WO2024048528A1 - Prophylactic agent and/or therapeutic agent for heart diseases and laminopathy - Google Patents

Prophylactic agent and/or therapeutic agent for heart diseases and laminopathy Download PDF

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WO2024048528A1
WO2024048528A1 PCT/JP2023/031020 JP2023031020W WO2024048528A1 WO 2024048528 A1 WO2024048528 A1 WO 2024048528A1 JP 2023031020 W JP2023031020 W JP 2023031020W WO 2024048528 A1 WO2024048528 A1 WO 2024048528A1
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lsmem2
nucleic acid
gene
substance
expression
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French (fr)
Japanese (ja)
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泰範 新谷
佑典 ▲高▼橋
一生 矢澤
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国立研究開発法人国立循環器病研究センター
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Publication of WO2024048528A1 publication Critical patent/WO2024048528A1/en

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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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    • A61K35/76—Viruses; Subviral particles; Bacteriophages
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to a mechanosignal transduction inhibitor, an anti-inflammatory agent, a preventive and/or therapeutic agent for heart disease, a preventive and/or therapeutic agent for laminopathy, etc., which contain a substance that suppresses the expression or function of LSMEM2.
  • LMNA lamin gene
  • Non-patent document 1 LMNA gene variant is found in about 10% of patients with dilated cardiomyopathy, and lamin cardiomyopathy is an intractable disease with high penetrance that is accompanied by severe heart failure and fatal arrhythmia.
  • Non-Patent Document 2 LMNA gene search is recommended for familial dilated cardiomyopathy complicated by conduction disorders, but no disease-specific treatment for lamin cardiomyopathy has been established.
  • Lamin which is a protein lining the nuclear envelope, is known to control gene expression by propagating mechanostress from outside the cell to the nucleus via the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex.
  • SUN1 KO mice that lack SUN1, a protein constituting the LINC complex improve the survival prognosis of lamin cardiomyopathy model mice (Non-Patent Document 3). It is thought to be important in the onset of the disease. However, SUN1 is ubiquitously expressed and KO mice exhibit infertility and hearing loss, making it difficult to use as a therapeutic target.
  • Non-patent Document 4 Although no obvious side effects were observed in mice, there remain concerns about its suitability to humans, as SUN1 KO mice exhibit infertility and hearing loss, and the original LINC complex function is destroyed. Furthermore, p38 MAPK signaling is known to be enhanced in lamin cardiomyopathy, and p38 ⁇ inhibitors have been reported to improve cardiac function in lamin cardiomyopathy model mice (Non-Patent Document 5). A p38 ⁇ inhibitor is currently undergoing phase 3 clinical trials for lamin cardiomyopathy, but p38 ⁇ KO mice are embryonic lethal, and there are also concerns about its safety in humans.
  • An object of the present invention is to provide a preventive and/or therapeutic agent for heart disease and laminopathy, and in particular, to provide an effective preventive and/or therapeutic agent for lamin cardiomyopathy.
  • the present inventors discovered that the intercalated disc is a receptor for mechano stress, and uniquely identified LSMEM2, a membrane protein localized in the intercalated disc and involved in mechanosignaling.
  • LSMEM2 knockout mice exhibited normal cardiac function at rest. Then, they discovered that mice produced by crossing LSMEM2 knockout mice with lamin cardiomyopathy model mice have improved cardiac function, and completed the present invention. Furthermore, we found that LSMEM2 knockout mice suppressed immune cell infiltration in a pressure overload heart failure model due to aortic coarctation, and that mice produced by crossing with lamin cardiomyopathy model mice had improved lower limb muscle strength. We demonstrated that targeted therapy can be applied not only to lamin cardiomyopathy but also to severe heart failure and laminopathy due to other etiologies.
  • the present invention is as follows.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, (c) The agent according to [1], which is a ribozyme nucleic acid for the transcription product of the LSMEM2 gene.
  • the agent is a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
  • a prophylactic and/or therapeutic agent for laminopathy which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
  • a mechanosignal transduction inhibitor containing a substance that suppresses the expression or function of LSMEM2 as an active ingredient, which is a prophylactic and/or therapeutic agent for laminopathy.
  • the present invention is as follows.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene,
  • the agent according to [A1] which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
  • [A3] The agent according to [A1], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • [A4] The agent according to any one of [A1] to [A3], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The agent according to [A5], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [A7] The agent according to [A5], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • [A8] The agent according to any one of [A5] to [A7], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [A9] A prophylactic and/or therapeutic agent for heart disease, which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The agent according to [A9], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [A11] The agent according to [A9], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • [A12] The agent according to any one of [A9] to [A11], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [A13] The agent according to any one of [A9] to [A12], wherein the heart disease is dilated cardiomyopathy.
  • An inhibitor of vesicular transport abnormality which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The agent according to [A14], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [A16] The agent according to [A14], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • [A17] The agent according to any one of [A14] to [A16], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • a prophylactic and/or therapeutic agent for laminopathy which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The agent according to [A18], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [A20] The agent according to [A18], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • [A21] The agent according to any one of [A18] to [A20], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [A22] The agent according to any one of [A18] to [A21], wherein the laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, or leukodystrophy.
  • [B1] A method for inhibiting mechanosignaling in a subject, which comprises administering to the subject a substance that suppresses the expression or function of LSMEM2.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B1].
  • [B4] The method according to any one of [B1] to [B3], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [B5] A method for suppressing inflammation in a subject, comprising administering to the subject a substance that suppresses the expression or function of LSMEM2.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B5]. [B7] The method according to [B5], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • [B8] The method according to any one of [B5] to [B7], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [B9] A method for preventing and/or treating heart disease in a subject, comprising administering to the subject a substance that suppresses the expression or function of LSMEM2.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B9].
  • [B12] The method according to any one of [B9] to [B11], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [B13] The method according to any one of [B9] to [B12], wherein the heart disease is dilated cardiomyopathy.
  • [B14] A method for suppressing abnormal vesicle transport in a subject, comprising administering to the subject a substance that suppresses the expression or function of LSMEM2.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B14].
  • [B17] The method according to any one of [B14] to [B16], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [B18] A method for preventing and/or treating laminopathy, which comprises administering to a subject a substance that suppresses the expression or function of LSMEM2.
  • a substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B18].
  • [B21] The method according to any one of [B18] to [B20], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • the laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, or leukodystrophy.
  • a composition comprising a substance that suppresses LSMEM2 expression or function for use in inhibiting mechanosignaling.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The composition according to [C1], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [C3] The composition according to [C1], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • composition according to any one of [C1] to [C3], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • composition comprising a substance that suppresses LSMEM2 expression or function for use in suppressing inflammation.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The composition according to [C5], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [C7] The composition according to [C5], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • composition according to any one of [C5] to [C7], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • a substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The composition according to [C9], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [C11] The composition according to [C9], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • composition according to any one of [C9] to [C11], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • a composition comprising a substance that suppresses LSMEM2 expression or function for use in suppressing vesicular transport abnormalities.
  • a substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The composition according to [C14], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
  • the composition according to [C14], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • composition according to any one of [C14] to [C16], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • composition comprising a substance that suppresses the expression or function of LSMEM2 for use in the prevention and/or treatment of laminopathy.
  • a substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The composition according to [C18], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [C20] The composition according to [C18], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, or leukodystrophy.
  • [D2] The substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The use according to [D1], which is (c) a ribozyme nucleic acid directed against the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [D3] The use according to [D1], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • [D4] The use according to any one of [D1] to [D3], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [D5] Use of a substance that suppresses the expression or function of LSMEM2 in the manufacture of a medicament for suppressing inflammation.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, (c) a ribozyme nucleic acid for the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the use according to [D5]. [D7] The use according to [D5], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • [D8] The use according to any one of [D5] to [D7], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [D9] Use of a substance that suppresses the expression or function of LSMEM2 in the manufacture of a medicament for the prevention and/or treatment of heart disease.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, The use according to [D9], which is (c) a ribozyme nucleic acid for the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene. [D11] The use according to [D9], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  • [D12] The use according to any one of [D9] to [D11], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [D13] The use according to any one of [D9] to [D12], wherein the heart disease is dilated cardiomyopathy.
  • [D14] Use of a substance that suppresses the expression or function of LSMEM2 in the manufacture of a medicament for suppressing abnormal vesicular transport.
  • the substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, (c) a ribozyme nucleic acid for the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the use according to [D14].
  • [D17] The use according to any one of [D14] to [D16], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [D18] Use of a substance that suppresses the expression or function of LSMEM2 in the manufacture of a medicament for preventing and/or treating laminopathy.
  • a substance that suppresses the expression of LSMEM2 is (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene, (b) antisense nucleic acid against the transcript of the LSMEM2 gene, (c) a ribozyme nucleic acid for the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the use according to [D18].
  • [D21] The use according to any one of [D18] to [D20], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
  • [D22] The use according to any one of [D18] to [D21], wherein the laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, or leukodystrophy.
  • a mechanosignaling inhibitor containing a substance that suppresses the expression or function of LSMEM2, thus making it possible to effectively treat lamin cardiomyopathy, severe heart failure due to other causes, and laminopathy.
  • FIG. 1 is a diagram showing the organ specificity of LSMEM2 expression by RT-PCR.
  • FIG. 2 is a diagram showing the results of RNA-seq of cardiomyocytes forcibly expressing LSMEM2.
  • FIG. 3 is a diagram showing that LSMEM2 is essential for mechanostress (developmental stimulus) dependent inflammatory signals.
  • FIG. 4 is a diagram showing the production and analysis of LSMEM2 knockout mice.
  • FIG. 5 shows that LSMEM2 KO improves survival rate, cardiac function, cardiac inflammatory signals, and skeletal muscle strength in a laminopathy model (LMNA H222P KI/KI).
  • FIG. 6 is a diagram showing the cardiac function improving effect of knockdown by MyoAAV2 shRNA LSMEM2.
  • FIG. 7 shows an analysis of LSMEM2 knockout in a pressure overload heart failure model.
  • LSMEM2 which is the target molecule of the mechanosignaling inhibitor of the present invention, is a single-transmembrane protein localized in the intercalated disc, which is a receptor for mechano stress, and in humans, isoform 1 (NP_694947.1) and Isoform 2 (NP_001291314.1) exists, and isoform 1 has an amino acid sequence consisting of 164 amino acids represented by SEQ ID NO: 2 translated from mRNA represented by SEQ ID NO: 1, of which positions 1 to 99 are The intracellular region, positions 100 to 122 are the transmembrane region, and positions 123 to 164 are the extracellular region.
  • Serine at position 50 is a phosphorylation site
  • asparagine at position 156 is a glycosylation site.
  • Isoform 2 (SEQ ID NO: 4) translated from mRNA represented by SEQ ID NO: 3 is one amino acid shorter than isoform 1 and consists of 163 amino acids.
  • LMEM2 is a protein that includes an amino acid sequence that is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2 (isoform 1) or SEQ ID NO: 4 (isoform 2).
  • proteins and peptides are described with the left end being the N-terminus (amino terminal) and the right end being the C-terminus (carboxyl terminal), in accordance with the convention for peptide markings.
  • amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4" means (a) Human LSMEM2 consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 in other warm-blooded animals (e.g., guinea pig, rat, mouse, chicken, rabbit, dog, pig, sheep, cow, monkey, etc.) ); or (b) human LSMEM2 consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or the amino acid sequence in a natural allelic variant or genetic polymorphism of the ortholog in (a) above. do.
  • LSMEM2 is human LSMEM2 consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a natural allelic variant or genetic polymorphism thereof.
  • LSMEM2 KO mice develop normally, have completely normal cardiac function, and have no problems with reproductive function.
  • gene therapy for LSMEM2 genome editing, antibody drugs, nucleic acid drugs (including but not limited to antisense, siRNA, miRNA, aptamers, etc.), low molecular compounds, etc. can be used without any modality limitations, such as mRNA, proteins, and downstream signals. Suppression of either of these is a promising drug discovery target for new treatments.
  • One aspect of the present invention is a mechanosignal transduction inhibitor that contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
  • a "substance that suppresses the expression of LSMEM2" refers to a substance that edits the LSMEM2 gene at the genome level, or acts at any stage such as the transcription level, post-transcriptional regulation level, protein translation level, or post-translational modification level. It may be something that does. Therefore, substances that suppress the expression of LSMEM2 include, for example, substances that inhibit the transcription of the LSMEM2 gene (e.g., antigene), substances that inhibit the processing of early transcripts into mRNA, and substances that inhibit the transport of mRNA to the cytoplasm.
  • substances that suppress the expression of LSMEM2 include, for example, substances that inhibit the transcription of the LSMEM2 gene (e.g., antigene), substances that inhibit the processing of early transcripts into mRNA, and substances that inhibit the transport of mRNA to the cyto
  • substances that inhibit translation of mRNA into protein e.g., antisense nucleic acids, miRNA
  • degrade mRNA e.g., siRNA, ribozymes, miRNA
  • substances that inhibit post-translational modification of initial translation products This includes substances that induce the decomposition of LSMEM2.
  • Any substance that acts at any stage can be used, but substances that bind complementary to mRNA and inhibit translation into protein or degrade mRNA are preferred.
  • the substance that specifically inhibits translation of LSMEM2 gene mRNA into protein (or degrades mRNA) preferably includes a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA or a portion thereof.
  • the nucleotide sequence complementary to the nucleotide sequence of the mRNA of the LSMEM2 gene has such a degree of complementarity that it can bind to the target sequence of the mRNA and inhibit its translation (or cleave the target sequence) under physiological conditions.
  • nucleotide sequences preferably have a homology of 95% or more, more preferably 97% or more, particularly preferably 98% or more.
  • nucleotide sequence complementary to the nucleotide sequence of mRNA of the LSMEM2 gene is a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions.
  • stringent conditions refer to, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6 ⁇ SSC (sodium Examples include hybridization in chloride/sodium citrate)/45°C, followed by one or more washings in 0.2x SSC/0.1% SDS/50 to 65°C; Hybridization conditions that provide stringency equivalent to that can be selected as appropriate.
  • LSMEM2 gene mRNA is human LSMEM2 (RefSeq Accession No. NM_153215.3 or NM_001304385.2) containing the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, or its ortholog in other warm-blooded animals. Further examples include mRNAs such as natural allelic variants or genetic polymorphisms thereof.
  • a part of the nucleotide sequence that is complementary to the nucleotide sequence of the mRNA of the LSMEM2 gene is capable of specifically binding to the mRNA of the LSMEM2 gene, and inhibits translation of protein from the mRNA (or inhibits the translation of the mRNA).
  • the portion complementary to the target sequence should be at least 10 bases or more, preferably 15 bases or more, more preferably 19 bases or more. It contains more than a base.
  • nucleic acids containing part of a nucleotide sequence complementary to the nucleotide sequence of mRNA of the LSMEM2 gene are preferably exemplified as nucleic acids containing part of a nucleotide sequence complementary to the nucleotide sequence of mRNA of the LSMEM2 gene.
  • Nucleic acid or its precursor having RNAi activity against LSMEM2 gene mRNA (b) Antisense nucleic acid against LSMEM2 gene mRNA, or (c) Ribozyme nucleic acid against LSMEM2 gene mRNA
  • the "substance that suppresses the expression of LSMEM2” may be a substance that suppresses the expression of LSMEM2 by modifying the sequence of the LSMEM2 gene in the genome using genome editing technology.
  • the following (d) is preferably exemplified.
  • the substance that suppresses the expression of LSMEM2 in the present invention is not limited to a nucleic acid or a nucleic acid sequence recognition module containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the LSMEM2 gene, or a part thereof, as described above, Other substances such as low-molecular compounds, peptides, cyclic peptides, proteins, medium molecules, and polymers may be used as long as they inhibit directly or indirectly.
  • the substances that inhibit the expression of LSMEM2 in the present invention are not limited to the above-mentioned antisense nucleic acids, siRNA, ribozymes, etc., but may include other substances (low molecular compounds, etc.) as long as they directly or indirectly inhibit the expression of LSMEM2. , peptide, cyclic peptide, protein, medium molecule, polymer, etc.). Examples of such low-molecular compounds include low-molecular-weight phosphorylation inhibitors that inhibit phosphorylation of serine at position 50 of LSMEM2.
  • nucleic acid having RNAi activity against LSMEM2 gene mRNA or its precursor double-stranded RNA consisting of an oligo RNA complementary to LSMEM2 gene mRNA and its complementary strand, so-called siRNA , is defined as being encompassed by nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of mRNA of the LSMEM2 gene, or a portion thereof.
  • siRNA can be designed based on the cDNA sequence information of the target gene, for example, according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)).
  • Examples of the target sequence of siRNA include, but are not limited to, AA+(N) 19 , AA+(N) 21 , or NA+(N) 21 (N is any base).
  • the location of the target sequence is also not particularly limited.
  • homology search software such as BLAST to check whether there is any homology in consecutive 16-17 base sequences in mRNA other than the target, and determine the specificity of the selected target sequence. Check.
  • the target sequence after AA (or NA) for which specificity has been confirmed is Two strands consisting of a sense strand having a 3'-terminal overhang of TT or UU at bases 19-21, and an antisense strand having a sequence complementary to the 19-21 bases and a 3'-terminal overhang of TT or UU.
  • Strand RNA may be designed as siRNA.
  • siRNA short hairpin RNA
  • an arbitrary linker sequence for example, about 5 to 25 bases
  • the sense strand and antisense strand are linked together. It can be designed by connecting via a linker sequence.
  • siRNA and/or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include, but are not limited to, siDESIGN Center provided by Dharmacon, siRNA Target Finder provided by GenScript, and the like.
  • siRNA or shRNA synthesized based on the hit siRNA sequence information can actually suppress LSMEM2 expression in cardiomyocytes to a therapeutically effective extent means that, for example, the expression of LSMEM2 mRNA in myocardial stem cells into which the nucleic acid has been introduced It can be verified by measuring the level.
  • the in vivo stability can be improved by adding various modifications to the constituent nucleotides of siRNA or shRNA, as described below. By improving the activity, the effect of suppressing the expression of LSMEM2 can be further sustained, and the desired therapeutic effect can be achieved.
  • siRNA and shRNA of the present invention are sequences consisting of at least 15 consecutive nucleotides within the region represented by nucleotide numbers 1 to 1434 in the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.
  • the siRNA of the present invention is Silencer® Select siRNA (LSMEM2 siRNA ID: s189327, sense (5' ⁇ 3'): GCACAGUGGAGGCUCACUATT (SEQ ID NO: 9), antisense e(5' ⁇ 3' ): UAGUGAGCUCCACUGUGCAG (SEQ ID NO: 10) (Thermo Fisher Scientific, Waltham, MA), and the shRNA is 5'-CTCTCACTGATGGCTTCATTT-3' (SEQ ID NO: 11).
  • microRNA that targets the mRNA of the LSMEM2 gene is also defined as a nucleic acid that includes a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the LSMEM2 gene, or a portion thereof.
  • miRNAs are involved in post-transcriptional control of gene expression by binding complementary to target mRNAs and inhibiting translation of the mRNAs, or by degrading the mRNAs.
  • pri-miRNA which is a primary transcription product
  • precursor-microRNA pre-miRNA
  • Drosha precursor-microRNA
  • miRNA can be searched using target prediction software provided free of charge on various websites. Examples of such sites include TargetScan, published by the Whitehead Institute in the United States, and DIANA-micro-T-CDS, published by the Alexander Fleming Biomedical Research Center in Greece. but not limited to.
  • search for miRNAs that target LSMEM2 mRNA using TarBase a database of miRNAs that has been experimentally proven to act on target mRNAs, published by the University of Tessari, Institut Pasteur, etc. You can also do that. Sequence information on these miRNAs and/or pre-miRNAs can be obtained using, for example, miRBase published by the University of Manchester, UK.
  • the nucleotide molecules constituting siRNA and/or shRNA, or miRNA and/or pre-miRNA may be natural RNA or DNA, but stability (chemical and/or enzymatic) and specific activity (affinity with RNA) Various chemical modifications can be included in order to improve the properties.
  • the phosphate residues of each nucleotide constituting the antisense nucleic acid are chemically modified with phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithionate etc. It can be substituted with a phosphate residue.
  • the base moiety pyrimidine, purine
  • siRNA is produced by synthesizing the sense and antisense strands of the target sequence on mRNA using an automatic DNA/RNA synthesizer, denaturing them in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute, and then It can be prepared by annealing at about 30 to about 70°C for about 1 to about 8 hours. Alternatively, it can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving this using dicer. miRNA and pre-miRNA can be synthesized with an automatic DNA/RNA synthesizer based on their sequence information.
  • a nucleic acid designed to be able to generate siRNA or miRNA against LSMEM2 gene mRNA in vivo also refers to a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of LSMEM2 gene mRNA or a portion thereof. Defined as encompassing. Examples of such nucleic acids include expression vectors constructed to express shRNA, siRNA, miRNA, or pre-miRNA described above.
  • shRNA is an oligonucleotide containing a nucleotide sequence in which the sense and antisense strands of the target sequence on mRNA are connected by inserting a spacer sequence of a length (for example, about 5 to 25 bases) between them to form an appropriate loop structure. It can be prepared by designing RNA and synthesizing it using an automatic DNA/RNA synthesizer.
  • vectors that express shRNA tandem type and stem-loop (hairpin) type.
  • the former is an expression cassette for the sense strand of siRNA and an expression cassette for the antisense strand linked in tandem, and each strand is expressed and annealed within the cell to form double-stranded siRNA (dsRNA). It is.
  • an shRNA expression cassette is inserted into a vector, and the shRNA is expressed within cells and processed by dicer to form dsRNA.
  • a pol II promoter eg, CMV immediate early promoter
  • a pol III promoter is generally used to ensure accurate transcription of short RNAs.
  • examples of pol III promoters include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoters, and human valine-tRNA promoters.
  • a sequence of four or more consecutive T's is used as a transcription termination signal.
  • Expression cassettes for miRNA and pre-miRNA can also be produced in the same manner as shRNA.
  • the siRNA, shRNA, miRNA or pre-miRNA expression cassette thus constructed is then inserted into a plasmid vector or viral vector.
  • vectors include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai viruses, and animal cell expression plasmids.
  • adeno-associated virus AAV
  • DNA encoding the Rep protein and capsid protein required for virus particle formation is inserted into a separate plasmid.
  • a plasmid containing genes (E1A, E1B, E2A, VA, and E4orf6) responsible for adenovirus helper functions necessary for AAV proliferation is constructed as an adenovirus helper plasmid.
  • recombinant AAV ie, AAV vector
  • AAV vector recombinant AAV
  • the host cell it is preferable to use a cell (for example, 293 cell, etc.) that can supply a part of the gene product (protein) of the gene responsible for the helper action, and when such a cell is used, There is no need for the adenovirus helper plasmid to carry a gene encoding a protein that can be supplied from the host cell. Since the produced AAV vector exists in the nucleus, the desired AAV vector is recovered by freezing and thawing host cells, and is separated and purified by density gradient ultracentrifugation using cesium chloride, column method, etc. A vector is prepared.
  • Antisense nucleic acid to mRNA of LSMEM2 gene refers to a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA or a part thereof, It has the function of suppressing protein synthesis by binding to mRNA by forming a specific and stable double strand.
  • Antisense nucleic acids include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (e.g., commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or containing special linkages, provided that the polymers contain bases such as those found in DNA and RNA. (contains nucleotides with a configuration that allows for pairing and attachment of bases).
  • They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA:RNA hybrids, and also unmodified polynucleotides (or unmodified oligonucleotides), known modified with additions, such as with labels known in the art, with caps, with methylation, with the substitution of one or more natural nucleotides by analogues, with intramolecular nucleotide modifications. those with uncharged bonds (e.g. methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), those with charged or sulfur-containing bonds (e.g.
  • phosphorothioate, phosphorodithioate, etc. substances, such as proteins (e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (e.g., monosaccharides, etc.), which have side chain groups, intercurrent containing compounds (e.g., acridine, psoralen, etc.), containing chelating compounds (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, modified It may be one that has a bond (for example, an ⁇ -anomer type nucleic acid).
  • proteins e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.
  • sugars e.g., monosaccharides, etc.
  • side chain groups intercurrent containing compounds (
  • nucleoside may include not only those containing purine and pyrimidine bases, but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified on the sugar moiety, for example, one or more hydroxyl groups may be substituted with a halogen, an aliphatic group, etc., or with a functional group such as an ether, an amine, etc. It may have been converted.
  • the antisense nucleic acid may be DNA or RNA, or may be a DNA/RNA chimera.
  • the antisense nucleic acid is DNA
  • the RNA:DNA hybrid formed by the target RNA and antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only a sequence in mRNA but also a sequence in the intron region of the early translation product of the LSMEM2 gene.
  • the intron sequence can be determined by comparing the genome sequence and the cDNA nucleotide sequence of the LSMEM2 gene using a homology search program such as BLAST or FASTA.
  • the target region of the antisense nucleic acid of the present invention is not particularly limited in length as long as hybridization of the antisense nucleic acid results in inhibition of translation into protein;
  • the sequence may be the entire sequence or a partial sequence, and examples include the short sequence of about 10 bases and the long sequence of the entire mRNA or initial transcription product.
  • oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred, but are not limited thereto.
  • 3' end palindromic region, or 3' end hairpin loop may be selected as preferred target regions for antisense nucleic acids, but are not limited thereto.
  • the target region of the antisense nucleic acid of the present invention is a continuous region within the region represented by nucleotide numbers 1 to 1434 in the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, as in the above-mentioned siRNA. Mention may be made of sequences consisting of at least 15 nucleotides.
  • the antisense nucleic acid of the present invention not only hybridizes with the mRNA and early transcripts of the LSMEM2 gene and inhibits its translation into protein, but also binds to these genes, which are double-stranded DNA, to form triple-stranded ( It may also be a gene (antigene) that can form a triplex (triplex) and inhibit transcription into RNA.
  • nucleotide molecules constituting the antisense nucleic acid may also be modified in the same manner as in the case of siRNA, etc., in order to improve stability, specific activity, etc.
  • the antisense oligonucleotide of the present invention is prepared by determining the target sequence of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of the LSMEM2 gene, and using a commercially available DNA/RNA automatic synthesizer (Applied Biosystems, Beckman). etc.), and can be prepared by synthesizing a complementary sequence thereto. Furthermore, antisense nucleic acids containing the various modifications described above can also be chemically synthesized by techniques known per se.
  • Ribozyme nucleic acid for LSMEM2 gene mRNA Another example of a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of LSMEM2 gene mRNA or a part thereof is a nucleic acid that specifically cleaves the mRNA within the coding region. Examples include ribozyme nucleic acids obtained. In a narrow sense, "ribozyme” refers to RNA that has an enzymatic activity that cleaves nucleic acids, but in this specification it is used as a concept that also includes DNA as long as it has sequence-specific nucleic acid cleavage activity.
  • the most versatile ribozyme nucleic acids are self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known.
  • the hammerhead type exhibits enzymatic activity with about 40 bases, and several bases at each end (about 10 bases in total) adjacent to the hammerhead structure are made into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA.
  • This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA, since it only uses RNA as a substrate.
  • the target sequence can be made into a single strand by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase.
  • a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase.
  • a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of LSMEM2 gene mRNA or a part thereof may be provided in a special form such as a liposome or microsphere, applied to gene therapy, or provided in an attached form. It can be done.
  • These additive forms include polycations such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids (which enhance interaction with cell membranes and increase uptake of nucleic acids). examples, phospholipids, cholesterol, etc.).
  • Preferred lipids to be added include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.).
  • nucleic acid can be attached to the 3' or 5' end of the nucleic acid, and can be attached via base, sugar, or intramolecular nucleoside linkages.
  • Other groups include capping groups specifically placed at the 3' or 5' ends of nucleic acids to prevent degradation by nucleases such as exonucleases and RNases.
  • capping groups include, but are not limited to, hydroxyl protecting groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol.
  • the LSMEM2 protein expression suppressing activity of these nucleic acids can be investigated using a transformant into which the LSMEM2 gene has been introduced, an in vivo or in vitro LSMEM2 gene expression system, or an in vivo or in vitro LSMEM2 protein translation system.
  • Nucleic acid sequence recognition module that specifically binds to the target region in the LSMEM2 gene (d-1) Substance that does not involve modification of the genome sequence
  • Conventional genome editing methods require sequence-independent DNA cutting ability.
  • a well-known method is to use an artificial nuclease that combines a molecule with sequence recognition ability and a molecule with sequence recognition ability.
  • ZFN zinc finger nuclease
  • TAL transcription activator-like effector
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • nuclease Cas Clustered Regularly Interspaced Short Palindromic Repeats
  • CRISPR-associated A method using the CRISPR-Cas9 system in combination with a protein family has been reported. Furthermore, an artificial nuclease has been reported in which a nuclease is linked to a PPR protein configured to recognize a specific nucleotide sequence by a series of PPR motifs consisting of 35 amino acids and recognizing one nucleobase. There is.
  • a specific target nucleotide sequence is recognized by a protein (ZF, TAL effector, PPR) or a complex of RNA and protein (CRISPR-Cas9). Therefore, the expression of LSMEM2 can be suppressed by binding to the target region in the LSMEM2 gene using these substances (nucleic acid sequence recognition modules) that impart sequence specificity to artificial nucleases.
  • nucleic acid sequence recognition module refers to a molecule or molecular complex that has the ability to specifically recognize and bind to a specific nucleotide sequence (ie, target nucleotide sequence) on a DNA strand. Binding of a nucleic acid sequence recognition module to a target nucleotide sequence allows an effector linked to the module to act specifically on a targeted site of DNA.
  • the nucleic acid sequence recognition module includes the CRISPR-Cas system.
  • the CRISPR-Cas system uses a complex of short CRISPR RNA (crRNA) complementary to a target nucleotide sequence and transactivating crRNA (tracrRNA), or a single synthetic RNA (guide RNA) that combines crRNA and tracrRNA.
  • crRNA short CRISPR RNA
  • tracrRNA transactivating crRNA
  • guide RNA single synthetic RNA
  • any sequence can be targeted simply by synthesizing an oligo DNA that can specifically hybridize with the complementary sequence of the target nucleotide sequence.
  • a nucleic acid sequence recognition module using CRISPR-Cas is provided as a complex of a Cas protein and an RNA molecule (guide RNA) consisting of a nucleotide sequence complementary to the target nucleotide sequence and tracrRNA necessary for recruiting the Cas protein.
  • a nucleic acid sequence recognition module using CRISPR-Cas is provided as a complex of crRNA, tracrRNA, and Cas, which includes an RNA sequence complementary to the target nucleotide sequence.
  • the Cas protein used in the present invention is not particularly limited as long as it belongs to the CRISPR system, but is preferably Cas9.
  • Cas9 include Cas9 (SpCas9) derived from Streptococcus pyogenes and Cas9 (StCas9) derived from Streptococcus thermophilus. Not limited. Preferably it is SpCas9.
  • SpCas9 Cas9 derived from Streptococcus pyogenes
  • StCas9 Cas9 derived from Streptococcus thermophilus.
  • it is SpCas9.
  • Cas with inactivated DNA cleaving activity dCas
  • the 10th Asp residue is converted to an Ala residue
  • the D10A mutant lacks the ability to cleave the opposite strand of the strand that forms a complementary strand with the guide RNA
  • the 840th His residue is converted to an Ala residue.
  • a double mutant of the H840A mutant that lacks the ability to cleave the guide RNA and the complementary strand can be used, but other mutant Cas can be used as well.
  • CRISPR-Cas When CRISPR-Cas is used as a nucleic acid sequence recognition module, when a complex of guide RNA and Cas binds to the target nucleotide sequence, the LSMEM2 gene is blocked by the complex, and the LSMEM2 gene cannot be expressed.
  • the target nucleotide sequence for the guide RNA ACTGGCTCCTAACCACGTGCAGG
  • SEQ ID NO: 5 used in the Examples below may be mentioned, although it is not particularly limited.
  • crRNA in the case of the LSMEM2 gene, crRNA can be designed, for example, by using CGG in the sequence as PAM and a reverse strand sequence of 20 nucleotides immediately upstream from it as a target nucleotide sequence.
  • the nucleic acid sequence recognition module includes zinc finger motifs, TAL effectors, PPR motifs, etc., as well as restriction enzymes, transcription factors, RNA polymerases, and other proteins that can specifically bind to DNA.
  • a fragment that includes a binding domain and does not have DNA double-strand cleavage ability can be used.
  • nuclease e.g., FokI
  • nucleic acid sequence recognition module such as a zinc finger motif, TAL effector, and PPR motif
  • the nuclease can be sterically It is also possible to block the LSMEM2 gene and thereby suppress the expression of LSMEM2.
  • two nucleic acid sequence recognition modules that specifically recognize a positive nucleotide sequence upstream from the target region and a reverse nucleotide sequence downstream from the target region are designed, and the end of the module (e.g. (C-terminus) can form a complex with a deactivated nuclease.
  • Nucleic acid sequence recognition modules such as zinc finger motifs, TAL effectors, and PPR motifs can be provided as fusion proteins with the above-mentioned nucleases, or can be provided as fusion proteins with protein binding domains such as SH3 domains, PDZ domains, GK domains, GB domains, etc.
  • a binding partner may be fused to a nucleic acid sequence recognition module and a nuclease, respectively, and provided as a protein complex through interaction between the protein binding domain and its binding partner.
  • inteins can be fused to the nucleic acid sequence recognition module and the nuclease, respectively, and the two can be linked by ligation after each protein is synthesized.
  • the nucleic acid sequence recognition module of the present invention forms a complex with a transcription repressor instead of a nuclease, and uses a sequence containing a cis element in the transcriptional regulatory region of the LSMEM2 gene as a target nucleotide sequence. It is also possible to further suppress transcription of the LSMEM2 gene while blocking the binding of transcription factors to the element.
  • transcription repressor refers to a protein or protein domain that has the activity of repressing the transcription of a target gene.
  • the transcriptional repressor used in the present invention is not particularly limited as long as it can suppress transcriptional activation of the LSMEM2 gene, but examples include KRAB, MBD2B, v-ErbA, SID (concatemer of SID (SID4X)). ), MBD2, MBD3, DNMT family (eg DNMT1, DNMT3A, DNMT3B), Rb, MeCP2, ROM2 and AtHD2A, preferably KRAB.
  • the protein derived from it is not particularly limited, but examples include KOX-1 (ZNF10), KOX8 (ZNF708), ZNF43, ZNF184, ZNF91, HPF4, HTF10, HTF34, etc. .
  • the contact between the nucleic acid sequence recognition module and the LSMEM2 gene is carried out in cells of a target mammal (e.g., human, mouse, rat, cow, dog, cat, monkey, etc., preferably human or mouse, more preferably human).
  • a target mammal e.g., human, mouse, rat, cow, dog, cat, monkey, etc., preferably human or mouse, more preferably human.
  • an effector such as a nuclease or a transcription repressor, the effector protein. Therefore, the nucleic acid sequence recognition module, or the nucleic acid sequence recognition module and the effector, can be used as a nucleic acid encoding a fusion protein thereof, or in a form that can form a complex in a host cell after being translated into a protein.
  • the nucleic acid may be DNA or RNA.
  • DNA it is preferably double-stranded DNA, and is provided in the form of an expression vector capable of expressing each component under the control of a functional promoter in mammalian cells.
  • RNA it is preferably single-stranded RNA.
  • CRISPR-Cas When CRISPR-Cas is used as a nucleic acid sequence recognition module, an expression vector encoding guide RNA and Cas protein is introduced into cells, and the guide RNA and Cas protein are expressed, thereby allowing the interaction between guide RNA and Cas protein to occur within the cell. Form a complex.
  • the guide RNA and Cas protein may be encoded on the same expression vector, or may be encoded on different expression vectors.
  • DNA encoding Cas can be cloned from cells that produce Cas by methods well known in the art.
  • the obtained Cas-encoding DNA can be inserted downstream of the promoter of an expression vector for mammalian cells.
  • the DNA encoding the guide RNA consists of an RNA sequence complementary to the target nucleotide sequence and a known tracrRNA sequence (for example, gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtggtgctttt; We designed an oligo DNA sequence that linked SEQ ID NO: 6) and used a DNA/RNA synthesizer.
  • nucleotide sequences are described as DNA sequences unless otherwise specified, but when the polynucleotide is RNA, thymine (T) shall be read as uracil (U) as appropriate.
  • DNA encoding guide RNA can also be inserted into expression vectors for mammalian cells.
  • the guide RNA and Cas may be encoded on the same expression vector, or may be encoded on different expression vectors.
  • the DNA encoding Cas and the DNA encoding the guide RNA are inserted downstream of separate promoters in the same expression vector.
  • RNA encoding Cas can also be prepared, for example, by using the above-described DNA encoding Cas as a template and transcribing it into mRNA using a known in vitro transcription system.
  • Guide RNA can also be chemically synthesized using a DNA/RNA synthesizer by designing an oligo RNA sequence in which an RNA sequence complementary to the target nucleotide sequence is linked to a known tracrRNA sequence. In this case, various modifications can be imparted to the ribonucleotides constituting the guide RNA to improve stability and membrane permeability.
  • crRNA and tracrRNA can be synthesized separately and annealed for use.
  • DNA encoding a nucleic acid sequence recognition module such as a zinc finger motif, TAL effector, or PPR motif can be obtained by any of the methods described above for each module.
  • DNA encoding a sequence recognition module such as a restriction enzyme, a transcription factor, or an RNA polymerase covers a region encoding a desired portion of the protein (a portion containing a DNA binding domain), for example, based on the cDNA sequence information thereof.
  • Cloning can be carried out by synthesizing oligo-DNA primers as described above, and amplifying by RT-PCR using total RNA or mRNA fractions prepared from cells producing the protein as a template.
  • DNA encoding effectors such as nucleases and transcriptional repressors can be obtained by synthesizing oligo DNA primers based on the cDNA sequence information of the effector to be used, and using total RNA or mRNA fractions prepared from cells that produce the effector. Cloning can be performed by amplifying by RT-PCR using as a template.
  • DNA encoding FokI can be cloned from Flavobacterium okeanokoites (IFO 12536)-derived mRNA by RT-PCR by designing appropriate primers for upstream and downstream of CDS based on the cDNA sequence.
  • the cloned DNA encoding the nucleic acid sequence recognition module can be used as is, or if desired, it can be digested with restriction enzymes, or an appropriate linker and/or nuclear export signal can be added.
  • a DNA encoding a cloned nucleic acid sequence recognition module is ligated with a DNA encoding the cloned nucleic acid sequence recognition module to encode a fusion protein.
  • DNA can be prepared.
  • nucleic acid sequence recognition module and the effector may be allowed to form a complex after translation within the host cell.
  • a linker and/or nuclear localization signal can be ligated to an appropriate position of one or both of the DNAs, if desired.
  • DNA encoding the nucleic acid sequence recognition module and DNA encoding the effector can be obtained by chemically synthesizing DNA strands, or by using synthesized partially overlapping oligo DNA short strands using PCR method or Gibson Assembly method. By connecting it, it is also possible to construct a DNA encoding the entire length.
  • the advantage of constructing a full-length DNA by chemical synthesis or in combination with the PCR method or the Gibson Assembly method is that the codons used can be designed over the entire length of the CDS depending on the host into which the DNA is introduced. When expressing heterologous DNA, an increase in protein expression can be expected by converting the DNA sequence to codons that are frequently used in the host organism.
  • codon usage frequency in the host for example, the genetic code usage frequency database published on the website of the Kazusa DNA Research Institute (public interest incorporated foundation) can be used, or literature describing the codon usage frequency in each host can be used. You may refer to it.
  • the obtained data and the DNA sequence to be introduced convert the codons used in the DNA sequence that are used less frequently in the host to codons that encode the same amino acid and are used more frequently. good.
  • animal virus vectors such as retroviruses, vaccinia viruses, and adenoviruses are used.
  • Promoters include SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. Any promoter that is functional in mammalian cells may be used, including but not limited to.
  • the expression vector may optionally contain an enhancer, a splicing signal, a terminator, a poly A addition signal, a selection marker such as a drug resistance gene, an origin of replication, and the like.
  • nucleic acid sequence recognition module By introducing an expression vector encoding a nucleic acid sequence recognition module (and, if desired, an effector) into a target cell of a mammal, the nucleic acid sequence recognition module or a complex of the module and the effector is produced in the target cell. It can be expressed and formed and contacted with the target nucleotide sequence on the LSMEM2 gene.
  • (d-2) Binding inhibitor accompanied by modification of genome sequence
  • DSB is generated within the LSMEM2 gene, and one or more of the LSMEM2 genes
  • the LSMEM2 gene can be disrupted by causing deletions or substitutions of nucleotides with other nucleotides, or insertions of one or more nucleotides into the gene.
  • the LSMEM2 gene is repaired by non-homologous end joining (NHEJ) after being cleaved within the target nucleotide sequence, but repair errors may result in the deletion of one or more nucleotides in the gene or the addition of other nucleotides. resulting in a substitution or insertion of one or more nucleotides into the gene.
  • NHEJ non-homologous end joining
  • Another method for introducing mutations into the LSMEM2 gene is to use DNA that contains sequences homologous to the upstream and downstream sequences adjacent to the target region in the LSMEM2 gene, and in which a mutation has been introduced into the target region (donor DNA). ) is introduced into a target mammalian cell together with a DNA encoding a complex of a nucleic acid sequence recognition module and a nuclease, thereby causing homologous recombination between the region containing the target region of the present invention and the donor DNA. By causing this, a desired mutation can be introduced into the target region.
  • the sequences homologous to the upstream and downstream sequences adjacent to the target region contained in the donor DNA are not particularly limited as long as they are long enough to cause homologous recombination. It may be a short sequence, or it may be a long homology arm spanning several kb. In the latter case, the donor DNA may be provided in the form of a targeting vector into which it has been inserted. "Targeting vector into which donor DNA has been inserted" is not just a targeting vector into which the same sequence as the above-mentioned donor DNA has been inserted, but also one which has a selection marker and/or recombinase target sequence between or outside the donor DNA. Also included.
  • the vector serving as the basic skeleton of the targeting vector is not particularly limited, as long as it is capable of self-replication in the cell to be transformed (eg, E. coli).
  • E. coli e.g. E. coli
  • commercially available pBluescript (Stratagene), pZErO1.1 (Invitrogen), pGEM-1 (Promega), etc. can be used.
  • Donor DNA can be introduced into cells in the form of either double-stranded DNA (circular double-stranded DNA, linear double-stranded DNA) or single-stranded DNA.
  • an enzyme capable of modifying the genome without DSB as an effector can be used in combination with a nucleic acid sequence recognition module.
  • Such enzymes include, for example, enzymes that substitute different bases by converting one substituent of a nucleobase into another (e.g., deaminase), and enzymes that catalyze abasic reactions and activate endogenous repair mechanisms. Examples include, but are not limited to, enzymes (eg, DNA glycosylase, etc.) that utilize errors to introduce mutations into abasic sites.
  • the Cas may be a mutant in which at least one DNA cleaving ability is inactivated (nCas), preferably a mutant in which both DNA cleaving abilities are inactivated ( dCas) is used. Details of using deaminase as an effector are described in, for example, International Publication No. 2015/133554, and details of using DNA glycosylase are described in, for example, International Publication No. 2016/072399.
  • VPS35 a component of the retromer complex that plays a central role in vesicular transport.
  • VPS35 is a central molecule in vesicle trafficking that controls vesicle destination by interacting molecules.
  • the "substance that suppresses the function of LSMEM2" may be any substance as long as it suppresses the function that contributes to mechanosignal transmission of LSMEM2 once it is functionally produced. For example, it binds to LSMEM2 and suppresses the function. Examples include substances that inhibit the movement of LSMEM2 to the cell membrane.
  • examples of substances that suppress the function of LSMEM2 include antibodies against LSMEM2.
  • the antibody may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to known antibody or antiserum production methods.
  • the isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG.
  • the antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding LSMEM2, and in addition to complete antibody molecules, for example, Fab, Fab', F ( ab') 2 , etc., genetically engineered conjugate molecules such as scFv, scFv-Fc, minibodies, and diabodies, or molecules with protein stabilizing effects such as polyethylene glycol (PEG). They may also be derivatives thereof.
  • CDR complementarity determining region
  • the antibody against LSMEM2 is used as a pharmaceutical to be administered to humans
  • the antibody is an antibody with a reduced risk of exhibiting antigenicity when administered to humans.
  • fully human antibodies, humanized antibodies, mouse-human chimeric antibodies, etc., and fully human antibodies are particularly preferred.
  • Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to conventional methods.
  • fully human antibodies can be produced from human-human (or mouse) hybridomas, but in order to stably provide large quantities of antibodies at low cost, human antibody-producing mice and phage display methods are required. It is desirable to manufacture using
  • the substance that suppresses the function of LSMEM2 may also be, for example, an aptamer (peptide aptamer or nucleic acid aptamer).
  • aptamer refers to a nucleic acid or peptide that can specifically bind to a specific molecule.
  • the nucleic acid may be DNA, RNA, or a chimera of DNA and RNA.
  • Aptamers can be screened or produced according to well-known methods (for example, Ellington et al., (1990), Nature, 346, 818-822; Tuerk et al., (1990) Science, 249, 505-510). Can be done.
  • the substance that suppresses the function of LSMEM2 may also be, for example, a substance that induces the decomposition of LSMEM2.
  • examples of the substance that induces the degradation of LSMEM2 include an LSMEM2 degrader using a targeted protein degradation technique.
  • a target protein degrader refers to a molecule capable of promoting the degradation of a target protein through the intracellular proteolytic machinery.
  • Such a target protein degrader can be produced according to a well-known method (for example, Mohammadsharif Tabebordbar et al., Cell. 2021 Sep 16; 184(19): 4919-4938.e22.).
  • the targeted protein degrader can include two parts: a part that specifically binds the target protein and another part that binds to the E3 ubiquitin ligase.
  • E3 ubiquitin ligase promotes ubiquitination of target proteins. As a result, degradation of target proteins by the proteasome system is promoted.
  • PIEZO sensor molecule
  • LSMEM2 is necessary and sufficient for mechanosignal (mechano stress (stretch stimulus) dependent inflammatory signal) transmission, so substances that suppress the expression or function of LSMEM2 are effective for mechanosignal transmission. It has the effect of inhibiting dilated cardiomyopathy, and can be used as a therapeutic agent targeting cardiomyocytes and the like to prevent and/or treat dilated cardiomyopathy.
  • mice produced by crossing LSMEM2 knockout mice with lamin cardiomyopathy model mice have improved cardiac function and lower limb muscle strength. Therefore, substances that suppress the expression or function of LSMEM2 can be used as preventive and/or therapeutic agents for laminopathy.
  • LSMEM2 KO mice develop normally, have completely normal cardiac function, and have no problems with reproductive function. Therefore, substances that suppress the expression or function of LSMEM2 have low toxicity to normal cells and have no side effects.
  • a drug containing a substance that suppresses the expression or function of LSMEM2 is useful as a mechanosignal transduction inhibitor, and as a preventive and/or therapeutic agent for heart disease, and also as a preventive and/or therapeutic agent for laminopathy. It is.
  • Treatment is used herein to include improvement of disease as well as improvement of prognosis.
  • prevention is used to include delaying the onset of a disease.
  • Only one type of substance that suppresses the expression or function of LSMEM2 may be used, or two or more types may be used in combination.
  • Two or more substances that suppress the expression or function of LSMEM2 may be formulated as separate pharmaceuticals, or may be combined in the same pharmaceutical composition.
  • each formulation may be administered at the same time or at intervals.
  • the administration routes may be the same or different.
  • the dosage mentioned below indicates the dosage of one kind of substance that suppresses the expression or function of LSMEM2, but even when two or more kinds of substances are used in combination, within a range that does not have an unfavorable effect on the subject of administration, Similar dosages can be used for each substance.
  • nucleic acids of the present invention can be used as anti-inflammatory agents and/or inhibitors of vesicular transport abnormalities, as well as preventive and/or therapeutic agents for heart diseases, and/or Alternatively, it can be used as a prophylactic and/or therapeutic agent for laminopathy.
  • the medicament containing the nucleic acid of the present invention can be administered to humans or non-human warm-blooded animals (e.g., rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, It can be administered orally or parenterally (eg, intravascularly, subcutaneously, etc.) to subjects such as chickens, etc.
  • non-human warm-blooded animals e.g., rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys
  • parenterally eg, intravascularly, subcutaneously, etc.
  • the nucleic acid of the present invention When used as an anti-inflammatory agent and/or an inhibitor of vesicular transport abnormalities, a preventive and/or therapeutic agent for heart disease, and/or a preventive and/or therapeutic agent for laminopathy, methods known per se can be used. It can be formulated and administered according to the following. That is, the nucleic acid of the present invention may be used alone or inserted in a functional manner into an appropriate expression vector for mammalian cells, such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. You can also do it. The nucleic acid can be administered as is or together with an adjuvant to promote uptake via a gene gun or a catheter such as a hydrogel catheter.
  • the nucleic acid may be formulated into a formulation (injection) alone or together with a carrier such as a liposome, and administered intravenously, subcutaneously, etc. .
  • the nucleic acid of the present invention may be administered as such or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent, or excipient.
  • Such pharmaceutical compositions are provided in dosage forms suitable for oral or parenteral administration.
  • compositions for parenteral administration include, for example, injections, suppositories, intranasal injections, etc.
  • Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. It may also include dosage forms such as agents.
  • Such injections can be prepared according to known methods. An injection can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous or oily liquid commonly used for injections.
  • aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil), etc.).
  • alcohol e.g., ethanol
  • polyalcohol e.g., propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily liquid for example, sesame oil, soybean oil, etc. are used, and benzyl benzoate, benzyl alcohol, etc. may be used in combination as a solubilizing agent.
  • the prepared injection solution is preferably filled into suitable ampoules.
  • compositions for oral administration include solid or liquid dosage forms, in particular tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups.
  • examples include formulations, emulsions, suspensions, and the like.
  • Such compositions may be manufactured by known methods and may contain carriers, diluents or excipients commonly used in the pharmaceutical field.
  • carriers and excipients for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • parenteral or oral pharmaceutical compositions described above are conveniently prepared in dosage unit form to suit the dosage of the active ingredient.
  • unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories.
  • the nucleic acid of the present invention is preferably contained, for example, in an amount of usually about 0.01 to 500 mg per unit dosage form.
  • the dosage of the above-mentioned medicine containing the nucleic acid of the present invention varies depending on the subject, target disease, symptoms, administration route, etc.; It is convenient to administer a single dose of nucleic acid, usually about 0.0001 to 20 mg/kg body weight, by intravenous injection about once every 1 day to 6 months. Similar amounts can be administered in other cases of parenteral administration and oral administration. If the symptoms are particularly severe, the dose may be increased depending on the symptoms.
  • the binding inhibitor of the present invention is a nucleic acid sequence recognition module used in genome editing technology (and an effector that forms a complex with the module), preferably the binding
  • the inhibitory substance is formulated in the form of an expression vector (hereinafter also referred to as "vector of the present invention") containing DNA encoding the inhibitory substance.
  • Vectors of the present invention include, for example, detoxified retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, poxviruses, polioviruses, Sindbis viruses, Sendai viruses, SV40, and immunodeficiency virus (HIV).
  • HIV immunodeficiency virus
  • Viral vectors such as the following can be used.
  • adenovirus or adeno-associated virus vectors are used.
  • Pharmaceutically acceptable carriers include, for example, excipients such as sucrose and starch, binders such as cellulose and methyl cellulose, disintegrants such as starch and carboxymethyl cellulose, lubricants such as magnesium stearate and Aerosil, citric acid, Flavoring agents such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinyl pyrrolid, dispersing agents such as surfactants, water, Examples include diluents such as physiological saline, base wax, etc., but are not limited thereto.
  • the agent of the present invention can further contain a reagent for nucleic acid introduction.
  • the nucleic acid introduction reagents include atelocollagen; liposome; nanoparticle; lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly(ethyleneimine) (PEI).
  • Cationic lipids such as, for example, can be used.
  • the agent of the present invention may be a pharmaceutical composition in which the vector is encapsulated in a liposome.
  • Liposomes are microscopic closed vesicles that have an internal phase surrounded by one or more lipid bilayers, and can typically retain water-soluble substances in the internal phase and lipid-soluble substances within the lipid bilayer.
  • the vector may be retained in the internal phase of the liposome or within the lipid bilayer.
  • the liposome used in the present invention may be a monolayer or a multilayer, and the particle size can be appropriately selected within the range of, for example, 10 to 1000 nm, preferably 50 to 300 nm. Considering deliverability to the target tissue, the particle size may be, for example, 200 nm or less, preferably 100 nm or less.
  • Methods for encapsulating the above-mentioned vectors in liposomes include, but are not limited to, the lipid film method (vortex method), reverse phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading method. Any known method can be selected as appropriate.
  • the agent of the present invention can be administered orally or parenterally to mammals (e.g., humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, monkeys). However, it is preferable to administer the drug parenterally.
  • mammals e.g., humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, monkeys.
  • sustained-release preparation such as a minipellet preparation
  • implant it near the affected area or it is also possible to continuously and gradually administer it to the affected area using an osmotic pump or the like.
  • Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions containing antioxidants. , a buffer, a bacteriostatic agent, a tonicity agent, etc. may be included. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, and the like.
  • the preparation can be packaged in containers such as ampoules or vials for unit doses or multiple doses.
  • the active ingredient and the pharmaceutically acceptable carrier can be lyophilized and stored by dissolving or suspending them in a suitable sterile vehicle immediately before use.
  • the content of the above vector in the pharmaceutical composition is, for example, about 0.1 to 100% by weight of the entire pharmaceutical composition.
  • the dosage of the agent of the present invention varies depending on the administration method, the type and severity of the target disease, and the circumstances of the recipient (sex, age, body weight, etc.), but for example, when administered systemically to adults, usually
  • a titer of virus can be administered, for example, in the range of about 1 ⁇ 10 3 pfu to 1 ⁇ 10 15 pfu per dose.
  • the agent of the present invention can be added to cells or tissues (e.g., cardiac muscle cells) collected from the subject to be administered, and intracellularly. It may also be an ex vivo preparation, in which the expression vector is introduced into the patient's body, and then returned to the body of the subject, preferably to the cardiac lesion.
  • methods for introducing genes into cells include lipofection, phosphate-calcium coprecipitation, and direct injection using a micro glass tube.
  • methods for introducing genes into tissues include gene introduction using encapsulated liposomes, gene introduction using electrostatic liposomes, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), and receptor-mediated method.
  • examples include a gene transfer method, a method in which an active ingredient is transferred into cells together with a carrier (metal particles) using a particle gun, a direct transfer method using naked-DNA, a method using a positively charged polymer, and the like.
  • compositions containing the above-mentioned antibodies, degraders, or low-molecular-weight compounds can be administered to humans or other warm-blooded animals (e.g., rats, rabbits, sheep, pigs, cows), either directly as a solution or as a pharmaceutical composition in an appropriate dosage form.
  • cats, dogs, monkeys, chickens, etc. can be administered orally or parenterally (eg, intravascularly, subcutaneously, etc.).
  • the above-mentioned antibodies, degraders, or low-molecular-weight compounds may be administered as such, or as a suitable pharmaceutical composition.
  • the pharmaceutical composition used for administration may contain the above-mentioned antibody or low-molecular-weight compound, or a salt thereof, and a pharmacologically acceptable carrier, diluent, or excipient.
  • Such pharmaceutical compositions are provided in dosage forms suitable for oral or parenteral administration.
  • compositions for parenteral administration include, for example, injections, suppositories, intranasal injections, etc.
  • Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. It may also include dosage forms such as agents.
  • Such injections can be prepared according to known methods. Injections can be prepared, for example, by dissolving, suspending, or emulsifying the antibody or low-molecular-weight compound of the present invention, or a salt thereof, in a sterile aqueous or oily liquid commonly used for injections.
  • aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil), etc.).
  • alcohol e.g., ethanol
  • polyalcohol e.g., propylene glycol, polyethylene glycol
  • nonionic surfactants eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil
  • oily liquid for example, sesame oil, soybean oil, etc. are used, and benzyl benzoate, benzyl alcohol, etc. may be used in combination as a solubilizing agent.
  • the prepared injection solution is preferably filled into suitable ampoules.
  • compositions for oral administration include solid or liquid dosage forms, in particular tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups.
  • examples include formulations, emulsions, suspensions, and the like.
  • Such compositions may be manufactured by known methods and may contain carriers, diluents or excipients commonly used in the pharmaceutical field.
  • carriers and excipients for tablets for example, lactose, starch, sucrose, and magnesium stearate are used.
  • parenteral or oral pharmaceutical compositions described above are conveniently prepared in dosage unit form to suit the dosage of the active ingredient.
  • unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories.
  • the antibody or low-molecular compound is usually contained in an amount of 0.1 to 500 mg per unit dosage form, particularly preferably 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms.
  • the dosage of the above-mentioned medicine containing the above-mentioned antibody or low-molecular compound or its salt varies depending on the subject, target disease, symptoms, administration route, etc., but for example, the antibody or low-molecular compound is usually administered as a single dose. About 0.0001 to 20 mg/kg body weight, low molecular weight compounds should be administered orally or parenterally 1 to 5 times a day, and antibodies should be administered by intravenous injection once a day to once every few months. It's convenient. Similar amounts can be administered in other cases of parenteral administration and oral administration. If the symptoms are particularly severe, the dose may be increased depending on the symptoms.
  • compositions may contain other active ingredients as long as they do not cause undesirable interactions when mixed with the above-mentioned antibodies or low-molecular-weight compounds.
  • active ingredients for example, antibacterial drugs, antifungal drugs, non-steroidal anti-inflammatory drugs, steroid drugs, anticoagulants, platelet antiaggregation drugs, thrombolytic drugs, immunomodulators, antiprotozoal drugs, antibiotics, antiviral drugs, antitussives and antitussives.
  • Suppressants sedatives, anesthetics, antiulcer drugs, antiarrhythmic drugs, antihypertensive diuretics, tranquilizers, antipsychotics, antitumor drugs, antihyperlipidemic drugs, muscle relaxants, antiepileptic drugs, antidepressants Medicines, antiallergic drugs, cardiotonic drugs, arrhythmia drugs, vasodilators, vasoconstrictors, antihypertensive diuretics, antidiabetic drugs, narcotic antagonists, vitamin drugs, vitamin derivatives, arthritis drugs, antirheumatic drugs, antiasthmatic drugs , Frequent urination/urinary incontinence treatment, atopic dermatitis treatment, allergic rhinitis treatment, vasopressor, proteolytic agent, protease inhibitor, anti-SIDS agent, anti-sepsis agent, anti-septic shock agent, endotoxin Antagonist or antibody, signal transduction inhibitor, inflammatory mediator action inhibitor, inflammatory mediator action
  • the drug can be used, for example, as an anti-inflammatory agent or as an inhibitor of vesicular transport abnormalities, to treat various inflammatory diseases (e.g., Crohn's disease, rheumatoid arthritis, Behcet's disease (ocular symptoms), ulcerative colitis, ankylosing spondylitis, psoriasis ( (including psoriatic arthritis), HIV infection, multiple myeloma, heart disease (e.g.
  • various inflammatory diseases e.g., Crohn's disease, rheumatoid arthritis, Behcet's disease (ocular symptoms), ulcerative colitis, ankylosing spondylitis, psoriasis (including psoriatic arthritis), HIV infection, multiple myeloma, heart disease (e.g.
  • congestive heart failure chronic heart failure, dilated cardiomyopathy, hypertrophic cardiomyopathy, myocardial ischemia, myocardial infarction, angina pectoris) , GVHD, giant cell arteritis (GCA), polymyalgia rheumatica (PMR), pigmented purpuric lichenoid dermatitis, sarcoidosis, Wegener's granuloma, pyoderma, Behcet's disease, TNF receptor-related periodicity TRAPS, SAPHO syndrome, Takayasu disease, myositis, Still's disease, periarteritis nodosa (PN), relapsing polychondritis, polymyositis scleroderma, hemophagocytic syndrome, pemphigus, Kawasaki disease atopic skin It can be used as a prophylactic and/or therapeutic agent for laminopathy (eg, muscular dystrophy, laminar cardiomyopathy, lipody
  • Mouse tissue RT-PCR RT-PCR was performed to examine the expression dynamics of LSMEM2 in mouse and human tissues. cDNA was examined for heart, brain, liver, skeletal muscle, testis, kidney, spleen, and lung using Mouse MTC Panel I (636745) from Clontech Laboratories. TaKaRa Ex Taq (Takara Bio Inc. Shiga, Japan) was used for PCR. Primers and reaction solutions were prepared as follows.
  • Mouse_RT-PCR_L agcttctgggtgtcttaccacatc (SEQ ID NO: 7)
  • Mouse_RT-PCR_R caatttgagcagagtctcctcttgtgtgc (SEQ ID NO: 8)
  • the PCR conditions were as follows.
  • RNA-seq of LSMEM2 forced expression cardiomyocytes Neonatal Rat Cardiomyocyte Primary Culture Hearts were removed from neonatal rats, loosened in Hank's balanced salt solution (Thermo Fisher Scientific, Waltham, Mass.), and the blood inside was washed. The dissected hearts were incubated overnight at 4°C in 0.25% Trypsin/EDTA (SIGMA, St. Louis, MO). The next day, D-MEM (in 10% FBS) was added and incubated at 37°C for 5 minutes. The supernatant was discarded, and collagenase was added and mixed by hand for 1 minute.
  • the cells were centrifuged at 3,000 g for 5 minutes, the supernatant was discarded, the cells were suspended in an appropriate amount of D-MEM, and aliquots were taken to count the number of cells. Finally, cells were seeded at an appropriate cell concentration depending on the experimental conditions.
  • RNA-seq analysis of cardiomyocytes Cultured cardiomyocytes were prepared at 5.0x10 6 cells/well and seeded in a 6cm dish.After 24 hours, the medium was changed and wild type LSMEM2, S37A LSMEM2, and Mock adenovirus vectors were added. Ta. After 48 hours, the medium was replaced with serum free D-MEM, and after 16 hours, myocardial RNA was collected and purified using RNA bee. Subsequently, RNA-seq data production itself was carried out at the Osaka University Research Institute for Microbial Diseases, and was generally carried out as follows.
  • RNA library was prepared from each group of RNA using TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego CA). Actually, mRNA was polyA purified using dT beads, ribosomal RNA was removed using Ribo-zero beads, and then an adapter sequence was added in one direction. Subsequently, RNA seq data was generated by sequencing an average of 15 million reads with a single read of 75 bp using Illumina's HiSeq 2500. Subsequently, the quality of the obtained read was confirmed by FASTQC. Next, mapping was performed using Tophat (Kim et al., 2013) to the reference genome Rnor6 of NCBI.
  • ddPCR Droplet Digital PCR
  • Il6 dRnoCPE5187348 (FAM), Tbp: dRnoCNS494181347 (HEX) Human IL6: dHsaCPE5036918, Human TBP: dHsaCPE5058363
  • the PCR conditions were as follows.
  • Cardiomyocyte Hydrostatic Pressure Loading Cultured cardiomyocytes were seeded on plates, and after 24 hours they were treated with 1% FBS DMEM, 25-mM HEPES-NaOH, pH 7.4 (Sigma-Aldrich, St. Louis, MO), 2-mM l- The medium was replaced with glutamine (Thermo Fisher Scientific, Waltham, Mass.), 1% P/S. Cardiomyocytes were placed in a pressure vessel and connected to a compressor supplying pressurized air regulated at 37 °C and 5% CO2 .
  • the air compression controller for adjusting the air flow rate can change the frequency from 0 to 0.25 Hz and the amplitude from 0 to 1 MPa, and this time it repeats 101 to 135 kPa at a frequency of 0.01 Hz. It was exposed for 12 hours under hydrostatic pressure conditions. Note that pressure was applied to the cells by compressing the volume of the medium.
  • Cardiomyocytes Hela cell stretch stimulation load Plasma treatment was performed in a stretch chamber STB-CH-04 (STREX, Osaka, Japan) using a tabletop vacuum plasma treatment device PC-400T (STREX, Osaka, Japan), and then iMatrix-511 (Nippi, Tokyo, Japan) was added to 1 mL of a laminin solution prepared at 4.8 ⁇ g/mL and incubated overnight at 37°C. The laminin solution was removed, and the cultured cardiomyocytes were adjusted to 1.3 ⁇ 10 6 cells/chamber and then seeded onto a stretch chamber.
  • the medium was replaced with serum free DMEM, set in an automatic extension device STB-1400 (STREX, Osaka, Japan), and grown at 0.5 Hz, 20%, and 24°C under conditions of a temperature of 37°C and 5% carbon dioxide. A time stretching stimulus was applied.
  • the siRNA against LSMEM2 included Silence® rSelect siRNA (LSMEM2 siRNA ID: s189327, sense (5' ⁇ 3'): GCACAGUGGAGGCUCACUATT (Sequence number 9) antisense (5' ⁇ 3'): UAGUGAGCCCUCCACUGUGCAG (SEQ ID NO: 10) (Thermo Fisher Scientific, Waltham, MA) was used. The final concentration was adjusted to 5 nM and introduced into neonatal rat cardiomyocytes 3 hours after seeding using Lipofectamine TM RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA).
  • IL6 mRNA was quantified in rat cardiomyocytes introduced with control siRNA (siCtrl) and rat cardiomyocytes in which LSMEM2 was knocked down by siRNA (siAIPID), which were subjected to stretch stimulation. IL6 expression was not induced by control siRNA, but was shown to be induced by siRNA against LSMEM2 (expressed as relative expression to Tbp mRNA; Fig. 3C).
  • Example 4 Production of Lsmem2 knockout mouse Production of knockout mice was requested to the Osaka University Animal Experiment Facility Reproductive Engineering Unit. Actually, Lsmem2 knockout mice were generated using the CRSPR/Cas9 system. Guide RNA was designed on exon 2 (Guide RNA sequence: ACTGGCTCCTAACCACGTGCAGG) (SEQ ID NO: 5) (FIG. 4A). A vector was created in which the designed Guide RNA was integrated into pX330 (Wyman et al., 2013), which can simultaneously express Cas9 and the target Guide RNA, and the vector was injected into a mouse fertilized egg. transplanted into pseudopregnant mice.
  • Guide RNA was designed on exon 2 (Guide RNA sequence: ACTGGCTCCTAACCACGTGCAGG) (SEQ ID NO: 5) (FIG. 4A).
  • a vector was created in which the designed Guide RNA was integrated into pX330 (Wyman et al., 2013), which can simultaneously express
  • mice with a 14 base deletion were lined up and maintained in a C57BL6J background.
  • LSMEM2 was detected by conventional Western blotting using mouse heart tissue, and it was confirmed that the LSMEM2 protein was lost in the knockout mice, that is, LSMEM2 knockout mice were formed (FIG. 4B).
  • Cardiac function analysis was performed at 3, 6, and 9 months of age using a Vevo 3100 imaging system, and LVEF (left ventricular ejection fraction) was evaluated. The number of individuals used is as shown in the figure. Lower limb muscle strength measurements were performed at 7 months of age. Lower limb muscle strength measurements were performed at 29 weeks of age using a 1300A 3-in-1 Whole Animal Muscle System (Aurora Scientific). was evaluated. The analysis used DMA software (Aurora Scientific). Comparisons between the two groups were statistically examined using a t-test.
  • AAV vector MyoAAV2 vector (Tabebordbar M. et al. Cell 2021) was produced by co-transfecting HEK293T cells with the transfer plasmid, Rep-cap plasmid, and helper plasmid, and the virus was isolated by cesium chloride density gradient ultracentrifugation. The particles were concentrated and purified.
  • MyoAAV2 U6-shRNA (LSMEM2) was produced.
  • the shRNA was designed as shLSMEM2 (5'-CTCTCACTGATGGCTTCATTT-3' (SEQ ID NO: 11)), which targets mouse LSMEM2.
  • shControl (5'-CCTAAGGTTAAGTCGCCCTCG-3' (SEQ ID NO: 12)), which does not target mouse genes, was designed. designed.
  • LSMEM2 U6-shRNA (hereinafter also referred to as shLSMEM2) and MyoAAV2 shControl at 7 ⁇ 10 11 genome copies (GC) were intravenously injected into the orbital venous plexus of 10-week-old LMNA KI mice. Cardiac function was evaluated.
  • Example 7 Mouse aortic arch coarctation model A model was created in which the aortic arches of wild-type mice and LSMEM2 knockout mice were ligated and a high pressure load was applied to the left ventricle. This model is a very common pressure overload heart failure model used in cardiovascular research. Cardiac function was evaluated using cardiac tissue 1 week after surgery and by echocardiographic observation over time at 1 and 4 weeks after surgery.
  • Heart tissue was removed one week after surgery, and immunostained using CD45 antibody, a surface marker for mouse leukocytes, in the same manner as in Figure 4 to examine inflammatory cell infiltration into the myocardial tissue.
  • CD45 antibody a surface marker for mouse leukocytes
  • FIG. 7A RNA-seq analysis was performed using heart tissue 1 week after surgery in the same manner as in FIG. 2.
  • Figure 7B a significant increase in expression of inflammation-related genes was observed one week after surgery, consistent with the results of CD45 staining, but in LSMEM2 knockout mice, these increases in expression were significantly suppressed.
  • a mechanosignaling inhibitor containing a substance that suppresses the expression or function of LSMEM2, thus making it possible to effectively treat lamin cardiomyopathy, severe heart failure due to other causes, and laminopathy.

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Abstract

The present invention provides a mechanosignaling inhibitor containing a substance capable of suppressing the expression or function of LSMEM2 as an active ingredient.

Description

心疾患及びラミノパチーの予防及び／又は治療剤Preventive and/or therapeutic agent for heart disease and laminopathy
　本発明は、ＬＳＭＥＭ２の発現又は機能を抑制する物質を含む、メカノシグナル伝達阻害剤、抗炎症剤、心疾患の予防及び／又は治療剤、ラミノパチーの予防及び／又は治療剤等に関する。 The present invention relates to a mechanosignal transduction inhibitor, an anti-inflammatory agent, a preventive and/or therapeutic agent for heart disease, a preventive and/or therapeutic agent for laminopathy, etc., which contain a substance that suppresses the expression or function of LSMEM2.
　わが国の心移植症例の原因疾患で最も多い拡張型心筋症（ＤＣＭ）において、ラミン遺伝子（ＬＭＮＡ）に病原性ｖａｒｉａｎｔを有する患者群（ラミン心筋症）の予後が特に悪いことが知られている（非特許文献１）。ＬＭＮＡ遺伝子ｖａｒｉａｎｔは拡張型心筋症患者のうち約１０％に見られ、ラミン心筋症は高い浸透率で重篤な心不全と致死的不整脈を合併する難病である（非特許文献２）。日本循環器学会の心筋症診療ガイドラインにおいて、家族性で伝導障害を合併する拡張型心筋症ではＬＭＮＡ遺伝子検索が勧められているが、ラミン心筋症に対する疾患特異的な治療法は確立されていない。 In dilated cardiomyopathy (DCM), which is the most common cause of heart transplantation in Japan, it is known that the prognosis of patients with a pathogenic variant in the lamin gene (LMNA) (lamin cardiomyopathy) is particularly poor ( Non-patent document 1). LMNA gene variant is found in about 10% of patients with dilated cardiomyopathy, and lamin cardiomyopathy is an intractable disease with high penetrance that is accompanied by severe heart failure and fatal arrhythmia (Non-Patent Document 2). In the Japanese Circulation Society's cardiomyopathy treatment guidelines, LMNA gene search is recommended for familial dilated cardiomyopathy complicated by conduction disorders, but no disease-specific treatment for lamin cardiomyopathy has been established.
　核膜の裏打ちタンパク質であるラミンはＬＩＮＣ（Ｌｉｎｋｅｒ　ｏｆ　Ｎｕｃｌｅｏｓｋｅｌｅｔｏｎ　ａｎｄ　Ｃｙｔｏｓｋｅｌｅｔｏｎ）複合体を介して細胞外からのメカノストレスを核へ伝搬することで遺伝子発現を制御していることが知られている。ＬＩＮＣ複合体構成タンパクであるＳＵＮ１を欠損させたＳＵＮ１　ＫＯマウスはラミン心筋症モデルマウスの生命予後を改善させることから（非特許文献３）、細胞外から核に誘導されるメカノストレスがラミン心筋症の発症に重要であると考えられている。しかしながら、ＳＵＮ１は発現がユビキタスであり、ＫＯマウスは不妊や難聴を呈するため治療標的とするには困難であると考えられていた。 Lamin, which is a protein lining the nuclear envelope, is known to control gene expression by propagating mechanostress from outside the cell to the nucleus via the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. SUN1 KO mice that lack SUN1, a protein constituting the LINC complex, improve the survival prognosis of lamin cardiomyopathy model mice (Non-Patent Document 3). It is thought to be important in the onset of the disease. However, SUN1 is ubiquitously expressed and KO mice exhibit infertility and hearing loss, making it difficult to use as a therapeutic target.
　近年、ＬＩＮＣ複合体の機能を欠失させるドミナントネガティブ作用のあるＳＵＮ１タンパク質を、ＡＡＶベクターを用いて心筋細胞特異的に発現させることによって、ラミン心筋症モデルマウスの生命予後が改善されることが報告された（非特許文献４）。マウスでは明らかな副作用は認めなかったとのことだが、ＳＵＮ１　ＫＯマウスは不妊や難聴を呈すること、本来のＬＩＮＣ複合体の機能を破壊してしまうという点において、ヒトへの適応には懸念が残る。また、ラミン心筋症ではｐ３８　ＭＡＰＫシグナル伝達が亢進していることが知られており、ｐ３８α阻害剤はラミン心筋症モデルマウスの心機能を改善させることが報告されている（非特許文献５）。ｐ３８α阻害剤はラミン心筋症に対して現在ｐｈａｓｅ３の臨床試験が行われているが、ｐ３８αのＫＯマウスは胎生致死であり、こちらもヒトへの安全性には懸念が残る。 In recent years, it has been reported that the survival prognosis of lamin cardiomyopathy model mice is improved by expressing the SUN1 protein, which has a dominant-negative effect that abolishes the function of the LINC complex, specifically in cardiomyocytes using an AAV vector. (Non-patent Document 4). Although no obvious side effects were observed in mice, there remain concerns about its suitability to humans, as SUN1 KO mice exhibit infertility and hearing loss, and the original LINC complex function is destroyed. Furthermore, p38 MAPK signaling is known to be enhanced in lamin cardiomyopathy, and p38α inhibitors have been reported to improve cardiac function in lamin cardiomyopathy model mice (Non-Patent Document 5). A p38α inhibitor is currently undergoing phase 3 clinical trials for lamin cardiomyopathy, but p38α KO mice are embryonic lethal, and there are also concerns about its safety in humans.
　従って、ラミン心筋症に対する有効な治療法開発には、大きなアンメットメディカルニーズがある。 Therefore, there is a large unmet medical need for the development of effective treatments for lamin cardiomyopathy.
　本発明の課題は、心疾患やラミノパチーの予防及び／又は治療剤を提供すること、特に、有効なラミン心筋症の予防及び／又は治療剤を提供することである。 An object of the present invention is to provide a preventive and/or therapeutic agent for heart disease and laminopathy, and in particular, to provide an effective preventive and/or therapeutic agent for lamin cardiomyopathy.
　本発明者らは介在板がメカノストレスの受容器であることを見出し、介在板に局在し、メカノシグナリングに関わる膜タンパク質ＬＳＭＥＭ２を独自に同定した。ＬＳＭＥＭ２ノックアウトマウスは安静時には正常心機能を呈した。そして、ＬＳＭＥＭ２ノックアウトマウスとラミン心筋症モデルマウスとの交配により生じたマウスは、心機能が改善されることを見出し、本発明を完成させた。更に、大動脈縮窄による圧負荷心不全モデルにおいてＬＳＭＥＭ２ノックアウトマウスは免疫細胞浸潤を抑制することや、ラミン心筋症モデルマウスとの交配により生じたマウスは、下肢筋力が改善されることも見出し、ＬＳＭＥＭ２を標的とする治療が、ラミン心筋症のみならず、他の病因による重症心不全やラミノパチーの治療法として適用できることを示した。 The present inventors discovered that the intercalated disc is a receptor for mechano stress, and uniquely identified LSMEM2, a membrane protein localized in the intercalated disc and involved in mechanosignaling. LSMEM2 knockout mice exhibited normal cardiac function at rest. Then, they discovered that mice produced by crossing LSMEM2 knockout mice with lamin cardiomyopathy model mice have improved cardiac function, and completed the present invention. Furthermore, we found that LSMEM2 knockout mice suppressed immune cell infiltration in a pressure overload heart failure model due to aortic coarctation, and that mice produced by crossing with lamin cardiomyopathy model mice had improved lower limb muscle strength. We demonstrated that targeted therapy can be applied not only to lamin cardiomyopathy but also to severe heart failure and laminopathy due to other etiologies.
　即ち、本発明は以下のとおりである。
［１］　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、メカノシグナル伝達阻害剤。
［２］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［１］に記載の剤。
［３］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体又はアプタマーである、［１］に記載の剤。
［４］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［１］～［３］のいずれかに記載の剤。
［５］　抗炎症剤である、［１］～［４］のいずれかに記載の剤。
［６］　心疾患の予防及び／又は治療剤である、［１］～［５］のいずれかに記載の剤。
［７］　心疾患が拡張型心筋症である、［６］に記載の剤。
［８］　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、小胞輸送異常の抑制剤。
［８－１］　小胞輸送異常の抑制剤である、ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、メカノシグナル伝達阻害剤。
［９］　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、ラミノパチーの予防及び／又は治療剤。
［９－１］　ラミノパチーの予防及び／又は治療剤である、ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、メカノシグナル伝達阻害剤。
［１０］　ラミノパチーが筋ジストロフィー、ラミン心筋症、リポジストロフィー、白質ジストロフィーから選択される、［９］に記載の剤。
［１０－１］　ラミノパチーの予防及び／又は治療剤である、ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、メカノシグナル伝達阻害剤。 That is, the present invention is as follows.
[1] A mechanosignaling inhibitor containing a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
[2] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
(c) The agent according to [1], which is a ribozyme nucleic acid for the transcription product of the LSMEM2 gene. (d) The agent is a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[3] The agent according to [1], wherein the substance that suppresses the function of LSMEM2 is an antibody or aptamer against LSMEM2.
[4] The agent according to any one of [1] to [3], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[5] The agent according to any one of [1] to [4], which is an anti-inflammatory agent.
[6] The agent according to any one of [1] to [5], which is a preventive and/or therapeutic agent for heart disease.
[7] The agent according to [6], wherein the heart disease is dilated cardiomyopathy.
[8] An inhibitor of vesicular transport abnormalities, which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
[8-1] A mechano-signaling inhibitor containing as an active ingredient a substance that suppresses the expression or function of LSMEM2, which is an inhibitor of vesicular transport abnormalities.
[9] A prophylactic and/or therapeutic agent for laminopathy, which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
[9-1] A mechanosignal transduction inhibitor containing a substance that suppresses the expression or function of LSMEM2 as an active ingredient, which is a prophylactic and/or therapeutic agent for laminopathy.
[10] The agent according to [9], wherein the laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, and leukodystrophy.
[10-1] A mechano-signaling inhibitor containing a substance that suppresses the expression or function of LSMEM2 as an active ingredient, which is a prophylactic and/or therapeutic agent for laminopathy.
　また、別の一態様において、本発明は以下の通りである。
［Ａ１］　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、メカノシグナル伝達阻害剤。
［Ａ２］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ａ１］に記載の剤。
［Ａ３］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ａ１］に記載の剤。
［Ａ４］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ａ１］～［Ａ３］のいずれかに記載の剤。
［Ａ５］　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、抗炎症剤。
［Ａ６］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ａ５］に記載の剤。
［Ａ７］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ａ５］に記載の剤。
［Ａ８］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ａ５］～［Ａ７］のいずれかに記載の剤。
［Ａ９］　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、心疾患の予防及び／又は治療剤。
［Ａ１０］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ａ９］に記載の剤。
［Ａ１１］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ａ９］に記載の剤。
［Ａ１２］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ａ９］～［Ａ１１］のいずれかに記載の剤。
［Ａ１３］　心疾患が拡張型心筋症である、［Ａ９］～［Ａ１２］のいずれかに記載の剤。
［Ａ１４］　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、小胞輸送異常の抑制剤。
［Ａ１５］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ａ１４］に記載の剤。
［Ａ１６］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ａ１４］に記載の剤。
［Ａ１７］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ａ１４］～［Ａ１６］のいずれかに記載の剤。
［Ａ１８］　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、ラミノパチーの予防及び／又は治療剤。
［Ａ１９］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ａ１８］に記載の剤。
［Ａ２０］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ａ１８］に記載の剤。
［Ａ２１］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ａ１８］～［Ａ２０］のいずれかに記載の剤。
［Ａ２２］　ラミノパチーが筋ジストロフィー、ラミン心筋症、リポジストロフィー、又は白質ジストロフィーから選択される、［Ａ１８］～［Ａ２１］のいずれかに記載の剤。
［Ｂ１］　対象にＬＳＭＥＭ２の発現又は機能を抑制する物質を投与することを含む、対象におけるメカノシグナル伝達を阻害する方法。
［Ｂ２］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｂ１］に記載の方法。
［Ｂ３］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｂ１］に記載の方法。
［Ｂ４］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｂ１］～［Ｂ３］のいずれかに記載の方法。
［Ｂ５］　対象にＬＳＭＥＭ２の発現又は機能を抑制する物質を投与することを含む、対象における炎症を抑制する方法。
［Ｂ６］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｂ５］に記載の方法。
［Ｂ７］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｂ５］に記載の方法。
［Ｂ８］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｂ５］～［Ｂ７］のいずれかに記載の方法。
［Ｂ９］　対象にＬＳＭＥＭ２の発現又は機能を抑制する物質を投与することを含む、対象における心疾患を予防及び／又は治療する方法。
［Ｂ１０］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｂ９］に記載の方法。
［Ｂ１１］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｂ９］に記載の方法。
［Ｂ１２］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｂ９］～［Ｂ１１］のいずれかに記載の方法。
［Ｂ１３］　心疾患が拡張型心筋症である、［Ｂ９］～［Ｂ１２］のいずれかに記載の方法。
［Ｂ１４］　対象にＬＳＭＥＭ２の発現又は機能を抑制する物質を投与することを含む、対象における小胞輸送異常を抑制するための方法。
［Ｂ１５］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｂ１４］に記載の方法。
［Ｂ１６］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｂ１４］に記載の方法。
［Ｂ１７］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｂ１４］～［Ｂ１６］のいずれかに記載の方法。
［Ｂ１８］　対象にＬＳＭＥＭ２の発現又は機能を抑制する物質を投与することを含む、ラミノパチーを予防及び／又は治療する方法。
［Ｂ１９］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｂ１８］に記載の方法。
［Ｂ２０］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｂ１８］に記載の方法。
［Ｂ２１］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｂ１８］～［Ｂ２０］のいずれかに記載の方法。
［Ｂ２２］　ラミノパチーが筋ジストロフィー、ラミン心筋症、リポジストロフィー、又は白質ジストロフィーから選択される、［Ｂ１８］～［Ｂ２１］のいずれかに記載の方法。
［Ｃ１］　メカノシグナル伝達の阻害における使用のための、ＬＳＭＥＭ２の発現又は機能を抑制する物質を含む、組成物。
［Ｃ２］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｃ１］に記載の組成物。
［Ｃ３］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｃ１］に記載の組成物。
［Ｃ４］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｃ１］～［Ｃ３］のいずれかに記載の組成物。
［Ｃ５］　炎症の抑制における使用のための、ＬＳＭＥＭ２の発現又は機能を抑制する物質を含む、組成物。
［Ｃ６］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｃ５］に記載の組成物。
［Ｃ７］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｃ５］に記載の組成物。
［Ｃ８］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｃ５］～［Ｃ７］のいずれかに記載の組成物。
［Ｃ９］　心疾患の予防及び／又は治療における使用のための、ＬＳＭＥＭ２の発現又は機能を抑制する物質を含む、組成物。
［Ｃ１０］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｃ９］に記載の組成物。
［Ｃ１１］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｃ９］に記載の組成物。
［Ｃ１２］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｃ９］～［Ｃ１１］のいずれかに記載の組成物。
［Ｃ１３］　心疾患が拡張型心筋症である、［Ｃ９］～［Ｃ１２］のいずれかに記載の組成物。
［Ｃ１４］　小胞輸送異常の抑制における使用のための、ＬＳＭＥＭ２の発現又は機能を抑制する物質を含む、組成物。
［Ｃ１５］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｃ１４］に記載の組成物。
［Ｃ１６］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｃ１４］に記載の組成物。
［Ｃ１７］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｃ１４］～［Ｃ１６］のいずれかに記載の組成物。
［Ｃ１８］　ラミノパチーの予防及び／又は治療における使用のための、ＬＳＭＥＭ２の発現又は機能を抑制する物質を含む、組成物。
［Ｃ１９］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｃ１８］に記載の組成物。
［Ｃ２０］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｃ１８］に記載の組成物。
［Ｃ２１］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｃ１８］～［Ｃ２０］のいずれかに記載の組成物。
［Ｃ２２］　ラミノパチーが筋ジストロフィー、ラミン心筋症、リポジストロフィー、又は白質ジストロフィーから選択される、［Ｃ１８］～［Ｃ２１］のいずれかに記載の組成物。
［Ｄ１］　メカノシグナル伝達の阻害用の医薬の製造におけるＬＳＭＥＭ２の発現又は機能を抑制する物質の使用。
［Ｄ２］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｄ１］に記載の使用。
［Ｄ３］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｄ１］に記載の使用。
［Ｄ４］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｄ１］～［Ｄ３］のいずれかに記載の使用。
［Ｄ５］　炎症の抑制用の医薬の製造におけるＬＳＭＥＭ２の発現又は機能を抑制する物質の使用。
［Ｄ６］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｄ５］に記載の使用。
［Ｄ７］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｄ５］に記載の使用。
［Ｄ８］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｄ５］～［Ｄ７］のいずれかに記載の使用。
［Ｄ９］　心疾患の予防及び／又は治療用の医薬の製造におけるＬＳＭＥＭ２の発現又は機能を抑制する物質の使用。
［Ｄ１０］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｄ９］に記載の使用。
［Ｄ１１］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｄ９］に記載の使用。
［Ｄ１２］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｄ９］～［Ｄ１１］のいずれかに記載の使用。
［Ｄ１３］　心疾患が拡張型心筋症である、［Ｄ９］～［Ｄ１２］のいずれかに記載の使用。
［Ｄ１４］　小胞輸送異常の抑制用の医薬の製造におけるＬＳＭＥＭ２の発現又は機能を抑制する物質の使用。
［Ｄ１５］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｄ１４］に記載の使用。
［Ｄ１６］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｄ１４］に記載の使用。
［Ｄ１７］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｄ１４］～［Ｄ１６］のいずれかに記載の使用。
［Ｄ１８］　ラミノパチーの予防及び／又は治療用の医薬の製造におけるＬＳＭＥＭ２の発現又は機能を抑制する物質の使用。
［Ｄ１９］　ＬＳＭＥＭ２の発現を抑制する物質が、
（ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
（ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
（ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
（ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
である、［Ｄ１８］に記載の使用。
［Ｄ２０］　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、［Ｄ１８］に記載の使用。
［Ｄ２１］　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、［Ｄ１８］～［Ｄ２０］のいずれかに記載の使用。
［Ｄ２２］　ラミノパチーが筋ジストロフィー、ラミン心筋症、リポジストロフィー、又は白質ジストロフィーから選択される、［Ｄ１８］～［Ｄ２１］のいずれかに記載の使用。 Moreover, in another aspect, the present invention is as follows.
[A1] A mechanosignaling inhibitor containing a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
[A2] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The agent according to [A1], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[A3] The agent according to [A1], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[A4] The agent according to any one of [A1] to [A3], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[A5] An anti-inflammatory agent containing a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
[A6] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The agent according to [A5], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[A7] The agent according to [A5], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[A8] The agent according to any one of [A5] to [A7], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[A9] A prophylactic and/or therapeutic agent for heart disease, which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
[A10] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The agent according to [A9], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[A11] The agent according to [A9], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[A12] The agent according to any one of [A9] to [A11], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[A13] The agent according to any one of [A9] to [A12], wherein the heart disease is dilated cardiomyopathy.
[A14] An inhibitor of vesicular transport abnormality, which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
[A15] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The agent according to [A14], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[A16] The agent according to [A14], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[A17] The agent according to any one of [A14] to [A16], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[A18] A prophylactic and/or therapeutic agent for laminopathy, which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
[A19] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The agent according to [A18], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[A20] The agent according to [A18], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[A21] The agent according to any one of [A18] to [A20], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[A22] The agent according to any one of [A18] to [A21], wherein the laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, or leukodystrophy.
[B1] A method for inhibiting mechanosignaling in a subject, which comprises administering to the subject a substance that suppresses the expression or function of LSMEM2.
[B2] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
(c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B1].
[B3] The method according to [B1], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[B4] The method according to any one of [B1] to [B3], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[B5] A method for suppressing inflammation in a subject, comprising administering to the subject a substance that suppresses the expression or function of LSMEM2.
[B6] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
(c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B5].
[B7] The method according to [B5], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[B8] The method according to any one of [B5] to [B7], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[B9] A method for preventing and/or treating heart disease in a subject, comprising administering to the subject a substance that suppresses the expression or function of LSMEM2.
[B10] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
(c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B9].
[B11] The method according to [B9], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[B12] The method according to any one of [B9] to [B11], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[B13] The method according to any one of [B9] to [B12], wherein the heart disease is dilated cardiomyopathy.
[B14] A method for suppressing abnormal vesicle transport in a subject, comprising administering to the subject a substance that suppresses the expression or function of LSMEM2.
[B15] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
(c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B14].
[B16] The method according to [B14], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[B17] The method according to any one of [B14] to [B16], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[B18] A method for preventing and/or treating laminopathy, which comprises administering to a subject a substance that suppresses the expression or function of LSMEM2.
[B19] A substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
(c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the method according to [B18].
[B20] The method according to [B18], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[B21] The method according to any one of [B18] to [B20], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[B22] The method according to any one of [B18] to [B21], wherein the laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, or leukodystrophy.
[C1] A composition comprising a substance that suppresses LSMEM2 expression or function for use in inhibiting mechanosignaling.
[C2] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The composition according to [C1], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[C3] The composition according to [C1], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[C4] The composition according to any one of [C1] to [C3], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[C5] A composition comprising a substance that suppresses LSMEM2 expression or function for use in suppressing inflammation.
[C6] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The composition according to [C5], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[C7] The composition according to [C5], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[C8] The composition according to any one of [C5] to [C7], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[C9] A composition containing a substance that suppresses the expression or function of LSMEM2 for use in the prevention and/or treatment of heart disease.
[C10] A substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The composition according to [C9], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[C11] The composition according to [C9], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[C12] The composition according to any one of [C9] to [C11], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[C13] The composition according to any one of [C9] to [C12], wherein the heart disease is dilated cardiomyopathy.
[C14] A composition comprising a substance that suppresses LSMEM2 expression or function for use in suppressing vesicular transport abnormalities.
[C15] A substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The composition according to [C14], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[C16] The composition according to [C14], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[C17] The composition according to any one of [C14] to [C16], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[C18] A composition comprising a substance that suppresses the expression or function of LSMEM2 for use in the prevention and/or treatment of laminopathy.
[C19] A substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The composition according to [C18], which is (c) a ribozyme nucleic acid for a transcription product of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[C20] The composition according to [C18], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[C21] The composition according to any one of [C18] to [C20], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[C22] The composition according to any one of [C18] to [C21], wherein the laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, or leukodystrophy.
[D1] Use of a substance that suppresses the expression or function of LSMEM2 in the manufacture of a medicament for inhibiting mechanosignaling.
[D2] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The use according to [D1], which is (c) a ribozyme nucleic acid directed against the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[D3] The use according to [D1], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[D4] The use according to any one of [D1] to [D3], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[D5] Use of a substance that suppresses the expression or function of LSMEM2 in the manufacture of a medicament for suppressing inflammation.
[D6] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
(c) a ribozyme nucleic acid for the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the use according to [D5].
[D7] The use according to [D5], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[D8] The use according to any one of [D5] to [D7], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[D9] Use of a substance that suppresses the expression or function of LSMEM2 in the manufacture of a medicament for the prevention and/or treatment of heart disease.
[D10] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
The use according to [D9], which is (c) a ribozyme nucleic acid for the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
[D11] The use according to [D9], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[D12] The use according to any one of [D9] to [D11], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[D13] The use according to any one of [D9] to [D12], wherein the heart disease is dilated cardiomyopathy.
[D14] Use of a substance that suppresses the expression or function of LSMEM2 in the manufacture of a medicament for suppressing abnormal vesicular transport.
[D15] The substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
(c) a ribozyme nucleic acid for the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the use according to [D14].
[D16] The use according to [D14], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[D17] The use according to any one of [D14] to [D16], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[D18] Use of a substance that suppresses the expression or function of LSMEM2 in the manufacture of a medicament for preventing and/or treating laminopathy.
[D19] A substance that suppresses the expression of LSMEM2 is
(a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
(b) antisense nucleic acid against the transcript of the LSMEM2 gene,
(c) a ribozyme nucleic acid for the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene, the use according to [D18].
[D20] The use according to [D18], wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
[D21] The use according to any one of [D18] to [D20], wherein the substance is provided in the form of one or more expression vectors encoding the substance.
[D22] The use according to any one of [D18] to [D21], wherein the laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, or leukodystrophy.
　本発明によれば、ＬＳＭＥＭ２の発現又は機能を抑制する物質を含むメカノシグナル伝達阻害剤が提供できるので、ラミン心筋症、他の病因による重症心不全及びラミノパチーに対する有効な治療が可能となる。 According to the present invention, it is possible to provide a mechanosignaling inhibitor containing a substance that suppresses the expression or function of LSMEM2, thus making it possible to effectively treat lamin cardiomyopathy, severe heart failure due to other causes, and laminopathy.
図１は、ＲＴ－ＰＣＲによりＬＳＭＥＭ２の発現の臓器特異性を示す図である。FIG. 1 is a diagram showing the organ specificity of LSMEM2 expression by RT-PCR. 図２は、ＬＳＭＥＭ２強制発現心筋細胞のＲＮＡ－ｓｅｑの結果を示す図である。FIG. 2 is a diagram showing the results of RNA-seq of cardiomyocytes forcibly expressing LSMEM2. 図３は、ＬＳＭＥＭ２がメカノストレス（進展刺激）依存的な炎症性シグナルに必須であることを示す図である。FIG. 3 is a diagram showing that LSMEM2 is essential for mechanostress (developmental stimulus) dependent inflammatory signals. 図４は、ＬＳＭＥＭ２ノックアウトマウスの作製及び解析を示す図である。FIG. 4 is a diagram showing the production and analysis of LSMEM2 knockout mice. 図５は、ＬＳＭＥＭ２　ＫＯがラミノパチーモデル（ＬＭＮＡ　Ｈ２２２Ｐ　ＫＩ／ＫＩ）の生存率、心機能、心臓炎症シグナル及び骨格筋力を改善することを示す図である。FIG. 5 shows that LSMEM2 KO improves survival rate, cardiac function, cardiac inflammatory signals, and skeletal muscle strength in a laminopathy model (LMNA H222P KI/KI). 図６は、ＭｙｏＡＡＶ２　ｓｈＲＮＡ　ＬＳＭＥＭ２によるノックダウンの心機能改善効果を示す図である。FIG. 6 is a diagram showing the cardiac function improving effect of knockdown by MyoAAV2 shRNA LSMEM2. 図７は、圧負荷心不全モデルにおけるＬＳＭＥＭ２ノックアウトの解析を示す図である。FIG. 7 shows an analysis of LSMEM2 knockout in a pressure overload heart failure model.
　本発明のメカノシグナル伝達阻害剤の標的分子であるＬＳＭＥＭ２は、メカノストレスの受容器である介在板に局在する１回膜貫通型のタンパク質であり、ヒトでは、ｉｓｏｆｏｒｍ　１（ＮＰ＿６９４９４７．１）及びｉｓｏｆｏｒｍ　２（ＮＰ＿００１２９１３１４．１）が存在し、ｉｓｏｆｏｒｍ　１は、配列番号１で表されるｍＲＮＡから翻訳される配列番号２で表される１６４アミノ酸からなるアミノ酸配列を有し、そのうち１～９９位が細胞内領域、１００～１２２位が膜貫通領域、１２３～１６４位が細胞外領域である。５０位のセリンはリン酸化部位であり、１５６位のアスパラギンはグリコシル化部位である。配列番号３で表されるｍＲＮＡから翻訳されるｉｓｏｆｏｒｍ　２（配列番号４）はｉｓｏｆｏｒｍ　１よりも１アミノ酸短く、１６３アミノ酸から成る。 LSMEM2, which is the target molecule of the mechanosignaling inhibitor of the present invention, is a single-transmembrane protein localized in the intercalated disc, which is a receptor for mechano stress, and in humans, isoform 1 (NP_694947.1) and Isoform 2 (NP_001291314.1) exists, and isoform 1 has an amino acid sequence consisting of 164 amino acids represented by SEQ ID NO: 2 translated from mRNA represented by SEQ ID NO: 1, of which positions 1 to 99 are The intracellular region, positions 100 to 122 are the transmembrane region, and positions 123 to 164 are the extracellular region. Serine at position 50 is a phosphorylation site, and asparagine at position 156 is a glycosylation site. Isoform 2 (SEQ ID NO: 4) translated from mRNA represented by SEQ ID NO: 3 is one amino acid shorter than isoform 1 and consists of 163 amino acids.
　本明細書において、「ＬＳＭＥＭ２」とは、配列番号２（ｉｓｏｆｏｒｍ　１）又は配列番号４（ｉｓｏｆｏｒｍ　２）で表されるアミノ酸配列と同一若しくは実質的に同一のアミノ酸配列を含むタンパク質である。本明細書において、タンパク質及びペプチドは、ペプチド標記の慣例に従って左端がＮ末端（アミノ末端）、右端がＣ末端（カルボキシル末端）で記載される。
　「配列番号２又は配列番号４で表されるアミノ酸配列と実質的に同一のアミノ酸配列」とは、
（ａ）配列番号２又は配列番号４で表されるアミノ酸配列からなるヒトＬＳＭＥＭ２の、他の温血動物（例えば、モルモット、ラット、マウス、ニワトリ、ウサギ、イヌ、ブタ、ヒツジ、ウシ、サルなど）におけるオルソログのアミノ酸配列；又は
（ｂ）配列番号２又は配列番号４で表されるアミノ酸配列からなるヒトＬＳＭＥＭ２若しくは上記（ａ）のオルソログの天然のアレル変異体若しくは遺伝子多型におけるアミノ酸配列を意味する。
　好ましくは、ＬＳＭＥＭ２は配列番号２又は配列番号４で表されるアミノ酸配列からなるヒトＬＳＭＥＭ２若しくはその天然のアレル変異体若しくは遺伝子多型である。 As used herein, "LSMEM2" is a protein that includes an amino acid sequence that is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 2 (isoform 1) or SEQ ID NO: 4 (isoform 2). In this specification, proteins and peptides are described with the left end being the N-terminus (amino terminal) and the right end being the C-terminus (carboxyl terminal), in accordance with the convention for peptide markings.
"Amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4" means
(a) Human LSMEM2 consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 in other warm-blooded animals (e.g., guinea pig, rat, mouse, chicken, rabbit, dog, pig, sheep, cow, monkey, etc.) ); or (b) human LSMEM2 consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or the amino acid sequence in a natural allelic variant or genetic polymorphism of the ortholog in (a) above. do.
Preferably, LSMEM2 is human LSMEM2 consisting of the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, or a natural allelic variant or genetic polymorphism thereof.
　ＬＳＭＥＭ２　ＫＯマウスは正常に発生し、心機能はまったく正常であり、生殖機能にも問題を認めない。即ちＬＳＭＥＭ２に対する遺伝子治療、ゲノム編集、抗体医薬、核酸医薬（アンチセンス、ｓｉＲＮＡ、ｍｉＲＮＡ、アプタマー等をいうがこれらに限られない）、低分子化合物などモダリティの制限なく、ｍＲＮＡ、タンパク質、下流のシグナルのいずれの抑制も新規治療法となる有望な創薬標的である。 LSMEM2 KO mice develop normally, have completely normal cardiac function, and have no problems with reproductive function. In other words, gene therapy for LSMEM2, genome editing, antibody drugs, nucleic acid drugs (including but not limited to antisense, siRNA, miRNA, aptamers, etc.), low molecular compounds, etc. can be used without any modality limitations, such as mRNA, proteins, and downstream signals. Suppression of either of these is a promising drug discovery target for new treatments.
　本発明の一態様は、ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、メカノシグナル伝達阻害剤である。本発明において「ＬＳＭＥＭ２の発現を抑制する物質」とは、ＬＳＭＥＭ２遺伝子をゲノムレベルで編集、或いは転写レベル、転写後調節のレベル、タンパク質への翻訳レベル、翻訳後修飾のレベル等のいかなる段階で作用するものであってもよい。従って、ＬＳＭＥＭ２の発現を抑制する物質としては、例えば、ＬＳＭＥＭ２遺伝子の転写を阻害する物質（例、アンチジーン）、初期転写産物からｍＲＮＡへのプロセッシングを阻害する物質、ｍＲＮＡの細胞質への輸送を阻害する物質、ｍＲＮＡからタンパク質への翻訳を阻害するか（例、アンチセンス核酸、ｍｉＲＮＡ）或いはｍＲＮＡを分解する（例、ｓｉＲＮＡ、リボザイム、ｍｉＲＮＡ）物質、初期翻訳産物の翻訳後修飾を阻害する物質、ＬＳＭＥＭ２の分解を誘導する物質などが含まれる。いずれの段階で作用するものであっても用いることができるが、ｍＲＮＡに相補的に結合してタンパク質への翻訳を阻害するか或いはｍＲＮＡを分解する物質が好ましい。 One aspect of the present invention is a mechanosignal transduction inhibitor that contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient. In the present invention, a "substance that suppresses the expression of LSMEM2" refers to a substance that edits the LSMEM2 gene at the genome level, or acts at any stage such as the transcription level, post-transcriptional regulation level, protein translation level, or post-translational modification level. It may be something that does. Therefore, substances that suppress the expression of LSMEM2 include, for example, substances that inhibit the transcription of the LSMEM2 gene (e.g., antigene), substances that inhibit the processing of early transcripts into mRNA, and substances that inhibit the transport of mRNA to the cytoplasm. substances that inhibit translation of mRNA into protein (e.g., antisense nucleic acids, miRNA) or degrade mRNA (e.g., siRNA, ribozymes, miRNA), substances that inhibit post-translational modification of initial translation products; This includes substances that induce the decomposition of LSMEM2. Any substance that acts at any stage can be used, but substances that bind complementary to mRNA and inhibit translation into protein or degrade mRNA are preferred.
　ＬＳＭＥＭ２遺伝子のｍＲＮＡからタンパク質への翻訳を特異的に阻害する（或いはｍＲＮＡを分解する）物質として、好ましくは、該ｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列又はその一部を含む核酸が挙げられる。
　ＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列とは、生理的条件下において、該ｍＲＮＡの標的配列に結合してその翻訳を阻害し得る（或いは該標的配列を切断する）程度の相補性を有するヌクレオチド配列を意味し、具体的には、例えば、該ｍＲＮＡのヌクレオチド配列と完全相補的なヌクレオチド配列（即ち、ｍＲＮＡの相補鎖のヌクレオチド配列）と、オーバーラップする領域に関して、９０％以上、好ましくは９５％以上、より好ましくは９７％以上、特に好ましくは９８％以上の相同性を有するヌクレオチド配列である。本発明における「ヌクレオチド配列の相同性」は、相同性計算アルゴリズムＮＣＢＩ　ＢＬＡＳＴ（Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｏｎ　Ｂａｓｉｃ　Ｌｏｃａｌ　Ａｌｉｇｎｍｅｎｔ　Ｓｅａｒｃｈ　Ｔｏｏｌ）を用い、以下の条件（期待値＝１０；ギャップを許す；フィルタリング＝ＯＮ；マッチスコア＝１；ミスマッチスコア＝－３）にて計算することができる。 The substance that specifically inhibits translation of LSMEM2 gene mRNA into protein (or degrades mRNA) preferably includes a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA or a portion thereof.
The nucleotide sequence complementary to the nucleotide sequence of the mRNA of the LSMEM2 gene has such a degree of complementarity that it can bind to the target sequence of the mRNA and inhibit its translation (or cleave the target sequence) under physiological conditions. Specifically, for example, with respect to a region that overlaps with a nucleotide sequence that is completely complementary to the nucleotide sequence of the mRNA (i.e., the nucleotide sequence of the complementary strand of the mRNA), 90% or more, The nucleotide sequences preferably have a homology of 95% or more, more preferably 97% or more, particularly preferably 98% or more. In the present invention, "nucleotide sequence homology" is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gap Allow; Filtering = ON; Match score = 1; mismatch score = -3).
　より具体的には、ＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列とは、配列番号１又は配列番号３で表されるヌクレオチド配列とストリンジェントな条件下でハイブリダイズするヌクレオチド配列である。ここで「ストリンジェントな条件」とは、例えば、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ，Ｊｏｈｎ　Ｗｉｌｅｙ　＆　Ｓｏｎｓ，６．３．１－６．３．６，１９９９に記載される条件、例えば、６×ＳＳＣ（ｓｏｄｉｕｍ　ｃｈｌｏｒｉｄｅ／ｓｏｄｉｕｍ　ｃｉｔｒａｔｅ）／４５℃でのハイブリダイゼーション、次いで０．２×ＳＳＣ／０．１％ＳＤＳ／５０～６５℃での一回以上の洗浄等が挙げられるが、当業者であれば、これと同等のストリンジェンシーを与えるハイブリダイゼーションの条件を適宜選択することができる。 More specifically, the nucleotide sequence complementary to the nucleotide sequence of mRNA of the LSMEM2 gene is a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 under stringent conditions. Here, "stringent conditions" refer to, for example, the conditions described in Current Protocols in Molecular Biology, John Wiley & Sons, 6.3.1-6.3.6, 1999, for example, 6×SSC (sodium Examples include hybridization in chloride/sodium citrate)/45°C, followed by one or more washings in 0.2x SSC/0.1% SDS/50 to 65°C; Hybridization conditions that provide stringency equivalent to that can be selected as appropriate.
　ＬＳＭＥＭ２遺伝子のｍＲＮＡの好ましい例としては、配列番号１又は配列番号３で表されるヌクレオチド配列を含むヒトＬＳＭＥＭ２（ＲｅｆＳｅｑ　Ａｃｃｅｓｓｉｏｎ　Ｎｏ．ＮＭ＿１５３２１５．３又はＮＭ＿００１３０４３８５．２）、或いは他の温血動物におけるそのオルソログ、更にはそれらの天然のアレル変異体若しくは遺伝子多型などのｍＲＮＡがあげられる。 A preferred example of LSMEM2 gene mRNA is human LSMEM2 (RefSeq Accession No. NM_153215.3 or NM_001304385.2) containing the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, or its ortholog in other warm-blooded animals. Further examples include mRNAs such as natural allelic variants or genetic polymorphisms thereof.
　「ＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列の一部」とは、ＬＳＭＥＭ２遺伝子のｍＲＮＡに特異的に結合することができ、且つ該ｍＲＮＡからのタンパク質の翻訳を阻害（或いは該ｍＲＮＡを分解）し得るものであれば、その長さや位置に特に制限はないが、配列特異性の面から、標的配列に相補的な部分を少なくとも１０塩基以上、好ましくは１５塩基以上、より好ましくは１９塩基以上含むものである。 "A part of the nucleotide sequence that is complementary to the nucleotide sequence of the mRNA of the LSMEM2 gene" is capable of specifically binding to the mRNA of the LSMEM2 gene, and inhibits translation of protein from the mRNA (or inhibits the translation of the mRNA). There are no particular restrictions on its length or position as long as it can be degraded (degraded), but from the perspective of sequence specificity, the portion complementary to the target sequence should be at least 10 bases or more, preferably 15 bases or more, more preferably 19 bases or more. It contains more than a base.
　具体的には、ＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列の一部を含む核酸として、以下の（ａ）～（ｃ）のものが好ましく例示される。
（ａ）ＬＳＭＥＭ２遺伝子のｍＲＮＡに対してＲＮＡｉ活性を有する核酸若しくはその前駆体
（ｂ）ＬＳＭＥＭ２遺伝子のｍＲＮＡに対するアンチセンス核酸、又は
（ｃ）ＬＳＭＥＭ２遺伝子のｍＲＮＡに対するリボザイム核酸 Specifically, the following (a) to (c) are preferably exemplified as nucleic acids containing part of a nucleotide sequence complementary to the nucleotide sequence of mRNA of the LSMEM2 gene.
(a) Nucleic acid or its precursor having RNAi activity against LSMEM2 gene mRNA (b) Antisense nucleic acid against LSMEM2 gene mRNA, or (c) Ribozyme nucleic acid against LSMEM2 gene mRNA
　本発明において「ＬＳＭＥＭ２の発現を抑制する物質」とは、ゲノム編集技術により、ゲノム中のＬＳＭＥＭ２遺伝子の配列を改変するなどにより、ＬＳＭＥＭ２の発現を抑制する物質であってもよい。このような物質として、以下の（ｄ）のものが好ましく例示される。
（ｄ）ＬＳＭＥＭ２遺伝子中の標的領域に特異的に結合する核酸配列認識モジュール In the present invention, the "substance that suppresses the expression of LSMEM2" may be a substance that suppresses the expression of LSMEM2 by modifying the sequence of the LSMEM2 gene in the genome using genome editing technology. As such a substance, the following (d) is preferably exemplified.
(d) Nucleic acid sequence recognition module that specifically binds to the target region in the LSMEM2 gene
　本発明におけるＬＳＭＥＭ２の発現を抑制する物質は、上記のようなＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列又はその一部を含む核酸や核酸配列認識モジュールに限定されず、ＬＳＭＥＭ２タンパク質の産生を直接的又は間接的に阻害する限り、低分子化合物、ペプチド、環状ペプチド、タンパク質、中分子、高分子などの他の物質であってもよい。 The substance that suppresses the expression of LSMEM2 in the present invention is not limited to a nucleic acid or a nucleic acid sequence recognition module containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the LSMEM2 gene, or a part thereof, as described above, Other substances such as low-molecular compounds, peptides, cyclic peptides, proteins, medium molecules, and polymers may be used as long as they inhibit directly or indirectly.
　本発明におけるＬＳＭＥＭ２の発現を阻害する物質は、上記のようなアンチセンス核酸、ｓｉＲＮＡ、リボザイムなどに限定されず、ＬＳＭＥＭ２の発現を直接的又は間接的に阻害する限り、他の物質（低分子化合物、ペプチド、環状ペプチド、タンパク質、中分子、高分子など）であってもよい。そのような低分子化合物としては、例えば、ＬＳＭＥＭ２の５０位のセリンのリン酸化を阻害する低分子量リン酸化阻害剤が挙げられる。 The substances that inhibit the expression of LSMEM2 in the present invention are not limited to the above-mentioned antisense nucleic acids, siRNA, ribozymes, etc., but may include other substances (low molecular compounds, etc.) as long as they directly or indirectly inhibit the expression of LSMEM2. , peptide, cyclic peptide, protein, medium molecule, polymer, etc.). Examples of such low-molecular compounds include low-molecular-weight phosphorylation inhibitors that inhibit phosphorylation of serine at position 50 of LSMEM2.
　上記で述べた（ａ）～（ｄ）の具体的な態様について、以下に示す。 Specific aspects of (a) to (d) described above are shown below.
（ａ）ＬＳＭＥＭ２遺伝子のｍＲＮＡに対してＲＮＡｉ活性を有する核酸若しくはその前駆体
　本明細書においては、ＬＳＭＥＭ２遺伝子のｍＲＮＡに相補的なオリゴＲＮＡとその相補鎖とからなる二本鎖ＲＮＡ、いわゆるｓｉＲＮＡは、ＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列又はその一部を含む核酸に包含されるものとして定義される。 (a) Nucleic acid having RNAi activity against LSMEM2 gene mRNA or its precursor In this specification, double-stranded RNA consisting of an oligo RNA complementary to LSMEM2 gene mRNA and its complementary strand, so-called siRNA , is defined as being encompassed by nucleic acids comprising a nucleotide sequence complementary to the nucleotide sequence of mRNA of the LSMEM2 gene, or a portion thereof.
　ｓｉＲＮＡは、標的遺伝子のｃＤＮＡ配列情報に基づいて、例えば、Ｅｌｂａｓｈｉｒら（Ｇｅｎｅｓ　Ｄｅｖ．，１５，１８８－２００（２００１））の提唱する規則に従って設計することができる。ｓｉＲＮＡの標的配列としては、例えばＡＡ＋（Ｎ）１９、ＡＡ＋（Ｎ）２１若しくはＮＡ＋（Ｎ）２１（Ｎは任意の塩基）等が挙げられるが、それらに限定されない。標的配列の位置も特に制限されるわけではない。選択された標的配列の候補群について、標的以外のｍＲＮＡにおいて１６－１７塩基の連続した配列に相同性がないかどうかを、ＢＬＡＳＴ等のホモロジー検索ソフトを用いて調べ、選択した標的配列の特異性を確認する。例えば、ＡＡ＋（Ｎ）１９、ＡＡ＋（Ｎ）２１若しくはＮＡ＋（Ｎ）２１（Ｎは任意の塩基）を標的配列とする場合、特異性の確認された標的配列について、ＡＡ（若しくはＮＡ）以降の１９－２１塩基にＴＴ若しくはＵＵの３’末端オーバーハングを有するセンス鎖と、該１９－２１塩基に相補的な配列及びＴＴ若しくはＵＵの３’末端オーバーハングを有するアンチセンス鎖とからなる２本鎖ＲＮＡをｓｉＲＮＡとして設計してもよい。また、ｓｉＲＮＡの前駆体であるショートヘアピンＲＮＡ（ｓｈＲＮＡ）は、ループ構造を形成しうる任意のリンカー配列（例えば、５－２５塩基程度）を適宜選択し、上記センス鎖とアンチセンス鎖とを該リンカー配列を介して連結することにより設計することができる。 siRNA can be designed based on the cDNA sequence information of the target gene, for example, according to the rules proposed by Elbashir et al. (Genes Dev., 15, 188-200 (2001)). Examples of the target sequence of siRNA include, but are not limited to, AA+(N) 19 , AA+(N) 21 , or NA+(N) 21 (N is any base). The location of the target sequence is also not particularly limited. For the selected target sequence candidate group, use homology search software such as BLAST to check whether there is any homology in consecutive 16-17 base sequences in mRNA other than the target, and determine the specificity of the selected target sequence. Check. For example, when the target sequence is AA+(N) 19 , AA+(N) 21 or NA+(N) 21 (N is any base), the target sequence after AA (or NA) for which specificity has been confirmed is Two strands consisting of a sense strand having a 3'-terminal overhang of TT or UU at bases 19-21, and an antisense strand having a sequence complementary to the 19-21 bases and a 3'-terminal overhang of TT or UU. Strand RNA may be designed as siRNA. In addition, for short hairpin RNA (shRNA), which is a precursor of siRNA, an arbitrary linker sequence (for example, about 5 to 25 bases) that can form a loop structure is appropriately selected, and the sense strand and antisense strand are linked together. It can be designed by connecting via a linker sequence.
　ｓｉＲＮＡ及び／又はｓｈＲＮＡの配列は、種々のｗｅｂサイト上に無料で提供される検索ソフトを用いて検索が可能である。このようなサイトとしては、例えば、Ｄｈａｒｍａｃｏｎが提供するｓｉＤＥＳＩＧＮ　Ｃｅｎｔｅｒ、ＧｅｎＳｃｒｉｐｔが提供するｓｉＲＮＡ　Ｔａｒｇｅｔ　Ｆｉｎｄｅｒ等が挙げられるが、これらに限定されない。ヒットしたｓｉＲＮＡ配列情報に基づいて合成したｓｉＲＮＡ又はｓｈＲＮＡが、実際に、治療上有効な程度に心筋細胞におけるＬＳＭＥＭ２発現を抑制し得ることは、例えば、該核酸を導入した心筋幹細胞におけるＬＳＭＥＭ２　ｍＲＮＡの発現レベルを測定することにより、検証することができる。尚、本来的にはＲＮＡｉ活性を有するものの、導入後ＬＳＭＥＭ２　ｍＲＮＡの発現抑制効果が減衰する場合には、後述するようにｓｉＲＮＡ又はｓｈＲＮＡの構成ヌクレオチドに種々の修飾を加えることで、その生体内安定性を向上させることにより、ＬＳＭＥＭ２の発現抑制効果をより持続させることが可能であり、所望の治療効果を奏し得る。 The sequences of siRNA and/or shRNA can be searched using search software provided free of charge on various websites. Examples of such sites include, but are not limited to, siDESIGN Center provided by Dharmacon, siRNA Target Finder provided by GenScript, and the like. The fact that siRNA or shRNA synthesized based on the hit siRNA sequence information can actually suppress LSMEM2 expression in cardiomyocytes to a therapeutically effective extent means that, for example, the expression of LSMEM2 mRNA in myocardial stem cells into which the nucleic acid has been introduced It can be verified by measuring the level. Although it originally has RNAi activity, if the effect of suppressing the expression of LSMEM2 mRNA is attenuated after introduction, the in vivo stability can be improved by adding various modifications to the constituent nucleotides of siRNA or shRNA, as described below. By improving the activity, the effect of suppressing the expression of LSMEM2 can be further sustained, and the desired therapeutic effect can be achieved.
　好ましい実施態様において、本発明のｓｉＲＮＡ及びｓｈＲＮＡは、配列番号１又は配列番号３で表されるヌクレオチド配列中、ヌクレオチド番号１～１４３４で示される領域内の、連続する少なくとも１５個のヌクレオチドからなる配列と相補的なヌクレオチド配列を含む。特に好ましい一実施態様において、本発明のｓｉＲＮＡは、Ｓｉｌｅｎｃｅｒ（登録商標）　Ｓｅｌｅｃｔ　ｓｉＲＮＡ（ＬＳＭＥＭ２　ｓｉＲＮＡ　ＩＤ：ｓ１８９３２７，ｓｅｎｓｅ（５’→３’）：ＧＣＡＣＡＧＵＧＧＡＧＧＣＵＣＡＣＵＡＴＴ（配列番号９）、ａｎｔｉｓｅｎｓｅ（５’→３’）：ＵＡＧＵＧＡＧＣＣＵＣＣＡＣＵＧＵＧＣＡＧ（配列番号１０）（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，Ｗａｌｔｈａｍ，ＭＡ）であり、ｓｈＲＮＡは、５’－ＣＴＣＴＣＡＣＴＧＡＴＧＧＣＴＴＣＡＴＴＴ－３’（配列番号１１）である。 In a preferred embodiment, the siRNA and shRNA of the present invention are sequences consisting of at least 15 consecutive nucleotides within the region represented by nucleotide numbers 1 to 1434 in the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3. Contains a nucleotide sequence complementary to In a particularly preferred embodiment, the siRNA of the present invention is Silencer® Select siRNA (LSMEM2 siRNA ID: s189327, sense (5'→3'): GCACAGUGGAGGCUCACUATT (SEQ ID NO: 9), antisense e(5'→3' ): UAGUGAGCUCCACUGUGCAG (SEQ ID NO: 10) (Thermo Fisher Scientific, Waltham, MA), and the shRNA is 5'-CTCTCACTGATGGCTTCATTT-3' (SEQ ID NO: 11).
　本明細書においては、ＬＳＭＥＭ２遺伝子のｍＲＮＡを標的とするマイクロＲＮＡ（ｍｉＲＮＡ）もまた、ＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列又はその一部を含む核酸に包含されるものとして定義される。ｍｉＲＮＡは、標的となるｍＲＮＡに相補的に結合してｍＲＮＡの翻訳を抑制するか、或いはｍＲＮＡを分解することにより、遺伝子発現の転写後制御に関与している。 As used herein, microRNA (miRNA) that targets the mRNA of the LSMEM2 gene is also defined as a nucleic acid that includes a nucleotide sequence complementary to the nucleotide sequence of the mRNA of the LSMEM2 gene, or a portion thereof. Ru. miRNAs are involved in post-transcriptional control of gene expression by binding complementary to target mRNAs and inhibiting translation of the mRNAs, or by degrading the mRNAs.
　ｍｉＲＮＡはまず、それをコードする遺伝子から一次転写産物であるｐｒｉｍａｒｙ－ｍｉｃｒｏＲＮＡ（ｐｒｉ－ｍｉＲＮＡ）が転写され、次いで、Ｄｒｏｓｈａにより特徴的なヘアピン構造を有する約７０塩基長のｐｒｅｃｕｒｓｏｒ－ｍｉｃｒｏＲＮＡ（ｐｒｅ－ｍｉＲＮＡ）にプロセッシングされた後、核から細胞質に輸送され、更に、Ｄｉｃｅｒ介在によるプロセシングにより成熟型ｍｉＲＮＡとなり、ＲＩＳＣに取り込まれて標的ｍＲＮＡに作用する。従って、ｍｉＲＮＡの前駆体として、ｐｒｅ－ｍｉＲＮＡやｐｒｉ－ｍｉＲＮＡ、好ましくはｐｒｅ－ｍｉＲＮＡを用いることもできる。 First, primary-microRNA (pri-miRNA), which is a primary transcription product, is transcribed from the gene encoding miRNA, and then precursor-microRNA (pre-miRNA), which is approximately 70 bases long and has a characteristic hairpin structure, is transcribed by Drosha. ), then transported from the nucleus to the cytoplasm, and further processed through Dicer-mediated processing to become mature miRNA, which is taken up by RISC and acts on target mRNA. Therefore, pre-miRNA or pri-miRNA, preferably pre-miRNA, can also be used as a precursor of miRNA.
　ｍｉＲＮＡは、種々のｗｅｂサイト上に無料で提供される標的予測ソフトを用いて検索が可能である。このようなサイトとしては、例えば、米国ホワイトヘッド研究所が公開しているＴａｒｇｅｔＳｃａｎ、ギリシアのアレクサンダー・フレミング生体医科学研究センターが公開しているＤＩＡＮＡ－ｍｉｃｒｏ－Ｔ－ＣＤＳ等が挙げられるが、これらに限定されない。或いは、テッサリ大学・パスツール研究所等が公開している、標的ｍＲＮＡに作用することが実験的に証明されているｍｉＲＮＡに関するデータベースであるＴａｒＢａｓｅを用いて、ＬＳＭＥＭ２　ｍＲＮＡを標的とするｍｉＲＮＡを検索することもできる。これらのｍｉＲＮＡ及び／又はｐｒｅ－ｍｉＲＮＡの配列情報は、例えば、英国マンチェスター大学が公開しているｍｉＲＢａｓｅを用いて取得することができる。 miRNA can be searched using target prediction software provided free of charge on various websites. Examples of such sites include TargetScan, published by the Whitehead Institute in the United States, and DIANA-micro-T-CDS, published by the Alexander Fleming Biomedical Research Center in Greece. but not limited to. Alternatively, search for miRNAs that target LSMEM2 mRNA using TarBase, a database of miRNAs that has been experimentally proven to act on target mRNAs, published by the University of Tessari, Institut Pasteur, etc. You can also do that. Sequence information on these miRNAs and/or pre-miRNAs can be obtained using, for example, miRBase published by the University of Manchester, UK.
　ｓｉＲＮＡ及び／又はｓｈＲＮＡ、或いはｍｉＲＮＡ及び／又はｐｒｅ－ｍｉＲＮＡを構成するヌクレオチド分子は、天然型のＲＮＡ若しくはＤＮＡでもよいが、安定性（化学的及び／又は対酵素）や比活性（ＲＮＡとの親和性）を向上させるために、種々の化学修飾を含むことができる。例えば、ヌクレアーゼなどの加水分解酵素による分解を防ぐために、アンチセンス核酸を構成する各ヌクレオチドのリン酸残基（ホスフェート）を、例えば、ホスホロチオエート（ＰＳ）、メチルホスホネート、ホスホロジチオネートなどの化学修飾リン酸残基に置換することができる。また、各ヌクレオチドの糖（リボース）の２’位の水酸基を、－ＯＲ（Ｒ＝ＣＨ３（２’－Ｏ－Ｍｅ）、ＣＨ２ＣＨ２ＯＣＨ３（２’－Ｏ－ＭＯＥ）、ＣＨ２ＣＨ２ＮＨＣ（ＮＨ）ＮＨ２、ＣＨ２ＣＯＮＨＣＨ３、ＣＨ２ＣＨ２ＣＮ等）に置換してもよい。更に、塩基部分（ピリミジン、プリン）に化学修飾を施してもよく、例えば、ピリミジン塩基の５位へのメチル基やカチオン性官能基の導入、或いは２位のカルボニル基のチオカルボニルへの置換などが挙げられる。 The nucleotide molecules constituting siRNA and/or shRNA, or miRNA and/or pre-miRNA, may be natural RNA or DNA, but stability (chemical and/or enzymatic) and specific activity (affinity with RNA) Various chemical modifications can be included in order to improve the properties. For example, in order to prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residues of each nucleotide constituting the antisense nucleic acid are chemically modified with phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. It can be substituted with a phosphate residue. In addition, the hydroxyl group at the 2' position of the sugar (ribose) of each nucleotide is changed to -OR (R=CH 3 (2'-O-Me), CH 2 CH 2 OCH 3 (2'-O-MOE), CH 2 CH 2 NHC(NH)NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introducing a methyl group or cationic functional group to the 5-position of the pyrimidine base, or replacing the carbonyl group at the 2-position with thiocarbonyl. can be mentioned.
　ＲＮＡの糖部のコンフォーメーションはＣ２’－ｅｎｄｏ（Ｓ型）とＣ３’－ｅｎｄｏ（Ｎ型）の２つが支配的であり、一本鎖ＲＮＡではこの両者の平衡として存在するが、二本鎖を形成するとＮ型に固定される。従って、標的ＲＮＡに対して強い結合能を付与するために、２’酸素と４’炭素を架橋することにより、糖部のコンフォーメーションをＮ型に固定したＲＮＡ誘導体であるＢＮＡ（ＬＮＡ）（Ｉｍａｎｉｓｈｉ，Ｔ．ｅｔ　ａｌ．，Ｃｈｅｍ．Ｃｏｍｍｕｎ．，１６５３－９，２００２；Ｊｅｐｓｅｎ，Ｊ．Ｓ．ｅｔ　ａｌ．，Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｓ，１４，１３０－４６，２００４）やＥＮＡ（Ｍｏｒｉｔａ，Ｋ．ｅｔ　ａｌ．，Ｎｕｃｌｅｏｓｉｄｅｓ　Ｎｕｃｌｅｏｔｉｄｅｓ　Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ，２２，１６１９－２１，２００３）もまた、好ましく用いられ得る。 There are two dominant conformations of the sugar part of RNA: C2'-endo (S type) and C3'-endo (N type), and in single-stranded RNA there is an equilibrium between the two, but in double-stranded RNA When formed, it is fixed into an N type. Therefore, in order to impart strong binding ability to the target RNA, BNA (LNA) (Imanishi , T.ET Al., Chem. Commun., 1653-9, 2002; JEPSEN, JEPSEN, JES.Et Al. OSIDES Nucleotides Nucleic Acids, 22, 1619-21, 2003) can also be preferably used.
　ｓｉＲＮＡは、ｍＲＮＡ上の標的配列のセンス鎖及びアンチセンス鎖をＤＮＡ／ＲＮＡ自動合成機でそれぞれ合成し、適当なアニーリング緩衝液中、約９０～約９５℃で約１分程度変性させた後、約３０～約７０℃で約１～約８時間アニーリングさせることにより調製することができる。また、ｓｉＲＮＡの前駆体となるｓｈＲＮＡを合成し、これをダイサー（ｄｉｃｅｒ）を用いて切断することにより調製することもできる。ｍｉＲＮＡ及びｐｒｅ－ｍｉＲＮＡは、それらの配列情報に基づいて、ＤＮＡ／ＲＮＡ自動合成機で合成することができる。 siRNA is produced by synthesizing the sense and antisense strands of the target sequence on mRNA using an automatic DNA/RNA synthesizer, denaturing them in an appropriate annealing buffer at about 90 to about 95°C for about 1 minute, and then It can be prepared by annealing at about 30 to about 70°C for about 1 to about 8 hours. Alternatively, it can also be prepared by synthesizing shRNA, which is a precursor of siRNA, and cleaving this using dicer. miRNA and pre-miRNA can be synthesized with an automatic DNA/RNA synthesizer based on their sequence information.
　本明細書においては、生体内でＬＳＭＥＭ２遺伝子のｍＲＮＡに対するｓｉＲＮＡ又はｍｉＲＮＡを生成し得るようにデザインされた核酸もまた、ＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列又はその一部を含む核酸に包含されるものとして定義される。そのような核酸としては、上記したｓｈＲＮＡ若しくはｓｉＲＮＡ又はｍｉＲＮＡ若しくはｐｒｅ－ｍｉＲＮＡを発現するように構築された発現ベクターなどが挙げられる。ｓｈＲＮＡは、ｍＲＮＡ上の標的配列のセンス鎖及びアンチセンス鎖を適当なループ構造を形成しうる長さ（例えば５～２５塩基程度）のスペーサー配列を間に挿入して連結したヌクレオチド配列を含むオリゴＲＮＡをデザインし、これをＤＮＡ／ＲＮＡ自動合成機で合成することにより調製することができる。ｓｈＲＮＡを発現するベクターには、タンデムタイプとステムループ（ヘアピン）タイプとがある。前者はｓｉＲＮＡのセンス鎖の発現カセットとアンチセンス鎖の発現カセットをタンデムに連結したもので、細胞内で各鎖が発現してアニーリングすることにより２本鎖のｓｉＲＮＡ（ｄｓＲＮＡ）を形成するというものである。一方、後者はｓｈＲＮＡの発現カセットをベクターに挿入したもので、細胞内でｓｈＲＮＡが発現しｄｉｃｅｒによるプロセシングを受けてｄｓＲＮＡを形成するというものである。プロモーターとしては、ｐｏｌＩＩ系プロモーター（例えば、ＣＭＶ前初期プロモーター）を使用することもできるが、短いＲＮＡの転写を正確に行わせるために、ｐｏｌＩＩＩ系プロモーターを使用するのが一般的である。ｐｏｌＩＩＩ系プロモーターとしては、マウス及びヒトのＵ６－ｓｎＲＮＡプロモーター、ヒトＨ１－ＲＮａｓｅ　Ｐ　ＲＮＡプロモーター、ヒトバリン－ｔＲＮＡプロモーターなどが挙げられる。また、転写終結シグナルとして４個以上Ｔが連続した配列が用いられる。ｍｉＲＮＡやｐｒｅ－ｍｉＲＮＡの発現カセットも、ｓｈＲＮＡと同様にして作製することができる。
　このようにして構築したｓｉＲＮＡ若しくはｓｈＲＮＡ又はｍｉＲＮＡ若しくはｐｒｅ－ｍｉＲＮＡ発現カセットを、次いでプラスミドベクターやウイルスベクターに挿入する。このようなベクターとしては、レトロウイルス、レンチウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス、センダイウイルスなどのウイルスベクターや、動物細胞発現プラスミドなどが用いられる。 As used herein, a nucleic acid designed to be able to generate siRNA or miRNA against LSMEM2 gene mRNA in vivo also refers to a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of LSMEM2 gene mRNA or a portion thereof. Defined as encompassing. Examples of such nucleic acids include expression vectors constructed to express shRNA, siRNA, miRNA, or pre-miRNA described above. shRNA is an oligonucleotide containing a nucleotide sequence in which the sense and antisense strands of the target sequence on mRNA are connected by inserting a spacer sequence of a length (for example, about 5 to 25 bases) between them to form an appropriate loop structure. It can be prepared by designing RNA and synthesizing it using an automatic DNA/RNA synthesizer. There are two types of vectors that express shRNA: tandem type and stem-loop (hairpin) type. The former is an expression cassette for the sense strand of siRNA and an expression cassette for the antisense strand linked in tandem, and each strand is expressed and annealed within the cell to form double-stranded siRNA (dsRNA). It is. On the other hand, in the latter, an shRNA expression cassette is inserted into a vector, and the shRNA is expressed within cells and processed by dicer to form dsRNA. Although a pol II promoter (eg, CMV immediate early promoter) can be used as the promoter, a pol III promoter is generally used to ensure accurate transcription of short RNAs. Examples of pol III promoters include mouse and human U6-snRNA promoters, human H1-RNase P RNA promoters, and human valine-tRNA promoters. Furthermore, a sequence of four or more consecutive T's is used as a transcription termination signal. Expression cassettes for miRNA and pre-miRNA can also be produced in the same manner as shRNA.
The siRNA, shRNA, miRNA or pre-miRNA expression cassette thus constructed is then inserted into a plasmid vector or viral vector. Examples of such vectors include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai viruses, and animal cell expression plasmids.
　例えばアデノ随伴ウイルス（ＡＡＶ）ベクターを調製する場合、まず、野生型のＡＡＶのゲノム配列のうち両端のＩＴＲを残し、それ以外のＲｅｐタンパク質及びカプシドタンパク質をコードするＤＮＡの代わりに、所望の核酸を挿入したベクタープラスミドを作製する。一方で、ウイルス粒子の形成に必要とされるＲｅｐタンパク質及びカプシドタンパク質をコードするＤＮＡは、別のプラスミドに挿入する。更に、ＡＡＶの増殖に必要なアデノウイルスのヘルパー作用を担う遺伝子（Ｅ１Ａ、Ｅ１Ｂ、Ｅ２Ａ、ＶＡ、及びＥ４ｏｒｆ６）を含むプラスミドを、アデノウイルスヘルパープラスミドとして作成する。これら３種のプラスミドを、宿主細胞にコトランスフェクションすることにより、該細胞内において組換えＡＡＶ（即ち、ＡＡＶベクター）が産生されるようになる。宿主細胞としては、前記ヘルパー作用を担う遺伝子の遺伝子産物（タンパク質）のうちの一部を供給し得る細胞（例えば、２９３細胞等）を用いることが好ましく、そのような細胞を用いる場合には、該宿主細胞より供給され得るタンパク質をコードする遺伝子を、前記アデノウイルスヘルパープラスミドに搭載する必要がない。産生されたＡＡＶベクターは核内に存在するため、宿主細胞を凍結融解して回収し、塩化セシウムを用いた密度勾配超遠心法やカラム法等により、分離及び精製を行うことにより、所望のＡＡＶベクターが調製される。 For example, when preparing an adeno-associated virus (AAV) vector, first leave the ITRs at both ends of the wild-type AAV genome sequence, and insert the desired nucleic acid in place of the DNA encoding the Rep protein and capsid protein. Create an inserted vector plasmid. On the other hand, DNA encoding the Rep protein and capsid protein required for virus particle formation is inserted into a separate plasmid. Furthermore, a plasmid containing genes (E1A, E1B, E2A, VA, and E4orf6) responsible for adenovirus helper functions necessary for AAV proliferation is constructed as an adenovirus helper plasmid. By cotransfecting these three plasmids into a host cell, recombinant AAV (ie, AAV vector) is produced within the cell. As the host cell, it is preferable to use a cell (for example, 293 cell, etc.) that can supply a part of the gene product (protein) of the gene responsible for the helper action, and when such a cell is used, There is no need for the adenovirus helper plasmid to carry a gene encoding a protein that can be supplied from the host cell. Since the produced AAV vector exists in the nucleus, the desired AAV vector is recovered by freezing and thawing host cells, and is separated and purified by density gradient ultracentrifugation using cesium chloride, column method, etc. A vector is prepared.
（ｂ）ＬＳＭＥＭ２遺伝子のｍＲＮＡに対するアンチセンス核酸
　本発明における「ＬＳＭＥＭ２遺伝子のｍＲＮＡに対するアンチセンス核酸」とは、該ｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列又はその一部を含む核酸であって、標的ｍＲＮＡと特異的かつ安定した二重鎖を形成して結合することにより、タンパク質合成を抑制する機能を有するものである。
　アンチセンス核酸は、２－デオキシ－Ｄ－リボースを含有しているポリデオキシリボヌクレオチド、Ｄ－リボースを含有しているポリリボヌクレオチド、プリン又はピリミジン塩基のＮ－グリコシドであるその他のタイプのポリヌクレオチド、非ヌクレオチド骨格を有するその他のポリマー（例えば、市販のタンパク質核酸及び合成配列特異的な核酸ポリマー）又は特殊な結合を含有するその他のポリマー（但し、該ポリマーはＤＮＡやＲＮＡ中に見出されるような塩基のペアリングや塩基の付着を許容する配置をもつヌクレオチドを含有する）などが挙げられる。それらは、二本鎖ＤＮＡ、一本鎖ＤＮＡ、二本鎖ＲＮＡ、一本鎖ＲＮＡ、ＤＮＡ：ＲＮＡハイブリッドであってもよく、更に非修飾ポリヌクレオチド（又は非修飾オリゴヌクレオチド）、公知の修飾の付加されたもの、例えば当該分野で知られた標識のあるもの、キャップの付いたもの、メチル化されたもの、１個以上の天然のヌクレオチドを類縁物で置換したもの、分子内ヌクレオチド修飾のされたもの、例えば非荷電結合（例えば、メチルホスホネート、ホスホトリエステル、ホスホルアミデート、カルバメートなど）を持つもの、電荷を有する結合又は硫黄含有結合（例、ホスホロチオエート、ホスホロジチオエートなど）を持つもの、例えばタンパク質（例、ヌクレアーゼ、ヌクレアーゼ・インヒビター、トキシン、抗体、シグナルペプチド、ポリ－Ｌ－リジンなど）や糖（例、モノサッカライドなど）などの側鎖基を有しているもの、インターカレント化合物（例、アクリジン、ソラレンなど）を持つもの、キレート化合物（例えば、金属、放射活性をもつ金属、ホウ素、酸化性の金属など）を含有するもの、アルキル化剤を含有するもの、修飾された結合を持つもの（例えば、αアノマー型の核酸など）であってもよい。ここで「ヌクレオシド」、「ヌクレオチド」及び「核酸」とは、プリン及びピリミジン塩基を含有するのみでなく、修飾されたその他の複素環型塩基をもつようなものを含んでいて良い。このような修飾物は、メチル化されたプリン及びピリミジン、アシル化されたプリン及びピリミジン、或いはその他の複素環を含むものであってよい。修飾されたヌクレオシド及び修飾されたヌクレオチドはまた糖部分が修飾されていてよく、例えば、１個以上の水酸基がハロゲンとか、脂肪族基などで置換されていたり、又はエーテル、アミンなどの官能基に変換されていてよい。 (b) Antisense nucleic acid to mRNA of LSMEM2 gene In the present invention, "antisense nucleic acid to mRNA of LSMEM2 gene" refers to a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of the mRNA or a part thereof, It has the function of suppressing protein synthesis by binding to mRNA by forming a specific and stable double strand.
Antisense nucleic acids include polydeoxyribonucleotides containing 2-deoxy-D-ribose, polyribonucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, Other polymers with non-nucleotide backbones (e.g., commercially available protein nucleic acids and synthetic sequence-specific nucleic acid polymers) or containing special linkages, provided that the polymers contain bases such as those found in DNA and RNA. (contains nucleotides with a configuration that allows for pairing and attachment of bases). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA:RNA hybrids, and also unmodified polynucleotides (or unmodified oligonucleotides), known modified with additions, such as with labels known in the art, with caps, with methylation, with the substitution of one or more natural nucleotides by analogues, with intramolecular nucleotide modifications. those with uncharged bonds (e.g. methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.), those with charged or sulfur-containing bonds (e.g. phosphorothioate, phosphorodithioate, etc.) substances, such as proteins (e.g., nucleases, nuclease inhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.) and sugars (e.g., monosaccharides, etc.), which have side chain groups, intercurrent containing compounds (e.g., acridine, psoralen, etc.), containing chelating compounds (e.g., metals, radioactive metals, boron, oxidizing metals, etc.), containing alkylating agents, modified It may be one that has a bond (for example, an α-anomer type nucleic acid). Here, "nucleoside", "nucleotide" and "nucleic acid" may include not only those containing purine and pyrimidine bases, but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified on the sugar moiety, for example, one or more hydroxyl groups may be substituted with a halogen, an aliphatic group, etc., or with a functional group such as an ether, an amine, etc. It may have been converted.
　上記の通り、アンチセンス核酸はＤＮＡであってもＲＮＡであってもよく、或いはＤＮＡ／ＲＮＡキメラであってもよい。アンチセンス核酸がＤＮＡの場合、標的ＲＮＡとアンチセンスＤＮＡとによって形成されるＲＮＡ：ＤＮＡハイブリッドは、内在性ＲＮａｓｅ　Ｈに認識されて標的ＲＮＡの選択的な分解を引き起こすことができる。従って、ＲＮａｓｅ　Ｈによる分解を指向するアンチセンスＤＮＡの場合、標的配列は、ｍＲＮＡ中の配列だけでなく、ＬＳＭＥＭ２遺伝子の初期翻訳産物におけるイントロン領域の配列であってもよい。イントロン配列は、ゲノム配列と、ＬＳＭＥＭ２遺伝子のｃＤＮＡヌクレオチド配列とをＢＬＡＳＴ、ＦＡＳＴＡ等のホモロジー検索プログラムを用いて比較することにより、決定することができる。 As mentioned above, the antisense nucleic acid may be DNA or RNA, or may be a DNA/RNA chimera. When the antisense nucleic acid is DNA, the RNA:DNA hybrid formed by the target RNA and antisense DNA can be recognized by endogenous RNase H and cause selective degradation of the target RNA. Therefore, in the case of antisense DNA directed to degradation by RNase H, the target sequence may be not only a sequence in mRNA but also a sequence in the intron region of the early translation product of the LSMEM2 gene. The intron sequence can be determined by comparing the genome sequence and the cDNA nucleotide sequence of the LSMEM2 gene using a homology search program such as BLAST or FASTA.
　本発明のアンチセンス核酸の標的領域は、該アンチセンス核酸がハイブリダイズすることにより、結果としてタンパク質への翻訳が阻害されるものであればその長さに特に制限はなく、タンパク質をコードするｍＲＮＡの全配列であっても部分配列であってもよく、短いもので約１０塩基程度、長いものでｍＲＮＡ若しくは初期転写産物の全配列が挙げられる。合成の容易さや抗原性、細胞内移行性の問題等を考慮すれば、約１０～約４０塩基、特に約１５～約３０塩基からなるオリゴヌクレオチドが好ましいが、それに限定されない。具体的には、ＬＳＭＥＭ２遺伝子の５’端ヘアピンループ、５’端６－ベースペア・リピート、５’端非翻訳領域、翻訳開始コドン、タンパク質コード領域、ＯＲＦ翻訳終止コドン、３’端非翻訳領域、３’端パリンドローム領域又は３’端ヘアピンループなどが、アンチセンス核酸の好ましい標的領域として選択しうるが、それらに限定されない。 The target region of the antisense nucleic acid of the present invention is not particularly limited in length as long as hybridization of the antisense nucleic acid results in inhibition of translation into protein; The sequence may be the entire sequence or a partial sequence, and examples include the short sequence of about 10 bases and the long sequence of the entire mRNA or initial transcription product. In consideration of ease of synthesis, antigenicity, intracellular distribution, etc., oligonucleotides consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases, are preferred, but are not limited thereto. Specifically, the 5' end hairpin loop, 5' end 6-base pair repeat, 5' end untranslated region, translation start codon, protein coding region, ORF translation stop codon, 3' end untranslated region of the LSMEM2 gene. , 3' end palindromic region, or 3' end hairpin loop may be selected as preferred target regions for antisense nucleic acids, but are not limited thereto.
　一実施態様において、本発明のアンチセンス核酸の標的領域として、上記ｓｉＲＮＡと同様に、配列番号１又は配列番号３で表されるヌクレオチド配列中、ヌクレオチド番号１～１４３４で示される領域内の、連続する少なくとも１５個のヌクレオチドからなる配列を挙げることができる。 In one embodiment, the target region of the antisense nucleic acid of the present invention is a continuous region within the region represented by nucleotide numbers 1 to 1434 in the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, as in the above-mentioned siRNA. Mention may be made of sequences consisting of at least 15 nucleotides.
　更に、本発明のアンチセンス核酸は、ＬＳＭＥＭ２遺伝子のｍＲＮＡや初期転写産物とハイブリダイズしてタンパク質への翻訳を阻害するだけでなく、二本鎖ＤＮＡであるこれらの遺伝子と結合して三重鎖（トリプレックス）を形成し、ＲＮＡへの転写を阻害し得るもの（アンチジーン）であってもよい。 Furthermore, the antisense nucleic acid of the present invention not only hybridizes with the mRNA and early transcripts of the LSMEM2 gene and inhibits its translation into protein, but also binds to these genes, which are double-stranded DNA, to form triple-stranded ( It may also be a gene (antigene) that can form a triplex (triplex) and inhibit transcription into RNA.
　アンチセンス核酸を構成するヌクレオチド分子もまた、安定性、比活性などを向上させるために、上記のｓｉＲＮＡ等の場合と同様の修飾を受けていてもよい。 The nucleotide molecules constituting the antisense nucleic acid may also be modified in the same manner as in the case of siRNA, etc., in order to improve stability, specific activity, etc.
　本発明のアンチセンスオリゴヌクレオチドは、ＬＳＭＥＭ２遺伝子のｃＤＮＡ配列若しくはゲノミックＤＮＡ配列に基づいてｍＲＮＡ若しくは初期転写産物の標的配列を決定し、市販のＤＮＡ／ＲＮＡ自動合成機（アプライド・バイオシステムズ社、ベックマン社等）を用いて、これに相補的な配列を合成することにより調製することができる。また、上記した各種修飾を含むアンチセンス核酸も、いずれも自体公知の手法により、化学的に合成することができる。 The antisense oligonucleotide of the present invention is prepared by determining the target sequence of mRNA or early transcript based on the cDNA sequence or genomic DNA sequence of the LSMEM2 gene, and using a commercially available DNA/RNA automatic synthesizer (Applied Biosystems, Beckman). etc.), and can be prepared by synthesizing a complementary sequence thereto. Furthermore, antisense nucleic acids containing the various modifications described above can also be chemically synthesized by techniques known per se.
（ｃ）ＬＳＭＥＭ２遺伝子のｍＲＮＡに対するリボザイム核酸
　ＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列又はその一部を含む核酸の他の例としては、該ｍＲＮＡをコード領域の内部で特異的に切断し得るリボザイム核酸が挙げられる。「リボザイム」とは、狭義には、核酸を切断する酵素活性を有するＲＮＡをいうが、本明細書では配列特異的な核酸切断活性を有する限りＤＮＡをも包含する概念として用いるものとする。リボザイム核酸として最も汎用性の高いものとしては、ウイロイドやウイルソイド等の感染性ＲＮＡに見られるセルフスプライシングＲＮＡがあり、ハンマーヘッド型やヘアピン型等が知られている。ハンマーヘッド型は約４０塩基程度で酵素活性を発揮し、ハンマーヘッド構造をとる部分に隣接する両端の数塩基ずつ（合わせて約１０塩基程度）をｍＲＮＡの所望の切断部位と相補的な配列にすることにより、標的ｍＲＮＡのみを特異的に切断することが可能である。このタイプのリボザイム核酸は、ＲＮＡのみを基質とするので、ゲノムＤＮＡを攻撃することがないという更なる利点を有する。ＬＳＭＥＭ２遺伝子のｍＲＮＡが自身で二本鎖構造をとる場合には、ＲＮＡヘリカーゼと特異的に結合し得るウイルス核酸由来のＲＮＡモチーフを連結したハイブリッドリボザイムを用いることにより、標的配列を一本鎖にすることができる［Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９８（１０）：５５７２－５５７７（２００１）］。更に、リボザイムを、それをコードするＤＮＡを含む発現ベクターの形態で使用する場合には、転写産物の細胞質への移行を促進するために、ｔＲＮＡを改変した配列を更に連結したハイブリッドリボザイムとすることもできる［Ｎｕｃｌｅｉｃ　Ａｃｉｄｓ　Ｒｅｓ．，２９（１３）：２７８０－２７８８（２００１）］。 (c) Ribozyme nucleic acid for LSMEM2 gene mRNA Another example of a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of LSMEM2 gene mRNA or a part thereof is a nucleic acid that specifically cleaves the mRNA within the coding region. Examples include ribozyme nucleic acids obtained. In a narrow sense, "ribozyme" refers to RNA that has an enzymatic activity that cleaves nucleic acids, but in this specification it is used as a concept that also includes DNA as long as it has sequence-specific nucleic acid cleavage activity. The most versatile ribozyme nucleic acids are self-splicing RNAs found in infectious RNAs such as viroids and virusoids, and hammerhead and hairpin types are known. The hammerhead type exhibits enzymatic activity with about 40 bases, and several bases at each end (about 10 bases in total) adjacent to the hammerhead structure are made into a sequence complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme nucleic acid has the additional advantage of not attacking genomic DNA, since it only uses RNA as a substrate. If the mRNA of the LSMEM2 gene has a double-stranded structure by itself, the target sequence can be made into a single strand by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to RNA helicase. [Proc. Natl. Acad. Sci. USA, 98(10):5572-5577 (2001)]. Furthermore, when a ribozyme is used in the form of an expression vector containing the DNA encoding it, a hybrid ribozyme may be created in which a modified tRNA sequence is further linked to promote the transfer of the transcript to the cytoplasm. [Nucleic Acids Res. , 29(13):2780-2788 (2001)].
　ＬＳＭＥＭ２遺伝子のｍＲＮＡのヌクレオチド配列と相補的なヌクレオチド配列又はその一部を含む核酸は、リポソーム、ミクロスフェアのような特殊な形態で供与されたり、遺伝子治療に適用されたり、付加された形態で与えられることができうる。こうして付加形態で用いられるものとしては、リン酸基骨格の電荷を中和するように働くポリリジンのようなポリカチオン体、細胞膜との相互作用を高めたり、核酸の取込みを増大せしめるような脂質（例、ホスホリピド、コレステロールなど）などの疎水性のものが挙げられる。付加するに好ましい脂質としては、コレステロールやその誘導体（例、コレステリルクロロホルメート、コール酸など）が挙げられる。こうしたものは、核酸の３’端又は５’端に付着させることができ、塩基、糖、分子内ヌクレオシド結合を介して付着させることができうる。その他の基としては、核酸の３’端又は５’端に特異的に配置されたキャップ用の基で、エキソヌクレアーゼ、ＲＮａｓｅなどのヌクレアーゼによる分解を阻止するためのものが挙げられる。こうしたキャップ用の基としては、ポリエチレングリコール、テトラエチレングリコールなどのグリコールをはじめとした当該分野で知られた水酸基の保護基が挙げられるが、それに限定されるものではない。 A nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence of LSMEM2 gene mRNA or a part thereof may be provided in a special form such as a liposome or microsphere, applied to gene therapy, or provided in an attached form. It can be done. These additive forms include polycations such as polylysine, which act to neutralize the charge on the phosphate backbone, and lipids (which enhance interaction with cell membranes and increase uptake of nucleic acids). examples, phospholipids, cholesterol, etc.). Preferred lipids to be added include cholesterol and its derivatives (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3' or 5' end of the nucleic acid, and can be attached via base, sugar, or intramolecular nucleoside linkages. Other groups include capping groups specifically placed at the 3' or 5' ends of nucleic acids to prevent degradation by nucleases such as exonucleases and RNases. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art including glycols such as polyethylene glycol and tetraethylene glycol.
　これらの核酸のＬＳＭＥＭ２タンパク質発現抑制活性は、ＬＳＭＥＭ２遺伝子を導入した形質転換体、生体内や生体外のＬＳＭＥＭ２遺伝子発現系、又は生体内や生体外のＬＳＭＥＭ２タンパク質翻訳系を用いて調べることができる。 The LSMEM2 protein expression suppressing activity of these nucleic acids can be investigated using a transformant into which the LSMEM2 gene has been introduced, an in vivo or in vitro LSMEM2 gene expression system, or an in vivo or in vitro LSMEM2 protein translation system.
（ｄ）ＬＳＭＥＭ２遺伝子中の標的領域に特異的に結合する核酸配列認識モジュール
（ｄ－１）ゲノム配列の改変を伴わない物質
　従来のゲノム編集の手法としては、配列非依存的なＤＮＡ切断能を有する分子と配列認識能を有する分子とを組み合わせた人工ヌクレアーゼを利用する方法がよく知られている。例えば、ジンクフィンガーＤＮＡ結合ドメインと非特異的なＤＮＡ切断ドメインとを連結した、ジンクフィンガーヌクレアーゼ（ＺＦＮ）、植物病原菌キサントモナス属が有するＤＮＡ結合モジュールである転写活性化因子様（ＴＡＬ）エフェクターと、ＤＮＡエンドヌクレアーゼとを連結したＴＡＬＥＮ、或いは、真正細菌や古細菌が持つ獲得免疫システムで機能する核酸配列ＣＲＩＳＰＲ（Ｃｌｕｓｔｅｒｅｄ　Ｒｅｇｕｌａｒｌｙ　Ｉｎｔｅｒｓｐａｃｅｄ　Ｓｈｏｒｔ　Ｐａｌｉｎｄｒｏｍｉｃ　Ｒｅｐｅａｔｓ）と、ＣＲＩＳＰＲとともに重要な働きを持つヌクレアーゼＣａｓ（ＣＲＩＳＰＲ－ａｓｓｏｃｉａｔｅｄ）タンパク質ファミリーとを組み合わせたＣＲＩＳＰＲ－Ｃａｓ９システムを利用する方法などが報告されている。更には、３５個のアミノ酸からなり１個の核酸塩基を認識するＰＰＲモチーフの連続によって、特定のヌクレオチド配列を認識するように構成されたＰＰＲタンパク質と、ヌクレアーゼとを連結した人工ヌクレアーゼも報告されている。 (d) Nucleic acid sequence recognition module that specifically binds to the target region in the LSMEM2 gene (d-1) Substance that does not involve modification of the genome sequence Conventional genome editing methods require sequence-independent DNA cutting ability. A well-known method is to use an artificial nuclease that combines a molecule with sequence recognition ability and a molecule with sequence recognition ability. For example, zinc finger nuclease (ZFN), which is a combination of a zinc finger DNA binding domain and a nonspecific DNA cleavage domain, a transcription activator-like (TAL) effector, which is a DNA binding module possessed by the plant pathogenic fungus Xanthomonas genus, and DNA TALEN linked with endonuclease, or the nucleic acid sequence CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which functions in the acquired immune system of eubacteria and archaea, and nuclease Cas (Clustered Regularly Interspaced Short Palindromic Repeats), which has an important function together with CRISPR. CRISPR-associated) A method using the CRISPR-Cas9 system in combination with a protein family has been reported. Furthermore, an artificial nuclease has been reported in which a nuclease is linked to a PPR protein configured to recognize a specific nucleotide sequence by a series of PPR motifs consisting of 35 amino acids and recognizing one nucleobase. There is.
　これらのゲノム編集技術においては、いずれもタンパク質（ＺＦ、ＴＡＬエフェクター、ＰＰＲ）又はＲＮＡとタンパク質との複合体（ＣＲＩＳＰＲ－Ｃａｓ９）により特定の標的ヌクレオチド配列を認識している。従って、人工ヌクレアーゼに配列特異性を付与するこれらの物質（核酸配列認識モジュール）を用いて、ＬＳＭＥＭ２遺伝子中の標的領域に結合させることで、ＬＳＭＥＭ２の発現を抑制することができる。 In all of these genome editing techniques, a specific target nucleotide sequence is recognized by a protein (ZF, TAL effector, PPR) or a complex of RNA and protein (CRISPR-Cas9). Therefore, the expression of LSMEM2 can be suppressed by binding to the target region in the LSMEM2 gene using these substances (nucleic acid sequence recognition modules) that impart sequence specificity to artificial nucleases.
　本発明において「核酸配列認識モジュール」とは、ＤＮＡ鎖上の特定のヌクレオチド配列（即ち、標的ヌクレオチド配列）を特異的に認識して結合する能力を有する分子又は分子複合体を意味する。核酸配列認識モジュールが標的ヌクレオチド配列に結合することにより、該モジュールに連結されたエフェクターがＤＮＡの標的化された部位に特異的に作用することを可能にする。 In the present invention, the term "nucleic acid sequence recognition module" refers to a molecule or molecular complex that has the ability to specifically recognize and bind to a specific nucleotide sequence (ie, target nucleotide sequence) on a DNA strand. Binding of a nucleic acid sequence recognition module to a target nucleotide sequence allows an effector linked to the module to act specifically on a targeted site of DNA.
　本発明の１つの態様において、核酸配列認識モジュールとしては、ＣＲＩＳＰＲ－Ｃａｓシステムが挙げられる。ＣＲＩＳＰＲ－Ｃａｓシステムは、標的ヌクレオチド配列に相補的な短鎖ＣＲＩＳＰＲ　ＲＮＡ（ｃｒＲＮＡ）とトランス活性化型ｃｒＲＮＡ（ｔｒａｃｒＲＮＡ）との複合体、又はｃｒＲＮＡとｔｒａｃｒＲＮＡとを組合せた単一の合成ＲＮＡ（ガイドＲＮＡ、ｇＲＮＡ）により目的のＤＮＡの配列を認識するので、標的ヌクレオチド配列の相補配列と特異的にハイブリッド形成し得るオリゴＤＮＡを合成するだけで、任意の配列を標的化することができる。 In one embodiment of the present invention, the nucleic acid sequence recognition module includes the CRISPR-Cas system. The CRISPR-Cas system uses a complex of short CRISPR RNA (crRNA) complementary to a target nucleotide sequence and transactivating crRNA (tracrRNA), or a single synthetic RNA (guide RNA) that combines crRNA and tracrRNA. , gRNA), any sequence can be targeted simply by synthesizing an oligo DNA that can specifically hybridize with the complementary sequence of the target nucleotide sequence.
　ＣＲＩＳＰＲ－Ｃａｓを用いた核酸配列認識モジュールは、標的ヌクレオチド配列に相補的なヌクレオチド配列、及びＣａｓタンパク質のリクルートに必要なｔｒａｃｒＲＮＡからなるＲＮＡ分子（ガイドＲＮＡ）とＣａｓタンパク質との複合体として提供される。また、他の態様として、ＣＲＩＳＰＲ－Ｃａｓを用いた核酸配列認識モジュールは、標的ヌクレオチド配列に相補的なＲＮＡ配列を含むｃｒＲＮＡ、及びｔｒａｃｒＲＮＡ、Ｃａｓの複合体として提供される。 A nucleic acid sequence recognition module using CRISPR-Cas is provided as a complex of a Cas protein and an RNA molecule (guide RNA) consisting of a nucleotide sequence complementary to the target nucleotide sequence and tracrRNA necessary for recruiting the Cas protein. . In another embodiment, a nucleic acid sequence recognition module using CRISPR-Cas is provided as a complex of crRNA, tracrRNA, and Cas, which includes an RNA sequence complementary to the target nucleotide sequence.
　本発明で使用されるＣａｓタンパク質は、ＣＲＩＳＰＲシステムに属するものであれば特に制限はないが、好ましくはＣａｓ９である。Ｃａｓ９としては、例えばストレプトコッカス・ピオゲネス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｐｙｏｇｅｎｅｓ）由来のＣａｓ９（ＳｐＣａｓ９）、ストレプトコッカス・サーモフィラス（Ｓｔｒｅｐｔｏｃｏｃｃｕｓ　ｔｈｅｒｍｏｐｈｉｌｕｓ）由来のＣａｓ９（ＳｔＣａｓ９）等が挙げられるが、それらに限定されない。好ましくはＳｐＣａｓ９である。ヒト臨床に利用することを考慮すれば、ＤＳＢを生じるのは好ましくないので、Ｃａｓとしては、ＤＮＡ切断活性を失活したもの（ｄＣａｓ）が好ましい。例えば、ＳｐＣａｓ９の場合、１０番目のＡｓｐ残基がＡｌａ残基に変換した、ガイドＲＮＡと相補鎖を形成する鎖の反対鎖の切断能を欠くＤ１０Ａ変異体、８４０番目のＨｉｓ残基がＡｌａ残基で変換した、ガイドＲＮＡと相補鎖の切断能を欠くＨ８４０Ａ変異体の二重変異体を用いることができるが、他の変異Ｃａｓも同様に用いることができる。 The Cas protein used in the present invention is not particularly limited as long as it belongs to the CRISPR system, but is preferably Cas9. Examples of Cas9 include Cas9 (SpCas9) derived from Streptococcus pyogenes and Cas9 (StCas9) derived from Streptococcus thermophilus. Not limited. Preferably it is SpCas9. When considering clinical use in humans, it is undesirable to generate DSB, so it is preferable to use Cas with inactivated DNA cleaving activity (dCas). For example, in the case of SpCas9, the 10th Asp residue is converted to an Ala residue, the D10A mutant lacks the ability to cleave the opposite strand of the strand that forms a complementary strand with the guide RNA, and the 840th His residue is converted to an Ala residue. A double mutant of the H840A mutant that lacks the ability to cleave the guide RNA and the complementary strand can be used, but other mutant Cas can be used as well.
　ＣＲＩＳＰＲ－Ｃａｓを核酸配列認識モジュールとして用いる場合、標的ヌクレオチド配列は、該配列にガイドＲＮＡとＣａｓの複合体が結合したときに、ＬＳＭＥＭ２遺伝子が該複合体によって遮断され、ＬＳＭＥＭ２遺伝子が発現できない状態にあれば、特に制限されないが、例えば、ＬＳＭＥＭ２遺伝子の場合、後述の実施例で使用されたガイドＲＮＡ（ＡＣＴＧＧＣＴＣＣＴＡＡＣＣＡＣＧＴＧＣＡＧＧ）（配列番号５）に対する標的ヌクレオチド配列が挙げられる。一方、ＬＳＭＥＭ２遺伝子の場合、例えば、配列中のＣＧＧをＰＡＭとして、その直前から上流側２０ヌクレオチドの逆鎖配列を標的ヌクレオチド配列として、ｃｒＲＮＡを設計することができる。 When CRISPR-Cas is used as a nucleic acid sequence recognition module, when a complex of guide RNA and Cas binds to the target nucleotide sequence, the LSMEM2 gene is blocked by the complex, and the LSMEM2 gene cannot be expressed. For example, in the case of the LSMEM2 gene, the target nucleotide sequence for the guide RNA (ACTGGCTCCTAACCACGTGCAGG) (SEQ ID NO: 5) used in the Examples below may be mentioned, although it is not particularly limited. On the other hand, in the case of the LSMEM2 gene, crRNA can be designed, for example, by using CGG in the sequence as PAM and a reverse strand sequence of 20 nucleotides immediately upstream from it as a target nucleotide sequence.
　本発明の他の態様においては、核酸配列認識モジュールとしては、ジンクフィンガーモチーフ、ＴＡＬエフェクター及びＰＰＲモチーフ等の他、制限酵素、転写因子、ＲＮＡポリメラーゼ等のＤＮＡと特異的に結合し得るタンパク質のＤＮＡ結合ドメインを含み、ＤＮＡ二重鎖切断能を有しないフラグメント等が用いられ得る。 In another aspect of the present invention, the nucleic acid sequence recognition module includes zinc finger motifs, TAL effectors, PPR motifs, etc., as well as restriction enzymes, transcription factors, RNA polymerases, and other proteins that can specifically bind to DNA. A fragment that includes a binding domain and does not have DNA double-strand cleavage ability can be used.
　ジンクフィンガーモチーフの作製の詳細については、特許第４９６８４９８号公報を参照することができる。ＴＡＬエフェクターの作製の詳細については、特表２０１３－５１３３８９号公報を参照することができる。ＰＰＲモチーフの作製の詳細については、特開２０１３－１２８４１３号公報を参照することができる。 For details on the production of zinc finger motifs, refer to Japanese Patent No. 4968498. For details on the production of the TAL effector, refer to Japanese Patent Publication No. 2013-513389. For details on the preparation of the PPR motif, refer to JP 2013-128413A.
　また、制限酵素、転写因子、ＲＮＡポリメラーゼ等のフラグメントを用いる場合、これらのタンパク質のＤＮＡ結合ドメインは周知であるので、該ドメインを含み、且つＤＮＡ二重鎖切断能を有しない断片を容易に設計し、構築することができる。 Furthermore, when using fragments of restriction enzymes, transcription factors, RNA polymerases, etc., since the DNA-binding domains of these proteins are well known, it is easy to design fragments that contain the domains and do not have the ability to cut DNA double-strands. and can be built.
　或いは、前記ＣＲＩＳＰＲ－ＣａｓのｄＣａｓの場合と同様に、ジンクフィンガーモチーフ、ＴＡＬエフェクター及びＰＰＲモチーフ等の核酸配列認識モジュールに、失活したヌクレアーゼ（例、ＦｏｋＩ）を組み合わせることにより、該ヌクレアーゼが立体的にＬＳＭＥＭ２遺伝子を遮断し、それによってＬＳＭＥＭ２の発現を抑制することもできる。この場合、例えば、標的領域より上流の正鎖ヌクレオチド配列と、該標的領域より下流の逆鎖ヌクレオチド配列とを特異的に認識する２つの核酸配列認識モジュールを設計し、該モジュールの末端（例、Ｃ末端）で失活したヌクレアーゼと複合体形成するようにすることができる。 Alternatively, as in the case of dCas of CRISPR-Cas, by combining a deactivated nuclease (e.g., FokI) with a nucleic acid sequence recognition module such as a zinc finger motif, TAL effector, and PPR motif, the nuclease can be sterically It is also possible to block the LSMEM2 gene and thereby suppress the expression of LSMEM2. In this case, for example, two nucleic acid sequence recognition modules that specifically recognize a positive nucleotide sequence upstream from the target region and a reverse nucleotide sequence downstream from the target region are designed, and the end of the module (e.g. (C-terminus) can form a complex with a deactivated nuclease.
　ジンクフィンガーモチーフ、ＴＡＬエフェクター及びＰＰＲモチーフ等の核酸配列認識モジュールは、上記ヌクレアーゼとの融合タンパク質として提供することもできるし、ＳＨ３ドメイン、ＰＤＺドメイン、ＧＫドメイン、ＧＢドメイン等のタンパク質結合ドメインとそれらの結合パートナーとを、核酸配列認識モジュールと、ヌクレアーゼとにそれぞれ融合させ、該タンパク質結合ドメインとその結合パートナーとの相互作用を介してタンパク質複合体として提供してもよい。或いは、核酸配列認識モジュールと、ヌクレアーゼとにそれぞれインテイン（ｉｎｔｅｉｎ）を融合させ、各タンパク質合成後のライゲーションにより、両者を連結することもできる。 Nucleic acid sequence recognition modules such as zinc finger motifs, TAL effectors, and PPR motifs can be provided as fusion proteins with the above-mentioned nucleases, or can be provided as fusion proteins with protein binding domains such as SH3 domains, PDZ domains, GK domains, GB domains, etc. A binding partner may be fused to a nucleic acid sequence recognition module and a nuclease, respectively, and provided as a protein complex through interaction between the protein binding domain and its binding partner. Alternatively, inteins can be fused to the nucleic acid sequence recognition module and the nuclease, respectively, and the two can be linked by ligation after each protein is synthesized.
　本発明の核酸配列認識モジュールは、ヌクレアーゼの代わりに、転写抑制因子と複合体を形成させ、ＬＳＭＥＭ２遺伝子の転写調節領域中のシスエレメントを含む配列を標的ヌクレオチド配列とすることにより、ＬＳＭＥＭ２遺伝子のシスエレメントへの転写因子の結合を遮断しつつ、ＬＳＭＥＭ２遺伝子の転写を更に抑制することもできる。本発明において、「転写抑制因子」とは、標的とする遺伝子の転写抑制活性を有するタンパク質又はタンパク質ドメインを意味する。 The nucleic acid sequence recognition module of the present invention forms a complex with a transcription repressor instead of a nuclease, and uses a sequence containing a cis element in the transcriptional regulatory region of the LSMEM2 gene as a target nucleotide sequence. It is also possible to further suppress transcription of the LSMEM2 gene while blocking the binding of transcription factors to the element. In the present invention, the term "transcription repressor" refers to a protein or protein domain that has the activity of repressing the transcription of a target gene.
　本発明で用いる転写抑制因子としては、ＬＳＭＥＭ２遺伝子の転写活性化を抑制することができるものであれば特に制限はないが、例えば、ＫＲＡＢ、ＭＢＤ２Ｂ、ｖ－ＥｒｂＡ、ＳＩＤ（ＳＩＤのコンカテマー（ＳＩＤ４Ｘ）を含む）、ＭＢＤ２、ＭＢＤ３、ＤＮＭＴファミリー（例：ＤＮＭＴ１、ＤＮＭＴ３Ａ、ＤＮＭＴ３Ｂ）、Ｒｂ、ＭｅＣＰ２、ＲＯＭ２及びＡｔＨＤ２Ａなどが挙げられ、好ましくは、ＫＲＡＢである。転写抑制因子としてＫＲＡＢを用いる場合に、その由来とするタンパク質は特に制限されないが、例えば、ＫＯＸ－１（ＺＮＦ１０）、ＫＯＸ８（ＺＮＦ７０８）、ＺＮＦ４３、ＺＮＦ１８４、ＺＮＦ９１、ＨＰＦ４、ＨＴＦ１０、ＨＴＦ３４などが挙げられる。 The transcriptional repressor used in the present invention is not particularly limited as long as it can suppress transcriptional activation of the LSMEM2 gene, but examples include KRAB, MBD2B, v-ErbA, SID (concatemer of SID (SID4X)). ), MBD2, MBD3, DNMT family (eg DNMT1, DNMT3A, DNMT3B), Rb, MeCP2, ROM2 and AtHD2A, preferably KRAB. When using KRAB as a transcription repressor, the protein derived from it is not particularly limited, but examples include KOX-1 (ZNF10), KOX8 (ZNF708), ZNF43, ZNF184, ZNF91, HPF4, HTF10, HTF34, etc. .
　上記核酸配列認識モジュールと、ＬＳＭＥＭ２遺伝子との接触は、対象である哺乳動物（例、ヒト、マウス、ラット、ウシ、イヌ、ネコ、サル等、好ましくはヒト又はマウス、より好ましくはヒト）の細胞に、該モジュール（ヌクレアーゼや転写抑制因子等のエフェクターと組み合わせて用いる場合は、更に該エフェクタータンパク質）をコードする核酸を導入することにより実施される。
　従って、核酸配列認識モジュール、又は核酸配列認識モジュール及びエフェクターは、それらの融合タンパク質をコードする核酸として、或いは、タンパク質に翻訳後、宿主細胞内で複合体形成し得るような形態で、各構成因子をコードする核酸として調製することが好ましい。ここで核酸は、ＤＮＡであってもＲＮＡであってもよい。ＤＮＡの場合は、好ましくは二本鎖ＤＮＡであり、哺乳動物細胞内で機能的なプロモーターの制御下に各構成因子を発現し得る発現ベクターの形態で提供される。ＲＮＡの場合は、好ましくは一本鎖ＲＮＡである。 The contact between the nucleic acid sequence recognition module and the LSMEM2 gene is carried out in cells of a target mammal (e.g., human, mouse, rat, cow, dog, cat, monkey, etc., preferably human or mouse, more preferably human). This is carried out by introducing a nucleic acid encoding the module (if used in combination with an effector such as a nuclease or a transcription repressor, the effector protein).
Therefore, the nucleic acid sequence recognition module, or the nucleic acid sequence recognition module and the effector, can be used as a nucleic acid encoding a fusion protein thereof, or in a form that can form a complex in a host cell after being translated into a protein. Preferably, it is prepared as a nucleic acid encoding. Here, the nucleic acid may be DNA or RNA. In the case of DNA, it is preferably double-stranded DNA, and is provided in the form of an expression vector capable of expressing each component under the control of a functional promoter in mammalian cells. In the case of RNA, it is preferably single-stranded RNA.
　ＣＲＩＳＰＲ－Ｃａｓを核酸配列認識モジュールとして用いる場合、ガイドＲＮＡ及びＣａｓタンパク質をコードする発現ベクターを細胞に導入し、該ガイドＲＮＡ及びＣａｓタンパク質を発現させることにより、細胞内でガイドＲＮＡとＣａｓタンパク質との複合体を形成する。ガイドＲＮＡ及びＣａｓタンパク質は、同一の発現ベクター上にコードされていてもよいし、異なる発現ベクター上に、それぞれコードされていてもよい。 When CRISPR-Cas is used as a nucleic acid sequence recognition module, an expression vector encoding guide RNA and Cas protein is introduced into cells, and the guide RNA and Cas protein are expressed, thereby allowing the interaction between guide RNA and Cas protein to occur within the cell. Form a complex. The guide RNA and Cas protein may be encoded on the same expression vector, or may be encoded on different expression vectors.
　ＣａｓをコードするＤＮＡは、当該技術分野で周知の方法により、Ｃａｓを産生する細胞からクローニングすることができる。得られたＣａｓをコードするＤＮＡは、哺乳動物細胞用の発現ベクターのプロモーターの下流に挿入することができる。 DNA encoding Cas can be cloned from cells that produce Cas by methods well known in the art. The obtained Cas-encoding DNA can be inserted downstream of the promoter of an expression vector for mammalian cells.
　一方、ガイドＲＮＡをコードするＤＮＡは、標的ヌクレオチド配列に相補的なＲＮＡ配列と既知のｔｒａｃｒＲＮＡ配列（例えば、ｇｔｔｔｔａｇａｇｃｔａｇａａａｔａｇｃａａｇｔｔａａａａｔａａｇｇｃｔａｇｔｃｃｇｔｔａｔｃａａｃｔｔｇａａａａａｇｔｇｇｃａｃｃｇａｇｔｃｇｇｔｇｇｔｇｃｔｔｔｔ；配列番号６）とを連結したオリゴＤＮＡ配列を設計し、ＤＮＡ／ＲＮＡ合成機を用いて、化学的に合成することができる。なお、本明細書においてヌクレオチド配列は、別段にことわりのない限りＤＮＡの配列として記載するが、ポリヌクレオチドがＲＮＡである場合は、チミン（Ｔ）をウラシル（Ｕ）に適宜読み替えるものとする。 On the other hand, the DNA encoding the guide RNA consists of an RNA sequence complementary to the target nucleotide sequence and a known tracrRNA sequence (for example, gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtggtgctttt; We designed an oligo DNA sequence that linked SEQ ID NO: 6) and used a DNA/RNA synthesizer. It can be chemically synthesized using In this specification, nucleotide sequences are described as DNA sequences unless otherwise specified, but when the polynucleotide is RNA, thymine (T) shall be read as uracil (U) as appropriate.
　ガイドＲＮＡをコードするＤＮＡも、哺乳動物細胞用の発現ベクターに挿入することができる。ガイドＲＮＡ及びＣａｓは、同一の発現ベクター上にコードされていてもよいし、異なる発現ベクター上に、それぞれコードされていてもよい。好適には、ＣａｓをコードするＤＮＡとガイドＲＮＡをコードするＤＮＡを、同一の発現ベクター中、別個のプロモーターの下流に挿入する。 DNA encoding guide RNA can also be inserted into expression vectors for mammalian cells. The guide RNA and Cas may be encoded on the same expression vector, or may be encoded on different expression vectors. Preferably, the DNA encoding Cas and the DNA encoding the guide RNA are inserted downstream of separate promoters in the same expression vector.
　ＣａｓをコードするＲＮＡは、例えば、上記したＣａｓをコードするＤＮＡを鋳型として、自体公知のインビトロ転写系にてｍＲＮＡに転写することにより調製することもできる。
　ガイドＲＮＡは、標的ヌクレオチド配列に相補的なＲＮＡ配列と既知のｔｒａｃｒＲＮＡ配列とを連結したオリゴＲＮＡ配列を設計し、ＤＮＡ／ＲＮＡ合成機を用いて、化学的に合成することもできる。この場合、ガイドＲＮＡを構成するリボヌクレオチドに、安定性や膜透過性を向上させるための種々の修飾を付与することができる。また、ｃｒＲＮＡとｔｒａｃｒＲＮＡとを別個に合成し、アニーリングして用いることもできる。 RNA encoding Cas can also be prepared, for example, by using the above-described DNA encoding Cas as a template and transcribing it into mRNA using a known in vitro transcription system.
Guide RNA can also be chemically synthesized using a DNA/RNA synthesizer by designing an oligo RNA sequence in which an RNA sequence complementary to the target nucleotide sequence is linked to a known tracrRNA sequence. In this case, various modifications can be imparted to the ribonucleotides constituting the guide RNA to improve stability and membrane permeability. Alternatively, crRNA and tracrRNA can be synthesized separately and annealed for use.
　ジンクフィンガーモチーフ、ＴＡＬエフェクター、ＰＰＲモチーフ等の核酸配列認識モジュールをコードするＤＮＡは、各モジュールについて上記したいずれかの方法により取得することができる。制限酵素、転写因子、ＲＮＡポリメラーゼ等の配列認識モジュールをコードするＤＮＡは、例えば、それらのｃＤＮＡ配列情報に基づいて、当該タンパク質の所望の部分（ＤＮＡ結合ドメインを含む部分）をコードする領域をカバーするようにオリゴＤＮＡプライマーを合成し、当該タンパク質を産生する細胞より調製した全ＲＮＡ若しくはｍＲＮＡ画分を鋳型として用い、ＲＴ－ＰＣＲ法によって増幅することにより、クローニングすることができる。 DNA encoding a nucleic acid sequence recognition module such as a zinc finger motif, TAL effector, or PPR motif can be obtained by any of the methods described above for each module. DNA encoding a sequence recognition module such as a restriction enzyme, a transcription factor, or an RNA polymerase covers a region encoding a desired portion of the protein (a portion containing a DNA binding domain), for example, based on the cDNA sequence information thereof. Cloning can be carried out by synthesizing oligo-DNA primers as described above, and amplifying by RT-PCR using total RNA or mRNA fractions prepared from cells producing the protein as a template.
　ヌクレアーゼや転写抑制因子等のエフェクターをコードするＤＮＡも、同様に、使用するエフェクターのｃＤＮＡ配列情報をもとにオリゴＤＮＡプライマーを合成し、当該エフェクターを産生する細胞より調製した全ＲＮＡ若しくはｍＲＮＡ画分を鋳型として用い、ＲＴ－ＰＣＲ法によって増幅することにより、クローニングすることができる。例えば、ＦｏｋＩをコードするＤＮＡはそのｃＤＮＡ配列をもとに、ＣＤＳの上流及び下流に対して適当なプライマーを設計し、Ｆｌａｖｏｂａｃｔｅｒｉｕｍ　ｏｋｅａｎｏｋｏｉｔｅｓ（ＩＦＯ　１２５３６）由来ｍＲＮＡからＲＴ－ＰＣＲ法によりクローニングできる。 Similarly, DNA encoding effectors such as nucleases and transcriptional repressors can be obtained by synthesizing oligo DNA primers based on the cDNA sequence information of the effector to be used, and using total RNA or mRNA fractions prepared from cells that produce the effector. Cloning can be performed by amplifying by RT-PCR using as a template. For example, DNA encoding FokI can be cloned from Flavobacterium okeanokoites (IFO 12536)-derived mRNA by RT-PCR by designing appropriate primers for upstream and downstream of CDS based on the cDNA sequence.
　クローン化された核酸配列認識モジュールをコードするＤＮＡは、そのまま、又は所望により制限酵素で消化するか、適当なリンカー及び／又は核移行シグナルを付加することができる。ヌクレアーゼや転写抑制因子等のエフェクターと組み合わせて用いる場合、クローン化された核酸配列認識モジュールをコードするＤＮＡを、前記クローン化された核酸配列認識モジュールをコードするＤＮＡとライゲーションして、融合タンパク質をコードするＤＮＡを調製することができる。或いは、核酸配列認識モジュールをコードするＤＮＡと、エフェクターをコードするＤＮＡに、それぞれ結合ドメイン若しくはその結合パートナーをコードするＤＮＡを融合させるか、両ＤＮＡに分離インテインをコードするＤＮＡを融合させることにより、核酸配列認識モジュールとエフェクターとが宿主細胞内で翻訳された後に複合体を形成できるようにしてもよい。これらの場合も、所望により一方若しくは両方のＤＮＡの適当な位置に、リンカー及び／又は核移行シグナルを連結することができる。 The cloned DNA encoding the nucleic acid sequence recognition module can be used as is, or if desired, it can be digested with restriction enzymes, or an appropriate linker and/or nuclear export signal can be added. When used in combination with an effector such as a nuclease or a transcription repressor, a DNA encoding a cloned nucleic acid sequence recognition module is ligated with a DNA encoding the cloned nucleic acid sequence recognition module to encode a fusion protein. DNA can be prepared. Alternatively, by fusing DNA encoding a nucleic acid sequence recognition module and DNA encoding an effector with DNA encoding a binding domain or its binding partner, respectively, or by fusing both DNAs with DNA encoding a separated intein, The nucleic acid sequence recognition module and the effector may be allowed to form a complex after translation within the host cell. In these cases as well, a linker and/or nuclear localization signal can be ligated to an appropriate position of one or both of the DNAs, if desired.
　核酸配列認識モジュールをコードするＤＮＡ、エフェクターをコードするＤＮＡは、化学的にＤＮＡ鎖を合成するか、若しくは合成した一部オーバーラップするオリゴＤＮＡ短鎖を、ＰＣＲ法やＧｉｂｓｏｎ　Ａｓｓｅｍｂｌｙ法を利用して接続することにより、その全長をコードするＤＮＡを構築することも可能である。化学合成又はＰＣＲ法若しくはＧｉｂｓｏｎ　Ａｓｓｅｍｂｌｙ法との組み合わせで全長ＤＮＡを構築することの利点は、該ＤＮＡを導入する宿主に合わせて使用コドンをＣＤＳ全長にわたり設計できる点にある。異種ＤＮＡの発現に際し、そのＤＮＡ配列を宿主生物において使用頻度の高いコドンに変換することで、タンパク質発現量の増大が期待できる。使用する宿主におけるコドン使用頻度のデータは、例えば（公財）かずさＤＮＡ研究所のホームページに公開されている遺伝暗号使用頻度データベースを用いることができ、又は各宿主におけるコドン使用頻度を記した文献を参照してもよい。入手したデータと導入しようとするＤＮＡ配列を参照し、該ＤＮＡ配列に用いられているコドンの中で宿主において使用頻度の低いものを、同一のアミノ酸をコードし使用頻度の高いコドンに変換すればよい。 DNA encoding the nucleic acid sequence recognition module and DNA encoding the effector can be obtained by chemically synthesizing DNA strands, or by using synthesized partially overlapping oligo DNA short strands using PCR method or Gibson Assembly method. By connecting it, it is also possible to construct a DNA encoding the entire length. The advantage of constructing a full-length DNA by chemical synthesis or in combination with the PCR method or the Gibson Assembly method is that the codons used can be designed over the entire length of the CDS depending on the host into which the DNA is introduced. When expressing heterologous DNA, an increase in protein expression can be expected by converting the DNA sequence to codons that are frequently used in the host organism. For data on codon usage frequency in the host used, for example, the genetic code usage frequency database published on the website of the Kazusa DNA Research Institute (public interest incorporated foundation) can be used, or literature describing the codon usage frequency in each host can be used. You may refer to it. By referring to the obtained data and the DNA sequence to be introduced, convert the codons used in the DNA sequence that are used less frequently in the host to codons that encode the same amino acid and are used more frequently. good.
　核酸配列認識モジュール及び／又はエフェクターをコードするＤＮＡが挿入される発現ベクターとしては、レトロウイルス、ワクシニアウイルス、アデノウイルスなどの動物ウイルスベクターなどが用いられる。
　プロモーターとしては、ＳＲαプロモーター、ＳＶ４０プロモーター、ＬＴＲプロモーター、ＣＭＶ（サイトメガロウイルス）プロモーター、ＲＳＶ（ラウス肉腫ウイルス）プロモーター、ＭｏＭｕＬＶ（モロニーマウス白血病ウイルス）ＬＴＲ、ＨＳＶ－ＴＫ（単純ヘルペスウイルスチミジンキナーゼ）プロモーターなど哺乳動物細胞で機能し得るプロモーターが用いられるが、これらに限定されない。 As the expression vector into which DNA encoding the nucleic acid sequence recognition module and/or effector is inserted, animal virus vectors such as retroviruses, vaccinia viruses, and adenoviruses are used.
Promoters include SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. Any promoter that is functional in mammalian cells may be used, including but not limited to.
　発現ベクターは、上記の他に、所望によりエンハンサー、スプライシングシグナル、ターミネーター、ポリＡ付加シグナル、薬剤耐性遺伝子等の選択マーカー、複製起点などを含有していてもよい。 In addition to the above, the expression vector may optionally contain an enhancer, a splicing signal, a terminator, a poly A addition signal, a selection marker such as a drug resistance gene, an origin of replication, and the like.
　核酸配列認識モジュール（及び所望により、更にエフェクター）をコードする発現ベクターを対象となる哺乳動物の標的細胞に導入することによって、該細胞内において核酸配列認識モジュール又は該モジュールとエフェクターとの複合体が発現・形成され、ＬＳＭＥＭ２遺伝子上の標的ヌクレオチド配列と接触させることができる。 By introducing an expression vector encoding a nucleic acid sequence recognition module (and, if desired, an effector) into a target cell of a mammal, the nucleic acid sequence recognition module or a complex of the module and the effector is produced in the target cell. It can be expressed and formed and contacted with the target nucleotide sequence on the LSMEM2 gene.
（ｄ－２）ゲノム配列の改変を伴う結合阻害物質
　上記（ｄ）において、ヌクレアーゼとして、ＤＳＢ活性を有する野生型酵素を用いることにより、ＬＳＭＥＭ２遺伝子内でＤＳＢを生じせしめ、ＬＳＭＥＭ２遺伝子の１以上のヌクレオチドの欠失又は他のヌクレオチドへの置換、或いは該遺伝子内への１以上のヌクレオチドの挿入をもたらすことにより、ＬＳＭＥＭ２遺伝子を破壊することができる。 (d-2) Binding inhibitor accompanied by modification of genome sequence In (d) above, by using a wild-type enzyme having DSB activity as a nuclease, DSB is generated within the LSMEM2 gene, and one or more of the LSMEM2 genes The LSMEM2 gene can be disrupted by causing deletions or substitutions of nucleotides with other nucleotides, or insertions of one or more nucleotides into the gene.
　ＬＳＭＥＭ２遺伝子は、標的ヌクレオチド配列内で切断された後、非相同末端連結（ＮＨＥＪ）により修復されるが、その際の修復エラーにより、該遺伝子の１以上のヌクレオチドの欠失又は他のヌクレオチドへの置換、或いは該遺伝子内への１以上のヌクレオチドの挿入を生じる。その結果、ＬＳＭＥＭ２遺伝子中に変異（例、フレームシフト変異）、を生じさせることができ、正しく翻訳されるＬＳＭＥＭ２　ｍＲＮＡの発現を抑制させることができる。 The LSMEM2 gene is repaired by non-homologous end joining (NHEJ) after being cleaved within the target nucleotide sequence, but repair errors may result in the deletion of one or more nucleotides in the gene or the addition of other nucleotides. resulting in a substitution or insertion of one or more nucleotides into the gene. As a result, mutations (eg, frameshift mutations) can be generated in the LSMEM2 gene, and the expression of correctly translated LSMEM2 mRNA can be suppressed.
　ＬＳＭＥＭ２遺伝子内に変異を導入する別の態様として、ＬＳＭＥＭ２遺伝子内の標的領域に隣接する上流及び下流の配列それぞれに相同な配列を含み、かつ該標的領域部分に変異が導入されたＤＮＡ（ドナーＤＮＡ）を、核酸配列認識モジュールとヌクレアーゼとの複合体をコードするＤＮＡとともに、対象となる哺乳動物細胞に導入することで、本発明の標的領域を含む領域と該ドナーＤＮＡとの間で相同組換えを引き起こすことにより、標的領域内に所望の変異を導入することができる。ドナーＤＮＡに含まれる、該標的領域に隣接する上流及び下流の配列それぞれに相同な配列は、相同組換えを生じるに十分な長さであれば、特に制限されず、それぞれ５０～１００ｍｅｒ程度の比較的短い配列であってもよいし、或いは、数ｋｂに及ぶ長いホモロジーアームであってもよい。後者の場合、ドナーＤＮＡはそれが挿入されたターゲティングベクターの形態で提供され得る。「ドナーＤＮＡが挿入されたターゲティングベクター」とは、単に上記ドナーＤＮＡと同一の配列が挿入されたターゲティングベクターにとどまらず、ドナーＤＮＡの間又は外側に、選択マーカー及び／又はリコンビナーゼ標的配列を有するものも含む。該ターゲティングベクターの基本骨格となるベクターは特に限定されず、形質転換を行う細胞（例えば、大腸菌）中で自己複製可能なものであればよい。例えば、市販のｐＢｌｕｓｃｒｉｐｔ（Ｓｔｒａｔａｇｅｎｅ社製）、ｐＺＥｒＯ１．１（Ｉｎｖｉｔｒｏｇｅｎ社）、ｐＧＥＭ－１（Ｐｒｏｍｅｇａ社）等が使用可能である。 Another method for introducing mutations into the LSMEM2 gene is to use DNA that contains sequences homologous to the upstream and downstream sequences adjacent to the target region in the LSMEM2 gene, and in which a mutation has been introduced into the target region (donor DNA). ) is introduced into a target mammalian cell together with a DNA encoding a complex of a nucleic acid sequence recognition module and a nuclease, thereby causing homologous recombination between the region containing the target region of the present invention and the donor DNA. By causing this, a desired mutation can be introduced into the target region. The sequences homologous to the upstream and downstream sequences adjacent to the target region contained in the donor DNA are not particularly limited as long as they are long enough to cause homologous recombination. It may be a short sequence, or it may be a long homology arm spanning several kb. In the latter case, the donor DNA may be provided in the form of a targeting vector into which it has been inserted. "Targeting vector into which donor DNA has been inserted" is not just a targeting vector into which the same sequence as the above-mentioned donor DNA has been inserted, but also one which has a selection marker and/or recombinase target sequence between or outside the donor DNA. Also included. The vector serving as the basic skeleton of the targeting vector is not particularly limited, as long as it is capable of self-replication in the cell to be transformed (eg, E. coli). For example, commercially available pBluescript (Stratagene), pZErO1.1 (Invitrogen), pGEM-1 (Promega), etc. can be used.
　ドナーＤＮＡは、二本鎖ＤＮＡ（環状二本鎖ＤＮＡ、直鎖状二本鎖ＤＮＡ）、一本鎖ＤＮＡのいずれの形態でも、細胞に導入され得る。 Donor DNA can be introduced into cells in the form of either double-stranded DNA (circular double-stranded DNA, linear double-stranded DNA) or single-stranded DNA.
　ＤＳＢは想定外のゲノム改変を伴うため、強い細胞毒性や染色体の転位などの副作用があり、遺伝子治療における信頼性を損なうおそれがある。そのため、ヌクレアーゼに代えて、エフェクターとしてＤＳＢを伴わずにゲノム改変が可能な酵素を、核酸配列認識モジュールと組み合わせて用いることができる。そのような酵素としては、例えば、核酸塩基の置換基を他の置換基に変換することで、異なる塩基に置換する酵素（例、デアミナーゼ等）、脱塩基反応を触媒し、内在の修復機構のエラーを利用して脱塩基部位に変異を導入する酵素（例、ＤＮＡグリコシラーゼ等）が挙げられるが、それらに限定されない。尚、この場合、核酸配列認識モジュールがＣＲＩＳＰＲ－Ｃａｓであれば、Ｃａｓとして、少なくとも一方のＤＮＡ切断能が失活した変異体（ｎＣａｓ）、好ましくは両方のＤＮＡ切断能が失活した変異体（ｄＣａｓ）が用いられる。エフェクターとしてデアミナーゼを用いる場合の詳細は、例えば国際公開第２０１５／１３３５５４号に、ＤＮＡグリコシラーゼを用いる場合の詳細は、例えば国際公開第２０１６／０７２３９９号に、それぞれ記載されている。 Because DSB involves unexpected genome modification, it has side effects such as strong cytotoxicity and chromosomal rearrangement, which may impair the reliability of gene therapy. Therefore, instead of a nuclease, an enzyme capable of modifying the genome without DSB as an effector can be used in combination with a nucleic acid sequence recognition module. Such enzymes include, for example, enzymes that substitute different bases by converting one substituent of a nucleobase into another (e.g., deaminase), and enzymes that catalyze abasic reactions and activate endogenous repair mechanisms. Examples include, but are not limited to, enzymes (eg, DNA glycosylase, etc.) that utilize errors to introduce mutations into abasic sites. In this case, if the nucleic acid sequence recognition module is CRISPR-Cas, the Cas may be a mutant in which at least one DNA cleaving ability is inactivated (nCas), preferably a mutant in which both DNA cleaving abilities are inactivated ( dCas) is used. Details of using deaminase as an effector are described in, for example, International Publication No. 2015/133554, and details of using DNA glycosylase are described in, for example, International Publication No. 2016/072399.
　理論に束縛されることはないが、伸展負荷がかかった心筋は、メカノストレス依存的に炎症性サイトカインを発現し、心筋内炎症を惹起させる。これが悪循環となり心機能を更に悪化、予後を悪化させるが、ＬＳＭＥＭ２抑制はこの悪循環を断ち切ることにより治療効果を発揮すると考えられる。また、ラミノパチーではＬＳＭＥＭ２が小胞輸送において中心的な役割を果たすレトロマー複合体の構成因子であるＶＰＳ３５と相互作用することが見出されている。ＶＰＳ３５は相互作用する分子によって小胞の行き先を制御する小胞輸送における中心的な分子である。一方、ラミノパチーモデル細胞ではメカノトランスダクションに関連した核膜タンパク質であるＳＵＮ１がゴルジ体にミスローカライズすることが報告されており（Ｃｅｌｌ．２０１２；１４９（３）：５６５－７７）、ラミノパチーの病態形成に小胞輸送の異常が関与していることが強く示唆されている。ＬＳＭＥＭ２欠損がラミノパチーの表現系を改善させることから、ラミノパチーの病態形成関与する小胞輸送変容とメカノストレス存在下でのＬＳＭＥＭ２依存的な小胞輸送変容がオーバーラップしていると考えられる。本発明において「ＬＳＭＥＭ２の機能を抑制する物質」とは、いったん機能的に産生されたＬＳＭＥＭ２のメカノシグナル伝達に寄与する機能を抑制する限りいかなるものでもよく、例えば、ＬＳＭＥＭ２に結合して前記機能を抑制する物質、ＬＳＭＥＭ２の細胞膜への移行を阻害する物質等が挙げられる。 Without being bound by theory, myocardium under stretching load expresses inflammatory cytokines in a mechanostress-dependent manner, causing intramyocardial inflammation. This becomes a vicious cycle, further deteriorating cardiac function and worsening prognosis, but LSMEM2 inhibition is thought to exert a therapeutic effect by breaking this vicious cycle. Furthermore, in laminopathies, LSMEM2 has been found to interact with VPS35, a component of the retromer complex that plays a central role in vesicular transport. VPS35 is a central molecule in vesicle trafficking that controls vesicle destination by interacting molecules. On the other hand, it has been reported that SUN1, a nuclear membrane protein related to mechanotransduction, mislocalizes to the Golgi apparatus in laminopathy model cells (Cell. 2012; 149(3):565-77). It has been strongly suggested that abnormalities in vesicle transport are involved in pathogenesis. Since LSMEM2 deficiency improves the phenotype of laminopathy, it is thought that the alterations in vesicle transport involved in the pathogenesis of laminopathy overlap with the changes in LSMEM2-dependent vesicle transport in the presence of mechano stress. In the present invention, the "substance that suppresses the function of LSMEM2" may be any substance as long as it suppresses the function that contributes to mechanosignal transmission of LSMEM2 once it is functionally produced. For example, it binds to LSMEM2 and suppresses the function. Examples include substances that inhibit the movement of LSMEM2 to the cell membrane.
　具体的には、ＬＳＭＥＭ２の機能を抑制する物質として、例えば、ＬＳＭＥＭ２に対する抗体が挙げられる。該抗体はポリクローナル抗体、モノクローナル抗体の何れであってもよい。これらの抗体は、自体公知の抗体又は抗血清の製造法に従って製造することができる。抗体のアイソタイプは特に限定されないが、好ましくはＩｇＧ、ＩｇＭ又はＩｇＡ、特に好ましくはＩｇＧが挙げられる。また、該抗体は、ＬＳＭＥＭ２を特異的に認識し結合するための相補性決定領域（ＣＤＲ）を少なくとも有するものであれば特に制限はなく、完全抗体分子の他、例えばＦａｂ、Ｆａｂ’、Ｆ（ａｂ’）２等のフラグメント、ｓｃＦｖ、ｓｃＦｖ－Ｆｃ、ミニボディー、ダイアボディー等の遺伝子工学的に作製されたコンジュゲート分子、或いはポリエチレングリコール（ＰＥＧ）等のタンパク質安定化作用を有する分子等で修飾されたそれらの誘導体などであってもよい。 Specifically, examples of substances that suppress the function of LSMEM2 include antibodies against LSMEM2. The antibody may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to known antibody or antiserum production methods. The isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG. Further, the antibody is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding LSMEM2, and in addition to complete antibody molecules, for example, Fab, Fab', F ( ab') 2 , etc., genetically engineered conjugate molecules such as scFv, scFv-Fc, minibodies, and diabodies, or molecules with protein stabilizing effects such as polyethylene glycol (PEG). They may also be derivatives thereof.
　好ましい一実施態様において、ＬＳＭＥＭ２に対する抗体はヒトを投与対象とする医薬品として使用されることから、該抗体（好ましくはモノクローナル抗体）はヒトに投与した場合に抗原性を示す危険性が低減された抗体、具体的には、完全ヒト抗体、ヒト化抗体、マウス－ヒトキメラ抗体などであり、特に好ましくは完全ヒト抗体である。ヒト化抗体及びキメラ抗体は、常法に従って遺伝子工学的に作製することができる。また、完全ヒト抗体は、ヒト－ヒト（若しくはマウス）ハイブリドーマより製造することも可能ではあるが、大量の抗体を安定に且つ低コストで提供するためには、ヒト抗体産生マウスやファージディスプレイ法を用いて製造することが望ましい。 In a preferred embodiment, since the antibody against LSMEM2 is used as a pharmaceutical to be administered to humans, the antibody (preferably a monoclonal antibody) is an antibody with a reduced risk of exhibiting antigenicity when administered to humans. , specifically, fully human antibodies, humanized antibodies, mouse-human chimeric antibodies, etc., and fully human antibodies are particularly preferred. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to conventional methods. In addition, fully human antibodies can be produced from human-human (or mouse) hybridomas, but in order to stably provide large quantities of antibodies at low cost, human antibody-producing mice and phage display methods are required. It is desirable to manufacture using
　ＬＳＭＥＭ２の機能を抑制する物質はまた、例えば、アプタマー（ペプチドアプタマーや核酸アプタマー）であってもよい。本明細書において、アプタマーは、特定の分子と特異的に結合することができる核酸やペプチドを意味する。アプタマーが核酸である場合、核酸はＤＮＡ、ＲＮＡまたはＤＮＡとＲＮＡのキメラのいずれであってもよい。アプタマーは、周知の方法（例えば、Ｅｌｌｉｎｇｔｏｎ　ｅｔ　ａｌ．，（１９９０），Ｎａｔｕｒｅ，３４６，８１８－８２２；Ｔｕｅｒｋ　ｅｔ　ａｌ．，（１９９０）Ｓｃｉｅｎｃｅ，２４９，５０５－５１０）に従って、スクリーニングや作製をすることができる。 The substance that suppresses the function of LSMEM2 may also be, for example, an aptamer (peptide aptamer or nucleic acid aptamer). As used herein, aptamer refers to a nucleic acid or peptide that can specifically bind to a specific molecule. When the aptamer is a nucleic acid, the nucleic acid may be DNA, RNA, or a chimera of DNA and RNA. Aptamers can be screened or produced according to well-known methods (for example, Ellington et al., (1990), Nature, 346, 818-822; Tuerk et al., (1990) Science, 249, 505-510). Can be done.
　ＬＳＭＥＭ２の機能を抑制する物質はまた、例えば、ＬＳＭＥＭ２の分解を誘導する物質であってもよい。ＬＳＭＥＭ２の分解を誘導する物質としては、例えば、標的タンパク質分解誘導技術（Ｔａｒｇｅｔｅｄ　Ｐｒｏｔｅｉｎ　Ｄｅｇｒａｄａｔｉｏｎ　Ｔｅｃｈｎｉｑｕｅ）を用いた、ＬＳＭＥＭ２デグレーダー等が挙げられる。本明細書において、標的タンパク質デグレーダーは、細胞内のタンパク質分解機構を通じて、標的タンパク質の分解を促進することができる分子を意味する。かかる標的タンパク質デグレーダーは、周知の方法（例えば、Ｍｏｈａｍｍａｄｓｈａｒｉｆ　Ｔａｂｅｂｏｒｄｂａｒ　ｅｔ　ａｌ．，　Ｃｅｌｌ．　２０２１　Ｓｅｐ　１６；１８４（１９）：４９１９－４９３８．ｅ２２．）に従って作製することができる。標的タンパク質デグレーダーを利用する一態様において、標的タンパク質デグレーダーは、標的タンパク質に特異的に結合する部分と、Ｅ３ユビキチンリガーゼに結合する別の部分との２つの部分を含み得る。Ｅ３ユビキチンリガーゼにより標的タンパク質のユビキチン化が促進される。その結果、プロテアソーム系による標的タンパク質の分解が促進される。 The substance that suppresses the function of LSMEM2 may also be, for example, a substance that induces the decomposition of LSMEM2. Examples of the substance that induces the degradation of LSMEM2 include an LSMEM2 degrader using a targeted protein degradation technique. As used herein, a target protein degrader refers to a molecule capable of promoting the degradation of a target protein through the intracellular proteolytic machinery. Such a target protein degrader can be produced according to a well-known method (for example, Mohammadsharif Tabebordbar et al., Cell. 2021 Sep 16; 184(19): 4919-4938.e22.). In one embodiment utilizing a targeted protein degrader, the targeted protein degrader can include two parts: a part that specifically binds the target protein and another part that binds to the E3 ubiquitin ligase. E3 ubiquitin ligase promotes ubiquitination of target proteins. As a result, degradation of target proteins by the proteasome system is promoted.
　本明細書において、「メカノシグナル伝達」とは、細胞が機械的刺激（例、液圧、伸展刺激など）を受けると、細胞の構成成分であるセンサー分子（ＰＩＥＺＯ）に構造変化が引き起こされ、そのことにより当該機械的刺激の情報が生化学的シグナルに変換されて、細胞による感知、解釈、及び応答等がもたらされるように他の媒介シグナル分子によって伝達されていくことを意味する。 As used herein, "mechanosignaling" refers to a process in which when a cell receives mechanical stimulation (e.g., hydraulic pressure, stretching stimulation, etc.), a structural change is caused in the sensor molecule (PIEZO), which is a component of the cell. This means that the information of the mechanical stimulus is converted into a biochemical signal and transmitted by other mediating signal molecules for sensing, interpretation, and response by the cell.
　後述の実施例に示されるように、ＬＳＭＥＭ２は、メカノシグナル（メカノストレス（伸展刺激）依存的な炎症性シグナル）伝達に必要十分であるので、ＬＳＭＥＭ２の発現若しくは機能を抑制する物質はメカノシグナル伝達を阻害する作用を有し、心筋細胞等の標的治療薬として拡張型心筋症の予防及び／又は治療剤として使用することができる。また、ＬＳＭＥＭ２ノックアウトマウスとラミン心筋症モデルマウスとの交配により生じたマウスは、心機能や下肢筋力が改善されることから、ＬＳＭＥＭ２の発現若しくは機能を抑制する物質はラミノパチーの予防及び／又は治療剤としても使用することができる。ＬＳＭＥＭ２　ＫＯマウスは正常に発生し、心機能はまったく正常であり、生殖機能にも問題を認めないことから、ＬＳＭＥＭ２の発現若しくは機能を抑制する物質は、正常細胞に対して低毒性であり、副作用のリスクが低いという更なる有利な効果を奏する。従って、ＬＳＭＥＭ２の発現若しくは機能を抑制する物質を含有する医薬は、メカノシグナル伝達阻害剤、ひいては心疾患の予防及び／又は治療剤として有用であり、並びにラミノパチーの予防及び／又は治療剤としても有用である。ここで「治療」とは、疾患の改善、並びに予後の改善を包含する意味で用いる。「予防」とは、疾患の発症遅延を包含する意味で用いる。 As shown in the Examples below, LSMEM2 is necessary and sufficient for mechanosignal (mechano stress (stretch stimulus) dependent inflammatory signal) transmission, so substances that suppress the expression or function of LSMEM2 are effective for mechanosignal transmission. It has the effect of inhibiting dilated cardiomyopathy, and can be used as a therapeutic agent targeting cardiomyocytes and the like to prevent and/or treat dilated cardiomyopathy. In addition, mice produced by crossing LSMEM2 knockout mice with lamin cardiomyopathy model mice have improved cardiac function and lower limb muscle strength. Therefore, substances that suppress the expression or function of LSMEM2 can be used as preventive and/or therapeutic agents for laminopathy. It can also be used as LSMEM2 KO mice develop normally, have completely normal cardiac function, and have no problems with reproductive function. Therefore, substances that suppress the expression or function of LSMEM2 have low toxicity to normal cells and have no side effects. The additional advantageous effect is that the risk of Therefore, a drug containing a substance that suppresses the expression or function of LSMEM2 is useful as a mechanosignal transduction inhibitor, and as a preventive and/or therapeutic agent for heart disease, and also as a preventive and/or therapeutic agent for laminopathy. It is. "Treatment" is used herein to include improvement of disease as well as improvement of prognosis. The term "prevention" is used to include delaying the onset of a disease.
　ＬＳＭＥＭ２の発現若しくは機能を抑制する物質は１種のみを用いてもよいし、２種以上を組み合わせて用いてもよい。２種以上の、ＬＳＭＥＭ２の発現若しくは機能を抑制する物質は、それぞれ別個の医薬として製剤化してもよいし、同一の医薬組成物中に配合してもよい。２種以上の、ＬＳＭＥＭ２の発現若しくは機能を抑制する物質が、それぞれ別個の医薬として製剤化される場合、各製剤を同時に投与してもよいし、時間をおいて投与してもよい。また、投与経路は同一であってもよいし、異なっていてもよい。後述する投与量は、１種の、ＬＳＭＥＭ２の発現若しくは機能を抑制する物質の投与量を示すが、２種以上の物質を組み合わせて用いる場合でも、投与対象に好ましくない影響を与えない範囲で、それぞれの物質について同様の投与量を用いることができる。 Only one type of substance that suppresses the expression or function of LSMEM2 may be used, or two or more types may be used in combination. Two or more substances that suppress the expression or function of LSMEM2 may be formulated as separate pharmaceuticals, or may be combined in the same pharmaceutical composition. When two or more substances that suppress the expression or function of LSMEM2 are formulated as separate pharmaceuticals, each formulation may be administered at the same time or at intervals. Moreover, the administration routes may be the same or different. The dosage mentioned below indicates the dosage of one kind of substance that suppresses the expression or function of LSMEM2, but even when two or more kinds of substances are used in combination, within a range that does not have an unfavorable effect on the subject of administration, Similar dosages can be used for each substance.
（１）ｓｉＲＮＡ及びその前駆体、アンチセンス核酸、リボザイム核酸を含有する医薬
　ＬＳＭＥＭ２遺伝子の転写産物に相補的に結合し、該転写産物からのタンパク質の翻訳を抑制することができるアンチセンス核酸（若しくはｍｉＲＮＡ）や、ＬＳＭＥＭ２遺伝子の転写産物における相同な（若しくは相補的な）塩基配列を標的として該転写産物を切断し得るｓｉＲＮＡ（若しくはリボザイム、ｍｉＲＮＡ）、更に該ｓｉＲＮＡやｍｉＲＮＡの前駆体であるｓｈＲＮＡやｐｒｅ－ｍｉＲＮＡなど（以下、包括的に「本発明の核酸」という場合がある）は、抗炎症剤及び／又は小胞輸送異常の抑制剤、ひいては心疾患の予防及び／又は治療剤、及び／又はラミノパチーの予防及び／又は治療剤として使用することができる。
　本発明の核酸を含有する医薬はそのまま液剤として、又は適当な剤型の医薬組成物として、ヒト又は非ヒト温血動物（例、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サル、ニワトリなど）などの対象に対して経口的又は非経口的（例、血管内投与、皮下投与など）に投与することができる。 (1) Medicine containing siRNA and its precursor, antisense nucleic acid, ribozyme nucleic acid Antisense nucleic acid (or miRNA), siRNA (or ribozyme, miRNA) that can target a homologous (or complementary) base sequence in the transcript of the LSMEM2 gene and cleave the transcript, shRNA that is a precursor of the siRNA or miRNA, Pre-miRNA etc. (hereinafter sometimes collectively referred to as "nucleic acids of the present invention") can be used as anti-inflammatory agents and/or inhibitors of vesicular transport abnormalities, as well as preventive and/or therapeutic agents for heart diseases, and/or Alternatively, it can be used as a prophylactic and/or therapeutic agent for laminopathy.
The medicament containing the nucleic acid of the present invention can be administered to humans or non-human warm-blooded animals (e.g., rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, It can be administered orally or parenterally (eg, intravascularly, subcutaneously, etc.) to subjects such as chickens, etc.
　本発明の核酸を抗炎症剤及び／又は小胞輸送異常の抑制剤、ひいては心疾患の予防及び／又は治療剤、及び／又はラミノパチーの予防及び／又は治療剤として使用する場合、自体公知の方法に従って製剤化し、投与することができる。即ち、本発明の核酸を単独で用いてもよいし、或いはレトロウイルスベクター、アデノウイルスベクター、アデノウイルスアソシエーテッドウイルスベクターなどの適当な哺乳動物細胞用の発現ベクターに機能可能な態様で挿入することもできる。該核酸は、そのままで、或いは摂取促進のための補助剤とともに、遺伝子銃やハイドロゲルカテーテルのようなカテーテルによって投与することができる。或いは、エアロゾル化して吸入剤として気管内に局所投与することもできる。
　更に、体内動態の改良、半減期の長期化、細胞内取り込み効率の改善を目的に、前記核酸を単独又はリポソームなどの担体とともに製剤（注射剤）化し、静脈、皮下等に投与してもよい。 When the nucleic acid of the present invention is used as an anti-inflammatory agent and/or an inhibitor of vesicular transport abnormalities, a preventive and/or therapeutic agent for heart disease, and/or a preventive and/or therapeutic agent for laminopathy, methods known per se can be used. It can be formulated and administered according to the following. That is, the nucleic acid of the present invention may be used alone or inserted in a functional manner into an appropriate expression vector for mammalian cells, such as a retrovirus vector, an adenovirus vector, or an adenovirus associated virus vector. You can also do it. The nucleic acid can be administered as is or together with an adjuvant to promote uptake via a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving the efficiency of cellular uptake, the nucleic acid may be formulated into a formulation (injection) alone or together with a carrier such as a liposome, and administered intravenously, subcutaneously, etc. .
　本発明の核酸は、それ自体を投与してもよいし、又は適当な医薬組成物として投与してもよい。投与に用いられる医薬組成物としては、本発明の核酸と薬理学的に許容され得る担体、希釈剤若しくは賦形剤とを含むものであってよい。このような医薬組成物は、経口又は非経口投与に適する剤形として提供される。 The nucleic acid of the present invention may be administered as such or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the nucleic acid of the present invention and a pharmacologically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions are provided in dosage forms suitable for oral or parenteral administration.
　非経口投与のための組成物としては、例えば、注射剤、坐剤、鼻腔内投与剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。このような注射剤は、公知の方法に従って調製できる。注射剤の調製方法としては、例えば、上記本発明の核酸を通常注射剤に用いられる無菌の水性液、又は油性液に溶解、懸濁又は乳化することによって調製できる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液等が用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン界面活性剤〔例、ポリソルベート８０、ＨＣＯ－５０（ｐｏｌｙｏｘｙｅｔｈｙｌｅｎｅ（５０ｍｏｌ）ａｄｄｕｃｔ　ｏｆ　ｈｙｄｒｏｇｅｎａｔｅｄ　ｃａｓｔｏｒ　ｏｉｌ）〕等と併用してもよい。油性液としては、例えば、ゴマ油、大豆油等が用いられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコール等を併用してもよい。調製された注射液は、適当なアンプルに充填されることが好ましい。直腸投与に用いられる坐剤は、上記核酸を通常の坐薬用基剤に混合することによって調製されてもよい。 Compositions for parenteral administration include, for example, injections, suppositories, intranasal injections, etc. Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. It may also include dosage forms such as agents. Such injections can be prepared according to known methods. An injection can be prepared, for example, by dissolving, suspending or emulsifying the nucleic acid of the present invention in a sterile aqueous or oily liquid commonly used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil), etc.). As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and benzyl benzoate, benzyl alcohol, etc. may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled into suitable ampoules. Suppositories for rectal administration may be prepared by mixing the nucleic acids described above with conventional suppository bases.
　経口投与のための組成物としては、固体又は液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。このような組成物は公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤若しくは賦形剤を含有していても良い。錠剤用の担体、賦形剤としては、例えば、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムが用いられる。 Compositions for oral administration include solid or liquid dosage forms, in particular tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Examples include formulations, emulsions, suspensions, and the like. Such compositions may be manufactured by known methods and may contain carriers, diluents or excipients commonly used in the pharmaceutical field. As carriers and excipients for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.
　上記の非経口用又は経口用医薬組成物は、活性成分の投与量に適合するような投薬単位の剤形に調製されることが好都合である。このような投薬単位の剤形としては、例えば、錠剤、丸剤、カプセル剤、注射剤（アンプル）、坐剤が挙げられる。本発明の核酸は、例えば、投薬単位剤形当たり通常０．０１～５００ｍｇ程度含有されていることが好ましい。 The parenteral or oral pharmaceutical compositions described above are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The nucleic acid of the present invention is preferably contained, for example, in an amount of usually about 0.01 to 500 mg per unit dosage form.
　本発明の核酸を含有する上記医薬の投与量は、投与対象、対象疾患、症状、投与ルートなどによっても異なるが、例えば、心疾患やラミノパチーの治療・予防において使用する場合には、本発明の核酸を１回量として、通常０．０００１～２０ｍｇ／ｋｇ体重程度を、１日～６ヶ月に１回程度、静脈注射により投与するのが好都合である。他の非経口投与及び経口投与の場合もこれに準ずる量を投与することができる。症状が特に重い場合には、その症状に応じて増量してもよい。 The dosage of the above-mentioned medicine containing the nucleic acid of the present invention varies depending on the subject, target disease, symptoms, administration route, etc.; It is convenient to administer a single dose of nucleic acid, usually about 0.0001 to 20 mg/kg body weight, by intravenous injection about once every 1 day to 6 months. Similar amounts can be administered in other cases of parenteral administration and oral administration. If the symptoms are particularly severe, the dose may be increased depending on the symptoms.
（２）核酸配列認識モジュールを含有する医薬
　本発明の結合阻害物がゲノム編集技術において利用される核酸配列認識モジュール（及び該モジュールと複合体を形成するエフェクター）である場合、好ましくは、該結合阻害物質は、それをコードするＤＮＡを含む発現ベクター（以下、「本発明のベクター」ともいう。）の形態で、製剤化される。本発明のベクターとしては、例えば、無毒化したレトロウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス、ワクシニアウイルス、ポックスウイルス、ポリオウイルス、シンドビスウイルス、センダイウイルス、ＳＶ４０、免疫不全症ウイルス（ＨＩＶ）等のウイルスベクターを用いることができる。好ましくはアデノウイルス又はアデノ随伴ウイルスベクターが挙げられる。 (2) Pharmaceutical containing a nucleic acid sequence recognition module When the binding inhibitor of the present invention is a nucleic acid sequence recognition module used in genome editing technology (and an effector that forms a complex with the module), preferably the binding The inhibitory substance is formulated in the form of an expression vector (hereinafter also referred to as "vector of the present invention") containing DNA encoding the inhibitory substance. Vectors of the present invention include, for example, detoxified retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, poxviruses, polioviruses, Sindbis viruses, Sendai viruses, SV40, and immunodeficiency virus (HIV). Viral vectors such as the following can be used. Preferably, adenovirus or adeno-associated virus vectors are used.
　医薬上許容される担体としては、例えば、ショ糖、デンプン等の賦形剤、セルロース、メチルセルロース等の結合剤、デンプン、カルボキシメチルセルロース等の崩壊剤、ステアリン酸マグネシウム、エアロジル等の滑剤、クエン酸、メントール等の芳香剤、安息香酸ナトリウム、亜硫酸水素ナトリウム等の保存剤、クエン酸、クエン酸ナトリウム等の安定剤、メチルセルロース、ポリビニルピロリド等の懸濁剤、界面活性剤等の分散剤、水、生理食塩水等の希釈剤、ベースワックス等が挙げられるが、それらに限定されるものではない。 Pharmaceutically acceptable carriers include, for example, excipients such as sucrose and starch, binders such as cellulose and methyl cellulose, disintegrants such as starch and carboxymethyl cellulose, lubricants such as magnesium stearate and Aerosil, citric acid, Flavoring agents such as menthol, preservatives such as sodium benzoate and sodium bisulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinyl pyrrolid, dispersing agents such as surfactants, water, Examples include diluents such as physiological saline, base wax, etc., but are not limited thereto.
　上記ベクターの標的細胞内への導入を促進するために、本発明の剤は、更に核酸導入用試薬を含むことができる。該核酸導入用試薬としては、アテロコラーゲン；リポソーム；ナノパーティクル；リポフェクチン、リプフェクタミン（ｌｉｐｏｆｅｃｔａｍｉｎｅ）、ＤＯＧＳ（トランスフェクタム）、ＤＯＰＥ、ＤＯＴＡＰ、ＤＤＡＢ、ＤＨＤＥＡＢ、ＨＤＥＡＢ、ポリブレン、或いはポリ（エチレンイミン）（ＰＥＩ）等の陽イオン性脂質等を用いることが出来る。 In order to promote the introduction of the vector into target cells, the agent of the present invention can further contain a reagent for nucleic acid introduction. The nucleic acid introduction reagents include atelocollagen; liposome; nanoparticle; lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly(ethyleneimine) (PEI). Cationic lipids such as, for example, can be used.
　好ましい一実施態様において、本発明の剤は、上記ベクターがリポソームに封入されてなる医薬組成物であり得る。リポソームは、１以上の脂質二重層により包囲された内相を有する微細閉鎖小胞であり、通常は水溶性物質を内相に、脂溶性物質を脂質二重層内に保持することができる。本明細書において「封入」という場合には、上記ベクターはリポソーム内相に保持されてもよいし、脂質二重層内に保持されてもよい。本発明に用いられるリポソームは単層膜であっても多層膜であってもよく、また、粒子径は、例えば１０～１０００ｎｍ、好ましくは５０～３００ｎｍの範囲で適宜選択できる。標的組織への送達性を考慮すると、粒子径は、例えば２００ｎｍ以下、好ましくは１００ｎｍ以下であり得る。 In a preferred embodiment, the agent of the present invention may be a pharmaceutical composition in which the vector is encapsulated in a liposome. Liposomes are microscopic closed vesicles that have an internal phase surrounded by one or more lipid bilayers, and can typically retain water-soluble substances in the internal phase and lipid-soluble substances within the lipid bilayer. When the term "encapsulation" is used herein, the vector may be retained in the internal phase of the liposome or within the lipid bilayer. The liposome used in the present invention may be a monolayer or a multilayer, and the particle size can be appropriately selected within the range of, for example, 10 to 1000 nm, preferably 50 to 300 nm. Considering deliverability to the target tissue, the particle size may be, for example, 200 nm or less, preferably 100 nm or less.
　上記ベクターのリポソームへの封入法としては、リピドフィルム法（ボルテックス法）、逆相蒸発法、界面活性剤除去法、凍結融解法、リモートローディング法等が挙げられるが、これらに限定されず、任意の公知の方法を適宜選択することができる。 Methods for encapsulating the above-mentioned vectors in liposomes include, but are not limited to, the lipid film method (vortex method), reverse phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading method. Any known method can be selected as appropriate.
　本発明の剤は、経口的に又は非経口的に、哺乳動物（例、ヒト、ラット、マウス、モルモット、ウサギ、ヒツジ、ウマ、ブタ、ウシ、サル）に対して投与することが可能であるが、非経口的に投与するのが望ましい。 The agent of the present invention can be administered orally or parenterally to mammals (e.g., humans, rats, mice, guinea pigs, rabbits, sheep, horses, pigs, cows, monkeys). However, it is preferable to administer the drug parenterally.
　また、徐放性の製剤（ミニペレット製剤等）を調製し患部近くに埋め込むことも可能であり、或いはオスモチックポンプ等を用いて患部に連続的に徐々に投与することも可能である。 It is also possible to prepare a sustained-release preparation (such as a minipellet preparation) and implant it near the affected area, or it is also possible to continuously and gradually administer it to the affected area using an osmotic pump or the like.
　非経口的な投与（例えば、皮下注射、筋肉注射、局所注入、腹腔内投与など）に好適な製剤としては、水性及び非水性の等張な無菌の注射液剤があり、これには抗酸化剤、緩衝液、制菌剤、等張化剤等が含まれていてもよい。また、水性及び非水性の無菌の懸濁液剤が挙げられ、これには懸濁剤、可溶化剤、増粘剤、安定化剤、防腐剤等が含まれていてもよい。当該製剤は、アンプルやバイアルのように単位投与量或いは複数回投与量ずつ容器に封入することができる。また、有効成分及び医薬上許容される担体を凍結乾燥し、使用直前に適当な無菌のビヒクルに溶解又は懸濁すればよい状態で保存することもできる。 Suitable formulations for parenteral administration (e.g., subcutaneous, intramuscular, local, intraperitoneal, etc.) include aqueous and non-aqueous isotonic sterile injectable solutions containing antioxidants. , a buffer, a bacteriostatic agent, a tonicity agent, etc. may be included. Also included are aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, and the like. The preparation can be packaged in containers such as ampoules or vials for unit doses or multiple doses. Alternatively, the active ingredient and the pharmaceutically acceptable carrier can be lyophilized and stored by dissolving or suspending them in a suitable sterile vehicle immediately before use.
　医薬組成物中の上記ベクターの含有量は、例えば、医薬組成物全体の約０．１ないし１００重量％である。 The content of the above vector in the pharmaceutical composition is, for example, about 0.1 to 100% by weight of the entire pharmaceutical composition.
　本発明の剤の投与量は、投与方法、対象とする疾患の種類、重篤度、投与対象の状況（性別、年齢、体重など）によって異なるが、例えば、成人に全身投与する場合、通常、ベクターがウイルスベクター粒子として投与される場合、１回につき、例えばウイルスの力価として約１×１０３ｐｆｕ～１×１０１５ ｐｆｕの範囲で投与され得る。 The dosage of the agent of the present invention varies depending on the administration method, the type and severity of the target disease, and the circumstances of the recipient (sex, age, body weight, etc.), but for example, when administered systemically to adults, usually When the vector is administered as a viral vector particle, a titer of virus can be administered, for example, in the range of about 1×10 3 pfu to 1×10 15 pfu per dose.
　上記物質が、それをコードするＤＮＡを含む発現ベクターの形態で提供される場合、本発明の剤は、投与対象から採取された細胞や組織（例、心筋細胞）に添加して、該細胞内に該発現ベクターを導入し、それを投与対象の体内、好ましくは心臓病変部に戻す、ｅｘ　ｖｉｖｏ製剤であってもよい。この場合、細胞への遺伝子導入法としては、リポフェクション法、リン酸－カルシウム共沈法；微小ガラス管を用いた直接注入法等が挙げられる。また、組織への遺伝子導入法としては、内包型リポソームによる遺伝子導入法、静電気型リポソームによる遺伝子導入法、ＨＶＪ－リポソーム法、改良型ＨＶＪ－リポソーム法（ＨＶＪ－ＡＶＥリポソーム法）、受容体介在性遺伝子導入法、パーティクル銃で担体（金属粒子）とともに有効成分を細胞に移入する方法、ｎａｋｅｄ－ＤＮＡの直接導入法、正電荷ポリマーによる導入法等が挙げられる。 When the above-mentioned substance is provided in the form of an expression vector containing DNA encoding the substance, the agent of the present invention can be added to cells or tissues (e.g., cardiac muscle cells) collected from the subject to be administered, and intracellularly. It may also be an ex vivo preparation, in which the expression vector is introduced into the patient's body, and then returned to the body of the subject, preferably to the cardiac lesion. In this case, methods for introducing genes into cells include lipofection, phosphate-calcium coprecipitation, and direct injection using a micro glass tube. In addition, methods for introducing genes into tissues include gene introduction using encapsulated liposomes, gene introduction using electrostatic liposomes, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), and receptor-mediated method. Examples include a gene transfer method, a method in which an active ingredient is transferred into cells together with a carrier (metal particles) using a particle gun, a direct transfer method using naked-DNA, a method using a positively charged polymer, and the like.
（３）ＬＳＭＥＭ２に対する抗体、ＬＳＭＥＭ２の発現若しくは機能を抑制する低分子化合物、又はＬＳＭＥＭ２デグレーダー等を含有する医薬
　ＬＳＭＥＭ２に対する抗体やＬＳＭＥＭ２デグレーダー、或いはＬＳＭＥＭ２の発現若しくは機能を抑制する低分子化合物は、ＬＳＭＥＭ２の産生又はそのメカノシグナル伝達に寄与する機能を阻害することができる。従って、これらの物質は、メカノシグナル伝達阻害剤、ひいては心疾患の予防及び／又は治療剤、及び／又はラミノパチーの予防及び／又は治療剤として使用することができる。
　上記の抗体、デグレーダー、又は低分子化合物を含有する医薬は、そのまま液剤として、又は適当な剤型の医薬組成物として、ヒト又は他の温血動物（例、ラット、ウサギ、ヒツジ、ブタ、ウシ、ネコ、イヌ、サル、ニワトリなど）に対して経口的又は非経口的（例、血管内投与、皮下投与など）に投与することができる。 (3) Pharmaceuticals containing antibodies against LSMEM2, low molecular weight compounds that suppress the expression or function of LSMEM2, or LSMEM2 degraders Antibodies against LSMEM2, low molecular weight compounds that suppress the expression or function of LSMEM2, etc. production or functions that contribute to mechanosignaling can be inhibited. Therefore, these substances can be used as mechanosignaling inhibitors, and thus as preventive and/or therapeutic agents for heart disease, and/or as preventive and/or therapeutic agents for laminopathy.
Pharmaceuticals containing the above-mentioned antibodies, degraders, or low-molecular-weight compounds can be administered to humans or other warm-blooded animals (e.g., rats, rabbits, sheep, pigs, cows), either directly as a solution or as a pharmaceutical composition in an appropriate dosage form. , cats, dogs, monkeys, chickens, etc.) can be administered orally or parenterally (eg, intravascularly, subcutaneously, etc.).
　上記の抗体、デグレーダー、又は低分子化合物は、それ自体を投与してもよいし、又は適当な医薬組成物として投与してもよい。投与に用いられる医薬組成物としては、上記の抗体若しくは低分子化合物又はその塩と薬理学的に許容され得る担体、希釈剤若しくは賦形剤とを含むものであってもよい。このような医薬組成物は、経口又は非経口投与に適する剤形として提供される。 The above-mentioned antibodies, degraders, or low-molecular-weight compounds may be administered as such, or as a suitable pharmaceutical composition. The pharmaceutical composition used for administration may contain the above-mentioned antibody or low-molecular-weight compound, or a salt thereof, and a pharmacologically acceptable carrier, diluent, or excipient. Such pharmaceutical compositions are provided in dosage forms suitable for oral or parenteral administration.
　非経口投与のための組成物としては、例えば、注射剤、坐剤、鼻腔内投与剤等が用いられ、注射剤は静脈注射剤、皮下注射剤、皮内注射剤、筋肉注射剤、点滴注射剤等の剤形を包含しても良い。このような注射剤は、公知の方法に従って調製できる。注射剤の調製方法としては、例えば、上記本発明の抗体若しくは低分子化合物又はその塩を通常注射剤に用いられる無菌の水性液、又は油性液に溶解、懸濁又は乳化することによって調製できる。注射用の水性液としては、例えば、生理食塩水、ブドウ糖やその他の補助薬を含む等張液等が用いられ、適当な溶解補助剤、例えば、アルコール（例、エタノール）、ポリアルコール（例、プロピレングリコール、ポリエチレングリコール）、非イオン界面活性剤〔例、ポリソルベート８０、ＨＣＯ－５０（ｐｏｌｙｏｘｙｅｔｈｙｌｅｎｅ（５０ｍｏｌ）ａｄｄｕｃｔ　ｏｆ　ｈｙｄｒｏｇｅｎａｔｅｄ　ｃａｓｔｏｒ　ｏｉｌ）〕等と併用してもよい。油性液としては、例えば、ゴマ油、大豆油等が用いられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコール等を併用してもよい。調製された注射液は、適当なアンプルに充填されることが好ましい。直腸投与に用いられる坐剤は、上記抗体又はその塩を通常の坐薬用基剤に混合することによって調製されても良い。 Compositions for parenteral administration include, for example, injections, suppositories, intranasal injections, etc. Injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. It may also include dosage forms such as agents. Such injections can be prepared according to known methods. Injections can be prepared, for example, by dissolving, suspending, or emulsifying the antibody or low-molecular-weight compound of the present invention, or a salt thereof, in a sterile aqueous or oily liquid commonly used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), nonionic surfactants (eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil), etc.). As the oily liquid, for example, sesame oil, soybean oil, etc. are used, and benzyl benzoate, benzyl alcohol, etc. may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled into suitable ampoules. Suppositories used for rectal administration may be prepared by mixing the above-described antibody or salt thereof with a conventional suppository base.
　経口投与のための組成物としては、固体又は液体の剤形、具体的には錠剤（糖衣錠、フィルムコーティング錠を含む）、丸剤、顆粒剤、散剤、カプセル剤（ソフトカプセル剤を含む）、シロップ剤、乳剤、懸濁剤等が挙げられる。このような組成物は公知の方法によって製造され、製剤分野において通常用いられる担体、希釈剤若しくは賦形剤を含有していても良い。錠剤用の担体、賦形剤としては、例えば、乳糖、でんぷん、蔗糖、ステアリン酸マグネシウムが用いられる。 Compositions for oral administration include solid or liquid dosage forms, in particular tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, capsules (including soft capsules), and syrups. Examples include formulations, emulsions, suspensions, and the like. Such compositions may be manufactured by known methods and may contain carriers, diluents or excipients commonly used in the pharmaceutical field. As carriers and excipients for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.
　上記の非経口用又は経口用医薬組成物は、活性成分の投与量に適合するような投薬単位の剤形に調製されることが好都合である。このような投薬単位の剤形としては、例えば、錠剤、丸剤、カプセル剤、注射剤（アンプル）、坐剤が挙げられる。抗体や低分子化合物は、投薬単位剤形当たり通常０．１～５００ｍｇ、とりわけ注射剤では５～１００ｍｇ、その他の剤形では１０～２５０ｍｇ含有されていることが好ましい。 The parenteral or oral pharmaceutical compositions described above are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. The antibody or low-molecular compound is usually contained in an amount of 0.1 to 500 mg per unit dosage form, particularly preferably 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms.
　上記の抗体若しくは低分子化合物又はその塩を含有する上記医薬の投与量は、投与対象、対象疾患、症状、投与ルートなどによっても異なるが、例えば、抗体若しくは低分子化合物を１回量として、通常０．０００１～２０ｍｇ／ｋｇ体重程度、低分子化合物であれば１日１～５回程度、経口又は非経口で、抗体であれば１日～数ヶ月に１回、静脈注射により投与するのが好都合である。他の非経口投与及び経口投与の場合もこれに準ずる量を投与することができる。症状が特に重い場合には、その症状に応じて増量してもよい。 The dosage of the above-mentioned medicine containing the above-mentioned antibody or low-molecular compound or its salt varies depending on the subject, target disease, symptoms, administration route, etc., but for example, the antibody or low-molecular compound is usually administered as a single dose. About 0.0001 to 20 mg/kg body weight, low molecular weight compounds should be administered orally or parenterally 1 to 5 times a day, and antibodies should be administered by intravenous injection once a day to once every few months. It's convenient. Similar amounts can be administered in other cases of parenteral administration and oral administration. If the symptoms are particularly severe, the dose may be increased depending on the symptoms.
　なお前記した各組成物は、上記抗体や低分子化合物との配合により好ましくない相互作用を生じない限り他の活性成分を含有してもよい。例えば、抗菌薬、抗真菌薬、非ステロイド性抗炎症薬、ステロイド薬、抗凝血薬、血小板凝集防止薬、血栓溶解薬、免疫調節薬、抗原虫薬、抗生物質、抗ウイルス薬、鎮咳・去たん薬、鎮静薬、麻酔薬、抗潰瘍薬、不整脈治療薬、降圧利尿薬、精神安定薬、抗精神病薬、抗腫瘍薬、抗高脂血症薬、筋弛緩薬、抗てんかん薬、抗うつ薬、抗アレルギー薬、強心薬、不整脈治療薬、血管拡張薬、血管収縮薬、降圧利尿薬、糖尿病治療薬、麻薬拮抗薬、ビタミン薬、ビタミン誘導体、関節炎治療薬、抗リウマチ薬、抗喘息薬、頻尿・尿失禁治療薬、アトピー性皮膚炎治療薬、アレルギー性鼻炎治療薬、昇圧薬、タンパク質分解薬、タンパク質分解酵素阻害薬、抗ＳＩＤＳ薬、抗セプシス薬、抗セプティックショック薬、エンドトキシン拮抗薬或いは抗体、シグナル伝達阻害薬、炎症性メディエーター作用抑制薬、炎症性メディエーター作用抑制抗体、炎症性メディエーター産生抑制薬、抗炎症性メディエーター作用抑制薬、抗炎症性メディエーター作用抑制抗体、抗炎症性メディエーター産生抑制薬、α１アドレナリン作動薬などが挙げられる。上記の抗体若しくは低分子化合物とそれらの他の薬剤とは、同時又は異なった時間に患者に投与すればよい。 Note that each of the above-mentioned compositions may contain other active ingredients as long as they do not cause undesirable interactions when mixed with the above-mentioned antibodies or low-molecular-weight compounds. For example, antibacterial drugs, antifungal drugs, non-steroidal anti-inflammatory drugs, steroid drugs, anticoagulants, platelet antiaggregation drugs, thrombolytic drugs, immunomodulators, antiprotozoal drugs, antibiotics, antiviral drugs, antitussives and antitussives. Suppressants, sedatives, anesthetics, antiulcer drugs, antiarrhythmic drugs, antihypertensive diuretics, tranquilizers, antipsychotics, antitumor drugs, antihyperlipidemic drugs, muscle relaxants, antiepileptic drugs, antidepressants Medicines, antiallergic drugs, cardiotonic drugs, arrhythmia drugs, vasodilators, vasoconstrictors, antihypertensive diuretics, antidiabetic drugs, narcotic antagonists, vitamin drugs, vitamin derivatives, arthritis drugs, antirheumatic drugs, antiasthmatic drugs , Frequent urination/urinary incontinence treatment, atopic dermatitis treatment, allergic rhinitis treatment, vasopressor, proteolytic agent, protease inhibitor, anti-SIDS agent, anti-sepsis agent, anti-septic shock agent, endotoxin Antagonist or antibody, signal transduction inhibitor, inflammatory mediator action inhibitor, inflammatory mediator action inhibitory antibody, inflammatory mediator production inhibitor, anti-inflammatory mediator action inhibitor, anti-inflammatory mediator action inhibitor, anti-inflammatory Examples include mediator production inhibitors and α1 adrenergic agonists. The above-mentioned antibodies or low-molecular-weight compounds and their other drugs may be administered to the patient at the same time or at different times.
　医薬は、例えば抗炎症剤又は小胞輸送異常の抑制剤として、種々の炎症性疾患（例えば、クローン病、関節リウマチ、ベーチェット病（眼症状）、潰瘍性大腸炎、強直性脊椎炎、乾癬（乾癬性関節炎も含む）、ＨＩＶ感染症、多発性骨髄腫、心疾患（例えば、うっ血性心不全、慢性心不全、拡張型心筋症、肥大型心筋症、心筋虚血症、心筋梗塞、狭心症）、ＧＶＨＤ、巨細胞動脈炎（ＧＣＡ）、リウマチ性多発筋痛症（ＰＭＲ）、色素性紫斑性苔癬様皮膚炎、サルコイドーシス、ヴェーゲナー肉芽腫、膿皮症、ベーチェット病、ＴＮＦ受容体関連周期性症候群（ＴＲＡＰＳ）、ＳＡＰＨＯ症候群、高安病、筋炎、スティル病、結節性動脈周囲炎（ＰＮ）、再発性多発軟骨炎、強皮症多発性筋炎、血球貪食症候群、天疱瘡、川崎病アトピー性皮膚炎）やラミノパチー（例えば、筋ジストロフィー、ラミン心筋症、リポジストロフィー、白質ジストロフィー）の予防及び／又は治療剤などとして使用することができる。 The drug can be used, for example, as an anti-inflammatory agent or as an inhibitor of vesicular transport abnormalities, to treat various inflammatory diseases (e.g., Crohn's disease, rheumatoid arthritis, Behcet's disease (ocular symptoms), ulcerative colitis, ankylosing spondylitis, psoriasis ( (including psoriatic arthritis), HIV infection, multiple myeloma, heart disease (e.g. congestive heart failure, chronic heart failure, dilated cardiomyopathy, hypertrophic cardiomyopathy, myocardial ischemia, myocardial infarction, angina pectoris) , GVHD, giant cell arteritis (GCA), polymyalgia rheumatica (PMR), pigmented purpuric lichenoid dermatitis, sarcoidosis, Wegener's granuloma, pyoderma, Behcet's disease, TNF receptor-related periodicity TRAPS, SAPHO syndrome, Takayasu disease, myositis, Still's disease, periarteritis nodosa (PN), relapsing polychondritis, polymyositis scleroderma, hemophagocytic syndrome, pemphigus, Kawasaki disease atopic skin It can be used as a prophylactic and/or therapeutic agent for laminopathy (eg, muscular dystrophy, laminar cardiomyopathy, lipodystrophy, leukodystrophy).
　以下、実施例等により、本発明を詳しく説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be explained in detail with reference to Examples, but the present invention is not limited thereto.
［実施例１］
マウス組織ＲＴ－ＰＣＲ
　ＬＳＭＥＭ２のマウス及びヒト組織における発現動態を見るためにＲＴ－ＰＣＲを行った。ｃＤＮＡはＣｌｏｎｔｅｃｈ　Ｌａｂｏｒａｔｏｒｉｅｓ社のＭｏｕｓｅ　ＭＴＣ　Ｐａｎｅｌ　Ｉ（６３６７４５）を使用し、心臓、脳、肝臓、骨格筋、精巣、腎臓、脾臓及び肺について検討した。ＰＣＲにはＴａＫａＲａ　Ｅｘ　Ｔａｑ（Ｔａｋａｒａ　Ｂｉｏ　Ｉｎｃ．Ｓｈｉｇａ，Ｊａｐａｎ）を使用した。プライマー、反応溶液は以下のように調製した。
使用したプライマー
Ｍｏｕｓｅ＿ＲＴ－ＰＣＲ＿Ｌ　ａｇｃｔｔｃｔｇｇｇｔｇｔｃｔｔａｃｃａｃｃａｔｃ（配列番号７）
Ｍｏｕｓｅ＿ＲＴ－ＰＣＲ＿Ｒ　ｃａａｔｔｔｇａｇｃａｇａｇｔｃｔｃｃｔｃｔｔｇｔｇｔｇｃ（配列番号８） [Example 1]
Mouse tissue RT-PCR
RT-PCR was performed to examine the expression dynamics of LSMEM2 in mouse and human tissues. cDNA was examined for heart, brain, liver, skeletal muscle, testis, kidney, spleen, and lung using Mouse MTC Panel I (636745) from Clontech Laboratories. TaKaRa Ex Taq (Takara Bio Inc. Shiga, Japan) was used for PCR. Primers and reaction solutions were prepared as follows.
Primer used Mouse_RT-PCR_L agcttctgggtgtcttaccacatc (SEQ ID NO: 7)
Mouse_RT-PCR_R caatttgagcagagtctcctcttgtgtgc (SEQ ID NO: 8)
　ＰＣＲ条件は以下のように行った。 The PCR conditions were as follows.
　その結果、ＬＳＭＥＭ２発現は心筋、骨格筋に限局することが示された（図１）。 The results showed that LSMEM2 expression was localized to cardiac and skeletal muscles (Figure 1).
［実施例２］
ＬＳＭＥＭ２強制発現心筋細胞のＲＮＡ－ｓｅｑ
新生仔ラット心筋細胞初代培養
　新生仔ラットから心臓を摘出し、Ｈａｎｋ’ｓ　ｂａｌａｎｃｅｄ　ｓａｌｔ　ｓｏｌｕｔｉｏｎ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，Ｗａｌｔｈａｍ，ＭＡ）内でほぐし、内部の血液を洗浄した。ほぐした心臓を０．２５％Ｔｒｙｐｓｉｎ／ＥＤＴＡ（ＳＩＧＭＡ、Ｓｔ．　Ｌｏｕｉｓ，ＭＯ）に４℃でＯｖｅｒｎｉｇｈｔインキュベートした。翌日、Ｄ－ＭＥＭ（ｉｎ　１０％　ＦＢＳ）を加え、３７℃で５分インキュベートした。上清を捨て、コラゲナーゼを加え１分間手で混合した。上清を捨ててフラスコに移し、コラゲナーゼ（Ｗｏｒｔｈｉｎｇｔｏｎ，ｔｙｐｅ　２）を加え、３７℃を維持した状態で、１５分間、スターラーを用いて撹拌した。３０００ｇ、５分間遠心した後に上清を捨てた後、Ｄ－ＭＥＭを３０ｍｌ加えて懸濁し、Ｐ１０　ｄｉｓｈに播種し、３７℃、７０分間インキュベートした（この過程で心臓線維芽細胞はｄｉｓｈに付着し、培養上清に心筋細胞が分離される）。上清でｄｉｓｈ表面を洗い流して心筋細胞を回収した。続いて３０００ｇ、５分間遠心し、上清を捨て必要に応じた量のＤ－ＭＥＭで懸濁し、一部をとって細胞数をカウントした。最終的に、実験条件に応じた適当な細胞濃度で播種した。 [Example 2]
RNA-seq of LSMEM2 forced expression cardiomyocytes
Neonatal Rat Cardiomyocyte Primary Culture Hearts were removed from neonatal rats, loosened in Hank's balanced salt solution (Thermo Fisher Scientific, Waltham, Mass.), and the blood inside was washed. The dissected hearts were incubated overnight at 4°C in 0.25% Trypsin/EDTA (SIGMA, St. Louis, MO). The next day, D-MEM (in 10% FBS) was added and incubated at 37°C for 5 minutes. The supernatant was discarded, and collagenase was added and mixed by hand for 1 minute. The supernatant was discarded, the mixture was transferred to a flask, collagenase (Worthington, type 2) was added, and the mixture was stirred using a stirrer for 15 minutes while maintaining the temperature at 37°C. After centrifugation at 3,000 g for 5 minutes and discarding the supernatant, 30 ml of D-MEM was added to suspend the cells, seeded on a P10 dish, and incubated at 37°C for 70 minutes (in this process, cardiac fibroblasts adhered to the dish). , cardiomyocytes are isolated in the culture supernatant). Cardiomyocytes were collected by washing the surface of the dish with the supernatant. Subsequently, the cells were centrifuged at 3,000 g for 5 minutes, the supernatant was discarded, the cells were suspended in an appropriate amount of D-MEM, and aliquots were taken to count the number of cells. Finally, cells were seeded at an appropriate cell concentration depending on the experimental conditions.
心筋細胞のＲＮＡ－ｓｅｑ解析
　培養心筋細胞を５．０ｘ１０６ｃｅｌｌ／ｗｅｌｌに調製して６ｃｍ　ｄｉｓｈに播種し、２４時間後にメディウムを交換し、Ｗｉｌｄ　ｔｙｐｅ　ＬＳＭＥＭ２、Ｓ３７Ａ　ＬＳＭＥＭ２及びＭｏｃｋのアデノウイルスベクターを加えた。４８時間後にメディウムをｓｅｒｕｍ　ｆｒｅｅのＤ－ＭＥＭに交換し、１６時間後にＲＮＡ　ｂｅｅを用いて心筋ＲＮＡを回収、精製した。続いて、ＲＮＡ－ｓｅｑのデータ産生自体は大阪大学微生物病研究所に依頼する形で行われたが、概略としては以下のように行われた。まず、それぞれの群のＲＮＡは、ＴｒｕＳｅｑ　Ｓｔｒａｎｄｅｄ　ｍＲＮＡ　Ｌｉｂｒａｒｙ　Ｐｒｅｐ　Ｋｉｔ（Ｉｌｌｕｍｉｎａ，Ｓａｎ　Ｄｉｅｇｏ　ＣＡ）によってＲＮＡライブラリーを調製した。実際には、ｍＲＮＡをｄＴビーズによってｐｏｌｙＡ精製し、Ｒｉｂｏ－ｚｅｒｏビーズによりｒｉｂｏｓｏｍａｌ　ＲＮＡを除去した後、１方向になるようにアダプター配列を付加した。続いて、Ｉｌｌｕｍｉｎａ　のＨｉＳｅｑ　２５００により、７５ｂｐのｓｉｎｇｌｅ　ｒｅａｄで平均１５００万ｒｅａｄをシークエンスすることにより、ＲＮＡ　ｓｅｑのデータを産生した。続いて、得られたｒｅａｄのクオリティを、ＦＡＳＴＱＣによって確認した。つぎに、Ｔｏｐｈａｔ（Ｋｉｍ　ｅｔ　ａｌ．，２０１３）によってｒｅｆｅｒｅｎｃｅ　ｇｅｎｏｍｅであるＮＣＢＩのＲｎｏｒ６にｍａｐｐｉｎｇを行った。ｍａｐｐｉｎｇした各検体の配列をＣｕｆｆｌｉｎｋｓ（Ｂａｒｅｎ　ｅｔ　ａｌ．，２０１０）を使用することによってアセンブリーを行い、更にＣｕｆｆｌｉｎｋｓから作成されたファイルを用いて発現変動遺伝子（ＦＰＲＫ）を検出するために、Ｃｕｆｆｄｉｆｆ（Ｔｒａｐｎｅｌｌ　ｅｔ　ａｌ．，２０１３）を使用した。更に、Ｃｕｆｆｄｉｆｆで作成・抽出された遺伝子群のリストがどのような機能と相互作用するのかを評価するために、ＤＡＶＩＤ（Ｈｕａｎｇ，Ｓｈｅｒｍａｎ，＆　Ｌｅｍｐｉｃｋｉ，２００９ｂ）（Ｈｕａｎｇ，Ｓｈｅｒｍａｎ，＆　Ｌｅｍｐｉｃｋｉ，２００９ａ）を用いてエンリッチメント解析を行った。 RNA-seq analysis of cardiomyocytes Cultured cardiomyocytes were prepared at 5.0x10 6 cells/well and seeded in a 6cm dish.After 24 hours, the medium was changed and wild type LSMEM2, S37A LSMEM2, and Mock adenovirus vectors were added. Ta. After 48 hours, the medium was replaced with serum free D-MEM, and after 16 hours, myocardial RNA was collected and purified using RNA bee. Subsequently, RNA-seq data production itself was carried out at the Osaka University Research Institute for Microbial Diseases, and was generally carried out as follows. First, an RNA library was prepared from each group of RNA using TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego CA). Actually, mRNA was polyA purified using dT beads, ribosomal RNA was removed using Ribo-zero beads, and then an adapter sequence was added in one direction. Subsequently, RNA seq data was generated by sequencing an average of 15 million reads with a single read of 75 bp using Illumina's HiSeq 2500. Subsequently, the quality of the obtained read was confirmed by FASTQC. Next, mapping was performed using Tophat (Kim et al., 2013) to the reference genome Rnor6 of NCBI. The sequence of each mapped sample was assembled using Cufflinks (Baren et al., 2010), and Cuffdiff (Trapnell et al., 2013) was used. Furthermore, in order to evaluate what kind of functions the list of gene groups created and extracted with Cuffdiff interacts with, we used DAVID (Huang, Sherman, & Lempicki, 2009b) (Huang, Sherman, & Lempicki, 2009a). Enrichment analysis was performed using
　その結果、心筋細胞でＬＳＭＥＭ２を強制発現させると、炎症関連の遺伝子発現が上昇することが示された（図２）。 The results showed that when LSMEM2 was forced to be expressed in cardiomyocytes, inflammation-related gene expression increased (Figure 2).
［実施例３］
Ｄｒｏｐｌｅｔ　ｄｉｇｉｔａｌ　ＰＣＲ（ｄｄＰＣＲ）法によるｍＲＮＡの定量
　ｄｄＰＣＲ　は、Ｂｉｏ－Ｒａｄ社の試薬・装置を用いて行った（Ｂｉｏ－Ｒａｄ　Ｌａｂｏｒａｔｏｒｉｅｓ，　Ｈｅｒｃｕｌｅｓ，　ＣＡ）。即ち、調製したサンプルとＤｒｏｐｌｅｔ　ｇｅｎｅｒａｔｏｒ　ｏｉｌをＱＸ２００　Ｄｒｏｐｌｅｔ　Ｇｅｎｅｒａｔｏｒで混合し、Ｄｒｏｐｌｅｔを作製した。作製したＤｒｏｐｌｅｔをサーマルサイクラーでＰＣＲし、ＱＸ２００　Ｄｒｏｐｌｅｔ　Ｒｅａｄｅｒでシグナルを検出した。Ｒａｔ　ＩＬ６及びＲａｔ　Ｔｂｐの発現の検出、Ｈｕｍａｎ　ＩＬ６及びＨｕｍａｎ　Ｔｂｐの発現の検出には以下のプローブを使用した。
Ｉｌ６：ｄＲｎｏＣＰＥ５１８７３４８（ＦＡＭ），Ｔｂｐ：ｄＲｎｏＣＮＳ４９４１８１３４７（ＨＥＸ）
Ｈｕｍａｎ　ＩＬ６：ｄＨｓａＣＰＥ５０３６９１８，Ｈｕｍａｎ　ＴＢＰ：ｄＨｓａＣＰＥ５０５８３６３ [Example 3]
Quantification of mRNA by Droplet Digital PCR (ddPCR) ddPCR was performed using reagents and equipment from Bio-Rad (Bio-Rad Laboratories, Hercules, CA). That is, the prepared sample and Droplet generator oil were mixed using QX200 Droplet Generator to produce a Droplet. The prepared Droplet was subjected to PCR using a thermal cycler, and the signal was detected using QX200 Droplet Reader. The following probes were used to detect the expression of Rat IL6 and Rat Tbp, and the expression of Human IL6 and Human Tbp.
Il6: dRnoCPE5187348 (FAM), Tbp: dRnoCNS494181347 (HEX)
Human IL6: dHsaCPE5036918, Human TBP: dHsaCPE5058363
　ＰＣＲ条件は以下のように行った。 The PCR conditions were as follows.
心筋細胞静水圧負荷
　培養心筋細胞をプレートに播種し、２４時間後に１％ＦＢＳ　ＤＭＥＭ，２５－ｍＭ　ＨＥＰＥＳ－ＮａＯＨ，ｐＨ　７．４（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ），２－ｍＭ　ｌ－ｇｌｕｔａｍｉｎｅ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，Ｗａｌｔｈａｍ，ＭＡ），１％Ｐ／Ｓに培地を交換した。心筋細胞を圧力容器に入れ、３７℃、５％ＣＯ２に調節された加圧空気を供給するコンプレッサーを接続した。更に空気の流量を調節するための空気圧縮コントローラーは、周波数を０から０．２５Ｈｚまで、振幅を０から１ＭＰａまで変化させることが可能であり、今回は１０１～１３５ｋＰａを０．０１Ｈｚの周期で繰り返す静水圧条件にて１２時間曝した。なお、圧力は培地の体積を圧縮することで細胞に加えた。 Cardiomyocyte Hydrostatic Pressure Loading Cultured cardiomyocytes were seeded on plates, and after 24 hours they were treated with 1% FBS DMEM, 25-mM HEPES-NaOH, pH 7.4 (Sigma-Aldrich, St. Louis, MO), 2-mM l- The medium was replaced with glutamine (Thermo Fisher Scientific, Waltham, Mass.), 1% P/S. Cardiomyocytes were placed in a pressure vessel and connected to a compressor supplying pressurized air regulated at 37 °C and 5% CO2 . Furthermore, the air compression controller for adjusting the air flow rate can change the frequency from 0 to 0.25 Hz and the amplitude from 0 to 1 MPa, and this time it repeats 101 to 135 kPa at a frequency of 0.01 Hz. It was exposed for 12 hours under hydrostatic pressure conditions. Note that pressure was applied to the cells by compressing the volume of the medium.
　静水圧を負荷した心筋細胞においてＩＬ６ｍＲＮＡの発現を定量したところ、ＩＬ６の発現が誘導されていないことが示された（図３Ａ；Ｔｂｐ　ｍＲＮＡに対する相対発現として表す）。 Quantification of IL6 mRNA expression in cardiomyocytes subjected to hydrostatic pressure showed that IL6 expression was not induced (Figure 3A; expressed as relative expression to Tbp mRNA).
心筋細胞、Ｈｅｌａ細胞伸展刺激負荷
　卓上型真空プラズマ処理装置ＰＣ－４００Ｔ（ＳＴＲＥＸ，Ｏｓａｋａ，Ｊａｐａｎ）によりストレッチチャンバーＳＴＢ－ＣＨ－０４（ＳＴＲＥＸ，Ｏｓａｋａ，Ｊａｐａｎ）にプラズマ処理を施し、その後にｉＭａｔｒｉｘ－５１１（Ｎｉｐｐｉ，Ｔｏｋｙｏ，Ｊａｐａｎ）を４．８μｇ／ｍＬとなるように調製したラミニン溶液１ｍＬを加え３７℃で一晩インキュベートした。ラミニン溶液を除き、培養心筋細胞を１．３ｘ１０６ｃｅｌｌ／ｃｈａｍｂｅｒに調整した培養心筋細胞をストレッチチャンバー上に播種した。播種から２４時間後ｓｅｒｕｍ　ｆｒｅｅ　ＤＭＥＭに培地を交換し、自動伸展装置ＳＴＢ－１４００（ＳＴＲＥＸ，Ｏｓａｋａ，Ｊａｐａｎ）にセットし、温度３７℃・二酸化炭素５％条件下で０．５Ｈｚ、２０％、２４時間の伸展刺激を与えた。 Cardiomyocytes, Hela cell stretch stimulation load Plasma treatment was performed in a stretch chamber STB-CH-04 (STREX, Osaka, Japan) using a tabletop vacuum plasma treatment device PC-400T (STREX, Osaka, Japan), and then iMatrix-511 (Nippi, Tokyo, Japan) was added to 1 mL of a laminin solution prepared at 4.8 μg/mL and incubated overnight at 37°C. The laminin solution was removed, and the cultured cardiomyocytes were adjusted to 1.3×10 6 cells/chamber and then seeded onto a stretch chamber. 24 hours after seeding, the medium was replaced with serum free DMEM, set in an automatic extension device STB-1400 (STREX, Osaka, Japan), and grown at 0.5 Hz, 20%, and 24°C under conditions of a temperature of 37°C and 5% carbon dioxide. A time stretching stimulus was applied.
　伸展刺激を負荷した心筋細胞においてＩＬ６ｍＲＮＡを定量したところ、ＩＬ６の発現が誘導されることが示された（図３Ｂ）。 Quantification of IL6 mRNA in cardiomyocytes loaded with stretch stimulation showed that IL6 expression was induced (Figure 3B).
ｓｉＲＮＡを用いた新生仔ラット心筋細胞でのＬＳＭＥＭ２ノックダウン
　ＬＳＭＥＭ２に対する　ｓｉＲＮＡには、Ｓｉｌｅｎｃｅ（登録商標）ｒＳｅｌｅｃｔ　ｓｉＲＮＡ（ＬＳＭＥＭ２　ｓｉＲＮＡ　ＩＤ：ｓ１８９３２７，ｓｅｎｓｅ（５’→３’）：ＧＣＡＣＡＧＵＧＧＡＧＧＣＵＣＡＣＵＡＴＴ（配列番号９）
ａｎｔｉｓｅｎｓｅ（５’→３’）：ＵＡＧＵＧＡＧＣＣＵＣＣＡＣＵＧＵＧＣＡＧ（配列番号１０）
（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，Ｗａｌｔｈａｍ，ＭＡ）を用いた。最終濃度が５ｎＭとなるように調製し、ＬｉｐｏｆｅｃｔａｍｉｎｅＴＭ　ＲＮＡｉＭＡＸ　Ｔｒａｎｓｆｅｃｔｉｏｎ　Ｒｅａｇｅｎｔ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ，Ｗａｌｔｈａｍ，ＭＡ）を用いて播種してから３時間後の新生仔ラット心筋細胞に導入した。 LSMEM2 knockdown in neonatal rat cardiomyocytes using siRNA The siRNA against LSMEM2 included Silence® rSelect siRNA (LSMEM2 siRNA ID: s189327, sense (5'→3'): GCACAGUGGAGGCUCACUATT (Sequence number 9)
antisense (5'→3'): UAGUGAGCCCUCCACUGUGCAG (SEQ ID NO: 10)
(Thermo Fisher Scientific, Waltham, MA) was used. The final concentration was adjusted to 5 nM and introduced into neonatal rat cardiomyocytes 3 hours after seeding using Lipofectamine ™ RNAiMAX Transfection Reagent (Thermo Fisher Scientific, Waltham, MA).
　伸展刺激を負荷した、コントロールｓｉＲＮＡを導入したラット心筋細胞（ｓｉＣｔｒｌ）及びｓｉＲＮＡによりＬＳＭＥＭ２をノックダウンしたラット心筋細胞（ｓｉＡＩＰＩＤ）においてＩＬ６　ｍＲＮＡを定量した。コントロールｓｉＲＮＡではＩＬ６の発現が誘導されなかったが、ＬＳＭＥＭ２に対するｓｉＲＮＡでは誘導されることが示された（Ｔｂｐ　ｍＲＮＡに対する相対発現として表す；図３Ｃ）。 IL6 mRNA was quantified in rat cardiomyocytes introduced with control siRNA (siCtrl) and rat cardiomyocytes in which LSMEM2 was knocked down by siRNA (siAIPID), which were subjected to stretch stimulation. IL6 expression was not induced by control siRNA, but was shown to be induced by siRNA against LSMEM2 (expressed as relative expression to Tbp mRNA; Fig. 3C).
［実施例４］
Ｌｓｍｅｍ２ノックアウトマウスの作製
　ノックアウトマウスの作製は大阪大学動物実験施設生殖工学ユニットに依頼した。実際には、ＣＲＳＰＲ／Ｃａｓ９システムを用いてＬｓｍｅｍ２ノックアウトマウスを作製した。Ｇｕｉｄｅ　ＲＮＡはエクソン２上に設計した（Ｇｕｉｄｅ　ＲＮＡ配列：ＡＣＴＧＧＣＴＣＣＴＡＡＣＣＡＣＧＴＧＣＡＧＧ）（配列番号５）（図４Ａ）。Ｃａｓ９及びｔａｒｇｅｔとなるＧｕｉｄｅ　ＲＮＡを同時に発現することが可能なｐＸ３３０（Ｗｙｍａｎ　ｅｔ　ａｌ．，２０１３）に、設計したＧｕｉｄｅ　ＲＮＡを組み込んだベクターを作製し、マウスの受精卵にインジェクションし、その受精卵を偽妊娠マウスに移植した。得られたＦ０マウスをＳａｎｇｅｒシークエンスにてＬＳＭＥＭ２遺伝子領域のｉｎ－ｄｅｌをチェックし、１４塩基の欠損をもつマウスをライン化し、Ｃ５７ＢＬ６Ｊバックグラウンドで継代維持した。マウス心臓組織を用いた通常のウェスタンブロット法によりＬＳＭＥＭ２を検出し、ノックアウトマウスでＬＳＭＥＭ２タンパク質が消失していること、即ちＬＳＭＥＭ２ノックアウトマウスができていることを確認した（図４Ｂ）。 [Example 4]
Production of Lsmem2 knockout mouse Production of knockout mice was requested to the Osaka University Animal Experiment Facility Reproductive Engineering Unit. Actually, Lsmem2 knockout mice were generated using the CRSPR/Cas9 system. Guide RNA was designed on exon 2 (Guide RNA sequence: ACTGGCTCCTAACCACGTGCAGG) (SEQ ID NO: 5) (FIG. 4A). A vector was created in which the designed Guide RNA was integrated into pX330 (Wyman et al., 2013), which can simultaneously express Cas9 and the target Guide RNA, and the vector was injected into a mouse fertilized egg. transplanted into pseudopregnant mice. The obtained F0 mice were checked for in-dels in the LSMEM2 gene region using Sanger sequencing, and mice with a 14 base deletion were lined up and maintained in a C57BL6J background. LSMEM2 was detected by conventional Western blotting using mouse heart tissue, and it was confirmed that the LSMEM2 protein was lost in the knockout mice, that is, LSMEM2 knockout mice were formed (FIG. 4B).
マウス心臓組織凍結切片の作製
　マウス心臓を摘出し、ＰＢＳで洗浄した後、Ｏ．Ｃ．Ｔ　Ｃｏｍｐｏｕｎｄを用いて包埋し、５分静置した後、液体窒素で冷却したイソペンタンの中で、心臓を凍結し、その後、クライオスタット（Ｌｅｉｃａ，Ｗｅｎｔｚｌｅｒ，Ｇｅｒｍａｎｙ）によって包埋した心臓をサンプルの厚さが８μｍになるように薄切し、プレパラートに貼付した後、風乾させ、－８０℃で保存した。 Preparation of frozen section of mouse heart tissue The mouse heart was removed, washed with PBS, and then washed with O. C. After embedding with T Compound and allowing it to stand for 5 minutes, the heart was frozen in isopentane cooled with liquid nitrogen, and then the embedded heart was adjusted to the thickness of the sample using a cryostat (Leica, Wentzler, Germany). It was sliced to a thickness of 8 μm, pasted on a slide, air-dried, and stored at -80°C.
マウス組織凍結切片を用いた免疫染色
　作製した凍結切片をアセトンで４℃、２０分間固定し、乾燥させた後、ＰＢＳで洗浄した。続いて、組織検体をブロッキングするために、ＰＢＳで洗浄し、５％ｇｏａｔ　ｓｅｒｕｍ／ＰＢＳを加え、室温で３０分静置した。内在するＩｇＧのブロッキングを行うために、ａｎｔｉ－ｍｏｕｓｅ　ＩｇＧ　Ｆａｂ　ｆｒａｇｍｅｎｔ（０．１ｍｇ／ｍｌ　ｉｎ　ＰＢＳ）を加え室温で１時間静置した。５％ｇｏａｔ　ｓｅｒｕｍ／ＰＢＳで２０００倍に希釈したＬＳＭＥＭ２モノクローナル抗体を加え、湿潤ボックスに入れ、４℃　ｏｖｅｒ　ｎｉｇｈｔで静置した。その後、二次抗体反応を行うために、検体をＰＢＳで洗浄した後、二次抗体を５００倍に希釈し、細胞核を染色するためのＨｏｅｃｈｓｔ３３３４２を加え、室温で４５分静置し、蛍光顕微鏡で観察した。結果として、ＬＳＭＥＭ２ノックアウトマウスで当該タンパク質が消失していることを確認した（図４Ｂ）。 Immunostaining using mouse tissue frozen sections The prepared frozen sections were fixed with acetone at 4°C for 20 minutes, dried, and then washed with PBS. Subsequently, in order to block the tissue specimen, it was washed with PBS, 5% goat serum/PBS was added, and it was allowed to stand at room temperature for 30 minutes. To block endogenous IgG, anti-mouse IgG Fab fragment (0.1 mg/ml in PBS) was added and allowed to stand at room temperature for 1 hour. LSMEM2 monoclonal antibody diluted 2000 times with 5% goat serum/PBS was added, placed in a humid box, and left standing at 4°C over night. After that, in order to perform a secondary antibody reaction, the specimen was washed with PBS, the secondary antibody was diluted 500 times, Hoechst 33342 for staining cell nuclei was added, left to stand at room temperature for 45 minutes, and then examined using a fluorescence microscope. Observed. As a result, it was confirmed that the protein was lost in LSMEM2 knockout mice (FIG. 4B).
　ヘマトキシリンエオジン染色によるマウス心臓組織切片の形態学的観察では、ＬＳＭＥＭ２ノックアウトマウスに明らかなフェノタイプは認めなかった（図４Ｃ）。 Morphological observation of mouse heart tissue sections using hematoxylin and eosin staining revealed no obvious phenotype in LSMEM2 knockout mice (Figure 4C).
［実施例５］
ラミノパチーモデルマウスとの交配実験
　ラミノパチーモデルマウスとしてＬＭＮＡ　Ｈ２２２Ｐノックイン（ＫＩ）マウス（Ｈｕｍ　Ｍｏｌ　Ｇｅｎｅｔ．２００５；１４（１）：１５５）を使用した。ＬＭＮＡ　Ｈ２２２Ｐ　ＫＩマウスとＬＳＭＥＭ２　ＫＯマウスを交配することで、ＬＭＮＡ　Ｈ２２２Ｐ　ＫＩ／ＫＩ，ＬＳＭＥＭ２－／－マウスを作製した。ＬＭＮＡ　Ｈ２２２Ｐ　ＫＩ／ＫＩ，ＬＳＭＥＭ２＋／＋マウスとＬＭＮＡ　Ｈ２２２Ｐ　ＫＩ／ＫＩ、ＬＳＭＥＭ２-／-マウスの生存期間の解析、心機能解析並びに下肢筋力測定を行った。心機能解析はＶｅｖｏ　３１００イメージングシステムを用いて３、６、９ヶ月齢で行い、ＬＶＥＦ（左室駆出率）を評価した。用いた個体数は図に記載している通りである。下肢筋力測定は７ヶ月齢で行った。下肢筋力測定は１３００Ａ　３－ｉｎ－１　Ｗｈｏｌｅ　Ａｎｉｍａｌ　Ｍｕｓｃｌｅ　Ｓｙｓｔｅｍ（Ａｕｒｏｒａ　Ｓｃｉｅｎｔｉｆｉｃ）を用いて２９週齢で行い、腓腹筋のＴｗｉｔｃｈ　ｆｏｒｃｅ，Ｔｅｔａｎｉｃ　ｆｏｒｃｅ，Ｆｏｒｃｅ－ｆｒｅｑｕｅｎｃｙを評価した。解析はＤＭＡソフトウェア（Ａｕｒｏｒａ　Ｓｃｉｅｎｔｉｆｉｃ）を用いた。２群の比較はｔ検定で統計学的検討を行った。 [Example 5]
Crossing experiment with laminopathy model mouse LMNA H222P knock-in (KI) mouse (Hum Mol Genet. 2005; 14 (1): 155) was used as a laminopathy model mouse. LMNA H222P KI/KI, LSMEM2−/− mice were generated by crossing LMNA H222P KI mice and LSMEM2 KO mice. Survival period analysis, cardiac function analysis, and lower limb muscle strength measurements were performed on LMNA H222P KI/KI, LSMEM2+/+ mice and LMNA H222P KI/KI, LSMEM2-/- mice. Cardiac function analysis was performed at 3, 6, and 9 months of age using a Vevo 3100 imaging system, and LVEF (left ventricular ejection fraction) was evaluated. The number of individuals used is as shown in the figure. Lower limb muscle strength measurements were performed at 7 months of age. Lower limb muscle strength measurements were performed at 29 weeks of age using a 1300A 3-in-1 Whole Animal Muscle System (Aurora Scientific). was evaluated. The analysis used DMA software (Aurora Scientific). Comparisons between the two groups were statistically examined using a t-test.
　その結果、ＬＳＭＥＭ２　ＫＯマウスはラミノパチーモデル（ＬＭＮＡ　Ｈ２２２Ｐ　ＫＩ／ＫＩ）の生存率、心機能、、心臓炎症シグナル及び骨格筋力をいずれも改善することも示された（図５）。 The results showed that LSMEM2 KO mice improved survival rate, cardiac function, cardiac inflammatory signals, and skeletal muscle strength in a laminopathy model (LMNA H222P KI/KI) (Figure 5).
［実施例６］
ＡＡＶベクターの作製
　ＨＥＫ２９３Ｔ細胞にトランスファープラスミド、Ｒｅｐ－ｃａｐプラスミド、ヘルパープラスミドをコトランスフェクトすることによって、ＭｙｏＡＡＶ２ベクター（Ｔａｂｅｂｏｒｄｂａｒ　Ｍ．　ｅｔ　ａｌ．　Ｃｅｌｌ　２０２１）を作製し、塩化セシウム密度勾配超遠心分離によってウイルス粒子の濃縮並びに精製を行った。骨格筋細胞特異的なＬＳＭＥＭ２のノックダウンを行うために、ＭｙｏＡＡＶ２　Ｕ６－ｓｈＲＮＡ（ＬＳＭＥＭ２）を作製した。ｓｈＲＮＡはマウスＬＳＭＥＭ２を標的としたｓｈＬＳＭＥＭ２（５’－ＣＴＣＴＣＡＣＴＧＡＴＧＧＣＴＴＣＡＴＴＴ－３’（配列番号１１）を設計した。コントロールとしてマウスの遺伝子を標的としないｓｈＣｏｎｔｒｏｌ（５’－ＣＣＴＡＡＧＧＴＴＡＡＧＴＣＧＣＣＣＴＣＧ－３’（配列番号１２））を設計した。 [Example 6]
Production of AAV vector MyoAAV2 vector (Tabebordbar M. et al. Cell 2021) was produced by co-transfecting HEK293T cells with the transfer plasmid, Rep-cap plasmid, and helper plasmid, and the virus was isolated by cesium chloride density gradient ultracentrifugation. The particles were concentrated and purified. In order to perform skeletal muscle cell-specific knockdown of LSMEM2, MyoAAV2 U6-shRNA (LSMEM2) was produced. The shRNA was designed as shLSMEM2 (5'-CTCTCACTGATGGCTTCATTT-3' (SEQ ID NO: 11)), which targets mouse LSMEM2. As a control, shControl (5'-CCTAAGGTTAAGTCGCCCTCG-3' (SEQ ID NO: 12)), which does not target mouse genes, was designed. designed.
ＡＡＶ投与実験
　ＭｙｏＡＡＶ２　Ｕ６－ｓｈＲＮＡ（ＬＳＭＥＭ２）（以下、ｓｈＬＳＭＥＭ２とも称することがある）とＭｙｏＡＡＶ２　ｓｈＣｏｎｔｒｏｌを７×１０１１ｇｅｎｏｍｅ　ｃｏｐｙ（ＧＣ）を１０週齢のＬＭＮＡ　ＫＩマウスの眼窩静脈叢より静注し、心機能を評価した。 AAV administration experiment MyoAAV2 U6-shRNA (LSMEM2) (hereinafter also referred to as shLSMEM2) and MyoAAV2 shControl at 7×10 11 genome copies (GC) were intravenously injected into the orbital venous plexus of 10-week-old LMNA KI mice. Cardiac function was evaluated.
　その結果、ＭｙｏＡＡＶ２　ｓｈＣｏｎｔｒｏｌ投与群では投与後８週、１６週、および２４週の時点で経時的な心機能の低下がみられたが、ｓｈＬＳＭＥＭ２投与群ではこの心機能低下が抑制された（図６、Ｃａｒｄｉａｃ　Ｆｕｎｃｔｉｏｎ）。また、投与２４週の時点における骨格筋機能において、ｓｈＬＳＭＥＭ２投与群では単収縮力（Ｔｗｉｔｃｈ　Ｆｏｒｃｅ）の向上が観察された（図６、Ｓｋｅｌｅｔａｌ　ｍｕｓｃｌｅ　Ｆｕｎｃｔｉｏｎ　ａｔ　ｐｏｓｔ　２４　Ｗ）。即ち後天的なＬＳＭＥＭ２発現抑制が、ラミノパチーモデルにおいて治療効果をもつことが示された。 As a result, in the MyoAAV2 shControl administration group, a decline in cardiac function over time was observed at 8 weeks, 16 weeks, and 24 weeks after administration, but this decline in cardiac function was suppressed in the shLSMEM2 administration group (Figure 6 , Cardiac Function). Furthermore, regarding skeletal muscle function at 24 weeks of administration, an improvement in twitch force was observed in the shLSMEM2 administration group (Figure 6, Skeletal muscle function at post 24W). That is, acquired suppression of LSMEM2 expression was shown to have a therapeutic effect in a laminopathy model.
［実施例７］
マウス大動脈弓縮窄モデル
　野生型マウス及びＬＳＭＥＭ２ノックアウトマウスの大動脈弓を結紮し、左室内に高圧負荷をかけたモデルを作製した。本モデルは循環器領域の研究で使用される圧負荷心不全モデルとして非常に一般的である。術後１週間の心臓組織を用いた検討と、術後１、４週と経時的な心エコー観察による心機能の評価を行った。 [Example 7]
Mouse aortic arch coarctation model A model was created in which the aortic arches of wild-type mice and LSMEM2 knockout mice were ligated and a high pressure load was applied to the left ventricle. This model is a very common pressure overload heart failure model used in cardiovascular research. Cardiac function was evaluated using cardiac tissue 1 week after surgery and by echocardiographic observation over time at 1 and 4 weeks after surgery.
　術後１週の心臓組織を摘出し、図４と同様の方法でマウス白血球の表面マーカーであるＣＤ４５抗体を用いて免疫染色を行い、心筋組織内への炎症細胞浸潤を検討した。野生型マウスでは術後１週間で顕著な心筋内炎症細胞浸潤が認められたが、ＬＳＭＥＭ２ノックアウトマウスではそれが有意に抑えられた（図７Ａ）。また、術後１週間の心臓組織を用いて図２と同様の方法によりＲＮＡ－ｓｅｑ解析を行った。野生型マウスでは、ＣＤ４５染色の結果と合致するように術後１週間で炎症関連の遺伝子群の有意な発現上昇が認められたが、ＬＳＭＥＭ２ノックアウトマウスではそれらの発現上昇が有意に抑えられた（図７Ｂ）。術後１、４週と心エコー観察による心機能の評価を行ったところ、野生型マウスでは経時的な心機能の低下がみられたが、ＬＳＭＥＭ２ノックアウトマウスではこの心機能低下が抑制された（図７Ｃ）。 Heart tissue was removed one week after surgery, and immunostained using CD45 antibody, a surface marker for mouse leukocytes, in the same manner as in Figure 4 to examine inflammatory cell infiltration into the myocardial tissue. In wild-type mice, significant intramyocardial inflammatory cell infiltration was observed one week after surgery, but this was significantly suppressed in LSMEM2 knockout mice (FIG. 7A). In addition, RNA-seq analysis was performed using heart tissue 1 week after surgery in the same manner as in FIG. 2. In wild-type mice, a significant increase in expression of inflammation-related genes was observed one week after surgery, consistent with the results of CD45 staining, but in LSMEM2 knockout mice, these increases in expression were significantly suppressed ( Figure 7B). When cardiac function was evaluated by echocardiography at 1 and 4 weeks after surgery, it was found that in wild-type mice there was a decline in cardiac function over time, but in LSMEM2 knockout mice, this decline in cardiac function was suppressed ( Figure 7C).
　本発明によれば、ＬＳＭＥＭ２の発現又は機能を抑制する物質を含むメカノシグナル伝達阻害剤が提供できるので、ラミン心筋症、他の病因による重症心不全及びラミノパチーに対する有効な治療が可能となる。 According to the present invention, it is possible to provide a mechanosignaling inhibitor containing a substance that suppresses the expression or function of LSMEM2, thus making it possible to effectively treat lamin cardiomyopathy, severe heart failure due to other causes, and laminopathy.
　本出願は、日本で出願された特願２０２２－１３６２７９（出願日：２０２２年８月２９日）を基礎としており、その内容は本明細書に全て包含されるものである。 This application is based on Japanese Patent Application No. 2022-136279 (filing date: August 29, 2022) filed in Japan, the contents of which are fully included in this specification.

Claims (9)

  1. 　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、メカノシグナル伝達阻害剤。 A mechanosignaling inhibitor containing a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
  2. 　ＬＳＭＥＭ２の発現を抑制する物質が、
    （ａ）ＬＳＭＥＭ２遺伝子の転写産物に対してＲＮＡｉ活性を有する核酸若しくはその前駆体、
    （ｂ）ＬＳＭＥＭ２遺伝子の転写産物に対するアンチセンス核酸、
    （ｃ）ＬＳＭＥＭ２遺伝子の転写産物に対するリボザイム核酸、又は
    （ｄ）ＬＳＭＥＭ２遺伝子中の標的ヌクレオチド配列に特異的に結合する核酸配列認識モジュール
    である、請求項１に記載の剤。     A substance that suppresses the expression of LSMEM2 is
    (a) A nucleic acid or its precursor having RNAi activity against the transcription product of the LSMEM2 gene,
    (b) antisense nucleic acid against the transcript of the LSMEM2 gene,
    The agent according to claim 1, which is (c) a ribozyme nucleic acid directed against the transcript of the LSMEM2 gene, or (d) a nucleic acid sequence recognition module that specifically binds to a target nucleotide sequence in the LSMEM2 gene.
  3. 　ＬＳＭＥＭ２の機能を抑制する物質が、ＬＳＭＥＭ２に対する抗体、デグレーダー又はアプタマーである、請求項１に記載の剤。 The agent according to claim 1, wherein the substance that suppresses the function of LSMEM2 is an antibody, degrader, or aptamer against LSMEM2.
  4. 　前記物質が、それをコードする１以上の発現ベクターの形態で提供される、請求項１～３のいずれか１項に記載の剤。 The agent according to any one of claims 1 to 3, wherein the substance is provided in the form of one or more expression vectors encoding it.
  5. 　抗炎症剤である、請求項１に記載の剤。 The agent according to claim 1, which is an anti-inflammatory agent.
  6. 　心疾患の予防及び／又は治療剤である、請求項１に記載の剤。 The agent according to claim 1, which is a preventive and/or therapeutic agent for heart disease.
  7. 　心疾患が拡張型心筋症である、請求項６に記載の剤。 The agent according to claim 6, wherein the heart disease is dilated cardiomyopathy.
  8. 　ＬＳＭＥＭ２の発現又は機能を抑制する物質を有効成分として含む、ラミノパチーの予防及び／又は治療剤。 A prophylactic and/or therapeutic agent for laminopathy, which contains a substance that suppresses the expression or function of LSMEM2 as an active ingredient.
  9. 　ラミノパチーが筋ジストロフィー、ラミン心筋症、リポジストロフィー、又は白質ジストロフィーから選択される、請求項８に記載の剤。 The agent according to claim 8, wherein the laminopathy is selected from muscular dystrophy, lamin cardiomyopathy, lipodystrophy, or leukodystrophy.
PCT/JP2023/031020 2022-08-29 2023-08-28 Prophylactic agent and/or therapeutic agent for heart diseases and laminopathy WO2024048528A1 (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010529966A (en) * 2007-06-08 2010-09-02 アシュラジェン インコーポレイテッド Genes and pathways regulated by miR-34 as targets for therapeutic intervention
WO2016209876A1 (en) * 2015-06-25 2016-12-29 The Medical College Of Wisconsin, Inc. Cardiomyocyte-specific biological markers and uses thereof
US20220196661A1 (en) * 2020-12-18 2022-06-23 Rebekah L. Gundry Cardiomyocyte-Specific Biological Markers and Uses Thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2010529966A (en) * 2007-06-08 2010-09-02 アシュラジェン インコーポレイテッド Genes and pathways regulated by miR-34 as targets for therapeutic intervention
WO2016209876A1 (en) * 2015-06-25 2016-12-29 The Medical College Of Wisconsin, Inc. Cardiomyocyte-specific biological markers and uses thereof
US20220196661A1 (en) * 2020-12-18 2022-06-23 Rebekah L. Gundry Cardiomyocyte-Specific Biological Markers and Uses Thereof

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