Application of AAV carrying anti-VEGF recombinant protein in prevention or treatment of proliferative diabetic retinopathy

Abstract

This invention relates to the application of an AAV carrying anti-VEGF recombinant protein in the prevention or treatment of proliferative diabetic retinopathy. After loading Fc-engineered VEGF antibody into an AAV vector, it can be used in drugs for the treatment of proliferative diabetic retinopathy. After injection, it can significantly improve the visual function of patients and reduce macular edema after a single dose. The anti-VEGF recombinant protein can be expressed for a long time, and can maintain the drug effect over a long period of time.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy technology, and in particular relates to the application of an anti-VEGF recombinant protein in the prevention or treatment of proliferative diabetic retinopathy. Background Technology

[0002] Diabetic retinopathy (DR) is a major microvascular complication of diabetes, easily causing vision loss in working-age individuals. The resulting vision decline and loss has become a significant public health problem. One in three diabetic patients suffers from DR, indicating a large patient population. DR is mainly divided into non-proliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR). In the NPDR stage, changes occur in retinal blood flow and vascular permeability, basement membrane thickening, pericyte loss, and the formation of acellular capillaries. Visual field examination reveals microaneurysms, venous beading, and intraretinal microvascular abnormalities. As the ischemia at the lesion site increases, it may develop into PDR, with a large number of new blood vessels appearing, leading to related complications, including vitreous hemorrhage and retinal traction detachment, which can result in blindness in severe cases. DR has become a significant factor affecting the normal work and study of diabetic patients, while PDR is the stage of the disease that most severely impacts their vision.

[0003] In the progression of PDR, various complications are prone to occur, including vitreous hemorrhage, fibrous membranes in PDR, preretinal hemorrhage, tractional retinal detachment, rhegmatogenous retinal detachment, vitreous hemorrhage combined with cataracts, and vitreous hemorrhage combined with iris neovascularization. Currently, vitrectomy is the primary treatment for PDR patients. Vitrectomy can remove the cloudy vitreous, hemorrhage, and fibrous membranes, allowing the detached retina to reposition itself and rapidly improving patient symptoms. However, postoperatively, vitreous hemorrhage and macular edema are prone to recurrence, and neovascular glaucoma and other symptoms are likely to occur. Anti-vascular endothelial growth factor (VEGF) drugs, including recombinant anti-VEGF antibodies or fusion proteins (collectively referred to as "recombinant proteins"), can control disease progression and reduce the occurrence of complications. However, on the one hand, these anti-VEGF drugs require long-term, repeated intravitreal injections, which bring more medical risks and increase the treatment burden on patients. On the other hand, after vitrectomy, the residence time of anti-VEGF drugs in the vitreous body is shortened, further weakening the efficacy and increasing the treatment burden. These reasons lead to poor adherence to continuous treatment in many patients, resulting in poor medium- and long-term treatment outcomes. By using a recombinant adeno-associated virus (rAAV) vector to carry the anti-VEGF recombinant protein gene expression sequence and administering it via intravitreal, subretinal, or suprachoroidal injection, this therapeutic protein can be expressed in the fundus for a long period, replacing the need for repeated injections of anti-VEGF drugs. This reduces the aforementioned treatment risks and burdens and may effectively alleviate the progression of neovascularization-related diseases, reduce the recurrence of vitreous hemorrhage, macular edema, and neovascular glaucoma. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of anti-VEGF recombinant protein in the prevention or treatment of proliferative diabetic retinopathy.

[0005] The technical solution adopted in this invention is: the application of AAV carrying anti-VEGF recombinant protein in drugs for the prevention or treatment of proliferative diabetic retinopathy.

[0006] Preferably, the AAV carrying the anti-VEGF recombinant protein can express the anti-VEGF recombinant protein, which is aflibercept, ranibizumab, bevacizumab, farexit antibody, conbercept, or a variant with a similarity of 99% or more.

[0007] Preferably, the AAV carrying the anti-VEGF recombinant protein can express the Fc optimized anti-VEGF protein.

[0008] Preferably, the dosage is 1x10 8 vg-1x10 13 vg / eye.

[0009] Preferably, the drug is administered to one or both eyes via intravitreal injection, subretinal injection, or suprachoroidal injection.

[0010] Preferably, the medication can reduce macular edema and inhibit the increase in the concentration of the target protein in the aqueous humor for at least 12 weeks after administration.

[0011] Preferably, the best corrected visual acuity (BCVA) improves by at least one ETDRS letter compared to the screening period (baseline); and / or, the central retinal thickness (CRT) improves by at least 25 μm compared to the screening period (baseline).

[0012] A drug for the prevention or treatment of proliferative diabetic retinopathy includes AAV carrying an anti-VEGF recombinant protein.

[0013] Preferably, the anti-VEGF recombinant protein includes a mutant with an Fc mutation site of H310E.

[0014] Preferably, the serotype of the AAV drug is AAV8.

[0015] Preferably, the promoter of the AAV drug is a panpromoter.

[0016] The advantages and positive effects of this invention are: the treatment of PDR by Fc-mutated AAV gene therapy drugs can significantly improve patients' visual function and reduce macular edema after a single dose; the target protein can be maintained for at least 12 weeks and maintain a stable concentration or show a slow decreasing trend over a longer period of time. Attached Figure Description

[0017] Figure 1 The effects of long-term AAV drug expression in non-human primates;

[0018] Figure 2 Change rate of leakage area; OD: right eye, OS: left eye;

[0019] Figure 3 Change rate of grayscale value due to leakage; OD: right eye, OS: left eye;

[0020] Figure 4 Target protein concentration in aqueous humor;

[0021] Figure 5 Visual acuity of subjects before and after drug administration

[0022] Figure 6 Changes in mean central retinal thickness in subjects before and after drug administration;

[0023] Figure 7 Optical coherence tomography (OCT) images of the retina of subjects before and after drug administration. Detailed Implementation

[0024] The embodiments of the present invention will now be described with reference to the accompanying drawings.

[0025] This invention relates to the application of an anti-VEGF recombinant protein in the prevention or treatment of proliferative diabetic retinopathy (PDR). The optimized target gene sequence is delivered to the patient's eye via AAV delivery of an optimized Fc-based VEGF receptor recombinant fusion protein or an anti-VEGF recombinant antibody, through subretinal, intravitreal, or suprachoroidal injection, achieving long-term stable expression of the target gene in the retina. This single-dose administration provides long-term effectiveness. It offers patients a safer, more convenient, and superior treatment method, improving fundus symptoms after PDR treatment, slowing disease progression, and reducing the number of re-treatment interventions required.

[0026] The optimized VEGF receptor recombinant fusion protein, comprising recombinant proteins of human vascular endothelial growth factor (VEGF) receptor 1 Domain 2 and receptor 2 Domain 3 with human immunoglobulin Fc fragment mutants, can be expressed in the eye at high concentrations and for extended periods. The anti-VEGF recombinant antibody is based on existing anti-VEGF antibodies, with the Fc fragment replaced by the IgG1 Fc fragment mutant described in Technical Solution 1. The anti-VEGF antibody includes, but is not limited to, bevacizumab, ranibizumab, and brolucizumab. The Fc fragment mutant of human IgG1 is obtained by undergoing at least one of the following mutations: T250A, L251A, M252L, I253A / D / P, S254A, T256A, L309A, H310L / V / A / D / E / Q, Q311A, L314A, M428L / I, H433L / V / A, N434L / V / A, H435L / V / A, Y436A, where the numbering is indicated by the EU index. Specific sequence information is disclosed as in patent CN117304307A.

[0027] Anti-VEGF protein can be expressed for a long time via a single subretinal administration using AAV gene therapy, solving the problem of frequent drug administration required for protein therapy and thus enabling the treatment of PDR. First, an AAV viral vector expression cassette for the anti-VEGF recombinant antibody is constructed. The cassette includes an ITR-CMV promoter-CMV enhancer-intron-signal peptide-Kozak sequence-VEGF receptor recombinant fusion protein / anti-VEGF recombinant antibody coding sequence-PolyA-ITR. The AAV-antiVEGF drug is then constructed by packaging with adeno-associated virus. In some embodiments of this invention, the serotype of the AAV drug is AAV8, and the promoter is a pan-promoter. For administration, the anti-VEGF protein can be directly prepared and injected into the eye at a dose of 0.1-20 mg / eye; alternatively, the AAV-packaged AAV-antiVEGF can be injected into the eye using an AAV vector at a dose of 1 x 10⁻⁶. 8 vg-1x10 13 Vg / eye. Can be administered to one or both eyes via intravitreal injection, subretinal injection, or suprachoroidal injection.

[0028] In some embodiments of the present invention, the Fc mutation site is preferably a mutant of H310E. A strain of AAV loaded with an anti-VEGF antibody containing the H310E mutation site is named Variant10. Variant10 is preferably used as a drug for the prevention or treatment of proliferative diabetic retinopathy. After a single dose, it can significantly improve the patient's visual function and reduce macular edema; the target protein can be maintained for at least 12 weeks, and maintain a stable concentration or show a slow decreasing trend over a longer period.

[0029] The present invention will now be described with reference to the accompanying drawings. Experimental methods not specifically described in terms of operation steps are performed in accordance with the corresponding product manuals. Unless otherwise specified, the instruments, reagents, and consumables used in the embodiments can be purchased from commercial companies.

[0030] Example 1: Construction of AAV-antiVEGF drug

[0031] The construction and preparation method of AAV-antiVEGF drugs refer to the construction process of variant10 in Example 7 of patent CN117304307A.

[0032] Example 2: Long-term expression experiment of AAV drug in monkeys

[0033] One male cynomolgus macaque was used, and 1×10⁻⁶ tbsp was injected into each eye. 9 vg / eye and 1×10 10AAV-antiVEGF intraocular injection of vg / eye was administered to one male rhesus monkey with 6 × 10⁶ injections in each eye. 10 AAV-antiVEGF intravitreal injection solution (vg / eye) was administered via a single subretinal injection at a volume of 50 μL / eye. Aqueous humor was collected from both eyes of the animals at 1, 3, 12, 18, 24, and 30 months post-treatment, with each sample containing approximately 50 μL.

[0034] The concentration of the target protein in the aqueous humor was detected by enzyme-linked immunosorbent assay (ELISA) as follows: VEGF165 was diluted to 200 ng / mL with CBS (pH 9.6±0.2), and 100 μL was added to each well of a high-adsorption 96-well plate. The plate was sealed and incubated overnight at 2–8 °C for coating. The coating solution was discarded, and 300 μL of blocking buffer (1% BSA) was added to each well. The plate was sealed and incubated at room temperature for 1–3 h. The protein reference standard RS001 Protein was diluted with diluent (1% BAST) to prepare a standard curve (STD) and quality control samples (QC). The standard curve concentrations were S1 (32 ng / mL), S2 (16 ng / mL), S3 (8 ng / mL), S4 (4 ng / mL), S5 (2 ng / mL), S6 (1 ng / mL), and S7 (0.5 ng / mL), while the quality control sample concentrations were HQC (24 ng / mL), MQC (5 ng / mL), LQC (1.5 ng / mL), and LLOQ (0.5 ng / mL). The sample concentrations were diluted to the standard curve range using diluent (1% BAST). The blocking buffer was discarded, and the plate was washed 5 times with a plate washer, adding 300 μL of washing buffer (PBST) to each well each time. After the final wash, residual liquid was patted dry on absorbent paper. 100 μL of the standard curve, quality control, and test samples were added to each well, with all samples in duplicate. The plates were sealed and incubated at room temperature for 1 h ± 5 min. The HRP-labeled goat anti-human IgG (Fc specific) antibody was diluted 1:3000 with diluent (1% BAST). Wash the plate 5 times with a plate washer, adding 300 μL of washing buffer (PBST) to each well each time. After the last wash, blot dry any remaining liquid on absorbent paper. Add 100 μL of HRP-labeled antibody to each well, seal the plate, and incubate at room temperature for 1 h ± 5 min. Remove the TMB single-component chromogenic solution and equilibrate to room temperature. Wash the plate 5 times with a plate washer, adding 300 μL of washing buffer to each well each time. After the last wash, blot dry any remaining liquid on absorbent paper. Add 100 μL of TMB chromogenic solution to each well, seal the plate, protect from light, and incubate at room temperature for 5–8 min. Stop the reaction by adding 50 μL of stop solution to each well. Within 20 min, read the OD at 450 nm using a microplate reader (with 650 nm as the reference wavelength).

[0035] The results are as follows Figure 1 As shown, 1×10⁻⁶ was injected subretinally into monkeys. 9 vg / eye~6×10 10Within 30 months after intravitreal injection of AAV-antiVEGF into vg / eye, the concentration of the target protein in the atrial fluid of monkeys remained stable without any downward trend.

[0036] Example 3: Long-term treatment effect in a non-human primate retinal PDR model

[0037] DL-AAA (DL-α-aminoadipic acid) is a selective glial cytotoxic agent that has been reported to inhibit glutamine synthetase activity, impairing broader Müller cell retinal homeostasis and leading to glial dysfunction and death, thereby disrupting the blood-retinal barrier. The retinal vascular system, retinal segmentation and basal boundary, the proportions of retinal neurons and glial cell subtypes, and the presence of the macula are homologous in monkeys and humans. The drug can cause damage to ocular glial cells, thereby disrupting retinal vessels, causing retinal vascular leakage, and inducing neovascularization, forming a persistent retinal neovascularization (PRNV) model. Since PDR disease is caused by retinal vascular leakage and neovascularization, the cynomolgus monkey model of DL-AAA is a good model for PDR disease.

[0038] Three adult male cynomolgus macaques (ID050063, ID013153, and ID070123) underwent a single intravitreal injection of DL-AAA to induce retinal neovascularization in both eyes (ID070123 only had its right eye modeled). After confirming successful modeling by fundus fluorescein angiography, the right eyes of animals ID050063 and ID013153, and both eyes of animal ID070123, were injected intravitreally with AAV-antiVEGF solution (6×10⁻⁶). 10 (vg / eye) In animals ID050063 and ID013153, the left eye was given a solvent control, with a single subretinal injection of 50 μL / eye. Fundus fluorescein angiography was performed at 2, 4, 6, 8, 10, 12, 16, 20, 24, 46, 53, 79, 105, and 127 weeks post-treatment.

[0039] The instrument model was Spectralis HRA. Before the examination, animals were intravenously injected with sodium fluorescein (20 mg / kg). Images were acquired within 1 minute (early phase, both eyes), 1-5 minutes (middle phase, both eyes), and 5-7 minutes (late phase, both eyes). ImageJ software was used to measure the area and grayscale value of sodium fluorescein leakage in different regions of the animal's fundus. The total leakage area and grayscale value were calculated, and the change rate was calculated using the pre-drug values ​​as a baseline.

[0040] Aqueous humor was collected from both eyes of the animals at 4, 6, 8, 53, 79, 105, and 127 weeks after drug administration, with each sample containing at least 50 μL / eye. The concentration of the target protein in the aqueous humor was detected by enzyme-linked immunosorbent assay (ELISA), using the same method as in Example 1. During the experiment, considering the animals' health and ethical requirements, animal ID050063 was followed up until 73 weeks after drug administration, ID070123 until 79 weeks after drug administration, and ID013153 was continuously followed up.

[0041] Change rate of fundus leakage area in animals Figure 2 As shown, the change rate of leakage grayscale value is as follows: Figure 3 As shown in the figure. Compared with the pre-treatment state, after administration of AAV-antiVEGF intraocular injection, the area and gray value of fundus leakage in the animals were reduced. Furthermore, compared with the solvent control group, AAV-antiVEGF intraocular injection significantly improved retinal neovascularization leakage in the animals. The concentration of the target protein in the aqueous humor of the animals is shown in the figure. Figure 4 As shown, 6×10⁶ cells were injected subretinally. 10 Within one year after intravitreal injection of AAV-antiVEGF for vg / eye, the concentration of the target protein in the aqueous humor showed a slow decreasing trend, and then the protein concentration remained stable until 2.5 years after the drug was administered.

[0042] Example 4: Clinical trial study of AAV-antiVEGF

[0043] To evaluate the preliminary safety and efficacy of a single unilateral subretinal injection of AAV-antiVEGF after or following vitrectomy in subjects with proliferative diabetic retinopathy (PDR).

[0044] The intravitreal injection containing AAV-antiVEGF was named RRG001 intravitreal injection. After screening and enrollment, the study eye underwent appropriate surgical procedures (such as vitrectomy, irrigation, etc.) based on the subject's ocular condition, combined with a single subretinal RRG001 intravitreal injection (day 0). The dosing regimen included a low-dose group and a high-dose group; the low-dose group received 3.0 × 10⁻⁶ doses. 9 vg / eye; high-dose group: 3.0 × 10 10Vg / eye, subretinal injection, single, unilateral injection, 100 μL or 150 μL per injection. RRG001 intravitreal injection solution is transported to the research center under cold chain conditions and stored at ≤-60°C, protected from light. Before injection, it is thawed at the research center and diluted to the appropriate dose according to the drug preparation procedure. Subretinal injection should be performed under aseptic conditions by a specialist with extensive vitrectomy experience and training in subretinal injection procedures, following the drug injection guidelines. A core follow-up period of 24 weeks follows injection. Upon completion of the 24-week trial follow-up or early withdrawal of the subject, the subject enters a long-term follow-up period, extending up to 5 years after administration of RRG001 intravitreal injection solution.

[0045] One participant in the clinical trial was followed up for 12 weeks. No DLT occurred during the DLT evaluation period, and no treatment-emergent adverse events (TEAEs) occurred, indicating good preliminary safety. The drug significantly improved the patient's visual function; the best corrected visual acuity (BCVA, with higher values ​​indicating better visual acuity) during the screening period was 52 letters, which significantly improved to 78 letters 7 days after administration and remained so until 12 weeks. Figure 5 Optical coherence tomography (OCT) imaging showed foveal edema in the patient during the screening period, specifically a central retinal thickness (CRT) of 385 μm, which decreased to 236 μm 7 days after drug administration and remained stable until 12 weeks. Figure 6 Four weeks after administration, the patient's retinal structure had returned to normal and remained so for up to 12 weeks. Figure 7 ).

[0046] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the patent coverage of the present invention.

Claims

1. Application of AAV carrying anti-VEGF recombinant protein in drugs for the prevention or treatment of proliferative diabetic retinopathy.

2. The application according to claim 1, characterized in that: AAV carrying anti-VEGF recombinant protein can express anti-VEGF recombinant protein, which is aflibercept, ranibizumab, bevacizumab, farexit antibody, conbercept or a variant with a similarity of ≥99%.

3. The application according to claim 2, characterized in that: AAV carrying anti-VEGF recombinant protein can express Fc optimized anti-VEGF protein.

4. The application according to any one of claims 1-3, characterized in that: The dosage is 1x10 8 vg-1x10 13 vg / eye.

5. The application according to claim 4, characterized in that: It can be administered to one or both eyes via intravitreal injection, subretinal injection, or suprachoroidal injection.

6. The application according to claim 5, characterized in that: After medication, macular edema can be reduced, and the inhibition of the increase in the concentration of target proteins in the aqueous humor can be maintained for at least 12 weeks.

7. The application according to claim 6, characterized in that: Best corrected visual acuity (BCVA) improved by at least one ETDRS letter compared to the screening period (baseline); and / or, central retinal thickness (CRT) improved by at least 25 μm compared to the screening period (baseline).

8. A drug for the prevention or treatment of proliferative diabetic retinopathy, characterized in that: This includes AAV carrying recombinant anti-VEGF protein.

9. The medicament for the prevention or treatment of proliferative diabetic retinopathy according to claim 8, characterized in that: Anti-VEGF recombinant proteins include mutants with the Fc mutation site H310E.

10. The medicament for the prevention or treatment of proliferative diabetic retinopathy according to claim 8, characterized in that: The serotype of AAV drugs is AAV8.

11. The medicament for the prevention or treatment of proliferative diabetic retinopathy according to claim 8, characterized in that: The promoter of AAV drugs is a panpromoter.

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