Application of NSUN2 in treatment of wet age-related macular degeneration

Abstract

The invention discloses an application of NSUN2 in treatment of wet age-related macular degeneration. Research finds that inhibition or silencing of the NSUN2 gene can significantly inhibit formation of ocular neovascularization, reduce ocular inflammatory response and effectively inhibit 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 invention provides a new strategy for the treatment of wet age-related macular degeneration.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of NSUN2 in the treatment of wet age-related macular degeneration. Background Art

[0002] Age-related macular degeneration (AMD), also known as age-related macular degeneration, is common in people over 60 years old. AMD is an important degenerative eye disease and the main cause of irreversible blindness. With the increasing aging of the population, the problems caused by AMD are becoming increasingly prominent. AMD is divided into two types: dry and wet. Among them, wet age-related macular degeneration is the main cause of blindness caused by AMD, accounting for more than 90% of AMD blindness. Wet age-related macular degeneration is characterized by choroidal neovascularization (CNV), which can lead to retinal exudation, submacular hemorrhage and subretinal fibrotic scar formation.

[0003] Studies have shown that vascular endothelial growth factor (VEGF) plays a key role in promoting neovascularization, which is one of the important causes of wet age-related macular degeneration. At present, anti-VEGF drugs are the first choice for the treatment of wet age-related macular degeneration, which can greatly improve the best corrected visual acuity of patients and maintain good visual acuity for a long time. However, there are still many problems with this drug, such as the inability to inhibit inflammation, which is also an important factor in the development of CNV. Recently, increasing evidence has shown that endothelial cells are not only blood channels, but also actively participate in regulating inflammation, such as atherosclerosis, cardiovascular disease, respiratory disease and sepsis. In addition, endothelial cell-specific Ihh deficiency leads to choroidal mast cell loss and altered inflammatory response, exacerbating visual dysfunction after retinal injury. Endothelial cells expressing AT1-R enhance the expression of MCP1 / CCL2, IL6, ICAM1 and VEGF. This increase leads to increased recruitment of macrophages and leads to CNV progression. Under hypoxic conditions, endothelial cells secrete higher levels of CSF1, which activates the PI3K / AKT / FOXO1 pathway in macrophages and promotes CNV progression. These findings emphasize the key role of endothelial cells in ocular inflammation. In addition, anti-VEGF has no inhibitory effect on subretinal fibrosis (SRF) in the late stage of wet age-related macular degeneration. According to a 10-year follow-up study, about 60%-70% of patients will still develop subretinal fibrosis after long-term anti-VEGF treatment. SRF will destroy the structure and function of the retina, and ultimately cause irreversible visual damage. Therefore, many patients have poor response to anti-VEGF treatment, and their vision recovery has not met expectations.

[0004] RNA methylation is a dynamic and reversible modification process that affects the stability of RNA and thus affects the immune response, metabolic process and other functions of cells. 5 C) is one of the main RNA methylation modes. It is catalyzed by NOP2 / Sun domain (NSUN) RNA methyltransferase or DNA methyltransferase 2 (DNMT2). 5 C is mainly induced by RNA methyltransferase NOP2 / Sun domain family member 2 (NSUN2). Abnormal expression of NSUN2 is associated with the occurrence and progression of various tumors.

[0005] Currently, the role of NSUN2 in eye diseases is not the same. In the 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 can improve retinal vascular leakage. These reports suggest that the role of NSUN2 in different eye diseases is different. There is no report on the role of NSUN2 in wet age-related macular degeneration.

[0006] In terms of inflammation, there are reports that NSUN2 knockout can reduce the inflammatory response of endothelial cells, but there are also reports that NSUN2 can protect against intestinal inflammation. This shows that the regulatory role of NSUN2 in inflammation is related to tissues / conditions. There are currently no reports of NSUN2 in ocular inflammation.

[0007] Currently, the function of NSUN2 in subretinal fibrosis has not been reported. Summary of the invention

[0008] The present invention discovered for the first time that inhibiting or silencing the NSUN2 gene can inhibit ocular neovascularization, reduce ocular inflammation and inhibit subretinal fibrosis formation. This result indicates that NSUN2 can be used as a potential target for the treatment of wet age-related macular degeneration.

[0009] Therefore, the first object of the present invention is to provide a use of a NSUN2 inhibitor in the preparation of a medicament for treating wet age-related macular degeneration.

[0010] The second object of the present invention is to provide a use of a NSUN2 inhibitor in the preparation of a drug for inhibiting the formation of subretinal fibrosis.

[0011] The third object of the present invention is to provide use of a NSUN2 inhibitor in the preparation of a medicament for inhibiting ocular inflammation.

[0012] Preferably, the NSUN2 inhibitor is an agent that inhibits or silences the NSUN2 gene.

[0013] Preferably, the agent for inhibiting or silencing the NSUN2 gene includes an agent for inhibiting or silencing the NSUN2 gene by means of RNA interference, gene knockout, chemical inhibition, miRNA-mediated silencing, transcription factor inhibition or epigenetic modification.

[0014] Preferably, the RNA interference agent 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, eye gels, eye patches, intraocular injections, ophthalmic microspheres, ocular implants, periocular injections and ophthalmic sustained-release preparations.

[0018] The present invention found that inhibiting or silencing the NSUN2 gene can significantly inhibit ocular neovascularization, reduce ocular inflammatory response, and effectively inhibit the development of subretinal fibrosis. This result indicates that NSUN2 can be used as a potential target for the treatment of wet age-related macular degeneration. The present invention provides a new strategy for the treatment of wet age-related macular degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 2Endothelial-specific knockout of NSUN2 inhibits angiogenesis and inflammation in the CNV model. A is RT-qPCR to detect the mRNA level of NSUN2 in WT and Nsun2 cKO mouse tissues. B is Western blot to detect the protein levels of NSUN2 and SELE in WT and Nsun2 cKO mouse tissues. C is the immunofluorescence image of IB4 (green) and IBA1 (red) of Nsun2 cKO choroid in the CNV model, scale bar: 100μm. D is the quantitative result of IB4. E is the quantitative result of IBA1. F is the non-vascular area of ​​the retina of Nsun2 cKO mice measured by IB4 staining in the OIR model, scale bar: 100μm. G is the quantitative result of the non-vascular area. H is the proportion of new blood vessels in the retina of Nsun2 cKO mice measured by IB4 staining in the OIR model, scale bar: 100μm. I is the quantitative result of the proportion of new blood vessels.

[0021] Figure 3 Targeting NSUN2 has the potential to treat wet age-related macular degeneration. A is a Western blot to detect the knockdown efficiency of shNsun2 in the choroid. B is an immunofluorescence image of IB4 (green) and F4 / 80 (red) in the choroid in the CNV model, scale bar: 100 μm. C is the quantitative result of IB4. D is the quantitative result of F4 / 80. DETAILED DESCRIPTION

[0022] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

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

[0024] Example 1

[0025] 1. Materials

[0026] 1) Plasmid

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

[0028] The designed sequence was synthesized by Tsingke Biotechnology and ligated with the pLKO.1 vector using T4 DNA ligase. All plasmids were transformed using 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 Part 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] 2. Methods

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

[0036] Nsun2 fl / fl The mice (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) were crossed with Cdh5-Cre mice (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) to generate Cdh5-Cre mice with NSUN2 knockout specifically in endothelial cells. + / - Nsun2 fl / fl (Nsun2-Cdh5-Cre) mice (referred to as Nsun2 cKO). PCR and sequencing confirmed that NSUN2 was successfully knocked out in endothelial cells.

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

[0038] Nsun2 fl / fl Mice (purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.) were cross-linked with CAG Cre ERT2 The mice (purchased from Jackson Laboratory) were crossed to generate mice with systemic conditional knockout of NSUN2, Cag-Cre + / - Nsun2 fl / fl (Nsun2-Cag-Cre) mice (referred to as Nsun2 KO). The mice were genotyped by PCR and sequencing.

[0039] 3. Laser-induced CNV Mouse Model

[0040] 1) Four laser photocoagulation spots were irradiated to the eyes of adult mice (spot size 75 μm, power 90 mW, duration 75 ms) in an area 1.5-2 mm from the optical disc diameter. Laser photocoagulation was performed at 12, 3, 6, and 9 positions, avoiding large retinal vessels.

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

[0042] 3) Collect eyes on day 7. The eyeballs were fixed in 4% PFA for 2 h, the cornea, lens, crystalline body and vitreous body were removed, and the choroid-RPE complex was separated. The following experiments were performed: the choroid-RPE complex was washed with PBS and incubated in Alexa Fluor 488 (1:500)-coupled isolectin GS-IB4 containing 1% donkey serum, 1% BSA and 0.1% Txiton X-100 at 4°C overnight; the choroid-RPE complex was rinsed with PBS and laid flat with the RPE facing up; images were taken using a fluorescence microscope, and the CNV area was analyzed using an image analysis software program. MethylFlash TM The m5C level of mRNA in the choroid-RPE complex was detected by 5-mC RNA methylation ELISA simple kit (Epigentek).

[0043] 4) The choroid-RPE complex was rinsed with PBS and laid flat with the RPE side facing up.

[0044] 5) Take images using a fluorescence microscope and analyze the CNV regions using an image analysis software program.

[0045] 4.OIR Model

[0046] On postnatal day 7 (P7), newborn mice were placed with their nursing mothers in an environment containing 75% oxygen concentration for 5 days, and then returned to a normal oxygen environment at P12 until P17. Eyeballs were collected at P12 and P17, and retinas were isolated for staining.

[0047] 5. Retinal Dissection, Processing, and Staining

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

[0049] 6. RNA Isolation and Extraction, cDNA Synthesis and Quantitative Real-time PCR

[0050] Mouse retina or choroid tissue was collected, total RNA was isolated from cells using TRIzol reagent (TIANGEN) and synthesized into cDNA using FastKing RT kit (TIANGEN) containing DNase, and qRT-PCR was performed using SYBR Green (ROCHE) and ABIQuantStudio 6Flex equipment (Life Technologies). The results were normalized with GAPDH transcripts. The relative fold change of gene expression was calculated using the delta-delta Ct method.

[0051] Table 3 Primers

[0052]

[0053]

[0054] 7. Western blot experiment

[0055] 1) Prepare liquid:

[0056] ①10×Running Buffer: add 144g glycine, 10g SDS powder, 30.3g Tris powder, ddHO 2 O as solvent, dilute to 1 L to prepare the storage solution. Dilute to 1× Running Buffer to prepare the electrophoresis working solution for use.

[0057] ②5×SDS Loading Buffer: Add SDS powder 4g, bromophenol blue 20mg, DTT 3.085g, Tris-HCL (1M pH 6.8) 10mL, glycerol 20mL, ddHO in sequence. 2 O as the solvent and make up to 40 mL.

[0058] ③10×Transfer Buffer: add 30.3g Tris powder, 144g glycine, ddHO 2 O as solvent, dilute to 1L to prepare the storage solution. Prepare 10× Transfer Buffer 100mL + methanol 200mL + ddHO 2 O 700mL formula is diluted into 1× Transfer Buffer to prepare the transfer working solution for use.

[0059] 2) Prepare separation gel and stacking gel:

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

[0061] Table 4 Preparation of 10% separation gel

[0062]

[0063]

[0064] ②Prepare 5% stacking gel (5 mL):

[0065] Table 5 Preparation of 5% stacking gel

[0066]

[0067] 3) Protein gel electrophoresis: Assemble the prepared gel and add the newly prepared electrophoresis solution into its tank, check for leakage, pull out the comb, add the marker and sample into the gel wells in sequence, add the electrophoresis solution into the electrophoresis tank, set the program to perform constant voltage electrophoresis at 80V for 30min and then switch to 120V for constant voltage electrophoresis for 1h.

[0068] 4) Transfer: First, use methanol to activate the PVDF membrane, and take out the gel in step 3), assemble the transfer clip in the order of the "sandwich" structure, and be careful not to leave bubbles during the assembly. Assemble the transfer device, add the transfer working solution, set the program to 250mA constant current for 2h, and place it on ice for transfer.

[0069] 5) Blocking: After the transfer, take out the membrane, add 1×TBST and place it on a shaker at room temperature for washing for 5 minutes, then discard it, and add 5% skim milk as blocking solution and place it on a shaker at room temperature for blocking. The blocking time is 1 hour.

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

[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 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 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 evenly, expose and develop on the machine, and save the results.

[0075] 3. 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 has an effect on the angiogenesis process, we first constructed a laser-induced choroidal neovascularization (CNV) model to detect the expression of NSUN2 ( Figure 1 Western blot showed that NSUN2 expression in the choroid was upregulated in the CNV model ( Figure 1 B). Since NSUN2 can catalyze the formation of RNAm5C, we used an m5C ELISA kit to further detect the total RNAm5C level, which was significantly increased in the choroid after CNV modeling ( Figure 1 C). 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 fl / fl Mice with CAG-Cre ERT2 Transgenic mice were crossed to generate inducible NSUN2 complete knockout mice (abbreviated as Nsun2 KO). Western blot analysis showed that after tamoxifen induction, NSUN2 was missing in mice ( Figure 1 D). NSUN2 deficiency significantly reduced IB4 in a mouse CNV model. + (endothelial cell marker), α-SMA + (fibrosis marker), Iba1 + and F4 / 80 + (microglia / macrophage markers) staining area ( Figure 1 EJ in ), showing that genetic deletion of NSUN2 suppresses wet age-related macular degeneration phenotypes in vivo, including inhibition of ocular neovascularization, decreased ocular inflammation, and suppressed subretinal fibrosis.

[0079] 2. To determine the function of NSUN2 in endothelial cells, we expressed Nsun2 fl / fl The mice were crossed with Cdh5-Cre transgenic mice to generate endothelial cell-specific NSUN2 knockout mice (called Nsun2 cKO). Both qRT-PCR and western blot showed the knockout effect of NSUN2 in endothelial cells ( Figure 2 AB in Figure 1). Using the laser-induced choroidal neovascularization (CNV) model of Nsun2 cKO mice, mice with specific knockout of NSUN2 in endothelial cells showed reduced IB4 and IBA1 staining, and alleviated angiogenesis and inflammation ( Figure 2CE in ). These data suggest that specific deletion of NSUN2 in endothelial cells can improve ocular neovascularization and inflammatory responses. Using the oxygen-induced Nsun2 cKO mouse retinopathy (OIR) model, the non-vascular area of ​​the retina of mice with specific knockout of NSUN2 in endothelial cells was significantly increased, and the proportion of neovascularization was reduced ( Figure 2 The results indicate that the specific deletion of NSUN2 in endothelial cells can inhibit the neovascularization of retinal diseases.

[0080] 3. To further study the potential role of NSUN2 in angiogenesis, we constructed a knockdown plasmid for NSUN2 and injected the plasmid into the fundus of CNV mice. Western blot showed the knockdown efficiency of shNsun2 in the choroid ( Figure 3 At the same time, the choroid of CNV mice was stained and it was found that the loss of NSUN2 significantly reduced IB4 in the mouse CNV model. + (endothelial cell marker), F4 / 80 + (macrophage marker) staining area ( Figure 3 BD in the retina), indicating that inhibition of NSUN2 can suppress angiogenesis and inflammatory responses in wet age-related macular degeneration.

[0081] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, 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 regarded as the protection scope of the present invention.

Claims

1. Application of NSUN2 inhibitors in the preparation of drugs for the treatment of wet age-related macular degeneration.

2. Application of NSUN2 inhibitors in the preparation of drugs for inhibiting the formation of subretinal fibrosis.

3. Application of NSUN2 inhibitors in the preparation of drugs for inhibiting ocular inflammation.

4. The use according to any one of claims 1 to 3, characterized in that: The NSUN2 inhibitor is an agent that inhibits or silences the NSUN2 gene.

5. The use according to claim 4, characterized in that: The reagents for inhibiting or silencing the NSUN2 gene include reagents for inhibiting or silencing the NSUN2 gene by means of RNA interference, gene knockout, chemical inhibition, miRNA-mediated silencing, transcription factor inhibition or epigenetic modification.

6. The use according to claim 5, characterized in that: The RNA interference reagent includes siRNA, dsRNA or shRNA targeting the NSUN2 gene.

7. The use according to claim 6, characterized in that: The sequence of the shRNA targeting the NSUN2 gene is shown in SEQ ID NO. 1-2.

8. The use according to claim 4, characterized in that: Inhibiting or silencing the NSUN2 gene can inhibit ocular neovascularization, reduce ocular inflammation and inhibit the formation of subretinal fibrosis.

9. The use according to any one of claims 1 to 3, characterized in that: The medicine is an ophthalmic preparation.

10. The use according to claim 9, characterized in that: The ophthalmic preparations include 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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