WO2013154128A1 - Nouvelle composition médicamenteuse pour le traitement de l'artériosclérose et méthode de criblage de médicaments pour le traitement de l'artériosclérose - Google Patents

Nouvelle composition médicamenteuse pour le traitement de l'artériosclérose et méthode de criblage de médicaments pour le traitement de l'artériosclérose Download PDF

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WO2013154128A1
WO2013154128A1 PCT/JP2013/060798 JP2013060798W WO2013154128A1 WO 2013154128 A1 WO2013154128 A1 WO 2013154128A1 JP 2013060798 W JP2013060798 W JP 2013060798W WO 2013154128 A1 WO2013154128 A1 WO 2013154128A1
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gene
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protein
amino acid
genbank
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陽子 山口
六嶋　正知
薫紀 山本
伊藤　剛
和彦 前川
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塩野義製薬株式会社
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    • 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
    • A61K31/7105—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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/113—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides

Definitions

  • the present invention relates to a pharmaceutical composition for the treatment of dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer's containing a substance that suppresses the expression of a specific gene.
  • the present invention includes CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, SGP2, MT , OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596, a substance that suppresses the expression of the gene or a substance that suppresses the activity of the protein encoded by these genes, dyslipidemia, hyperlipidemia, arteriosclerosis Therapeutic pharmaceutical composition, as well as CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, Z
  • LDL low-density lipoprotein
  • HDL high-density lipoprotein
  • a cholesterol reverse transfer system known as a biological mechanism for recovering excess cholesterol in cells to the liver and maintaining normal cholesterol levels in the living body. Plays an important role. HDL plays a central role in the reverse cholesterol transport system, and it is now widely recognized that HDL is one of the defense factors against arteriosclerosis. That is, since a drug that enhances HDL function is expected to be extremely useful clinically as a therapeutic agent for arteriosclerotic diseases, research for searching for substances that increase plasma HDL levels is being conducted.
  • Lipoproteins such as HDL are generally composed of lipids and protein components called apolipoproteins.
  • apolipoproteins called apolipoprotein A1 (hereinafter referred to as “ApoA1”) are the main components. Therefore, as one of the most effective methods for exploring substances that increase plasma HDL levels, it is assumed that the concentration of ApoA1 in the blood, which is the main component of HDL, is increased. It has been clarified that there is a direct correlation between mRNA levels in the liver and blood ApoA1 protein and HDL levels (Non-patent Document 1).
  • RNA oligonucleotide strands of about 21 bases complementary to a desired gene (mRNA) and double-stranded RNA (small RNA interfering RNA) (siRNA) consisting of the antisense RNA oligonucleotide strand have been developed, and can be arbitrarily used in mammalian cells. It has become possible to suppress the expression of these genes (Non-patent Document 6).
  • mRNA desired gene
  • siRNA small RNA interfering RNA
  • siRNA and antisense oligo technologies are actively used in life science research, but there are no disease treatment siRNA or antisense oligos sold as pharmaceuticals.
  • the present inventors significantly increase the secretion of ApoA1 from the human liver-derived cell line by suppressing gene expression by introducing a siRNA library covering all human genes into the human liver-derived cell line.
  • 26 genes CNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK10, SLA2, CFT1, T1139 , UBE2NL, FLJ16734, and FLJ38596 genes), thereby completing the present invention.
  • the present invention provides the following [1] to [14].
  • [1] CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, TLA1, FTMT, ECR9C
  • An agent for increasing ApoA1 secretion comprising a substance that suppresses the expression of the FLJ16734 or FLJ38596 gene or suppresses the activity of the protein encoded by these genes.
  • nucleic acid that suppresses gene expression is siRNA, an antisense oligonucleotide, or an expression vector thereof.
  • [7] (1) A step of bringing a cell into contact with a test substance, (2) CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, EPPK1, EPPK1, EPPK1, EPPK1, Measuring the expression level of the FTMT, GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596 gene, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; and (3) a test substance Selecting the test substance as a substance that increases the secretion of ApoA
  • [8] (1) A step of contacting a cell with a test substance, (2) CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, EPPK1, EPPK1, EPPK1, Measuring the expression level of the FTMT, GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596 gene, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; and (3) a test substance When the expression of the gene in the cell given the test substance is lower than the expression of the gene in the cell not given the test substance, the test substance is treated with dyslipidemia, hyperlipidemia, arteriosclerosis.
  • a method for screening a therapeutic substance for dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer's disease comprising a step of selecting as a therapeutic substance for Alzheimer's disease.
  • a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or (2) A polypeptide having an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1 have been deleted, substituted, and / or added and having an activity of binding to an agonist for GPR139 protein
  • a method for screening an ApoA1 secretagogue which is characterized by using.
  • Contacting a polypeptide having an amino acid sequence in which amino acids are deleted, substituted, and / or added and having GPR139 activity (2) comparing the binding activity of the agonist and the polypeptide contacted with the test substance with the binding activity of the agonist and the polypeptide not contacted with the test substance; and (3) Increase the secretion of ApoA1 in the test substance when the binding activity of the agonist and polypeptide that contacted the test substance is changed compared to the binding activity of the agonist and polypeptide that does not contact the test substance
  • a screening method for an ApoA1 secretion increasing substance comprising a step of selecting as a substance.
  • [11] (1) an agonist for GPR139 protein, a test substance, and (i) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or (ii) one or several amino acid sequences represented by SEQ ID NO: 1 Contacting a cell expressing a polypeptide having an amino acid sequence in which an amino acid is deleted, substituted, and / or added and having GPR139 activity; (2) a step of comparing the intracellular cAMP amount or calcium ion concentration in the cell contacted with the test substance with the intracellular cAMP amount or calcium ion concentration in the control cell not contacted with the test substance, and (3) adding the test substance When the intracellular cAMP level or calcium ion concentration in a given cell is changed compared to the intracellular cAMP level or calcium ion concentration in a cell not given the test substance, the test substance increases the secretion of ApoA1.
  • a method for screening for an ApoA1 secretion increasing substance comprising a step of selecting as a substance to be caused.
  • a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or (2) A polypeptide having an amino acid sequence in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1 have been deleted, substituted, and / or added and having GPR139 activity is used. Screening method for therapeutic substances for dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer's disease.
  • an agonist for GPR139 protein a test substance, and (i) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or (ii) one or several amino acid sequences represented by SEQ ID NO: 1
  • Contacting a polypeptide having an amino acid sequence in which amino acids are deleted, substituted, and / or added and having GPR139 activity (2) comparing the binding activity of the agonist and the polypeptide contacted with the test substance with the binding activity of the agonist and the polypeptide not contacted with the test substance; and (3) When the binding activity between the agonist and polypeptide that is contacted with the test substance is changed compared to the binding activity between the agonist and polypeptide that is not contacted with the test substance, the test substance is treated with dyslipidemia, high
  • a method for screening a therapeutic substance for dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer's disease comprising a step of selecting as a therapeutic substance for treating lipemia, arteriosclerosis or Alzheimer's.
  • [14] (1) an agonist for GPR139 protein, a test substance, and (i) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or (ii) one or several amino acid sequences represented by SEQ ID NO: 1 Contacting a cell having an amino acid sequence in which an amino acid is deleted, substituted, and / or added, and expressing a polypeptide having GPR139 activity; (2) a step of comparing the intracellular cAMP amount or calcium ion concentration in the cell contacted with the test substance with the intracellular cAMP amount or calcium ion concentration in the control cell not contacted with the test substance, and (3) adding the test substance When the intracellular cAMP amount or calcium ion concentration in the obtained cells is changed as compared with the intracellular cAMP amount or calcium ion concentration in cells not given the test substance, the test substance is treated with dyslipidemia, A screening method for a therapeutic substance for dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer's disease, comprising a step of selecting as a
  • UBE2NL, FLJ16734 or FLJ38596 gene By suppressing the expression of UBE2NL, FLJ16734 or FLJ38596 gene, it is possible to enhance the secretion of ApoA1, and substances that inhibit the expression of these genes are dyslipidemia, hyperlipidemia, arteriosclerosis or It can be used as an active ingredient of a pharmaceutical composition for treating Alzheimer's.
  • the pharmaceutical composition according to the present invention provides a therapeutic agent having a new mechanism of action that specifically suppresses the expression of a specific gene, and includes conventional lipid metabolism disorders, hyperlipidemia, arteriosclerosis or It is characterized by low toxicity compared to Alzheimer's therapeutic agents.
  • a method of screening for a therapeutic substance for dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer's using the UBE2NL, FLJ16734 or FLJ38596 gene as a target.
  • the present invention in one aspect, CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, SGP2, FT , OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596
  • the present invention provides a pharmaceutical composition comprising a substance that suppresses the expression of a gene or suppresses the activity of a protein encoded by these genes.
  • Such pharmaceutical compositions are suitable for the treatment of, for example, dyslipidemia, hyperlipidemia, arteriosclerosis, atherosclerosis, hypercholesterolemia, hypertriglyceridemia, diabetes, obesity, syndrome X and / or Alzheimer. Can be used.
  • CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, LA2S , FTMT, GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 and FLJ38596 were found as novel target molecules for dyslipidemia, hyperlipidemia, arteriosclerosis and Alzheimer.
  • CTNNB1 gene means a gene encoding CTNNB1 protein.
  • the nucleotide sequence of the human CTNNB1 gene and the amino acid sequence of the human CTNNB1 protein are known.
  • the nucleotide sequence of the human CTNNB1 gene and the amino acid sequence of the human CTNNB1 protein are registered in GenBank (GenBank Accession No. NM_001904) and published.
  • the human CTNNB1 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of an amino acid sequence registered in the above-mentioned GenBank.
  • the human CTNNB1 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “CTNNB1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • CTNNB1 protein is also called ⁇ -catenin and is an intracellular molecule having two functions.
  • the first function is to bind to the intracellular domain of the intercellular adhesion molecule cadherin and link cadherin to the cytoskeleton (The Journal of Biological Chemistry, 270, 34, 20201-20206, 1995).
  • the other is a function of binding to a transcription factor in the nucleus as a Wnt signal transduction molecule and activating transcription of its target gene (Molecular and Cellular Biology, Vol. 18, No. 5, pp. 2474-2485, 1998). Year).
  • the relationship between the CTNNB1 gene and protein and ApoA1, arteriosclerosis, etc. is not known.
  • NFE2L2 gene means a gene encoding NFE2L2 protein.
  • the nucleotide sequence of the human NFE2L2 gene and the amino acid sequence of the human NFE2L2 protein are known.
  • the nucleotide sequence of the human NFE2L2 gene and the amino acid sequence of the human NFE2L2 protein are registered in GenBank (GenBank Accession No. NM_006164) and published.
  • the human NFE2L2 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • the human NFE2L2 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “NFE2L2 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% of the nucleotide sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “TXN gene” means a gene encoding a TXN protein.
  • the base sequence of the human TXN gene and the amino acid sequence of the human TXN protein are known.
  • the base sequence of the human TXN gene and the human TXN protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_003329) and published.
  • the human TXN protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human TXN proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human TXN gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “TXN gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • CUL1 gene means a gene encoding CUL1 protein.
  • the base sequence of the human CUL1 gene and the amino acid sequence of the human CUL1 protein are known.
  • the base sequence of the human CUL1 gene and the human CUL1 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_003592) and published.
  • the human CUL1 protein includes not only a protein consisting of the amino acid sequence registered in the above GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, and are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human CUL1 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human CUL1 gene includes not only a gene consisting of a base sequence registered in the above GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “CUL1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • RAE1 gene means a gene encoding RAE1 protein.
  • the nucleotide sequence of the human RAE1 gene and the amino acid sequence of the human RAE1 protein are known.
  • the nucleotide sequence of the human RAE1 gene and the amino acid sequence of the human RAE1 protein are registered in GenBank (GenBank Accession No. NM_001015885) and published.
  • the human RAE1 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • the human RAE1 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “RAE1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • TNFRSF10C gene means a gene encoding TNFRSF10C protein.
  • the nucleotide sequence of the human TNFRSF10C gene and the amino acid sequence of the human TNFRSF10C protein are known.
  • the nucleotide sequence of the human TNFRSF10C gene and the human TNFRSF10C protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_003841) and published.
  • the human TNFRSF10C protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • the human TNFRSF10C gene includes not only a gene consisting of the base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “TNFRSF10C gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “BCLAF1 gene” means a gene encoding a BCLAF1 protein.
  • the base sequence of the human BCLAF1 gene and the amino acid sequence of the human BCLAF1 protein are known.
  • the base sequence of the human BCLAF1 gene and the human BCLAF1 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_014739) and published.
  • the human BCLAF1 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human BCLAF1 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the aforementioned GenBank.
  • the human BCLAF1 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “BCLAF1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • TNIP1 gene means a gene encoding TNIP1 protein.
  • the nucleotide sequence of the human TNIP1 gene and the amino acid sequence of the human TNIP1 protein are known.
  • the nucleotide sequence of the human TNIP1 gene and the amino acid sequence of the human TNIP1 protein are registered in GenBank (GenBank Accession No. NM_006058) and published.
  • the human TNIP1 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human TNIP1 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human TNIP1 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “TNIP1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “STK38 gene” means a gene encoding STK38 protein.
  • the nucleotide sequence of the human STK38 gene and the amino acid sequence of the human STK38 protein are known.
  • the nucleotide sequence of the human STK38 gene and the amino acid sequence of the human STK38 protein are registered in GenBank (GenBank Accession No. NM_007271) and published.
  • the human STK38 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, and are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human STK38 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human STK38 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “STK38 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “ZC3H13 gene” means a gene encoding a ZC3H13 protein.
  • the base sequence of the human ZC3H13 gene and the amino acid sequence of the human ZC3H13 protein are known.
  • the base sequence of the human ZC3H13 gene and the amino acid sequence of the human ZC3H13 protein are registered in GenBank (GenBank Accession No. NM_015070) and published.
  • the human ZC3H13 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • the human ZC3H13 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “ZC3H13 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “C14orf122 gene” means a gene encoding a C14orf122 protein.
  • the base sequence of the human C14orf122 gene and the amino acid sequence of the human C14orf122 protein are known.
  • the base sequence of the human C14orf122 gene and the human C14orf122 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_016049) and published.
  • the human C14orf122 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • the human C14orf122 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a mutant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “C14orf122 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • C4orf18 gene means a gene encoding C4orf18 protein.
  • the nucleotide sequence of the human C4orf18 gene and the amino acid sequence of the human C4orf18 protein are known.
  • the nucleotide sequence of the human C4orf18 gene and the amino acid sequence of the human C4orf18 protein are registered in GenBank (GenBank Accession No. NM_001031700) and published.
  • the human C4orf18 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • the human C4orf18 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “C4orf18 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “ARS2 gene” means a gene encoding ARS2 protein.
  • the nucleotide sequence of the human ARS2 gene and the amino acid sequence of the human ARS2 protein are known.
  • the nucleotide sequence of the human ARS2 gene and the amino acid sequence of the human ARS2 protein are registered in GenBank (GenBank Accession No. NM_015908) and published.
  • the human ARS2 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, and are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human ARS2 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human ARS2 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “ARS2 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • C4orf20 gene means a gene encoding C4orf20 protein.
  • the base sequence of the human C4orf20 gene and the amino acid sequence of the human C4orf20 protein are known.
  • the base sequence of the human ARS2 gene and the human C4orf20 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_018359) and published.
  • the human C4orf20 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of an amino acid sequence registered in the above-mentioned GenBank.
  • the human C4orf20 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a mutant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “C4orf20 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • RC74 gene means a gene encoding RC74 protein.
  • the base sequence of the human RC74 gene and the amino acid sequence of the human RC74 protein are known.
  • the base sequence of the human RC74 gene and the amino acid sequence of the human RC74 protein are registered in GenBank (GenBank Accession No. NM_018250) and published.
  • the human RC74 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human RC74 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human RC74 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “RC74 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “EP400 gene” means a gene encoding the EP400 protein.
  • the base sequence of the human EP400 gene and the amino acid sequence of the human EP400 protein are known.
  • the base sequence of the human EP400 gene and the human EP400 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_015409) and published.
  • the human EP400 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, and are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human EP400 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above-mentioned GenBank.
  • the human EP400 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “EP400 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • RUFY1 gene means a gene encoding RUFY1 protein.
  • the nucleotide sequence of the human RUFY1 gene and the amino acid sequence of the human RUFY1 protein are known.
  • the nucleotide sequence of the human RUFY1 gene and the amino acid sequence of the human RUFY1 protein are registered in GenBank (GenBank Accession No. NM — 025158) and published.
  • the human RUFY1 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, and are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human RUFY1 proteins have a function equivalent to that of a protein comprising an amino acid sequence registered in the above-described GenBank.
  • the human RUFY1 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “RUFY1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • EPPK1 gene means a gene encoding EPPK1 protein.
  • the base sequence of the human EPPK1 gene and the amino acid sequence of the human EPPK1 protein are known.
  • the base sequence of the human EPPK1 gene and the amino acid sequence of the human EPPK1 protein are registered in GenBank (GenBank Accession No. NM_031308) and published.
  • the human EPPK1 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • the human EPPK1 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “EPPK1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • EPPK1 is also called Epiplakin1, and is known to be a gene belonging to the plakin family that plays a role in linking and binding cytoskeletal fibers to each other in adhesion junctions related to plasma membranes (Genes to Cells, 13 Vol., 667-678, 2008). From the experimental results of knockdown with siRNA, EPPK1 protein is presumed to maintain the keratin intermediate fiber and to be involved in keratinocyte differentiation (Journal of Cell Science, Vol. 118, No. 4, pp. 781-793 ,Year 2005). However, the relationship between the EPPK1 gene and protein and ApoA1, arteriosclerosis, etc. is not known.
  • SLA2 gene means a gene encoding SLA2 protein.
  • the base sequence of the human SLA2 gene and the amino acid sequence of the human SLA2 protein are known.
  • the base sequence of the human SLA2 gene and the human SLA2 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_032214) and published.
  • the human SLA2 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, which are caused by mutations based on polymorphism or mutation, are included.
  • these mutant human SLA2 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human SLA2 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “SLA2 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “FTMT gene” means a gene encoding FTMT protein.
  • the nucleotide sequence of the human FTMT gene and the amino acid sequence of the human FTMT protein are known.
  • the nucleotide sequence of the human FTMT gene and the amino acid sequence of the human FTMT protein are registered in GenBank (GenBank Accession No. NM_177478) and published.
  • the human FTMT protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, and are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human FTMT proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human FTMT gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a mutant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “FTMT gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • GPR139 gene means a gene encoding GPR139 protein.
  • the base sequence of the human GPR139 gene and the amino acid sequence of the human GPR139 protein are known.
  • the base sequence of the human GPR139 gene and the human GPR139 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_001002911) and published.
  • the human GPR139 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, and are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human GPR139 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human GPR139 gene includes not only a gene consisting of the base sequence registered in the above-mentioned GenBank but also a mutant that can occur in a human individual, for example, from the base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “GPR139 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • GPR139 protein is a G protein-coupled receptor expressed mainly in the central nervous system and is thought to play an important role in motor behavior (Biochemical and Biophysical Research Communications, Vol.331, pp.363-369). ,Year 2005). There is a report that the ⁇ subunit of the conjugated G protein is Gs type (Journal of Biomolecular Screening, Vol.14, No.7, 789-797, 2009), and a report that it is Gq / 11 type. (Biochemical and Biophysical Research Communications, 331, 363-369, 2005). However, the relationship between GPR139 gene and protein and ApoA1, arteriosclerosis, etc. is not known.
  • the “OR10AG1 gene” means a gene encoding the OR10AG1 protein.
  • the nucleotide sequence of the human OR10AG1 gene and the amino acid sequence of the human OR10AG1 protein are known.
  • the nucleotide sequence of the human OR10AG1 gene and the amino acid sequence of the human OR10AG1 protein are registered in GenBank (GenBank Accession No. NM_001005491) and published.
  • the human OR10AG1 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • the human OR10AG1 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “OR10AG1 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “C1QTNF9 gene” means a gene encoding a C1QTNF9 protein.
  • the base sequence of the human C1QTNF9 gene and the amino acid sequence of the human C1QTNF9 protein are known.
  • the base sequence of the human C1QTNF9 gene and the human C1QTNF9 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_178540) and published.
  • the human C1QTNF9 protein includes not only a protein consisting of the amino acid sequence registered in the above GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above GenBank.
  • the human C1QTNF9 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, for example, from a base sequence registered in the above-mentioned GenBank. And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “C1QTNF9 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has 99.5% or more, 99.7% or more, or 99.9% or more identity is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “UBE2NL gene” means a gene encoding the UBE2NL protein.
  • the base sequence of the human UBE2NL gene and the amino acid sequence of the human UBE2NL protein are known.
  • the base sequence of the human UBE2NL gene and the human UBE2NL protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_001012989) and published.
  • the human UBE2NL protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of the amino acid sequence registered in the above-mentioned GenBank.
  • the human UBE2NL gene includes not only a gene consisting of the base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “UBE2NL gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “FLJ16734 gene” means a gene encoding the FLJ16734 protein.
  • the nucleotide sequence of the human FLJ16734 gene and the amino acid sequence of the human FLJ16734 protein are known.
  • the nucleotide sequence of the human FLJ16734 gene and the amino acid sequence of the human FLJ16734 protein are registered in GenBank (GenBank Accession No. AK131514) and published.
  • the human FLJ16734 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of an amino acid sequence registered in the above-mentioned GenBank.
  • Proteins in which one amino acid or several amino acids are deleted, substituted and / or added in the protein, and are caused by mutations based on polymorphisms or mutations are included.
  • these mutant human FLJ16734 proteins have functions equivalent to those of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the human FLJ16734 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “FLJ16734 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • FLJ38596 gene means a gene encoding FLJ38596 protein.
  • the base sequence of the human FLJ38596 gene and the amino acid sequence of the human FLJ38596 protein are known.
  • the base sequence of the human FLJ38596 gene and the human FLJ38596 protein amino acid sequence are registered in GenBank (GenBank Accession No. NM_001039905) and published.
  • the human FLJ38596 protein includes not only a protein consisting of the amino acid sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual, and consists of an amino acid sequence registered in the above-mentioned GenBank.
  • the human FLJ38596 gene includes not only a gene consisting of a base sequence registered in the above-mentioned GenBank but also a variant that can occur in a human individual. For example, from the base sequence registered in the above-mentioned GenBank And a gene in which one or several bases are deleted, substituted, and / or added, and is generated by a mutation based on polymorphism or mutation.
  • the “FLJ38596 gene” is 80% or more, for example, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, 99% with respect to the base sequence registered in the above GenBank.
  • the variant which consists of a nucleotide sequence which has the identity of 99.5% or more, 99.7% or more, or 99.9% or more is included.
  • the identity of the base sequence can be determined using a known algorithm such as BLAST or FASTA.
  • these mutant genes encode a protein having a function equivalent to that of the protein consisting of the amino acid sequence registered in the above GenBank.
  • the “substance that suppresses gene expression” refers to a substance that suppresses the transcription of the target gene mRNA, a substance that degrades the transcribed mRNA, or a substance that suppresses the translation of the protein from the mRNA.
  • examples of such substances include siRNA, antisense oligonucleotides or ribozymes, or expression vectors thereof.
  • siRNA and its expression vector are preferable, and siRNA is particularly preferable.
  • “substances that suppress gene expression” include proteins, peptides, and other small molecules.
  • the target genes are CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, MT1, SLP2, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596 gene.
  • the “substance that suppresses the activity of the protein” is not particularly limited as long as it is a substance that suppresses the function inherent to the target protein.
  • examples of such substances include antibodies and antagonists.
  • a substance that suppresses the activity of GPR139 protein is an antibody that inhibits the increase in intracellular cAMP level by inhibiting the binding of endogenous ligand to GPR139 protein, or inhibits the increase in intracellular calcium concentration Or antagonist.
  • RNA is an RNA molecule having a double-stranded RNA portion consisting of about 15 to about 40 bases, and cleaves the mRNA of the target gene having a sequence complementary to the antisense strand of the siRNA. And has a function of suppressing the expression of the target gene.
  • siRNA in the present invention is CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, SLA, GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596 two mRNAs consisting of a sense RNA strand consisting of a sequence homologous to the RNA sequence in the mRNA and an antisense RNA strand consisting of a sequence complementary to the sense RNA sequence RNA comprising a single-stranded RNA portion.
  • the design and production of such siRNAs and mutant siRNAs described below are within the skill of the artisan.
  • the length of the double-stranded RNA portion is about 15 to about 40 bases, preferably 15 to 30 bases, more preferably 15 to 25 bases, still more preferably 18 to 23 bases, and most preferably 19 to 21 bases as a base. It is.
  • the end structure of the sense strand or antisense strand of siRNA is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it may have a blunt end or a protruding end (overhang) It is preferable that the 3 ′ end protrudes.
  • the siRNA having an overhang consisting of several bases, preferably 1 to 3 bases, more preferably 2 bases, at the 3 ′ end of the sense RNA strand and the antisense RNA strand suppresses the expression of the target gene. In many cases, the effect is large, which is preferable.
  • the type of the overhanging base is not particularly limited, and may be either a base constituting RNA or a base constituting DNA.
  • the siRNA in which 1 to several nucleotides are deleted, substituted, inserted and / or added in one or both of the sense strand or the antisense strand of the siRNA is also a therapeutic agent for treating arteriosclerosis according to the present invention. It can be used in a composition.
  • the 1 to several bases are not particularly limited, but are preferably 1 to 4 bases, more preferably 1 to 3 bases, and most preferably 1 to 2 bases.
  • Such mutations include those in which the number of bases in the overhang portion at the 3 ′ end is 0 to 3, or the base sequence in the overhang portion at the 3 ′ end is changed to another base sequence, or Those in which the length of the sense RNA strand differs from that of the antisense RNA strand by 1 to 3 bases due to insertion, addition or deletion, or in which the base is replaced with another base in the sense strand and / or antisense strand For example, but not limited to. However, it is necessary that the sense strand and the antisense strand can hybridize in these mutant siRNAs, and that these mutant siRNAs have the same ability to suppress gene expression as siRNA having no mutation.
  • the siRNA may be a molecule having a closed structure at one end, for example, an siRNA having a hairpin structure (Short hairpin RNA; shRNA).
  • shRNA is a RNA containing a sense strand RNA of a specific sequence of a target gene, an antisense strand RNA consisting of a sequence complementary to the sense strand RNA, and a linker sequence that connects both strands.
  • the sense strand portion and the antisense strand portion Hybridize to form a double stranded RNA portion.
  • siRNA does not show a so-called off-target effect in clinical use.
  • the off-target effect refers to the action of suppressing the expression of another gene that is partially homologous to the siRNA used in addition to the target gene.
  • NCBI National Center for Biotechnology Information
  • RNA of the present invention In order to produce the siRNA of the present invention, a known method such as a method using chemical synthesis or a method using a gene recombination technique can be appropriately used.
  • double-stranded RNA can be synthesized by a conventional method based on sequence information.
  • an expression vector incorporating a sense strand sequence or an antisense strand sequence is constructed, and the sense strand RNA or antisense strand RNA generated by transcription after introducing the vector into a host cell. It can also be produced by acquiring each of the above.
  • Desired double-stranded RNA can also be prepared.
  • a part of the nucleic acid constituting the siRNA may be DNA.
  • the siRNA may be a nucleic acid in which all or part of the nucleic acid constituting the siRNA is modified as long as it has the activity of suppressing the expression of the target gene.
  • the modified nucleic acid means a nucleic acid having a structure different from that of a natural nucleic acid, in which a nucleoside (base site, sugar site) and / or internucleoside binding site is modified.
  • modified nucleoside constituting the modified nucleic acid
  • examples of the “modified nucleoside” constituting the modified nucleic acid include abasic nucleoside; arabino nucleoside, 2′-deoxyuridine, ⁇ -deoxyribonucleoside, ⁇ -L-deoxyribonucleoside, and other sugars
  • examples include nucleosides having modifications; peptide nucleic acids (PNA), peptide nucleic acids to which phosphate groups are bound (PHONA), locked nucleic acids (LNA), morpholino nucleic acids and the like.
  • PNA peptide nucleic acids
  • PONA peptide nucleic acids to which phosphate groups are bound
  • LNA locked nucleic acids
  • morpholino nucleic acids and the like.
  • nucleoside having a sugar modification examples include substituted pentose monosaccharides such as 2′-O-methylribose, 2′-deoxy-2′-fluororibose, and 3′-O-methylribose; 1 ′, 2′-deoxyribose Arabinose; substituted arabinose sugars; nucleosides with hexose and alpha-anomeric sugar modifications are included.
  • These nucleosides may be modified bases with modified base sites. Examples of such modified bases include pyrimidines such as 5-hydroxycytosine, 5-fluorouracil, 4-thiouracil; purines such as 6-methyladenine and 6-thioguanosine; and other heterocyclic bases.
  • modified internucleoside linkage constituting the modified nucleic acid
  • examples of the “modified internucleoside linkage” constituting the modified nucleic acid include, for example, alkyl linker, glyceryl linker, amino linker, poly (ethylene glycol) linkage, methylphosphonate internucleoside linkage; methylphosphonothioate, phosphotriester , Phosphothiotriester, phosphorothioate, phosphorodithioate, triester prodrug, sulfone, sulfonamide, sulfamate, formacetal, N-methylhydroxylamine, carbonate, carbamate, morpholino, boranophosphonate, phosphoramidate, etc.
  • Non-natural internucleoside linkages include, for example, alkyl linker, glyceryl linker, amino linker, poly (ethylene glycol) linkage, methylphosphonate internucleoside linkage; methylphosphonothioate
  • the sequences described in SEQ ID NOs: 2 to 157 in the sequence listing can be preferably used.
  • Tables 1 and 2 show the nucleotide sequences of these siRNAs. In Tables 1 and 2, the uppercase letters indicate the sense RNA sequence and the antisense RNA sequence, and the lowercase letters indicate the 3 ′ end overhang sequence.
  • the first sequence in Table 1 is a double-stranded siRNA composed of a sense strand shown in SEQ ID NO: 2 and an antisense strand shown in SEQ ID NO: 3, and tt at the 3 ′ end of SEQ ID NO: 2 And tc at the 3 ′ end of SEQ ID NO: 3 is an overhang sequence. Since these siRNAs significantly enhance the secretion of ApoA1, the effect of the pharmaceutical composition for treating dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer containing these siRNAs is great.
  • Oligonucleotides complementary to the mRNA of the target gene are called “antisense oligonucleotides”, and the function of the mRNA is suppressed by forming a double strand with the gene (mRNA) targeted by the antisense oligonucleotide.
  • Antisense oligonucleotides are not limited to those that are completely complementary to the target gene (mRNA), but may contain some mismatches as long as they can be stably hybridized with mRNA.
  • Antisense oligonucleotides may be modified. By applying an appropriate modification, the antisense oligonucleotide becomes difficult to be degraded in vivo, and the expression of the target gene can be more stably inhibited.
  • modified oligonucleotides include S-oligo type (phosphorothioate type), C-5 thiazole type, D-oligo type (phosphodiester type), M-oligo type (methyl phosphonate type), peptide nucleic acid
  • modified antisense oligonucleotides such as phosphodiester bond type, C-5 propynyl pyrimidine type, 2-O-propyl ribose, 2′-methoxyethoxy ribose type.
  • the antisense oligonucleotide may be one in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a sulfur atom.
  • Such an antisense oligonucleotide is particularly excellent in nuclease resistance and affinity for RNA.
  • Examples of the antisense oligonucleotide in which at least a part of the oxygen atom constituting the phosphate group is substituted or modified with a sulfur atom include oligonucleotides such as S-oligo type.
  • An antisense oligonucleotide (or a derivative thereof) can be synthesized by a conventional method, and can be easily synthesized by, for example, a commercially available DNA synthesizer (for example, manufactured by AppliedBiosystems).
  • Examples of the synthesis method include a solid phase synthesis method using phosphoramidite and a solid phase synthesis method using hydrogen phosphonate.
  • RNA having an enzyme activity that cleaves nucleic acid refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligoDNA having the base sequence of the enzyme active site also has a nucleic acid cleaving activity. In the book, it is used as a concept including DNA as long as it has sequence-specific nucleic acid cleavage activity. Specifically, the ribozyme can specifically cleave mRNA or an initial transcription product encoding a target gene within the coding region (including an intron portion in the case of the initial transcription product). The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and hammerhead type and hairpin type are known.
  • the hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends adjacent to the portion having the hammerhead structure (about 10 bases in total) are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, it should be a hybrid ribozyme further linked with a tRNA-modified sequence. (Nucleic Acids Res., 29 (13): 2780-2788 (2001)).
  • the substance that suppresses the expression of the target gene may be a nucleic acid molecule such as siRNA, antisense oligonucleotide, or ribozyme, and an expression vector encoding the nucleic acid molecule.
  • the oligonucleotide or polynucleotide encoding the nucleic acid molecule must be operably linked to a promoter capable of exhibiting promoter activity in the mammalian cells to be administered.
  • the promoter to be used is not particularly limited as long as it can function in the mammal to be administered, but for example, polIII promoter (eg, tRNA promoter, U6 promoter, H1 promoter), mammalian promoter (eg, CMV promoter, CAG promoter, SV40 promoter) and the like.
  • the expression vector preferably contains a transcription termination signal, ie a terminator region, downstream of the oligo (poly) nucleotide encoding the nucleic acid molecule.
  • selection marker genes for selection of transformed cells are also included.
  • the basic backbone vector used as the expression vector is not particularly limited, and examples thereof include a plasmid vector and a viral vector.
  • Suitable vectors for administration to mammals such as humans include viral vectors such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poxvirus, poliovirus, Sindbis virus, Sendai virus, etc. .
  • siRNA expression vectors examples include siRNA, antisense oligonucleotide or ribozyme expression vectors. Among them, siRNA expression vectors are preferable.
  • the nucleic acid sequences encoded by the siRNA expression vectors of the present invention are preferably those shown in Tables 1 and 2.
  • the siRNAs listed in Tables 1 and 2 remarkably enhance the secretion of ApoA1, so that the effect of the pharmaceutical composition for treating dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer containing these siRNAs is large. It is.
  • a substance that suppresses the expression of the UBE2NL, FLJ16734, or FLJ38596 gene or suppresses the activity of the protein encoded by these genes can be used as an active ingredient of a pharmaceutical composition.
  • the pharmaceutical composition of the present invention can be used as a pharmaceutical composition for the treatment of dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer by administering the pharmaceutical composition in vivo.
  • a single expression suppressing substance may be used as an active ingredient, or a plurality of gene expression suppressing substances may be used as active ingredients.
  • the target genes of the plurality of gene expression inhibitors may be different genes.
  • the expression-suppressing substance of the pharmaceutical composition of the present invention is siRNA
  • one or more siRNA may be used as an active ingredient.
  • the pharmaceutical composition of the present invention may contain siRNA for GPR139 gene and siRNA for EPPK1 gene.
  • the type of disease that is the target of treatment of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include dyslipidemia, hyperlipidemia, arteriosclerosis, atherosclerosis, hypercholesterolemia Symptom, hypertriglyceridemia, diabetes, obesity, syndrome X and / or Alzheimer.
  • the pharmaceutical composition of the present invention can employ both oral and parenteral dosage forms.
  • the pharmaceutical composition of the present invention can be formulated according to a conventional method, and may contain a pharmaceutically acceptable carrier or additive.
  • Such carriers and additives include water, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, carboxymethyl.
  • additives examples include additives.
  • the additive is selected from the above alone or in appropriate combination depending on the dosage form of the pharmaceutical composition of the present invention.
  • the dosage form in the case of oral administration, it can be administered as a tablet, capsule, fine granule, powder, granule, solution, syrup or the like, or in an appropriate dosage form.
  • pulmonary dosage forms for example, those using a nephriser etc.
  • nasal dosage forms for example, transdermal dosage forms (for example, ointments, creams), injection dosage forms and the like
  • injection dosage form it can be administered systemically or locally by intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection or the like.
  • the expression inhibitor is introduced into a phospholipid endoplasmic reticulum such as a liposome.
  • the endoplasmic reticulum can be used as the pharmaceutical composition of the present invention.
  • the dosage of the pharmaceutical composition of the present invention varies depending on age, sex, symptoms, administration route, administration frequency, and dosage form.
  • the administration method is appropriately selected depending on the age and symptoms of the patient.
  • the effective dose is 0.01 ⁇ g to 1000 mg, preferably 0.1 ⁇ g to 100 ⁇ g, per kg body weight.
  • the therapeutic agent is not limited to these doses.
  • the invention in a further aspect, (I) CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, TLA1, FTMT, ECR1, C9
  • a method for treating dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer's comprising a step of administering a substance that suppresses the expression of the FLJ16734 or FLJ38596 gene or suppresses the activity of the protein encoded by these genes , (Ii) CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, used for the treatment of dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer Substance
  • the present invention in another aspect, CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, SLA, Providing a method for screening therapeutic agents for dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer's using index of inhibition of GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596 gene expression or antagonist activity of GPR139 protein To do.
  • test substance to be subjected to the screening method may be any known compound and novel compound, for example, nucleic acid, carbohydrate, lipid, protein, peptide, low molecular organic compound, compound prepared using combinatorial chemistry technology
  • libraries random peptide libraries prepared by solid phase synthesis and phage display methods, or natural components derived from microorganisms, animals and plants, marine organisms, and the like.
  • the screening method of the present invention comprises: (1) a step of bringing cells into contact with a test substance; (2) CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, EPPK1, EPPK1, EPPK1, EPPK1, Measuring the expression level of the FTMT, GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596 gene, and comparing the expression level with the expression level of the gene in a control cell not contacted with the test substance; and (3) a test substance A substance that increases secretion of ApoA1 or a dyslipidemia when the expression of the gene in a cell to which the test substance is given is lower than the expression of the gene in a cell that is not given the test substance, Selecting
  • the cells are CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, LA2S , FTMT, GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596 means a cell capable of measuring the expression.
  • cells and a test substance are placed in contact with each other in a culture medium.
  • CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, EPPK1, SLA2, EFTAG, GPR139, ETL1, GPR139, E1 Cells that can measure FLJ38596 gene expression are CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, E400 , SLA2, FTMT, GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596 gene, for example, a cell in which the expression level of a transcription product or translation product can be evaluated directly
  • a cell capable of directly evaluating the expression level of the gene product can be a cell capable of naturally expressing the gene, while a cell capable of indirectly evaluating the expression level of the gene product includes: Examples include cells that allow reporter assay for the gene transcription regulatory region.
  • the cells capable of measuring the expression of the gene animal cells such as mice, rats, hamsters, guinea pigs, rabbits, dogs, monkeys or human mammalian cells can be used, and human-derived cells are particularly preferable.
  • a cell enabling a reporter assay for a gene transcription regulatory region is a cell containing a target gene transcription regulatory region and a reporter gene operably linked to the region.
  • the target gene transcription regulatory region and the reporter gene can be inserted into an expression vector.
  • the transcriptional regulatory region of the target gene is not particularly limited as long as it can control the expression of the target gene. For example, a region from the transcription start point to about 2 kbp upstream, or one or more bases in the base sequence of the region Examples include a region consisting of a base sequence deleted, substituted or added and having the ability to control the transcription of the target gene.
  • the reporter gene may be any gene that encodes a detectable protein or an enzyme that produces a detectable substance.
  • the GFP green fluorescent protein
  • GUS ⁇ -glucuronidase
  • LUC luciferase
  • CAT Chloramphenicol acetyltransferase
  • a cell into which a gene transcription regulatory region and a reporter gene operably linked to the region are introduced can be used for quantitative analysis of the expression level of the reporter gene as long as the transcriptional regulatory function of the target gene can be evaluated. As long as it is, it is not particularly limited.
  • the culture medium in which the cells and the test substance are brought into contact is appropriately selected depending on the type of cells used.
  • a minimal essential medium (MEM) containing about 5-20% fetal calf serum, Dulbecco's modification Minimum essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like.
  • the culture conditions are also appropriately determined according to the type of cells to be used.
  • the pH of the medium is about 6 to about 8
  • the culture temperature is usually about 30 to about 40 ° C.
  • the culture time is About 12 to about 144 hours.
  • step (2) of the above method first, CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400 in cells contacted with the test substance , RUFY1, EPPK1, SLA2, FTMT, GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734 or FLJ38596 gene is measured.
  • the expression level can be measured by a method known per se in consideration of the type of cells used.
  • cells capable of measuring the expression of the gene include CTNNB1, NFE2L2, TXN, CUL1, RAE1, TNFRSF10C, BCLAF1, TNIP1, STK38, ZC3H13, C14orf122, C4orf18, ARS2, C4orf20, RC74, EP400, RUFY1, LA2S , FTMT, GPR139, OR10AG1, C1QTNF9, UBE2NL, FLJ16734, or FLJ38596 gene is used in a method known per se for a gene product, for example, a transcription product or a translation product. It can be measured.
  • the expression level of the transcript can be measured by preparing total RNA from cells and performing RT-PCR, Northern blotting, or the like.
  • the expression level of the translation product can be measured by preparing an extract from the cells and using an immunological technique.
  • an immunological technique a radioisotope immunoassay (RIA method), an ELISA method (Methods in Enzymol. 70: 419-439 (1980)), a fluorescent antibody method, or the like can be used.
  • the expression level can be measured based on the signal intensity of the reporter.
  • step (3) of the above method the expression level of the gene in the cell contacted with the test substance is compared with the expression level of the gene in the control cell not contacted with the test substance.
  • the comparison of the expression level is preferably performed based on the presence or absence of a significant difference.
  • the expression level of the gene in the control cell not contacted with the test substance is the expression level measured at the same time, even if the expression level was measured in advance, compared to the measurement of the expression level of the gene in the cell contacted with the test substance.
  • the expression level is preferably measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment.
  • the screening method of the present invention comprises: (1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or (2) an amino acid in which one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1 have been deleted, substituted and / or added
  • the screening method of the present invention comprises: (1) an agonist for GPR139 protein, a test substance and 1: a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or 2: deletion of one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1, Contacting a polypeptide having a substituted and / or added amino acid sequence and having GPR139 activity; (2) a step of comparing the binding activity between the agonist contacted with the test substance and the polypeptide with the binding activity between the agonist contacted with the test substance and the polypeptide; and (3) the agonist and polypeptide contacted with the test substance.
  • a test substance as a substance that increases the secretion of ApoA1 when the binding activity is changed compared to the binding activity of an agonist that does not contact the test substance and the polypeptide, and a substance that increases the secretion of ApoA1
  • the agonist for GPR139 protein in step (1) of the above method can be appropriately selected, and examples thereof include LP-360924 reported in a paper (Journal of Biomolecular Screening, Vol. 14, No. 7). 789-797, 2009)
  • the polypeptide having the amino acid sequence represented by SEQ ID NO: 1 includes an isolated polypeptide, a polypeptide in a membrane fraction, and an intracellular polypeptide.
  • a polypeptide having GPR139 activity has a binding activity to an endogenous ligand for GPR139 protein, and has an action of increasing the amount of cAMP in a cell by coupling to G ⁇ s, or coupled to G ⁇ q. Means a polypeptide having an action of increasing intracellular calcium concentration.
  • the screening method of the present invention comprises: (1) an agonist for GPR139 protein, a test substance and 1: a polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or 2: deletion of one or several amino acids of the amino acid sequence represented by SEQ ID NO: 1, Contacting a cell having a substituted and / or added amino acid sequence and expressing a polypeptide having GPR139 activity; (2) a step of comparing the intracellular cAMP amount or calcium ion concentration in the cell contacted with the test substance with the intracellular cAMP amount or calcium ion concentration in the control cell not contacted with the test substance, and (3) adding the test substance When the intracellular cAMP level or calcium ion concentration in a given cell is changed compared to the intracellular cAMP level or calcium ion concentration in a cell not given the test substance, the test substance increases the secretion of ApoA1.
  • the culture medium in which the cells expressing the GPR139 protein are contacted with the agonist and the test substance is appropriately selected according to the type of cells used, for example, about 5 Minimal essential medium (MEM) containing ⁇ 20% fetal calf serum, Dulbecco's modified minimal essential medium (DMEM), RPMI 1640 medium, 199 medium, and the like.
  • MEM Minimal essential medium
  • DMEM Dulbecco's modified minimal essential medium
  • RPMI 1640 medium 199 medium
  • the culture conditions are also appropriately determined according to the type of cells to be used.
  • the pH of the medium is about 6 to about 8
  • the culture temperature is usually about 30 to about 40 ° C.
  • the culture time is About 12 to about 144 hours.
  • step (2) of the above method first, the intracellular cAMP amount or calcium ion concentration in the cell contacted with the agonist and the test substance is measured.
  • the intracellular cAMP amount or calcium ion concentration can be measured by a method known per se in consideration of the type of cells used.
  • the intracellular cAMP amount or calcium ion concentration in the cell contacted with the test substance is compared with the intracellular cAMP amount or calcium ion concentration in the control cell not contacted with the test substance.
  • the comparison of intracellular cAMP amount or calcium ion concentration in step (3) of the above method is preferably performed based on the presence or absence of a significant difference.
  • the intracellular cAMP amount or calcium ion concentration in the control cells not contacted with the test substance is the intracellular cAMP amount or calcium ion concentration measured in advance with respect to the intracellular cAMP amount or calcium ion concentration in the cells contacted with the test substance.
  • the intracellular cAMP amount or calcium ion concentration measured simultaneously may be used, but the intracellular cAMP amount or calcium ion concentration measured simultaneously from the viewpoint of the accuracy and reproducibility of the experiment is preferable.
  • Negative Control # 1 siRNA (NT-1; manufactured by Ambion) was dispensed in the same amount on each plate as a negative control, and used for hit criteria setting.
  • Opti-MEM I Gabco
  • siRNA introduction reagent RNAiMAX Invitrogen 0.14 ⁇ l were mixed and kept at room temperature for 5 minutes, then 384-well plate dispensed with the above siRNA Added to.
  • siRNA focus library (3 siRNAs / gene) for the above 179 genes was prepared, and a reproducibility test was performed according to the same protocol as in Example 1. As a result, out of 3 siRNAs for the same gene, 2 or more siRNAs with HTRF Ratio of 120% or more compared to the negative control were selected, and 27 genes that increase ApoA1 secretion in HepG2 cells were obtained. .
  • the amount of ApoB secreted when each gene was knocked down with siRNA was evaluated as a counter assay.
  • siRNA 3 siRNAs / gene
  • the secretion amount of ApoB was measured using ApoB HTRF assays (manufactured by Cisbio) in the same protocol as in Example 1. No increase in ApoB secretion was observed for the 26 genes shown below (115% or less of the negative control). From the above experimental results, the 26 genes were identified as a novel target having an ApoA1 secretion enhancing action.
  • RNAiMAX manufactured by Invitrogen
  • Opti-MEM I and 1.1 ⁇ l of RNAiMAX were mixed and held at room temperature for 5 minutes, and then added to the 96-well plate into which the aforementioned siRNA was dispensed.
  • the secretion amount of ApoA1 was measured by the HTRF method using the same protocol as in Example 1.
  • Table 4 shows that for CTNNB1, EPPK1, and GPR139, the secretion amount of ApoA1 increased to 110% or more with respect to the negative control in siRNA of 2 or more sequences out of 3 sequences.
  • Antisense oligonucleotide sequence design Design the base sequence of antisense oligonucleotides for three new target genes.
  • the base sequence information of each gene for example, information registered in GenBank of the National Center for Biological Information (human GPR139, NM_001002911; human EPPK1, NM_0313086; human CTNNB1, NM_001904) can be used.
  • GenBank GenBank of the National Center for Biological Information
  • the chain length is 13 bases
  • the selection criteria are not limited to the above as long as a sequence having an expression suppressing effect on the target nucleic acid can be selected.
  • Examples of antisense oligonucleotides for each new target designed according to the above criteria are shown in Tables 5 to 11, but the antisense oligo is not limited to these as long as it has an effect of suppressing expression on the target nucleic acid. .
  • These antisense oligonucleotides may contain a modified base, and the base may be bound by a bond other than a phosphodiester bond.
  • At least one of the bases shown in lowercase letters in Tables 5 to 11 may be LNA (Locked Nucleic Acid), and at least one of the bonds between the bases shown in uppercase letters may be a phosphorothioate bond.
  • LNA Locked Nucleic Acid
  • Specific examples of such modified antisense oligonucleotides include those in which all the bases shown in lower case letters in Tables 5 to 11 are LNA, and the bonds between the bases shown in upper case letters are phosphorothioate bonds. It is not limited to these. Sequences are written in the 5 ′ to 3 ′ direction.
  • Phosphinate oligonucleotides are prepared using the method described in US Pat. No. 5,508,270.
  • Alkyl phosphonate oligonucleotides are prepared using the method described in US Pat. No. 4,469,863.
  • 3′-deoxy-3′-methylene phosphonate oligonucleotides are prepared using the methods described in US Pat. No. 5,610,289 or US Pat. No. 5,562,050.
  • Phosphoramidite oligonucleotides are prepared using the methods described in US Pat. No. 5,256,775 or US Pat. No. 5,366,878.
  • Alkylphosphonothioate oligonucleotides are prepared using the methods described in WO94 / 17093 or WO94 / 02499.
  • a 3′-deoxy-3′-aminophosphoramidate oligonucleotide is prepared using the method described in US Pat. No. 5,476,925.
  • Phosphotriester oligonucleotides are prepared using the methods described in US Pat. No. 5,032,243.
  • Borano phosphate oligonucleotides are prepared using the methods described in US Pat. No. 5,130,302 or US Pat. No. 5,177,198.
  • an oligonucleotide for example, LNA having a modified nucleoside is prepared.
  • the target nucleic acid expression suppression effect of an antisense oligonucleotide can be evaluated in various cell types if the target nucleic acid is present at a measurable level.
  • the expression suppression effect can be determined by a conventional method using, for example, a quantitative PCR method or Northern blot analysis. In the following, an example using HepG2, which is a human liver cancer-derived cell line, is described, but the cell type is not particularly limited as long as the target nucleic acid is expressed.
  • a 24-well plate consisting of D-MEM (Sigma Aldrich) and 10% Fetal Bovine Serum, dispense 500 ⁇ l of a culture solution containing 25,000 HepG2 cells, and in a 37 ° C, 5% CO2 environment Incubate overnight to allow cells to adhere.
  • RNA is extracted using RNeasy Plus Mini Kit (Qiagen) and quantitative PCR is performed using QuantiTect SYBR Green RT-PCR Kit (Qiagen).
  • ⁇ -actin is measured, and the relative expression level is calculated by the comparative Ct method using the same gene as an internal standard.
  • a primer for quantitative PCR of each gene for example, a product of Takara Bio Inc. (catalog number; CTNNB1, HA135644; EPPK1, HA114366; GPR139, HA033632; ⁇ -actin, HA067803) can be used. Compared with the negative control, it is possible to examine the degree of expression decrease of each gene and evaluate the expression suppression effect of each antisense oligonucleotide.
  • the ApoA1 secretion enhancing action of antisense oligonucleotide For antisense oligonucleotides that have been confirmed to suppress the expression of the target nucleic acid, the ApoA1 secretion enhancing action on cells is evaluated.
  • the ApoA1 secretion enhancing action can be routinely determined, for example, by measuring the amount of ApoA1 protein in the culture supernatant sample by the HTRF method, as in the above-described examples relating to the primary screening.
  • examples using HepG2 cells are provided, but other cell types can be used as long as antisense oligonucleotides can be introduced into the cell types to be selected.
  • a culture solution containing 40,000 HepG2 cells consisting of D-MEM (Sigma Aldrich) and 2% Fetal Bovine Serum was dispensed and cultured overnight at 37 ° C in a 5% CO2 environment.
  • Adhere. Add a mixture of antisense oligonucleotide 18 pmol, 100 ⁇ l of Opti-MEM I, and 2 ⁇ l of Lipofectamine 2000 (Invitrogen) whose expression suppression effect has been confirmed above, and incubate for several hours. In addition, prepare a sample without antisense as a negative control. After exchanging the medium, the cells are further cultured for 96 hours, and the culture supernatant is recovered.
  • ApoA1 HTRF assays (Cisbio) were used to measure the amount of ApoA1 protein by the HTRF method, and the amount of ApoA1 secretion increased in the antisense-treated group compared to the negative control group. Evaluate what to do.
  • the present invention can be used in the field of pharmaceuticals, particularly in the field of development and production of therapeutic agents for dyslipidemia, hyperlipidemia, arteriosclerosis or Alzheimer's.

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Abstract

La présente invention a trait à : l'utilisation, comme cible de traitement de la dyslipidémie, de l'hyperlipidémie, de l'artériosclérose ou de la maladie d'Alzheimer, d'un gène codant pour les protéines CTNNB1 humaine, NFE2L2 humaine, TXN humaine, CUL1 humaine, RAE1 humaine, TNFRSF10C humaine, BCLAF1 humaine, TNIP1 humaine, STK38 humaine, ZC3H13 humaine, C14orf122 humaine, C4orf18 humaine, ARS2 humaine, C4orf20 humaine, RC74 humaine, EP400 humaine, RUFY1 humaine, EPPK1 humaine, SLA2 humaine, FTMT humaine, GPR139 humaine, OR10AG1 humaine, C1QTNF9 humaine, UBE2NL humaine, FLJ16734 humaine ou FLJ38596 humaine ; un agent de traitement de la dyslipidémie, de l'hyperlipidémie, de l'artériosclérose ou de la maladie d'Alzheimer ciblant les gènes ; et une méthode de criblage à la recherche d'agents de traitement de la dyslipidémie, de l'hyperlipidémie, l'artériosclérose ou de la maladie d'Alzheimer ciblant les gènes.
PCT/JP2013/060798 2012-04-11 2013-04-10 Nouvelle composition médicamenteuse pour le traitement de l'artériosclérose et méthode de criblage de médicaments pour le traitement de l'artériosclérose WO2013154128A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006522831A (ja) * 2003-04-09 2006-10-05 アルナイラム ファーマシューティカルズ インコーポレイテッド ｉＲＮＡ複合体
JP2008535474A (ja) * 2005-02-10 2008-09-04 アメリカ合衆国 β−カテニンスプライス変異体を用いた癌の診断および処置の方法
WO2008109460A2 (fr) * 2007-03-02 2008-09-12 Nastech Pharmaceutical Company Inc. Composés d'acide nucléique pour inhiber l'expression du gène ctnnb1 et utilisations de ceux-ci
WO2012018754A2 (fr) * 2010-08-02 2012-02-09 Merck Sharp & Dohme Corp. Inhibition à médiation par interférence arn de caténine (protéine associée à cadhérine), expression du gène bêta 1 (ctnnb1) à l'aide de petit acide nucléique interférent (sian)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006522831A (ja) * 2003-04-09 2006-10-05 アルナイラム ファーマシューティカルズ インコーポレイテッド ｉＲＮＡ複合体
JP2008535474A (ja) * 2005-02-10 2008-09-04 アメリカ合衆国 β−カテニンスプライス変異体を用いた癌の診断および処置の方法
WO2008109460A2 (fr) * 2007-03-02 2008-09-12 Nastech Pharmaceutical Company Inc. Composés d'acide nucléique pour inhiber l'expression du gène ctnnb1 et utilisations de ceux-ci
WO2012018754A2 (fr) * 2010-08-02 2012-02-09 Merck Sharp & Dohme Corp. Inhibition à médiation par interférence arn de caténine (protéine associée à cadhérine), expression du gène bêta 1 (ctnnb1) à l'aide de petit acide nucléique interférent (sian)

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