Application of medicine for knocking out or reducing RBM39 gene expression in preparation of medicine for treating graft versus host disease

Abstract

The invention discloses an application of a medicine for knocking out or reducing RBM39 gene expression in preparation of a medicine for treating graft-versus-host disease (GVHD), the research finds that RBM39 can be used as a target for treating GVHD for the first time, and after the expression of RBM39 is knocked out or reduced, proliferation of T cells can be inhibited, apoptosis of the T cells can be promoted, differentiation of Th1 and Th17 can be inhibited, and the purpose of treating autoimmune diseases can be achieved; and the targeted RBM39 does not cause the weight loss of animals, has no obvious toxic or side effect, and can be used for clinically treating GVHD.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to the application of a drug for knocking out or reducing the expression of RBM39 gene in the preparation of a drug for treating graft-versus-host disease. BACKGROUND

[0002] Graft-versus-host disease (GVHD) is a pathological state caused by immune cells in the graft attacking host cells, which mainly occurs after hematopoietic stem cell transplantation. After hematopoietic stem cell transplantation, the immune cells (T cells are the main pathogenic cells) of the donor recognize the antigens of the host. The antigen presenting cells (APCs) of the host, such as dendritic cells, present their own antigens to the T cells of the donor, causing the donor T cells to be activated by the host antigens and start to proliferate and differentiate. These activated T cells include CD4 + helper T cells and CD8 + cytotoxic T cells. The activated T cells release a large amount of inflammatory cytokines such as TNF-α, IFN-γ, IL-2 and IL-17A. T cells and released cytokines attack multiple tissues and organs of the host, including the skin, liver and digestive tract, causing symptoms of acute and chronic GVHD. Current clinical treatment of GVHD mainly controls the progression of the patient's disease course by using broad-spectrum immunosuppressive drugs. Such drugs are usually non-specific targeted drugs, which have strong toxic side effects on Treg cells and normal tissues with strong proliferation and metabolism, such as the intestinal tract, liver, etc. in addition to the inhibition of inflammatory immune cells. Therefore, a target that can specifically inhibit T cell function without affecting the function of other normal cells is the key to developing high-specificity, low-toxicity GVHD-targeted treatment drugs. SUMMARY

[0003] Therefore, one of the purposes of the present application is to provide an application of a drug for knocking out or reducing the expression of RBM39 gene in the preparation of a drug for treating GVHD.

[0004] To achieve the above-mentioned purpose, the present application provides the following technical solutions: 1. An application of a drug for knocking out or reducing the expression of RBM39 gene in the preparation of a drug for treating GVHD.

[0005] Preferably, in the present application, the GVHD is T cell-mediated GVHD.

[0006] Preferably, in the present application, the RBM39 gene expression is knocked out by using a drug-induced gene knockout.

[0007] Preferably, in the present application, the drug for reducing the expression of RBM39 gene is Indisulam.

[0008] Preferably, in this invention, knocking out or reducing the RBM39 gene is used to treat GVHD by inhibiting T cell proliferation and promoting T cell apoptosis.

[0009] Preferably, the present invention describes that knocking out or reducing the RBM39 gene to treat GVHD inhibits the differentiation of Th1 and Th17 cells and promotes the differentiation of Treg cells.

[0010] The beneficial effects of this invention are as follows: This invention discloses that RBM39 can serve as a target for treating GVHD. Knocking out or reducing the expression of RBM39 can treat GVHD. The treatment process is achieved by inhibiting T cell proliferation and activation, promoting T cell apoptosis, and inhibiting Th1 and Th17 differentiation. Furthermore, the use of this drug does not lead to a decrease in animal body weight; the weight change is no different from that of normal control mice. Histological sections of multiple organs show that the drug has no significant toxic side effects on any organ. This invention provides a new target for GVHD treatment and has important significance for the clinical treatment of GVHD. Attached Figure Description

[0011] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 The changes in body weight in the control and experimental groups on day 1 and day 29 after using small molecule drugs to specifically reduce RBM39 expression (A: changes in body weight in the control and experimental groups; B: changes in body weight percentage in the control and experimental groups). Figure 2 To reduce RBM39 expression and H&E staining of mouse organs; Figure 3 To demonstrate that using small molecule drugs to specifically reduce RBM39 expression can inhibit T cell proliferation and promote apoptosis of activated T cells (A: Flow cytometry plot of the effect of different concentrations of drug-specific RBM39 degradation on T cell proliferation; B: Statistical graph of the effect of different concentrations of drug-specific RBM39 degradation on T cell proliferation; C: Flow cytometry plot of the effect of different concentrations of drug-specific RBM39 degradation on T cell apoptosis; D: Statistical graph of the effect of different concentrations of drug-specific RBM39 degradation on T cell apoptosis). Figure 4 To illustrate the treatment of GVHD in mice by gene knockout to reduce RBM39 expression (A, B: Changes in body weight (A) and body weight percentage (B) after GVHD induction in wild-type and RBM39 knockout mice; C: Disease severity score after GVHD induction in wild-type and RBM39 knockout mice; D: Mice mortality curve after GVHD induction in wild-type and RBM39 knockout mice; E: Appearance of wild-type and RBM39 knockout mice after GVHD induction); Figure 5 To illustrate the treatment of GVHD in mice by specifically reducing RBM39 expression with a small molecule drug, the following graphs are presented: A, B: Changes in body weight (A) and body weight percentage (B) after GVHD induction in the control group and the RBM39-specifically degraded group; C: Disease severity score after GVHD induction in the control group and the RBM39-specifically degraded group; D: Mice mortality curves after GVHD induction in the control group and the RBM39-specifically degraded group; E: Appearance of mice after GVHD induction in the control group and the RBM39-specifically degraded group. Figure 6 To investigate the infiltration of immune cells in peripheral lymphoid organs of mice after treating GVHD with a small molecule drug that specifically reduces RBM39 expression (CD45.1 levels in lymph nodes (A) and spleen (B) of mice in the control group and the group treated with the drug-specifically degraded RBM39 group after GVHD induction). + (Proportion and number of immune cells) Figure 7 To investigate the proliferation of T cells in peripheral lymphoid organs of mice after treating GVHD with a small molecule drug that specifically reduces RBM39 expression (CTV in lymph nodes (A) and spleen (B) after GVHD induction in control and drug-specifically degraded RBM39 groups), - CD45.1 + T cell percentage). Detailed Implementation

[0012] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0013] RBM39 gene knockout mice were obtained from Cyagen Biotech Ltd., catalog number: S-CKO-03525.

[0014] Indisulam, a small molecule drug that degrades RBM39 protein, was obtained from Shanghai Hanxiang Biotechnology Co., Ltd., CAS: 165668-41-7.

[0015] Example 1: Effects of using small molecule drugs to specifically reduce RBM39 expression on body weight or organs in normal mice. Preparation of cyclodextrin: Dissolve hydroxypropyl β-cyclodextrin (pharmaceutical grade) in sterile physiological saline at a concentration of 0.2 g / ml.

[0016] Indisulam preparation: Dilute Indisulam (100 mg / ml) with the above HPCD at a volume ratio of 1:19.

[0017] Injection: Normal mice were intraperitoneally injected with 100 μl of hydroxypropyl β-cyclodextrin (HPCD) (control group) or Indisulam (experimental group) every other day for four consecutive weeks. Each mouse was weighed and the results were recorded before injection (day 1) and four weeks after injection (day 29). Figure 1 As shown in the figure. The results showed that there was no significant difference in body weight or the ratio of body weight to body weight on day 1 between the experimental group and the control group 29 days after injection.

[0018] Then, heart, liver, spleen, lung, kidney, large intestine, and small intestine from both the control and experimental groups were subjected to H&E staining. The H&E staining was performed by a biotechnology company, including dehydration, trimming, embedding, sectioning, staining, and mounting. Finally, the samples were examined under a microscope. The results are as follows: Figure 2 As shown in the figure. The results showed no significant difference between the experimental group and the control group, indicating that the drug had no obvious toxic side effects on any organ.

[0019] Example 2: Effects of specifically reducing RBM39 expression on T cell proliferation and apoptosis Using small molecule drugs to target and reduce RBM39 in vitro: (1) Coating culture plate: The procedure is the same as in I.

[0020] (2) Sampling of mouse lymph organs: Select wild-type mice aged 8-10 weeks and follow the same steps as in I.

[0021] (3) Cell proliferation markers: The procedure is the same as in I.

[0022] (4) In vitro expansion culture: Take an appropriate volume of culture medium (RPMI 1640 + 10% FBS + 1% P / S + 1% β-mercaptoethanol), add Anti-CD28 (2 μg / ml) and IL-2 (2 μg / ml) and mix well for later use; prepare culture medium containing different concentration gradients (1.25 μM, 2.5 μM, 5 μM) of Indisulam according to the experimental design, and use culture medium containing a final concentration of less than 1% DMSO for the control group. Plate the required number of cells and the corresponding culture medium, and incubate statically in a 5% CO2 cell culture incubator at 37℃ for 4 days. (5) Flow cytometry staining and instrumentation: The procedure is the same as in I; The results are as follows Figure 3 The results showed that the Indisulam treatment group inhibited T cell proliferation and promoted T cell apoptosis, and the effect was concentration-dependent.

[0023] Example 4: Establishment and Treatment of Graft-versus-Host Disease (GVHD) Model Recipient mouse irradiation pretreatment: BALB / c mice aged 7-8 weeks were purchased and observed for their overall condition after entering the room. After confirming that there were no abnormalities, the mice were allowed to acclimatize for one week. One day before modeling, all mice received a whole-body irradiation dose of 5.5 Gy. After irradiation, they were fed antibiotic solution (ciprofloxacin: 20 μg / ml) for one week.

[0024] GVHD modeling: Preparation of single-cell suspensions of donor mouse bone marrow cells: Wild-type mice aged 8-10 weeks were prepared and euthanized by cervical dislocation. They were placed on a foam board lined with 70% alcohol-soaked paper towels. The skin was cut open to fully expose the limbs. The humerus (upper limb), femur (lower limb), and fibula (lower limb) were harvested. The muscles were separated, and the epiphyses on both sides of the bones were cut open to reveal the red bone marrow. Using a 20ml syringe, 1×PBS was drawn and repeatedly rinsed with the bone marrow until it turned white, indicating complete rinsing. The bone marrow cell suspension was obtained from the above sites and centrifuged (500g, 5min). The supernatant was discarded, and an appropriate volume of erythrocyte lysis buffer was added. Lysis was performed at room temperature for 5min. After lysis, an appropriate volume of 1×PBS buffer was added, and the cells were transferred to a cell culture chamber for cell counting at a rate of 5×10⁶ cells per mouse. 6 Obtain the corresponding number of cells for each bone marrow cell count for future use.

[0025] Preparation of donor mouse spleen CD4 + T cell suspension: Mice aged 8-10 weeks were prepared (wild-type mice were used for experiments specifically reducing RBM39 protein levels with small molecule drugs, and drug-induced knockout mice were used for drug-induced reduction of RBM39 levels). Mice were euthanized by cervical dislocation and placed on a foam board lined with 70% alcohol-soaked paper towels. The mouse skin was cut open, and the spleen was harvested. The spleen was ground and filtered through a nylon cell filter in 1×PBS buffer, centrifuged (500g, 5min), and the supernatant was discarded. An appropriate volume of erythrocyte lysis buffer was added, and lysis was performed at room temperature for 5min. After lysis, an appropriate volume of 1×PBS buffer was added. In a clean bench in the cell culture room, using a CD4+ T cell sorting kit, the spleen single-cell suspension was separated and counted according to the manufacturer's instructions to obtain spleen CD4+ T cells. + T cell suspension, at a rate of 0.5 × 10⁶ cells per mouse. 6 Spleen CD4 + Obtain the corresponding number of T cells for future reference.

[0026] Inoculated mice: BALB / c mice were anesthetized with an anesthetic, and the anesthesia was terminated after the mice’s respiratory rate slowed down. The cells to be used in steps (1) and (2) were mixed with an appropriate volume of 1×PBS, and 100 ml of cell suspension was drawn up with a 1 ml insulin syringe and injected into the orbital venous plexus of BALB / c mice.

[0027] Weigh and score Starting from the day of modeling, BALB / c mice were weighed every other day. After signs of disease appeared, the mice were weighed, scored, and their survival status was recorded daily. The scoring criteria are shown in Table 2.

[0028] Table 2. Clinical scoring criteria for graft-versus-host disease (5) Treatment of graft-versus-host disease by specifically reducing RBM39 protein levels with small molecule drugs: A graft-versus-host disease (GVHD) model was constructed using wild-type mice according to Example 4. Starting from the day of graft reception, BALB / c mice in the experimental group were intraperitoneally injected with Indisulam at a dose of 25 mg / kg for 6 consecutive days. The control group received the same volume of Indisulam solvent hydroxypropyl β-cyclodextrin intraperitoneally. The survival of BALB / c mice was observed daily from the day of model establishment, and they were weighed and scored 3 times per week. Data were recorded to create weight curves, disease score curves, and survival curves. Experimental results are as follows: Figure 4 The Indisulam group mice showed delayed progression of GVHD and significantly improved survival rate.

[0029] (6) Using drug-induced gene knockout mice to reduce RBM39 levels to treat graft-versus-host disease: Using the drug-induced gene knockout mice from Example 2, graft-versus-host disease was constructed according to Example 4. Two days before receiving the graft, recipient mice were intraperitoneally injected with Tamoxifen (20 mg / ml, prepared with corn oil) at a dose of 75 mg / kg, for five consecutive days. From the day of modeling, the survival of BALB / c mice was observed daily, and they were weighed and scored three times a week. Data were recorded to create weight data, disease score data, and survival data curves. The experimental results are as follows: Figure 5 Lowering RBM39 levels can effectively delay the progression of GVHD and improve the survival rate of mice.

[0030] Example 5: Pathological analysis of recipient mice in acute graft-versus-host disease: According to the operation steps of Example 4, CD45.1 mice were selected to prepare a suspension of CD4+ T cells in the spleen. At the same time, cell proliferation labeling was performed according to (4) in Experiment 2: the recipient mice were treated for 5 consecutive days from the day they received irradiation (the experimental group received Indisulam treatment to specifically reduce the level of RBM39 protein, and the control group received solvent hydroxypropyl β-cyclodextrin treatment). The mice were sacrificed on the fourth day after receiving the graft, and the spleen and peripheral lymph nodes of the mice were taken for lymphocyte separation. The cell status was analyzed by flow cytometry.

[0031] The specific steps of flow cytometry (FACS) are as follows: 1. Cell harvesting: ① Lymph node cell acquisition: The mice were euthanized by cervical dislocation and placed on a foam board lined with 70% alcohol disinfectant paper towels. The outer skin of the mice was cut open, and two lymph nodes each from the axilla, armpit, and groin were taken. The lymph nodes were ground and filtered in 1×PBS buffer using a nylon cell filter, centrifuged (500g, 5min), and then resuspended in 1×PBS buffer.

[0032] ② Spleen cell acquisition: Take spleen, follow the same steps as above, grind and filter, centrifuge (500g, 5min), resuspend in 1ml ACK Lysing Buffer, let stand at room temperature for 5min to lyse red blood cells, centrifuge (500g, 5min) and resuspend in 1×PBS buffer.

[0033] 2. Cell death staining: Centrifuge the cell suspension (500g, 5min), discard the supernatant, resuspend each sample in 100μl of the cell death staining solution (zombie yellow: 1×PBS buffer 1:1000), incubate at room temperature in the dark for 15min, centrifuge (500g, 5min), and resuspend in 1×PBS buffer.

[0034] 3. Cell surface staining: Prepare a surface antibody premix solution according to the ratio of surface antibody to FACS Buffer (0.5% FBS-PBS) 1:400. Centrifuge the suspension (500g, 5min), discard the supernatant, resuspend each sample in 50μl of surface antibody premix solution, incubate on ice in the dark for 30min, centrifuge (500g, 5min) and resuspend in FACS Buffer.

[0035] 4. Apoptosis staining: Dilute the apoptosis dye with Annexin V Binding Buffer at a ratio of 1:100, resuspend the cells in 50 μL per tube, and incubate at room temperature in the dark for 15 min. After staining, add an appropriate volume of Annexin V Binding Buffer to stop the apoptosis staining. Transfer the liquid to a flow cytometer tube and wait for it to be used. Important Note: For CTV-labeled cells, a certain amount of cells should be collected on the day of labeling for the above flow cytometry staining to confirm the CTV labeling status. When staining cells after the culture is completed, the CTV voltage should be kept consistent with the initial voltage.

[0036] 5. Cytokine / Transcription Factor Staining: Centrifuge the cell suspension (500g, 5min), discard the supernatant, and resuspend each sample in 50μl of the corresponding cytokine / transcription factor fixative. Incubate on ice in the dark for 30min. Centrifuge again (10000g, 1min), and resuspend each sample in 200μl of the corresponding Wash Buffer. Centrifuge again (10000g, 1min), and resuspend each sample in 50μl of cytokine / transcription factor staining solution (cytokine antibody:cytokine Wash Buffer 1:200, transcription factor antibody:transcription factor Wash Buffer 1:100). Incubate on ice in the dark for 30min. Centrifuge again (10000g, 1min), and resuspend each sample in 200μl of the corresponding Wash Buffer. (If simultaneously staining cytokines and transcription factors, a transcription factor kit can be used alone.) 6. Sample collection: Centrifuge the stained suspension (500g, 5min for unfixed, 10000g, 1min for fixed), discard the supernatant, resuspend in 150μl of FACS, and add 5ul of CountBright™ absolute counting beads to each tube. Filter the solution through a nylon mesh into the flow cytometry sample tube and perform flow cytometry fluorescence detection using a flow cytometer.

[0037] Experimental results are as follows Figure 6 As shown, specifically reducing RBM39 levels significantly reduced CD45.1 levels in the spleen and lymph nodes of recipient mice. + CD4 + T cell count and proportion; Figure 7 As shown, specifically reducing RBM39 levels can significantly inhibit CD45.1 in the spleen and lymph nodes. + CD4 + T cell proliferation;

[0038] Therefore, RBM39 can serve as a target for the treatment of GVHD. Knocking out or reducing the expression of RBM39 can inhibit the proliferation and activation of T cells, promote T cell apoptosis, and inhibit the differentiation of Th1 and Th17 cells.

[0039] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. Application of drugs that knock out or reduce RBM39 gene expression in the preparation of drugs for treating graft-versus-host disease.

2. The application according to claim 1, characterized in that: The graft-versus-host disease is GVHD mediated by T cells.

3. The application according to claim 1, characterized in that: The RBM39 gene expression was knocked out using drug-induced gene knockout.

4. The application according to claim 1, characterized in that: The drug used to reduce RBM39 gene expression is Indisulam.

5. The application according to claim 1, characterized in that: Knocking out or reducing the RBM39 gene is used to treat graft-versus-host disease by inhibiting T cell proliferation and promoting T cell apoptosis.

6. The application according to claim 1, characterized in that: Knocking out or reducing the RBM39 gene to treat graft-versus-host disease inhibits the differentiation of Th1 and Th17 cells and promotes the differentiation of Treg cells.

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