Use of nsun2 in the treatment of wet age-related macular degeneration

Abstract

The application discloses application of NSUN2 in treatment of wet age-related macular degeneration. The application research finds that inhibition or silencing of the NSUN2 gene can significantly inhibit ocular neovascularization, reduce ocular inflammatory response, and effectively inhibit the development of subretinal fibrosis, and the result shows that the NSUN2 can be used as a potential target for treating wet age-related macular degeneration. Therefore, the application provides a new strategy for treating wet age-related macular degeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of NSUN2 in the treatment of wet age-related macular degeneration. Background Technology

[0002] Age-related macular degeneration (AMD), also known as age-related macular degeneration, primarily affects people over 60 years of age. AMD is a significant degenerative eye disease and a leading cause of irreversible blindness. With the increasing aging of the population, the problems caused by AMD are becoming increasingly prominent. AMD is classified into dry and wet types, with wet AMD being the leading cause of blindness, accounting for over 90% of AMD-related blindness cases. Wet AMD is characterized by choroidal neovascularization (CNV), which can lead to retinal exudates, submaculatic hemorrhage, and subretinal fibrotic scarring.

[0003] Studies have shown that vascular endothelial growth factor (VEGF) plays a crucial role in promoting angiogenesis, which is a significant contributing factor to the pathogenesis of wet age-related macular degeneration (AMD). Currently, anti-VEGF drugs are the first-line treatment for wet AMD, significantly improving patients' best-corrected visual acuity and maintaining good vision for extended periods. However, these drugs still have several limitations, such as their inability to suppress inflammation, a major factor in CNV development. Recent evidence increasingly suggests that endothelial cells are not merely blood vessels but also actively participate in regulating inflammation, such as in atherosclerosis, cardiovascular disease, respiratory diseases, and sepsis. Furthermore, endothelial cell-specific Ihh deficiency leads to choroidal mast cell loss and altered inflammatory responses, exacerbating visual impairment following retinal damage. Endothelial cells expressing AT1-R enhance the expression of MCP1 / CCL2, IL6, ICAM1, and VEGF. This increase leads to increased macrophage recruitment and contributes to CNV progression. Under hypoxic conditions, endothelial cells secrete higher levels of CSF1. CSF1 activates the PI3K / AKT / FOXO1 pathway in macrophages, promoting CNV progression. These findings highlight the crucial role of endothelial cells in ocular inflammation. Furthermore, anti-VEGF therapy has no inhibitory effect on late-stage subretinal fibrosis (SRF) in wet age-related macular degeneration. A 10-year follow-up study found that approximately 60%-70% of patients still develop SRF after long-term anti-VEGF treatment. SRF damages retinal structure and function, ultimately causing irreversible visual impairment. Therefore, many patients experience poor responses to anti-VEGF treatment and unsatisfactory visual recovery.

[0004] RNA methylation is a dynamic and reversible modification process that affects cellular immune responses, metabolic processes, and other functions by influencing RNA stability. 5-Methylcytosine (m...) 5 C) is one of the main RNA methylation pathways. It is catalyzed by NOP2 / Sun domain (NSUN) RNA methyltransferase or DNA methyltransferase 2 (DNMT2). 5 C is primarily induced by NOP2 / Sun domain family member 2 (NSUN2) of RNA methyltransferases. Aberrant expression of NSUN2 is associated with the occurrence and progression of various tumors.

[0005] Currently, the role of NSUN2 in eye diseases varies. In a corneal epithelial injury model, NSUN2 accelerates corneal epithelial healing by increasing 5-methylcytosine (m5C) modification of UHRF1 mRNA. In a mouse model of diabetic retinopathy (DR), knocking down NSUN2 improves retinal vascular leakage. These reports suggest that the role of NSUN2 differs in different eye diseases. Currently, there are no reports on the role of NSUN2 in wet age-related macular degeneration.

[0006] Regarding inflammation, some reports indicate that NSUN2 knockout can reduce inflammatory responses in endothelial cells, while others demonstrate that NSUN2 can protect against intestinal inflammation. This suggests that the regulatory role of NSUN2 in inflammation is tissue / condition-dependent. Currently, there are no reports on the effects of NSUN2 on ocular inflammation.

[0007] There are currently no reports on the function of NSUN2 in subretinal fibrosis. Summary of the Invention

[0008] This invention is the first to discover that inhibiting or silencing the NSUN2 gene can suppress ocular neovascularization, reduce ocular inflammation, and inhibit subretinal fibrosis. These results suggest that NSUN2 may serve as a potential target for the treatment of wet age-related macular degeneration.

[0009] Therefore, the first objective of this invention is to provide the use of NSUN2 inhibitors in the preparation of medicaments for the treatment of wet age-related macular degeneration.

[0010] The second objective of this invention is to provide the use of NSUN2 inhibitors in the preparation of drugs that inhibit the formation of subretinal fibrosis.

[0011] A third objective of this invention is to provide the use of NSUN2 inhibitors in the preparation of drugs for inhibiting ocular inflammation.

[0012] Preferably, the NSUN2 inhibitor is a reagent that inhibits or silences the NSUN2 gene.

[0013] Preferably, the reagents for inhibiting or silencing the NSUN2 gene include those that inhibit or silence the NSUN2 gene through RNA interference, gene knockout, chemical inhibition, miRNA-mediated silencing, transcription factor inhibition, or epigenetic modification.

[0014] Preferably, the RNA interference reagent includes siRNA, dsRNA, or shRNA targeting the NSUN2 gene.

[0015] Preferably, the sequence of the shRNA targeting the NSUN2 gene is shown in SEQ ID NO.1-2.

[0016] Preferably, the drug is an ophthalmic preparation.

[0017] Preferably, the ophthalmic preparations include eye drops, eye ointments, eye sprays, ophthalmic gels, eye patches, intraocular injections, ophthalmic microspheres, ophthalmic implants, periocular injections, and sustained-release ophthalmic preparations.

[0018] This invention reveals that inhibiting or silencing the NSUN2 gene can significantly suppress ocular neovascularization, reduce ocular inflammation, and effectively inhibit the development of subretinal fibrosis. These results suggest that NSUN2 may serve as a potential therapeutic target for wet age-related macular degeneration (AMD). This invention provides a novel strategy for the treatment of wet AMD. Attached Figure Description

[0019] Figure 1 This diagram illustrates the phenotype of NSUN2 gene deletion suppressing wet age-related macular degeneration in the CNV model. A is a schematic diagram of the CNV model. B shows the expression level of NSUN2 in the choroidal tissue of the CNV model detected by Western blot. C shows the m5C level of mRNA in the choroidal tissue of the CNV model detected by m5C ELISA. D shows the knockout efficiency of NSUN2 in the choroidal tissue of Nsun2 KO mice detected by Western blot. E shows the immunofluorescence images of IB4 (green) and α-SMA (red) in the choroid of Nsun2 KO mice in the CNV model, scale bar: 100 μm. F shows the quantitative results of IB4. G shows the quantitative results of α-SMA. H shows the immunofluorescence images of Iba1 (red) and F4 / 80 (red) in the choroid of Nsun2 KO mice in the CNV model, scale bar: 100 μm. I shows the quantitative results of Iba1. J shows the quantitative results of F4 / 80.

[0020] Figure 2This study aimed to investigate the endothelial-specific knockout of NSUN2 to inhibit angiogenesis and inflammation in a CNV model. A shows the mRNA level of NSUN2 in WT and Nsun2 cKO mouse tissues detected by RT-qPCR. B shows the protein levels of NSUN2 and SELE in WT and Nsun2 cKO mouse tissues detected by Western blot. C shows immunofluorescence images of IB4 (green) and IBA1 (red) in the choroid of Nsun2 cKO mice in the CNV model (scale bar: 100 μm). D shows the quantitative results of IB4. E shows the quantitative results of IBA1. F shows the area of ​​the non-vascular region of the retina in Nsun2 cKO mice measured by IB4 staining in the OIR model (scale bar: 100 μm). G shows the quantitative results of the non-vascular area. H shows the proportion of neovascularization in the retina of Nsun2 cKO mice measured by IB4 staining in the OIR model (scale bar: 100 μm). I shows the quantitative results of the proportion of neovascularization.

[0021] Figure 3 Targeting NSUN2 has the potential to treat wet age-related macular degeneration. In the figures, A shows the knockdown efficiency of shNsun2 in the choroid as detected by Western blot. B shows immunofluorescence images of IB4 (green) and F4 / 80 (red) in the choroid in a CNV model, scale bar: 100 μm. C shows the quantitative results of IB4. D shows the quantitative results of F4 / 80. Detailed Implementation

[0022] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the reagents and materials used are commercially available unless otherwise specified.

[0024] Example 1

[0025] I. Materials

[0026] 1) Plasmid

[0027] The shRNA targeting NSUN2 was designed using pLKO.1 as a vector via the Sigma website.

[0028] The designed sequences were synthesized by Tsingke Biotechnology and ligated to the pLKO.1 vector using T4 DNA ligase. All plasmids were transformed with DH5α and extracted using a plasmid extraction kit (TIANGEN, DP117TA).

[0029] Table 1 shRNA sequence information

[0030]

[0031] 2) Proteins and antibodies

[0032] Table 2 Proteins and Antibodies

[0033] name Item number company GS-IB4 I21413 Thermofisher ERG ab92513 Abcam F4 / 80 MCA497G Biorad Iba-1 019-19741 wako anti-NSUN2 20854-1-AP Proteintech anti-β-ACTIN RM2001 Beijing Ray Antibody Biotech

[0034] II. Methods

[0035] 1. Construction of Nsun2-Cdh5-Cre conditional knockout mice

[0036] Nsun2 fl / fl Mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were crossed with Cdh5-Cre mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to produce Cdh5-Cre mice with NSUN2 specifically knocked out in endothelial cells. + / - Nsun2 fl / fl (Nsun2-Cdh5-Cre) mice (designated Nsun2 cKO). PCR and sequencing confirmed successful knockout of NSUN2 in endothelial cells.

[0037] 2. Construction of Nsun2-Cag-Cre conditional knockout mice

[0038] Nsun2 fl / fl Mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were compared with CAG Cre ERT2 Cag-Cre mice (purchased from Jackson Laboratory) were created by crossbreeding to produce mice with systemic conditional knockout of NSUN2. + / - Nsun2 fl / fl (Nsun2-Cag-Cre) mice (abbreviated as Nsun2 KO). Genotyping of mice was performed using PCR and sequencing technologies.

[0039] 3. Laser-induced CNV mouse model

[0040] 1) Irradiate four laser photocoagulation spots (spot size 75μm, power 90mw, duration 75ms) onto the eyes of adult mice within an area 1.5-2mm from the diameter of the optical disc. Perform laser photocoagulation at positions 12, 3, 6, and 9, avoiding large retinal vessels.

[0041] 2) After laser photocoagulation, inject shNSUN2 or shCTRL plasmid-PEI complex (1 μg / μL) into the vitreous cavity.

[0042] 3) Eyes were collected on day 7. The eyeballs were fixed under 4% PFA for 2 hours. The cornea, lens, lenticule, and vitreous humor were removed, and the choroid-RPE complex was separated. The following experiments were performed: The choroid-RPE complex was washed with PBS and incubated overnight at 4°C in Alexa Fluor 488 (1:500) conjugated with 1% donkey serum, 1% BSA, and 0.1% Txiton X-100, along with the hemagglutinin GS-IB4. The choroid-RPE complex was rinsed with PBS and laid flat with the RPE side up. Images were taken using a fluorescence microscope, and the CNV region was analyzed using image analysis software. MethylFlash was used. TM The Epigentek Simple ELISA Kit for 5-mC RNA Methylation was used to detect the m5C level of mRNA in the choroid-RPE complex.

[0043] 4) Rinse the choroid-RPE complex with PBS and lay it flat with the RPE side up.

[0044] 5) Images were captured using a fluorescence microscope, and the CNV region was analyzed using image analysis software.

[0045] 4. OIR Model

[0046] On day 7 after birth (P7), newborn mice were placed in an environment with 75% oxygen concentration along with their nursing mothers for 5 days of continuous exposure. They were then returned to a normal oxygen environment at P12 until P17. Eyeballs were collected at P12 and P17, and the retinas were separated and stained.

[0047] 5. Retinal detachment, treatment, and staining

[0048] Eyeballs were collected, enucleated in PBS, and fixed with 4% PFA for 30 min at room temperature. The retina was dissected, softened in PBS containing 5% donkey serum and 0.5% Triton X-100 for 1 h, and then incubated overnight at 4°C with allogeneic proteins GS-IB4, ERG, F4 / 80 (1:200), and Iba-1 (1:100) bound to primary antibody Alexa Fluor 488 (1:500). After rinsing with PBS, the retina was incubated with the corresponding secondary antibody at room temperature for 2 h. The retina was then analyzed using confocal fluorescence microscopy after being laid flat.

[0049] 6. RNA isolation and extraction, cDNA synthesis and quantitative real-time PCR

[0050] Mouse retinal or choroidal tissues were collected, and total RNA was isolated from cells using TRIzol reagent (TIANGEN). cDNA was synthesized from this RNA using a DNase-containing FastKing RT kit (TIANGEN). qRT-PCR was performed using a SYBR Green (ROCHE) and ABI QuantStudio 6Flex instrument (Life Technologies). Results were normalized to GAPDH transcripts. Relative folding changes in gene expression were calculated using the delta-delta Ct method.

[0051] Table 3 Primers

[0052]

[0053]

[0054] 7. Western blot experiment

[0055] 1) Preparing liquids:

[0056] ①10×Running Buffer: Add 144g glycine, 10g SDS powder, and 30.3g Tris powder sequentially, using ddH2O as the solvent, and bring the volume to 1L to prepare a storage solution. Dilute to 1×Running Buffer to prepare the electrophoresis working solution for use.

[0057] ②5×SDS Loading Buffer: Add 4g of SDS powder, 20mg of bromophenol blue, 3.085g of DTT, 10mL of Tris-HCl (1M pH 6.8), and 20mL of glycerol in sequence, and use ddH2O as a solvent to make up to 40mL.

[0058] ③ 10× Transfer Buffer: Add 30.3g Tris powder and 144g glycine sequentially, using ddH2O as the solvent, and bring the volume to 1L to prepare a storage solution. Dilute 10× Transfer Buffer 100mL + methanol 200mL + ddH2O 700mL to prepare 1× Transfer Buffer as the working solution for membrane transfer.

[0059] 2) Preparation of separating gel and stacking gel:

[0060] ① Prepare 10% separating gel (10 mL):

[0061] Table 4 shows the preparation of 10% separating gel.

[0062]

[0063]

[0064] ②Prepare a 5% concentrate (5 mL):

[0065] Table 5 shows the preparation of a 5% concentrate gel.

[0066]

[0067] 3) Protein gel electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis solution to the tank. Check for leakage. Remove the comb and add the marker and sample to the gel wells in sequence. Add electrophoresis solution to the electrophoresis tank and set the program to 80V for 30 minutes of constant voltage electrophoresis. Then switch to 120V for 1 hour of constant voltage electrophoresis.

[0068] 4) Transfer: First, activate the PVDF membrane with methanol, and remove the gel from step 3). Assemble the transfer clamps in a "sandwich" structure, taking care not to leave air bubbles during assembly. Assemble the transfer apparatus, add the transfer working solution, set the program to a constant current of 250 mA for 2 hours, and place it on ice for transfer.

[0069] 5) Blocking: Take out the membrane after the transfer is completed, add 1×TBST and place it on a shaker at room temperature for 5 minutes to clean it. Discard the solution and add 5% skim milk as the blocking solution. Place it on a shaker at room temperature for blocking for 1 hour.

[0070] 6) Primary antibody incubation: Discard the blocking solution, add 1×TBST and place on a shaker at room temperature to wash until the blocking solution is clean. Discard the liquid, add the primary antibody prepared using 5% BSA as antibody diluent, and incubate overnight on a shaker in a 4°C cold storage.

[0071] 7) Wash the membrane: Wash the membrane three times with 1×TBST, 10 min each time.

[0072] 8) Secondary antibody incubation: Discard the solution, add the secondary antibody prepared with 5% skim milk at a volume ratio of 1:5000, and incubate on a shaker at room temperature for 1 hour.

[0073] 9) Same as step 7).

[0074] 10) Exposure and development: Prepare the developer (A solution: B solution = 1:1 volume ratio, prepare and use immediately). Place the film on the plate, add developer to the film, shake well, expose and develop on the machine, and save the results.

[0075] III. Results

[0076] All data are presented as mean ± standard deviation (SD). *: p < 0.05, **p < 0.01, ***: p < 0.001.

[0077] 1. To determine whether NSUN2 affects angiogenesis, we first constructed a laser-induced choroidal angiogenesis (CNV) model to detect NSUN2 expression. Figure 1 A). Western blot showed upregulation of NSUN2 expression in the choroid in the CNV model ( Figure 1 (B in the original text). Since NSUN2 can catalyze the formation of RNAm5C, we used an m5C ELISA kit to further detect the total RNAm5C level. This level was significantly increased in the choroid membrane after CNV modeling. Figure 1 (C in the text). Therefore, these results suggest that NSUN2-mediated RNAm5C modification may play a role in CNV.

[0078] To understand the potential role of NSUN2 in angiogenesis, we will use Nsun2 fl / fl Mice and CAG Cre ERT2 Transgenic mice were crossed to generate induced NSUN2 complete knockout mice (Nsun2 KO). Western blot analysis showed that NSUN2 was absent in the mice after tamoxifen induction. Figure 1 (D in the text). In the mouse CNV model, NSUN2 deficiency significantly reduced IB4. + (endothelial cell markers), α-SMA + (fibrosis marker), Iba1 + and F4 / 80 + (Microglia / macrophage markers) staining areas ( Figure 1 The study (EJ) indicates that the deletion of the NSUN2 gene suppresses the wet age-related macular degeneration phenotype in vivo, including inhibiting ocular neovascularization, reducing ocular inflammation, and inhibiting subretinal fibrosis.

[0079] 2. To determine the function of NSUN2 in endothelial cells, we used Nsun2 fl / fl Mice were crossed with Cdh5-Cre transgenic mice to generate endothelial cell-specific NSUN2 knockout mice (designated Nsun2 cKO). Both qRT-PCR and Western blot showed the knockout effect of NSUN2 in endothelial cells. Figure 2 (AB in the text). Using a laser-induced Nsun2 cKO mouse model of choroidal neovascularization (CNV), reduced IB4 and IBA1 staining in endothelial cells of NSUN2-specific knockout mice alleviated angiogenesis and inflammation. Figure 2(CE in the text). These data indicate that specific loss of NSUN2 in endothelial cells can improve ocular neovascularization and inflammatory responses. Using an oxygen-induced Nsun2 cKO mouse retinopathy (OIR) model, the area of ​​the non-vascular region of the retina in mice with NSUN2-specific knockout in endothelial cells was significantly increased, and the proportion of neovascularization was reduced (CE in the text). Figure 2 The results (FI) indicate that specific loss of NSUN2 in endothelial cells can inhibit neovascularization in retinopathy.

[0080] 3. To further investigate the potential role of NSUN2 in angiogenesis, we constructed a knockdown plasmid of NSUN2 and injected it into the fundus of CNV mice. Western blot analysis showed the knockdown efficiency of shNsun2 in the choroid. Figure 3 (A) Simultaneously, staining the choroid of CNV mice revealed that NSUN2 deficiency significantly reduced IB4 in the mouse CNV model. + (endothelial cell markers), F4 / 80 + (Macrophage markers) staining areas ( Figure 3 The results (BD) indicate that inhibiting NSUN2 can suppress angiogenesis and inflammatory response in wet age-related macular degeneration.

[0081] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of an NSUN2 inhibitor in the preparation of a medicament for treating wet age-related macular degeneration; the NSUN2 inhibitor is an shRNA targeting the gene with the nucleotide sequence as shown in SEQ ID NO. 1 and SEQ ID NO.

2. NSUN2 gene.

2. Use according to claim 1, characterized in that, The NSUN2 inhibitor can inhibit ocular neovascularization, reduce ocular inflammation, and inhibit subretinal fibrosis formation.

3. Use according to claim 1 or 2, characterized in that, The drug is an ophthalmic preparation.

4. Use according to claim 3, characterized in that, The ophthalmic preparation includes eye drops, eye ointments, eye sprays, eye gels, eye patches, intraocular injections, ophthalmic microspheres, ocular implants, periocular injections, and ophthalmic sustained-release preparations.

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