Novel screening method for myositis therapeutic agent

Abstract

To provide a novel screening method that enables the discovery of agents for treating, preventing, or ameliorating myositis.SOLUTION: Provided is a screening method for a nucleic acid compound targeting the RasGRP4 gene, including the steps of: introducing a nucleic acid compound targeting the RasGRP4 gene into monocyte cells or macrophage cells; culturing the resulting cells; and measuring the amount of mRNA per unit volume present in the culture.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present application relates to a screening method for creating a therapeutic agent for myositis. [Background technology]

[0002] Polymyositis / dermatomyositis (PM / DM) is an autoimmune disease in which inflammation occurs throughout the body, mainly in the muscles, skin, and lungs, causing muscle weakness, mainly in the proximal muscles. PM / DM is often accompanied by interstitial pneumonia, which can lead to a poor prognosis. The number of PM / DM patients in Japan is 17,000 (2010), and the prevalence is reported to be 13.2 per 100,000 (see, for example, Non-Patent Document 1). Since the exact cause of the disease is unknown, the standard treatment is mainly the administration of nonspecific immunosuppressants, including glucocorticoids and other immunosuppressants.

[0003] Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a guanine exchange factor that activates Ras and is highly expressed in myeloid cells such as monocytes, macrophages, neutrophils, and mast cells. Histological analysis of muscle biopsy samples from PM patients has shown that the expression levels of MHC class I molecules in muscle cells and the amounts of cytotoxic factors (perforin and granzymes) in CD8-positive T cells are increased (see non-patent literature 2), and that CD8-positive T cell clones proliferate in the peripheral blood of PM patients and infiltrate into muscle tissue (see non-patent literature 3, 4, and 5). It has been reported that the expression level of RasGRP4 in peripheral blood mononuclear cells derived from PM / DM patients is significantly higher than that in healthy subjects (see Non-Patent Document 6). It has been reported that in muscle tissue of mice with myositis induced by immunization with C protein, CD11b-positive cells include monocytes and macrophages, and the number of these cells is similar to that of T cells (see Non-Patent Document 7). T cells are part of the immune system. T cells react with foreign substances (antigens) in lymph nodes and other secondary lymphoid organs. After the foreign substances (antigens) are processed by antigen-presenting cells, T cells can recognize the antigens. Known antigen-presenting cells include dendritic cells, macrophages, and B cells. As a substance that inhibits the expression of the RasGRP4 gene, siRNA (Patent Document 1, Patent Document 2, Non-Patent Document 8) has been reported. It has been reported that administration of RasGRP4 siRNA into the ankle joint significantly reduces ankle arthritis scores and ankle diameter in collagen-induced arthritis rats compared to controls, and also reduces bone and cartilage destruction (see Non-Patent Document 8). It has also been reported that RasGRP4 knockout mice have reduced sodium dodecyl sulfate-induced colitis compared to wild-type mice (see Non-Patent Document 9). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2019-131502 A [Patent Document 2] JP 2020-015678 A [Non-patent literature]

[0005] [Non-Patent Document 1] Mod Rheumatol. 2014,24(3),pp 477-80 [Non-Patent Document 2] J Rheumatol. 1996,23(7),pp 1135-42 [Non-Patent Document 3] J Immunol. 2001,167(7),pp 4051-8 [Non-Patent Document 4] J Exp Med. 1995,181(5),pp 1863-8 [Non-Patent Document 5] J Clin Invest. 1993,91(6),pp 2880-6 [Non-Patent Document 6] Arthritis Res Ther. 2011,13(5),R154 [Non-Patent Document 7] Arthritis Rheumatol. 2007,56(4),pp 1304-14 [Non-Patent Document 8] Arthritis Rheumatol. 2015,67(2),pp 396-407 [Non-Patent Document 9] J Biol Chem. 2012,287(24),pp 20047-55 Summary of the Invention [Problem to be solved by the invention]

[0006] Existing drugs for the treatment of PM / DM have problems such as side effects and the emergence of treatment resistance, so there is a need for new drugs to treat, prevent, or improve myositis. An object of the present application is to provide a novel screening method that makes it possible to provide a drug for treating, preventing, or ameliorating myositis. [Means for solving the problem]

[0007] As mentioned above, the importance of CD8 positive T cells has been suggested in muscle disorders. On the other hand, RasGRP4 expressed in monocytes and macrophages may induce muscle damage via antigen-specific lymphocyte activation. Suppression of RasGRP4 expression in monocytes and macrophages suppresses muscle damage mediated by antigen-specific lymphocyte activation, and is expected to be effective in treating myositis.

[0008] The present inventors induced myositis in RasGRP4-deficient mice and normal mice, and measured the severity of myositis and limb muscle strength. The results showed that myositis was milder in RasGRP4-deficient mice than in normal mice. Therefore, drugs that inhibit RasGRP4 are expected to be able to treat, prevent and / or ameliorate myositis (particularly polymyositis / dermatomyositis). Furthermore, as a result of intensive research, the present inventors have found a method for screening drugs that inhibit the expression of the RasGRP4 gene by measuring the amount of RasGRP4 mRNA in monocytes and / or macrophages. They have also found a method for inducing myositis in normal mice and measuring the amount of RasGRP4 mRNA / histological score indicating the size of the lesion / limb muscle strength, thereby completing the present invention. That is, the present invention is characterized as follows.

[0009] 1. A method for screening a nucleic acid compound targeting the RasGRP4 gene, comprising: introducing a nucleic acid compound targeting the RasGRP4 gene into a monocyte or macrophage cell; Culturing the obtained cells; and Measuring the amount of mRNA present per unit volume in the culture. A method comprising: 2. 2. The method of claim 1, wherein the cell is a monocyte cell. 3. 3. The method according to claim 2, wherein the monocytic cells are human monocytic leukemia cells. 4. 3. The method according to claim 3, wherein the human monocytic leukemia cells are THP1 cells. 5. 2. The method of claim 1, wherein the cell is a macrophage cell. 6. 6. The method according to 5., wherein the macrophage cells are derived from mouse bone marrow or mouse peritoneal cavity. 7. The method according to 1., wherein the cells are a mouse monocyte-macrophage cell line. 8. 8. The method according to claim 7, wherein the mouse monocyte-macrophage cell line is RAW264.7 or J774A.1. 9. 9. The method according to any one of 1. to 8., wherein the step of introducing is carried out using a nucleic acid compound introduction reagent or an electroporation method. 10. A method for screening a nucleic acid compound targeting the RasGRP4 gene, comprising: Administering a nucleic acid compound targeting the RasGRP4 gene to the myositis-induced mouse; and Measuring the amount of mRNA per unit volume A method comprising: 11. 11. The method according to claim 10, comprising the step of producing a myositis-induced mouse by administering bupivacaine or a skeletal muscle C protein fragment to a normal mouse. 12. A method for screening for a compound for treating, preventing, and / or ameliorating myositis, comprising: administering the compound to myositis-induced mice; and Measuring the limb muscle strength of the mouse and / or measuring the size of the lesion. A method comprising: 13. 13. The method according to 12., comprising the step of producing a myositis-induced mouse by administering bupivacaine or a skeletal muscle C protein fragment to a normal mouse. 14. The method, wherein the compound is a nucleic acid compound that targets the RasGRP4 gene. 15. A pharmaceutical composition for treating, preventing and / or ameliorating myositis, comprising as an active ingredient a nucleic acid compound targeting the RasGRP4 gene or a pharmacologically acceptable salt thereof. 16. 15. The pharmaceutical composition described in 15., wherein the myositis is polymyositis and / or dermatomyositis. 17. 17. The pharmaceutical composition according to claim 15 or 16, wherein the nucleic acid compound is a siRNA. 18. 17. The pharmaceutical composition according to claim 15 or 16, wherein the nucleic acid compound is an antisense oligonucleotide. Effect of the Invention

[0010] The present application provides a novel screening method that enables provision of a drug for treating, preventing, or ameliorating myositis, and therefore a medicament that can treat, prevent, and / or ameliorate myositis. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of histological score measurement in RasGRP4-deficient mice in Evaluation Example 5, which is one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In the present invention, "RasGRP4" (RAS Guanyl Releasing Protein 4) means any protein of RasGRP4. "RasGRP4 mRNA" or "RasGRP4 gene mRNA" or "RasGRP4 mRNA" means mRNA encoding RasGRP4 protein, for example, as represented by SEQ ID NO: 1. "RasGRP4 pre-mRNA" or "RasGRP4 gene pre-mRNA" or "RasGRP4 pre-mRNA" means pre-mRNA encoding RasGRP4 protein, for example, as represented by SEQ ID NO: 2. For both "RasGRP4 mRNA" and "RasGRP4 pre-mRNA", thymine in the sequence listing is usually replaced with uracil.

[0013] The nucleic acid compound targeting the RasGRP4 gene in the present invention may be any nucleic acid compound having the function of targeting the RasGRP4 gene, and examples thereof include, but are not limited to, siRNA, antisense oligonucleotide, aptamer, miRNA target nucleic acid, miRNA mimic, CpG oligo, decoy, or improved drug delivery versions thereof. An "antisense oligonucleotide" (ASO) is an oligonucleotide that produces an antisense effect. Examples include, but are not limited to, DNA, gapmers, and mixmers, and may be RNA or an oligonucleotide designed to normally produce an antisense effect. ASOs are typically single-stranded antisense oligonucleotides, consisting of 10 to 30 bases, preferably 12 to 25 bases, and more preferably 14 to 20 bases. "siRNA" (small interfering RNA) is a low molecular weight double-stranded oligoribonucleotide. siRNA is involved in a phenomenon called RNA interference (RNAi) and suppresses gene expression by destroying the target mRNA. siRNA typically consists of 15 to 30 base pairs, preferably 21 to 23 base pairs. The 3' ends of the two nucleic acid strands often have two overhanging deoxythymidines (commonly written as tt, dTdT, etc.). The nucleic acid compound targeting the RasGRP4 gene in the present invention is preferably an siRNA, an antisense oligonucleotide, an miRNA mimic, a decoy, or an improved drug delivery product thereof, more preferably an siRNA or an ASO, or an improved drug delivery product thereof, and particularly preferably an ASO.

[0014] 1. In vitro screening method In one embodiment of the present invention, the method for screening a nucleic acid compound targeting the RasGRP4 gene of the present invention comprises: introducing a nucleic acid compound targeting the RasGRP4 gene into a monocyte or macrophage cell; Culturing the obtained cells; and Measuring the amount of mRNA present per unit volume in the culture. Includes.

[0015] The cells in the method of the present invention are monocytes, preferably mammalian monocytes, preferably mouse or human monocytes. More preferably, they are mouse bone marrow or mouse peritoneal, more preferably primary mouse bone marrow or primary mouse peritoneal. They may be chronic or acute monocytic leukemia cells, more preferably human monocytic leukemia cells, even more preferably THP1 cells. They may also be primary human monocytic cells, or human peripheral blood mononuclear cells. In the method of the present invention, the cells, such as monocytic leukemia cells, are cultured by conventional methods known to those skilled in the art, preferably in RPMI-1640 medium.

[0016] The cells in the method of the present invention may be macrophage cells, such as mammalian macrophage cells, preferably mouse or human macrophage cells. More preferably, they are mouse bone marrow or mouse peritoneal macrophage cells, more preferably primary mouse bone marrow or primary mouse peritoneal macrophage cells. They may be primary human macrophage cells. They may also be mouse monocyte-macrophage cell lines (e.g., RAW264.7 or J774A.1) or acute myeloid leukemia cells (e.g., TF-1 cells), but are not limited thereto.

[0017] In the method of the present invention, the macrophage cells are cultured by conventional methods known to those skilled in the art, preferably in OptiMEM medium.

[0018] The introduction step in the method of the present invention may be carried out using a nucleic acid compound introduction reagent or electroporation, but is not limited thereto.

[0019] The nucleic acid compound may be introduced into monocytes or macrophage cells with or without the use of a transfection reagent. When a transfection reagent is used, examples of the transfection reagent include Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific), Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific), Lipofectamine LTX Reagent (Thermo Fisher Scientific), Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific), and ViaFect Transfection Reagent (Promega).

[0020] Furthermore, the introduction of nucleic acid compounds into monocytes or macrophage cells may or may not involve the use of electroporation. Electroporation is a physical transfection method in which an electric pulse is used to temporarily form pores in the cell membrane, and a substance such as nucleic acid is allowed to pass through the pores into the cells. This method can be performed using a gene transfer device. Examples of such devices include Nucleofector (registered trademark) (manufactured by LONZA).

[0021] The nucleic acid compound is introduced into monocytes or macrophage cells at, for example, 30 to 40°C, preferably 35 to 40°C, and more preferably 36 to 38°C. Culture is also carried out at the same temperature. After introduction, the culture period until the expression level of the RasGRP4 gene is measured is, for example, 1 day to 1 week, and preferably 1 to 3 days. The culture temperature is, for example, 30 to 40°C, preferably 35 to 40°C, and more preferably 36 to 38°C.

[0022] Instead of the monocytes or macrophages, screening can be performed in a similar manner using "cells expressing RasGRP4." The "cells expressing RasGRP4" are not particularly limited as long as they are cells in which "RasGRP4" can be detected by real-time PCR or the like, and examples of such cells include monocytes, macrophages, neutrophils, mast cells, and the like.

[0023] The method of the present invention preferably includes an evaluation step, which can be carried out, for example, by using the amount of mRNA as an indicator, comparing the amount of mRNA of a housekeeping gene with the amount of mRNA of the RasGRP4 gene, and / or by quantifying the amount of mRNA of the RasGRP4 gene by real-time PCR, but is not limited to these.

[0024] A specific example is the following real-time PCR method. First, RNA is isolated from cells using an RNeasy kit (QIAGEN), then reverse transcribed using a QuantiTect Reverse Transcription Kit (QIAGEN) to produce cDNA. The expression level of the RasGRP4 gene can then be measured using quantitative real-time PCR with SYBR Green 1 (LightCycler96 system, Roche).

[0025] Further, the following real-time PCR method can be mentioned. First, RNA was isolated from cells using the RNeasy kit (QIAGEN), and then reverse transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). Using the cDNA, the expression level of the RasGRP4 gene can be measured by quantitative real-time PCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific).

[0026] The degree of decrease in the expression level of RasGRP4 mRNA is not particularly limited because it depends on the concentration of the nucleic acid compound, but for example, a decrease of 20% or more, preferably 40% or more, and more preferably 50% or more is observed, which is preferable as a candidate for a drug for treating, preventing, and / or improving myositis. The concentration of the nucleic acid compound is not particularly limited, but can be appropriately set within the range of 0.1 nM to 10 μM (for example, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM).

[0027] The method of the present invention may include a step of comparing the expression level of RasGRP4 mRNA in the case where the nucleic acid compound is introduced as described above with the expression level of RasGRP4 mRNA in the above cell in the case where the nucleic acid compound is not introduced. Usually, a nucleic acid compound having an effect of reducing the expression level of RasGRP4 gene is selected as a candidate for a drug for treating, preventing and / or improving myositis. It is preferable to select a nucleic acid compound that reduces the expression level of RasGRP4 gene at a lower concentration as a candidate for a drug for treating, preventing and / or improving myositis. Furthermore, the selected candidates may be subjected to screening to confirm their therapeutic, preventive and / or ameliorative effects, if necessary.

[0028] 2. In vivo screening method The method of the present invention is a method for screening a nucleic acid compound targeting the RasGRP4 gene, comprising the steps of: Administering a nucleic acid compound targeting the RasGRP4 gene to the myositis-induced mouse; and Measuring the amount of mRNA per unit volume The method may include: The above method may include a step of preparing a myositis-induced mouse in advance, for example, by administering bupivacaine or a skeletal muscle C protein fragment to a normal mouse.

[0029] The method of the present invention is a method for screening a compound for treating, preventing, and / or ameliorating myositis, comprising the steps of: administering the compound to myositis-induced mice; and Measuring the limb muscle strength of the mouse and / or measuring the size of the lesion. The method may include: In the above method, the compound may be a nucleic acid compound that targets the RasGRP4 gene. The limb muscle strength measurement method and the method for measuring the size of the lesion can be used to evaluate not only nucleic acid compounds that target the RasGRP4 gene, but also compounds such as small molecules. Furthermore, the above method may include a step of preparing a myositis-induced mouse in advance, for example, by administering bupivacaine or skeletal muscle C protein fragment to a normal mouse.

[0030] Normal mice include, but are not limited to, C57BL / 6J mice.

[0031] The skeletal muscle C-protein fragment is preferably dissolved in Freund's complete adjuvant containing heat-killed Mycobacterium butyricum. The skeletal muscle C-protein fragment is preferably administered intradermally in the base of the tail and extremities.

[0032] The method of administration of the nucleic acid compound is not particularly limited, but subcutaneous administration is preferred. The dosage of the nucleic acid compound is not particularly limited, but is, for example, 1 to 500 mg / kg, preferably 3 to 300 mg / kg, 5 to 200 mg / kg, 10 to 100 mg / kg, and particularly preferably 30 to 60 mg / kg.

[0033] The expression level of RasGRP4 mRNA, the size of the lesion, and the muscle strength of the limbs can be used as indicators of evaluation.

[0034] The expression level of RasGRP4 mRNA can be measured in the same manner as in the above-mentioned in vitro screening method. Lesion size can be measured as a histological score, see Sci Rep. 2018;8:5498. Limb muscle strength can be measured using a mouse grip strength tester (eg, MK-380M).

[0035] The above-mentioned indicators are evaluated, for example, 2 to 100 days after immunization, preferably 3 to 60 days, more preferably 4 to 30 days, even more preferably 5 to 20 days, and particularly preferably 10 to 20 days.

[0036] The histological score of myositis can be assessed by sampling the quadriceps and / or flexor muscles and scoring the extent of lesions in each muscle, which show mononuclear cell infiltration and myocyte necrosis, for example, by hematoxylin-eosin (HE) staining. Scores will range from 1 to 6 as follows: 1 point: 1 muscle fiber 2 points: 2-5 muscle fibers 3 points: 6-15 muscle fibers 4 points: 16-30 muscle fibers 5 points: 31-100 muscle fibers 6 points: 100 or more muscle fibers If there are multiple lesions on one cross section and the scores are different, the lesion with the highest score is used, and if there are two lesions with the same score, 0.5 is added to that score. As the score per muscle, the average of the scores of multiple cross sections can be used, for example, the average of the scores of 1 to 5 cross sections, preferably the average of the scores of 2 cross sections. The sum of the quadriceps and femoral flexor muscles can be used as the score for each individual.

[0037] The method of the present invention may include a step of comparing the expression level of RasGRP4 mRNA, histological score and / or limb muscle strength when a nucleic acid compound is administered as described above with the expression level of RasGRP4 mRNA, histological score and / or limb muscle strength when a nucleic acid compound is not administered. Generally, a nucleic acid compound having the effect of decreasing the expression level of the RasGRP4 gene is selected as a candidate for a drug for treating, preventing and / or ameliorating myositis. Furthermore, compounds having the effect of reducing the size of the lesions are selected as candidates for drugs for treating, preventing and / or ameliorating myositis. Furthermore, compounds that have the effect of maintaining limb muscle strength at a value close to that of normal mice are selected as candidates for drugs for treating, preventing and / or ameliorating myositis. It is preferable to select a nucleic acid compound having the above-mentioned effect at a lower concentration and / or a compound having a greater effect as a candidate for a drug for treating, preventing and / or improving myositis.

[0038] 3. Medicines capable of treating, preventing and / or ameliorating myositis The method of screening nucleic acid molecules targeting the RasGRP4 gene of the present invention provides compounds (e.g., nucleic acid compounds) for treating, preventing and / or ameliorating diseases in which inhibition of RasGRP4 gene expression is effective, such as myositis, particularly polymyositis and / or dermatomyositis. The pharmaceutical composition obtained by the method of screening for a nucleic acid molecule targeting the RasGRP4 gene of the present invention preferably contains, as a nucleic acid compound, an siRNA or an antisense oligonucleotide, or an improved drug delivery version thereof.

[0039] The pharmaceutical composition containing the nucleic acid compound of the present invention or its pharmacologically acceptable salt can be formulated by a known pharmaceutical method. For example, it can be used enterally (orally, etc.) or parenterally as an injection, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating agent, pellet, troche, sublingual, chewable agent, buccal agent, paste, syrup, suspension, elixir, emulsion, liniment, ointment, plaster, cataplasm, transdermal preparation, lotion, inhalant, aerosol, suppository, etc.

[0040] In preparing these formulations, the composition may be appropriately combined with a carrier that is pharmacologically or acceptable as a food or beverage, specifically, sterile water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, pH regulator, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonicity agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, solubilizing agent, or other additives.

[0041] The administration form of the pharmaceutical composition containing the nucleic acid compound or its pharmacologically acceptable salt obtained by the screening method of the present invention is not particularly limited, and includes enteral (oral, etc.) or parenteral administration. More preferred are intravenous administration, intraarterial administration, intraarticular administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, intrathecal administration, intraventricular administration, intranasal administration, intravitreal administration, etc., and administration by infusion, more preferred are intravenous administration and subcutaneous administration, and particularly preferred is subcutaneous administration.

[0042] The preparation of pharmaceutical compositions containing the nucleic acid compound of the present invention or its pharmacologically acceptable salt can be carried out by known conventional methods.For example, the preparation of the nucleic acid compound of the present invention or its pharmacologically acceptable salt, which is prepared by adding additives such as buffer, isotonicity agent, etc., is completely dissolved in water for injection, and then sterilized by filtration.The preparation is filled into a sterilized syringe to produce a prefilled syringe preparation, filled into a sterilized vial to produce an injection preparation, and filled into a sterilized vial and then lyophilized to produce a preparation for preparation when used.Instead of sterilization by filtration, final sterilization such as autoclaving or gamma irradiation can be carried out.

[0043] Various mammalian diseases can be treated, prevented and / or ameliorated by the pharmaceutical composition comprising the nucleic acid compound of the present invention or its pharmacologically acceptable salt. For example, mammalian diseases can be treated, prevented and / or ameliorated, including, but not limited to, humans, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats, rabbits, chimpanzees or other rodent species such as bovines, ovines, equines, canines, felines, and mice. The mammal is particularly preferably a human.

[0044] When the pharmaceutical composition containing the nucleic acid compound of the present invention or its pharmacologically acceptable salt is administered or ingested to a mammal, including a human, the dosage or intake is preferably an effective amount. The effective amount means an amount of the compound sufficient to bring about a desired pharmacological effect in an individual who requires the drug, and varies depending on the age, weight, symptoms, health condition, and composition formulation of the individual to be treated, prevented, and / or improved, and is appropriately selected. The dosage or intake of the nucleic acid compound is preferably 0.0001 mg / kg / day to 100 mg / kg / day.

[0045] Preferred methods for using a pharmaceutical composition containing the nucleic acid compound of the present invention or a pharmacologically acceptable salt thereof include the following. A method for regulating expression of the RasGRP4 gene, comprising the step of contacting a cell with the nucleic acid compound of the present invention or a pharmacologically acceptable salt thereof. A method for regulating expression of the RasGRP4 gene in a mammal, comprising the step of administering to the mammal a pharmaceutical composition comprising the nucleic acid compound of the present invention or a pharmacologically acceptable salt thereof. Use of the nucleic acid compound of the present invention or a pharmacologically acceptable salt thereof for controlling expression of the RasGRP4 gene in a mammal. Use of the nucleic acid compound of the present invention or a pharmacologically acceptable salt thereof for producing a drug for controlling expression of the RasGRP4 gene in a mammal. EXAMPLES

[0046] The present invention will be described in more detail below with reference to examples, but the embodiments of the present invention are not limited to the following examples. In the examples, "Compd. No." means compound number.

[0047] In the chemical structure notations in the examples, unless otherwise specified, "(L)" means an LNA (β-D-methyleneoxy BNA) nucleoside, "(V)" means a 2'-O-MCE nucleoside, lowercase letters mean deoxyribonucleosides, uppercase letters (excluding "L" in (L) and "V" in (V)) mean ribonucleosides, "^" means a phosphorothioate bond, "5(x)" means that the nucleobase of the deoxyribonucleoside is 5-methylcytosine, and the "5" in "5(V)" and "5(L)" means that the nucleobase of the nucleoside is 5-methylcytosine.

[0048] In addition, in the notation of chemical structures in the examples, when there is no "^" between two adjacent nucleosides, the internucleoside bond between the two nucleosides is a phosphodiester bond. For example, in the notation "5(L)A(L)", the internucleoside bond between 5-methylcytosine and A is a phosphodiester bond, and in the notation "5(L)g", the internucleoside bond between 5-methylcytosine and g is a phosphodiester bond.

[0049] (Preparation of Nucleic Acid Compounds) The nucleic acid compounds listed in Table 1 were purchased from Thermo Fisher Scientific. siRNA-1 was an siRNA against human RasGRP4, and its catalog number was S41861. siRNA-2 was an siRNA against mouse RasGRP4, and its catalog number was S107413. [Table 1]

[0050] (Production example) The nucleic acid compounds listed in Table 2 were prepared using an automatic nucleic acid synthesizer nS-8II (Gene Design) or NTS M-8-SE (Nihon Techno Service). [Table 2] In addition, "C16-" in compound ASO-3 means that the part obtained by removing a hydrogen atom from the hydroxyl group at the 5' end is represented by the following formula: [ka] (wherein * represents the bonding position with respect to the oligonucleotide; that is, * represents the oxygen atom of the hydroxyl group at the 5'-terminus).

[0051] [Evaluation Example 1-1] Residual rate of human RasGRP4 mRNA in THP1 cells (human monocytic leukemia cell line) The effect of the nucleic acid compounds of Tables 1 and 2 that target RasGRP4 nucleic acid on RasGRP4 RNA transcripts was tested in vitro. The THP-1 cell suspension, adjusted to 2,000,000 cells / mL with phosphate buffered saline (PBS), was dispensed in 1 mL portions into 1.5 mL tubes, and the PBS was removed after centrifugation. The cells were then resuspended in 0.1 mL of Nucleofector solution V (LONZA, included in Nucleofector Kit V) containing the nucleic acid compound (150 pmol), transferred to a cuvette, and the nucleic acid compound was introduced into the cells using Nucleofector (LONZA, program: V-001). Next, 0.5 mL of pre-warmed RPMI-1640 medium (Sigma, hereafter referred to as RP10 medium) containing 10% heat-inactivated fetal bovine serum (Biological Industries) and penicillin / streptomycin (Gibco) was added, and the cells were transferred to a 6-well plate containing 1 mL of RP10 medium and incubated. After 48 hours, RNA was isolated from the cells using an RNeasy kit (QIAGEN), and then reverse transcribed to cDNA using a QuantiTect Reverse Transcription Kit (QIAGEN). Using the cDNA, the expression level of the RasGRP4 gene was measured by quantitative real-time PCR (LightCycler96 system, Roche) using SYBR Green 1. In the real-time PCR, the mRNA amount of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase [GAPDH] was also quantified at the same time, and the expression level of RasGRP4 was evaluated as the amount of RasGRP4 mRNA relative to the amount of GAPDH mRNA. The results are shown in Table 3 as the percentage expression of RasGRP4 relative to the untreated control cells.

[0052] [Table 3]

[0053] [Evaluation Example 1-2] Residual rate of human RasGRP4 mRNA in THP1 cells (human monocytic leukemia cell line) The effect of the nucleic acid compounds in Table 2 that target RasGRP4 nucleic acid on RasGRP4 RNA transcripts was tested in vitro. The THP-1 cell suspension, adjusted to 2,000,000 cells / mL with phosphate buffered saline (PBS), was dispensed in 1 mL portions into 1.5 mL tubes, and the PBS was removed after centrifugation. The cells were then resuspended in 0.1 mL of Nucleofector solution V (LONZA, included in Nucleofector Kit V) containing the nucleic acid compound (150 pmol), transferred to a cuvette, and the nucleic acid compound was introduced into the cells using Nucleofector (LONZA, program: V-001). Next, 0.5 mL of pre-warmed RPMI-1640 medium (Sigma, hereafter referred to as RP10 medium) containing 10% heat-inactivated fetal bovine serum (Biological Industries) and penicillin / streptomycin (Gibco) was added, and the cells were transferred to a 6-well plate containing 1 mL of RP10 medium and incubated. After 48 hours, RNA was isolated from the cells using an RNeasy kit (QIAGEN), and then reverse transcribed to cDNA using a QuantiTect Reverse Transcription Kit (QIAGEN). Using the cDNA, the expression level of the RasGRP4 gene was measured by quantitative real-time PCR (LightCycler96 system, Roche) using SYBR Green 1. In the real-time PCR, the mRNA amount of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase [GAPDH] was also quantified at the same time, and the expression level of RasGRP4 was evaluated as the amount of RasGRP4 mRNA relative to the amount of GAPDH mRNA. The results are shown in Table 4 as the percentage expression of RasGRP4 relative to the untreated control cells.

[0054] [Table 4]

[0055] [Evaluation Example 2] Residual rate of mouse RasGRP4 mRNA in primary mouse bone marrow-derived macrophages Bone marrow-derived macrophages collected from C57BL / 6J mice (8 weeks old, male and female) were cultured for 7 days, then seeded in a 12-well plate at 250,000 cells / mL per well and cultured overnight. The next day, nucleic acid compound (10 pmol) and Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) were incubated at room temperature for 20 minutes in OptiMEM medium (Sigma). 0.1 mL of the solution was added to the wells in which the cells were seeded and incubated overnight at 37°C. The next day, the medium was replaced with fresh culture medium and incubated at 37°C. After 48 hours, RNA was isolated from the cells using an RNeasy kit (QIAGEN), and then reverse transcribed using a QuantiTect Reverse Transcription Kit (QIAGEN) to produce cDNA. The RasGRP4 gene expression level was measured by quantitative real-time PCR (LightCycler96 system, Roche) using the cDNA prepared with SYBR Green 1. In the real-time PCR, the amount of mRNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase [GAPDH] was also quantified at the same time, and the amount of RasGRP4 mRNA relative to the amount of GAPDH mRNA was evaluated as the expression level of RasGRP4. The results are shown in Table 5 as the percentage expression of RasGRP4 relative to the untreated control cells. [Table 5]

[0056] [Evaluation Example 3] Residual rate of mouse RasGRP4 mRNA in RAW264.7 cells (mouse macrophage cell line) RAW264.7 cells were seeded in a 96-well plate at a density of 20,000 cells / well, and after about 24 hours, nucleic acid compounds were added using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). After about 48 hours, RNA was isolated from the cells using RNeasy Mini kit (QIAGEN), and then reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). Using the cDNA, the expression level of RasGRP4 gene was measured by quantitative real-time PCR using TaqMan Gene Expression Assays (Thermo Fisher Scientific). In real-time PCR, the amount of mRNA of the housekeeping gene HPRT was also quantified at the same time, and the amount of RasGRP4 mRNA relative to the amount of HPRT mRNA was evaluated as the expression level of RasGRP4.

[0057] [Evaluation Example 4] Residual rate of mouse RasGRP4 mRNA in muscle tissue of myositis-induced mice C protein-induced myositis (CIM), a model of polymyositis, was induced in C57BL / 6J mice (8-10 weeks old, female). CIM was induced by intradermal administration of recombinant human skeletal muscle C protein fragments (200 μg / body) together with complete Freund's adjuvant (CFA) at four sites, namely, at both sides of the tail base and at both sides of the hind paw base, intradermal administration of CFA at both sides of the forepaw base, and intraperitoneal administration of pertussis toxin (250 ng / body). On the 7th day after antigen immunization, C16-ASO-2 was administered subcutaneously once at 50 mg / kg, and 7 days after administration, the mice were dissected and the quadriceps muscles were removed. The negative control group received a subcutaneous administration of 10 ml / kg of PBS, the administration vehicle. The quadriceps muscle was soaked in TRI Reagent (Cosmo Bio), and the tissue was disrupted using a TissueLyser II. Bromochloropropane was added and mixed, and the aqueous layer was collected after centrifugation. RNA was isolated using a KingFisher Apex (Thermo Fisher Scientific). The RasGRP4 gene expression level was then measured using One-step PrimeScriptIII RT-qPCR Mix (Takara Bio). In real-time PCR, the mRNA amount of the housekeeping gene Hypoxanthine Phosphoribosyltransferase 1 [HPRT1] was also quantified at the same time, and the amount of RasGRP4 mRNA relative to the amount of HPRT1 mRNA was evaluated as the expression level of RasGRP4. As a result, the mRNA amount in the C16-ASO-2-treated group was 20-40% of that in the negative control group, demonstrating the inhibitory effect of RasGRP4 gene expression in vivo.

[0058] [Evaluation Example 5] Effect on myositis in RasGRP4-deficient mice C protein-induced myositis (CIM), a model of polymyositis, was induced in RasGRP4-deficient mice (8-10 weeks old, n=13) and wild-type C57BL / 6J mice (n=12). CIM was induced by intradermal administration of recombinant human skeletal muscle C protein fragment (200 μg / body) together with complete Freund's adjuvant (CFA) at four sites, namely, at the base of the tail on both sides and at the base of both hind limbs, intradermal administration of CFA at the base of both forelimbs, and intraperitoneal administration of pertussis toxin (250 ng / body). As the primary endpoint, the mice were euthanized 14 days after immunization, and the quadriceps and femoral flexors were collected and the severity of myositis was evaluated using a histological score (Sci Rep.2018;8:5498.). The histological score represents the size of the lesion. Specifically, two cross-sectional sections were prepared from different locations per muscle, and the severity of myositis was evaluated by scoring the extent of the lesion area where mononuclear cell infiltration and muscle cell necrosis were observed by HE staining. The scores were as follows: 1 point: equivalent to 1 muscle fiber, 2 points: equivalent to 2-5 muscle fibers, 3 points: equivalent to 6-15 muscle fibers, 4 points: equivalent to 16-30 muscle fibers, 5 points: equivalent to 31-100 muscle fibers, and 6 points: equivalent to 100 muscle fibers or more. If there were multiple lesions in one cross-section with different scores, the lesion with the highest score was used. If there were two lesions with the same score, 0.5 was added to the score. The total score of the quadriceps and femoral flexor muscles was used as the histological score of the individual. As a secondary outcome measure, limb muscle strength was measured using a mouse grip strength tester (MK-380M) before immunization, 6 days after immunization, and 14 days after immunization. Histological scores were analyzed using the Mann-Whitney U test, and limb muscle strength was analyzed using the Welch t test.

[0059] The histological score measurement results for each individual are shown in Figure 1. In the figure, "Score" indicates the histological score. The average histological score was 5.69 in wild-type mice and 1.52 in RasGRP4-deficient mice. Compared to wild-type mice, the histological scores were significantly lower in RasGRP4-deficient mice. This shows that polymyositis was milder in RasGRP4-deficient mice.

[0060] Table 6 shows the average limb muscle strength of wild-type mice (Wild Type) and RasGRP4-deficient mice (RasGRP4 KO) before immunization (Day 0), 6 days after immunization (Day 6), and 14 days after immunization (Day 14). [Table 6]

[0061] Wild-type mice showed a significant decrease in limb muscle strength 14 days after immunization. On the other hand, in RasGRP4-deficient mice, limb muscle strength 14 days after immunization was significantly higher than that in wild-type mice, indicating that CIM-induced muscle weakness was alleviated in RasGRP4-deficient mice. [Industrial Applicability]

[0062] The screening method of the present invention is useful for screening for therapeutic agents for myositis that target the RasGRP4 gene.

Claims

1. A method for screening a nucleic acid compound targeting the RasGRP4 gene, comprising: introducing a nucleic acid compound targeting the RasGRP4 gene into a monocyte or macrophage cell; Culturing the obtained cells; and Measuring the amount of mRNA present per unit volume in the culture. A method comprising:

2. The method of claim 1 , wherein the cell is a monocyte cell.

3. The method of claim 2 , wherein the monocytic cells are human monocytic leukemia cells.

4. The method of claim 3, wherein the human monocytic leukemia cells are THP1 cells.

5. The method of claim 1 , wherein the cell is a macrophage cell.

6. The method of claim 5 , wherein the macrophage cells are derived from mouse bone marrow or mouse peritoneal cavity.

7. The method of claim 1 , wherein the cells are a murine monocyte-macrophage cell line.

8. The method of claim 7, wherein the murine monocyte-macrophage cell line is RAW264.7 or J774A.

1.

9. The method according to claim 1 , wherein the step of introducing is carried out using a nucleic acid compound introduction reagent or an electroporation method.

10. A method for screening a nucleic acid compound targeting the RasGRP4 gene, comprising: administering a nucleic acid compound targeting the RasGRP4 gene to the myositis-induced mice; and Measuring the amount of mRNA per unit volume A method comprising:

11. The method according to claim 10, comprising the step of producing a myositis-induced mouse by administering bupivacaine or skeletal muscle C protein fragment to a normal mouse.

12. A method for screening for a compound for treating, preventing, and / or ameliorating myositis, comprising: administering the compound to myositis-induced mice; and Measuring the limb muscle strength of the mouse and / or measuring the size of the lesion. A method comprising:

13. The method according to claim 12, comprising the step of producing a myositis-induced mouse by administering bupivacaine or skeletal muscle C protein fragment to a normal mouse.

14. The method, wherein the compound is a nucleic acid compound that targets the RasGRP4 gene.

15. A pharmaceutical composition for treating, preventing and / or ameliorating myositis, comprising as an active ingredient a nucleic acid compound targeting the RasGRP4 gene or a pharmacologically acceptable salt thereof.

16. The pharmaceutical composition of claim 15, wherein the myositis is polymyositis and / or dermatomyositis.

17. The pharmaceutical composition according to claim 15 or 16, wherein the nucleic acid compound is an siRNA.

18. The pharmaceutical composition of claim 15 or 16, wherein the nucleic acid compound is an antisense oligonucleotide.

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