Application of NCOA3 polyQ structural domain as target spot in preparation of medicine for relieving eye abnormal hyperplasia diseases
By targeting the NCOA3 polyQ domain, inhibiting its function and blocking its mediated angiogenesis, drugs to alleviate ocular proliferative diseases were developed. This solved the problem of insufficient targets in existing technologies and achieved significant reduction in corneal neovascularization and downregulation of gene expression.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANTONG UNIV
- Filing Date
- 2026-02-11
- Publication Date
- 2026-05-12
Smart Images
Figure CN122005802A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biomedical technology, and in particular to the application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate ocular proliferative diseases. Background Technology
[0002] Ocular vascular proliferative disorders are a general term for diseases characterized by abnormal angiogenesis, dilation, or leakage in the intraocular or ocular surface tissues, affecting the cornea, conjunctiva, iris, retina, and choroid. Normally, the cornea is avascular to maintain transparency and refractive function. When factors such as infection, inflammation, trauma, hypoxia, and immune responses disrupt the corneal microenvironment, limbal vessels can invade the central cornea, forming neovascularization. This leads to corneal edema, opacity, lipid deposition, and scarring, resulting in decreased vision or even blindness, and significantly reducing the success rate of corneal transplantation. Besides the cornea, pathological angiogenesis in the fundus is also a significant cause of irreversible visual impairment. Conditions such as diabetic retinopathy, retinopathy of prematurity, and retinal vein occlusion are often accompanied by ischemic and hypoxic-driven abnormal vascular proliferation and leakage. Wet age-related macular degeneration is characterized by choroidal neovascularization, which is prone to leakage and hemorrhage, damaging the macular structure and causing a rapid decline in central vision. The aforementioned diseases are often characterized by insidious, progressive, and recurrent nature. Once bleeding, exudation, or fibrotic scarring occurs, they often cause long-term or even permanent visual impairment. Therefore, inhibiting pathological angiogenesis is an important direction for ophthalmic treatment and blindness prevention.
[0003] Pathological angiogenesis is driven by multiple factors and processes, involving endothelial cell proliferation and migration, basement membrane degradation, abnormal pericyte coverage, inflammatory cell infiltration, and extracellular matrix remodeling. The VEGF / VEGFR pathway plays a crucial role in various ocular neovascular diseases. Anti-VEGF drugs are widely used in wet age-related macular degeneration and diabetic macular edema, but there are still issues with efficacy and tolerability, the need for repeated intravitreal injections leading to risks of infection / bleeding / intraocular pressure elevation, and compliance problems. Furthermore, long-term inhibition of VEGF may affect normal vascular homeostasis and neuroprotection. In ocular surface diseases such as corneal neovascularization, there are also shortcomings in delivery, sustained action, and recurrence control. In addition to VEGF, inflammatory mediators such as TNF-α, IL-1β, and IL-6, as well as signaling pathways such as Notch, Ang / Tie, and HIF, also participate in pathogenesis. Single pathway intervention often fails to achieve long-term stable efficacy, and there is an urgent need to find more upstream regulatory targets. Summary of the Invention
[0004] The purpose of this application is to address the problems of insufficient target points and lack of treatment methods for pathological angiogenesis in the middle eye in the existing technology.
[0005] To solve the above-mentioned technical problems, this application provides the following technical solution:
[0006] Application of NCOA3 polyQ domain as a target in the preparation of drugs to alleviate ocular proliferative diseases.
[0007] Preferably, the drug is used to inhibit the function of the NCOA3 polyQ domain and block the angiogenesis-related effects mediated by the NCOA3 polyQ domain.
[0008] Preferably, the ocular proliferative disorders include corneal pathological proliferative disorders.
[0009] Preferably, the ocular proliferative disorders include diabetic retinopathy, early-onset thermal retinopathy, and retinal vein occlusion, which are often accompanied by ischemic and hypoxic-driven abnormal vascular proliferation and leakage.
[0010] This application also provides a drug for alleviating ocular abnormal proliferative disorders, the drug being used to inhibit the function of the NCOA3 polyQ domain and block the angiogenesis-related effects mediated by the NCOA3 polyQ domain.
[0011] Preferably, the drug comprises at least one of a nucleic acid intervention agent targeting the NCOOA3 polyQ domain, a gene editing intervention system, and a small molecule compound.
[0012] Preferably, the drug also includes other medically acceptable adjuvants.
[0013] Compared with the prior art, this application has the following beneficial effects:
[0014] This application reveals for the first time the direct association between the NCOA3 polyQ domain and pathological angiogenesis in ocular vascular dysplasia. Functional evidence from mice lacking the Ncoa3 polyQ domain demonstrates that polyQ domain deficiency significantly reduces corneal pathological neovascularization, providing a new direction for developing ophthalmic anti-angiogenic targeted drugs against the NCOA3 polyQ domain. Specifically, in an adult mouse corneal micropocket model, numerous neovascularizations were observed growing from the limbus towards the microspheres in WT mice, while Ncoa3... wt / △Q Reduced neovascularization at the limbus of the mouse cornea, and Ncoa3 △Q / △Q The mouse cornea showed the least amount of neovascularization; whole-corneal CD31 immunofluorescence revealed Ncoa3 wt / △Q Decreased CD31 positive signal in mouse corneal tissue, and Ncoa3 △Q / △QThe smallest CD31-positive area was observed in mice, an indicator that directly reflects the density and extent of neovascularization, demonstrating that the loss of the polyQ domain in NCOOA3 can improve corneal neovascularization burden at the tissue level. Furthermore, corneal tissue qPCR analysis showed that, compared to WT mice, the mRNA expression of vascular marker genes Pecam1 and Cdh5 was significantly lower in NCOOA3. wt / △Q Decreased in mice, and in Ncoa3 △Q / △Q Further downregulation was observed in mice. These results, at the macroscopic, tissue, and molecular levels, collectively support the NCOA3 polyQ domain as a crucial structural basis for regulating corneal pathological angiogenesis, and suggest its potential clinical value as a drug development target. Attached Figure Description
[0015] Figure 1 This is a slit-lamp image (A) of the cornea taken on the 7th day after surgery in a mouse corneal micropocket model according to an embodiment of the present invention; wherein the control group is WT mice and the experimental group is Ncoa3 mice. wt / △Q Mice and Ncoa3 △Q / △Q Mice; In WT mice, numerous neovascularizations were observed growing from the limbus towards the granulation zone in the cornea, while Ncoa3... wt / △Q Reduced blood vessels at the limbus of the mouse cornea, and Ncoa3 △Q / △Q The mouse cornea has the fewest neovascularizations.
[0016] Figure 2 This is an example of a whole-corneal CD31 immunofluorescence map (A) and a statistical map of the area of CD31-positive regions (B) in one embodiment of the present invention; compared with WT mice, Ncoa3 wt / △Q Decreased CD31 positive signal in mouse corneal tissue, and Ncoa3 △Q / △Q The area of CD31-positive corneal tissue in mice was further reduced.
[0017] Figure 3 This is a diagram showing the results of qPCR detection of corneal tissue vascular marker gene expression in one embodiment of the present invention. Figure (A) shows the Pecam1 mRNA expression results, and Figure (B) shows the Cdh5 mRNA expression results: compared with WT mice, Ncoa3... wt / △Q The expression of Pecam1 and Cdh5 mRNA was decreased in mouse corneal tissue, and also decreased in Ncoa3. △Q / △Q Further downregulation was observed in mice. Detailed Implementation
[0018] This application provides the use of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate ocular proliferative diseases, wherein the drugs are used to inhibit the function of the NCOA3 polyQ domain and block the angiogenesis-related effects mediated by the NCOA3 polyQ domain.
[0019] The ocular proliferative disorders include corneal pathological diseases.
[0020] Preferably, the ocular proliferative disorders include diabetic retinopathy, early-onset thermal retinopathy, and retinal vein occlusion, which are often accompanied by ischemic and hypoxic-driven abnormal vascular proliferation and leakage.
[0021] Furthermore, based on the above applications, this application also provides a drug for alleviating ocular abnormal proliferative disorders, wherein the drug is used to inhibit the function of the NCOA3 polyQ domain and block the angiogenesis-related effects mediated by the NCOA3 polyQ domain.
[0022] In one embodiment, the drug comprises at least one of a nucleic acid intervention agent targeting the NCOA3 polyQ domain, a gene editing intervention system, and a small molecule compound.
[0023] The drug also includes other medically acceptable adjuvants.
[0024] The above content will be explained in conjunction with specific verification experiments:
[0025] I. Experimental Materials and Sources
[0026] 1. Matrigel matrix was purchased from Corning.
[0027] 2. The body dissecting microscope was purchased from Olympus Corporation, Japan;
[0028] 3. The laser confocal microscope was purchased from Leica GmbH, Germany;
[0029] 4. The RT-PCR instrument was purchased from Thermo Fisher Scientific, USA;
[0030] 5. Rat Anti-Mouse CD31 was purchased from BD.
[0031] 6. The slit-lamp microscope was purchased from Suzhou Liuliu Vision Technology Co., Ltd.
[0032] II. Experimental Conclusions
[0033] In this application, an Ncoa3 polyQ domain-deficient mouse model was prepared using an embryonic stem cell homologous recombination gene targeting strategy (WT and Ncoa3 were screened to obtain the model). wt / △Q Ncoa3 △Q / △Based on the three types of experimental mice, a mouse corneal micropocket pathological angiogenesis model was constructed. Subsequently, experiments were carried out in sequence, including mouse corneal micropocket model construction, whole corneal CD31 immunofluorescence experiment, corneal tissue RNA extraction and real-time quantitative PCR (qPCR) experiment. The detection results of the three types of experiments and the corresponding figures were combined to verify the core regulatory role of the NCOA3 polyQ domain in ocular pathological angiogenesis.
[0034] Specifically, this application established a corneal pathological angiogenesis model using a mouse corneal micropocket model. On the 7th day post-surgery, the gross morphology of the cornea was observed and photographed using a slit lamp. Figure 1 This study verified that the loss of the Ncoa3 polyQ domain significantly inhibited the overall formation of corneal neovascularization, and the inhibitory effect was positively correlated with the degree of domain loss. Results showed that numerous neovascularizations growing from the limbus towards the microspheres were observed in the cornea of WT mice, indicating that Ncoa3... wt / △Q Neovascularization was significantly reduced at the limbus of the mouse cornea, while Ncoa3... △Q / △Q The amount of neovascularization in the cornea of the mice was the lowest among the three mouse types.
[0035] The corneal tissue of model mice was stained with CD31 immunofluorescence throughout the cornea, and the area of the positive region was quantified. Figure 2 This study validated that the loss of the Ncoa3 polyQ domain can reduce the density and extent of corneal neovascularization at the tissue level, directly improving the corneal neovascularization burden. Results showed that, compared with WT mice, Ncoa3... wt / △Q CD31 positive signal was significantly reduced in mouse corneal tissue, Ncoa3 △Q / △Q The area of CD31-positive corneas in mice was further reduced.
[0036] The mRNA expression levels of vascular marker genes Pecam1 and Cdh5 in corneal tissue were detected by extracting RNA from corneal tissue and performing real-time quantitative PCR. Figure 3 A represents Pecam1, and B represents Cdh5), verifying that the deletion of the Ncoa3 polyQ domain can downregulate the expression of genes related to pathological angiogenesis in the eye at the molecular level. The results showed that, compared with WT mice, Ncoa3... wt / △Q The mRNA expression of Pecam1 and Cdh5 in mouse corneal tissue showed a significant decreasing trend, and the expression levels of these two genes were significantly lower in Ncoa3. △Q / △Q It was further downregulated in mice.
[0037] III. Some Experimental Steps
[0038] Experiment 1: Construction of a mouse corneal micropocket model
[0039] (1) Preparation of microspheres: Mix pre-melted matrigel with DMEM culture medium at a ratio of 1:1 on ice, and add 50 U / mL Heparin at the same time. Drop 10 μL / sphere onto aluminum foil, place at -20℃, and after it becomes gel-like, use fine tweezers to roll it into particles of about 1 mm, and store at -20℃ for later use;
[0040] (2) Weigh the mice, anesthetize them with 0.01 mL / g 1% sodium pentobarbital, and then place the anesthetized mice under a stereomicroscope and instill promecaine into the eyeballs for surface anesthesia.
[0041] (3) Gently squeeze the mouse’s eye socket with forceps to make the eyeball protrude. Make a linear incision in the corneal stroma with a scalpel. Insert the tip of the forceps vertically along the incision and carefully separate the corneal layer from the anterior chamber to form a wedge-shaped bag of about 2 mm.
[0042] (4) Gently pick up the prepared microspheres with sharp tweezers and place them on the surface of the eyeball near the incision. Use a tissue needle to slowly push the microspheres into the microcapsules.
[0043] (5) Apply erythromycin eye ointment to the surface of the mouse eyeball and carefully transfer the mouse into a warming box preheated to 36°C to wait for it to wake up; 7 days after the operation, use a slit lamp to photograph the cornea and observe the angiogenesis.
[0044] Experiment 2: Whole corneal CD31 immunofluorescence
[0045] (1) The mice were euthanized by cervical dislocation, the eyeballs were quickly removed and washed in pre-cooled PBS, the cornea was removed under a stereomicroscope while the limbus was preserved, and the cornea was transferred to 4% PFA for overnight fixation.
[0046] (2) Remove 4% PFA and rinse three times with pre-cooled PBS on a shaker for 20 min each time;
[0047] (3) Transfer the cornea to 1 mL of proteinase K solution (20 μg / mL, diluted with 10 mM pH7.4 Tris-HCl) and digest at room temperature for 5 min;
[0048] (4) Transfer to 1 mL of 100% methanol and allow to permeate at room temperature for 30 min;
[0049] (5) Remove methanol and wash three times with TBST (0.3% Triton X-100) for 30 min each time;
[0050] (6) Add 3% BSA prepared by TBST and seal at room temperature for 2 h;
[0051] (7) Add primary antibody Rat anti-CD31 (BD, 1:100) and incubate overnight at 4°C;
[0052] (8) Perform TBST cleaning 3 times, 10 minutes each time;
[0053] (9) Add secondary antibody Goat anti-Rat 488 (Jackson Immuno Research, 1:1000) and incubate at room temperature in the dark for 2 h;
[0054] (10) TBST cleaning 3 times, 20 min each time;
[0055] (11) Place the cornea in a pre-cooled PBS culture dish, cut it into four pieces, transfer it to a glass slide, and seal the slide with the cornea cup facing down;
[0056] (12) Seal the nail polish edges, store at 4°C in the dark, and take pictures under a fluorescence microscope.
[0057] Experiment 3: Corneal tissue RNA extraction and real-time quantitative PCR (qPCR)
[0058] 1) Tissue RNA extraction
[0059] (1) Weigh 50-100 mg of corneal tissue into an enzyme-free abrasive tube, cut it on ice, add 2-3 enzyme-free abrasive beads, add 1 mL of Trizol and grind for 5 min, and then transfer the lysis buffer to a new enzyme-free centrifuge tube.
[0060] (2) Let stand at room temperature for 15 min;
[0061] (3) Add 200 μL of chloroform, shake vigorously for 30 s, and let stand in an ice bath for 10 min;
[0062] (4) Centrifuge at 12,000 rpm for 15 min at 4℃, and transfer about 500 μL of the upper aqueous phase to a new EP tube;
[0063] (5) Add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 min;
[0064] (6) Centrifuge at 4℃ and 12000 rpm for 15 min, then discard the supernatant;
[0065] (7) Wash with pre-cooled 75% ethanol, centrifuge at 7500 rpm for 5 min at 4°C, and repeat once;
[0066] (8) Discard the supernatant, air dry at room temperature until the precipitate is clear, and add 20-50 μL of DEPC water to dissolve the RNA;
[0067] (9) Determine RNA concentration and purity.
[0068] 2) Tissue RNA extraction
[0069] (1) Follow the instructions of the Vazyme reverse transcription kit: calculate the volume required for 1 μg of RNA based on the RNA concentration;
[0070] (2) Prepare a 0.2 mL enzyme-free centrifuge tube, add the volume corresponding to 1 μg RNA, 4 μL 4×g DNAwiper Mix, and then add RNase-free ddH2O to make up to 16 μL. Mix gently with a pipette and incubate in a 42°C water bath for 2 min.
[0071] (3) After the water bath, remove the centrifuge tubes and add 4 μL of 5×HisScript II qRT SuperMix II to each tube. Mix gently with a pipette.
[0072] (4) Place the centrifuge tube into the PCR instrument and set the reverse transcription reaction conditions as follows: 50 °C for 15 min, 85 °C for 5 s;
[0073] (5) After the reverse transcription reaction is completed, store the cDNA at -20°C for later use.
[0074] 3) qPCR detection
[0075] qPCR system:
[0076]
[0077] The primer sequences are as follows:
[0078]
[0079] In summary, this application validates the NCOA3 polyQ domain as a crucial structural basis for regulating ocular pathological angiogenesis. Its functional loss significantly reduces the burden of corneal neovascularization (manifested as reduced neovascularization under slit-lamp examination and a decrease in the total area of CD31-positive cornea), and downregulates the mRNA expression of vascular endothelial-related marker genes Pecam1 and Cdh5 at the molecular level. This in vivo evidence clearly establishes the key association between the NCOA3 polyQ domain and the pathological angiogenesis process in the development of ocular vascular dysplastic diseases, providing a theoretical basis and experimental support for the development of ophthalmic anti-angiogenic therapeutics targeting the NCOA3 polyQ domain. Therefore, the intervention strategy targeting the NCOA3 polyQ domain proposed in this application can be used to prepare ophthalmic therapeutic agents (e.g., small molecule compounds, peptides / proteins, nucleic acid intervention drugs such as siRNA / antisense oligonucleotides, and gene editing / gene regulation intervention systems, etc.) with the potential to provide new and transformative therapeutic directions for ocular vascular dysplastic diseases such as corneal neovascularization.
Claims
1. Application of NCOA3 polyQ domain as a target in the preparation of drugs to alleviate ocular proliferative diseases.
2. The application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate ocular proliferative diseases according to claim 1, characterized in that: The drug is used to inhibit the function of the NCOOA3 polyQ domain and block the angiogenesis-related effects mediated by the NCOOA3 polyQ domain.
3. The application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate ocular proliferative diseases according to claim 2, characterized in that: The aforementioned ocular proliferative disorders include corneal pathological proliferative diseases.
4. The application of the NCOA3 polyQ domain as a target in the preparation of drugs to alleviate ocular proliferative diseases, as described in claim 3, is characterized in that: The ocular proliferative disorders include diabetic retinopathy, early-onset thermal retinopathy, and retinal vein occlusion, which are often accompanied by abnormal vascular proliferation and leakage driven by ischemia and hypoxia.
5. A drug for relieving abnormal proliferative changes in the eye, characterized in that: The drug is used to inhibit the function of the NCOOA3 polyQ domain and block the angiogenesis-related effects mediated by the NCOOA3 polyQ domain.
6. The drug for relieving ocular abnormal proliferative disorders according to claim 5, characterized in that: The drug includes at least one of the following: a nucleic acid intervention agent targeting the NCOOA3 polyQ domain, a gene editing intervention system, and a small molecule compound.
7. The drug for relieving ocular abnormal proliferative disorders according to claim 6, characterized in that: The drug also includes other medically acceptable adjuvants.