Application of recombinant adeno-associated virus in preparation of medicine for treating angiogenesis-related eye diseases

Abstract

The invention relates to an application of a recombinant adeno-associated virus in preparation of a medicine for treating angiogenesis-related eye diseases, and particularly relates to application of a medicine composition containing the recombinant adeno-associated virus to eyes of a subject, and the recombinant adeno-associated virus comprises nucleotide of fusion protein for encoding an anti-vascular endothelial growth factor. The dose of the recombinant adeno-associated virus administered is 108 to 1013 vg / eye.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy, and in particular to a method for treating angiogenesis-related eye diseases in a subject. Background Art

[0002] The formation of new blood vessels is the key to the development and spread of many diseases. Many diseases of the eye 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, proliferation and angiogenesis. Many studies have shown that excessive expression of VEGF can induce pathological neovascular eye diseases. Due to the importance of VEGF signaling to angiogenesis, blocking VEGF or VEGF receptors to inhibit angiogenesis has an important therapeutic effect on diseases related to angiogenesis, including cancer and retinal vascular disease. In the past decade, some anti-VEGF drugs for the treatment of ocular neovascular eye diseases have been developed, such as pegaptanib (Macugen), bevacizumab (Avastin), ranibizumab (Lucentis), and aflibercept (Eylea).

[0003] However, due to the short half-life of currently marketed drugs, repeated administration is required, which increases the burden on doctors and patients. Therefore, gene therapy is expected to achieve long-term drug efficacy due to its continuous expression of drugs, such as adeno-associated virus vector (AAV) gene therapy tools. Summary of the invention

[0004] The present invention provides an effective method for treating angiogenesis-related ocular diseases in a subject.

[0005] 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 Up to 10 13 vg / eye.

[0006] 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.

[0007] In some preferred embodiments, the anti-vascular endothelial growth factor (VEGF) fusion protein comprises the amino acid sequence of SEQ ID NO:1.

[0008] In some preferred embodiments, the recombinant adeno-associated virus (rAAV) comprises:

[0009] (i) rAAV capsid protein, wherein the capsid protein is AAV8 capsid protein or a mutant thereof; and

[0010] (ii) a polynucleotide expression cassette comprising, from 5' to 3':

[0011] (a) AAV 5'ITR;

[0012] (b) CBA promoter;

[0013] (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;

[0014] (d) rabbit-globin polyadenylation signal;

[0015] (e) AAV 3'ITR.

[0016] In some specific embodiments, the nucleic acid encoding the anti-vascular endothelial growth factor (VEGF) protein has a nucleotide sequence as described in SEQ ID NO:2.

[0017] 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'.

[0018] In some specific embodiments, the pharmaceutical composition is provided in the form of a rAAV vector active ingredient dispersed in a buffer, which includes a physiologically compatible aqueous buffer, a surfactant, and an optional excipient.

[0019] In some specific embodiments, the rAAV dose administered is 3*10 8 Up to 1*10 13 vg / eye.

[0020] In some specific embodiments, the rAAV dose administered is 3*10 8 Up to 1*10 12 vg / eye.

[0021] In some specific embodiments, the rAAV dose administered is 3*10 9 Up to 5*1011 vg / eye.

[0022] In some specific embodiments, the rAAV dose administered is 3*10 9 Up to 1*10 11 vg / eye.

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

[0024] 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.

[0025] 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 a 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.

[0026] In some specific embodiments, the eye diseases or disorders associated with angiogenesis (VEGF) described in the present invention 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.

[0027] 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, a rhesus monkey, a cynomolgus monkey, etc.

[0028] 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 a fusion protein against vascular endothelial growth factor (VEGF), the rAAV administered at a dose of 10 8 Up to 10 13 vg / eye.

[0029] 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.

[0030] In some preferred embodiments, the recombinant adeno-associated virus (rAAV) comprises:

[0031] (i) rAAV capsid protein, wherein the capsid protein is AAV8 capsid protein or a mutant thereof; and

[0032] (ii) a polynucleotide expression cassette comprising, from 5' to 3':

[0033] (a) AAV 5'ITR;

[0034] (b) CBA promoter;

[0035] (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;

[0036] (d) rabbit-globin polyadenylation signal;

[0037] (e) AAV 3'ITR.

[0038] In some specific embodiments, the nucleic acid encoding the anti-vascular endothelial growth factor (VEGF) protein has a nucleotide sequence as set forth in SEQ ID NO: 2. 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'.

[0039] In some specific embodiments, the pharmaceutical composition is provided in the form of a rAAV vector active ingredient dispersed in a buffer, which includes a physiologically compatible aqueous buffer, a surfactant, and an optional excipient.

[0040] In some specific embodiments, the rAAV dose administered is 3*10 8 Up to 1*10 13 vg / eye. In some specific embodiments, the rAAV dose administered is 3*10 8 Up to 1*10 12 vg / eye. In some specific embodiments, the rAAV dose administered is 3*109 Up to 5*10 11 vg / eye. In some specific embodiments, the rAAV dose administered is 3*10 9 Up to 1*10 11 vg / eye. In some specific embodiments, the rAAV dose administered is 1*10 10 Up to 3*10 10 vg / eye.

[0041] 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*10 12 / eye, 5*10 12 / eye, 6*1012 / eye, 7*10 12 / eye, 8*10 12 / eye, 9*10 12 / eye, 1*10 13 / Eye.

[0042] 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 a 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.

[0043] 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 membrane of the human subject. Choroidal, subretinal or intravitreal injection should result in the delivery of the transgenic product (anti-vascular endothelial growth factor (VEGF) protein) to the retina, vitreous humor and / or aqueous humor. Specifically, the expression of the transgenic product by retinal cells, such as rods, cones, retinal pigment epithelium, horizontal, bipolar, amacrine, ganglion and / or Muller cells, results in the delivery and maintenance of the transgenic product in the retina, vitreous humor and / or aqueous humor.

[0044] 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 1 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 ≥50μm from the highest value, or subretinal or intraretinal fluid decreases 30% from the highest value); 3) The best corrected visual acuity (BCVA) of the sentinel subject's study eye is 63 to 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 to 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.

[0045] 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 other diseases other than AMD (including pathological myopia, etc.); or those whose study eye, as determined by the researcher, has non-nAMD diseases that affect the examination of the macular area or central vision (including diabetic retinopathy, central vein occlusion, etc.); 2) those whose study eye has implants (except artificial lenses), opacities of the refractive medium that affect fundus examination, or pupil constriction; 3) those whose study eye has scars, fibrosis, or atrophy under the fovea that affect the evaluation of the test results; 4) those whose study eye has had or currently has retinal detachment (including rhegmatogenous retinal detachment, tractional retinal detachment, exudative retinal detachment, etc.); 5) those whose study eye currently has Patients with severe dry eyes who are currently receiving medication; 6) Patients with tear drainage system diseases (including tear duct obstruction or tear duct stenosis) that require medication or surgical treatment in the study eye; 7) Patients with glaucoma and ocular hypertension (intraocular pressure ≥ 25 mmHg during the screening period) that are not controlled by medication in the study eye; 8) Patients with intraocular tumors (including choroidal melanoma, etc.) in the study eye; 9) Patients with active ocular infections (including viruses, bacteria and fungi) in the study eye or fellow eye, such as blepharitis, infectious conjunctivitis, keratitis, scleritis, endophthalmitis, dacryocystitis, etc.; 9) Patients with active ocular infections (including viruses, bacteria and fungi) 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 diseases other than anti-VEGF ophthalmic injection drugs (including intraocular hormone use, etc.) within 6 months before the study eye screening; 11) Patients with retinal toxicity (including chloroquine, hydroxychloroquine, etc.) within 6 months before the study eye screening; 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 study eye screening; 14) Patients with allergic reactions or allergic history to fluorescein sodium; 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 those who are currently taking anticoagulants or antiplatelet drugs and who the researchers believe need to be excluded; 17) Those with hypertension whose blood pressure is still not controlled after antihypertensive treatment during the screening period: SBP / DBP ≥ 160 / 100 mmHg; 18) Those with poor blood sugar control during the screening period (glycosylated hemoglobin value ≥ 10%); 19) Those with liver and kidney function tests during the screening period: ALT, AST>2×ULN, or Cr>ULN and clinically significant as determined by the researchers; 20) Those who have had thrombotic events (including acute myocardial infarction, ischemic stroke, etc.) within 6 months before screening; 21) Those with a history of chemotherapy or radiotherapy for malignant tumors within 5 years before screening; 22) Those who have had or are currently suffering from systemic immune diseases or are receiving immunosuppressants or immunopotentiators;23) 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) Active hepatitis B virus (surface antigen positive and HBV DNA ≥ 2000IU / mL), hepatitis C virus, human immunodeficiency virus, Treponema pallidum pathogenic test positive during the screening period, or those who are unwilling to undergo human immunodeficiency virus and Treponema pallidum pathogenic test; 25) Active herpes virus infection during the screening period; 26) Those 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) Those who have participated in other clinical trials within 3 months before screening (if the half-life of the trial drug is long, and its 7 half-lives are greater than 3 months, then 7 half-lives) ; 29) Those who are considered unsuitable for participation in this trial by the researchers. ; BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Percentage of level 4 light spots in the negative control group and each dosage group in Example 2

[0047] Figure 2 Representative schematic diagram of fluorescein angiography in the negative control group and each dose group in Example 2

[0048] Figure 3 Diagram of changes in retinal thickness of the negative control group and each dose group in Example 2 DETAILED DESCRIPTION

[0049] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0050] Example 1 Preparation of recombinant adeno-associated virus vector (rAAV)

[0051] 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 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 described in SEQ ID NO: 1. The coding sequence of the Conbercept fusion protein has the nucleotide sequence described in SEQ ID NO: 2. The recombinant plasmid contains the nucleotide sequence described in SEQ ID NO: 3 from 5' to 3' between the inverted terminal repeats (ITRs).

[0052] The recombinant adeno-associated virus vector (rAAV) was prepared by a three-plasmid co-transfection process known in the art. Specifically, three plasmids (pAAV-Conbercept: pAAV 8-RC-Kan: pHelper-Kan) were first added to the HBS buffer solution and vortexed to mix; then the transfection reagent was added, vortexed to mix, and incubated at room temperature for 20 to 30 minutes; then added to the HEK293 cell solution to be transfected. After transfection, the cells were cultured at 36.5°C and 5.0% CO. 2 , 120rpm, harvest the cell fluid for 72h, and detect the rAAV genome titer.

[0053] Example 2: Drug efficacy experiment of rhesus monkey retinal administration

[0054] Animal models

[0055] Rhesus monkeys, 24 in total.

[0056] Age: 2.5-5 years old; Weight: 3.70-5.90kg at purchase, average 4.70kg.

[0057] Sample solution:

[0058] The rAAV prepared in Example 1 was prepared with 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.

[0059] Experimental Grouping

[0060] 1 negative control group (Vehicle / buffer group), 3 animals.

[0061] 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 10vg / eye group, 1×10 10 In the vg / eye group, one animal was excluded from each group due to drug administration, and only two animals were actually included in the experimental statistics. The other groups had three animals in each group.

[0062] Dosage

[0063] 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 of rAAV injection solution and buffer solution were used. 28 days after administration, binocular laser damage was performed on the Bruch's membrane of the outer retina of rhesus monkeys to establish an animal model similar to human choroidal neovascularization (CNV).

[0064] Modeling method: Rhesus monkeys were anesthetized by intravenous injection of 2.5% sodium pentobarbital (25 mg / kg) and / or 5% ketamine (10 mg / kg) (the anesthetic dose can be adjusted appropriately according to the monkey's anesthesia condition). During anesthesia, a small amount of Diclofenac was dripped from time to time to keep the cornea moist. After dilating the pupils with Midori (compound tropicamide eye drops), the rhesus monkey's head was fixed in front of an ophthalmic laser photocoagulation instrument, and the macular area was photocoagulated through a panretinal microscope. Photocoagulation avoided the fovea, and 6 points were irradiated in each eye.

[0065] Laser parameters: spot diameter 50μm, energy 0.5~0.7W, exposure time 0.05s.

[0066] Photocoagulation criteria: Bubbles can be seen, indicating that Bruch's membrane is broken.

[0067] Experimental observation

[0068] 1) Fundus photography and fluorescein angiography

[0069] Testing time: once during the adaptation period, and once at 4, 6, and 8 weeks after administration;

[0070] Examination method: The monkeys to be examined are anesthetized with 5% ketamine (intramuscular injection, 10 mg / kg), and the mydriasis (eye drops, 1-2 drops / eye) is administered with Midori (compound tropicamide eye drops). After fundus photography is performed with a fundus camera, a rapid push of fluorescein sodium injection (20 mg / kg) is given from the saphenous vein of the monkey's lower limb (or other suitable sites), and fundus images are taken with a fundus camera in the early (within about 1 minute) and late (after about 5 minutes) after angiography. During the test, ketamine (about 5-25 mg / mouse) can be injected intramuscularly or sodium pentobarbital (about 10-80 mg / mouse) can be administered intravenously to supplement and maintain anesthesia, and 1-2 drops of ofloxacin eye ointment can be applied after the operation to prevent infection.

[0071] Evaluation indicators:

[0072] (1) Fluorescent spot rating

[0073] Grading standards for the light spots photographed by fluorescence angiography after modeling:

[0074] Level 1: The light spot does not show high fluorescence;

[0075] Grade 2: The spot has high fluorescence but no fluorescein leakage;

[0076] Grade 3: The spot has high fluorescence, with slight leakage of fluorescein, which does not exceed the edge of the spot;

[0077] Grade 4: The light spot has high fluorescence, with significant fluorescein leakage that exceeds the edge of the light spot.

[0078] The number of level 4 spots was counted, and the level of fundus laser spots was recorded in each examination after modeling.

[0079] 2) Optical coherence tomography (OCT) examination method: The monkey to be examined was anesthetized by intravenous injection of 2.5% sodium pentobarbital (25 mg / kg) and / or 5% ketamine (10 mg / kg) (the anesthetic dose can be adjusted appropriately according to the monkey's anesthesia condition), and the mydriasis (compound tropicamide eye drops) was applied (eye drops, 1-2 drops / eye), and the monkey's fundus was scanned by optical coherence tomography using an OCT instrument. After the operation, 1-2 drops of ofloxacin eye ointment were applied to prevent infection.

[0080] 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.

[0081] Evaluation indicators:

[0082] (1) Retinal thickness: the thickness from the nerve fiber layer to Bruch's membrane;

[0083] (2) Change in retinal thickness = retinal thickness after modeling - retinal thickness before modeling.

[0084] Experimental Results

[0085] 1) Fundus photography and fluorescein angiography results

[0086] 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 Tables 1 and Figure 1 The representative schematic diagrams of fluorescein angiography in each group are shown in Figure 2 .

[0087] The above results show that in the case of buffer control, obvious neovascularization fluorescein leakage was observed 14 days after modeling, and there was a slight decrease in fluorescein leakage from 14 to 28 days after modeling. After different doses of rAAV were given, the proportion of the fourth-order spot decreased significantly, indicating that the 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 cynomolgus monkeys.

[0088] Table 1

[0089]

[0090] 2) Optical coherence tomography (OCT) examination

[0091] 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 shown.

[0092] The results showed that there was no significant difference in retinal thickness between the groups of animals before modeling. OCT examination of each group of animals 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.

[0093]

[0094] Example 3 Phase I / II Clinical Trial

[0095] Phase 1 of this example mainly evaluates the tolerability of subretinal injection of different doses of AAV-VEGF ophthalmic injection in patients with neovascular (wet) age-related macular degeneration (nAMD), preliminarily evaluates the efficacy of KH631 ophthalmic injection in the treatment of nAMD, and mainly evaluates overall safety at 24 weeks. Phase 2 is mainly based on the results of Phase 1 tolerability, safety and preliminary efficacy evaluation, and 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, and mainly evaluates effectiveness at 52 weeks. The purpose of the long-term follow-up period is to evaluate the long-term safety and effectiveness 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 were willing to be visited at the time specified in the trial; 2) aged 50 to 85 years (including the borderline value), diagnosed with neovascular (wet) age-related macular degeneration (nAMD); 3) currently receiving anti-VEGF ophthalmic injection treatment and at least 1 anti-VEGF ophthalmic injection treatment within 6 months before the screening period; 4) previous OCT examinations proved that 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 sentinel subject's study eye was 63 to 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 was 78 to 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 was ≥19 letters (approximately equivalent to 20 / 400 on the Snellen visual acuity 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 other 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 researchers; 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 scars, fibrosis, or atrophy under the fovea of ​​the study eye that affect 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 in the study eye who are currently receiving medication ; 6) The study eye currently has tear drainage system diseases that require medication or surgical treatment (including tear duct obstruction or tear duct stenosis, etc.); 7) The study eye currently has glaucoma and ocular hypertension (intraocular pressure ≥ 25 mmHg during the screening period) that are not controlled by medication; 8) The study eye currently has intraocular tumors (including choroidal melanoma, etc.); 9) The study eye or the fellow eye currently has active ocular 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 has active ocular 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 treatment for nAMD other than anti-VEGF ophthalmic injections within 6 months before screening (including intraocular hormone use, etc.); 11) Patients who used retinal toxic drugs (including chloroquine, hydroxychloroquine, etc.) within 6 months before the screening of the study eye; 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 fluorescein sodium; 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 the researcher believes Patients who are 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 researchers; 20) Patients who have experienced thrombotic events (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 previous or current systemic immune diseases or those who are receiving immunosuppressants or immunopotentiators;23) 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) Active hepatitis B virus (surface antigen positive and HBVDNA ≥ 2000IU / mL), hepatitis C virus, human immunodeficiency virus, Treponema pallidum pathogenic test positive during the screening period, or those who are unwilling to undergo human immunodeficiency virus and Treponema pallidum pathogenic test; 25) Active herpes virus infection during the screening period; 26) Those 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) Those who have participated in other clinical trials within 3 months before screening (if the half-life of the trial drug is long, and its 7 half-life time is greater than 3 months, then it is 7 half-life time); 29) Those who are considered unsuitable to participate in this trial by the researchers. ;

[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) within 24 weeks of subretinal injection of different doses of KH631 ophthalmic injection. Monitor the change of subjects' BCVA compared with baseline at week 52.

[0102] Secondary endpoints and evaluation time: Observe or monitor the change of BCVA from baseline, CRT from baseline, retinal leakage area from baseline, number of rescue treatments, visual function scale, biodistribution, detachment evaluation, aqueous humor, VEGF-A concentration in blood, proportion of subjects with geographic atrophy, and proportion of subjects without subretinal and intraretinal fluid at weeks 52 and 104. Monitor the cellular and humoral immunity of subjects at days 1-7 and weeks 2, 3, 4, 12, 24, 52, 76 and 104.

Claims

1. Use of a recombinant adeno-associated virus (rAAV) in the preparation of a drug for treating angiogenesis-related eye diseases, characterized in that: The invention comprises administering to the eye of a subject a pharmaceutical composition comprising rAAV, wherein the rAAV comprises a nucleotide encoding a fusion protein of an anti-vascular endothelial growth factor (VEGF), wherein the dose of the administered rAAV is 10 8 Up to 10 13 vg / eye.

2. The use 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 use 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 use 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 use 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 use according to claim 1, characterized in that: The rAAV dose administered was 3*10 8 Up to 10 13 vg / eye, preferably 3*10 8 Up to 10 12 vg / eye, preferably 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.

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

8. The use according to claim 1, characterized in that Administer subretinal.

9. The use according to claim 8, characterized in that The rAAV is injected into the subretinal space by using a subretinal drug delivery device.

10. The use 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.

11. The use according to claim 1, characterized in that The subject is a human.

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

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

14. The use according to claim 11, 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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