Application of TFEB in preparation of medicine for treating allergic rhinitis

Abstract

The invention belongs to the technical field of biological medicines, discloses application of TFEB in preparation of a medicine for treating allergic rhinitis, and particularly discloses application of a TFEB inhibitor in preparation of a medicine for treating and / or preventing rhinitis. The invention discloses the application of the TFEB inhibitor in prevention and treatment of rhinitis for the first time, proliferation and functional effects of ILC2 cells can be inhibited through targeted down-regulation of TFEB by the inhibitor, so that the purpose of treating rhinitis is achieved, namely allergic rhinitis is treated by inhibiting proliferation and functional effects of ILC2 cells through targeted TFEB.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of TFEB in preparing medicines for allergic rhinitis. Background Art

[0002] Allergic rhinitis (AR) is characterized by an immunoglobulin E (IgE)-promoted immune response to inhaled airborne allergens. Epidemiological data suggest that AR affects more than 10% of the global population, and recent studies report an increasing prevalence of the disease.

[0003] Type II innate lymphocytes (ILC2) are innate immune cells, mainly distributed in mucosal tissues, and their differentiation and function mainly depend on specific transcription factors GATA3 and RORα. There are few ILC2s in peripheral blood, but they are crucial to the occurrence of type 2 inflammatory responses and are an indispensable prerequisite for the occurrence of allergic diseases. Their activation mainly depends on cytokines such as IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) derived from the nasal epithelium. Type 2 cytokines (IL-4, IL-5 and IL-13) are the main inflammatory mediators that regulate allergic reactions.

[0004] Transcription factor EB (TFEB) is a transcription factor that binds to the Eμ heavy chain immunoglobulin enhancer. Recently, TFEB has been identified as a key transcriptional activator and lysosomal gene in autophagy. Phosphorylation of TFEB by mTORC1, ERK2, and GSK3 signaling pathways is closely related to its function. TFEB plays a role in mediating innate immunity by directly regulating proinflammatory / cytoprotective genes and upregulating the autophagy / lysosomal system after intracellular infection and phagocytosis. In AR, how TFEB-mediated autophagy regulates the metabolism and function of ILC2s and thus participates in the pathogenesis of AR has not been reported. Summary of the invention

[0005] The first aspect of the present invention aims to provide a use of a TFEB inhibitor.

[0006] The second aspect of the present invention aims to provide a sgRNA.

[0007] The purpose of the third aspect of the present invention is to provide biological materials related to the sgRNA of the second aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a product.

[0009] The purpose of the fifth aspect of the present invention is to provide the application of the sgRNA of the second aspect of the present invention, the biomaterial of the third aspect of the present invention or the product of the fourth aspect of the present invention.

[0010] The sixth aspect of the present invention aims to provide the use of TFEB as a target in the development of drugs for preventing and treating rhinitis.

[0011] In order to achieve the above object, the technical solution adopted by the present invention is:

[0012] The first aspect of the present invention provides a use of a TFEB inhibitor in any one of (1) to (7):

[0013] (1) Preparation of drugs for treating and / or preventing rhinitis;

[0014] (2) inhibiting the expression of IL-5 and / or IL-13;

[0015] (3) preparing an agent for inhibiting the expression of IL-5 and / or IL-13;

[0016] (4) inhibit the proliferation of ILC2 cells;

[0017] (5) preparing a reagent for inhibiting the proliferation of ILC2 cells;

[0018] (6) inhibiting the expression of ILC2-specific transcription factors GATA3 and / or RORα;

[0019] (7) Prepare an agent for inhibiting the expression of ILC2-specific transcription factors GATA3 and / or RORα.

[0020] In some embodiments of the present invention, the TFEB inhibitor is at least one of a substance that inhibits TFEB activity, a substance that degrades TFEB, a substance that reduces / knocks down the expression level of TFEB, and a substance that knocks out TFEB.

[0021] In some embodiments of the present invention, the TFEB inhibitor is a substance that reduces / knocks down the expression level of TFEB.

[0022] In some embodiments of the present invention, the TFEB inhibitor is at least one of a1) to a5):

[0023] a1) sgRNA, siRNA, dsRNA, miRNA, ribozyme or shRNA targeting TFEB;

[0024] a2) a nucleic acid molecule encoding the sgRNA, siRNA, dsRNA, miRNA, ribozyme or shRNA targeting TFEB described in a1);

[0025] a3) an expression cassette, a vector or a transgenic cell line comprising the nucleic acid molecule described in a2);

[0026] a4) Small molecule drugs that target and inhibit TFEB;

[0027] a5) TFEB inhibitory antibodies that block TFEB protein and / or TFEB protein intermediates.

[0028] In some embodiments of the present invention, the TFEB inhibitor is at least one of a1) to a5):

[0029] a1) sgRNA targeting TFEB;

[0030] a2) a nucleic acid molecule encoding the sgRNA targeting TFEB described in a1);

[0031] a3) an expression cassette, a vector or a transgenic cell line comprising the nucleic acid molecule described in a2);

[0032] a4) Small molecule drugs that target and inhibit TFEB;

[0033] a5) TFEB inhibitory antibodies that block TFEB protein and / or TFEB protein intermediates.

[0034] In some embodiments of the present invention, the TFEB inhibitory antibody is selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a surface remodeled antibody, a reconstructed antibody, a fully human antibody or an antigen-binding portion thereof specific for TFEB.

[0035] In some embodiments of the present invention, the TFEB inhibitory antibody is a monoclonal antibody specific for TFEB.

[0036] In some embodiments of the present invention, the small molecule drug targeted to inhibit TFEB includes Eltrombopag (EO).

[0037] In some embodiments of the present invention, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:3.

[0038] In some embodiments of the present invention, (1) the rhinitis includes allergic rhinitis.

[0039] In some embodiments of the present invention, the inhibition of IL-5 and / or IL-13 expression in (2) to (3) is the inhibition of IL-5 and / or IL-13 expression in ILC2 cells.

[0040] In some embodiments of the present invention, the drug in (1) is in the form of an injection, a spray, a nasal drop, an inhaler or an oral agent.

[0041] According to a second aspect of the present invention, an sgRNA is provided, wherein the nucleotide sequence of the sgRNA is shown in SEQ ID NO:3.

[0042] The third aspect of the present invention provides a biological material related to the sgRNA of the second aspect of the present invention, wherein the biological material comprises any one of b1) to b12):

[0043] b1) a nucleic acid molecule encoding the sgRNA of the second aspect of the present invention;

[0044] b2) an expression cassette comprising the nucleic acid molecule described in b1);

[0045] b3) a vector comprising the nucleic acid molecule described in b1);

[0046] b4) a vector comprising the expression cassette described in b2);

[0047] b5) a transgenic cell line comprising the nucleic acid molecule described in b1);

[0048] b6) a transgenic cell line comprising the expression cassette described in b2);

[0049] b7) a transgenic cell line comprising the vector described in b3);

[0050] b8) a transgenic cell line comprising the vector described in b4);

[0051] b9) a recombinant microorganism containing the nucleic acid molecule described in b1);

[0052] b10) a recombinant microorganism containing the expression cassette described in b2);

[0053] b11) a recombinant microorganism containing the vector described in b3);

[0054] b12) A recombinant microorganism containing the vector described in b4).

[0055] In some embodiments of the invention, the transgenic animal cell line does not contain reproductive material.

[0056] In some embodiments of the present invention, the expression cassette comprises a 5' transcription control region, an open reading frame encoding the fusion protein of the first aspect of the present invention, a translation control signal, a 3' untranslated region (3'UTR) and a transcription termination signal.

[0057] In some embodiments of the present invention, the 5' transcription control region comprises a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeping" promoter, may be used), a transcription start site, an enhancer and / or a silencer element.

[0058] In some embodiments of the invention, the 3'UTR may encode an AU-rich element, which is a common regulator of mRNA stability through the 3'-5' exosome pathway and is usually located in the 3'UTR. The AU-rich element may contain one or more repeats of the sequence AUUUA. It may also contain one or more so-called US2B elements having the sequence AUAUAU.

[0059] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0060] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a viral vector.

[0061] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

[0062] In some embodiments of the present invention, the non-viral vector includes at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0063] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0064] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0065] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new species of plants or animals.

[0066] In some embodiments of the present invention, the prokaryotic cells include Escherichia coli, Streptomyces, Bacillus subtilis and other bacteria well known in the art that can be used to express target proteins.

[0067] In some embodiments of the present invention, the eukaryotic cell comprises at least one of a yeast cell, a mammalian cell, a plant cell and an insect cell.

[0068] The fourth aspect of the present invention provides a product, comprising the sgRNA of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, or a TFEB inhibitory antibody that targets and blocks TFEB protein and / or TFEB protein intermediates.

[0069] In some embodiments of the present invention, the TFEB inhibitory antibody is selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a surface remodeled antibody, a reconstructed antibody, a fully human antibody or an antigen-binding portion thereof specific for TFEB.

[0070] In some embodiments of the present invention, the TFEB inhibitory antibody is a monoclonal antibody specific for TFEB.

[0071] In some embodiments of the present invention, the product includes a drug, a reagent and / or a kit.

[0072] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0073] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an absorption promoter, a surfactant, an antioxidant, an excipient, a stabilizer, a flavoring agent, a sustained-release agent, a metal complexing agent, an inert gas, a preservative, a filler, a local analgesic, a pH regulator, and an isotonic or isotonic regulator.

[0074] In some embodiments of the present invention, the diluent includes but is not limited to: at least one of starch, sugar, cellulose, and inorganic salts. The wetting agent includes but is not limited to: at least one of water, ethanol, and propanol. The adhesive includes but is not limited to: at least one of starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, and polyethylene glycol. The disintegrant includes but is not limited to: at least one of microcrystalline cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked povidone, surfactants (such as sodium dodecylbenzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate, etc.), and effervescent disintegrants. The lubricant includes but is not limited to: at least one of talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, micropowder silica gel, calcium carbonate, sodium bicarbonate, and polyethylene glycol. The color and flavor regulator includes but is not limited to: at least one of pigments, flavors, sweeteners, mucilage agents, and deodorants. The solvent includes but is not limited to: at least one of water, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, ethyl acetate, and vegetable oil (such as soybean oil, castor oil, peanut oil, blending oil, etc.). The solubilizer includes but is not limited to: at least one of Tweens, benzyl alcohols, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates. The cosolvent includes but is not limited to: at least one of organic acids and their salts, amide and amine compounds, inorganic salts, polyethylene glycol, povidone, and glycerol. The emulsifier includes but is not limited to: at least one of Spans, Tweens, benzyl alcohols, benzyl alcohols, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxide, silicon dioxide, and bentonite. The suspending agent includes but is not limited to: at least one of glycerol, syrup, gum arabic, gum tragacanth, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol, and thixotropic glue. The antioxidant includes but is not limited to: at least one of sulfites, pyrosulfites, bisulfites, ascorbic acid, gallic acid and its esters. The metal complexing agent includes but is not limited to: at least one of disodium ethylenediaminetetraacetic acid and polycarboxylic acid compounds; the inert gas includes but is not limited to: at least one of nitrogen and carbon dioxide. The preservative includes but is not limited to: at least one of parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils. The filler includes at least one of starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose or glucose. The flavoring agent includes at least one of aspartame, sucralose, flavor, stevioside, acesulfame potassium, citric acid or saccharin sodium. The local analgesic includes, but is not limited to, at least one of benzyl alcohol, chlorobutanol, lidocaine, and procaine.The pH regulator includes, but is not limited to, at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, and citrate. The isotonic or isotonic regulator includes, but is not limited to, at least one of glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

[0075] In some embodiments of the present invention, the drug can be administered orally, parenterally (e.g., intravenously, intramuscularly, intraperitoneally), intracranially, intrathecally, intraarterially (e.g., via the carotid artery), rectally, cutaneously, nasally, vaginally, by inhalation, on the skin (patch), or ophthalmically, and the effective dose administered is a pharmaceutically acceptable dose.

[0076] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is selected from tablets (such as ordinary tablets, double-layer tablets, multi-layer tablets, sustained-release tablets, single-chamber controlled-release tablets, double-chamber controlled-release tablets, microporous controlled-release tablets, sublingual tablets, orally rapidly disintegrating tablets, dispersible tablets, enteric-coated tablets), pills, patches, powders, suspensions, gels, emulsions, creams, granules, solutions, nanoparticles, drops, capsules (such as ordinary capsules, sustained-release capsules, controlled-release capsules, capsules containing micropellets or small tablets, pH-dependent capsules containing micropellets or small tablets, gastrointestinal composite capsules), enemas, suppositories, granules, injections, sprays and injections.

[0077] The fifth aspect of the present invention provides the use of the sgRNA of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, or the product of the fourth aspect of the present invention in any one of (1) to (7):

[0078] (1) Preparation of medicines for treating and / or preventing rhinitis;

[0079] (2) inhibiting the expression of IL-5 and / or IL-13;

[0080] (3) preparing an agent for inhibiting the expression of IL-5 and / or IL-13;

[0081] (4) inhibit the proliferation of ILC2 cells;

[0082] (5) preparing a reagent for inhibiting the proliferation of ILC2 cells;

[0083] (6) inhibiting the expression of ILC2-specific transcription factors GATA3 and / or RORα;

[0084] (7) Prepare an agent for inhibiting the expression of ILC2-specific transcription factors GATA3 and / or RORα.

[0085] In some embodiments of the present invention, (1) the rhinitis includes allergic rhinitis.

[0086] In some embodiments of the present invention, the inhibition of IL-5 and / or IL-13 expression in (2) to (3) is the inhibition of IL-5 and / or IL-13 expression in ILC2 cells.

[0087] A sixth aspect of the present invention provides the use of TFEB as a target in the development of drugs for preventing and treating rhinitis.

[0088] In some embodiments of the present invention, the rhinitis comprises allergic rhinitis.

[0089] The beneficial effects of the present invention are:

[0090] The present invention discloses for the first time the use of TFEB inhibitors in the prevention and treatment of rhinitis. By targeting and downregulating TFEB with the inhibitor, the proliferation and functional effects of ILC2 cells can be inhibited, thereby achieving the purpose of treating rhinitis, that is, allergic rhinitis is treated by targeting TFEB to inhibit the proliferation and functional effects of ILC2 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0091] Figure 1 Figure 2 shows the differences in the proportion and functional phenotype of ILC2 in the nasal mucosa between AR and control groups; A is a comparison of the proportion of ILC2 cells in the nasal mucosa of AR patients (10 cases) and normal controls (10 cases) detected by flow cytometry, B is a statistical graph of the flow cytometry detection results of A, C is a comparison of the proportion of IL-5+ILC2 and IL-13+ILC2 cells in the nasal mucosa of AR patients and normal control groups detected by flow cytometry, D to E are statistical graphs of the results of flow cytometry detection of the proportion of IL-5+ILC2 (D) and IL-13+ILC2 cells (E) in the nasal mucosa of AR patients and normal control groups.

[0092] Figure 2 Figure 3 Correlation between the expression of autophagosomes and autophagy-related proteins in nasal mucosal ILC2 of AR and control patients and the proportion and functional phenotype of ILC2 cells; A is the comparison of the number of autophagosomes in the nasal mucosa of AR patients and normal controls by transmission electron microscopy, B is the comparison of the expression of autophagy-related proteins (LC3II, P62, ATG5 and TFEB) in nasal mucosal ILC2 of AR patients and normal controls by Western blot, C is the correlation between the expression of autophagy-related proteins (LC3II, P62, ATG5) in nasal mucosal ILC2 of AR and the proportion of ILC2 cells, D is the difference in the expression of TFEB in nasal mucosal ILC2 of AR and control groups by immunofluorescence, the scale bar is 1:800, HC, normal control group, AR, patients with allergic rhinitis, * represents P < 0.05.

[0093] Figure 3To investigate the difference in TFEB expression in ILC2 of nasal mucosa in AR patients and normal controls and its correlation with the proportion of IL-5+ILC2 and IL-13+ILC2 cells in nasal mucosa.

[0094] Figure 4 To overexpress or knock down the expression level of TFEB gene in ILC2 cells.

[0095] Figure 5 The proliferation and functional changes of ILC2 after lentiviral vector intervention of TFEB expression in ILC2. In the figure, TFEB-OV is the TFEB overexpression group, TFEB-KD is the TFEB knockout group, and * represents P < 0.05.

[0096] Figure 6 The therapeutic effect and immune regulation of TFEB antagonist on AR mouse model; A is the comparison of Der p1-specific IgE content in each group, B is the comparison of the number of sneezes recorded within 15 minutes in each group, C is the comparison of the number of nose scratching recorded within 15 minutes in each group, D is the comparison of ILC2 content in nasal mucosa in each group, E is the comparison of IL-5+ILC2 content in nasal mucosa in each group, and F is the comparison of IL-13+ILC2 content in nasal mucosa in each group. DETAILED DESCRIPTION

[0097] The present invention is further described in detail below through specific examples.

[0098] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0099] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0100] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0101] Example 1

[0102] 1) Patient inclusion

[0103] According to the 2022 AR diagnosis and treatment guidelines of the Chinese Medical Association, the inclusion criteria for AR patients are as follows: typical perennial nasal symptoms (nasal congestion, nasal itching, sneezing and runny nose for at least 1 year), age over 18 years old, skin prick and serum specific IgE tests indicate mite allergy (at least one of house dust mite or dust mite); Exclusion criteria are as follows: recent (within 2 weeks) systemic or nasal use of glucocorticoids; accompanied by upper respiratory tract infection; accompanied by respiratory diseases such as acute and chronic sinusitis, cystic fibrosis, and asthma; accompanied by severe systemic diseases. The normal control group is patients aged 18 years or older, with negative allergens in skin prick and serum specific IgE tests, no respiratory diseases and systemic diseases, and who are scheduled to undergo partial inferior turbinate resection. The patients were from the Women and Children's Medical Center Affiliated to Guangzhou Medical University. With the informed consent of the patients and the approval of the Ethics Committee, peripheral blood (20mL) and nasal mucosa were collected from the two groups of patients for standby use.

[0104] 2) Sample preparation, human peripheral blood mononuclear cell (PBMC) preparation and ILC2 sorting

[0105] ①Human peripheral blood: Fresh heparin-anti-venous blood (20 mL) from AR patients was thoroughly mixed with RPMI-1640 medium at a ratio of 1:1, then slowly added to a test tube containing an appropriate amount of lymphocyte separation fluid (Ficoll), and centrifuged at 2200 rpm for 25 minutes at room temperature. PBMCs were collected, washed, and resuspended in RPMI-1640. Peripheral blood lineage negative (Lin) cells were sorted from PBMCs using a lineage cell depletion kit (Miltenyi Biotec). - ) cells (CD2, CD3, CD14, CD16, CD19, CD56, CD235a and FcεRI), and then stained with PE-labeled CRTH2 and PE-Cy7-labeled CD127 and sorted by flow cytometry to obtain Lin - CD127 + CRTH2 + , that is, ILC2 cells are obtained.

[0106] ② Human nasal mucosa: One to three nasal scrapings were performed on the inferior turbinate of the patient using a Rhino probe (Arlington Scientific, Springville, Utah), and each sample was placed in RPMI medium for later use. Li was sorted from PBMCs using a lineage cell removal kit (Miltenyi Biotec). n- Cells (CD2, CD3, CD14, CD16, CD19, CD56, CD235a and FcεRI), and Lin was further sorted by flow cytometry - CD127 + CRTH2+ , that is, ILC2 cells are obtained.

[0107] The above flow cytometry sorting process is as follows: PBMCs were purified using Ficoll-Paque PLUS (Fenghao, China). To reduce nonspecific binding, cells were pre-incubated with 5% fetal bovine serum (FBS, Fenghao, China) on ice for 10 minutes and then stained with a fixable viability dye (Fenghao, China) to exclude non-viable cells. ILC2 cells are defined as follows: human ILC2 cells: Lin - CD127 + CRTH2 + , among which Lin - Markers include CD2, CD3, CD14, CD16, CD19, CD56, CD235a and FcεRI. Mouse ILC2 cells: Lin - ST2 + CD45 + CD90.2 + CD25 + Sca1 + .

[0108] Flow cytometry was used to detect and compare ILC2 cells in the nasal mucosa of AR patients and normal controls. The results showed differences in the proportions of IL-5+ILC2 and IL-13+ILC2 cells. The proportions of ILC2, IL-5+ILC2 and IL-13+ILC2 derived from the nasal mucosa of AR patients were significantly higher than those in the normal control group, suggesting the importance of ILC2 cell proliferation and function in the development of AR.

[0109] 3) Transmission electron microscopy

[0110] Tissues (nasal mucosa) or cells were fixed with 4% glutaraldehyde solution overnight, then treated with 1% OsO4 (osmium tetroxide) in 0.1 mol / L dicarbonate buffer containing 0.1% CaCl2 for 2 h (4°C), and then stained with uranyl acetate and dehydrated with increasing concentrations of ethanol. Ultrathin sections were embedded in epoxy resin at 60°C for 48 h and cut into sections. The autophagosomes of ILC2 were counted using a transmission electron microscope (model H-7100FA; Hitachi, Tokyo, Japan) after staining with 0.1% lead citrate and 4% uranyl acetate.

[0111] Transmission electron microscopy was used to compare the number of autophagosomes in ILC2 of AR patients and normal controls. The results are shown in the figure. The autophagy of ILC2 derived from the nasal mucosa of AR patients was significantly stronger than that of the normal control group. The number of autophagosomes in ILC2 increased ( Figure 2 (A).

[0112] 4) Western Blot

[0113] Western Blot was used to detect the expression of autophagy-related proteins (LC3II, P62, ATG5, and TFEB) in nasal mucosal ILC2 of AR patients and normal controls, and the correlation between the expression of autophagy-related proteins (LC3II, P62, ATG5) and the proportion of ILC2 cells in AR nasal mucosa ILC2 was further analyzed. The experimental process of Western Blot was as follows: the sample was treated with cell lysis buffer, and protease inhibitors were added at the same time, and the supernatant was obtained by centrifugation. The protein sample was added to the polyacrylamide gel, and the protein was separated in the gel by the electric field. The protein separated by electrophoresis was transferred from the gel to the membrane using the wet transfer method. The membrane was treated with blocking buffer to prevent nonspecific binding. The membrane was incubated with specific antibodies (primary antibodies, LC3II, P62, ATG5, and TFEB) to allow the primary antibody to bind to the target protein, and then the antibody bound to the primary antibody (sheep anti-human IgG (1:10000), Thermo Fisher, AH1109925) was added to form an antibody complex. The color was developed using chemiluminescence, and then imaged using a fluorescence imager for quantitative analysis.

[0114] The results showed that the expression of autophagy-related proteins LC3II, P62, ATG5 and TFEB in ILC2 was significantly upregulated compared with the normal control group ( Figure 2 B and C).

[0115] 5) Immunofluorescence

[0116] Immunofluorescence technique was used to detect the difference in the expression of transcription factor EB (TFEB) in nasal mucosal ILC2 between the AR group and the control group, and the correlation between the expression of TFEB in AR nasal mucosal ILC2 and the proportion of IL-5+ILC2 cells and IL-13+ILC2 cells in the nasal mucosa was further analyzed. Immunofluorescence staining kit (P0188, Beyotime Biotechnology) was used, and the experimental process was as follows: the sample tissue cell suspension was centrifuged and smeared, and the cells or tissues were fixed with a fixative and then treated with a permeabilizer. Use blocking solution to block cells or tissues, add primary antibodies (TFEB (3μg / mL), GATA3 (5μg / mL), DAPI (0.1μg / mL), all purchased from R&D system), incubate at room temperature or overnight at 4°C, then add secondary antibodies (NL001 (1:200), NL002 (1:200), NL003 (1:200), all purchased from R&D system) labeled with the corresponding species of the primary antibody, and incubate at room temperature in the dark. After each antibody incubation, wash several times with PBS buffer, add autofluorescence quencher, and seal with anti-fluorescence quenching sealing agent. Observe and take pictures under a fluorescence microscope, and record the experimental results.

[0117] The results showed that TFEB expression was positively correlated with the proportion and functional phenotype of ILC2 cells, IL-5+ILC2 cells, and IL-13+ILC2 cells in the nasal mucosa ( Figure 2 Medium D and Figure 3 )

[0118] Example 2

[0119] (1) Regulate (overexpress / knockdown) TFEB expression in ILC2 cells isolated from the peripheral blood of AR patients using lentiviral vectors. The specific process is as follows:

[0120] ILC2 cells (sorted in Example 1) were revived and cultured in advance to ensure that the cells were in a good growth state. The TFEB regulatory sequence was inserted into the appropriate site of the lentiviral vector to construct an expression vector. It was roughly as follows: primers were designed based on the human TFEB gene sequence in the NCBI database, and EcoRI and BamHI restriction sites and protective bases were added to both ends by aligning the sequences. The upstream of the primer sequence was 5'-CGGAATTCTGATGGCGTCACGCATAGGGTTGC-3' (SEQ ID NO: 1), and the downstream was 5'-CGGGATCCCGCAGCACATCGCCCTCCTCCATG-3' (SEQ ID NO: 2). Among them, the nucleotide sequence of the sgRNA used for knockdown expression was 5'-CATTGACAACATTATGCGTC-3' (SEQ ID NO: 3). The constructed expression vector was transfected into specific packaging cells. One day before transfection, HEK 293T cells in good growth state were seeded in 6-well plates. When the cell confluence reached about 70%, 105μg of TFEB-DNA3.1-Flag-PCDH, 3.75μg of packaging plasmid pSPAX2 and 1.5μg of pMD2G were added to 500μL Optimum, mixed to prepare DNA mixture, and allowed to stand at room temperature for 5 minutes. At the same time, 500μL Optimum and 20μL Lipofactamine2000 were mixed, allowed to stand at room temperature for 20 minutes, and then the DNA mixture was mixed with them and evenly dropped into HEK 293T cells. The cells were placed at 37℃ and 5% After 6 hours of transfection in a CO2 environment, the culture medium was replaced with fresh complete medium containing 10% FBS. After 48 hours of continuous culture, the cell supernatant was collected, filtered using a 0.45 μm filter, and centrifuged at 4°C 12000 r / min for 5 minutes to obtain TFEB overexpression lentivirus. The TFEB knockdown lentivirus was obtained in the same way. Virus particles were produced on a large scale through cell culture and virus amplification processes.

[0121] 2×10 6THP-1 cells were inoculated into 6-well plates, and when the cells grew to a confluence of 50% to 60%, a corresponding volume of virus stock solution was added according to the virus infection multiplicity and the number of cells; after 24 hours of infection, the virus-free adaptive culture medium was replaced, and the cells were cultured in a cell culture incubator for another 24 hours, and the virus-infected cells were screened using an adaptive culture medium containing ampicillin (2 μg / ml), and the cells were cultured for 3 to 4 days until very few THP-1 cells survived, so that the THP-1 containing the TFEB-DNA3.1-Flag-PCDH10 lentivirus was stably passaged, thereby obtaining a THP-1 cell line with high expression of the TFEB gene, namely the overexpression group (over expression, OV), a THP-1 cell line transfected with a TFEB gene knockdown, namely the knockdown group (Gene knock-down, KD), and a THP-1 cell line transfected with an empty plasmid without the TFEB gene as the control group.

[0122] The expression level of TFEB gene in ILC2 cells was detected by RT-PCR and Western blot to determine whether ILC2 cells were knocked down or overexpressed.

[0123] like Figure 4 As shown, ILC2 cells were successfully knocked down or overexpressed TFEB.

[0124] (2) CFSE detection of ILC2 cell proliferation after TFEB knockdown

[0125] CFSE was used to detect the proliferation of ILC2 cells after TFEB knockdown, as follows: CFSE reagent (Abmole, catalog number M5117) was taken out of the refrigerator, and opened after being cooled to room temperature. CFSE was dissolved in DMSO to prepare a storage solution, and then diluted in serum-free DMEM culture medium to prepare a working solution. The cell concentration was adjusted to 1*10 5 / mL, add an appropriate amount of CFSE working solution (take out 1mL from 10mM CFSE prepared with DMSO), gently stir to mix, and incubate in a 37℃ incubator for 20 minutes. After the incubation, centrifuge to remove the supernatant, add PBS solution to wash the cells, and repeat this operation 2 to 3 times to remove excess CFSE. Add the washed cells to RPMI 1640 culture medium to make a cell suspension, and then place it in a culture dish for culture. Analyze the CFSE fluorescence intensity of the cells to track cell division.

[0126] The results are as follows Figure 5 As shown, after overexpressing TFEB expression in ILC2s sorted from the peripheral blood of AR patients using a lentiviral vector, the proliferation and functional effects of ILC2s were enhanced. Conversely, knocking down TFEB expression inhibited the proliferation and functional effects of ILC2s.

[0127] (3) RT-PCR detection of differential expression of ILC2-specific transcription factors GATA3 and RORα

[0128] RT-PCR was used to detect the expression differences of ILC2-specific transcription factors GATA3 and RORα. The detection process is as follows:

[0129] 30 mg of tissue was taken and ground with liquid nitrogen, and total RNA was extracted using an RNA extraction kit (Guangzhou Feiyang Bioengineering Co., Ltd.) according to the instructions. 260 / A 280 Subsequent experiments can be performed when the pH is between 1.8 and 2.1. 1 μg of total RNA was reverse transcribed to synthesize cDNA, and amplified according to the conditions in the instruction manual (the reagents were purchased from Toyobo Biotechnology Co., Ltd.), and detected in the Aoolied Biosustems 7 system. GADPH was used as the internal reference to calibrate the initial amount of the sample, and 2 -△△Ct The relative expression levels of ILC2-specific transcription factors GATA3 and RORα were calculated by the method.

[0130] RT-PCR test results Figure 5 After knocking down TFEB expression in ILC2s sorted from the peripheral blood of AR patients using a lentiviral vector, the expression of ILC2-specific transcription factors GATA3 and RORα was downregulated, while overexpression of TFEB enhanced the expression of ILC2-specific transcription factors GATA3 and RORα.

[0131] (4) ELISA detection of IL-5 expression differences in ILC2 cells

[0132] ELISA was used to detect the expression difference of IL-5 in ILC2 cells. The specific detection process is as follows:

[0133] Take 30 mg of tissue and add appropriate amount of RIPA (tissue / cell lysis buffer) according to the instructions and grind it thoroughly on ice. The lysed sample was centrifuged at 12,000 r / min for 5 minutes and the supernatant was taken. The protein concentration was then determined using the BCA method (Biyuntian BCA protein concentration determination kit). The protein expression levels of IL-5 and IL-13 in all samples were detected using an enzyme-linked detection kit (Shanghai Jianglai Biotechnology Co., Ltd.), and the operation steps were carried out according to the instructions of the kit.

[0134] ELISA test results Figure 5 After TFEB expression was overexpressed in ILC2s isolated from the peripheral blood of AR patients using a lentiviral vector, IL-5 expression was enhanced, while knocking down TFEB expression inhibited IL-5 expression.

[0135] The above experimental conclusions suggest that by targeting and inhibiting the expression of TFEB in ILC2, the proliferation of ILC2 cells and the expression of their main inflammatory cytokine IL-5 can be significantly inhibited, thereby significantly inhibiting the occurrence and development of allergic inflammation.

[0136] Example 3

[0137] 1) Experimental mice

[0138] Eight-week-old female BALB / c mice were randomly divided into a normal group, an AR group, and an anti-TFEB group, with 6 mice in each group. The AR group and the anti-TFEB group were used to establish sensitized AR models by inducing ovalbumin (OVA). The sensitized AR model was established as follows: the mice were intraperitoneally injected with 0.5 mL of a saline suspension containing 100 μg OVA (0.2 mg / mL, Invivogen, USA) and 2 mg aluminum hydroxide (4 mg / mL, Sigma-Aldrich, USA) on days 0, 2, 4, 6, 8, 10, and 12, and 10% OVA was instilled into the nasal cavity at 10 μL per mouse on days 14 to 27. The normal group was injected with saline at the corresponding time points. The establishment of the sensitized AR model was confirmed by recording the number of scratching and sneezing of the mice to determine whether the model was successfully established. The AR group and the anti-TFEB group were instilled with 10% OVA at 10 μL per mouse on days 14 to 27. The normal group was injected with saline at the corresponding time points. The anti-TFEB group was intraperitoneally injected with 0.4 mg / mL anti-TFEB (anti-TFEB antibody purchased from Abcam, USA, catalog number ab245349) at a dose of 10 mL / kg 30 minutes before nasal instillation of OVA on days 14, 18, 22, and 26. The number of nasal scratching and sneezing was recorded within 10 minutes after the last administration in all groups, and samples were collected the next day. All mice were anesthetized and blood was collected from the eye sockets. The blood samples were anticoagulated with sodium heparin. After euthanasia, alveolar lavage fluid and nasal lavage fluid were collected, and the complete nasal mucosa and lungs were taken.

[0139] The confirmation of the injection concentration of anti-TFEB antibodies mainly relies on preliminary experiments. The inventors injected concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.0 mg / mL, and 2.0 mg / mL. Comparison showed that 0.4 mg / mL had the best effect on improving the symptoms of AR mice, and no adverse events occurred in mice at all concentrations.

[0140] 2) Isolation of ILC2 cells in mouse nasal mucosa: Sensitized AR model mice were euthanized, and then the entire mouse nose was peeled and peeled, and cut into small pieces with scissors. The slices were digested with 1 mg / mL collagenase IV and 10 U / mL DNase I with continuous stirring at 37°C for 40 minutes. After filtering with a 70 μm cell strainer, the cells were resuspended in 40% Percoll medium and gently added to 80% Percoll medium. Centrifuge at 400 × g for 15 minutes at room temperature. Mononuclear cells (MNCs) were collected from the interface between 40% and 80% Percoll medium. The MNCs were then washed twice with ice-cold PBS. Lin was sorted from PBMCs using a lineage cell removal kit (Miltenyi Biotec). - Cells (CD2, CD3, CD14, CD16, CD19, CD56, CD235a and FcεRI) were further sorted by flow cytometry (the experimental process was the same as in Example 1). - ST2 + CD45 + , that is, mouse-derived ILC2 cells were obtained.

[0141] 3) Isolation of ILC2 cells from mouse peripheral blood: Fresh heparinized venous blood was added to a test tube containing lymphocyte separation fluid (Ficoll), centrifuged for 25 minutes (2200rpm), PBMCs were collected, washed, and suspended in RPMI-1640. The sorting strategy was the same as that of mouse nasal mucosa. Mouse-derived ILC2 cells were obtained.

[0142] The results are as follows Figure 6 As shown in the figure, TFEB antagonists (anti-TFEB antibodies) have therapeutic effects on AR (AR symptoms are relieved after TFEB administration) and immune regulation. Specifically, the anti-TFEB group had a higher level of Der p 1-specific IgE ( Figure 4 Middle A), nasal mucosal ILC2 cells ( Figure 4 Middle D), nasal mucosal IL-5+ILC2 cells ( Figure 4 E) and IL-13+ILC2 cells ( Figure 4 The content of F) in mice sneezes ( Figure 4 Middle B) and the number of nose scratching ( Figure 4 C) was also significantly reduced.

[0143] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of a TFEB inhibitor in any one of (1) to (7): (1) Preparation of medicines for treating and / or preventing rhinitis; (2) inhibiting the expression of IL-5 and / or IL-13; (3) preparing an agent for inhibiting the expression of IL-5 and / or IL-13; (4) inhibit the proliferation of ILC2 cells; (5) preparing a reagent for inhibiting the proliferation of ILC2 cells; (6) inhibiting the expression of ILC2-specific transcription factors GATA3 and / or RORα; (7) Prepare an agent for inhibiting the expression of ILC2-specific transcription factors GATA3 and / or RORα.

2. The use according to claim 1, characterized in that The TFEB inhibitor is at least one of a substance that inhibits TFEB activity, a substance that degrades TFEB, a substance that reduces / knocks down the expression level of TFEB, and a substance that knocks out TFEB.

3. The use according to claim 2, characterized in that: The TFEB inhibitor is at least one of a1) to a5): a1) sgRNA, siRNA, dsRNA, miRNA, ribozyme or shRNA targeting TFEB; a2) a nucleic acid molecule encoding the sgRNA, siRNA, dsRNA, miRNA, ribozyme or shRNA targeting TFEB described in a1); a3) an expression cassette, a vector or a transgenic cell line comprising the nucleic acid molecule described in a2); a4) Small molecule drugs that target and inhibit TFEB; a5) TFEB inhibitory antibodies that block TFEB protein and / or TFEB protein intermediates; Preferably, the nucleotide sequence of the sgRNA is as shown in SEQ ID NO:

3.

4. The use according to claim 3, characterized in that: The TFEB inhibitory antibody is selected from a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a surface remodeled antibody, a reconstructed antibody, a fully human antibody or an antigen-binding portion thereof specific for TFEB.

5. The use according to any one of claims 1 to 4, characterized in that: (1) The rhinitis includes allergic rhinitis.

6. A sgRNA, the nucleotide sequence of which is shown in SEQ ID NO:

3.

7. A biological material related to the sgRNA according to claim 6, wherein the biological material comprises any one of b1) to b12): b1) a nucleic acid molecule encoding the sgRNA according to claim 6; b2) an expression cassette comprising the nucleic acid molecule described in b1); b3) a vector comprising the nucleic acid molecule described in b1); b4) a vector comprising the expression cassette described in b2); b5) a transgenic cell line comprising the nucleic acid molecule described in b1); b6) a transgenic cell line comprising the expression cassette described in b2); b7) a transgenic cell line comprising the vector described in b3); b8) a transgenic cell line comprising the vector described in b4); b9) a recombinant microorganism containing the nucleic acid molecule described in b1); b10) a recombinant microorganism containing the expression cassette described in b2); b11) a recombinant microorganism containing the vector described in b3); b12) A recombinant microorganism containing the vector described in b4).

8. A product comprising the sgRNA of claim 6, the biomaterial of claim 7, or a TFEB inhibitory antibody that targets and blocks TFEB protein and / or TFEB protein intermediates.

9. Use of the sgRNA of claim 6, the biomaterial of claim 7 or the product of claim 8 in any one of (1) to (7): (1) Preparation of medicines for treating and / or preventing rhinitis; (2) inhibiting the expression of IL-5 and / or IL-13; (3) preparing an agent for inhibiting the expression of IL-5 and / or IL-13; (4) inhibit the proliferation of ILC2 cells; (5) preparing a reagent for inhibiting the proliferation of ILC2 cells; (6) inhibiting the expression of ILC2-specific transcription factors GATA3 and / or RORα; (7) preparing an agent for inhibiting the expression of ILC2-specific transcription factors GATA3 and / or RORα; Preferably, (1) the rhinitis includes allergic rhinitis.

10. Application of TFEB as a target in the development of drugs for the prevention and treatment of rhinitis.

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