A method for screening a drug capable of preventing and / or treating posterior segment disease of the eye

Abstract

This invention relates to the field of biotechnology and discloses a method for screening drugs capable of preventing and / or treating posterior segment diseases of the eye. The method includes: first, decompressing the anterior chamber of an experimental animal; then, pre-establishing a "perforation-closure" channel in the sclera of the experimental animal; next, injecting the drug to be screened into the suprachoroidal space of the experimental animal through the "perforation-closure" channel in the sclera; and finally, evaluating the effectiveness of the drug in preventing and / or treating posterior segment diseases based on changes in the eye condition of the experimental animal. This invention reduces the incidence of ocular surface inflammation and improves safety by lowering intraocular pressure and creating a pre-closed scleral incision to prevent backflow of the injected AAV vector; it also provides a broader expression range and longer duration of action. Studies have shown that injecting shRNA targeting the TGF-β2 gene using the method of this invention inhibits myopia progression in a guinea pig model. The method provided by this invention is beneficial for screening gene therapy drugs related to posterior segment diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for screening drugs that can prevent and / or treat diseases of the posterior segment of the eye. Background Technology

[0002] Posterior segment diseases (including inflammatory diseases of the choroid or retina, choroidal neovascularization-related diseases, and hereditary retinal degeneration) place high demands on drug delivery pathways. Traditional systemic administration is limited by the blood-retinal barrier, making it difficult to achieve adequate exposure in the posterior segment and potentially causing systemic adverse reactions. Intravitreal injection, as a local intraocular drug delivery method, involves injecting the drug directly into the vitreous cavity (the core region of the posterior segment) using a syringe. The drug then diffuses to reach the outer layers of tissue, such as the retina and choroid, and is currently considered the "gold standard" delivery method for treating posterior segment diseases. However, its penetration efficiency, distribution within the vitreous body, and clearance kinetics are limited for certain drug molecules.

[0003] Existing animal models for posterior segment diseases include non-human primates, pigs, rabbits, rats, mice, and guinea pigs. Guinea pigs, with their retinas containing both cone and rod cells, and a relatively high proportion of cone cells, and a retinal structure similar to humans, are well-suited for establishing models of retinal degenerative diseases (such as retinitis pigmentosa) and photoretinopathy. Furthermore, guinea pigs have abundant choroidal vessels, and their blood-retinal barrier (BRB) structure and function are similar to humans, making them suitable for studying posterior segment diseases such as choroidal neovascularization (CNV) and retinal vascular diseases. However, because guinea pigs have larger eyeballs than mice and rats (3-4 mm), subretinal injection is not possible under vertical illumination. Additionally, their eyeball size (8-9 mm) is much smaller than that of rabbits, monkeys, and humans, and their compact intraocular structure makes it impossible to perform internal illumination to place optical fibers inside the eye. Therefore, guinea pigs currently lack suitable gene delivery methods for screening disease-related therapeutic drugs.

[0004] The suprachoroidal space (SCS) is a potential gap between the sclera and choroid. Drug delivery via the SCS can create localized high concentrations in the choroid, choroidal capillary layer, or outer retina, while relatively reducing the risk of anterior segment and systemic exposure. It is considered an important alternative route for posterior segment local drug delivery. Currently, there are studies on suprachoroidal injection of drugs in humans and large animals, but reliable drug delivery remains challenging due to the small eyes of rodents. Furthermore, the key to the efficacy and safety of drug delivery lies not only in the delivery route itself, but also in the selection of the genetic elements and capsid of the delivery vehicle. The delivery route and delivery vehicle together determine core attributes such as delivery specificity, expression levels of the target gene and drug, onset and duration of action, and safety.

[0005] AAV (adeno-associated virus vector) has been widely used as a gene delivery vector, and in gene therapy and research, it is often used to achieve targeted expression through tissue-specific promoters. The VMD2 (Bestrophin-1) promoter can specifically target retinal pigment epithelial cells (RPE), and the targeting of drug delivery can be improved by binding related promoters to AAV vectors.

[0006] Currently, Hejri A et al. have developed a high-precision microneedle-based injector for choroidal drug delivery in rats and guinea pigs. However, due to the use of a specialized microneedle injector and the lack of targeted injection of gene-delivered drugs into posterior segment cells, the efficacy, safety, and durability of the delivered drugs have not been verified. Therefore, there is an urgent need to develop a drug screening method suitable for guinea pig animal models for the prevention and / or treatment of posterior segment diseases, providing a new technical solution for drug screening research on posterior segment diseases. Summary of the Invention

[0007] This invention provides a method for screening drugs that can prevent and / or treat posterior segment diseases of the eye, in order to address the problems of drug reflux, insufficient cell targeting, unstable injection success rate, and insufficient persistence and safety of gene delivery.

[0008] In a first aspect, the present invention provides a method for screening drugs capable of preventing and / or treating posterior segment diseases of the eye, the method comprising: firstly performing anterior chamber decompression on an experimental animal, then pre-establishing a "perforation-closure" channel in the sclera of the experimental animal, then injecting the drug to be screened into the suprachoroidal space of the experimental animal through the "perforation-closure" channel in the sclera of the experimental animal, and finally evaluating the effectiveness of the drug to be screened in preventing and / or treating posterior segment diseases of the experimental animal based on changes in the eye condition of the experimental animal.

[0009] In one optional embodiment, the anterior chamber decompression of the experimental animal includes: making a micro-incision in the peripheral cornea of ​​the experimental animal using a corneal scalpel to enter the anterior chamber, so that the aqueous humor in the anterior chamber of the experimental animal is slowly released until the intraocular pressure (IOP) drops to below 5 mmHg.

[0010] In one alternative embodiment, the amount of aqueous humor released is 3-6 μL.

[0011] In one optional embodiment, the pre-establishment of a "penetration-closure" channel in the sclera of the experimental animal includes: using an insulin needle to make an incision and puncture at a position 1.5-1.8 mm posterior to the limbus of the experimental animal, thereby forming a pre-established "penetration-closure" channel in the sclera of the experimental animal; during the insulin needle puncture, the angle between the needle and the tangent of the sclera of the experimental animal is controlled to be 10-20°.

[0012] In one alternative implementation, after the insulin needle puncture is completed, the needle is withdrawn 0.2-0.5 mm to facilitate the closure of the superficial needle tract, preserving the potential channel direction toward the posterior pole.

[0013] In one alternative implementation, the experimental animal is a normal experimental animal or an animal model of posterior segment disease.

[0014] In one alternative embodiment, the posterior segment disease animal model includes at least one of the following: negative lens-induced defocus myopia model, age-related macular degeneration (AMD) model, diabetic retinopathy (DR) model, retinitis pigmentosa (RP) model, choroidal ischemia model, ischemic optic neuropathy model, and glaucoma model.

[0015] In one alternative implementation, the posterior segment disease animal model includes a negative lens-induced defocus myopia model.

[0016] In one alternative implementation, the experimental animals include normal guinea pigs or animal models of posterior segment diseases obtained using guinea pigs.

[0017] In one alternative implementation, the guinea pig is 3 weeks old.

[0018] In one alternative implementation, the drugs to be screened include drugs for the prevention and / or treatment of posterior segment diseases of the eye.

[0019] In one alternative embodiment, the drug comprises at least one of an anti-vascular endothelial factor (VEGF) drug, a complement factor inhibitor, and a gene therapy drug.

[0020] In one alternative embodiment, the anti-VEGF drug comprises an anti-VEGF monoclonal antibody; the anti-VEGF monoclonal antibody comprises ranibizumab or an active analogue thereof.

[0021] In one alternative implementation, the complement factor inhibitor comprises antibodies against C5 and C3.

[0022] In one alternative embodiment, the gene therapy drug includes at least one of a drug for preventing and / or treating posterior segment diseases by expressing a target gene and a drug for preventing and / or treating posterior segment diseases by inhibiting a target gene.

[0023] In one alternative implementation, the drug for preventing and / or treating posterior segment diseases by inhibiting target genes includes small nucleic acid drugs.

[0024] In one alternative implementation, the small nucleic acid drug includes at least one of siRNA, miRNA, and shRNA.

[0025] In one alternative implementation, the small nucleic acid drug targets myopia-related genes.

[0026] In one alternative implementation, the myopia-related gene includes transforming growth factor TGF-β2.

[0027] In one alternative implementation, the small nucleic acid drug comprises shRNA that targets the myopia-related gene TGF-β2.

[0028] In one alternative embodiment, the gene therapy drug is loaded onto a gene delivery vector.

[0029] In one alternative implementation, the gene delivery vector includes at least one of an AAV vector and a lentiviral vector.

[0030] In one alternative embodiment, the gene delivery vector carries a gene expressing the EGFP fluorescent protein.

[0031] In one alternative embodiment, the AAV vector carries a gene expressing the EGFP fluorescent protein.

[0032] In one alternative implementation, the AAV vector carries a cell-specific promoter.

[0033] In one alternative implementation, the cell-specific promoter includes VMD2, a promoter that targets retinal pigment epithelial cells (RPE).

[0034] In one alternative embodiment, the viral capsid of the AAV vector includes at least one of AAV8, AAV2.7m8, and AAV8-BP2.

[0035] In one alternative embodiment, the viral capsid of the AAV vector is AAV8.

[0036] In an optional implementation, the method further includes: animal preparation and baseline assessment, anesthesia and local anesthesia and ocular surface protection, surgical site disinfection and exposure, and conjunctival and Tenon treatment prior to anterior chamber decompression of the experimental animal.

[0037] In one alternative implementation, the experimental animals are treated with conjunctiva and Tenon before anterior chamber decompression and pre-positioning of a "perforation-closure" passage.

[0038] In one alternative implementation, the AAV carrier is injected into the suprachoroidal space of the experimental animal through a "penetration-closure" channel in the sclera. After the injection, reflux control and hemostasis, conjunctival closure and postoperative medication, as well as immediate confirmation and follow-up recording are performed.

[0039] In one optional embodiment, the injection dose of the AAV carrier is 10. 9 vg-10 10 vg / only.

[0040] In one optional embodiment, the injection rate of the AAV carrier is 0.5-1.0 μL / s.

[0041] In one optional embodiment, the injection rate of the AAV carrier is 0.7-0.8 μL / s.

[0042] In one optional embodiment, the injection conditions of the AAV carrier are as follows: the osmotic pressure is controlled at 300±30mOsm / kg, the pH is controlled at 6.8-7.6, the AAV carrier is filtered using a 0.22μm filter membrane, and it is pyrogen-free.

[0043] In one optional embodiment, the injection titer of the AAV carrier is ≥1×10⁻⁶. 12 vg / mL.

[0044] In one optional embodiment, the needle is left in place for 5-10 seconds after the drug to be screened is injected; the conjunctiva is then closed by suturing after the needle is left in place.

[0045] In an alternative implementation, the method further includes the management of deviations and complications.

[0046] In one alternative implementation, a 29G insulin needle is used to pre-set the "penetration-closure" channel; a 34G microneedle is used for suprachoroidal injection.

[0047] In one alternative embodiment, the changes in the ocular condition of the experimental animals include at least one of the following: incidence of conjunctival hyperemia, duration of conjunctival hyperemia, incidence of symblepharon, incidence of ocular atrophy, incidence of retinal detachment, and changes in intraocular pressure.

[0048] Secondly, the present invention also provides the application of the method in screening drugs that can prevent and / or treat diseases of the posterior segment of the eye.

[0049] In one alternative implementation, the posterior segment disease includes at least one of choroidal or retinal inflammatory diseases, choroidal neovascularization-related diseases, and hereditary retinal degeneration.

[0050] The technical solution of this invention has the following advantages: This invention provides a method for screening drugs that can prevent and / or treat posterior segment diseases of the eye. The method includes: first, decompressing the anterior chamber of an experimental animal; then, pre-establishing a "perforation-closure" channel in the sclera of the experimental animal; then, injecting the drug to be screened into the suprachoroidal space of the experimental animal through the "perforation-closure" channel in the sclera; and finally, evaluating the effectiveness of the drug in preventing and / or treating posterior segment diseases based on changes in the ocular condition of the experimental animal. This invention prevents the reflux of injected AAV viral vectors by lowering intraocular pressure and creating a pre-closed scleral incision, reducing ocular surface inflammation caused by reflux, improving ocular condition, and enhancing the safety of the experimental method; at the same time, it allows AAV vectors carrying target genes or small nucleic acid drugs to express a wider range and for a longer duration, sustaining expression for more than 24 weeks. Studies have shown that injecting shRNA targeting the TGF-β2 gene using the method of this invention inhibits the progression of myopia in a guinea pig model. The method provided by this invention is beneficial for screening gene therapy drugs related to posterior segment diseases.

[0051] Furthermore, by injecting a retinal pigment epithelial (RPE)-specific AAV vector, the expression intensity is controllable, improving the targeting, effectiveness, and persistence of the injection. The AAV vector described in this invention carries the VMD2 promoter, a key sequence regulating the transcription of the VMD2 gene (encoding bestrophin protein). Its core characteristic is its RPE specificity, enabling highly specific and persistent expression in the RPE cell layer without relying on a strong broad-spectrum promoter.

[0052] Furthermore, this invention selects AAV8 as the viral capsid of the AAV vector, which, in synergy with the VMD2 promoter, enhances the transduction efficiency and specificity of guinea pig RPE cells.

[0053] Furthermore, the method described in this invention utilizes a parameterized suprachoroidal (SCS) injection protocol and quality control standards suitable for guinea pig microphthalmia to target RPE cells for gene delivery, achieving high reproducibility and low complications, and ensuring optimal efficacy and safety of the AAV vector. This invention standardizes the relevant parameters of anterior chamber decompression, pre-positioning of the "perforation-closure" channel, and suprachoroidal injection of the AAV vector into a standard procedure, establishing a "functional one-way valve effect" and "pre-reserved compliance" from a tissue mechanics and fluid dynamics perspective. This significantly reduces the backflow of the AAV viral vector, ensuring precise injection and retention of the drug solution in the suprachoroidal space. By employing a dual-needle strategy using a 29G insulin needle and a 34G microneedle, the approach and injection process are separated, resulting in greater precision. A 1.5-1.8 mm posterior approach to the limbus and a 10-20° needle angle to the scleral tangent define the geometric boundary, matching the scleral thickness and bulbar curvature of guinea pigs, reducing the risk of accidental intrusion into the vitreous humor or choroidal perforation. Quantitative anterior chamber decompression allows for compliance during small-volume injections, weakening the instantaneous IOP peak and reducing tissue mechanical stress and the risk of hemorrhage. Simultaneously, intraocular pressure is controlled to below 5 mmHg during anterior chamber decompression, at which point the injected medication will not reflux due to increased intraocular pressure. AAV is injected through a "perforation-closure" channel, with the needle remaining in place for 5-10 minutes after injection. The method utilizes Tenon / scleral rebound and pressure differential rebalancing to form a functional "one-way valve effect," significantly suppressing needle tract reflux and superficial extravasation, thus avoiding safety issues caused by AAV virus reflux. Precise control of AAV injection dosage and rate ensures posterior pole directional spreading and stable resistance. A verifiable layer confirmation and postoperative quality control system is established, using "grayish-white crescent-shaped directional elevation and surface gloss change" as a positive criterion for SCS entry, supplemented by OCT confirmation of the sclera-choroid separation zone, standardizing the microscopic signs of the drug injection process. Fixed follow-up after drug injection (0 h, 2-4 h, 24 h post-AAV injection) and collection of indicators (IOP, anterior segment / vitreous reaction, extravasation / reflux records) form a closed-loop quality control and batch-to-batch comparable data framework.

[0054] Using the drug screening method provided by this invention, the success rate of AAV vector injection via suprachoroidal injection reached over 95%, with improved targeting, covering 50-75% of the posterior pole. Simultaneously, it avoided drug reflux, reducing the immune response of the AAV viral vector and the occurrence of ocular surface inflammation. Studies showed that the EGFP gene carried by the AAV vector was continuously expressed for more than 24 weeks, improving the persistence of gene delivery. Furthermore, this invention exhibits high safety, with the incidence of synechiae, phthisis bulbi, and retinal detachment all less than 5%, and the probability of increased intraocular pressure after injection was reduced by approximately 30% compared to direct suprachoroidal injection. This invention, by injecting an AAV vector carrying the VMD2 promoter, combined with a parameterized SCS injection method for guinea pig microphthalmia and standardized quality control, provides high effective intraocular volume fraction, directional spreading, and reproducibility with low complications. The present invention provides a method for screening drugs that can prevent and / or treat posterior segment diseases of the eye. Targeting the characteristics of guinea pig eyes, it solves the problem of reliable drug delivery to guinea pig SCS, and simultaneously achieves efficient, specific and stable gene delivery to RPE cells. It provides a highly efficient, targeted, safe, persistent, and highly reproducible drug screening method, which can be effectively used for drug screening to prevent and / or treat choroidal neovascularization-related lesions and hereditary retinal degeneration, among other posterior segment diseases of the eye. Attached Figure Description

[0055] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0056] Figure 1 This is a schematic diagram of the anatomy of a guinea pig's eye; Figure 2 This is a map of the recombinant AAV8-VMD2-EGFP vector plasmid; Figure 3 These are images of EGFP fluorescence in the fundus of guinea pigs under different injection methods; Figure 3 In the table, (A) and (B) are fundus imaging results after intrachoroidal injection of AAV8-VMD2-EGFP vector in the experimental group; (C) is fundus imaging result after intravitreal injection of AAV8-VMD2-EGFP vector in the control group 3; and (D) is fundus imaging result after subretinal injection of AAV8-VMD2-EGFP vector in the control group 4. Figure 4 This is a diagram showing the modeling results of defocus myopia; Figure 4In the middle, (A) the results of axial length measurement of guinea pigs after 3 weeks of binocular negative lens induction; (B) the changes in body weight of guinea pigs after 3 weeks of binocular negative lens induction. Figure 5 This is a graph showing the axial length measurement results of guinea pigs after the TGF-β2 gene was knocked down; Figure 6 This is a diagram showing the changes in refractive power in guinea pigs after the TGF-β2 gene was knocked down. Detailed Implementation

[0057] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0058] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0059] The reagents, equipment, and instruments used in the embodiments of this invention are as follows: The AAV vector was purchased from Charles River, a commercial CDMO supplier; the guinea pigs were purchased from the National Rodent Laboratory Animal Seed Center (China National Institutes for Food and Drug Control). 1) Magnification and illumination: The surgical microscope (20-25×) was purchased from the ZEISS OPMI series, and the head-mounted illuminator was purchased from the HEINE ML4 / HRP headlamp system; 2) Microsurgical instruments: eyelid opener, toothed forceps / toothless forceps, microsurgical needle holder, and microsurgical scissors were all purchased from Geuder, Germany; 3) Injection system: 10 μL Hamilton microsyringe (with 34G microneedle, beveled needle tip) purchased from Hamilton Company (USA) 7000 series; 29G insulin needle purchased from BD Ultra-Fine series. 4) Anterior chamber puncture instruments: 15-degree puncture knife purchased from Alcon. 5) Intraocular pressure monitoring: The rebound tonometer was purchased from Icare (Finland) TonoLab / TonoVet series; 6) Hemostasis and irrigation: 3M medical sterile gauze, sterile cotton swabs, irrigation device; 7) Image confirmation: The handheld direct ophthalmoscope was purchased from Welch Allyn; 8) Suture material: Vicryl 8-0 absorbable sutures were purchased from Ethicon (Johnson & Johnson), along with a micro needle holder and suture cutter.

[0060] Example 1: A method for screening drugs that can prevent and / or treat posterior segment diseases of the eye. This embodiment provides a method for screening drugs that can prevent and / or treat posterior segment diseases (taking the screening of AAV carrier drugs as an example). The specific method is as follows (see the schematic diagram of the guinea pig eyeball anatomy). Figure 1 (as shown) 1. Animal preparation and baseline assessment (1) Prepare 3-week-old tricolor guinea pigs weighing 150-180 g, and number and weigh them; (2) Slit-lamp and indirect retinoscopy to screen for corneal / anterior chamber / vitreous / fundus abnormalities; (3) Intraocular pressure (IOP) measured by rebound tonometer (the average of 3 measurements in the same eye is recorded as T0).

[0061] 2. Anesthesia and local anesthesia, ocular surface protection (1) Anesthesia induction is performed using 3-4% (v / v) concentration of isoflurane, and anesthesia maintenance is performed using 1-2% (v / v) concentration of isoflurane; (2) Topical anesthesia: Use promecaine eye drops, once every 5 minutes, for a total of 3 times; (3) Use artificial tears to keep the corneal surface moist.

[0062] 3. Disinfection and exposure of the surgical area (1) Disinfect the skin with 5% (w / w) povidone-iodine and disinfect the conjunctival sac with 0.5% (w / w) povidone-iodine. After disinfection for 30 seconds, rinse with 0.9% (g / 100mL) physiological saline. (2) Place a guinea pig-specific eyelid opener to expose the surgical area above the eyeball (12 o'clock position).

[0063] 4. Conjunctival and Tenon treatment (1) Make a T-shaped conjunctival incision behind the limbus at the 12 o'clock position; (2) Bluntly dissect the Tenon tissue to expose the sclera and avoid visible blood vessels and tendon attachments.

[0064] 5. Anterior chamber decompression (with provisions for compliance) (1) Use a 15° corneal scalpel to make a micro-incision in the peripheral part of the cornea to enter the anterior chamber; (2) Slowly release 3-6 μL of aqueous humor to reduce IOP to below 5 mmHg; (3) Retest IOP after 1-2 minutes and record the result.

[0065] 6. Pre-installation of "perforated-closed" passage (1) Needles: 29G insulin needle with the bevel facing down; (2) Approach point: 1.5-1.8 mm behind the limbus, at the 12 o'clock position, avoiding blood vessels and tendons; (3) Angle: 10-20° with the tangent of the sclera; (4) Action: Slowly advance until a slight "breakthrough" sensation is felt, then enter the suprachoroidal space. Then withdraw slightly by 0.2-0.5 mm to promote the closure of the superficial needle channel, preserving the potential channel direction towards the posterior pole.

[0066] 7. AAV Loading and Air Management (1) Thaw the AAV vector and filter it using a 0.22 μm filter membrane to ensure it is pyrogen-free; control the proportion of solid particles of the AAV vector to be ≥70%, host DNA to be ≤10 ng / dose (or ≤100 pg / μg DNA) or host cell protein to be ≤100 ng / dose, and verify the shearing / ITR integrity by sequencing or qPCR.

[0067] (2) Using a 10 μL Hamilton syringe (connected with a 34G needle), draw 5 μL of AAV carrier solution (titer ≥ 1 × 10⁻⁶). 12 (vg / mL, 300±30 mOsm / kg, pH 6.8-7.6), gently push to the needle tip to form microdroplets, and expel the microbubbles.

[0068] 8. Suprachoroidal endovascular injection (fluid control) (1) Advance to the previous layer at the original angle of the channel preset in step 6, without breaking through the new tissue; (2) Inject 5 μL of AAV carrier solution at a constant rate, controlling the injection speed to be 0.7-0.8 μL / s; (3) Stop the injection when you observe “grayish-white crescent-shaped directional bulges” and changes in surface gloss, and the injection resistance is small and stable. (4) After injection, leave the needle in place for 5-10 seconds, then withdraw the needle very slowly.

[0069] 9. Reflux control and hemostasis If bleeding occurs, apply gentle pressure with a cotton swab for 10-20 seconds to stop the bleeding at a point away from the needle insertion site.

[0070] 10. Conjunctival closure and postoperative medication (1) Use Vicryl 8-0 absorbable sutures to suture with 1-2 interrupted stitches, with good edge alignment and no eversion; (2) Apply compound hormone eye ointment to the operated eye after surgery (2-3 times a day for 3 consecutive days).

[0071] 11. Immediate confirmation and follow-up records (1) Immediately: Directly examine the SCS strip-like bulges and record the approach direction and extent; (2) Follow-up: IOP was retested at 0 hours, 2-4 hours and 24 hours after surgery. Anterior segment and vitreous reaction scores, extravasation or reflux signs were recorded. When conditions permit, optical coherence tomography (OCT) was used to confirm scleral-choroidal interface separation.

[0072] 12. Management of Deviations and Complications (1) Accidental entry into the vitreous body: stop injection immediately, record the case and remove it from the main analysis, and make improvements in the future, such as reducing the angle or shortening the exposed length of the needle tip; (2) Backflow or leakage: If backflow or leakage occurs, make improvements, extend the needle retention time to 10 seconds, reduce the rate to 0.5-0.7 μL / s, and recheck the "penetration-closure" quality; (3) Abnormal intraocular pressure: If abnormal intraocular pressure occurs, retest after 2-4 hours. If necessary, intervention to lower intraocular pressure can be performed. Brimonidine eye drops can be used twice a day and retested every other day.

[0073] Experiment Example 1: Feasibility Verification Based on AAV-VMD2-EGFP Targeting RPE This experimental example verifies the feasibility of the method described in Example 1 by injecting recombinant AAV viral vector (AAV-VMD2-EGFP). The specific steps are as follows: 1. Construction of the AAV-VMD2-EGFP recombinant viral vector: Using a commercially available AAV vector (purchased from Charles River, a commercial CDMO supplier) as a template, the VMD2 promoter sequence and EGFP protein expression gene were inserted to construct the AAV-VMD2-EGFP recombinant viral vector; the plasmid map of the recombinant AAV viral vector AAV-VMD2-EGFP is shown below. Figure 2 As shown, the viral capsid serotype is AAV8; the nucleotide sequence of VMD2-EGFP is shown in SEQ ID NO.1. 2. An experimental group and a control group were set up. The injection method for the guinea pigs in the experimental group was the same as that in Example 1. The control group was as follows: Control group 1: Based on the experimental group, the injected recombinant AAV virus vector was replaced with PBS buffer, and all other conditions were the same.

[0074] Control Group 2: Twenty guinea pigs aged 2-3 weeks were anesthetized with isoflurane, and the surgical area was exposed. A 34G microneedle (connected to a 10 μL Hamilton microsyringe) was used to directly puncture the sclera into the suprachoroidal space approximately 1.5-2.0 mm posterior to the limbus at a near-vertical angle (approximately 80-90° angle with the scleral tangent), without anterior chamber decompression or pre-established "puncture-closure" channels. The AAV viral vector was the same as in the experimental group, with an injection volume of 5 μL and an injection rate of 1.0-1.5 μL / s. Postoperatively, a compound steroid eye ointment was applied to the operated eye (2-3 times daily for 3 days).

[0075] Control Group 3: Twenty guinea pigs aged 2-3 weeks were used for intravitreal injection. After isoflurane anesthesia and full mydriasis, a 33G microsyringe was used under surgical microscope to insert the needle into the vitreous cavity at an angle of approximately 45° towards the center of the eyeball, about 2 mm posterior to the limbus, avoiding contact with the lens. The AAV viral vector was the same as in the experimental group, with an injection volume of 5-10 μL and a slow injection rate (approximately 1-2 μL / s). After injection, the needle was slowly withdrawn, and the puncture site was gently pressed to prevent leakage. Post-injection fundus examination confirmed the absence of significant hemorrhage or retinal damage.

[0076] Control Group 4: Twenty 2-3 week old guinea pigs were anesthetized with isoflurane and their pupils were fully dilated. Subretinal injection was performed under surgical microscopy using a trans-scleral approach. The procedure was as follows: the sclera was exposed, and a 34G microsyringe was used to puncture the sclera and choroid 2 mm posterior to the limbus into the choroidal cavity. The puncture was then slowly advanced into the subretinal space opposite the puncture point. The AAV viral vector was the same as in the experimental group, with an injection volume of 5 μL, inducing local retinal detachment. After injection, the needle was slowly withdrawn, and the diffusion of the filtering bleb was observed. Post-injection, fundus examination confirmed the subretinal layer and recovery of the local detachment.

[0077] 3. Detection of EGFP gene expression in RPE cells (1) Imaging was performed using a small animal fundus imaging system (Phoenix Micron IV, equipped with a long-wavelength excitation fluorescence module). The injected AAV-VMD2-EGFP recombinant viral vector carried the EGFP gene as an indicator protein. After injection, the guinea pigs in the experimental and control groups were kept under anesthesia, and compound tropicamide eye drops were used to ensure that the pupils were fully dilated. The anesthetized guinea pigs were fixed in a lateral decubitus position on the imaging platform (injection eye facing upward), coupling agent (0.9% (g / 100mL) physiological saline) was applied to the corneal surface, and the imaging lens was gently placed close to the corneal surface and the focus was adjusted until the retina was clear.

[0078] (2) First, color fundus photographs were acquired in white light mode to record the distribution of the optic disc, blood vessels, and the overall morphology of the retina. Then, the image was captured using long-wavelength excitation light (ICG mode: excitation light 790nm, emission light 835nm) in fluorescence mode. Fluorescence distribution was observed and recorded in real time at time points immediately after injection (0-5 minutes), 15 minutes, 30 minutes, 1 hour, and 24 hours. Images of the posterior pole and peripheral retina were acquired with the optic disc as the center. The distribution pattern, range, boundary clarity, and spatiotemporal changes of the fluorescence signal were observed, as well as the presence of subretinal fluid accumulation, detachment, leakage, regurgitation, or vascular staining. Delivery efficiency was assessed by measuring the area of ​​high-signal fluorescence regions and evaluating the uniformity and directionality of signal distribution. Regurgitation and extravasation were assessed by checking for abnormal fluorescence signals in the subconjunctival, intravitreal, and retinal vessels. ImageJ or dedicated software was used to quantify the fluorescence area and intensity for data analysis.

[0079] Experimental results: The results are as follows Figure 3 As shown. Figure 3 In Figures (A) and (B), no significant retinal detachment or flame-shaped hemorrhage was observed in the experimental group of guinea pigs. A band-shaped high-signal area with clear boundaries was visible at the posterior pole of the optic disc, consistent with the predetermined suprachoroidal space approach, suggesting that the recombinant AAV viral vector spread directionally along the inner surface of the sclera within the suprachoroidal space. The high-signal area contrasted clearly with the surrounding background, and no patchy leakage or large areas of high-reflectivity shadows were observed, indicating concentrated layering and limited extravasation. No linear leakage signs consistent with the course of blood vessels were observed (excluding retinal vascular leakage as the primary cause). Figure 3 As shown in Figure (C), fundus imaging of guinea pigs in control group 3 showed no EGFP fluorescence signal, indicating restricted posterior transfection; Figure 3 As shown in Figure (D), fundus imaging of guinea pigs in control group 4 showed no obvious fluorescence signal, with a signal coverage area of ​​<10%.

[0080] 4. Detection of transfection efficiency and ocular inflammation-related indicators (1) The guinea pigs were subjected to systematic eye examination using a slit-lamp microscope and a handheld indirect ophthalmoscope. The experimental group and the control group were kept under anesthesia at key time points such as 0 hours, 2-4 hours and 24 hours after the operation, and the pupils were fully dilated with compound tropicamide eye drops. (2) Fix the guinea pig on the examination table and adjust the slit lamp to an appropriate magnification (10-16×). First, use diffuse light illumination mode to observe the anterior segment of the eye, assess the degree of conjunctival hyperemia, corneal transparency, whether there is edema or epithelial defects, whether the anterior chamber depth is stable, and whether the aqueous humor is clear. Carefully observe whether there is aqueous humor flare (Tyndall phenomenon), fibrinous exudate, hypopyon or hemopyon. At the same time, check the iris morphology, pupillary light reflex and lens transparency. (3) Next, widen the slit lamp beam and focus it on the anterior part of the vitreous body to observe whether the vitreous cavity is clear, whether there are inflammatory cells, cord-like opacities, hemorrhage or foreign bodies; (4) Then, using a handheld indirect ophthalmoscope in conjunction with a head-mounted illuminator or slit lamp light source, examine the fundus through a fully dilated pupil, systematically observe the color, boundary and course of blood vessels of the optic disc, whether the retina is flat, whether there is hemorrhage (including fluffy, flame-shaped, and round hemorrhage), exudation, edema or detachment, whether the macular reflex is normal, and whether the choroidal contour is flat, whether there are signs of bulging or detachment. (5) Record and photograph the examination results at each time point in detail, and use a standardized scoring system to assess the severity of anterior segment inflammation (0-4 grade) and fundus pathological changes to ensure comparability between different time points and different individuals.

[0081] Experimental Results: The results are shown in Tables 1-3. As shown in Table 1, the success rate of direct injection into the suprachoroidal space in control group 2 was low, only about 30% (6 eyes successfully entered the SCS layer, confirmed by ophthalmoscopy with grayish-white bulges and subsequent histological examination). The main reasons for failure were severe reflux (approximately 50% of eyes showed obvious needle leakage and subconjunctival effusion) and accidental entry into the vitreous cavity (approximately 20%). In addition, there was a high level of ocular surface inflammation, with approximately 40% of eyes showing moderate conjunctival hyperemia and anterior segment inflammation, and some eyes showing a tendency for mild symblepharon. Although this method is relatively simple to operate, the lack of decompression compliance and functional one-way valve effect leads to large dose dispersion, uncertain layer location, and low safety, failing to achieve reliable and reproducible guinea pig SCS gene delivery.

[0082] As shown in Table 2, control group 3 was administered via intravitreal injection. This method is relatively simple to perform, has a high success rate (nearly 100%), and few complications. However, because the AAV carrier mainly diffuses within the vitreous cavity, its transduction or delivery efficiency to the outer retina, especially retinal pigment epithelial cells (RPE), is extremely low due to the limitations imposed by the vitreoretinal barrier and vitreous clearance dynamics. This makes it difficult to achieve specific high expression or sufficient exposure of RPE cells, and thus cannot effectively meet the requirements for drug screening targeting RPE.

[0083] As shown in Table 2, control group 4 underwent subretinal injection. Although this method can achieve high local transduction efficiency near the injection point, the overall success rate is low (20%, meaning only about 4 eyes achieved effective subretinal delivery) due to the limitation of the guinea pig's eyeball, which prevents direct visualization of the injection point. The main reasons for failure are the difficulty in judging the needle tip position, which easily leads to accidental entry into the vitreous cavity, suprachoroidal space, or direct scleral perforation; even if the injection is successful, the infection / expression range is very limited, confined to the local area formed by the injection (usually covering <20%-30% of the posterior pole, forming an isolated "island" expression area), making it difficult to achieve extensive and uniform coverage of the posterior pole; in addition, this method is very likely to cause local retinal detachment, hemorrhage, inflammation, or permanent tissue damage.

[0084] As shown in Table 3, no local irritation was caused by PBS injection into the suprachoroidal space in control group 1, indicating that the provided injection method has a certain degree of safety.

[0085] In summary, taking the injection of the recombinant viral vector AAV8-VMD2-EGFP as an example, the experimental group achieved a success rate of over 95% by injecting the AAV vector into the suprachoroidal space. The injection targeting was improved, covering 50%-75% of the posterior pole. Simultaneously, it avoided drug reflux, reducing the immune response of the AAV viral vector and the occurrence of ocular surface inflammation. Studies showed that the EGFP gene carried by the AAV vector was continuously expressed for over 24 weeks, improving the persistence of gene delivery. Furthermore, this invention exhibits high safety, with the incidence of synechiae, ocular atrophy, and retinal detachment all less than 10%. The probability of increased intraocular pressure after injection was reduced by approximately 30% compared to direct suprachoroidal injection (control group 2). By injecting the AAV vector carrying the VMD2 promoter, combined with a parameterized SCS injection method targeting guinea pig microphthalmia and standardized quality control, the experimental group provided high effective intraocular volume fraction, directional spreading, and reproducibility with low complications.

[0086] Table 1. Comparison of injection effects between the experimental group and the control group 2

[0087] Table 2. Comparison of the effects of intravitreal injection, subretinal injection, and suprachoroidal injection.

[0088] Table 3. Local irritation caused by SCS procedure in control group 1.

[0089] Experimental Example 2: Feasibility Validation of RPE Targeting Based on AAV8-VMD2-TGF-β2 shRNA This experimental example verifies the effectiveness of the method described in Example 1 by further injecting the gene therapy drug (AAV8-VMD2-TGF-β2 shRNA). The specific steps are as follows: 1. shRNA synthesis A shRNA sequence was designed targeting the TGF-β2 gene. The nucleotide sequence of the TGF-β2-shRNA is shown in SEQ ID NO.2. The target gene TGF-β2 is located in the q41 region of chromosome 1 (NC_000001.11: 218,345,336..218,444,619), and its Ensembl database number is ENSCPOG00000007647.

[0090] 2. Construction of AAV8-VMD2-TGF-β2 shRNA recombinant viral vector Based on the recombinant AAV viral vector AAV-VMD2-EGFP, as follows Figure 2 The target site of the plasmid map shown is inserted with the TGF-β2-shRNA sequence synthesized in step 1 to construct the AAV8-VMD2-TGF-β2 shRNA recombinant viral vector.

[0091] 3. Construction of the posterior segment animal model (1) A negative lens-induced defocus myopia (LIM) model was used. Negative lenses with a diopter of -30D (D) were fixed in front of the left and right eyes of the experimental group guinea pigs (3-week-old healthy spotted guinea pigs). The negative lenses were made of polymethyl methacrylate (PMMA) and had a diameter of 15.0 mm. The lenses were attached to the edge of the guinea pig's eye socket with medical tape to ensure that the guinea pig could open and blink freely while wearing them. (2) During the experiment, check the wearing position, center positioning and surface smoothness of the lens every morning; if the lens falls off, is misaligned or contaminated, adjust or replace the lens. (3) Continue induction for 3 weeks, remove the lens at a fixed time each week, and perform ocular biometrics and fundus imaging on the animals, and then put it back on; (4) Verify whether the model has been successfully constructed by detecting the axial length and refractive power of the guinea pigs. If the axial length and refractive power of the experimental group are significantly different from those of the control group, it means that the model has been successfully constructed. The specific operation is as follows in step 5.

[0092] 4. Suprachoroidal injection The AAV viral vector was injected into the suprachoroidal space, as described in Example 1. The experimental group was the guinea pig model of negative lens-induced defocus myopia constructed in step 3 above. Recombinant AAV viral vector (AAV8-VMD2-TGF-β2 shRNA) was injected into the suprachoroidal space to target RPE knockdown of TGF-β2 gene expression; the control group was injected with the same type of AAV viral vector carrying the Scramble sequence.

[0093] 5. Validation of the posterior segment animal model (1) Measurement of axial length: After local anesthesia of the guinea pig model with 0.5% promecaine hydrochloride, the axial length of the guinea pig's eye was measured using an ophthalmic ultrasound diagnostic instrument (A-mode ultrasound; probe frequency: 11MHz). The sound velocity settings were as follows: cornea and aqueous humor 1557.5m / s, lens 1723.3m / s, vitreous cavity 1540m / s. The measurement results were the average of 5 independent operations. Axial length is defined as the distance from the corneal apex to the inner limiting membrane of the retina.

[0094] (2) Refractive power measurement: The refractive status was measured in a dark room using a strip retinoscope. Fifteen minutes before the retinoscopy, compound tropicamide eye drops (0.5% tropicamide / 0.5% phenylephrine hydrochloride) were administered three times (5 minutes apart) to fully dilate the pupils. The refractive power in the horizontal and vertical axes was recorded and the average value was taken. The average of the three measurements was then used.

[0095] Experimental results: The results are as follows Figures 4-6 As shown, compared with the negative control group guinea pigs, the axial length of guinea pigs wearing negative lenses in both eyes (NLIAE) increased by 0.23±0.04 mm after 3 weeks of induction (P<0.0001). Figure 4 China A and Figure 5 Increased refractive power ( Figure 6 While axial elongation was observed, negative lens wearing did not affect the weight of guinea pigs (P>0.2 at all time points). Figure 4 (B). The above indicates that the posterior segment animal model was successfully constructed.

[0096] 6. Targeted RPE knockdown of TGF-β2 inhibits myopia progression The methods for measuring axial length and detecting refractive error are the same as those described in step 5 above.

[0097] Experimental results: The results are as follows Figure 5 and Figure 6 As shown. Figure 5The results showed that the axial length elongation was significant. At baseline, the axial length of all groups remained around 8.00±0.02 mm, with no statistically significant difference between groups (P>0.05). After 5 weeks of induction intervention, the axial length of the defocus myopia group (LIM) rapidly increased from baseline to 8.86±0.03 mm, significantly higher than the 8.40±0.01 mm of the normal control group, confirming the successful induction of the guinea pig myopia model by defocus stimulation. In contrast, the experimental group receiving AAV8-VMD2-TGF-β2 shRNA injection into the suprachoroidal space had an axial length of only 8.65±0.03 mm at week 5. These results indicate that AAV-mediated VMD2 promoter-specific knockdown of the TGF-β2 gene in the RPE layer can effectively inhibit defocus-induced rapid axial elongation, demonstrating that gene intervention targeting the RPE layer is an effective means to delay the progression of high myopia.

[0098] Figure 6 The results showed refractive error drift. At baseline, all animals were hyperopic, with a mean refractive error of approximately +3.44 ± 0.42 D, and there was no statistically significant difference between groups (P > 0.05). After 5 weeks of induction intervention, the refractive error of the defocus myopia group decreased from baseline to -5.25 ± 0.80 D, demonstrating significant myopic drift and confirming that defocus stimulation successfully induced a high myopia model. In contrast, the experimental group receiving AAV8-VMD2-TGF-β2 shRNA injection into the choroidal space had a refractive error of only -2.25 ± 0.80 D at week 5, with a much lower degree of myopia progression than the defocus-only group. These results indicate that AAV-mediated VMD2 promoter-specific knockdown of the TGF-β2 gene in the RPE layer can significantly delay the progression of defocus-induced refractive errors, further demonstrating from a functional perspective that gene intervention targeting the RPE layer is an effective means of preventing and treating high myopia.

[0099] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for screening drugs capable of preventing and / or treating posterior segment diseases of the eye, characterized in that, The method includes: first, decompressing the anterior chamber of the experimental animal; then, pre-establishing a "perforation-closure" channel in the sclera of the experimental animal; then, injecting the drug to be screened into the suprachoroidal space of the experimental animal through the "perforation-closure" channel in the sclera of the experimental animal; and finally, evaluating the effectiveness of the drug to be screened in preventing and / or treating posterior segment diseases based on changes in the eye condition of the experimental animal.

2. The method according to claim 1, characterized in that, The procedure for decompressing the anterior chamber of experimental animals includes: using a corneal scalpel to make a micro-incision in the peripheral part of the cornea of ​​the experimental animal to enter the anterior chamber, so that the aqueous humor in the anterior chamber of the experimental animal is slowly released until the intraocular pressure (IOP) drops to below 5 mmHg; The method of pre-establishing a "perforation-closure" channel in the sclera of experimental animals includes: using an insulin needle to make an incision 1.5-1.8 mm behind the limbus of the experimental animal and performing puncture, so that a pre-established "perforation-closure" channel is formed in the sclera of the experimental animal; when the insulin needle is punctured, the angle between the needle and the tangent of the sclera of the experimental animal is controlled at 10-20°.

3. The method according to claim 1 or 2, characterized in that, The experimental animals were either normal experimental animals or animal models of posterior segment diseases. Optionally, the posterior segment disease animal model includes at least one of the following: negative lens-induced defocus myopia model, age-related macular degeneration model, diabetic retinopathy model, retinitis pigmentosa model, choroidal ischemia model, ischemic optic neuropathy model, and glaucoma model. Optionally, the posterior segment disease animal model includes a negative lens-induced defocus myopia model.

4. The method according to claim 3, characterized in that, The experimental animals include normal guinea pigs or animal models of posterior segment diseases obtained using guinea pigs.

5. The method according to any one of claims 1-4, characterized in that, The drugs to be screened include drugs used for the prevention and / or treatment of posterior segment diseases of the eye; Optionally, the drug includes at least one of an anti-vascular endothelial factor (VEGF) drug, a complement factor inhibitor, and a gene therapy drug; Optionally, the gene therapy drug includes at least one of drugs that prevent and / or treat posterior segment diseases by expressing a target gene and drugs that prevent and / or treat posterior segment diseases by inhibiting a target gene; Optionally, the drugs that prevent and / or treat posterior segment diseases by inhibiting target genes include small nucleic acid drugs; Optionally, the small nucleic acid drug includes at least one of siRNA, miRNA, and shRNA; Optionally, the small nucleic acid drug targets myopia-related genes; Optionally, the myopia-related genes include transforming growth factor TGF-β2.

6. The method according to claim 5, characterized in that, The gene therapy drug is loaded onto a gene delivery vector; Optionally, the gene delivery vector includes at least one of an AAV vector and a lentiviral vector; Optionally, the AAV vector carries a cell-specific promoter; Optionally, the cell-specific promoter includes the VMD2 promoter, which targets retinal pigment epithelial cells (RPE).

7. The method according to claim 6, characterized in that, The viral capsid of the AAV vector includes at least one of AAV8, AAV2.7m8, and AAV8-BP2; Optionally, the viral capsid of the AAV vector is AAV8.

8. The method according to claim 6 or 7, characterized in that, The injection dose of the AAV carrier is 10. 9 vg-10 10 vg / unit; the injection rate of the AAV carrier is 0.5-1.0 μL / s; Optionally, the injection rate of the AAV vector is 0.7-0.8 μL / s.

9. The method according to any one of claims 1-8, characterized in that, The needle is left in place for 5-10 seconds after the drug to be screened is injected. After the needle is left in place, the conjunctiva is closed by suturing.

10. The use of the method according to any one of claims 1-9 in screening for drugs capable of preventing and / or treating posterior segment diseases of the eye.

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