal route.

9. The method according to claim 1, characterized in that: Administer subretinal.

10. The method according to claim 9, characterized in that The rAAV is injected into the subretinal space by using a subretinal drug delivery device.

11. The method according to claim 1, characterized in that: The eye diseases or conditions associated with angiogenesis (VEGF) are age-related macular degeneration, retinal neovascularization, choroidal neovascularization, diabetic retinopathy, proliferative diabetic retinopathy, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, diabetic retinal ischemia, ischemic retinopathy and diabetic retinal edema.

12. The method according to claim 1, characterized in that The subject is a human.

13. The method according to claim 1, characterized in that The human subject has a best corrected visual acuity (BCVA) of 20 / 32 to 20 / 400.

14. The method according to claim 1, characterized in that The subject is a non-human mammal; preferably, the non-human mammal is a non-human primate.

15. The method according to claim 12, characterized in that The anti-vascular endothelial growth factor (VEGF) protein is produced by human photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells and / or retinal pigment epithelial cells in the external limiting membrane of the human subject.

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